2016-2

Volume 33 Issue 2 June 2016 80 TL

ISSN 1300-7777

Review Article

The Role of Angiogenesis in Haemophilic Arthropathy: Where Do We Stand and Where Are We Going?

Alexandra Agapidou, et al.; Thessaloniki, Greece

Research Articles

Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and

Primary Myelofibrosis

İpek Yönal, et al.; İstanbul, Turkey

D-index: A New Scoring System in Febrile Neutropenic Patients for Predicting Invasive Fungal Infections

Gülden Yılmaz, et al.; Ankara, Turkey

Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher

Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

Türker Bilgen, et al.; Antalya, Tekirdağ, Turkey

The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin

Hadil A. Al Otair, et al.; Riyadh, Saudi Arabia

Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems

Özlen Bektaş, et al.; Ankara, Turkey

Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It?

Olga Meltem Akay, et al.; Eskişehir, Turkey

Immature Reticulocyte Fraction and Absolute Neutrophil Count as Predictor of Hemopoietic Recovery in Patients

with Acute Lymphoblastic Leukemia on Remission Induction Chemotherapy

Shan E. Rauf, et al.; Rawalpindi, Pakistan

The Prognostic Significance of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia

Nergiz Erkut, et al.; Trabzon, Turkey

Investigation of Rho-Kinase Expressions and Polymorphisms in Mantle Cell Lymphoma Patients

Didar Yanardağ Açık, et al.; Gaziantep, Turkey

Prospective Audit of Blood Donor Selection Process in a Tertiary Care Hospital of a Developing Country

Naila Raza; Karachi, Pakistan

Cover Picture:

Dilek Argon

Fog Covering the Bosphorus, İstanbul

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Editor-in-Chief

Reyhan Küçükkaya

İstanbul Bilim University, İstanbul, Turkey

Associate Editors

Ayşegül Ünüvar

İstanbul University, İstanbul, Turkey

Cengiz Beyan

Gülhane Military Medical Academy,

Ankara, Turkey

Hale Ören

Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

Selami Koçak Toprak

Ankara University, Ankara, Turkey

Semra Paydaş

Çukurova University, Adana, Turkey

Assistant Editors

A. Emre Eşkazan

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

Ali İrfan Emre Tekgündüz

Dr. A. Yurtaslan Ankara Oncology Training

and Research Hospital, Ankara, Turkey

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

İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya

Gazi University, Ankara, Turkey

International Review Board

Nejat Akar

Görgün Akpek

Serhan Alkan

Çiğdem Altay

Koen van Besien

Ayhan Çavdar

M. Sıraç Dilber

Ahmet Doğan

Peter Dreger

Thierry Facon

Jawed Fareed

Gösta Gahrton

Dieter Hoelzer

Marilyn Manco-Johnson

Andreas Josting

Emin Kansu

Winfried Kern

Nigel Key

Korgün Koral

Abdullah Kutlar

Luca Malcovati

Robert Marcus

Jean Pierre Marie

Ghulam Mufti

Gerassimos A. Pangalis

Antonio Piga

Ananda Prasad

Jacob M. Rowe

Jens-Ulrich Rüffer

Norbert Schmitz

Orhan Sezer

Anna Sureda

Ayalew Tefferi

Nükhet Tüzüner

Catherine Verfaillie

Srdan Verstovsek

Claudio Viscoli

Past Editors

Erich Frank

Orhan Ulutin

Hamdi Akan

Aytemiz Gürgey

Senior Advisory Board

Yücel Tangün

Osman İlhan

Muhit Özcan

Teoman Soysal

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

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Publishing

Services

GALENOS PUBLISHER

Molla Gürani Mah. Kaçamak Sk. No: 21, 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 : İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara / Turkey

Phone : +90 312 490 98 97

Fax : +90 312 490 98 68

E-mail : info@tjh.com.tr

ISSN: 1300-7777

Turkish Society of Hematology Editorial Board

Ahmet Muzaffer Demir, President

Güner Hayri Özsan, General Secretary

T. Tiraje Celkan, Vice President

M. Cem Ar, Research Secretary

E. Naci Tiftik, Treasurer

Meltem Yüksel, Member

İlknur Kozanoğlu, Member

Online Manuscript Submission

http://mc.manuscriptcentral.com/tjh

Web page

www.tjh.com.tr

Owner on behalf of the Turkish Society of Hematology

Türk Hematoloji Derneği adına yayın sahibi

Ahmet Muzaffer Demir

Üç ayda bir yayımlanan İngilizce süreli yayındır.

International scientific journal published quarterly.

Publishing Manager

Sorumlu Yazı İşleri Müdürü

Güner Hayri Özsan

Management Address

Yayın İdare Adresi

Türk Hematoloji Derneği

İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya,

Ankara / Turkey

Publishing House / Yayınevi

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey

Tel: +90 212 621 99 25 Faks: +90 212 621 99 27

E-posta: info@galenos.com.tr

Baskı: Özgün Ofset Ticaret Ltd. Şti.

Yeşilce Mah. Aytekin Sk. No: 21 34418 4. Levent / İSTANBUL

Printing Date / Basım Tarihi

15.05.2016

Cover Picture

Dilek Argon is currently working at Academic Hospital, Division of

Hematology, İstanbul, Turkey.

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.

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AIMS AND SCOPE

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

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

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

periodical encompassing subjects relevant to hematology.

The Editorial Board of The Turkish Journal of Hematology adheres to the

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

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

Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and

Strengthening the Reporting of Observational Studies in Epidemiology

(STROBE).

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

hematological research of the highest scientific quality and clinical relevance.

Additionally, educational material, reviews on basic developments, editorial

short notes, images in hematology, and letters from hematology specialists

and clinicians covering their experience and comments on hematology

and related medical fields as well as social subjects are published. As of

December 2015, The Turkish Journal of Hematology does not accept case

reports. Important new findings or data about interesting hematological

cases may be submitted as a brief report.

General practitioners interested in hematology and internal medicine

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

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

Hematology is indexed, as follows:

- PubMed Medline

- PubMed Central

- Science Citation Index Expanded

- EMBASE

- Scopus

- CINAHL

- Gale/Cengage Learning

- EBSCO

- DOAJ

- ProQuest

- Index Copernicus

- Tübitak/Ulakbim Turkish Medical Database

- Turk Medline

Impact Factor: 0.360

Subscription Information

The Turkish Journal of Hematology is sent free-of-charge to members

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

Hematologists, other medical specialists that are interested in hematology,

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

published volumes are available in full text free-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 Diz 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.

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TURKISH JOURNAL OF HEMATOLOGY

INSTRUCTIONS TO 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 kind

of reviewing system.

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

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

each section of the structured abstract with the appropriate subheading

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

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

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

assistance should be provided on the title page.

Original Manuscripts

Title Page

Title: The title should provide important information regarding the

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

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

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

of ferrous succinate tablet formulations without correction for baseline

circadian changes in iron concentration in healthy Chinese male subjects:

A single-dose, randomized, 2-period crossover study. Clin Ther. 2011; 33:

2054-2059).

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

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

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

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

should be followed by a superscript number that corresponds to their

institution, which is listed separately. Please provide contact information

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

telephone and fax numbers.

Running Head: The running head should not be more than 40 characters,

including spaces, and should be located at the bottom of the title page.

Word Count: A word count for the manuscript, excluding abstract,

acknowledgments, figure and table legends, and references, should be

provided not exceed 2500 words. The word count for an abstract should

be not exceed 300 words.

Conflict-of-Interest Statement: To prevent potential conflicts of

interest from being overlooked, this statement must be included in each

manuscript. In case there are conflicts of interest, every author should

complete the ICMJE general declaration form, which can be obtained at:

http://www.icmje.org/coi_disclose.pdf.

Abstract and Keywords: The second page should include an abstract

that does not exceed 300 words. For manuscripts sent by authors in

Turkey, a title and abstract in Turkish are also required. As most readers

read the abstract first, it is critically important. Moreover, as various

electronic databases integrate only abstracts into their index, important

findings should be presented in the abstract.

Objective: The abstract should state the objective (the purpose of the

study and hypothesis) and summarize the rationale for the study.

Materials and Methods: Important methods should be written

respectively.

Results: Important findings and results should be provided here.

Conclusion: The study’s new and important findings should be

highlighted and interpreted.

Other types of manuscripts, such as reviews, perspectives, and

editorials, will be published according to uniform requirements.

Provide 3-10 keywords below the abstract to assist indexers. Use

terms from the Index Medicus Medical Subject Headings List

(for randomized studies a CONSORT abstract should be provided (http://

www.consort-statement.org).

Introduction: The introduction should include an overview of the

relevant literature presented in summary form (one page), and what ever

remains interesting, unique, problematic, relevant, or unknown about

the topic must be specified. The introduction should conclude with the

rationale for the study, its design, and its objective(s).

Materials and Methods: Clearly describe the selection of observational

or experimental participants, such as patients, laboratory animals, and

controls, including inclusion and exclusion criteria and a description of the

source population. Identify the methods and procedures in sufficient detail

to allow other researchers to reproduce your results. Provide references

to established methods (including statistical methods), provide references

to brief modified methods, and provide the rationale for using them and

an evaluation of their limitations. Identify all drugs and chemicals used,

including generic names, doses, and routes of administration. The section

should include only information that was available at the time the plan

or protocol for the study was devised (http://www.strobe-statement.org/

fileadmin/Strobe/uploads/checklists/STROBE_checklist_v4_combined.

pdf).

Statistics: Describe the statistical methods used in enough detail to

enable a knowledgeable reader with access to the original data to verify

the reported results. Statistically important data should be given in the

text, tables and figures. Provide details about randomization, describe

treatment complications, provide the number of observations, and specify

all computer programs used.

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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 parentheses.

Number references consecutively according to the order in which they

first appear in the text. Journal titles should be abbreviated according to

the style used in Index Medicus (consult List of Journals Indexed in Index

Medicus). Include among the references any paper accepted, but not yet

published, designating the journal and followed by, in press.

Examples of References:

1. List all authors.

Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow

transplantation from unrelated donors for patients with aplastic anemia

after failure immunosuppressive therapy. Blood 2006;108:1485-1491.

2. Organization as author

Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of

syngeneic bone marrow graft without preconditioning in post-hepatitis

marrow aplasia. Lancet 1977;2:742-744.

3. Book

Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger,

1961.

4. Book Chapter

Perutz MF. Molecular anatomy and physiology of hemoglobin. In:

Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin:

Genetics, Pathophysiology, Clinical Management. New York, Cambridge

University Press, 2000.

5. Abstract

Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin)

stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract).

6. Letter to the Editor

Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further

definition of 20q deletion in myeloid leukemia using fluorescence in situ

hybridization. Blood 1994;84:2821-2823.

7. Supplement

Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant.

2005;9(Suppl 7):81-86

Brief Reports

Abstract length: Not to exceed 150 words.

Article length: Not to exceed 1200 words.

Introduction: State the purpose and summarize the rationale for the study.

Materials and Methods: Clearly describe the selection of the observational

or experimental participants. Identify the methods and procedures in

sufficient detail. Provide references to established methods (including

statistical methods), provide references to brief modified methods, and

provide the rationale for their use and an evaluation of their limitations.

Identify all drugs and chemicals used, including generic names, doses, and

routes of administration.

Statistics: Describe the statistical methods used in enough detail to

enable a knowledgeable reader with access to the original data to verify

the reported findings/results. Provide details about randomization,

describe treatment complications, provide the number of observations,

and specify all computer programs used.

Results: Present the findings/results in a logical sequence in the text,

tables, and figures. Do not repeat all the findings/results in the tables and

figures in the text; emphasize and/or summarize only those that are most

important.

Discussion: Highlight the new and important findings/results of the

study and the conclusions they lead to. Link the conclusions with the

goals of the study, but avoid unqualified statements and conclusions not

completely supported by your data.

Invited Review Articles

Abstract length: Not to exceed 300 words.


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

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have published research articles on therel evant subject. The study’s new and

important findings should be highlighted and interpreted in the Conclusion

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

Images in Hematology

Article length: Not exceed 200 words.

Authors can submit for consideration an illustration and photos that is

interesting, instructive, and visually attractive, along with a few lines

of explanatory text and references. Images in Hematology can include

no more than 200 words of text, 5 references, and 3 figure or table. No

abstract, discussion or conclusion are required but please include a brief

title.

Letters to the Editor


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

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

Tables

Supply each table on a separate file. Number tables according to the order

in which they appear in the text, and supply a brief caption for each. Give

each column a short or abbreviated heading. Write explanatory statistical

measures of variation, such as standard deviation or standard error of

mean. Be sure that each table is cited in the text.

Figures

Figures should be professionally drawn and/or photographed. Authors

should number figures according to the order in which they appear in the

text. Figures include graphs, charts, photographs, and illustrations. Each

figure should be accompanied by a legend that does not exceed 50 words.

Use abbreviations only if they have been introduced in the text. Authors

are also required to provide the level of magnification for histological

slides. Explain the internal scale and identify the staining method used.

Figures should be submitted as separate files, not in the text file. 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 1

author.

Contributor’s Statement

All submissions should contain a contributor’s statement page. Each

manuscript should contain substantial contributions to idea and design,

acquisition of data, or analysis and interpretation of findings. All persons

designated as an author should qualify for authorship, and all those that

qualify should be listed. Each author should have participated sufficiently

in the work to take responsibility for appropriate portions of the text.

Acknowledgments

Acknowledge support received from individuals, organizations, grants,

corporations, and any other source. For work involving a biomedical

product or potential product partially or wholly supported by corporate

funding, a note stating, “This study was financially supported (in part)

with funds provided by (company name) to (authors’ initials)”, must be

included. Grant support, if received, needs to be stated and the specific

granting institutions’ names and grant numbers provided when applicable.

Authors are expected to disclose on the title page any commercial or other

associations that might pose a conflict of interest in connection with the

submitted manuscript. All funding sources that supported the work and

the institutional and/or corporate affiliations of the authors should be

acknowledged on the title page.

Ethics

When reporting experiments conducted with humans indicate that

the procedures were in accordance with ethical standards set forth by

the committee that oversees human experimentation. Approval of

research protocols by the relevant ethics committee, in accordance with

international agreements (Helsinki Declaration of 1975, revised 2002

available at http://www.wma.net/e/policy/b3.htm, “Guide for the Care and

use of Laboratory Animals” www.nap.edu/catalog/5140.html/), is required

for all experimental, clinical, and drug studies. Patient names, initials, and

hospital identification numbers should not be used. Manuscripts reporting

the results of experimental investigations conducted with humans must

state that the study protocol received institutional review board approval

and that the participants provided informed consent.

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

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

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

To publish data and findings/results that do not exist. Duplication: Use of

data from another publication, which includes re-publishing a manuscript

in different languages. Salamisation: To create more than one publication

by dividing the results of a study preternaturally.

We disapprove of such unethical practices as plagiarism, fabrication,

duplication, and salamisation, as well as efforts to influence the

review process with such practices as gifting authorship, inappropriate

acknowledgements, and references. Additionally, authors must respect

participant right to privacy.

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

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

that are presented in an electronic environment are not accepted prepublished

work. Authors in such situation must declare this status on the

first page of the manuscript and in the cover letter.

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

We use iThenticate to screen all submissions for plagiarism before

publication.

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Turkish Journal of Hematology uses plagiarism screening service to verify

the originality of content submitted before publication.

Conditions of Publication

All authors are required to affirm the following statements before their

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

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

submitted elsewhere while under consideration by The Turkish Journal

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

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

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

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

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

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

analysis and interpretation of the data, 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 that has been altered, revised,

amended, or otherwise changed. Our original copyright form must be

used as is.

Units of Measurement

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Example for CBC.

Table 1. Distribution of patients according to French-

American-British and World Health Organization 2001

classification systems.

Variables n=101

MDS FAB classification

RA 48 (47.5%)

RARS 17 (16.8%)

RAEB-I 22 (21.8%)

RAEB-II 14 (13.9%)

MDS 2001 WHO classification

MDS: Myelodysplastic syndrome, WHO: World Health Organization, FAB: French-

American-British, RAEB: refractory anemia with excess blast

Source: http://www.vetstream.com/felis/Corporate/993fhtm/ha-mat.htm

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A-VII

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A-VIII

CONTENTS

Review Article

88 The Role of Angiogenesis in Haemophilic Arthropathy: Where Do We Stand and Where Are We Going?

Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou

Research Articles

94 Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis

İpek Yönal, Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz, Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın

102 D-index: A New Scoring System in Febrile Neutropenic Patients for Predicting Invasive Fungal Infections

Gülden Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan

107 Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/

Deletion (δβ) 0 Mutation in Antalya

Türker Bilgen, Özden Altıok Clark, Zeynep Öztürk, M. Akif Yeşilipek, İbrahim Keser

112 The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin

Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer, Abdul Kareem Al Momen, Mashael Al Shaikh,

Farja Al Gahtani, Zohair A. Al Aseri, Hossam AH Abdelrazik

119 Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems

Özlen Bektaş, Ayşegül Üner, Eylem Eliaçık, Burak Uz, Ayşe Işık, Sezgin Etgül, Süreyya Bozkurt, İbrahim Celalettin Haznedaroğlu,

Hakan Göker, Nilgün Sayınalp, Salih Aksu, Haluk Demiroğlu, Osman İlhami Özcebe, Yahya Büyükaşık

127 Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It?

Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş

131 Immature Reticulocyte Fraction and Absolute Neutrophil Count as Predictor of Hemopoietic Recovery in Patients with

Acute Lymphoblastic Leukemia on Remission Induction Chemotherapy

Shan E. Rauf, Saleem Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan

135 The Prognostic Significance of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia

Nergiz Erkut, Ahmet Menteşe, Hasan Mücahit Özbaş, Nilay Ermantaş, Ayşegül Sümer, Asım Örem, Mehmet Sönmez

141 Investigation of Rho-Kinase Expressions and Polymorphisms in Mantle Cell Lymphoma Patients

Didar Yanardağ Açık, Mehmet Yılmaz, İbrahim Sarı, Serdar Öztuzcu, Zeynel A. Sayıner, Salih Subari, Abdullah T. Demiryürek

148 Prospective Audit of Blood Donor Selection Process in a Tertiary Care Hospital of a Developing Country

Naila Raza

Brief Reports

153 Regulatory T Cells in Patients with Idiopathic Thrombocytopenic Purpura

Alev Akyol Erikçi, Bülent Karagöz, Oğuz Bilgi

157 Serum Zinc Levels in Iron Deficient Women: A Case-Control Study

Onur Özhan, Neslihan Erdem, İsmet Aydoğdu, Ali Erkurt, İrfan Kuku

A-IX

Case Report

160 Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal Masses and Hematuria: A Case Report

Şiyar Erdoğmuş, Serkan Aktürk, Zeynep Kendi Çelebi, Saba Kiremitçi, Gülşah Kaygusuz, Namık Kemal Altınbaş, Evren Üstüner,

Kenan Keven

Letters to the Editor

164 A Comparison of Healthy Infants and Adults with Respect to Indirect Microparticle Activity and the Parameters of the Thrombin

Generation Test

Filiz Şimşek Orhon, Nejat Akar, Yonca Eğin, Betül Ulukol, Sevgi Başkan

165 Comment: In Response to “Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell

Lymphoma - De Novo Occurrence or Transformation”

Burak Uz, Kadir Acar

167 Tumor Necrosis Factor and Splenectomy

İrfan Yavaşoğlu

Images in Hematology

168 Auer Rod in a Neutrophil in a Nonmalignant Condition

Harish Chandra, Smita Chandra, Vibha Gupta, Divyaa Mahajan

169 Precursor B-Cell Lymphoblastic Lymphoma Presenting as a Spinal Mass at Initial Diagnosis

Oğuzhan Erol, Çiğdem Tokyol, Feyzullah Akyüz, Nuran Ahu Baysal, Mehmet Sezgin Pepeler

171 Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Should Be Kept in Mind in Children with Febrile Neutropenia,

Oral Cavity Lesions, and Skin Rash

Eda Ataseven, Şebnem Yılmaz Bengoa, Hale Ören

A-X

REVIEW

DOI: 10.4274/tjh.2016.0031

Turk J Hematol 2016;33:88-93

The Role of Angiogenesis in Haemophilic Arthropathy: Where Do

We Stand and Where Are We Going?

Hemofilik Artropatide Anjiyogenezin Rolü: Neredeyiz ve Nereye Gidiyoruz?

Alexandra Agapidou 1 , Thomas Stavrakis 1 , Efthymia Vlachaki 2 , Panagiotis Anagnostis 1 , Sophia Vakalopoulou 1

1Aristotle University, Hippokration Hospital, Second Propaedeutic Department of Internal Medicine, Thessaloniki, Greece

2Aristotle University, Hippokration Hospital, Second Department of Internal Medicine, Thessaloniki, Greece

Abstract

Haemophilia is an inherited bleeding disorder that can lead to

degenerative joint arthropathy due to recurrent bleeding episodes

affecting the musculoskeletal system of the patient. The cause of

bleeding can be either traumatic or spontaneous. The pathogenesis

of haemophilic arthropathy is unclear as many factors like iron,

inflammatory cytokines, and angiogenic factors contribute to this

process. Blood into joints can deteriorate the bone to such an extent

that the patient experiences pain, reduction of the range of movement,

and deformity of the joint, conditions that could have a great impact on

quality of life. Over the years, management of haemophilic arthropathy

has changed. Nowadays, early diagnosis with high resolution imaging

like magnetic resonance imaging along with application of prophylaxis

regimens can reduce the extent of damage to the joints. However, not

all haemophilia patients have access to these interventions as cost may

be prohibitive for some of them. The need for new, easy, and costeffective

strategies with the ability to identify early changes could

be beneficial and could make a difference in the management of

haemophilic arthropathy. Understanding the mechanism of processes

like angiogenesis in the mechanism of developing arthropathy could

be innovative for these patients and could help in the detection of new

early diagnostic and therapeutic markers.

Keywords: Angiogenesis, Haemophilic arthropathy, Vascular

endothelial growth factor, Haemophilia

Öz

Hemofili, hastanın kas ve iskelet sistemini etkileyen, tekrarlayan

kanama atakları ile dejeneratif eklem artropatisine neden olan,

kalıtsal bir kanama bozukluğudur. Kanama travma sonrasında

ya da kendiliğinden olabilir. Hemofilik artropatinin patogenezi

kesin bilinmemekle birlikte, demir, yangı sitokinleri ve anjiyogenik

faktörlerin sürece katkıları vardır. Eklem içine kanama kemiği bir

düzeye kadar bozabilir ve hasta, yaşam kalitesi üzerine büyük

bir etkisi olan ağrı, hareket kısıtlılığı ve eklem deformitesi gibi

durumları yaşar. Yıllar içerisinde hemofilik artropatinin yönetiminde

değişiklikler olmuştur. Günümüzde manyetik rezonans görüntüleme

gibi yüksek çözünürlüklü görüntüleme yöntemleri ile erken tanı ve

profilaksi rejimlerinin kullanılması eklemlerdeki harabiyetin derecesini

azaltmaktadır. Ancak bunların ücretleri bazı hastalar açısından

sınırlayıcı olabileceğinden, tüm hastalar bu müdahalelere ulaşamaz.

Erken dönemdeki değişiklikleri tespit edebilen yeni, kolay ve maliyet

etkin stratejiler yararlı olabilir ve hemofilik artropatinin yönetiminde

bir değişiklik yapabilir. Artropati gelişim mekanizmalarından

anjiyogenez gibi süreçlerin mekanizmasının anlaşılması bu hastalar

için bir yenilik olabilir ve yeni erken tanısal ve terapötik belirteçlerin

bulunmasına yardımcı olabilir.

Anahtar Sözcükler: Anjiyogenez, Hemofilik artropati, Vasküler

endoteliyal büyüme faktörü, Hemofili

Introduction

Haemophilia A and B are X-linked inherited disorders respectively

caused by the deficiency of coagulation factor VIII or IX [1]. Lack

of those clotting factors (CFs) leads to an increased tendency to

bleed at various intensities, according to the percentage of the

missing CF. The system that is mainly affected by these recurrent

bleeding episodes is the musculoskeletal system. Repeated

joint bleeds can cause progressive destruction of the cartilage,

resulting in a decreased range of motion due to pain and

stiffness. This condition is known as haemophilic arthropathy

or haemophilic joint disease (HJD) and it has a progressively

negative impact on patients’ quality of life. Haemophilia is found

to be associated with decreased bone mass in both adults and

children [2]. Haemarthrosis, formed after repeated joint bleeds,

could be prevented by providing prophylaxis to these patients

by means of administering the missing CF from an early age

and in a standard regimen. However, this requires good venous

access and skills from the patient’s point of view along with

highly specialised and properly organised structures from the

Address for Correspondence/Yazışma Adresi: Alexandra AGAPIDOU, M.D.,

Aristotle University, Hippokration Hospital, Second Propaedeutic

Department of Internal Medicine, Thessaloniki, Greece

E-mail : alekagapidou@yahoo.gr

Received/Geliş tarihi: January 19, 2016

Accepted/Kabul tarihi: April 15, 2016

88


Agapidou A, et al: The Role of Angiogenesis in Haemophilic Arthropathy

health provider’s side. If a patient cannot receive prophylactic

treatment, there are various other ways of chronic joint pain

relief, like applying interventions such as synovectomy and

arthroplasty. These are invasive surgical procedures that may be

frustrating for the patient. It is important to realise that the

mechanism underlying progressive haemophilic arthropathy is

multifactorial and still remains unclear. Availability of an easy,

quick, and low-cost test with high specificity for diagnosing HJD

would be beneficial for both patients and health providers. Use

of the Fracture Risk Assessment Tool for assessing fracture risk,

regular bone mineral density assessment, and supplemented

calcium, vitamin D, and, in specific cases, bisphosphonate

intake, as well as long-term prophylactic factor replacement

therapy, were suggested as means of prevention of bone loss

[2]. Furthermore, various inflammatory and angiogenetic

processes have been implicated in early joint bleeding and in

the pathogenesis of HJD. Achieving a deeper knowledge of HJD

could potentially lead to earlier diagnosis and treatment in

patients with haemophilia.

Haemophilic Arthropathy

Structure of the Synovial Joint

The synovial joint belongs to the group of joints that have to

bear a great amount of movement. In such joints, the bony

surfaces are covered with articular cartilage and are connected

by ligaments. The joint may be divided by an articular disc or

meniscus, which is continuous in the periphery with the fibrous

capsule while its free surfaces are covered by the synovial

membrane. Synovial joints facilitate movement by bringing

articulating bones into contact (Figure 1).

The components of a synovial joint are the synovial cavity,

which is the space between the bones filled with synovial fluid,

and the articular capsule, which surrounds the joint and unites

the articulating bones. The articular capsule also consists of

two layers: the outer fibrous membrane, which may contain

ligament, and the inner synovial membrane that secretes the

lubricating synovial fluid. The bones of the synovial joint are

covered by a layer of cartilage that functions to absorb tension

and reduce friction during movement [3]. The articular capsule

is highly innervated but is lacking blood vessels. The surrounding

blood network provides the necessary nutritional supply [4].

HJD is the end result of a number of changes occurring in

every component of the joint after repeated bleeding episodes.

Bleeding into the synovial joint exposes synovial cells to blood,

which is toxic for the joint. Morris et al. proposed that iron

plays a substantial role in the development of haemophilic

synovitis [5]. Studies by Wen et al. showed that iron is also

involved in myelocytomatosis viral oncogene (MYC) and

mouse double minute-2 (MDM2) homolog expression, which

causes proliferation of the synovium and active inflammation

[6]. Roosendaal and Lafeber observed that iron increases the

expression of proinflammatory cytokines like interleukin-6

(IL-6), interleukin 1-β (IL-1β), and tumour necrosis factor-α

(TNF-α) in synovial cells [7]. Histologically, it was shown that

synovial inflammation incorporates three characteristics: 1)

hypertrophy of the villi, 2) increased number of inflammatory

cells, and 3) increased vascularisation [7,8,9].

Synovial Angiogenesis

Angiogenesis is a normal process during wound healing and

embryogenesis. It is also considered part of the pathophysiologic

mechanisms implicated in diseases like rheumatoid arthritis

(RA), osteoarthritis, systemic lupus erythematosus, and

carcinogenesis. It is regulated by various inducers and inhibitors.

During inflammation, the inducers/promoters prevail over

inhibitors (Figure 2).

Angiogenesis takes place mainly in the bone marrow and

in vascular stem cells. Angiogenetic factors like vascular

endothelial growth factor (VEGF), angiopoietin-1 (Ang-1),

angiopoietin-2 (Ang-2), and fibroblast growth factor (FGF)

participate in endothelial cell proliferation.

In 2008, it was shown that mice with factor IX deficiency

experienced delayed wound healing and increased wound

angiogenesis along with subcutaneous haematoma formation

days after induction of a wound. It was suggested that tissue

damage induces coagulation and inflammation response [10].

For angiogenesis to be triggered, a series of events have to take

place. Mediators from the synovium activate endothelial cells,

which release proteolytic enzymes that act on the endothelial

basement membrane and the perivascular extracellular matrix.

Figure 1. Synovial joint.

89

Agapidou A, et al: The Role of Angiogenesis in Haemophilic Arthropathy Turk J Hematol 2016;33:88-93

The endothelial cells participate in the formation of primary

sprouts. The lumen of the sprouts facilitate the formation of

“capillary loops” followed by the synthesis of new basement

membrane and new capillaries [11].

RA is among the inflammatory disorders in which increased

angiogenesis is observed as well. By shedding light on the

understanding of the relationship between angiogenesis and

inflammatory arthritis, the role of new vessel formation in

haemophilic arthropathy could be more easily comprehended.

Angiogenesis is induced by several conditions like hypoxia and

injury where proangiogenic molecules are secreted by tissues.

Endothelial cell proliferation and migration are followed by

capillary tube formation, deposition of basement membrane,

and migration of smooth muscle cells. Anastomoses are created

and the flow of blood is established [12].

In RA, inflammatory cells like macrophages, lymphocytes,

mast cells, and fibroblasts, along with their soluble products

including pro-inflammatory cytokines, TNF-α, IL-1, and IL-8,

are all promoters of angiogenesis. One of the major endothelial

growth factors found in the synovium of patients with RA is

VEGF. In RA synovium, IL-1 and TNF-α facilitate the fibroblast

expression of VEGF [13,14]. VEGF induces the endothelial cell

decay-accelerating factor, which acts protectively for the cells

against activated complement components and may regulate

endothelial proliferation and angiogenesis [15].

Direct measurements confirm that the intra-articular

environment is hypoxic in inflammatory arthritis. Hypoxia is

often a feature of inflammation and is a strong inducer of VEGF.

Tissue hypoxia in the rheumatoid joint results in increased VEGF

messenger ribonucleic acid (mRNA) stability [16] and enhanced

VEGF gene transcription through the binding of hypoxiainducible

transcription factors such as hypoxia-induced factor-1

(HIF) and HIF-2 that are overexpressed in the synovial lining

and stromal cells of RA patients relative to synovial tissues from

individuals without arthritis [17].

Zimmermann in 1923 introduced the term ‘pericyte’ to describe

a periendothelial support cell wrapped around the length of

micro-vessels [18]. While it is known that pericytes are present

in the microcirculation, their functional roles and importance

in microvascular physiology has not been fully investigated.

Recently pericytes have become a research area of growing

interest as potential targets for pro- or antiangiogenic therapies.

Vascular pericytes elongate around endothelial cells and their

function is to assist in the regulation of vessel stabilisation

and in endothelial cell proliferation. During angiogenesis,

signals from the pericyte to the endothelial cell and vice versa

are critical for the formation of the capillary sprout. Studies

found that pericytes may have a leading role in newly formed

capillaries. This implicates their role in endothelial cell guidance.

During angiogenesis, pericytes are involved in recruitment and

direct interaction with endothelial cells [19]. Moreover, they

participate in the development of newly formed endothelial

cells [20]. Pericyte development is usually controlled by plateletderived

growth factor-B (PDGF), secreted by endothelial cells

[21].

It was found that pericytes are directly involved in the process

of angiogenesis as increasing pericyte coverage via Ang-2

inhibition can potentially represent an antiangiogenic tumour

therapy [22,23]. Advancing the understanding of pericytes and

the ability to develop pericyte-related therapies is a challenging

and very promising process.

Angiogenesis and Haemophilia

Angiogenesis is a natural process considered as a physiologic

response to inflammation, hypoxia, and malignancy. It may

be mediated by various factors including growth factors,

proinflammatory cytokines, chemokines, extracellular matrix

Figure 2. Angiogenesis in normal state and inflammation state.

Figure 3. Angiogenesis and hypoxia in rheumatoid arthritis. RA:

Rheumatoid arthritis.

90



molecules, matrix-degrading proteolytic enzymes, cellular

adhesion molecules, and others [24]. One of the most important

growth factors in terms of angiogenesis is VEGF. HIF acts by

upregulating VEGF [17,25]. Furthermore, prostaglandin and

nitric oxide act via VEGF in neovascularisation. There is an

interaction between VEGF and Ang-1 that acts in favour of the

newly formed vessels as a form of protection and stabilisation.

On the contrary, Ang-2 antagonises Ang-1 and has a negative

impact on neovascularisation (Figure 3).

VEGF and PDGF are involved in the development of

inflammatory joint disease, potentially by favouring cytokinerelated

cartilage destruction and not synthetic cell responses

associated with growth factor activity. Expression of VEGF

is increased in individuals with inflammatory joint disease

compared with normal controls. Among individuals with

polyarthritis, concentrations of VEGF and its receptor, Ang-1,

have been found to be related to inflammatory markers and

bone destruction. Based on that, VEGF could play a role in

haemophilic arthropathy [26].

There are a variety of factors that work in an inhibitory manner

regarding formation of new vessels. Among them are cytokines

like interferon-α (IFN-α), IFN-γ, IL-4, IL-12, and leukaemia

inhibitory factor (LIF). Protease inhibitors like tissue inhibitor

of metalloproteinase (TIMPS), plasminogen activator inhibitors

(PAIs), and thrombospondin-1 inhibit capillary and new vessel

formation [11,27].

Acharya et al. [28] observed that there is involvement of

angiogenesis in the development of haemophilic synovitis. Sera

from haemophilic subjects with joint arthropathy induced an

angiogenic response in endothelial cells that was ceased by

blocking VEGF while peripheral blood mononuclear cells from

these subjects stimulated synovial cell proliferation, which was

blocked by a humanised anti-VEGF antibody (bevacizumab).

Human synovial cells, when incubated with haemophilic sera,

could elicit upregulation of hypoxia-inducible factor-1A (HIF-

1A) mRNA, indicating that hypoxia plays an important role in

the neoangiogenesis process.

The onset of endothelial proliferation is based on the equilibrium

between positive and negative regulators [29]. Once there is

a stimulus from VEGF-A, endothelial cells became activated,

proliferate, and form new vessels. However, these newly formed

vessels are extremely sensitive and prone to bleeding. In order

to protect them, PDGF stimulates pericytes, which migrate to

the place of the angiogenesis and embrace the vessels with

dendritic processes, forming a stabilising coat. The presence of

pericytes is considered a sign of maturity.

In another study, Zetterberg et al. [30] observed that VEGF

is increased in synovial tissue from haemophilic patients.

In this study, synovial tissue was obtained when HJD was

already established. The increased VEGF in synovial cells from

these patients showed that HJD is characterised by active

angiogenesis. Vessels from HJD and control synovial tissue

were found to be covered by pericytes, indicating that most of

the vessels were mature. Even though the number of patients

with different stages of HJD in this study was too low to

understand how angiogenesis develops over time in HJD, there

were indications that end-stage HJD is characterised by a

chronic proinflammatory, proangiogenic drive, while the vessel

formation is relatively slow, permitting vessels to mature and

develop pericyte “protection”.

Tattersall et al. [31] observed that macrophages enhance

angiogenesis, increasing the number and length of endothelial

sprouts, a property called “angiotrophism”. Polarising

macrophages in a proinflammatory manner could increase their

angiotrophic stimulation of vessel sprouting. This increase was

found to be dependent on macrophage Notch signalling. JAG1

expression and Notch signalling are essential for the growth of

both endothelial cells and pericytes.

In a recent study by Yi et al. [32], it was demonstrated that

annexin a2 (Axna2) could promote the progression of RA. Axna2

plays an important role in pannus formation in RA. Cytological

analysis showed that the Axna2/Axna2 receptor (Axna2/Axna2R)

axis promoted new vessel formation by activation of the

Hedgehog (HH) signalling pathway and increased the Patched

(Ptc) and Smoothened (Smo) expression in order to upregulate

the expression of the downstream metalloproteinases (MMPs),

VEGF, and Ang-2. These results suggest that the effect of Axna2

might provide a new potential measure for treatment of RA and

potentially HJD.

New Potential Therapies: A Step to the Future

Cancer and inflammation research trials have been targeting

angiogenic mediator and inhibitor pathways regarding the

development of new therapeutic agents. There have been

attempts to target VEGF by using synthetic VEGF and VEGFR

inhibitors, anti-VEGF antibodies, and inhibitors of VEGF and

VEGFR signalling, primarily in colorectal, lung, renal, and liver

cancers. Bevacizumab, a human monoclonal antibody to VEGF,

has been used in the treatment of various types of cancer [11].

Vatalanib, a VEGFR protein kinase inhibitor, inhibited knee

arthritis in rabbits [33].

YC-1, a superoxide-sensitive stimulator of soluble guanylyl

cyclase originally developed to treat hypertension and

thrombosis, is also a HIF-1 inhibitor [34]. Microtubule

destabilisers, such as 2-ME, as well as paclitaxel, an anticancer

agent, also diminish HIF-1α expression and activity [35].

Infliximab treatment in combination with methotrexate

reduced synovial VEGF expression and vascularity [36,37]. Anti-

91

Agapidou A, et al: The Role of Angiogenesis in Haemophilic Arthropathy Turk J Hematol 2016;33:88-93

TNF therapy in arthritic patients reduced Ang-1 but stimulated

Ang-2 expression [38]. Recently, the anti-IL-6 receptor antibody

tocilizumab decreased serum levels of VEGF [39]. Thalidomide,

currently used in multiple myeloma treatment but also tried in

lupus and RA, is a potent TNF-α antagonist and angiogenesis

inhibitor [27,40]. Thalidomide could suppress both synovitis and

angiogenesis [27], suggesting that its antiangiogenic effects

may be, in part, VEGF-independent. Fumagillin is a natural

product of Aspergillus fumigatus. TNP-470 and PPI2458 are

synthetic derivatives of fumagillin that inhibit methionine

aminopeptidase-2, an enzyme involved in angiogenesis [41].

It would be extremely interesting to learn if some of these

treatment options could be applied to haemophilia patients in

the future and if they could have an impact on the development,

progression, and treatment of synovitis and haemophilic joint

arthropathy.

Conclusion

Further studies will have to clarify the mechanisms and

circumstances that may be responsible for modulating the

contribution of angiogenesis to HJD. It is very intriguing to

consider the possibility that angiogenetic factors may play a

crucial role in the pathogenesis of arthropathy seen in patients

suffering from haemophilia. Finally, the possibility that there

may be potential markers enabling identification of the onset

as well as the progression of haemophilic synovitis deserves

further investigation.

Acknowledgements

Many thanks to Dr. Emma Fosbury, BA, MBBS, MRCP, FRCPath,

Clinical Research Fellow at the Katharine Dormandy Haemophilia

Centre and Thrombosis Unit, Royal Free London NHS Foundation

Trust, London, UK, for her kind editing of the English language

of the manuscript.

Authorship Contributions

Surgical and Medical Practices: Alexandra Agapidou, Thomas

Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia

Vakalopoulou; Concept: Alexandra Agapidou, Thomas Stavrakis,

Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou;

Design: Alexandra Agapidou, Thomas Stavrakis, Efthymia

Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Data

Collection or Processing: Alexandra Agapidou, Thomas Stavrakis,

Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou;

Analysis or Interpretation: Alexandra Agapidou, Thomas


Vakalopoulou; Literature Search: Alexandra Agapidou, Thomas


Vakalopoulou; Writing: Alexandra Agapidou, Thomas Stavrakis,

Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou.

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

of interest, including specific financial interests, relationships,

and/or affiliations relevant to the subject matter or materials

included.

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93

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0136


Impact of JAK2V617F Mutational Status on Phenotypic Features

in Essential Thrombocythemia and Primary Myelofibrosis

Esansiyel Trombositemi ve Primer Miyelofibroziste JAK2V617F Mutasyonunun Fenotipik Etkileri

İpek Yönal, Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz, Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın

İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

Abstract

Objective: The JAK2V617F mutation is present in the majority

of patients with essential thrombocythemia (ET) and primary

myelofibrosis (PMF). The impact of this mutation on disease

phenotype in ET and PMF is still a matter of discussion. This study aims

to determine whether there are differences in clinical presentation

and disease outcome between ET and PMF patients with and without

the JAK2V617F mutation.

Materials and Methods: In this single-center study, a total of

184 consecutive Philadelphia-negative chronic myeloproliferative

neoplasms, 107 cases of ET and 77 cases of PMF, were genotyped for

JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which

involves allele-specific polymerase chain reaction.

Results: ET patients positive for JAK2V617F mutation had higher

hemoglobin (Hb) and hematocrit (Hct) levels, lower platelet counts,

and more prevalent splenomegaly at diagnosis compared to patients

negative for the JAK2V617F mutation, but rates of major thrombotic

events, arterial thrombosis, and venous thrombosis were comparable

between the groups. At presentation, PMF patients with JAK2V617F

mutation had significantly higher Hb and Hct levels and leukocyte

counts than patients without the mutation. Similar to the findings of

ET patients, thromboembolic rates were similar in PMF patients with

and without theJAK2V617F mutation. For ET and PMF patients, no

difference was observed in rates of death with respect to JAK2V617F

mutational status. Moreover, leukemic transformation rate was not

different in our PMF patients with and without JAK2V617F mutation.

Conclusion: We conclude that JAK2V617F-mutated ET patients express

a polycythemia vera-like phenotype and JAK2V617F mutation in PMF

patients is associated with a more pronounced myeloproliferative

phenotype.

Keywords: JAK2V617F mutation, Essential thrombocythemia, Primary

myelofibrosis

Öz

Amaç: Esansiyel trombositemi (ET) ve primer miyelofibrozis (PMF)

tanılı hastaların büyük çoğunluğunda JAK2V617F mutasyonu

bulunmaktadır. ET ve PMF’de bu mutasyonun hastalık fenotipi üzerine

etkisi halen tartışılmaktadır. Bu çalışmada, JAK2V617F mutasyonunu

taşıyan ve taşımayan ET ve PMF hastalarının başvuru sırasındaki klinik

parametreler ve hastalık seyri açısından karşılaştırılması amaçlanmıştır.

Gereç ve Yöntemler: Tek merkezli olan bu çalışmada, 107 ET

ve 77 PMF olmak üzere toplam 184 Philadelphia-negatif kronik

miyeloproliferatif neoplazili hastada bir allel spesifik polimeraz zincir

reaksiyonu olan JAK2 Ipsogen MutaScreen kullanılarak JAK2V617F

mutasyonu taranmıştır.

Bulgular: JAK2V617F mutasyonunu taşıyan ET hastalarında, mutasyon

bulunmayanlara göre tanı sırasındaki hemoglobin (Hb) ve hematokrit

(Hct) düzeyleri anlamlı olarak daha yüksek, trombosit sayısı daha düşük

ve splenomegali oranları daha yüksek bulunmuştur. Fakat her iki grup

arasında majör trombotik olay, arteriyel tromboz ve venöz tromboz

açısından fark saptanmamıştır. JAK2V617F mutasyonu bulunan PMF

hastalarında ise mutasyon taşımayan gruba göre başvuru anındaki Hb,

Hct ve lökosit değerleri anlamlı olarak daha yüksek saptanmıştır. PMF

hastalarında, ET hastalarında olduğu gibi tromboembolik olayların

JAK2V617F mutasyonundan bağımsız olduğu görülmüştür. ET ve PMF

hastalarında JAK2V617F mutasyonu varlığında ölüm oranında farklılık

gözlenmemiştir. Bunun yanında JAK2V617F mutasyonunu taşıyan ve

taşımayan PMF hastaları arasında lösemik dönüşüm oranı açısından

anlamlı bir fark bulunmamıştır.

Sonuç: Bu çalışmanın sonucunda JAK2V617F mutasyonunu taşıyan

ET hastalarında polisitemia vera benzeri fenotipin ortaya çıktığı

ve bu mutasyonun varlığında PMF hastalarının daha belirgin bir

miyeloproliferatif fenotiple ilişkili olduğu söylenebilir.

Anahtar Sözcükler: JAK2V617F mutasyonu, Esansiyel trombositemi,

Primer miyelofibrozis

Address for Correspondence/Yazışma Adresi: İpek YÖNAL, M.D.,

İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine,

Division of Hematology, İstanbul, Turkey

E-mail: ipekyon@istanbul.edu.tr

Received/Geliş tarihi: March 30, 2014

Accepted/Kabul tarihi: July 14, 2014

94


Yönal İ, et al: Impact of JAK2V617F Mutational Status

Introduction

Philadelphia-negative chronic myeloproliferative neoplasms

(Ph-negative MPNs) are a heterogeneous group including 3 major

diseases: polycythemia vera (PV), essential thrombocythemia

(ET), and primary myelofibrosis (PMF). Thrombotic events are the

major cause of morbidity and mortality in ET. Other complications

include hemorrhage and progression to myelofibrosis or acute

myeloid leukemia [1,2]. PMF is characterized by a worse life

expectancy and a progressive disease course. The disease presents

with classically severe anemia, massive splenomegaly, and acute

leukemia [3]. JAK2V617F mutation is present in more than 95%

of PV patients and approximately 50%-60% of ET and PMF

patients [4]. Several studies investigated the clinical relevance

of JAK2V617F mutation in ET and PMF patients [5,6,7,8,9,10].

In ET, overall survival (OS) or leukemia-free survival was found

not to be affected by the presence of JAK2V617F mutation,

while the influence of JAK2V617F on thrombosis or fibrotic

transformation remained less clear [5,7,11,12]. Conflicting

results have been reported regarding the impact on OS,

leukemic transformation rate, and need for chemotherapy or

splenectomy in the presence of JAK2V617F mutation [8,9,10,13].

We previously evaluated the clinical and laboratory correlates

in 184 patients with Ph-negative MPNs according to the allele

burden of JAK2V617F mutation (unpublished data). Herein,

we investigate the usefulness of JAK2V617F mutational status

for explaining phenotypic variability using the same group of

patients, which includes a relatively large series of Ph-negative

MPN patients.

Materials and Methods

A total of 184 consecutive Ph-negative MPN patients, 107 with ET

and 77 with PMF, admitted to the Division of Hematology of the

İstanbul University İstanbul Medical Faculty from 1995 to 2013

were included in the study. ET and PMF patients were diagnosed

based on WHO criteria [14]. Informed consent was obtained

from all participants according to the local ethics committee

guidelines. Complete clinical history, blood count, lactate

dehydrogenase (LDH) level, and thrombotic or hemorrhagic

complications were recorded. Spleen longitudinal diameters

of ≥130 mm to 160 mm and of ≥160 mm on ultrasound were

considered as mild and massive splenomegaly, respectively. A

scale of 0-3 was used to grade reticulin fibrosis on bone marrow

trephine biopsies [15]. The Dynamic International Prognostic

Scoring System (DIPSS) plus was used for risk stratification

in PMF [16]. Unfavorable karyotypes in PMF were defined as

complex karyotype or sole or 2 abnormalities that included +8,

-7/7q-, i(17q), inv(3), -5/5q-, 12p-, or 11q23 rearrangement

[17]. Patients were genotyped for the JAK2V617F mutation by

JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille,

France), which is a TaqMan allelic discrimination assay that

contains fluorescent probes specific for wild-type (617V) and

mutant (617F) alleles [18].

Statistical Analysis

Data were processed using SPSS 16 (SPSS Inc., Chicago,

IL, USA). Continuous variables were summarized as mean

[standard deviation (SD)]. Chi-square statistics were used to

compare categorical variables among the different patient

groups categorized according to the JAK2V617F mutational

status. Analysis of continuous variables among the groups was

performed using the Mann-Whitney U test. A p-value of less

than 0.050 was considered to indicate statistical significance;

all tests were 2-tailed.

Results

A total of 184 patients (107 with ET and 77 with PMF) were

included. Bone marrow fibrosis was detected in 90.7% (97 in

107) of ET and 100% of PMF patients. In ET patients, the grade

of bone marrow fibrosis was scaled as follows: grade 0, 9.3%;

grade 1, 62.7%; grade 2, 25.2%; and grade 3, 2.8%. All PMF

patients had bone marrow fibrosis (grade 2 in 20.8% and grade

3 in 79.2%).

JAK2V617F mutation was identified in 64 of 107 ET (59.8%) and

58 of 77 PMF (75.3%) patients (p=0.028). Clinical and laboratory

correlates of ET patients according to JAK2V617F mutational

status are summarized in Tables 1 and 2.

JAK2V617F-positive and -negative ET patients showed no

significant differences with respect to sex and age at diagnosis.

ET patients with JAK2V617F mutation presented with higher

hemoglobin (Hb) and hematocrit (Hct) levels and lower platelet

count at diagnosis compared to patients without mutation

(p=0.001, p=0.001, and p=0.043, respectively). The leukocyte

count and LDH levels were similar for the 2 groups.

The 2 groups showed no significant difference with respect

to mean spleen size. However, JAK2V617F-positive ET patients

presented with more prevalent splenomegaly at diagnosis

compared to patients without the mutation (p=0.044).

ET patients with JAK2V617F mutation showed a higher, albeit

not statistically significant, rate of bleeding events compared

to the JAK2V617F-negative group (15.6% and 7%, respectively;

p=0.298).

ET patients with and without JAK2V617F mutation showed no

significant difference with respect to the degree of bone marrow

fibrosis, prevalence of hydroxyurea use, and rate of splenectomy.

In addition, no significant differences were observed in the use

of other medical treatments in any of the categories (p>0.050).

Duration of follow-up in patients with and without JAK2V617F

mutation was 69.7 months (SD: 63.7) and 70.1 months (SD:

56.9), respectively (p=0.675). During follow-up, 3 of 64 (4.7%)

JAK2V617F-positive ET and 2 of 43 (4.7%) JAK2V617F-negative

ET patients succumbed to their disease (p=1.000).

95



Clinical and laboratory parameters of PMF patients classified

according to genotype are outlined in Tables 3 and 4.

The rate of female patients was higher in the JAK2V617Fnegative

group compared to the JAK2V617F-positive group

(84.2% and 46.6%, respectively; p=0.009). PMF patients with

and without JAK2V617F mutation showed no significant

differences with respect to age at diagnosis. At initial diagnosis,

PMF patients with the JAK2V617F mutation presented with

significantly higher Hb and Hct levels and leukocyte counts

compared to those without the mutation (p=0.005, p=0.034,

and p=0.046, respectively). Platelet count and LDH level did

not differ between the 2 groups.

The mean spleen size showed no significant difference among

any of the categories, although PMF patients with JAK2V617F

mutation showed a trend towards higher prevalence of massive

splenomegaly at diagnosis compared to patients without

mutation (p=0.193 and p=0.090, respectively).

JAK2V617F-positive PMF patients showed a trend towards a

higher prevalence of bleeding events compared to JAK2V617Fnegative

PMF patients (24.1% and 5.3%, respectively; p=0.090).

There was no significant difference in the prevalence of total

thrombotic events, arterial thrombosis, and venous thrombosis

between JAK2V617F-positive and -negative PMF patients.

The degree of reticulin fibrosis, prevalence of hydroxyurea use,

rate of allogeneic hematopoietic stem cell transplantation

(AHSCT), and history of splenectomy did not differ in any of

the categories. In addition, the 2 groups showed no significant

differences in the use of other medical treatments (p>0.050).

No significant difference was observed in the distribution of

karyotype categories and DIPSS-Plus risk stratification between

JAK2V617F-positive and -negative PMF patients.

Duration of follow-up in PMF patients with and without

JAK2V617F mutation was 42 months (SD: 46.9) and 56.6

months (SD: 48.7), respectively (p=0.165). At the end of the data

collection period, 11 of 58 (19%) PMF patients with JAK2V617F

mutation succumbed to their disease, while the rate of death

in patients without JAK2V617F mutation was 15.8% (p=1.000).

During follow-up, rate of leukemic transformation was similar

between the 2 categories.

Discussion

In our relatively large series of patients with Ph-negative MPNs,

including 107 ET patients with a mean follow-up duration of

more than 5 years and 77 PMF patients with a mean follow-up

duration of more than 3 years, we documented that JAK2V617F

mutation correlates with disease phenotype in adult Turkish

patients with ET and PMF.

Our results suggest that JAK2V617F positivity in ET induces a

phenotype resembling PV. Confirming previous observations,

we found that ET patients with JAK2V617F mutation presented

with higher Hb and Hct levels and lower platelet counts

compared to unmutated patients [5,6,7,19,20,21,22]. Contrary

to some previous reports yet consistent with the findings

of Kittur et al. [5] and Pich et al. [22], our ET patients with

JAK2V617F mutation showed no difference in leukocyte count

at diagnosis as opposed to patients without the mutation [6,21].

Furthermore, in contrast to some previous reports but consistent

Table 1. Clinical and laboratory features between JAK2V617F-mutated and -unmutated patients among 107 patients with

essential thrombocythemia.

ET JAK2V617F-mutated, mean [SD] JAK2V617F-unmutated, mean [SD] p-value

Number of patients 64 43 -

Age at diagnosis 49.7 [14.9] 51.7 [15.7] 0.565

Females (%) 38 (59.4%) 20 (46.5%) 0.266

Leukocytes at diagnosis (mm 3 ) 10.196 [4.138] 9.593 [3.434] 0.483

Hb at diagnosis (g/dL) 13.6 [1.8] 12.4 [1.9] 0.001

Hct at diagnosis (%) 40.7 [5.37] 36.8 [5.21] 0.001

Platelet count at diagnosis (mm 3 ) 874.782 [320.867] 1055.116 [495.928] 0.043

LDH at diagnosis (U/L) 453.2 [150] 462.1 [159.7] 0.927

Spleen size at diagnosis (mm) 141.7 [37.26] 132.07 [23.86] 0.126

Bone marrow fibrosis, n (%) 64 (100%) 43 (100%) 0.522

0 7 (10.9%) 3 (7%) -

1 42 (65.6%) 25 (58.1%) -

2 14 (21.9%) 13 (30.2%) -

3 1 (1.6%) 2 (4.7%) -

Follow-up duration (months) 69.7 [63.7] 70.1 [56.9] 0.675

ET: Essential thrombocythemia, Hb: hemoglobin, Hct: hematocrit, LDH: lactate dehydrogenase, SD: standard deviation.

96



with the study of Vannucchi et al. [11], we observed a higher

prevalence of splenomegaly in ET patients with JAK2V617F

mutation than in patients without the mutation [5,6,7,20,21].

Data on ET regarding the impact of JAK2V617F mutational

status on thrombotic events are conflicting. In the study by

Campbell et al., JAK2V617F mutation in ET was associated with

an increased frequency of venous thromboembolism, but not

with arterial thrombosis [6]. In the study by Kittur et al., the

presence of JAK2V617F mutation was found to be significantly

associated with increased incidence of venous thrombosis

during follow-up, but not with major thrombosis, arterial

thrombosis, and venous thrombosis at diagnosis [5]. In contrast,

Antonioli et al. reported that there was no correlation between

thrombotic events and JAK2V617F mutation in ET patients [20].

In another study, there was no difference between ET patients

with JAK2V617F mutation or wild-type alleles with respect to

the frequency of major thrombotic events and major arterial

and venous thrombosis, either at diagnosis or during follow-up

[21]. Similar to the aforementioned study in ET patients, the

presence of JAK2V617F mutation made no significant difference

in the frequency of vascular complications at presentation [7].

In the current study, we observed no significant difference in

the frequency of major thrombotic events, arterial thrombosis,

and venous thrombosis between JAK2V617F-positive and

-negative ET patients. In the study by Pich et al., ET patients

with JAK2V617F mutation were younger than those without

mutation [22]. Conversely, in several studies, the presence of

JAK2V617F mutation was significantly associated with older

age at diagnosis [5,7,11,21,23,24,25]. Some studies revealed no

difference in age between JAK2V617F-positive and -negative

ET patients [20,26]. In our study group, we found no significant

difference in age among ET patients with and without JAK2V617F

mutation. Moreover, in the current study, we did not determine

an association between JAK2V617F mutation and sex, consistent

with previous reports [5,7,11,20,21,23,24,25,26]. Alvarez-Larrán

et al. reported that the presence of JAK2V617F mutation in ET

patients was associated with increased LDH levels [25]. On the

contrary, in another study, JAK2V617F mutation in ET did not

correlate with LDH level [21]. Our ET patients with JAK2V617F

mutation did not show differences in LDH level as compared

to wild-type patients. To our knowledge, there is limited

information about the association between JAK2V617F mutation

and histological changes in bone marrow biopsy of ET patients.

In a series of 103 ET patients, Pich et al. reported no significant

impact of JAK2V617F mutation on bone marrow fibrosis [22]. In

the current study, the presence of JAK2V617F mutation in ET did

not correlate with the degree of reticulin fibrosis. Several studies

investigated the association between JAK2V617F mutation in ET

and major hemorrhages [7,11,20,21,25]. Confirming the findings

of the aforementioned studies, our ET patients with mutant and

wild-type alleles showed no differences in the rate of bleeding

complications [7,11,20,21,25]. Some previous studies reported

that cytoreductive therapy requirement did not differ between

ET patients with and without JAK2V617F mutation [7,21,23,24].

This finding is in line with our data showing that the prevalence

of hydroxyurea use and other medical treatments was similar

between JAK2V617F-mutated and -unmutated ET patients

[7,21,23,24]. In ET patients, OS was shown not to be influenced

by the presence of JAK2V617F mutation [5,7]. Confirming this

observation, the death rate did not differ in our ET patients with

and without JAK2V617F mutation.

In our series of 77 PMF patients, we found a significant

association between JAK2V617F mutation and the expression

of a more pronounced myeloproliferative phenotype. In PMF

patients, JAK2V617F mutational status contributed to laboratory

abnormalities, including higher Hb level and leukocyte count,

but its association with platelet count is inconsistent [19]. Our

PMF patients with JAK2V617F mutation had higher Hb and Htc

levels and leukocyte counts at diagnosis than those without the

mutation. In contrast, in our PMF patients, platelet count at

initial diagnosis did not differ with respect to the JAK2V617F

mutation. Barosi et al. demonstrated the association between

JAK2V617F mutational status and development of marked

splenomegaly [9]. On the other hand, in this population,

several other groups did not show any correlation between

the presence of JAK2V617F mutation and spleen size [8,10]. In

the study by Guglielmelli et al., JAK2V617F mutated and wildtype

patients did not differ from each other as regards the

presence of palpable splenomegaly greater than 15 cm from

the left costal margin [27]. In our study, the mean spleen size

did not significantly differ between JAK2V617F-positive and

-negative PMF patients, although PMF patients with JAK2V617F

mutation showed a trend towards higher prevalence of massive

splenomegaly at diagnosis compared to patients without

mutation. In PMF patients, the relationship of JAK2V617F

mutation and thrombosis is controversial. In the study by

Barosi et al., there was no significant difference in the rate of

major thrombotic events between JAK2V617F-mutated and

-unmutated PMF patients [9]. In a series of 199 PMF patients,

Tefferi et al. showed no significant difference in the prevalence

of thrombosis between JAK2V617F-positive and -negative PMF

patients, whereas in another series of 117 PMF patients, Tefferi

et al. reported the association of the presence of JAK2V617F

mutation with history of thrombosis [8,13]. In the current study,

the prevalence of total thrombotic events, arterial thrombosis,

and venous thrombosis did not significantly differ among PMF

patients with and without JAK2V617F mutation. Several studies

have shown that ET patients with mutant alleles and wild-type

alleles showed no significant difference with respect to age and

sex [8,10,27]. In the current study, the presence of JAK2V617F

mutation in PMF patients was not associated with age. However,

in our study, the rate of females was higher among JAK2V617Fnegative

PMF patients than JAK2V617F-positive PMF. We did

97



Table 2. Clinical and laboratory features between JAK2V617F-mutated and -unmutated patients among 107 patients with

essential thrombocythemia (continued).

ET JAK2V617F-mutated, n (%) JAK2V617F-unmutated, n (%) p-value


Splenomegaly group 64 (100%) 43 (100%) 0.044

No splenomegaly 34 (53.1%) 33 (76.8%) -

Mild splenomegaly 17 (26.6%) 5 (11.6%) -

Massive splenomegaly 13 (20.3%) 5 (11.6%) -

Bleeding 10 (15.6%) 3 (7%) 0.298

Hydroxyurea 57 (89.1%) 35 (81.4%) 0.273

History of splenectomy 1 (1.6%) 1 (2.3%) 1.000

Thrombosis 26 (40.6%) 15 (34.9%) 0.692

Thrombosis group 64 (100%) 43 (100%) 0.219

No thrombosis 38 (59.4%) 28 (65.1%) -

Arterial 11 (17.2%) 10 (23.3%) -

Venous 14 (21.9%) 4 (9.3%) -

Arterial and venous 1 (1.5%) 1 (2.3%) -

Death 3 (4.7%) 2 (4.7%) 1.000

ET: Essential thrombocythemia.

Table 3. Clinical and laboratory features between JAK2V617F-positive and -negative patients among 77 primary myelofibrosis

patients.

PMF JAK2V617F-mutated, mean [SD] JAK2V617F-unmutated, mean [SD] p-value


Age at diagnosis 58.1 [13.7] 52.8 [16] 0.120

Females (%) 27 (46.6%) 16 (84.2%) 0.009

Leukocytes at diagnosis (mm 3 ) 16.134 [14.633] 9.726 [7.875] 0.046

Hb at diagnosis (g/dL) 11.03 [2.2] 9.4 [1.3] 0.005

Hct at diagnosis (%) 32.9 [7.39] 29.4 [4.81] 0.034

Platelet count at diagnosis (mm 3 ) 423.691 [353.469] 464.526 [396.324] 0.832

LDH at diagnosis (U/L) 843 [405] 782 [364] 0.836

Spleen size at diagnosis (mm) 202.19 [44.2] 183.7 [37.3] 0.193

Bone marrow fibrosis, n (%) 58 (100%) 19 (100%) 0.330

2 14 (24.1%) 2 (10.5%) -

3 44 (75.9%) 17 (89.5%) -

Follow-up duration (months) 42 [46.9] 56.6 [48.7] 0.165

PMF: Primary myelofibrosis, Hb: hemoglobin, Hct: hematocrit, LDH: lactate dehydrogenase, SD: standard deviation.

not find a significant difference in LDH level between PMF

patients with and without JAK2V617F mutation, in accordance

with some previous reports [8,10,27]. In a study involving 117

patients with PMF, the presence of JAK2V617F mutation did not

correlate with degree of reticulin fibrosis [8]. Consistent with

the study by Tefferi et al., the degree of reticulin fibrosis did not

differ between our PMF patients when stratified by JAK2V617F

mutational status [8]. There is limited information regarding

the relevance of JAK2V617F on bleeding complications in PMF

patients. Tefferi et al. did not determine a statistically significant

correlation between JAK2V617F mutation and bleeding history

[8]. However, we observed a trend towards higher prevalence of

bleeding events in JAK2V617F-positive PMF patients compared

to JAK2V617F-negative PMF patients (24.1% and 5.3%,

respectively). In the study by Barosi et al., JAK2V617F mutational

status was associated with an increased requirement for

splenectomy and greater need of cytoreductive therapy in PMF

patients [9]. However, in the study by Tefferi et al. involving 199

patients with PMF, no significant correlation was found between

the presence of JAK2V617F mutation and need for cytoreductive

therapy or splenectomy [13]. Confirming the finding of Tefferi

et al., in our study, the presence of JAK2V617F mutation in

98



Table 4. Clinical and laboratory features between JAK2V617F-positive and -negative patients among 77 primary myelofibrosis

patients (continued).

PMF JAK2V617F-mutated, n (%) JAK2V617F-unmutated, n (%) p-value


Splenomegaly group 58 (100%) 19 (100%) 0.090

No splenomegaly 0 1 (5.3%) -

Mild splenomegaly 11 (19%) 6 (31.6%) -

Massive splenomegaly 47 (81%) 12 (63.2%) -

Bleeding 14 (24.1%) 1 (5.3%) 0.090

Hydroxyurea 54 (93.1%) 18 (94.7%) 1.000

History of splenectomy 3 (5.2%) 1 (5.3%) 1.000

AHSCT 2 (3.4%) 1 (5.3%) 1.000

Karyotype 58 (100%) 19 (100%) 0.274

Normal 49 (84.5%) 18 (94.7%) -

Favorable 7 (12.1%) 0 -

Unfavorable 2 (3.4%) 1 (5.3%) -

DIPSS-Plus 58 (100%) 19 (100%) 0.143

Low risk 11 (19%) 4 (21.1%) -

Intermediate-1 22 (37.9%) 5 (26.3%) -

Intermediate-2 17 (29.3%) 10 (52.6%) -

High risk 8 (13.8%) 0 -

Thrombosis 8 (13.8%) 3 (15.8%) 1.000

Thrombosis group 58 (100%) 19 (100%)

No thrombosis 50 (86.2%) 16 (84.2%) -

Arterial 4 (6.9%) 3 (15.8%) -

Venous 3 (5.2%) 0 -

Arterial and venous 1 (1.7%) 0 -

Leukemic transformation 3 (5.2%) 1 (5.3%) 1.000

Death 11 (19%) 3 (15.8%) 1.000

PMF: Primary myelofibrosis, AHSCT: allogeneic hematopoietic stem cell transplantation, DIPSS: Dynamic International Prognostic Scoring System.

PMF had no impact on the need for cytoreductive treatment or

requirement for splenectomy [13]. Several studies investigated

the association of JAK2V617F mutation in PMF patients with

prognostic scoring systems [8,10,13,27]. In a series of 186 PMF

patients, the number of JAK2V617F-positive patients in the low

risk category of the Dupriez scoring system was significantly

higher compared with JAK2V617F-negative patients [27].

Campbell et al. reported that Dupriez prognostic scores tended

to be lower for patients positive for JAK2V617F mutation [10].

On the contrary, several groups reported no correlation between

JAK2V617F mutation and Dupriez prognostic score [8,13]. To

analyze whether the JAK2V617F mutational status correlated

with prognostic scoring systems, we evaluated the distribution

of patients in the different risk categories of the DIPSS-Plus

[16]. We found no significant difference in the DIPSS-Plus risk

stratification between JAK2V617F-positive and -negative PMF

patients. Several studies revealed that in PMF, the presence of

JAK2V617F mutation showed no correlation with presence or

distribution of cytogenetic abnormalities [8,9,10]. Confirming

the aforementioned studies, in our population, we observed no

significant difference in the distribution of karyotype categories

between JAK2V617F-positive and -negative groups. Divergent

results were reported regarding the effect of JAK2V617F

mutation on OS and leukemic transformation rate in PMF

patients [8,9,10,13]. We did not observe any differences in the

rates of death and leukemic transformation in PMF patients

with and without JAK2V617F mutation.

Collectively, according to the results of our study, JAK2V617F

mutation may identify distinct disease phenotypes of ET and

PMF patients.

Acknowledgment

We thank the Molecular Hematology Laboratory staff of the

İstanbul University İstanbul Medical Faculty for their assistance

with sample handling.

99



Ethics

Ethics Committee Approval: The study was approved by the Local

Ethics Commitee of İstanbul University İstanbul Medical Faculty

(file number: 2012/1571-1245), Informed Consent: Informed

consent was obtained from all patients for being included in

the study.


Design the Research: İpek Yönal, Meliha Nalçacı, Akif Selim

Yavuz, Fatma Deniz Sargın; Concept: İpek Yönal, Meliha Nalçacı,

Akif Selim Yavuz; Supply Samples: İpek Yönal; Analyze the Data:

İpek Yönal; Literature Search: İpek Yönal; Draft the Article: İpek

Yönal, Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz;

Perform the Laboratory Work: Aynur Dağlar-Aday, Başak

Akadam-Teker, Ceylan Yılmaz; Help in Acquisition of Data:

Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz; Revise

the Article: Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz

Sargın; Writing: İpek Yönal, Aynur Dağlar-Aday, Başak Akadam-

Teker, Ceylan Yılmaz, Meliha Nalçacı, Akif Selim Yavuz, Fatma

Deniz Sargın.

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

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

affiliations relevant to the subject matter or materials included.

Financial Disclosure: The study was supported by the İstanbul

University Scientific Research Foundation (project number:

30427).

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101

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0070


D-index: A New Scoring System in Febrile Neutropenic Patients

for Predicting Invasive Fungal Infections

D-index: Febril Nötropenik Hastalarda İnvazif Mantar Enfeksiyonlarının Tanımlanmasında

Yeni Bir Skorlama Sistemi

Gülden Yılmaz 1 , Belgin Coşkun 1 , Atilla Elhan 2 , Alpay Azap 1 , Hamdi Akan 3

1Ankara University Faculty of Medicine, Department of Clinical Microbiology and Infectious Diseases, Ankara, Turkey

2Ankara University Faculty of Medicine, Department of Biostatistics, Ankara, Turkey

3Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Objective: Neutropenia is a critical risk factor for invasive fungal

infections (IFIs). We retrospectively performed this study to assess

the performance of the D-index, a new test that combines both the

duration and the severity of neutropenia, in predicting IFIs among

patients with acute myelogenous leukemia.

Materials and Methods: Fifteen patients with IFIs and 28 patients

who did not develop IFIs were enrolled in the study. The D-index was

defined as the area over the neutrophil curve, whereas the cumulative-

D-index (c-D-index) was the area over the neutrophil curve from the

start of neutropenia until the first clinical manifestation of IFI.

Results: The D-index and the c-D-index tended to be significantly

higher in patients with IFIs, with medians of 10,150 (range: 4000-

22,000) and 5300 (range: 2300-22,200), respectively (p=0.037 and

p=0.003, respectively). The receiver operating characteristic analyses

showed that there was a cutoff point of 3875 for the D-index in

predicting IFI; the sensitivity, specificity, and positive and negative

predictive values were 100%, 67.9%, 35.4%, and 100%, respectively.

There was also a cutoff point of 4225 for the c-D-index in predicting

IFI; the sensitivity, specificity, and positive and negative predictive

values for the c-D-index were 93.3%, 71.4%, 36.6%, and 98.4%.

Conclusion: The D-index and especially the c-D-index could be useful

tools with high negative predictive value to exclude as well as to

predict IFIs in the management of neutropenic patients.

Keywords: Neutropenia, D-index, Cumulative-D-index, Hematological

malignancies, Invasive fungal infections

Öz

Amaç: İnvaziv fungal enfeksiyonların (İFE) gelişiminde nötropeni

önemli bir risk faktörüdür. Biz bu çalışmayı, akut miyeloid lösemi

olup, İFE gelişen hastalarda, nötropeni süresini ve sayısını birlikte

değerlendiren yeni bir test olan D-indeks’in performansını ölçmek için

geriye dönük olarak yaptık.

Gereç ve Yöntemler: Çalışmaya 50 tane İFE gelişen hasta, 28 tane

İFE gelişmeyen hasta alındı. D-indeks nötrofil eğrisinin üzerinde kalan

alan olarak, kümülatif-D-indeks (k-D-indeks) ise, nötropeninin ilk

başladığı günden İFE’nin belirtilerinin başladığı ilk güne kadar çizilen

nötrofil eğrisinin üzerinde kalan alan olarak belirlendi.

Bulgular: D-indeks ve k-D-indeks İFE gelişen hastalarda yüksek olma

eğilimindedir. D-indeks ve k-D-indeks için değerler ortalama 10,150

(aralık: 4000-22,000) ve 5300 (aralık: 2300-22,200) olup, sırası ile

p=0,037 ve p=0,003 saptandı. Yapılan analizlerde D-indeks için eşik

değerin 3875 olup, duyarlılık, özgüllük, pozitif ve negatif prediktif

değerleri sırası ile %100, %67,9, %35,4 ve %100 saptandı. k-D-index

için eşik değer 4225 olup, duyarlılık, özgüllük, pozitif ve negatif

prediktif değeri sırası ile %93,3, %71,4, %36,6 ve %98,4 saptandı.

Sonuç: D-indeks ve özellikle k-D-indeks nötropenik hastaları günlük

değerlendirmede kullanılabilecek bir testtir. Negatif prediktif değerinin

yüksek olması nedeni ile İFE olan hastaları erken yakalamanın yanı

sıra, İFE’yi dışlamak için de etkili bulunmuştur.

Anahtar Sözcükler: Nötropeni, D-indeks, Kümülatif-D-indeks,

Hematolojik malignite, İnvaziv fungal enfeksiyon

Introduction

Invasive fungal infections (IFIs) are major life-threatening

infections among immunocompromised patients with

hematologic malignancies. Although there has been significant

progress in the management of febrile neutropenic cancer

patients related to increasing protective measures and

antifungal agents, neutropenia is still a critical risk factor

for IFI. Profound (<100 neutrophils/µL) and prolonged (>10

days) neutropenia is associated with a higher risk of invasive

aspergillosis [1,2,3,4,5,6,7].

Address for Correspondence/Yazışma Adresi: Gülden YILMAZ, M.D.,

Ankara University Faculty of Medicine, Department of Clinical Microbiology and Infectious Diseases, Ankara, Turkey

Phone : +90 312 508 27 15

E-mail : drguldeny@yahoo.com.tr

Received/Geliş tarihi: February 14, 2014

Accepted/Kabul tarihi: May 21, 2014

102


Yılmaz G, et al: D-index

Several scoring systems have been developed to categorize

febrile neutropenic patients into risk groups. These systems

usually take neutropenia duration into account. Recently,

Portugal et al. developed indexes called the D-index and the

cumulative-D-index (c-D-index), which take into account both

the duration and the intensity of neutropenia to predict the IFI

risk [8]. We performed this study to assess the performance of

these new tests in predicting IFIs among patients with acute

myelogenous leukemia (AML).


Patients

The Department of Adult Hematology of Ankara University’s

Faculty of Medicine, a 56-bed institution, is one of the

main regional centers of hematology and bone marrow

transplantation in Ankara. Patients with newly diagnosed AML

receiving first induction or with relapsed or refractory AML,

and who developed neutropenia at this center between March

2011 and March 2012, were included in the study. Among

these patients, 15 patients with IFIs and 28 patients who did

not develop IFIs were enrolled. We selected controls with the

same underlying disease and leukemia status. IFIs were classified

as possible, probable, or proven according to the European

Organization for Research and Treatment of Cancer/Invasive

Fungal Infections Cooperative Group and the National Institute

of Allergy and Infectious Diseases Mycoses Study Group (EORTC/

MSG) revised criteria [9]. Only the proven and probable cases

were included in the study (proven: 2, probable: 13). Clinical and

epidemiological data were collected by structured survey forms

during daily infectious disease consultation visits. The patients

who developed IFIs were compared with controls regarding age,

sex, underlying disease, comorbidities, type of chemotherapy,

antibacterial and antifungal prophylaxis, mortality rate, duration

of neutropenia, profound neutropenia, D-index, and c-D-index.

The study was approved by the local ethics committee.

D-index and Cumulative-D-index Calculation

The absolute neutrophil count was recorded in patients and

controls. The D-index is an index based on a graph showing

the absolute neutrophil counts over the course of the episode

of neutropenia (Figure 1). It is geometrically the area over

the neutrophil curve. The D-index was calculated as the

difference between the observed area under curve (AUC) (A o )

and the expected neutrophil area (A e ) if the patient did not

develop neutropenia (D-index: A e -A o ). A o was calculated by

the trapezoidal method, while Ae is the product of 500 and

the number of days with neutropenia (A e : 500/µL x days with

neutropenia). An XLA add-in, developed by Usansky et al., was

used to apply the trapezoidal method [10].

We also calculated the c-D-index, which is from the start of

neutropenia until the date of first clinical manifestation of IFI in

patients. The date of first clinical manifestation was defined by

3 specialists (2 from the department of infectious diseases and

one from the department of hematology), and then their results

were compared with each other. The clinical manifestations

were cough, nasal discharge, pleuritic chest pain, hemoptysis,

skin nodules, and stomachache with elevated liver enzymes.

Power Analysis

The D-index was considered as the primary outcome variable for

this study. Group sample sizes of 25 and 15 achieved 82% power

to detect a difference of 5000 between the null hypothesis that

both group means were 4000 and the alternative hypothesis

that the mean of group 2 was 9000 with estimated group

standard deviations of 5000 and 5000 and with a significance

level (alpha) of 0.05 using a 2-sided Mann-Whitney U test,

assuming that the actual distribution was normal.


Mean ± standard deviation, median (minimum-maximum),

or percentage values are given as descriptive statistics as

applicable. Dichotomous variables were compared using the chisquare

or Fisher’s exact test. Test of normality was assessed by

Shapiro-Wilk test. Comparison of continuous variables between

fungal and control groups was analyzed by Mann-Whitney U

test. A receiver operating characteristic (ROC) curve analysis

was performed to evaluate the ability of the D-index and c-Dindex

to predict IFI. Positive and negative predictive values were

calculated by using cutoff values obtained from ROC analysis.

A multiple logistic regression was performed to identify the

independent risk factors of outcome variable and the adjusted

odds ratio (OR) was calculated. SPSS 15.0 for Windows was

used for statistical analysis. A p-value of less than 0.05 was

considered significant.

Results

A total of 15 patients with IFIs and 28 controls were enrolled

during the 1-year study. The clinical and epidemiological data

of the patients are shown in Table 1. Those that developed IFIs

were older than the controls. The lung was the most common

site of fungal infection (86.7%). There were no significant

differences between patients and controls regarding sex, status

of underlying disease, chemotherapies, and comorbidities. All

patients were given fluconazole prophylaxis. The duration and

the severity of neutropenia were significantly higher in IFI

patients. Consequently, the D-index and the c-D-index tended

to be significantly higher in patients with IFIs, with a median

of 10,150 (range: 4000-22,000) and 5300 (range: 2300-22,200),

respectively.

103



Table 1. Patient demographics and clinical characteristics.

Cases (n=15) Controls (n=28) p

Age, years (mean ± SD) 52.5±15.1 42.5±14.6 0.042

Sex (n, %)

Male

Female

Status of underlying disease (n, %)

Induction

Relapse

Refractory

Bone marrow transplantation (n, %) 4 26.7 6 21.4 0.719

Chemotherapy regimen (n, %)

Daun+Ara-C

EMA

Cyclosporine

Comorbidity (n, %) 1 6.7 3 10.7 1.000

Duration of neutropenia (<500/µL), median (minimum-maximum) 13 (6-53) 8.5 (5-22) 0.004

Duration of profound neutropenia (<100/µL), median (minimum-maximum) 9 (3-22) 4.5 (0-16) 0.005

D-index, median (minimum-maximum) 10,150 (4000-22,000) 3200 (1350-9200) 0.037

Cumulative-D-index, median (minimum-maximum) 5300 (2300-22,200) 3200 (1350-9200) 0.003

SD: Standard deviation

7

8

8

5

2

12

2

1

46.7

53.3

53.3

33.3

13.3

80

13.3

6.7

15

13

23

3

2

24

2

2

53.6

46.4

82.1

10.7

7.1

85.7

7.1

7.1

0.666

0.122

0.801

No. of Neutrophils, mm 3

500

Neutrophil curve

400

300

200

D-index

100

0

1 2 3 4 5 6 7 8

AUC

9 10 11 12

Duration of Neutropenia (days)

1.0

0.8

ROC Curve

Figure 1. The D-index is an index based on a graph showing

absolute neutrophil counts over the course of the episode of

neutropenia [8].

The ROC analyses showed that both the D-index and the c-Dindex

could be used to predict IFIs [AUC ± standard error (SE):

0.914±0.042, p<0.001 and AUC ± SE: 0.779±0.074, p=0.003,

respectively; Figures 2 and 3]. There was a cutoff point of 3875

for the D-index in predicting IFIs; the sensitivity, specificity,

and positive and negative predictive values were 100%, 67.9%,

35.4%, and 100%, respectively. There was also a cutoff point

of 4225 for the c-D-index in predicting IFI; the sensitivity,

specificity, and positive and negative predictive values for the

c-D-index were 93.3%, 71.4%, 36.6%, and 98.4%.

Discussion

Fungal infections are responsible for most of the deaths from

infections in febrile neutropenic patients with hematological

malignancies, with mortality rates of 50%-80%. Although

the initiation of appropriate antifungal therapy is crucial

and associated with improved outcomes, the management of

Sensitivity

0.6

0.4

0.2

0.0

0.0 0.2 0.4 0.6 0.8 10

1- Specificity

Figure 2. Receiver operating characteristic analyses for the

D-index.

antifungal treatment in this heterogeneous population is a

matter of great research [3,6,11,12,13,14].

Empirical antifungal therapy, which is the administration of

systemic antifungals for persistent and recurrent fever 96 h after

broad-spectrum antibacterial treatment, has been the standard

of care for many years. Since the fever-based approach increased

antifungal usage, preemptive or diagnostic-driven antifungal

therapy, which is usually guided by clinical or radiological signs

and serum biomarkers (galactomannan, 1,3-beta-D-glucan,

104



1.0

ROC Curve

respectively. However, the D-index and c-D-index still tended to

be significantly higher for the IFI group than the controls when

age and sex were adjusted [OR: 1.54, 95% confidence interval

(CI): 1.28-2.19].

Sensitivity

0.8

0.6

0.4

0.2

0.0

0.0 0.2 0.4 0.6

1- Specificity

Diagonal segments are produced by ties

0.8 1.0

Figure 3. Receiver operating characteristic analyses for the

cumulative-D-index.

polymerase chain reaction), was defined [15]. Studies comparing

these 2 approaches reported that the rate of antifungal usage

was reduced and no increase in mortality was observed with

diagnostic-driven antifungal therapy. However, the success of

this strategy depends on the availability and the performance

of the tests predicting IFI [15,16,17,18].

IFIs are difficult to predict and diagnose. Host factors are

important for predicting IFIs as well, as they are the determinants

of the outcome [6]. Neutropenia, one of the host factors, is still

a significant risk factor, and resolution of neutropenia has a key

role in complete recovery from an IFI [6,19]. The duration and

also the severity of neutropenia are critical, but there was no

practical tool that combined both the duration and the severity

of neutropenia in its evaluation approach.

Recently, Portugal et al. developed the D-index and c-D-index,

simple indexes to calculate, which combine both the duration

and the intensity of neutropenia [8]. They reported that these

indexes were superior to the duration of neutropenia for

predicting IFI. Shortly afterwards, Kimura et al. also showed

that early pulmonary infections in hematopoietic stem cell

transplantation recipients tended to occur in patients with

higher D-index and c-D-index scores [20]. In accordance

with these results, higher D-index and c-D-index scores were

associated with IFIs in our study. We presume that the c-Dindex

score in particular, available earlier than the D-index

score, has the ability to discriminate among patients with the

same duration but different severities of neutropenia according

to IFI development.

Previous studies documented increased risks for fungal

infections in older patients [11,21,22]. In this study, univariate

analysis showed that the median age was higher for patients

with IFIs than the controls, at 52.5±15.1 and 42.5±14.6 years,

Although 20% of the stem cell transplantation centers in Turkey

use diagnostic-driven approaches, empirical treatment is still

the main approach [23]. This means that a significant proportion

of patients are receiving antifungal therapy unnecessarily and

we need helpful tools to assess the risk of IFI besides chest

computed tomography scan and the use of serum biomarkers.

The galactomannan test is the only available serum biomarker

at our center. It is performed twice weekly but the results are

reported with a 1-week delay. Hence, the c-D-index could be

integrated with other parameters to promote diagnostic-driven

therapy in such centers.

The negative predictive values of the D-index and c-D-index

for IFI prevalence of 15% was 100% (95% CI: 89.8-102.0) and

98.4% (95% CI: 87.2-101.6) using the cutoff values of 3875 and

4225, respectively. The high negative predictive values suggest

that this new tool should work to exclude invasive fungal

infections. Serum biomarkers such as galactomannan and betaglucan

for fungal infections have some false positives. Thus,

when interpreting the results in these situations, a c-D-index of

less than 4225 supports the false positivity of other biomarkers

and suggests that antifungal therapy could be delayed.

This study has some limitations. The first is the small number of

patients. Since April 2012, AML patients with induction therapy

have started to receive antifungal prophylaxis (posaconazole)

regularly at our center and the impact of this prophylaxis

could not be assessed in this study. The second limitation is the

underlying disease, due to the fact that only patients with AML

were evaluated. Thus, we could not investigate the applicability

of this new test with other malignancies.

Conclusion

In conclusion, this study confirms that the D-index, and in

particular the c-D-index, could be useful tools to exclude as

well as to predict IFIs. These cheap and simple tests stand out

with high negative predictive values in daily management of

neutropenic patients.

Ethics

Ethics Committee Approval: The study was approved by the

local ethics committee, Informed Consent: N/A.


Medical Practices: Belgin Coşkun; Concept: Gülden Yılmaz,

Design: Alpay Azap, Hamdi Akan; Data Collection or Processing:

Belgin Coşkun, Gülden Yılmaz, Atilla Elhan; Analysis or

105



Interpretation: Gülden Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay

Azap, Hamdi Akan; Literature Search: Gülden Yılmaz, Belgin

Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan; Writing: Gülden

Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan.




included.

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106

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0242


Gap-PCR Screening for Common Large Deletional Mutations

of β-Globin Gene Cluster Revealed a Higher Prevalence of the

Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

β-Globin Gen Kümesini İçine Alan Büyük Delesyonel Mutasyonların Gap-PCR ile Taranması Türk Tipi

İnversiyon/Delesyon (δβ)0 Mutasyonunun Antalya’da Yüksek Sıklıkta Olduğunu Gösterdi

Türker Bilgen 1,2 , Özden Altıok Clark 3 , Zeynep Öztürk 4 , M. Akif Yeşilipek 4 , İbrahim Keser 1

1Akdeniz University Faculty of Medicine, Department of Medical Biology and Genetics, Antalya, Turkey

2Namık Kemal University Central Research Laboratory (NABİLTEM), Tekirdağ, Turkey

3Akdeniz University Faculty of Medicine, Department of Medical Genetics, Antalya, Turkey

4Akdeniz University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Antalya, Turkey

Abstract

Objective: Although the calculated carrier frequency for point

mutations of the β-globin gene is around 10% for Antalya Province,

nothing is known about the profile of large deletional mutations

involving the β-globin gene. In this study, we aimed to screen

common deletional mutations in the β-globin gene cluster in patients

for whom direct DNA sequencing was not able to demonstrate the

mutation(s) responsible for the disease phenotype.

Materials and Methods: Thirty-one index cases selected with a

series of selection events among 60 cases without detected β-globin

gene mutation from 580 thalassemia-related cases tested by direct

sequencing over the last 4 years in our diagnostic center were

screened for the most common 8 different large deletional mutations

of the β-globin gene cluster by gap-PCR.

Results: We detected 1 homozygous and 9 heterozygous novel

unrelated cases for the Turkish inversion/deletion (δβ) 0 mutation in

our series of 31 cases. Our study showed that the Turkish inversion/

deletion (δβ) 0 mutation per se accounts for 16.6% of the unidentified

causative alleles and also accounts for 1.5% of all detected mutations

over the last 4 years in our laboratory.

Conclusion: Since molecular diagnosis of deletional mutations in

the β-globin gene cluster warrants different approaches, it deserves

special attention in order to provide prenatal diagnosis and prevention

opportunities to the families involved. We conclude that the Turkish

inversion/deletion (δβ) 0 , as the most prevalent deletional mutation

detected so far, has to be routinely tested for in Antalya, and the gap-

PCR approach has valuable diagnostic potential in the patients at risk.

Keywords: Deletional mutations, Turkish inversion/deletion (δβ) 0

mutation, Gap-PCR, β-Globin gene cluster

Address for Correspondence/Yazışma Adresi: Türker BİLGEN, PhD.,

Namık Kemal University Central Research Laboratory

(NABİLTEM), Tekirdağ, Turkey

E-mail : tbilgen@nku.edu.tr, tbilgen@akdeniz.edu.tr

Öz

Amaç: Beta-globin genindeki nokta mutasyonlarının sıklığı Antalya

bölgesi için yaklaşık %10 olarak belirlenmiş olmasına rağmen, betaglobin

genini içine alan büyük delesyonel tip mutasyonların profili

hakkında hiçbir şey bilinmemektedir. Bu çalışmada, DNA dizi analizi

yöntemiyle beta-globin geninde hastalığın oluşmasından sorumlu

mutasyon(lar) tespit edilememiş talasemili olgularda beta-globin gen

kümesinde yaygın görülen büyük delesyonel mutasyonları taramayı

amaçladık.

Gereç ve Yöntemler: Son dört yıl boyunca tanı merkezimizde DNA

dizi analizi yöntemiyle test edilmiş, talasemiyle ilişkilendirilen 580 olgu

arasından öncelikle beta-globin geni mutasyonu belirlenememiş 60

olgu seçildi. Bu 60 olgu arasından bir seri seleksiyon işlemi uygulanarak

nihai olarak belirlenmiş 31 hasta, beta-globin gen kümesinde en

yaygın görülen sekiz farklı büyük delesyonel tip mutasyon için gap-

PCR yöntemiyle tarandı.

Bulgular: Otuz bir olgudan oluşan serimiz içerisinde, Türk tipi

inversiyon/delesyon (δβ) 0 mutasyonu açısından heterozigot olan

dokuz yeni olgu ve homozigot olan bir yeni olgu belirlendi. Çalışmamız

Türk tipi inversiyon/delesyon (δβ) 0 mutasyonunun, laboratuvarımızda

son dört yıl boyunca tespit edilmiş tüm mutasyonların %1,5’ini ve

DNA dizi analizi yöntemiyle mutasyon tespit edilemeyen alellerin ise

%16,6’sını oluşturduğunu gösterdi.

Sonuç: Beta-globin gen kümesinde delesyonel tip mutasyonlar farklı

moleküler yöntemlerle tespit edilebilir. Bu durum prenatal teşhis ve

hastalığı önleme fırsatı sağlayabildiği için özel bir ilgi gerektirmektedir.

Sonuç olarak, toplumumuzda şu ana kadar belirlenmiş en sık görülen

delesyonel tip mutasyon olan Türk tipi inversiyon/delesyon (δβ) 0

mutasyonu Antalya’da rutin olarak test edilmelidir ve gap-PCR

yöntemi risk altındaki hastalar için önemli bir tanı potansiyeline

sahiptir.

Anahtar Sözcükler: Delesyonel mutasyonlar, Türk tipi inversiyon/

delesyon (δβ) 0 mutasyonu, Gap-PCR, Beta-globin gen kümesi

Received/Geliş tarihi: June 19, 2014

Accepted/Kabul tarihi: October 28, 2014

107

Bilgen T, et al: Screening of β-Globin Gene Cluster Deletions


Introduction

Beta-thalassemia (β-thal) is generally caused by point mutations

in the β-globin gene. However, there are at least 80 different

large deletional mutations in the β-globin gene cluster described

in the human hemoglobin variant (HbVar) database. While only

the β-globin gene is partially or completely removed in some of

those deletions, the δ-globin gene or δ- and γ-globin genes are

deleted in addition to the β-globin gene in some others [1,2].

It was also stated that 10% of the β-globin gene mutations

are large deletions causing phenotypes associated with β-thal

[3]. The phenotypes produced by deletions in the β-globin gene

cluster are classified according to the gene(s) involved, such as

β-thal, δβ-thal, εγδβ-thal, and hereditary persistence of fetal

hemoglobin (HPFH) [4]. Despite general carrier frequency for

β-globin gene mutations being reported at 2% for Turkey and at

as high as 10% for Antalya Province, large deletional mutations

in the β-globin gene cluster have rarely been reported so far

and there is no systemic study on mutation profiles of large

deletional mutations in the β-globin gene cluster in Turkey

[4,5,6,7,8,9]. On the other hand, the number of studies on

variety and allelic frequencies of large deletions in the β-globin

gene cluster has been growing recently [2,3,10,11,12]. Previous

studies revealed that HPFH-1, HPFH-2, HPFH-3, Sicilian (δβ) 0 -

thal, Chinese G γ( A γδβ) 0 -thal, Hb Lepore, Asian-Indian inversiondeletion

G γ( A γδβ) 0 -thal, and Turkish inversion-deletion (δβ) 0 -

thal mutations are among the most recurrent large deletional

mutations in the β-globin gene cluster [10,13].

Detection of large deletions of the β-globin gene cluster has

recently become an important issue because of its significance

in evaluation of unresolved thalassemia-related cases and

in disease prevention. On the other hand, commonly used

diagnostic tests targeting point mutations and small insertionsdeletions

of the β-globin gene are not suitable for detection

of large deletional mutations. Therefore, molecular detection

of large deletions needs different approaches in the laboratory.

Researchers have recently applied strategies like Southern

blotting, FISH, quantitative polymerase chain reaction (PCR),

multiplex ligation-dependent probe amplification (MLPA), and

gap-PCR for molecular detection of large deletional mutations

of the β-globin gene cluster [10,12,14,15,16]. Among them,

gap-PCR is a fast and reliable method allowing us to detect

the previously characterized mutations [3,13]. In this study,

we screened patients in whom we were not able to find the

underlying β-globin gene mutation(s) by direct DNA sequencing

for the 8 different common deletional mutations of the β-globin

gene cluster by gap-PCR.


Patients

Among the 580 patients who were tested in our diagnostic

laboratory for β-globin gene mutations by direct DNA

sequencing between July 2008 and July 2012, a total of 60

unrelated patients who had either no causative β-globin gene

mutation(s) by sequencing or no detectable PCR amplification

for the β-globin gene were initially selected. Being homozygous

for all common intragenic single-nucleotide polymorphisms

detected by sequence analyses was then used as the second

inclusion criterion for its potential to indicate hemizygosity.

Finally, the 31 most probable candidates were screened by gap-

PCR for the 8 different known deletions of the β-globin gene

cluster. Out of these 31 patients included in the study, 21 had

a mild phenotype without any β-globin gene mutation, while

the remaining 10 were moderately to seriously affected by the

disease with either one or no detected causative mutations.

All hematological and clinical findings were collected with

the informed consent of the patients. Hematological indices

were obtained with an automated cell counter (Abbott Cell

DYN3700; Abbott Laboratories, Abbott Park, IL, USA). The HbA2

and HbF levels were measured by high-performance liquid

chromatography (VARIANT; Bio-Rad Laboratories, Hercules, CA,

USA).

Sequence Analyses and Gap-PCR Screening for the 8 Known

Deletional Mutations of the β-Globin Gene

Following the isolation of genomic DNA with a commercial kit

(AxyPrep Blood Genomic DNA Miniprep Kit; Axygen Biosciences

Inc., Union City, CA, USA), the β-globin gene was amplified as

2 PCR fragments (from the -101 position to the Poly-A signal)

using 30-50 ng of genomic DNA in 25-µL reaction volumes.

The PCR mixture contained 12.5 µL of 2X PCR master mix

and 5 pmol of each primer (GML, Wollerau, Switzerland). The

sequencing was performed using the BigDye Terminator v3.1

Cycle Sequencing Kit and an ABI Prism 3130 Genetic Analyzer

(Applied Biosystems, Foster City, CA, USA).

The deletional mutations were chosen by taking into account

ethnic background and according to the published frequencies

[10]. Gap-PCR protocols and the primers for the deletional

mutations HPFH-1, HPFH-2, HPFH-3, Sicilian (δβ) 0 -thal, Chinese

G γ( A γδβ) 0 -thal, Hb Lepore, Asian-Indian inversion-deletion

G γ( A γδβ) 0 -thal, and Turkish inversion-deletion (δβ) 0 -thal were

used as previously described elsewhere [13].

Results

Among the 8 different known deletions of the β-globin gene

cluster mentioned above, only the Turkish inversion-deletion

(δβ) 0 mutation was detected in 10 patients in our series. We

found that 9 were heterozygous and 1 was homozygous for the

Turkish inversion-deletion (δβ) 0 mutation. The hematological

indices and molecular findings of 7 heterozygous patients and 1

homozygous patient are summarized in Table 1. Hematological

indices were not available for 2 heterozygous patients, males

108



as a technical limitation of gap-PCR; in addition, its usage is

limited to known deletional mutations. Gap-PCR analyses of the

parents of the patient would help to clarify such a situation;

however, we were not able to perform this analysis in this family.

Figure 1. Representative samples of Turkish-type inversion/

deletion (δβ) 0 mutation detected by gap-PCR. For reaction A

testing the upstream breakage of the mutation, the upper band

(742 bp) corresponds to normal results and the lower band (432

bp) to the mutation. Case 4 and Case 5 are heterozygous as both

have normal and mutation-related polymerase chain reaction

fragments. For reaction B testing the downstream breakage of

the mutation, the upper band (700 bp) corresponds to normal

results and the lower band (489 bp) to the mutation. Cases 4,

5, 6, and 7 show both normal and mutation-related polymerase

chain reaction fragments, confirming that they are heterozygous

for the mutation. NS: Normal sample, N: normal, M: mutation,

252x91 mm (72x72 dpi).

of 24 and 16 years old. Sequence analyses of mutation-related

gap-PCR bands of 10 patients showed that there was no

variation in sequence or at breakpoints of the Turkish inversiondeletion

(δβ) 0 mutation. Sequence analyses determined that

the exact breakpoints positions were 5.255,764 and 5.244,281

for upstream deletion (11,484 bp) and 5.236,654 and 5.235,062

(1592 bp) for downstream deletion according to NCBI reference

sequence NC_000011.9, chromosome 11 GRCh37.p13 primary

assembly.

Discussion

While nearly 25 different β-globin gene mutations have been

reported for Antalya Province as well as for Turkey so far, the

large deletional-type mutations of the β-globin gene cluster

have not been systematically investigated [4,6,17]. It has been

suggested that 10% of the causative alleles cannot be easily

detected by routine methods in β-thal-associated phenotypes

[18]. This proportion in our survey was similar to the literature.

Large deletional mutations might be somewhat responsible for

this challenge. In this regard, the Turkish inversion/deletion

(δβ)0 mutation per se accounts for approximately 16.6% of

the unidentified causative alleles and accounts for 1.5% of

all detected mutations from the last 4 years in our laboratory.

Among the 10 new unrelated cases of the Turkish inversiondeletion

(δβ)0 mutation detected in this study, only one

seemed to be homozygous. The gap-PCR technique is able to

reliably detect the heterozygous state of this type of mutation

by showing both normal and mutation-related bands on

agarose gel (Figure 1). On the other hand, this technique does

not exclude the possibility of the presence of another larger

deletional mutation such as the second mutation in the patient

found as homozygous in our study. This should be considered

It has been demonstrated that the deletions in the β-globin

gene cluster may cause HPFH, which is characterized by high

HbF levels reducing the disease severity [19]. While the patients

with deletions including the δ- and β-globin genes tend to

have mild phenotypes, patients with larger deletions involving

γ-globin genes have severe clinical phenotypes because of the

lack of the compensatory effect of fetal Hb [20]. Furthermore,

recent studies on hemoglobin switching events have revealed

that there is a binding site between the δ- and γ-globin genes

for BCL11, which is a repressor of γ-globin genes. The deletion

of this cis-acting element seems to be related to higher HbF

levels [9]. The Turkish type of inv/del (δβ) 0 thalassemia was first

characterized at the molecular level by Kulozik et al. in a Turkish

patient living in Germany with normal HbA2 and elevated HbF

levels in 1992 [21]. It was also associated with elevated HbF

and normal HbA2 levels in another later study [22]. Our study

revealed that 7 out of 9 patients carrying the Turkish inv/del

(δβ) 0 had elevated HbF levels, while the remaining 2 had normal

HbF levels. This controversial observation can be explained by

other factors that may modify the hematological expression of

this mutation. Such a situation was reported in δβ-thalassemia

before by Öner et al. [23]. This phenomenon shows that a small

proportion of the carriers of the Turkish inv/del (δβ) 0 mutation

may not have elevated HbF levels, which should be considered

in case selection for mutation screening.

Another important point is that the molecular detection

of large deletional mutations in the β-globin gene cluster is

extremely important for families at risk and seeking prevention.

Because their detection requires special attention, this type of

mutation may sometimes compromise the prenatal diagnosis

in laboratories used to focusing on point mutations and small

ins/del-type mutations of the β-globin gene. Despite not

being useful for previously uncharacterized deletions, gap-

PCR is the easiest and most precise way of detecting previously

characterized recurrent deletions. For these reasons, and taking

into account the relatively higher incidence of the Turkish-type

inv/del (δβ) 0 mutation in Antalya Province, we suggest that it is

worthwhile to screen for this mutation in Turkish patients when

the first-line diagnostic tests such as sequencing and strip assay

fail to detect the causative mutation(s).

Gap-PCR is the cheapest and fastest method for the detection

of large deletional mutations. Nevertheless, the approach

has specific requirements for being used as a diagnostic tool,

such as positive controls, and the targeted mutation has to be

109



Table 1. Hematological findings of the patients with Turkish inversion-deletion (δβ) 0 mutation.

Case

Age

(years)/Sex

β-Globin Gene

Mutation(s)

HbA2*

HbF (%)

Hb

(g/dL)

MCV

(fL)

MCHC

(g/dL)

MCH

(pg/cell)

RBC

(10 6 /µL)

RDW

(%)

(δβ)-Thalassemia 1 11/M Turk inv-del (δβ)0/N 2.6 11.2 66.5 32.0 21.3 5.28 19.2

Heterozygotes

13.5

2 35/M Turk inv-del (δβ) 0 /N 2.3 13.4 73.2 31.7 23.2 5.77 23.9

11.8

3 55/F Turk inv-del (δβ) 0 /N 2.6 11.7 68.1 27.6 18.8 5.9 21.9

7.4

4 34/M Turk inv-del (δβ) 0 /N 2.9 14.5 65.2 31.7 20.7 7.03 22.5

9.8

5 13/M Turk inv-del (δβ) 0 /N 2.8 11.8 68.5 30.50 20.9 5.66 17.3

7.7

6 14/M Turk inv-del (δβ) 0 /N 2.7 11.9 62.2 31.5 19.6 6.1 16.2

1.9

7 27/M Turk inv-del (δβ) 0 /N 2.4

0

14.1 64.4 32.2 20.7 6.8 54.8

(δβ)-Thalassemia 8 48/M Turk inv-del (δβ) 0 /N

Turk inv-del (δβ)0

0

100

13 74.8 31.7 23.7 5.5 22.3

F: Female, M: male, Hb: hemoglobin, WBC: white blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin

concentration, RBC: red blood cell, RDW: Red blood cell distribution width.

*Normal HbA2 levels (between 1.5% and 3.8%) according to laboratory reference values.

previously well described. Having positive controls is important

for optimization and validation of gap-PCR. Without welloptimized

protocols, gap-PCR should not be used as a routine

diagnostic method. In addition to the possibility of false

negativity, positive results should also be confirmed by family

study when the parents are available. We had positive controls

for the Turkish-type inv/del (δβ) 0 mutation prior to this study,

but not for the other types of mutations that we screened.

This could be considered as a limitation of our study. The other

patients in whom we could detect none of the deletions screened

in our study are strong candidates for screening for either other

previously described but rarer or completely novel deletional

mutations. Therefore, there is need for further analyses in order

to resolve these cases. MLPA and array comparative genomic

hybridization methods are strong tools to investigate possible

novel and rare deletional mutations. MLPA is currently the

more commonly used approach for detection of large deletions

affecting a particular region of the genome, but its coverage

is limited to the probe set designed. We are planning a MLPA

study for the patients who had no positive findings in our gap-

PCR screening. On the other hand, not only the patients whose

mutation(s) were not identified but also even homozygous

patients for one particular parental β-globin gene mutation

detected by sequencing or strip assay should be investigated for

deletional mutations in order to find out the exact second hit

leading to thalassemia intermedia or major phenotypes.

Ethics

Ethics Committee Approval: Retrospective study, Informed

Consent: It was taken.


Surgical and Medical Practices: M. Akif Yeşilipek; Concept:

Türker Bilgen; Design: Türker Bilgen; Data Collection or

Processing: Türker Bilgen, Özden Altıok Clark, Zeynep Öztürk, M.

Akif Yeşilipek, İbrahim Keser; Analysis or Interpretation: Türker

Bilgen, İbrahim Keser; Literature Search: Türker Bilgen; Writing:

Türker Bilgen, İbrahim Keser.




included.

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111

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0312


The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients

Receiving Prophylactic Enoxaparin

Profilaktik Enoksaparin Alan Sepsis Hastalarında Doku Faktör Yolak İnhibitörü Düzeyleri

Hadil A. Al Otair 1 , Abdel Galil M. Abdel Gader 2 , Syed M. Khurshid 1 , Abdulaziz H. Alzeer 1 , Abdul Kareem Al Momen 3 , Mashael Al Shaikh 4 ,

Farja Al Gahtani 3 , Zohair A. Al Aseri 5 , Hossam A.H. Abdelrazik 5

1King Saud University College of Medicine, King Khalid University Hospital, Department of Critical Care, Riyadh, Saudi Arabia

2King Saud University College of Medicine, King Khalid University Hospital, Department of Physiology, Riyadh, Saudi Arabia

3King Saud University College of Medicine, King Khalid University Hospital, Department of Medicine, Riyadh, Saudi Arabia

4King Saud University College of Medicine, King Khalid University Hospital, Department of Pharmacy, Riyadh, Saudi Arabia

5King Saud University College of Medicine, King Khalid University Hospital, Department of Emergency, Riyadh, Saudi Arabia

Abstract

Objective: Sepsis syndrome is usually accompanied by activation

of blood coagulation mechanisms. Earlier studies found deficiencies

of the 3 main natural anticoagulants, antithrombin, protein C, and

protein S. However, none of these inhibitors block tissue factor,

the prime trigger of coagulation during sepsis that is controlled

specifically by the tissue factor pathway inhibitor (TFPI). The aim of

this study was to characterize the fluctuations in the levels of natural

anticoagulants, particularly TFPI, in the course of sepsis and to find

out their association with the anticoagulant action of the lowmolecular-weight

heparin enoxaparin.

Materials and Methods: We studied 51 consecutive patients with

sepsis. Blood samples were collected from patients at baseline (0 h) and

at 4, 12, and 24 h after enoxaparin administration. The following assays

were undertaken using commercial kits: activated partial thromboplastin

time, prothrombin time, thrombin time, total and free TFPI, protein C

and protein S, antithrombin, fibrinogen, and anti-factor Xa.

Results: Before enoxaparin administration, there was significant

prolongation of the prothrombin time and activated partial thromboplastin

time, and this remained the case in the 3 subsequent samples. There was

marked reduction in the levels of antithrombin, protein C, and total and

free protein S to below control values throughout the study. In contrast,

plasma levels of both total and free TFPI were markedly elevated and

increased after enoxaparin therapy. Anti-factor Xa levels were within

the therapeutic range throughout. There was no difference in TFPI levels

between those patients who died and those who survived.

Conclusion: Sepsis triggered marked release of TFPI from

endothelial cells. This persisted and was increased further following

the administration of enoxaparin. In contrast, there was marked

consumption of the natural coagulation inhibitors antithrombin,

protein C, and protein S. These results go some way towards explaining

why the therapeutic use of recombinant TFPI fails to correct sepsisassociated

coagulopathy.

Keywords: Coagulation, Sepsis, Enoxaparin

Öz

Amaç: Sepsis sendromuna genellikle kan pıhtılaşma sisteminin

aktivasyonu eşlik eder. İlk çalışmalar ana doğal 3 antikoagülan olan

antitrombin, protein C ve protein S eksikliği bulmuştur. Bununla birlikte,

bu inhibitörlerin hiç biri doku faktörü bloke etmez, sepsis sırasındaki

koagülasyon tetiklenişi özelllikle doku faktör yolak inhibitörü (DFYİ)

ile kontrol edilir. Bu çalışmanın amacı sepsis sırasındaki doğal

antikoagülan ve özellikle DFYİ düzeyi dalgalanmalarını karakterize

etmek ve bunların düşük moleküler ağırlıklı heaprin enoksaparinin

antikoagülan eylemi ile ilişkilerini öğrenmekti.

Gereç ve Yöntemler: Ardışık 51 sepsis hastası çalışmaya alındı. Taban

(0 saat) ve enoksaparin verimesinden 4, 12, 24 saat sonra kan örnekleri

alındı. Aşağıdaki deneyler ticari kitleri kullanılarak yapılmıştır; parsiyel

tromboplastin zamanı, protrombin zamanı, trombin zamanı, toplam ve

serbest DFYİ, protein C ve protein S, antitrombin, fibrinojen, ve aktif

anti-faktör Xa.

Bulgular: Enoksaparin uygulamadan önce ptorombin zamanı ve aktif

parsiyel protrombin zamanında önemli uzama vardı. Bu durum sonraki

3 örneklemde de devam etti. Çalışma boyunca antitrombin, protein C,

toplam ve serbest protein S seviyeleri değerlerinde kontrollere göre

belirgin bir azalma oldu. Buna karşılık, hem toplam hem de serbest

plazma DFYİ değerleri belirgin biçimde yükseldi ve enoksaparin

tedavisinden sonra arttı. Anti faktör Xa düzeyleri terapötik aralık

içindeydi. Vefat eden ve sağ kalan hastalar arasında DFYİ düzeyi

açısından fark yoktu.

Sonuç: Sepsis, endotel hücrelerinden belirgin DFYİ salınımı ile tetiklenir.

Bu, enoksaparin uygulmasını takiben kalıcı olmuş ve daha da artmıştır.

Bunun aksine, doğal koagülasyon inhibitörleri antitrombin, protein

C ve protein S’nin belirgin tüketimi vardı. Bu sonuçlar, tedavi amaçlı

rekombinant DFYİ kullanımının sepsis ilişkili koagülopatiyi düzeltmek

için neden başarısız olduğunu doğru biçimde açıklamaktadır.

Anahtar Sözcükler: Koagülasyon, Sepsis, Enoksaparin

Address for Correspondence/Yazışma Adresi: Hadil A. AL OTAIR, M.D.,

King Saud University College of Medicine, King Khalid University Hospital,

Department of Critical Care, Riyadh, Saudi Arabia

Phone : +96611-4692253 E-mail : hadil.alotair@live.com

Received/Geliş tarihi: August 03, 2014

Accepted/Kabul tarihi: January 15, 2015

112


Al Otair AH, et al: Tissue Factor Pathway Inhibitor in Patients with Sepsis

Introduction

Sepsis syndrome results from a host reaction to infection that

triggers the systemic inflammatory response syndrome, which,

on one hand, activates procoagulation mechanisms, and, on the

other, shuts down fibrinolysis, leading to the formation of fibrin

microthrombi in microcirculation and multiple organ failure

[1,2]. In its worst form, the interaction between inflammation

and the coagulation system may lead to the development of

disseminated intravascular coagulation [3,4].

Over the last 3 decades, numerous reports have emerged that

describe disturbances in the measured levels of coagulation

parameters in patients with sepsis [5,6,7,8,9]. Much emphasis

has been focused on the deficiencies of the 3 main natural

coagulation inhibitors: antithrombin (AT), activated protein C,

and tissue factor pathway inhibitor (TFPI) [9,10,11,12]. This led

to numerous clinical therapeutic trials of administering these

inhibitors to patients with sepsis. Some success was initially

obtained with the administration of activated protein C, but

later on, the PROWESS-SHOCK trial showed an increased risk of

bleeding with the use of activated protein C, with no mortality

benefit. Similarly, trials with AT and recombinant TFPI generated

disappointing results [13,14].

The resultant procoagulant state associated with sepsis has

also been recognized as an important risk factor for venous

thromboembolism in critically ill patients [14,15]. Therefore,

deep vein thrombosis prophylaxis is considered of utmost

importance and is practiced with vigilance in intensive care units

(ICU) using unfractionated heparin and low-molecular-weight

heparin (LMWH) [16,17]. LMWH exerts its antithrombotic effect

mainly by inhibiting activated factor X (FXa) and to a lesser

degree AT [18]. Nevertheless, failure of deep vein thrombosis

prophylaxis in critically ill patients has been well described

[17,19]. The reason for this is thought to be multifactorial and

one possible proposed explanation could be related to lower

anticoagulant effect (as assessed by anti-FXa activity) in these

patients, despite appropriate LMWH dosage [20].

The recent availability of more precise assay techniques for

the measurement of the natural anticoagulants, particularly

total and free TFPI and protein S, encouraged us to monitor

the fluctuations of natural anticoagulants in patients with

sepsis, in a way that no study has done before, to find a

possible explanation for why past trials administering natural

anticoagulants to patients with sepsis failed.

Therefore, the aim of this study was to assess the levels of natural

anticoagulants, particularly total and free TFPI, in patients with

sepsis and septic shock and to find out the association between

these fluctuations and the anticoagulant action of the LMWH

enoxaparin.


Study Population

Fifty-one consecutive patients were studied; 29 were male and

22 female, with a mean age of 51±20.8 years. All were admitted

to the ICU of King Khalid University Hospital, Riyadh, with sepsis

or septic shock. Sepsis is defined as systemic inflammatory

response syndrome due to infection [1,2]. Septic shock is

defined as severe sepsis-induced hypotension that persists

despite adequate fluid resuscitation [1,6]. Exclusion criteria

were patients younger than 18 years old, body weight of <45

kg or >148 kg, renal insufficiency (creatinine clearance of <30

mL/min), active bleeding, platelet count of <75,000 mm3, INR of

>2, activated partial thromboplastin time (APTT) of >2 times the

upper normal, therapeutic anticoagulation, pregnancy, porcine

hypersensitivity, and administration of unfractionated heparin

or LMWH prior to enrollment in the study. Controls (n=42)

were healthy individuals (28 males) selected from blood donors,

academic staff, and volunteers from the general public. Their

ages ranged from 21 to 62 years (mean: 47.4). They were not

taking any form of medication at the time of blood sampling.

The study was approved by the Institutional Review Board of

the College of Medicine-King Saud University. Written informed

consent was obtained from all patients or their next of kin.

Data Collection

A data entry form was used for the collection of patients’

demographic data and clinical information as well as laboratory

results.

Enoxaparin (Clexane R, Aventis Pharma, Frankfurt, Germany),

which is a LMWH (4500 Da) isolated from porcine intestinal

mucosa and used as sodium salt, was injected subcutaneously

at a dose of 0.5 mg/kg in the thighs of all eligible patients after

obtaining the baseline blood samples within 1 h of the diagnosis

of sepsis [20,21].

Measurements of coagulation tests for APTT, prothrombin time

(PT), and thrombin time (TT), as well as the levels of natural

anticoagulants including total and free TFPI, protein S, protein

C, and AT, were repeated 4, 12, and 24 h after the administration

of enoxaparin.

Blood Collection and Processing

A total of 9.5 mL of blood was carefully collected into vacutainer

tubes containing 0.5 mL of sodium citrate (3.8%, 0.129 mol/L;

Terumo, Tokyo, Japan) at 0 h, before the administration of the

first enoxaparin dose (the baseline sample), and at 4, 12, and 24

h thereafter. Blood samples were mixed gently and transferred

immediately to the Coagulation Research Laboratory, Physiology

Department, College of Medicine, King Saud University.

113



The blood sample tubes were centrifuged at 3000 rpm (1000×g)

for 15 min in a refrigerated (4-6 °C) centrifuge (Jouan Centrifuge

Series, France). Platelet-poor plasma was separated using plastic

pipettes and aliquots and immediately stored at -80 °C, until

analysis in batches at a later date. Before assays were performed

plasma specimens were thawed at 37 °C for 15 min.

Laboratory Assays

Coagulation screening tests included APTT, PT, and TT. PT was

measured using a Stago STA Analyzer and STA Neoplastine

CI 5 (freeze-dried rabbit brain thromboplastin with heparin

inhibitor). For APTT, the STA PTT Automate 5 Kit was used. TT was

measured using the STA thrombin kits with calcium thrombin

reagent (approximately 1.5 NIH U/mL, freeze dried). The

coefficient of variation (CV) varied from 5% for APTT to 2% for

PT and TT. Plasma fibrinogen was measured by a turbidimetric

method [22] and the CV varied between 6% and 8%. Anti-FXa

was assayed by a colorimetric kit (Rotachrom HBPM/LMWH

Assay, Diagnostica Stago, Asnières-sur-Seine, France).

Coagulation inhibitors were assayed using an automated

coagulometer (Stago STAT 4) and reagents were supplied

by Diagnostica Stago, Asnières-sur-Seine, France: TFPI

[Asserachrom Enzyme-Linked Immunosorbent Assay (ELISA) Kit]

[23], total and free protein S (Asserachrom Protein S ELISA Kit),

protein C (Asserachrom Protein C ELISA Kit), and AT (Stachrom

Antithrombin Kit), with CV of 5% or less for total TFPI, free TFPI,

total protein S, free protein S, and protein C and 4% for AT.

STA-Liquid Anti-Xa for use with the STA Compact (Diagnostica

Stago, France) was used for the quantitative determination of

the potentiating effect of LMWH on antithrombin by recording

the anti-FXa activity in plasma using a chromogenic substrate.

Results were expressed as percent activity and according to the

manufacturer’s instructions.

Statistical Methods

The Mann-Whitney U test was used to compare means for 2

independent groups. The chi-square test or Fisher’s test was used

as appropriate to compare the percentages for 2 categorical

variables. A p-value of less than 0.05 indicated statistical

significance. SPSS 15 for Windows was used for the analysis

and for the drawing of the bar graphs.

Results

Pneumonia was the most common diagnosis (37.2%) in the

study population, followed by urosepsis and abdominal sepsis

(11.8% each). Thirteen patients (25.5%) developed septic shock

and were started on vasopressors. Nine (69.2%) of them died

during hospitalization, 10 died during the first week, and 6

patients died 2 weeks later. None died during the study period.

This accounted for a mortality rate of 31.4% (Table 1). A definite

infective organism was identified in 22 patients.

To facilitate comparisons between subjects and to reduce the

day-to-day variation in individuals, the results of each test were

expressed as percentage of normal pooled plasma.

On arrival to the Accident and Emergency Department and

before receiving any treatment, the baseline blood tests

showed prolongation of PT and APTT; the TT did not fluctuate

significantly. Significant prolongation of both PT and APTT

persisted in the 3 subsequent samples (4, 8, and 24 h), with

the APTT prolongation getting worse in the subsequent samples

(Figure 1).

The plasma fibrinogen levels were significantly elevated above

normal control values at baseline and in the 3 subsequent

samples (local laboratory reference values: 150-400 mg/dL)

(Figure 2).

There was significant reduction in the levels of the natural

anticoagulants AT, protein C, and total and free protein S below

control values from baseline and in the 3 subsequent samples (4,

12, and 24 h) (Figure 3).

Table 1. Descriptive statistics of study population.

n=51

Age (years) 51±20.8

Sex

Male

Female

29

28

BMI (kg/m 2 ) 29±3

APACHE II

Mean ± SD

Range

Diagnosis

Abdominal sepsis

Pneumonia

Bronchiectasis

Diabetic foot

Urosepsis

Central nervous system infections

Others

Comorbid conditions

Hematological conditions

Malignancy

Diabetic mellitus

Hypertension

Ischemic heart disease

Cerebral vascular accident

Chronic kidney disease

Chronic liver disease

Transplant

Others

24±4

16-29

6

19

4

2

6

3

11

4

3

12

12

5

6

2

2

3

2

Vasopressor support 13

Death 16

APACHE II: Acute Physiology and Chronic Health Evaluation II, BMI: body mass index,

SD: standard deviation.

114



In contrast to the above 3 natural coagulation inhibitors, the

plasma levels of total and free TFPI were markedly elevated above

control values (local laboratory reference value: 60.7±16.9 ng/

mL) throughout the study period. The mean level of total TFPI

was 73.0±39.0 ng/mL at baseline, and it remained significantly

elevated at 4 h (101.9±55.5 ng/mL), 12 h (91.2±55.1 ng/mL),

and 24 h (85.7±55.5 ng/mL). A similar trend was noted in

the fluctuations of free TFPI, whose levels were also elevated

significantly, but much more so than total TFPI, to almost 4 times

the control levels upon arrival to the Accident and Emergency

Department (30.0±17.1 ng/mL) (Figure 4).

When the patients who had sepsis (n=38) were compared to

patients with septic shock (n=13), we noted higher PT after

4 h and higher free TFPI after 4 and 12 h of enoxaparin

administration (Table 2).

Table 2. Comparison between the hemostatic variables in patients with sepsis and septic shock.

Patients with sepsis n=38 Patients with septic shock n=13 p-value

PT

(s)

APTT

(s)

TT

(s)

Fibrinogen

(mg/dL)

AT III

(%)

Protein C

(%)

Protein S-total

(%)

Protein S-free

(%)

TFPI-total

(ng/mL)

TFPI-free

(ng/mL)

0 h 17.14±6.23 17.92±3.61 0.096

4 h 17.27±3.82 19.55±3.70 0.020*

12 h 16.74±3.17 18.07±3.76 0.166

24 h 15.84±2.22 16.68±3.58 0.988

0 h 43.69±13.53 43.15±8.43 0.834

4 h 50.50±14.33 51.90±8.62 0.380

12 h 45.68±10.26 49.08±10.61 0.209

24 h 45.21±15.57 49.55±13.17 0.340

0 h 18.81±13.27 16.56±2.15 0.854

4 h 19.75±9.65 17.37±2.50 0.895

12 h 16.35±2.37 16.67±1.99 0.508

24 h 15.77±2.46 18.20±6.62 0.221

0 h 617.93±244.43 606.57±183.42 0.907

4 h 642.44±198.64 630.64±159.77 0.736

12 h 696.81±162.90 600.08±194.75 0.144

24 h 673.79±160.72 674.50±200.68 0.814

0 h 78.17±21.23 76.01±22.59 0.734

4 h 80.96±23.23 71.57±25.06 0.321

12 h 80.33±19.75 70.08±20.54 0.072

24 h 78.25±20.25 77.07±21.77 0.937

0 h 62.35±28.33 62.21±23.38 0.982

4 h 64.30±28.93 58.85±26.55 0.478

12 h 67.66±24.18 61.50±23.27 0.254

24 h 64.08±22.21 68.07±25.42 0.738

0 h 61.41±21.37 57.92±15.25 0.604

4 h 61.90±34.25 53.81±19.36 0.439

12 h 60.55±20.33 53.16±18.48 0.132

24 h 64.52±23.41 61.64±24.15 0.649

0 h 44.89±17.06 43.21±15.67 0.907

4 h 39.71±15.14 41.81±21.51 0.937

12 h 44.07±18.15 40.50±19.34 0.594

24 h 43.82±19.61 47.0±19.05 0.707

0 h 77.12±37.53 86.78±35.30 0.417

4 h 94.00±47.58 117.42±53.07 0.084

12 h 85.15±49.15 97.61±44.63 0.157

24 h 79.91±43.88 146.00±214.23 0.598

0 h 29.48±16.52 38.57±16.28 0.589

4 h 36.40±15.52 50.71±16.28 0.004*

12 h 29.80±16.54 43.30±19.66 0.049*

24 h 27.78±15.79 34.53±21.60 0.478

PT: Prothrombin time, APTT: activated partial prothrombin time, TT: thrombin time, AT: antithrombin, TFPI: tissue factor pathway inhibitor.

115



Percentage (%)

200

180

160

140

120

100

80

60

40

20

0

Baseline 4 Hours 12 Hours 24 Hours

PT%

APPT% TT%

PT: Prothrombin time, APTT: activated partial prothrombin time, TT: thrombin time

Figure 1. Comparison of PT%, APTT%, and TT% at baseline and at

4, 12, and 24 h after the administration of enoxaparin.

Fibrogen mg/dL

900.0

800.0

700.0

600.0

500.0

400.0

300.0

200.0

100.0

0.0

Baseline 4 Hours 12 Hours 24 Hours Controls

Figure 2. Comparison of fibrinogen at baseline and at 4, 12, and

24 h after the administration of enoxaparin.

Percentage (%)

120

100

80

60

40

20

0

Baseline 4 Hours 12 Hours 24 Hours Controls

Prot C AT

Prot S Total Prot 5 FREE

Figure 3. Comparison of plasma levels of protein C, AT, and total

and free protein S at baseline and at 4, 12, and 24 h after the

administration of enoxaparin.

ng/mL

Prot C: Protein C, AT: Anti thrombin, Prot S Total: Protein S total, Prot S FREE: Protein S free

180

160

140

120

100

80

60

40

20

0

Baseline

TFPI T

4 Hours 12 Hours 24 Hours

TFPI F

TPFI T: Tissue factor pathway inhibitor-Total, TPFI F: Tissue factor pathway inhibitor-Free

Figure 4. Comparison of the plasma levels of total and free TFPI

at baseline and at 4, 12, and 24 h after the administration of

enoxaparin.

The plasma level of anti-FXa at 4 h was 0.52±0.11 IU/mL, at 12 h

was 0.5±0.07 IU/mL, and at 24 h was 0.59±0.11 IU/mL; all were

within the prophylactic range (0.2-0.5 IU/mL) [24].

Comparing the measured hemostatic variables in survivors and

nonsurvivors, there were only 3 isolated significant findings:

lower levels in nonsurvivors of TT (15.9±2.48% in nonsurvivors

versus 21.74±10.2% in survivors, p=0.04), AT (74.36±17.6%

in nonsurvivors versus 106.5±22.59%, p=0.01), and protein C

(62.51±21.19% in nonsurvivors versus 90.0±26.87%, p=0.03) in

the 24-h samples.

Discussion

The findings of the current study revealed marked derangement

of the coagulation system in patients with sepsis and septic

shock, in the form of significant prolongation of results of

both the screening tests of the intrinsic (APTT) and extrinsic

(PT) coagulation pathways that persisted after enoxaparin

administration. There was also very significant consumption

of the natural anticoagulants protein C, AT, and total and free

protein S. On the other hand, we noted with much interest that

the baseline levels of both total and free TFPI were elevated

above healthy control values and increased further after the

administration of enoxaparin.

Numerous previous studies have examined the fluctuations

of the circulating levels of hemostatic parameters in septic

syndrome. In this respect, natural coagulation inhibitors AT,

activated protein C, TFPI, and thrombomodulin received much

attention and almost all studies found marked reduction in

their blood levels [3,4]. Our study is in accordance with these

studies and showed lower levels of AT, protein C, and total and

free protein S at baseline and 4, 12, and 24 h after enoxaparin

administration.

AT is the main inactivator of thrombin and also inhibits the

activated forms of FIX, FX, and XI. Protein C, in the presence

of protein S, inhibits the activated forms of FVIII and FV. In

our patients, the levels of these inhibitors remained below

control values throughout the study period, which suggested

their consumption in the face of the activated coagulation in

these septic patients. However, none of these 3 inhibitors act on

tissue factor, the prime trigger of coagulation in vivo [25] and

whose expression in septic patients is markedly enhanced by

proinflammatory cytokines on the surface of endothelial cells

and monocytes [9,11,25,26].

The prime and specific physiological inhibitor of tissue factor is

TFPI, which is a proteinase inhibitor generated mainly from the

microvascular endothelium and that circulates in 2 forms: 80%

bound to lipoproteins and 20% in the physiologically active free

form [26,27]. TFPI also inhibits FXa directly and indirectly by

blocking action of the FVIIa/TF complex [10,27].

116



In an early report, Gando et al. [25], who measured both TF and

TFPI daily for 4 days, concluded that tissue factor production

is not balanced by concurrent production of TFPI and that

underlies the resulting activation of the coagulation system.

The design of the current study is different from that of Gando

et al. [25] and we have undertaken multiple measurements over

the first 24 h of admission, which we think is a critical period

in the management of septic patients. We also carried out more

detailed measurements of both total TFPI and free TPFI [26].

We noted with much interest the remarkable elevation in the

levels of both total and free TFPI above the healthy control

levels on admission, indicating that the prime inhibitor of the

tissue factor does in fact show a very active response to the

presumed excessive sepsis-induced generation of tissue factor.

The administration of prophylactic doses of enoxaparin resulted

in significant inhibition of FXa at 4, 12, and 24 h and was

associated with further rise in the circulating levels of both

forms of TFPI. Free TFPI exhibited more remarkable elevation

(4 times the control levels) than total TFPI. This was taken to

indicate that sepsis, in its own right, must be a strong trigger to

the release of TFPI from the vascular endothelium. This release

process must have approached its maximum degree following

the administration of enoxaparin, which is known to be most

potent in the mobilization and release of TFPI from the vascular

endothelium as compared to other LMWHs [28,29]. Interestingly,

some studies have reported not only reduced levels of natural

coagulation inhibitors but also impairment of their function

[2,28]. If this is indeed the case, and until the mechanism of

this impairment of function is delineated, no benefit should

be expected of the therapeutic uses of genetically engineered

recombinant natural coagulation inhibitors.

In the present study, we noted a trend towards higher TFPI

levels and particularly free TFPI in patients with septic shock as

compared to patients with sepsis. This could represent a more

exaggerated release of TFPI in these patients with more severe

disease. However, the number of patients in this group was

small and perhaps future studies with larger number of patients

with septic shock are needed to confirm these observations.

Unlike other studies, in which most of the patients were receiving

vasopressors [30,31], we found that enoxaparin administered in

prophylactic doses resulted in significant inhibition of FXa. This

suggests the presence of additional factors that contribute to

the failure of deep venous thrombosis prophylaxis in patients

with sepsis. One possibility could be the lower levels of Na as

described before [9,10,11,12] and confirmed by our findings.

In conclusion, the main finding of the current study is the

remarkable elevation in the plasma levels of both total and

free TFPI in septic patients at baseline. The levels of both forms

of the inhibitor remained elevated throughout the first 24 h

with further elevation after enoxaparin administration. This

observation would help to explain why the administration of

recombinant TFPI did not affect the course and outcome of

sepsis and septic shock.

Acknowledgment

This study was supported by the College of Medicine Research

Centre and the Deanship of Scientific Research of King Saud

University, Riyadh, Saudi Arabia. All authors disclosed that there

is no conflict of interest and that this study was not sponsored

by any drug company. We are also grateful to Mr. M.A. Hamid

and Mr. Lugman El-Sid for their technical assistance, and to

the nurses of the Accident and Emergency and ICUs of the

Department of Critical Care, King Khalid University Hospital,

Riyadh, for the collection of blood samples.

Ethics


Institutional Review Board of the College of Medicine-King

Saud University. Informed Consent: A written informed consent

was obtained from all patients or their next of kin.


Concept: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Abdulaziz

H. Alzeer, Mashael Al Shaikh; Design: Hadil A. Al Otair, Abdel Galil

M. Abdel Gader, Abdulaziz H. Alzeer, Mashael Al Shaikh; Data

Collection or Processing: Hadil A. Al Otair, Abdel Galil M. Abdel

Gader, Syed M. Khurshid, Abdulaziz H. Alzeer, Abdul Kareem Al

Momen, Mashael Al Shaikh, Farja Al Gahtani, Zohair A. Al Aseri,

Hossam A.H. Abdelrazik; Analysis or Interpretation: Hadil A. Al

Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz

H. Alzeer, Abdul Kareem Al Momen, Mashael Al Shaikh, Farja Al

Gahtani, Zohair A. Al Aseri, Hossam A.H. Abdelrazik; Literature

Search: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M.

Khurshid, Abdulaziz H. Alzeer; Writing: Hadil A. Al Otair, Abdel

Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer.




included.

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118

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0455


Comparison of Myelodysplastic Syndrome Prognostic Scoring

Systems

Miyelodisplastik Sendrom Prognostik Skorlama Sistemlerinin Kıyaslanması

Özlen Bektaş 1 , Ayşegül Üner 2 , Eylem Eliaçık 1 , Burak Uz 1 , Ayşe Işık 1 , Sezgin Etgül 1 , Süreyya Bozkurt 2 , İbrahim Celalettin Haznedaroğlu 1 ,

Hakan Göker 1 , Nilgün Sayınalp 1 , Salih Aksu 1 , Haluk Demiroğlu 1 , Osman İlhami Özcebe 1 , Yahya Büyükaşık 1

1Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey

2Hacettepe University Faculty of Medicine, Department of Pathology, Ankara, Turkey

Abstract

Objective: Myelodysplastic syndrome (MDS) is a clonal hematopoietic

stem cell disease. Patients are at risk of developing cytopenias or

progression to acute myeloid leukemia. Different classifications and

prognostic scoring systems have been developed. The aim of this study

was to compare the different prognostic scoring systems.

Materials and Methods: One hundred and one patients who were

diagnosed with primary MDS in 2003-2011 in a tertiary care university

hospital’s hematology department were included in the study.

Results: As the International Prognostic Scoring System (IPSS), World

Health Organization Classification-Based Prognostic Scoring System

(WPSS), MD Anderson Prognostic Scoring System (MPSS), and revised

IPSS (IPSS-R) risk categories increased, leukemia-free survival and

overall survival decreased (p<0.001). When the IPSS, WPSS, MPSS,

and IPSS-R prognostic systems were compared by Cox regression

analysis, the WPSS was the best in predicting leukemia-free survival

(p<0.001), and the WPSS (p<0.001) and IPSS-R (p=0.037) were better

in predicting overall survival.

Conclusion: All 4 prognostic systems were successful in predicting

overall survival and leukemia-free survival (p<0.001). The WPSS was

found to be the best predictor for leukemia-free survival, while the

WPSS and IPSS-R were found to be the best predictors for overall

survival.

Keywords: Myelodysplastic syndrome, International Prognostic

Scoring System, MD Anderson Prognostic Scoring System, World

Health Organization Classification-Based Prognostic Scoring System,

Revised International Prognostic Scoring System

Öz

Amaç: Miyelodisplastik sendrom (MDS) klonal bir hematopoetik kök

hücre hastalığıdır. Hastalarda sitopeni veya akut miyeloid lösemi

gelişmesi riski söz konusudur. Farklı sınıflandırma ve prognostik

skorlama sistemleri geliştirilmiştir. Bu çalışmanın amacı, farklı

prognostik skorlama sistemlerinin karşılaştırılmasıdır.

Gereç ve Yöntemler: Üçüncü basamak üniversite hastanesi,

hematoloji bölümünde 2003-2011 yılları arasında tanı alan 101 primer

MDS hastası çalışmaya dahil edildi.

Bulgular: Uluslararası Prognostik Skorlama Sistemi (UPSS), Dünya

Sağlık Örgütü Sınıflandırması Bazlı Prognostik Skorlama Sistemi

(DPSS), MD Anderson Prognostik Skorlama Sistemi (MPSS) ve

yeniden düzenlenmiş UPSS (UPSS-D) risk kategorileri arttıkça

lösemisiz sağkalım ve toplam sağkalım azalıyordu (p<0,001). UPSS,

DPSS, MPSS ve UPSS-R Cox regresyon analizi ile karşılaştırıldığında,

DPSS’nin lösemisiz sağkalımı (p<0,001), DPSS (p<0,001) ve UPSS-D’nin

(p=0.037) toplam sağkalımı daha iyi öngördüğü tespit edildi.

Sonuç: Dört prognostik skorlama sistemi de toplam sağkalımı ve

lösemisiz sağkalımı başarılı şekilde öngörüyordu (p<0.001). DPSS’nin

lösemisiz sağkalımın, DPSS ve UPSS-D’nin toplam sağkalımın en iyi

öngöreni olduğu tespit edildi.

Anahtar Sözcükler: Miyelodisplastik sendrom, Uluslararası Prognostik

Skorlama Sistemi, MD Anderson Prognostik Skorlama Sistemi, Dünya

Sağlık Örgütü Sınıflandırması Bazlı Prognostik Skorlama Sistemi,

Yeniden Düzenlenmiş Uluslararası Prognostik Skorlama Sistemi

Address for Correspondence/Yazışma Adresi: Özlen BEKTAŞ, M.D.,

Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Phone : +90 532 543 05 75

E-mail : ozlenbektas@hotmail.com

Received/Geliş tarihi: November 20, 2014

Accepted/Kabul tarihi: December 23, 2014

119

Bektaş Ö, et al: Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems


Introduction

Myelodysplastic syndromes (MDSs) are a heterogeneous group

of clonal hematopoietic stem cell disorders with heterogeneous

morphological, clinical, and survival characteristics. Common

features include cytopenia(s), dysplasia of one or more major

myeloid series, ineffective hematopoiesis, and an increased risk

of acute myeloid leukemia [1].

In 1982, the first classification of MDS was developed by the

French-American-British (FAB) group. This was a morphological

classification based on the degree of dysplasia and blasts in the

bone marrow, without biological basis [2]. The World Health

Organization (WHO) rearranged the classification of MDS-FAB in

2001 and 2008. Several parameters with prognostic significance

were added in the 2008 version: number of cytopenias, dysplasia

in one or more series, and presence of genetic abnormalities [3].

Following diagnosis and classification of MDS, prognostic staging

should be made to plan the treatment [4]. The International

MDS Risk Analysis Workshop developed the International

Prognostic Scoring System (IPSS), recognizing the bone marrow

blast percentage, cytogenetic status, and number and degree of

cytopenias as the most important prognostic markers in MDS [5].

The IPSS is the most widely used prognostic scoring system [4]. It

was designed based on untreated and primary MDS patients [5].

The WHO category, cytogenetics, and transfusion requirements

were identified as the most important prognostic indicators

in MDS and the WHO Classification-Based Prognostic Scoring

System (WPSS) was developed by Malcovati et al. [6]. The WPSS

was also designed based on untreated patients and it does not

include secondary MDS patients. Kantarjian et al. developed a

new classification model to overcome the limitations existing in

both prior prognostic systems of MDS, which includes patients’

performance status, age, number and degree of cytopenias,

cytogenetics, bone marrow blast percentage, and transfusion

needs [7]. The MD Anderson Prognostic Scoring System (MPSS) is

a system that can be applied to primary and secondary MDS and

chronic myelomonocytic leukemia (CMML). The International

Working Group for Prognosis in MDS project was initiated due

to limitations of the IPSS and the revised IPSS (IPSS-R) was

developed. The IPSS-R considers bone marrow blast percentage,

cytogenetics, and number and degree of cytopenias. This

prognostic system also does not include secondary MDS and

was developed based on untreated patients [8].

In this study, we aimed to compare the different prognostication

systems and determine the most appropriate system for routine

clinical practice.


One hundred and one patients who were diagnosed with

primary MDS during 2003-2011 and suitable for all of the

prognostication systems were included in the study. We used

101 routinely managed patients regardless of whether they

were on MDS-specific treatment or not. Patient information

was accessed from patient chart reviews. Each patient was

categorized according to the MDS-FAB and 2001 WHO

classification systems according to their bone marrow

aspiration and biopsy specimens. We did not use 2008 WHO

classification since the WPSS was validated only for the 2001

WHO classification system. Criteria for inclusion were: age

>18 years, primary MDS patients, and marrow and peripheral

blood blast counts of <20%. Exclusion criteria were: CMML,

secondary MDS, and marrow or peripheral blood blast counts

of ≥20%.

To analyze the prognosis, we used 4 different prognostic

systems: the IPSS, WPSS, MPSS, and IPSS-R. Leukemia

transformation and death were recorded as events and the first

developed event was recorded. Event-free survival was defined

as the duration from the time of diagnosis until the time of

developing an event or the last follow-up time, leukemia-free

survival (LFS) was defined as the duration from the time of

diagnosis until the time of developing leukemia (marrow or

peripheral blood blast count of ≥20%) or the last follow-up

time, and overall survival (OS) was defined as the duration

from the time of diagnosis until death or the last follow-up

time. Last follow-up date and condition were recorded as the

last condition.

This investigation was approved by the Local Ethics Committee

of Hacettepe University.

Statistical Methods

Data analysis was performed using SPSS 11.5 for Windows.

Continuous data were presented as mean ± standard deviation

or median (range). Categorical data were presented as numbers

and percentages. For the IPSS, WPSS, MPSS, and IPSS-R, the

LFS, OS, and life expectancy were evaluated with Kaplan-Meier

survival analysis using the log-rank test. Life expectancy; 1-,

3-, and 5-year survival rates; and 95% confidence intervals

(CIs) were calculated for each variable category. The prediction

capacities of the IPSS, WPSS, MPSS, and IPSS-R for LFS and

OS were compared with multivariate Cox proportional hazard

regression analysis. For each variable, the hazard ratios and

95% CIs were calculated.

A value of p<0.05 was considered statistically significant.

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Results

Patient Characteristics

The present study consisted of 101 patients; 44 of them (43.6%)

were male and 57 (56.4%) were female. The mean age of the

patients was 64±14.77 years. Transfusion support was given

to 26 (23%) patients; hypomethylating agents were used in

21.2% of patients (n=24; 23 of them were on 5-azacytidine

and 1 was on decitabine), lenalidomide in 0.9% of patients

(n=1), and erythropoietin in 2.7% of patients (n=3); and 4.4%

(n=5) of patients had undergone allogeneic bone marrow

transplantation. The follow-up period for the patients ranged

between 0 and 92 months with an average of 21.2 months.

Cytogenetic classification of the patients according to the

IPSS was as follows: 66 (58.4%) of good risk, 17 (15%) of

intermediate risk, and 18 (15.9%) of poor risk. MDS subgroup

distributions according to both the MDS-FAB classification and

the 2001 WHO classification are shown in Table 1.

Patients were evaluated by 4 different prognostic systems.

Accordingly, the risk distributions of the patients are shown in

Table 2.

During the follow-up period, 34.7% (n=35) of patients

experienced an event. The first event was leukemic

transformation in 20.8% of the patients (n=21), while death

was the first event in 13.9% (n=14) of the patients. Median

time to event was 15.25 months. Median leukemic progression

time was 8.28 months. Total death rate was 29.7% (n=30).

The estimated OS and LFS durations were 55.93±10.19 and

56.52±10.29 months, respectively.

In all 101 patients the average life expectancy was 55.9 months

(95% CI: 45.77-66.09), and 1-, 3-, and 5-year OS rates were

found as 77.5%, 57.5%, and 57.5%, respectively. The OS and

median survival times were significantly reduced as the degree

of risk increased regardless of which classification system was

used (p<0.001) (Table 3, Figures 1a, 1b, 1c, 1d).

In all 4 classification systems, the LFS was reduced as the degree

of risk increased (p<0.001). The 1-, 3-, and 5-year leukemia

survival rates in all subjects were 76%, 60.1%, and 60.1%,

respectively, and the average LFS time was found to be 56.52

months (95% CI: 46.2-66.8) (Table 4, Figures 2a, 2b, 2c, 2d).

When the efficacies of the IPSS, WPSS, MPSS, and IPSS-R

prognostic systems in predicting LFS were compared, the WPSS

showed the best performance (p<0.001, hazard ratio [HR]: 2.1,

95% CI: 1.543-2.858). The WPSS (p<0.001, HR: 2.461, 95% CI:

1.812-3.343) and IPSS-R (p=0.037, HR: 1.460, 95% CI: 1.024-

2.081) systems were better than the others in predicting OS.

Table 1. Distribution of patients according to French-

American-British and World Health Organization 2001

classification systems.

Variables n=101

MDS FAB classification

RA 48 (47.5%)

RARS 17 (16.8%)

RAEB-I 22 (21.8%)

RAEB-II 14 (13.9%)

MDS 2001 WHO classification

RA 11 (10.9%)

RARS 6 (5.9%)

RCMD 34 (33.7%)

RCMD-RS 9 (8.9%)

5q (-) 5 (5%)

RAEB-I 21 (20.8%)

RAEB-II 15 (14.9%)

MDS: Myelodysplastic syndrome, WHO: World Health Organization, FAB: French-

American-British, RAEB: refractory anemia with excess blasts, RARS: refractory

anemia with ring sideroblasts, RA: refractory anemia, RCMD-RS: refractory cytopenia

with multilineage dysplasia and ring sideroblasts, RCMD: refractory cytopenia with

multilineage dysplasia.

Table 2. Distribution of patients by risk groups.

IPSS WPSS MPSS IPSS-R

Very low risk NA 8 (7.9%) NA 18 (17.8%)

Low risk 31 (30.7%) 31 (30.7%) 28 (27.7%) 23 (22.8%)

Intermediate (or Int-I) risk 41 (40.6%) 25 (24.8%) 31 (30.7%) 25 (24.8%)

Intermediate-II risk 20 (19.8%) NA 24 (23.8%) NA

High risk 9 (8.9%) 26 (25.7%) 18 (17.8%) 18 (17.8%)

Very high risk NA 11 (10.9%) NA 17 (16.8%)

IPSS: International Prognostic Scoring System, MPSS: MD Anderson Prognostic Scoring System, WPSS: World Health Organization Classification-Based Prognostic Scoring System,

IPSS-R: Revised International Prognostic Scoring System, NA: not applicable.

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a

b

c

d

Figure 1. Kaplan-Meier curves show rates of overall survival (OS) for International Prognostic Scoring System (IPSS) (a), World Health

Organization-Based Prognostic Scoring System (WPSS) (b), MD Anderson Prognostic Scoring System (MPSS) (c), and Revised International

Prognostic Scoring System (IPSS-R) (d).

a

b

c

d

Figure 2. Kaplan-Meier curves show rates of leukemia-free survival (LFS) for International Prognostic Scoring System (IPSS) (a), World

Health Organization Classification-Based Prognostic Scoring System (WPSS) (b), MD Anderson Prognostic Scoring System (MPSS) (c), and

Revised International Prognostic Scoring System (IPSS-R) (d).

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Table 3. Overall survival according to International Prognostic Scoring System, World Health Organization Classification-Based

Prognostic Scoring System, MD Anderson Prognostic Scoring System, and Revised International Prognostic Scoring System.

Variables Survival Rates (%) Mean Survival Log-rank p-value

1 year 3 years 5 years

IPSS 37.10 <0.001

Low 100.0 85.9 85.9 68.38 (58.68-78.08)

Intermediate-I 81.6 76.2 76.2 65.05 (52.72-77.38)

Intermediate-II 47.9 8.0 8.0 21.51 (8.66-34.36)

High 35 0 0 10.17 (7.33-13.01)

WPSS 60.42 <0.001

Very low 100.0 75 75 56.72 (40.81-72.63)

Low 96.3 89.4 89.4 76.39 (58.26-94.51)

Intermediate 88.9 83 83 72.15 (58.25-86.05)

High 62.2 0 0 20.05 (15.02-25.08)

Very high 0 0 0 7.26 (5.66-8.86)

MPSS 44.02 <0.001

Low 100.0 100.0 100.0 70.85 (63.85-77.84)

Intermediate-I 100 75.2 75.2 70.23 (56.98-83.48)

Intermediate-II 56.3 30.8 30.8 27.85 (15.46-40.24)

High 24.3 8.1 8.1 17.45 (3.87-31.04)

IPSS-R 56.56 <0.001

Very low 100.0 80.0 80.0 47.00 (38.93-55.06)

Low 94.7 94.7 94.7 69.75 (61.73-77.76)

Intermediate 90 73.8 73.8 69.33 (50.43-88.23)

High 51.3 0 0 20.46 (14.00-26.92)

Very high 38.2 9.5 9.5 11.33 (7.41-15.25)

General 77.5 57.5 57.5 55.93 (45.77-66.09) 72.84 0.000


IPSS-R: Revised International Prognostic Scoring System.

Discussion

The current prognostication systems have been criticized for

some specific properties. They were developed in untreated

cohorts and they have generally not been tested in treated

cohorts except for the IPSS-R. Neukirchen et al. demonstrated

the value of the IPSS-R for patients treated with induction

chemotherapy and/or allogeneic stem cell transplantation

in their validation study [9]. Currently there are many widely

available treatment alternatives in MDS. Therefore, we thought

that these systems should be tested in a modern routinely

managed MDS cohort. The IPSS and IPSS-R are mostly criticized

because they were developed in untreated patient cohorts

that do not reflect current patient profiles [10]. The MPSS is

mainly criticized for the inclusion of secondary and therapyrelated

MDS and MDS/myeloproliferative disease cases, which

are now considered separate entities [11,12]. The WPSS was

initially criticized for arbitrariness of transfusion dependence.

However, it was revised to include stable hemoglobin thresholds

instead of this arbitrary definition [13]. It is still criticized for

low reproducibility of WHO classification of subentities with

low blast counts.

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Table 4. Leukemia-free survival according to International Prognostic Scoring System, World Health Organization Classification-

Based Prognostic Scoring System, MD Anderson Prognostic Scoring System, and Revised International Prognostic Scoring

System.

Variables Survival Rates (%) Mean Survival Log-rank p-value

1 year 3 years 5 years

IPSS 37.16 0.000

Low 100.0 85.9 85.9 68.38 (58.68-78.08)

Intermediate-I 82.4 76.9 76.9 64.99 (52.53-77.46)

Intermediate-II 35.9 9.0 9.0 18.73 (4.38-33.08)

High 38.1 0 0 9.32 (5.58-13.07)

WPSS 47.87 0.000

Very low 100.0 75.0 75.0 56.72 (40.81-72.63)

Low 96.3 89.4 89.4 76.00 (57.34-94.66)

Intermediate 90.9 84.8 84.8 73.19 (60.07-86.31)

High 52.9 0 0 16.93 (11.25-22.61)

Very high 0 0 0 6.42 (4.41-8.43)

MPSS 39.41 0.000

Low 100.0 100.0 100.0 70.20 (64.50-64.50)

Intermediate-I 96.7 78.9 78.9 71.15 (3.10-56.69)

Intermediate-II 49.9 31.2 31.2 26.06 (4.62-12.08)

High 26.8 8.9 8.9 17.34 (2.09-30.09)

IPSS-R 54.34 0.000

Very low 100.0 80.0 80.0 47.00 (38.93-55.06)

Low 94.7 94.7 94.7 69.44 (61.17-77.71)

Intermediate 90.9 74.6 74.6 69.52 (60.60-88.45)

High 37.5 0 0 17.17 (9.47-24.86)

Very high 35.3 0 0 9.86 (5.64-14.09)

General 76.0 60.1 60.1 56.52 (46.23-66.81) 84.30 0.000


IPSS-R: Revised International Prognostic Scoring System.

In spite of these critiques, there is no doubt that these systems

are useful in routine clinical practice. But which one(s) deserve

the most credit?

In our study 101 MDS patients appropriate for all prognostic

systems were evaluated with the IPSS, WPSS, MPSS, and IPSS-R.

The median age of the patients was 64 years, which is lower

than in Western populations; younger age at diagnosis was also

seen in some Asian countries, as Matsuda et al. and Kuendgen et

al. demonstrated [1,14]. This is the first study to compare these

4 prognostic scoring systems in MDS. All 4 prognostic systems

were successful in predicting OS and LFS (p<0.001). When the

systems were compared, the WPSS was found to be the best

predictor for LFS, while the WPSS and IPSS-R were found to

be the best predictors for OS. Equal efficacies of IPSS-R and

WPSS in our practice implies that our hematopathologists

are quite capable of separating single-lineage dysplasia from

multilineage dysplasia and refractory anemia with excess blasts

(RAEB)-I from RAEB-II. Unfortunately, this capability may not

be available in every setting.

124



There are several studies that compared prognostic scoring

systems. Voso et al. compared the IPSS, WPSS, and IPSS-R in their

IPSS-R validation study and found that the IPSS-R predicted OS

better than the other systems [15]. Reis-Alves et al. showed that

only IPSS-R score was an independent risk factor in terms of OS

in their comparison of the IPSS, WPSS, and IPSS-R [16].

In our study, the WPSS and IPSS-R may have estimated OS

better since the hemoglobin cut-off was accepted as lower

than in the other systems in both these scoring systems

(<9 g/dL in males and <8 g/dL in females for WPSS; 8-10 g/

dL [1 point] and <8 g/dL [1.5 point] for IPSS-R). This may be

especially true for low-risk patients since the main predictor of

mortality is marrow failure in low-risk patients and leukemic

transformation in high-risk patients. When the advanced age

and frailty of many MDS patients are taken into consideration,

the lower hemoglobin threshold may better reflect the impact

of anemia on health. In our study, the WPSS was found to

be best in reflecting LFS. This may be due to the fact that it

depends on the MDS-WHO classification. This classification

reflects leukemia transformation risk very well [17,18].

The MPSS is a dynamic scoring system like the WPSS and

predicts survival at any time during follow-up. It can be used

for chronic myelomonocytic leukemia and secondary MDS if

prognostic assessment is required [7].

This study has some handicaps inherent to its retrospective

nature. Additionally, it would be better to include a higher

number of patients in future analyses.

Ethics

Ethics Committee Approval: This investigation was approved by

the Local Ethics Committee of Hacettepe University, Informed

Consent: Not applicable (retrospective study).


Pathological Processing: Ayşegül Üner; Cytogenetic Processing:

Süreyya Bozkurt; Data Collection or Processing: Özlen Bektaş,

Ayşegül Üner, Eylem Eliaçık, Burak Uz, Ayşe Işık, Sezgin Etgül,

Süreyya Bozkurt, İbrahim Celalettin Haznedaroğlu, Hakan

Göker, Nilgün Sayınalp, Salih Aksu, Haluk Demiroğlu, Osman

İlhami Özcebe, Yahya Büyükaşık; Analysis or Interpretation:

Özlen Bektaş, Yahya Büyükaşık; Literature Search: Özlen Bektaş,

Yahya Büyükaşık; Writing: Özlen Bektaş, Yahya Büyükaşık.




included.

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AL, D’Andrea M, D’Addosio A, Leone G, Venditti A. Revised International

Prognostic Scoring System (IPSS) predicts survival and leukemic evolution of

myelodysplastic syndromes significantly better than IPSS and WHO Prognostic

Scoring System: validation by the Gruppo Romano Mielodisplasie Italian

Regional Database. J Clin Oncol 2013;31:2671-2677.

16. Reis-Alves SC, Traina F, Harada G, Campos PM, Saad ST, Metze K, Lorand-

Metze I. Immunophenotyping in myelodysplastic syndromes can add

prognostic information to well-established and new clinical scores. PLoS

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17. Vardiman JW. Hematopathological concepts and controversies in the

diagnosis and classification of myelodysplastic syndromes. Hematology

Am Soc Hematol Educ Program 2006;199-204.

18. Cermak J, Michalova K, Brezinova J, Zemanova Z. A prognostic impact

of separation of refractory cytopenia with multilineage dysplasia

and 5q-syndrome from refractory anemia in primary myelodysplastic

syndrome. Leuk Res 2003;27:221-229.

126

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0213


Platelet Dysfunction in Patients with Chronic Myeloid Leukemia:

Does Imatinib Mesylate Improve It?

Kronik Miyelositer Lösemili Hastalarda Trombosit Disfonksiyonu: İmatinib Mesilat Düzeltir mi?

Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş

Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey

Abstract

Objective: The aim of this study was to investigate the effects of

imatinib mesylate on platelet aggregation and adenosine triphosphate

(ATP) release in chronic myeloid leukemia patients.

Materials and Methods: Platelet aggregation and ATP release induced

by 5.0 mM adenosine diphosphate, 0.5 mM arachidonic acid, 1.0 mg/

mL ristocetin, and 2 µg/mL collagen were studied by whole blood

platelet lumi-aggregometer in 20 newly diagnosed chronic myeloid

leukemia patients before and after imatinib mesylate treatment.

Results: At the time of diagnosis, 17/20 patients had abnormal

platelet aggregation results; 8 (40%) had hypoactivity, 6 (30%) had

hyperactivity, and 3 (15%) had mixed hypo- and hyperactivity. Repeat

platelet aggregation studies were performed after a mean of 19

months (min: 5 months-max: 35 months) in all patients who received

imatinib mesylate during this period. After therapy, 18/20 (90%)

patients had abnormal laboratory results; 12 (60%) had hypoactive

platelets, 4 (20%) had mixed hypo- and hyperactive platelets, and 2

(10%) had hyperactive platelets. Three of the 8 patients with initial

hypoactivity remained hypoactive, while 2 developed a mixed picture,

2 became hyperactive, and 1 normalized. Of the 6 patients with initial

hyperactivity, 4 became hypoactive and 2 developed a mixed pattern.

All of the 3 patients with initial hypo- and hyperactivity became

hypoactive. Finally, 2 of the 3 patients with initial normal platelets

became hypoactive while 1 remained normal. There was a significant

decrease in ristocetin-induced platelet aggregation after therapy

(p<0.001), while platelet aggregation and secretion induced by other

agonists showed no difference after treatment (p>0.05).

Conclusion: These findings indicate that a significant proportion of

chronic myeloid leukemia patients have different patterns of platelet

function abnormalities and imatinib mesylate has no effect on these

abnormalities, with a significant impairment in ristocetin-induced

platelet aggregation.

Keywords: Platelet aggregation, Chronic myeloid leukemia, Imatinib

mesylate

Öz

Amaç: Bu çalışmanın amacı kronik miyelositer lösemili hastalarda

imatinib mesilatın trombosit agregasyonu ve adenozin trifosfat (ATP)

salınımı üzerine etkilerini araştırmaktır.

Gereç ve Yöntemler: Yirmi yeni tanı almış kronik miyelositer

lösemili hastada imatinib mesilat tedavisi öncesi ve sonrası 5,0 mM

adenozin difosfat, 0,5 mM araşidonik asit, 1,0 mg/mL ristosetin ve 2

µg/mL kollagen ile indüklenen trombosit agregasyon ve ATP salınımı

çalışılmıştır.

Bulgular: Tanı sırasında, 17/20 hasta anormal trombosit agregasyon

sonuçlarına sahip idi; sekizinde (%40) hipoaktivite, altısında (%30)

hiperaktivite ve üçünde (%15) miks hipo- ve hiperaktivite saptandı.

Ortalama 19 ay (min: 5 ay-maks: 35 ay) imatinib mesilat kullanımı

sonrası tüm hastalarda trombosit agregasyon testleri tekrarlandı.

Tedavi sonrası, 18/20 (%90) hasta anormal laboratuvar sonuçlarına

sahip idi; 12’si (%60) hipoaktif trombositler, dördü (%20) miks hipo- ve

hiperaktif trombositler ve ikisi (%10) hiperaktif trombositlere sahip idi.

Başlangıçta hipoaktivitesi olan sekiz hastanın üçü hipoaktif kalır iken

ikisi miks bir görüntü geliştirdi, ikisi hiperaktif oldu ve biri normalize

oldu. Başlangıçta hiperaktivitesi olan altı hastanın dördü hipoaktif

oldu ve ikisi miks patern geliştirdi. Başlangıçta hipo- ve hiperaktivitesi

olan üç hastanın tamamı hipoaktif oldu. Son olarak, başlangıçta

normal trombositleri olan üç hastanın ikisi hipoaktif olur iken biri

normal kaldı. Ristosetin ile indüklenen trombosit agregasyonunda

tedavi sonrası anlamlı azalma (p<0,001) olur iken diğer agonistler

ile indüklenen trombosit agregasyon ve sekresyonu tedavi sonrası

farklılık göstermedi (p>0,05).

Sonuç: Bulgularımız kronik miyelositer lösemili hastaların önemli bir

çoğunluğunun farklı paternde trombosit fonksiyon anormalliklerine

sahip olduğunu ve imatinib mesilatın ristosetin ile indüklenen

trombosit fonksiyonunda azalma dışında bu anormallikler üzerinde

etkisi olmadığını göstermiştir.

Anahtar Sözcükler: Trombosit agregasyonu, Kronik miyelositer

lösemi, İmatinib mesilat

Address for Correspondence/Yazışma Adresi: Olga Meltem AKAY, M.D.,

Osmangazi University Faculty of Medicine,

Department of Hematology, Eskişehir, Turkey

E-mail : olga.akay@hotmail.com

Received/Geliş tarihi: May 28, 2014

Accepted/Kabul tarihi: September 30, 2014

127

Akay OM, et al: Platelet Dysfunction in Chronic Myeloid Leukemia and Imatinib Mesylate


Introduction

Imatinib mesylate (Gleevec or Glivec, Novartis, Basel,

Switzerland) is the first tyrosine kinase (TK) inhibitor active

against BCR-ABL, c-ABL, ARG, PDGF-r, and c-KIT. TKs are

important signaling enzymes for the cellular regulation of

proliferation, differentiation, survival, function, and motility,

and various tumors overexpress TKs, leading to uncontrolled

mitogenic signals to neoplastic cells [1]. Imatinib mesylate has

considerable antineoplastic activity in patients with chronic

myeloid leukemia (CML) and some solid tumors [2].

Thromboembolic and bleeding complications are the leading

causes of morbidity and mortality in myeloproliferative

neoplasms, particularly polycythemia vera and essential

thrombocythemia, although these occur least frequently in

patients with CML [3]. Abnormalities of platelet function arising

from the clonal proliferation of hematopoietic cells including

megakaryocyte precursors are regarded as the main origin of

thrombo-hemorrhagic episodes [4]. Considering the reduction

of BCR-ABL+ clones in response to imatinib mesylate and the

recovery of normal hematopoietic stem and progenitor cells in

the bone marrow [5], we performed platelet aggregation studies

in CML patients who were treated with imatinib mesylate to

investigate the effect of this drug on platelet function.


Patients

A total of 20 newly diagnosed chronic-phase CML patients who

started treatment with imatinib were enrolled. The diagnosis

of CML was made by the demonstration of Philadelphia

chromosome positivity and compatible hematological findings

in peripheral blood and bone marrow. Imatinib was used as the

fırst-line treatment in all patients. It was started at a dose of

400 mg daily. Dose modifications were allowed according to

toxicity and treatment efficacy, ranging from 200 to 800 mg.

Sample Collection

Venous blood was collected from patients under light tourniquet

through 19-gauge needles into vacutainers (Becton Dickinson).

A 3-mL di-potassium EDTA (1.5 mg/mL) sample was collected

fırst followed by two 4.5-mL 3.2% tri-sodium citrate (0.105 M)

vacutainers. The collection was performed early in the morning

following a light breakfast. Subjects with known bleeding or

other systemic disorders such as renal, hepatic, and endocrine

diseases, and those who had taken aspirin or other nonsteroidal

anti-inflammatory agents within 10 days prior to blood

sampling were excluded. Automated cell counts were performed

on the EDTA sample tube with a Beckman Coulter Gen-S SM

(USA) automated blood-counting device.

Whole Blood Platelet Lumi-Aggregometry

Whole blood platelet lumi-aggregometry studies were

performed on the citrate tubes at the time of diagnosis and

repeated following imatinib therapy in all patients. Platelet

aggregation (measured as the increase in impedance) and

release were simultaneously measured using a whole blood

lumi-aggregometer (Model 560-Ca, Chrono-log Corporation,

USA) according to the manufacturer’s instructions. The agonists

used and their final concentrations were, in sequence, adenosine

diphosphate (ADP; Chrono Par 384, Chrono-log Corporation), 5

µM; arachidonic acid (AA; Chrono Par 390), 0.5 mM; ristocetin

(Chrono Par 396), 1.0 mg/mL; and collagen (Chrono Par 385), 2

µg/mL. Platelet function testing on all samples was completed

within 2 h of collection. Our laboratory reference ranges for

platelet aggregation (ohm) and adenosine triphosphate (ATP)

release (nmol) were 10-22 ohm and 0.3-2 nmol for ADP, 10-28

ohm and 0.6-3 nmol for AA, 10-32 ohm and 0.3-2 nmol for

collagen, and 3-19 ohm for ristocetin.

Statistics

Statistical analysis was performed using IBM SPSS 20.0 (IBM

Corp., Armonk, NY, USA). The Shapiro-Wilk test was used to test

normality for continuous variables. Nonnormally distributed

variables were compared with the Wilcoxon test for paired data

and presented as medians (quartiles). P<0.05 was accepted as

statistically significant.

Results

The median age of the total group of patients was 43 years

(range: 29-71); there were 10 females and 10 males. Median

platelet counts before and after treatment were 339.50x10 9 /L

(range: 227.50-527.25) and 225.50x10 9 /L (range: 171.00-

259.25), respectively.

As described by Manoharan et al. [6], platelets were considered

to be hyperactive if at least one result (aggregation or ATP

release with one agonist) was above the reference range and

hyporeactive if at least one result (aggregation or ATP release

with one agonist) was below the reference range. Mixed hypoand

hyperactive platelets were considered present when at least

one result (aggregation or ATP release) was below and above the

reference range, respectively.

At the time of diagnosis, 17/20 patients had abnormal platelet

aggregation results; 8 (40%) had platelet hypoactivity, 6 (30%)

had platelet hyperactivity, and 3 (15%) had mixed hypo- and

hyperactivity.

After a mean of 19 months (min: 5 months-max: 35 months),

repeat platelet aggregation studies were performed in all patients

who received imatinib during this period. A major molecular

response was achieved in 17 (85%) of the patients at the time

128



of retesting. After imatinib therapy, 18/20 (90%) patients had

abnormal laboratory results; 13 (65%) had hypoactive platelets,

3 (15%) had mixed hypoa and hyperactive platelets, and 2

(10%) had hyperactive platelets. Three of the 8 patients with

initial hypoactivity remained hypoactive, while 2 developed a

mixed picture, 2 became hyperactive, and 1 normalized. Of the

6 patients with initial hyperactivity, 5 became hypoactive and

1 developed a mixed pattern. All of the 3 patients with initial

hypo- and hyperactivity became hypoactive. Finally, 2 of the 3

patients with initial normal platelets became hypoactive while

1 remained normal (Figure 1).

When we compared pretreatment and post treatment platelet

aggregation values induced by ADP, AA, ristocetin, and collagen,

we found that there was a significant decrease in ristocetininduced

platelet aggregation (p<0.001) after treatment, while

pre- and post treatment platelet aggregation responses to the

other agonists were not significantly different (p>0.05). There

was also no significant difference between pretreatment and

post treatment platelet secretion values induced by ADP, AA, and

collagen (Table 1). We did not notice a significant correlation

between platelet count and platelet aggregation and secretion

results induced by any of the agonists used. Moreover, platelet

responses showed no correlation with the length of time on

imatinib and none of the studied patients experienced bleeding.

Discussion

In the present study, we demonstrated that a significant

proportion of CML patients (85%) have different patterns of

platelet dysfunction and imatinib therapy has neither a positive

nor a negative impact on these functional defects.

The number of studies investigating the effect of CML therapy

on platelet abnormalities is very limited. In one study including

6 CML patients, plasma levels of beta-TG and PF4 were not

reduced and platelet aggregation did not improve following

normalization of leukocyte and platelet counts after busulfan

or hydroxyurea [7]. In contrast, Barbui et al. [8] reported

a normalization of spontaneous platelet aggregation and

improvement of collagen-induced aggregation but a persistent

dense granule storage deficiency after busulfan therapy. Data

evaluating the effects of imatinib on platelet function are also

still limited. In a recent study by Quintas-Cardama et al. [9], 5 of

the 15 evaluable CML patients on imatinib had normal platelet

aggregation and 10 (66%) had impaired AA-induced platelet

aggregation, including 2 (13%) with impaired epinephrineinduced

aggregation.

Our results suggest that imatinib does not have either a positive

or a negative impact on platelet function, with a significant

impairment in ristocetin-induced platelet aggregation. It is

known that, during ristocetin-induced platelet aggregation,

ristocetin binds to the platelet surface through its phenolic

groups. Being positively charged, the bound ristocetin reduces

the net negative charge on the platelet surface and permits

a closer contact between platelets. This, in turn, permits the

von Willebrand factor to bridge between platelets, resulting in

agglutination. Imatinib, either by altering ristocetin’s phenolic

Figure 1. Platelet aggregation (ohm) and secretion (nmol) values

induced by agonists before and after imatinib therapy in CML

patients (n=20). Reference interval for impedance (ohm) or

release (nmol) of the agonist is shown by dotted lines in each

chart. ADP: Adenosine diphosphate, AA: arachidonic acid.

Table 1. Comparison of platelet aggregation and secretion

results induced by agonists before and after imatinib

therapy in chronic myeloid leukemia patients (n=20).

Before therapy After therapy p-value

Median

(25%-75%)

Median

(25%-75%)

Platelet aggregation

ADP (ohm) 14.5 (8.5-21) 8.5 (4.25-16) 0.079

AA (ohm) 12 (9-19.75) 13 (3.25-18.75) 0.380

Ristocetin (ohm) 13 (9.25-22.75) 5.5 (3-8.75) 0.002

Collagen (ohm) 26 (20-28.75) 26 (20-28.75) 0.251

Platelet secretion

ADP (nmol) 0.65 (0.31-1.09) 0.5 (0.3-0.94) 0.481

AA (nmol) 1.4 (0.9-1.9) 0.83 (0.55-1.16) 0.370

Collagen (nmol) 0.55 (0.3-0.84) 0.67 (0.47-1.07) 0.083

ADP: Adenosine diphosphate, AA: arachidonic acid

129



groups or by occupying its binding sites on the platelet surface,

may cause a decrease in ristocetin-induced platelet aggregation.

Moreover, since ristocetin does bind to the platelet, the decrease

in platelet counts after imatinib treatment may be another

explanation for impaired ristocetin-induced platelet aggregation

results. However, these hypotheses must be confirmed with

further in vitro studies [10,11].

We speculated that myelosuppression under imatinib treatment

depends on the fact that after reduction of clonal hematopoiesis

in response to treatment, normal hematopoietic stem and

progenitor cells have to recover from preexisting suppression by

the malignant clone and re-expand in the bone marrow. This may

be related to changes in the growth pattern of megakaryocytes

and a certain improvement of platelet function and activation

after therapy. However, imatinib treatment did not improve

most of the patterns of platelet function abnormalities while

significantly increasing hypoactivity of platelets. The existence

of platelet function abnormalities even in patients who achieved

a major molecular response with imatinib in our study led us

to assume that normal hematopoiesis is not fully restored in

a substantial portion of CML patients despite the achievement

of the desired response. Imatinib mesylate is designed as a TK

inhibitor active against BCR-ABL [12,13], but it also inhibits

other TKs, such as PDGF-r and c-KIT [14,15]. Inhibitory effects

of the drug on platelet function may be partly explained by the

inhibition of platelet TKs.

Conclusion

In conclusion, these findings indicate that a significant

proportion of CML patients have different patterns of platelet

function abnormalities, which must be further investigated.

However, one important limitation of this study is that we

only studied platelet function tests in CML patients without

a control group including imatinib-treated non-CML patients

such as patients with gastrointestinal stromal tumors or

hypereosinophilic syndrome. Subsequent studies should

investigate how imatinib changes the function of platelets in

patients with normal hematopoiesis.

Ethics

Ethics Committee Approval: Eskişehir Osmangazi University

Ethics Committee, Informed Consent: It was taken.


Medical Practices: Olga Meltem Akay; Concept: Olga Meltem

Akay; Design: Olga Meltem Akay, Zafer Gülbaş; Data Collection

or Processing: Olga Meltem Akay, Zafer Gülbaş; Analysis or

Interpretation: Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş;

Literature Search: Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş;

Writing: Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş.




included.

References

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P, Oehler L. In vivo effects of imatinib mesylate on human haematopoietic

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1984;64:1-12.

4. Wehmeier A, Schneider W. Megakaryocytes and platelets as the main cause

for vascular events in chronic myeloproliferative disorders. Hamostaseologie

1996;16:151-163.

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hematopoiesis and immune activation. Stem Cells 2005;23:1082-1088.

6. Manoharan A, Gemmell R, Brighton T, Dunkley S, Lopez K, Kyle P. Thrombosis and

bleeding in myeloproliferative disorders: identification of at-risk patients with

whole blood platelet aggregation studies. Br J Haematol 1999;105:618-625.

7. Wehmeier A, Scharf RE, Fricke S, Schneider W. A prospective study of hemostatic

parameters in relation to the clinical course of myeloproliferative disorders. Eur J

Haematol 1990;45:191-197.

8. Barbui T, Bassan R, Viero P, Cortelazzo S, Dini E. Platelet function after busulfan in

chronic myeloproliferative disorders. Haematologica 1983;68:469-477.

9. Quintas-Cardama A, Han X, Kantarjian H, Cortes J. Tyrosine kinase inhibitorinduced

platelet dysfunction in patients with chronic myeloid leukemia. Blood

2009;114: 261-263.

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agglutination. Effects of structural modification of ristocetin and vancomycin. J

Clin Invest 1977;60:302-312.

11. Kattlove HE, Gomez MH. Studies on the mechanism of ristocetin-induced platelet

aggregation. Blood 1975;45:91-96.

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kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:3-7.

13. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J,

Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth

of BCR-ABL positive cells. Nat Med 1996;2:561-566.

14. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, Lydon NB.

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mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp

Ther 2000;295:139-145.

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Blood 2000;96:925-932.

130

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0379


Immature Reticulocyte Fraction and Absolute Neutrophil Count

as Predictor of Hemopoietic Recovery in Patients with Acute

Lymphoblastic Leukemia on Remission Induction Chemotherapy

Remisyon İndüksiyon Kemoterapisi Alan Akut Lenfoblastik Lösemi Hastalarında

Hematopoietik Toparlanmanın Öngörülmesinde İmmatür Retikülosit Fraksiyonu ve Mutlak

Nötrofil Sayısı

Shan E. Rauf, Saleem Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan

Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan

Abstract

Objective: Acute lymphoblastic leukemia (ALL) encompasses a group

of lymphoid neoplasms that are more common in children and

arise from B-and T-lineage lymphoid precursor cells. The immature

reticulocyte fraction (IRF), a new routine parameter in hematology

analyzers, can give an indication of hemopoietic recovery like absolute

neutrophil count (ANC). The purpose of this study was to evaluate IRF

in excess of 5% was considered as IRF recovery.

Materials and Methods: In this descriptive study, 2.5 to 3 mL of EDTA

blood of 45 ALL patients undergoing the remission induction phase

of their treatment was sampled and analyzed with a Sysmex XE-5000

on day 1 and every second day thereafter until the day of recovery.

ANC of >0.5x109/L on the day corresponding to the first of the three

consecutive counts was considered as the day of ANC recovery. IRF

recovery was an IRF in excess of 5%.

Results: The mean age of the patients was 12.04±5.30 years; 25

patients (55.6%) were male and 20 patients (44.4%) were female. On

day 1 of induction remission, the mean IRF value was 9.68±1.41, while

the mean ANC value was 0.077±0.061. Mean recovery day for IRF was

11.84±7.44 and mean recovery day for ANC was 17.67±8.77 (twotailed

p-value <0.0001 with 95% confidence interval). By day 28, out

of 45 patients 36 (80%) showed ANC recovery, while 41 (91%) showed

IRF recovery. The remaining patients who had not shown recovery by

day 28 were further followed up and all of them showed recovery of

both parameters by day 39.

Conclusion: This study concluded that postinduction bone marrow

hemopoietic recovery was earlier by IRF than ANC in children with

ALL on chemotherapy.

Keywords: Acute lymphoblastic leukemia, Lymphoid cell neoplasm,

Hematopoiesis, Chemotherapy

Öz

Amaç: Akut lenfoblastik lösemi (ALL) çocuklarda daha sık görülen ve

B- ve T-lenfoid öncül hücre dizilerinden kaynaklanan bir grup lenfoid

neoplaziyi kapsamaktadır. Hematolojik incelemede yeni rutin bir

parametre olan immatür retikülosit fraksiyonu (İRF), mutlak nötrofil

sayısı (MNS) gibi hematopoietik toparlanma hakkında fikir oluşturabilir.

Bu çalışmanın amacı remisyon indüksiyon kemoterapisi almakta olan

ALL hastalarında İRF ve MNS toparlanmasını değerlendirmektir.

Gereç ve Yöntemler: Bu tanımlayıcı çalışmada, remisyon indüksiyon

tedavisi alan 45 ALL hastasından tedavinin birinci günü başlayarak,

toparlanmaya kadar günaşırı 2,5-3 mL EDTA’lı tüpe kan alınmış

ve Sysmex XE-5000 ile inceleme yapılmıştır. MNS toparlaması,

ardışık üç sayımda MNS değerinin >0,5x109/L olduğu günlerden ilki

olarak tanımlanmıştır. İRF toparlanması İRF’nin %5’i aşması olarak

belirlenmiştir.

Bulgular: Hastaların ortalama yaşı 12,04±5,30 yıldı; 25 hasta (%55,6)

erkek ve 20 hasta (%44,4) kadındı. İndüksiyon tedavisinin birinci

gününde, ortalama İRF değeri 9,68±1,41 iken, ortalama MNS değeri

0,077±0,061 idi. İRF için ortalama toparlanma süresi 11,84±7,44

gün ve MNS için ortalama toparlanma süresi ise 17,67±8,77 gündü

(p değeri <0,0001, %95 güven aralığı içinde). Yirmi sekizinci günde,

45 hastanın 36’sında (%80) MNS toparlanması varken 41’inde (%91)

İRF toparlanması bulunmaktaydı. Yirmi sekizinci günde toparlanması

bulunmayan hastaların takibine devam edildi ve 39. günde bu

hastaların tamamında her iki parametre açısından da toparlanma

tespit edildi.

Sonuç: Bu çalışma kemoterapi alan ALL’li çocuklarda indüksiyon

tedavisi sonrasında kemik iliği hematopoietik toparlanmanın İRF’de

MNS’ye göre daha erken olduğunu göstermiştir.

Anahtar Sözcükler: Akut lenfoblastik lösemi, Lenfoid hücreli neoplazi,

Hematopoiez, Kemoterapi

Address for Correspondence/Yazışma Adresi: Shan E. RAUF, M.D.,

Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan

Phone : 92-333-563 19 29

E-mail : shan.e.rauf673@gmail.com

Received/Geliş tarihi: September 24, 2014

Accepted/Kabul tarihi: April 20, 2015

131

Rauf SE, et al: IRF and ANC as Predictors of Hemopoietic Recovery in ALL Patients


Introduction

Leukemia is the most common malignancy of childhood and

acute lymphoblastic leukemia (ALL) accounts for up to 75% to

80% of the leukemia cases in the world [1]. In the Pakistani

population, the frequency of ALL in children and adults combined

is 32% of all malignancies [2]. The first case of leukemia in an

adult was reported in 1845 by John Hughes Bennett and in

children by Henry Fuller in 1846 [2]. Over the last 50 years many

new modalities of diagnosis and treatment of leukemia have

evolved, leading to improved survival [3,4].

Chemotherapy is the initial treatment of choice in most patients

of ALL and is divided into the following stages: remission

induction, consolidation or intensification, and maintenance

(continuation) therapy, with central nervous system prophylaxis

generally provided in each stage. The aim of remission induction

therapy is to induce a complete remission. The initial response

to remission induction therapy is one of the most important

prognostic factors in ALL [5]. The cytotoxic chemotherapeutic

agents cause marrow suppression, making patients prone to

anemia, bleeding, and infections. The main cause of death in

two-thirds of the patients is infection, mostly fungal [6].

During the period of marrow suppression, extensive monitoring

of blood counts is required to assess hemopoietic recovery. The

hemopoietic recovery can be assessed by conventional parameters

like absolute neutrophil count (ANC) recovery or the newer

but less commonly used parameter of immature reticulocyte

fraction (IRF) recovery. The ANC has >96% sensitivity to predict

bone marrow recovery after chemotherapy [7]. The IRF is now

being widely used in different centers as an early predictor for

hemopoietic recovery in place of the more traditional parameter

of ANC recovery, which appears later in the induction phase.

According to a prior study, IRF shows early bone marrow

recovery in 78% of cases as compared to ANC [8] and has a

sensitivity of 92% [9]. Reticulocytes reflect the erythropoietic

activity of the bone marrow and were traditionally assessed by

manual microscopic method; however, this method is subject to

high variability. Today it is measured more objectively by flow

cytometry-based hematology analyzers, which measure the

messenger ribonucleic acid (RNA) content and the maturity of

reticulocytes. The fluorescence of reticulocytes is dependent on

the RNA content of the reticulocytes. Immature reticulocytes

with higher RNA content will have maximum fluorescence,

while reticulocytes with lower RNA content produce minimum

fluorescence. On the basis of fluorescence intensity signals,

reticulocytes are classified into 3 maturation stages: lowfluorescence

reticulocytes (LFRs), medium-fluorescence

reticulocytes (MFRs), and high-fluorescence reticulocytes

(HFRs). The IRF is the combination of HFRs and MFRs and its

fraction in excess of 5% is postulated as a reliable marker for

hemopoietic recovery [8].

Flow cytometry-based hematology analyzers are now being

used in most of the large diagnostic centers of Pakistan,

whereas assessment of hemopoietic recovery is still based on

the conventional parameter of ANC. Unfortunately, due to

lack of published data in regard to the importance of the IRF

during treatment of ALL patients, this parameter is not being

used effectively to monitor patients’ marrow status and so far

has not been used as a protocol in Pakistan. The aim of this

study is to evaluate IRF as an earlier indicator of bone marrow

recovery than ANC in patients with ALL on remission induction

chemotherapy.


This descriptive study was carried out in the Department

of Hematology at the Armed Forces Institute of Pathology,

Rawalpindi, over a period of 1 year from January 2013 to

January 2014. Sampling was done based on a consecutive non

probability sampling technique. All diagnosed ALL patients

undergoing remission induction chemotherapy of both genders

were included. Remission induction as per the UKALL 2003

protocol was given, comprising dexamethasone, vincristine,

L-asparaginase, and intrathecal methotrexate. Relapsead

patients and those undergoing reinduction were excluded.

For each included patient, 2.5 to 3 mL of EDTA anticoagulated

blood was sampled and analyzed on day 1 and every second

day thereafter until the day of recovery. For each sample of

blood, complete blood counts along with differential leukocyte

count (for calculation of ANC) and reticulocyte parameters

were noted after running the sample on the flow cytometrybased

hematology analyzer Sysmex XE-5000. ANC of more

than >0.5x10 9 /L on the day corresponding to the first of three

consecutive scores was considered as ANC recovery. IRF recovery

was an IRF (MFR+HFR) in excess of 5%.

As a control, 2.5 to 3 mL of EDTA anticoagulated blood of

normal healthy individuals was also examined for reticulocyte

parameters on the Sysmex XE-5000. All the collected data were

analyzed with SPSS 19.0. The mean and standard deviation were

calculated for quantitative variables like age, ANC, and IRF, and

comparisons of means were carried out by paired samples t-test.

For qualitative variables like gender, frequency and percentage

was calculated.

Results

A total of 45 patients were included in this study. The majority of

the patients were 11-20 years of age. Mean age of the patients

was 12.04±5.30 years, and 25 patients (55.6%) were male and

20 patients (44.4%) were female.

Mean IRF value on day 1 of induction remission was 9.68±1.41,

mean ANC value on day 1 of induction remission was 0.077±0.061,

mean recovery day for IRF was 11.84±7.44, and mean recovery

132



day for ANC was 17.67±8.77 (two-tailed p-value <0.0001, 95%

confidence interval). Mean values of different variables of ANC

and IRF are shown in Table 1. Out of 45 patients, 40 (88.9%)

patients showed earlier IRF recovery as compared to ANC. Four

(8.9%) patients had the same day of recovery by both IRF and

ANC while one (2.2%) had a later IRF recovery than ANC. The

recovery days of each patient for both ANC and IRF are shown

in Figure 1. By day 28, 41 (91%) patients showed IRF recovery,

and ANC recovery was seen in 36 (80%) patients. All those

patients not showing recovery by day 28 were further followed

up and all of them showed recovery by day 39, as shown in Table

1 and Figure 1.

Discussion

Chemotherapy for ALL has not changed much over the

years, except for a few variations in different centers. The

chemotherapeutic agents used in remission induction therapy

usually cause severe myelosuppression in these patients. This

critical period is variable in patients and requires critical care,

supportive therapy, and regular monitoring. Many patients

succumb to severe sepsis and bleeding in this period. Recovery

of bone marrow from myelosuppression is an indicator of likely

hematological remission. The ANC has traditionally been used

as an early predictor of bone marrow recovery. However, with

the advent of the latest flow cytometry-based hematological

analyzers, the IRF is being increasingly used for this purpose.

The IRF is an accurate and reliable parameter easily obtained

from automated cell counters such as the Sysmex XE-Series [9].

Several studies showed that the immature reticulocytes detected

by flow cytometry are earlier indicators of bone marrow recovery

than the detection of ANC in post chemotherapy patients with

acute leukemia [10,11].

Although our study was limited in time and sample size, we

were able to reach similar conclusions to those published by

George et al., who found that immature reticulocytes indicate

engraftment, and the use of immature reticulocytes might

enable the cessation of antibiotics and growth factors, which

could lead to earlier discharge from the hospital and cost

savings [9].

The Spanish Multicentric Study Group for hemopoietic recovery

also concluded that a rise in IRF indicates hemopoietic recovery

[12] and IRF recovery was seen in 91.2% of ALL patients on

remission induction before ANC recovery. Luczynski et al. in

their study stated that IRF was the first sign of hemopoietic

recovery and might be used as a parameter of bone marrow

function in clinical studies [13].

Das et al. in 2006 also showed IRF as the earlier predictor of

bone marrow recovery as compared to ANC in childhood

malignancies [10]. The median day for IRF recovery was 21, while

for ANC recovery it was 23 [12]. In a study done in Bangladesh,

IRF showed 78% similar or earlier recovery in patients with ALL

on remission induction chemotherapy. The mean day for IRF

recovery was 16.6±4.6 while it was 23.3±5.7 for ANC recovery,

indicating that IRF is an earlier predictor of bone marrow

hemopoietic recovery than ANC [8].

Figure 1. Recovery day of each patient by both immature

reticulocyte fraction and absolute neutrophil count.

In our study, mean recovery days were 11.84±7.44 for IRF and

17.67±8.77 for ANC, showing earlier IRF recovery by an average

of 6 days, while it was shown to be 4 days earlier by Grazziutti

et al. [14]. This prediction of early recovery by the simple and

reproducible parameter of IRF can have significant impact on

the management of patients.

Conclusion

This study concluded that the IRF shows earlier hemopoietic

recovery as compared to the current practice of ANC for the

monitoring of ALL patients on remission induction chemotherapy.

Table 1. Mean values of different variables.

Variable Number Minimum Maximum Mean ± SD Two-tailed p-value

IRF on day 1 of remission induction 45 7.0 12.1 9.68±1.41 -

ANC on day 1 of remission induction 45 0.01 0.3 0.077±0.061 -

Day of recovery for IRF 45 4 34 11.84±7.44 <0.0001

Day of recovery for ANC 45 8 39 17.67±8.77 <0.0001

IRF: Immature reticulocyte fraction, ANC: absolute neutrophil count, SD: standard deviation.

133



This early laboratory indicator of hemopoietic recovery will

guide clinicians to make early and important therapeutic

decisions in such patients. Nowadays, the IRF is offered by

most new hematology analyzers. Moreover, this test is a simple,

quick, reproducible, and reliable parameter in the automated

hematology analyzers.

Authorship Contribution

Concept: Shan E. Rauf, Saleem Ahmed Khan; Design: Shan E.

Rauf, Saleem Ahmed Khan; Data Collection and Processing:

Shan E. Rauf, Nabeel Khan Afridi; Analysis and Interpretation:

Shan E. Rauf, Nadir Ali; Literature Search: Shan E. Rauf, Saleem

Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon,

Ammara Arslan; Writing: Shan E. Rauf, Saleem Ahmed Khan,

Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan.




included.

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lymphoblastic leukemia after induction therapy-3 year experience at a

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W, Niemeyer C, Henze G, Feldges A, Zintl F, Kornhuber B, Ritter J, Welte K,

Gadner H, Riehm H. Improved outcome in childhood acute lymphoblastic

leukemia despite reduced use of anthracyclines and cranial radiotherapy:

results of trial ALL-BFM 90. German-Austrian-Swiss ALL-BFM Study Group.

Blood 2000;95:3310-3322.

6. Asim M, Zaidi A, Ghafoor T, Qureshi Y. Death analysis of childhood acute

lymphoblastic leukemia; an experience at Shoukat Khanum Memorial Cancer

Hospital and Research Centre, Pakistan. J Pak Med Assoc 2011;61:666-670.

7. Hijiya N, Onciu M, Howard SC, Zhang Z, Cheng C, Sandlund JT, Khyzer EP,

Behm FG, Pui CH. Utility of automated counting to determine absolute

neutrophil counts and absolute phagocyte counts for pediatric cancer

treatment protocols. Cancer 2004;101:2681-2686.

8. Yesmin MS, Sultana T, Roy CK, Rehman MQ, Ahmed ANN. Immature

reticulocyte fraction as a predictor of bone marrow recovery in children with

acute lymphoblastic leukemia on remission induction phase. Bangladesh

Med Res Counc Bull 2011;37:57-60.

9. George P, Wyre RM, Bruty SJ, Sweetenham JW, Duncombe AS. Automated

immature reticulocyte counts are early markers of engraftment following

autologous PBSC transplantation in patients with lymphoma. J Hematother

Stem Cell Res 2000;9:219-223.

10. Das R, Dip NB, Garewal G, Marwaha RK, Vohra H. Automated reticulocyte

response is a good predictor of bone-marrow recovery in pediatric

malignancies. Pediatr Hematol Oncol 2006;3:299-305.

11. Norhana JF, De Souza CA, Vigorito AC. Immature reticulocytes as an early

predictor of engraftment in autologous and allogeneic bone marrow

transplantation. Clin Lab Haematol 2003;25:47-54.

12. [No authors listed.] Flow cytometric reticulocyte quantification in the

evaluation of hematopoietic recovery. Spanish Multicentric Study Group

for Hematopoietic Recovery. Eur J Haematol 1994;53:293-297.

13. Luczynski W, Ratomski K, Wysocka J, Krawczuk-Rybuk M, Jankiewicz P.

Immature reticulocyte fraction (IRF)--an universal marker of hemopoiesis

in children with cancer? Adv Med Sci 2006;51:188-190.

14. Grazziutti ML, Dong L, Miceli MH, Cottler-Fox M, Krishna SG, Fassas

A, van Rhee F, Barlogie BM, Anaissie EJ. Recovery from neutropenia can

be predicted by the immature reticulocyte fraction several days before

neutrophil recovery in autologous stem cell transplant recipients. Bone

Marrow Transplant 2006;37:403-409.

134

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0405


The Prognostic Significance of Soluble Urokinase Plasminogen

Activator Receptor in Acute Myeloid Leukemia

Akut Miyeloid Lösemili Hastalarda Solubl Ürokinaz Plazminojen Aktivatör Reseptörünün

Prognozdaki Önemi

Nergiz Erkut1,2, Ahmet Menteşe3,4, Hasan Mücahit Özbaş2, Nilay Ermantaş2, Ayşegül Sümer4, Asım Örem4, Mehmet Sönmez2

1Kanuni Training and Research Hospital, Clinic of Hematology, Trabzon, Turkey

2Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey

3Karadeniz Technical University, Vocational School of Health Sciences, Program of Medical Laboratory Techniques, Trabzon, Turkey

4Karadeniz Technical University Faculty of Medicine, Department of Medical Biochemistry, Trabzon, Turkey

Abstract

Objective: The soluble urokinase plasminogen activator receptor

(suPAR) is a soluble form of the urokinase plasminogen activator

receptor expressed in various immune and cancer cells. The levels of

suPAR have been demonstrated to correlate with prognosis in various

cancers. This study was intended to investigate serum suPAR levels

and their effect on prognosis in patients with acute myeloid leukemia

(AML).

Materials and Methods: Thirty newly diagnosed patients with AML

and 29 healthy individuals were enrolled. Serum suPAR levels were

analyzed by enzyme-linked immunosorbent assay.

Results: Serum suPAR levels were significantly higher in patients with

AML than in healthy individuals (9±5.9 ng/mL and 2.4±1.4 ng/mL,

respectively; p<0.001). Positive correlation was determined between

suPAR levels and white blood cell counts (p<0.01). Serum suPAR

levels were lower in patients who achieved complete response than in

patients not achieving complete response (5.5±2.2 ng/mL and 12±6.6

ng/mL, respectively; p<0.001). The median overall survival was longer

in patients with serum suPAR levels below 6.71 ng/mL than in those

with serum suPAR levels above 6.71 ng/mL (12.6±13.2 months and

1.71±0.6 months, respectively; p=0.02). Multivariate Cox regression

analysis showed that suPAR had independent prognostic value (95%

confidence interval: 1.029-6.259; p<0.05) in AML.

Conclusion: Serum suPAR levels can be used as a prognostic marker

in AML.

Keywords: Soluble urokinase plasminogen activator receptor, Acute

myeloid leukemia, Prognosis

Öz

Amaç: Solubl ürokinaz plazminojen aktivatör reseptörü (süPAR)

çeşitli immün sistem ve kanser hücrelerinde eksprese edilen ürokinaz

plazminojen aktivatör reseptörün çözünür formudur. Çeşitli

kanserlerde süPAR düzeyinin prognoz ile ilişkili olduğu gösterilmiştir.

Bu çalışmada akut miyeloid lösemili (AML) hastalarda süPAR düzeyi ve

prognoz üzerine olan etkisinin araştırılması planlandı.

Gereç ve Yöntemler: Çalışmaya yeni tanı almış 30 AML’li hasta ve

29 sağlıklı birey dahil edildi. Serum süPAR düzeyi enzyme-linked

immunosorbent assay yöntemi ile analiz edildi.

Bulgular: Serum süPAR düzeyi AML’li hastalarda sağlıklı bireylere

göre önemli derecede daha yüksek tespit edildi (9±5,9 ng/mL, 2,4±1,4

ng/mL, sırasıyla, p<0,001). süPAR düzeyi ile lökosit sayısı arasında

pozitif bir korelasyon izlendi (p<0,01). Serum süPAR düzeyi, tam

remisyona giren hastalarda tam remisyona girmeyen hastalara göre

daha düşüktü (5,5±2,2 ng/mL, 12±6,6 ng/mL, sırasıyla, p<0,001).

Toplam yaşam süresi, serum süPAR düzeyi 6,71 ng/mL’nin altında olan

hastalarda, 6,71 ng/mL üstünde olanlara göre daha uzundu (12,6±13,2

ay, 1,71±0,6 ay, sırasıyla, p=0,02). AML’de çok değişkenli Cox regresyon

analizi süPAR düzeyinin bağımsız prognostik değere sahip olduğunu

gösterdi (%95 güven aralığı: 1,029-6,259; p<0,05).

Sonuç: AML’li hastalarda serum süPAR düzeyi prognostik bir belirteç

olarak kullanılabilir.

Anahtar Sözcükler: Solubl ürokinaz plazminojen aktivatör reseptörü,

Akut miyeloid lösemi, Prognoz

Address for Correspondence/Yazışma Adresi: Nergiz ERKUT, M.D.,

Kanuni Training and Research Hospital, Clinic of Hematology, Trabzon, Turkey

Phone : +90 462 341 56 56

E-mail : drnusta@hotmail.com

Received/Geliş tarihi: October 10, 2014

Accepted/Kabul tarihi: May 04, 2015

135

Erkut N, et al: Levels of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia


Introduction

Acute myeloid leukemia (AML) is a heterogeneous neoplastic

disorder characterized by uncontrolled proliferation of

hematopoietic stem cells [1]. Although 70%-80% of patients

younger than 60 years of age achieve complete remission (CR),

only 30%-40% obtain long-term survival. Moreover, CR is only

observed in 10%-15% of elderly patients [2]. The pathogenesis

of AML involves various disorders, such as mutations in

transcription factors or epigenetic modifiers, aberrant signaling

pathways, overexpression of the multidrug resistance gene,

abnormal immune function, and abnormalities in the bone

marrow microenvironment [3]. Prognostic factors include

advanced age, poor performance status, high white blood cell

(WBC) count, existence of prior myelodysplastic syndrome

and myeloproliferative disease, previous history of cytotoxic

therapy, and particularly cytogenetics and molecular genetic

changes [4,5].

The urokinase plasminogen activator receptor (uPAR) is a

glycoprotein consisting of 274 amino acids with a molecular

weight of 55-60 kDa attached to the plasma membrane via

a glycosylphosphatidylinositol anchor protein [6]. uPAR is

expressed in neutrophils, lymphocytes, monocytes, macrophages,

fibroblasts, and endothelial and some tumor cells [7,8,9]. The

soluble urokinase plasminogen activator receptor (suPAR) is a

soluble form of uPAR found in serum, plasma, urine, and other

body fluids [10]. suPAR affects cancer progression through

adhesion, migration, chemotaxis, proteolysis, and invasion [11].

Several studies have demonstrated that suPAR increases in some

cancers and is associated with poor prognosis [12]. This study

was intended to investigate serum suPAR levels and their effect

on prognosis in patients with AML.


Thirty newly diagnosed patients with AML and 29 healthy

individuals presenting to the Deparment of Hematology, Faculty

of Medicine, Karadeniz Technical University between January 2009

and July 2011 were enrolled in this study. The eligibility criterion

was age between 18 and 80 years. Patients with a history of solid

cancer or other hematological cancer, the presence of active

infection, or active inflammatory disease were excluded. Venous

blood specimens collected from both patient and control groups

were placed into biochemical separator-containing tubes. Blood

samples were centrifuged at 3000 rpm for 10 min and serum

was stored at -80 °C for investigation of suPAR levels.

All AML patients were diagnosed according to the World Health

Organization classification system [13] and categorized into

three groups (i.e. low risk, intermediate risk, and high risk)

according to the National Comprehensive Cancer Network

guidelines [14].

Patients aged ≤60 years or 61-65 years with good performance

status were treated with the standard regimen [cytarabine, 24-h

continuous intravenous (IV) infusion, 100 mg/m 2 , days 1-7;

idarubicin, 30-min IV infusion, 12 mg/m2, days 1-3]. Patients

with acute promyelocytic leukemia were treated with all-transretinoic

acid (ATRA) plus idarubicin therapy (ATRA, orally, 45

mg/m2 per day in two divided doses until CR was achieved;

idarubicin, 30-min IV infusion, 12 mg/m2, days 2, 4, 6, and

8). Elderly patients were treated with low-dose chemotherapy

[low-dose cytarabine, subcutaneous (SC), 10 mg/m2, twice a day,

days 1-10; or 5-azacytidine, SC, 75 mg/m2, days 1-7]. Remission

status was evaluated after the completion of cancer therapy

according to conventional criteria. Patients were followed for

2 years, monthly for the first year and every third month in the

following year.

Measurement of Soluble Urokinase Plasminogen Activator

Receptor Levels

The levels of serum suPAR were determined by enzyme-linked

immunosorbent assay kit (ViroGates A/S, Denmark) according to

the manufacturer’s protocols. The absorbance of samples was

measured at 450 nm using a VERSA max tunable microplate

reader (designed by Molecular Devices, USA). The results were

expressed as ng/mL. The minimum detection limit of the assay

was estimated to be 0.1 ng/mL.


All analyses were carried out using SPSS 21.0. Descriptive

statistical analysis was performed for all studied variables. Data

were tested for normal distribution using the Kolmogorov-

Smirnov test. Statistical comparisons between the patient and

control groups were carried out using the Mann-Whitney test

and chi-square test. The associations between serum suPAR levels

and hemoglobin (Hb) or hematocrit levels and white blood cell

(WBC) or platelet count were examined by Spearman correlation

analysis. The area under the receiver operating characteristic

(ROC) curve was used to compare the discriminative power of

suPAR levels in the diagnosis of AML. Estimates of overall survival

(OS) were calculated using the Kaplan-Meier method. The logrank

test was used to analyze the effect on survival time of each

variable. The Cox regression model was applied for multivariate

analysis. Linear regression analysis was used to investigate the

relationship between serum suPAR levels and sex, patient age,

WBC count, French-American-British (FAB) classification, and

Fms-like tyrosine receptor kinase-3 (FLT-3) mutation. A value of

p<0.05 was considered statistically significant.

Results

Thirty patients with AML and 29 healthy controls were included

in the study. There were no statistical differences in term of

age or sex between the two groups. Risk groups included 6

136



patients at good risk, 19 at intermediate risk, and 5 at poor

risk. At the end of the 2-year follow-up, 26 patients had died

and 4 survived. Fourteen patients exhibited CR after remissioninduction

chemotherapy, while CR was not achieved in the other

16. Table 1 shows the general characteristics and laboratory

findings for both patients and healthy individuals.

Serum suPAR levels were significantly higher in patients with

AML than in healthy individuals (9±5.9 ng/mL and 2.4±1.4 ng/

mL, respectively; p<0.001) (Figure 1). Positive correlation was

determined between suPAR levels and WBC count in patients

with AML (p<0.01) (Figure 2), whereas there was no correlation

between suPAR levels and Hb levels or platelet count. There was

no significant difference in serum suPAR levels between patients

aged ≤60 and >60 years (7.6±4.4 ng/mL and 12.3±8 ng/mL,

respectively; p>0.05). Serum suPAR levels were lower in patients

who achieved CR than in patients not achieving CR (5.5±2.2 ng/

mL and 12±6.6 ng/mL, respectively; p<0.001) (Figure 3).

In AML patients, the area under the ROC curve for suPAR was

0.938 [95% confidence interval (CI): 0.843-0.984]. For the

optimum diagnostic cut-off value of 2.79 ng/mL, the sensitivity

and specificity were 96.67% and 79.31%, respectively (Figure 4).

30.00

25.00

20.00

15.00

10.00

5.00

0.00

Figure 1. Soluble urokinase plasminogen activator receptor

concentrations in serum from acute myeloid leukemia patients

and healthy controls. The dotted line indicates the mean value

plus 3 standard deviations of healthy control serum (6.6 ng/mL).

suPAR: Soluble urokinase plasminogen activator receptor, AML:

acute myeloid leukemia, SD: standard deviation.

The median OS of AML patients was 4.16 months (range: 0-32.9

months). In the Kaplan-Meier analysis and the Cox regression

model, patients with high serum suPAR levels showed a trend

toward poorer survival (p=0.02). The median OS was longer in

patients with serum suPAR levels below 6.71 ng/mL than in

those with serum suPAR levels above 6.71 ng/mL (12.6±13.2

months and 1.71±0.6 months, respectively; p=0.02) (Figure 5).

WBC count had no significant effect on OS (p=0.9) (Figure 6).

In linear regression analysis, sex, patient age, WBC count, FAB

classification (i.e. M2, M3, M4, M5), and FLT-3 mutation were

not associated with serum suPAR levels (p>0.05). Multivariate

Cox regression analysis showed that suPAR had independent

prognostic value (95% CI: 1.029-6.259; p<0.05) in AML. When

the suPAR cut-off level was considered as 6.71 ng/mL, mortality

risk was 2.5-fold higher in patients with levels above the cut-off

limit.

30.00

25.00

20.00

15.00

10.00

5.00

0.00

Figure 3. Soluble urokinase plasminogen activator receptor

concentrations in serum from acute myeloid leukemia patients

with no complete remission and acute myeloid leukemia patients

with complete remission. The dotted line indicates the mean value

plus 3 standard deviations of serum of acute myeloid leukemia

patients with complete remission (12.1 ng/mL). suPAR: Soluble

urokinase plasminogen activator receptor, AML: acute myeloid

leukemia, SD: standard deviation, CR: complete remission.

30.00

25.00

20.00

15.00

10.00

5.00

0.00

Figure 2. Correlations between soluble urokinase plasminogen

activator receptor levels and white blood cell count in acute

myeloid leukemia patients. WBC: White blood cell, suPAR: soluble

urokinase plasminogen activator receptor, AML: acute myeloid

leukemia.

Figure 4. The receiver operating characteristic curves of acute

myeloid leukemia patients according to soluble urokinase

plasminogen activator receptor levels. suPAR: Soluble urokinase

plasminogen activator receptor, AUC: area under the curve.

137



Table 1. Characteristics of acute myeloid leukemia patients.

Figure 5. Kaplan-Meier curves of acute myeloid leukemia patients

according to soluble urokinase plasminogen activator receptor

levels. suPAR: Soluble urokinase plasminogen activator receptor.

Figure 6. Kaplan-Meier curves of acute myeloid leukemia patients

according to white blood cell count. WBC: White blood cell.

Discussion

The urokinase-mediated plasminogen activation (uPA) system

plays an important role in tissue remodeling, angiogenesis,

proteolysis, migration, chemotaxis, invasion, and metastasis

[15,16,17]. The uPA system consists of uPA, uPAR, plasminogen,

and plasminogen activator inhibitor [18]. In vitro studies have

shown that suPAR is associated with cell adhesion, migration,

and proliferation [19,20]. Elevated suPAR levels have been

determined in solid cancers such as ovarian [21], endometrial,

cervical [22], breast [23], stomach [24], colon [25], and non-small

cell lung cancer [26]. Positive associations between serum suPAR

levels and soluble serum CD138, creatinine, β2 microglobulin,

stage of disease, and extramedullary bone marrow involvement

have been reported in patients with multiple myeloma [27].

In acute leukemia, circulating blast cells provide an important

advantage for studying proteins expressed on the tumor

Parameters n=30

Median age (minimum-maximum), years 52 (24-80)

≤60 years, n (%) 22 (73.3)

>60 years, n (%) 8 (26.7)

Sex, n (%)

Female 17 (56.7)

Male 13 (43.3)

Risk groups, n (%)

Good risk 6 (20)

Intermediate risk 19 (63.3)

Poor risk 5 (16.7)

Classification of AML, n (%)

AML with recurrent genetic abnormalities 7 (23.3)

t(15;17)(q22;q12); PML-RARA 3 (10)

t(8;21)(q22;q22); RUNX1-RUNX1T1 2 (6.7)

t(16;16)(p13.1q22); CBFB-MYH11 1 (3.3)

Inv(3)(q21q26.2); RPN1-EVI1 1 (3.3)

AML with MDS-related changes 5 (16.7)

AML, therapy-related 1 (3.3)

AML not otherwise categorized 16 (53.2)

AML minimally differentiated 1 (3.3)

AML without maturation 1 (3.3)

AML with maturation 7 (23.3)

Acute myelomonocytic leukemia 6 (20)

Acute monocytic leukemia 1 (3.3)

Other 1 (3.3)

Laboratory findings at baseline, median (minimum-maximum)

WBC count (x10 9 /L) 62 (3.6-218)

Hb (g/dL) 9.9 (5-17.5)

Platelets (x10 9 /L) 64 (9-198)

FLT-3 mutation, n (%) 6 (20)

Treatment regimen, n (%)

Cytarabine plus idarubicin (10 CR, 11 without CR) 21 (70)

ATRA plus idarubicin (3 CR) 3 (10)

5-azacitidine (1 CR, 4 without CR) 5 (16.7)

Low-dose cytarabine (1 without CR) 1 (3.3)

Remission-induction chemotherapy response, n (%)

CR 14 (46.7)

No CR 16 (53.3)

AML: Acute myeloid leukemia, MDS: myelodysplastic syndrome, WBC: white blood

cell, Hb: hemoglobin, FLT-3: Fms-like tyrosine receptor kinase, ATRA: all-trans-retinoic

acid, CR: complete response.

138



cell surface. Lanza et al. demonstrated that uPAR (CD87)

expression increased in patients with AML and was associated

with mucocutaneous infiltration, hepatosplenomegaly,

lymphadenopathy, and central nervous system involvement

[28]. The levels of suPAR in the plasma of mice during the

growth of xenografted cell lines were significantly related to

tumor volume [29]. Mustjoki et al. reported that increased

suPAR levels were correlated with number of circulating tumor

cells in AML and that serum suPAR levels decreased rapidly after

cytotoxic treatment [30]. Aref et al. further demonstrated that

serum suPAR levels were significantly higher in AML patients as

compared to controls [31]. Similarly, in our study, serum suPAR

levels significantly increased in patients with AML compared to

healthy individuals. In addition, suPAR was observed to possess

high sensitivity and specificity in patients with AML in ROC

analysis. There was a positive correlation between suPAR levels

and number of circulating WBCs. Therefore, we think that the

production of suPAR is related to blast cells in the peripheral

circulation.

Lomholt et al. demonstrated that elevated suPAR levels were

independent prognostic factors in patients with colorectal

cancer [32]. Another study showed that high suPAR levels were

associated with poor outcome in patients with breast cancer

independent of tumor size, estrogen receptor status, and lymph

node status [23]. On the other hand, Begum et al. reported

that preoperative plasma suPAR levels were not correlated

with prognosis for stage III ovarian cancer patients [33]. In our

study, serum suPAR levels were significantly higher in patients

who did not achieve CR than in patients achieving CR. More

importantly, high suPAR levels were associated with poor

prognosis in patients with AML. When the suPAR cut-off level

was considered as 6.71 ng/mL, mortality risk was 2.5-fold higher

in patients with levels above the cut-off limit. Sex, patient age,

WBC count, FAB classification (i.e. M2, M3, M4, M5) and FLT-3

mutation were not associated with serum suPAR levels (p>0.05).

Serum suPAR levels were an independent prognostic indicator

for the OS of patients with AML.

Conclusion

In conclusion, our study indicates that suPAR increases in

patients with AML and this situation is associated with poorer

survival. suPAR can thus be used as a diagnostic and prognostic

biomarker in AML and may help in the developing of specific

therapeutic targets. However, further studies are required to

assess the clinical relevance of suPAR.

Ethics


Local Ethics Committee of the Karadeniz Technical University

Faculty of Medicine, and was conducted in accordance with the

Declaration of Helsinki. Informed consent was taken from all

patients and healthy subjects.


Concept: Nergiz Erkut, Mehmet Sönmez; Design: Nergiz Erkut,

Mehmet Sönmez; Data Collection or Processing: Nergiz Erkut,

Ahmet Menteşe, Hasan Mücahit Özbaş, Nilay Ermantaş, Ayşegül

Sümer, Asım Örem, Mehmet Sönmez; Analysis or Interpretation:

Nergiz Erkut, Ahmet Menteşe; Literature Search: Nergiz Erkut,


Sümer, Asım Örem, Mehmet Sönmez; Writing: Nergiz Erkut,


Sümer, Asım Örem, Mehmet Sönmez.




included.

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140

RESEARCH ARTICLE

DOI: 10.4274/tjh.2015.0193


Investigation of Rho-Kinase Expressions and Polymorphisms in

Mantle Cell Lymphoma Patients

Mantle Hücreli Lenfoma Hastalarında Rho-Kinaz Ekspresyonları ve Polimorfizmlerinin

Araştırılması

Didar Yanardağ Açık 1 , Mehmet Yılmaz 1 , İbrahim Sarı 2 , Serdar Öztuzcu 3 , Zeynel A. Sayıner 4 , Salih Subari 4 , Abdullah T. Demiryürek 5

1Gaziantep University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Gaziantep, Turkey

2Gaziantep University Faculty of Medicine, Department of Pathology, Gaziantep, Turkey

3Gaziantep University Faculty of Medicine, Department of Medical Biology, Gaziantep, Turkey

4Gaziantep University Faculty of Medicine, Department of Internal Medicine, Gaziantep, Turkey

5Gaziantep University Faculty of Medicine, Department of Medical Pharmacology, Gaziantep, Turkey

Abstract

Objective: Mantle cell lymphoma (MCL) is a rare but aggressive form of

B-cell non-Hodgkin lymphoma characterized by excessive expression

of cyclin D1. Intracellular signaling enzyme Rho-kinase (ROCK) can

contribute to cellular migration, proliferation, and differentiation, as

well as tumor development and metastasis. However, ROCK gene and

protein expressions or polymorphisms have never been investigated in

MCL patients. The purpose of this study was to investigate the role of

ROCK gene and protein expressions in MCL patients. We also examined

ROCK2 gene polymorphisms in this study.

Materials and Methods: A total of 60 patients with MCL and 60

healthy controls were included in this retrospective study. Hematoxylin

and eosin-stained lymph node tissue slides in the entire archive were

reevaluated and used for immunohistochemistry, gene expression, and

polymerase chain reaction studies.

Results: In immunohistochemical studies, there were significant

increases in ROCK1 (p=0.0009) and ROCK2 (p<0.0001) protein

expressions in MCL patients when compared with the control group.

Although a marked increase in ROCK1 gene expression (p=0.0215) was

noted, no significant change was observed in ROCK2 gene expression

in MCL patients. Seven ROCK2 polymorphisms were studied, but the

results showed no significant differences between the groups.

Conclusion: This is the first study to show that ROCK1 gene and ROCK

protein expressions may contribute to the development of MCL.

Keywords: Lymphoma, Expression, Polymorphism, Rho-kinase

Öz

Amaç: Mantle hücreli lenfoma (MHL) siklin D1’in aşırı ekspresyonuyla

karakterize B-hücreli Hodgkin dışı lenfomanın nadir fakat agresif

bir şeklidir. İntraselüler sinyal enzimi olan Rho-kinaz (ROCK), hücre

migrasyonu, proliferasyonu, farklılaşması yanında tümör gelişimi ve

metastazına da katkıda bulunur. Fakat MHL hastalarında ROCK gen

ve protein ekspresyonları veya polimorfizmleri araştırılmamıştır.

Bu çalışmanın amacı, MHL hastalarında ROCK gen ve protein

ekspresyonlarının rolünü araştırmaktı. Biz bu çalışmada ROCK2 gen

polimorfizmleri de araştırdık.

Gereç ve Yöntemler: Bu retrospektif çalışmaya 60 MHL hastası ve 60

sağlıklı kontrol dahil edildi. Bütün arşivde hematoksilin ve eosin boyalı

lenf düğümü kesitleri yeniden incelendi ve immünohistokimya, gen

ekspresyonu ve polimeraz zincir reaksiyonu çalışmaları için kullanıldı.

Bulgular: İmmünohistokimyasal çalışmada, kontrol grubuyla

karşılaştırıldığında MHL hastalarında ROCK1 (p=0,0009) ve ROCK2

protein ekspresyonlarında (p<0,0001) anlamlı artış vardı. MHL

hastalarında ROCK1 gen ekspresyonunda (p=0,0215) anlamlı artış

bulunmasına karşın ROCK2 gen ekspresyonunda anlamlı değişiklik

gözlenmedi. Yedi ROCK2 polimorfizmi çalışıldı, fakat sonuçlar gruplar

arasında anlamlı farklılıklar göstermedi.

Sonuç: Bu çalışma, ROCK1 gen ve ROCK protein ekspresyonlarının

MHL gelişimine katkısı olabileceğini gösteren ilk çalışmadır.

Anahtar Sözcükler: Lenfoma, Ekspresyon, Polimorfizm, Rho-kinaz

Address for Correspondence/Yazışma Adresi: Didar YANARDAĞ AÇIK, M.D.,

Gaziantep University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Gaziantep, Turkey

Phone : +90 532 157 76 56

E-mail : didaryanardag@gmail.com


Accepted/Kabul tarihi: August 24, 2015

141

Yanardağ Açık D, et al: Rho-Kinase in Mantle Cell Lymphoma


Introduction

Mantle cell lymphoma (MCL) is an aggressive B-cell-type non-

Hodgkin lymphoma characterized by cyclin D1 overexpression

and occurs more commonly in advanced ages and in males

[1,2]. MCL is a rare subtype accounting for about 6% of all

non-Hodgkin lymphoma cases. During the development of

MCL, via t(11;14) (q13;q32) translocation, the BCL1 gene

(11q13) and immunoglobulin heavy chain gene (14q32) come

together, and hence BCL1 is upregulated. As a consequence

of this translocation, excessive synthesis of cyclin D1 protein

promotes the cell cycle progression (to S-/G2-phase) via cyclindependent

kinase 4 and 6 activation in an uncontrolled manner.

A minority (about 5%) of cases are cyclin D1-negative, and

these cases often exhibit high expression of cyclin D2 or D3

[3]. Phenotypically, MCL is positive for the B-cell markers CD5,

CD20, and CD79a. In MCL cells, CD10 and CD23 are usually

negative. There is also overexpression of SOX-11 in the nuclei

in most cases [4]. Overexpression of SOX-11 has been described

as a diagnostic marker for MCL, with the absence of SOX-11 a

characteristic of indolent MCL [5].

No single genetic lesion that can give rise to MCL has been

identified. Molecular studies including single nucleotide

polymorphisms (SNPs) have revealed a large number of

chromosomal alterations in MCL [6,7,8,9]. Several copy number

aberrations have been found to be correlated with genomic

complexity in MCL cases [10].

Most patients are diagnosed at an advanced stage, and

extranodal sites are often involved [11]. Even though patients

with MCL often respond to therapy, the responses are usually

partial and most patients eventually relapse [12]. There is

currently no proven curative therapy and no standard of care

has been established for initial or subsequent lines of therapy.

Therefore, ideal treatment regimens for MCL are still being

investigated and studies indicate that intracellular signaling

pathways may be important targets in the treatment of MCL.

Rho-kinase (ROCK) signaling has been implicated in various

cellular functions downstream of Rho GTPases. Rho GTPases

are important regulators of cancer cell proliferation, survival,

invasion, and metastasis. More recently, crucial functions of Rho

GTPases in the regulation of tumor stroma, including endothelial

cells, immune cells, and cancer-associated fibroblasts, as

well as in the formation of microvesicles, have been reported

[13]. ROCK is a serine-threonine protein kinase with multiple

downstream effects. Two isoforms of ROCK protein, ROCK1

and ROCK2, have been characterized. The ROCK isoforms are

encoded by separate genes on human chromosomes 18q11

(ROCK1) and 2p24 (ROCK2) [14,15]. ROCK is substantially

involved in a wide range of fundamental cellular functions,

such as proliferation, differentiation, adhesion, contraction,

metabolism, and apoptosis. ROCK signaling enhances myosinmediated

contractility and drives amoeboid migration, which

is associated with certain types of carcinoma, lymphomas,

and leukemia [15,16,17]. Increased expression of the ROCK

proteins promotes tumor cell proliferation and contributes to

the metastatic behavior of some cancers [15]. Several of the

ROCK substrates are prominent players in the development of

cancer and its associated phenotypes. For example, the tumor

suppressor phosphatase and tensin homolog (PTEN), which is

frequently inactivated in melanoma, as well as c-Jun N-terminal

Kinase (JNK)-interacting protein-3, an inhibitor of JNK signaling

that is upregulated in melanoma, are inhibited by ROCK

phosphorylation [17]. It has been shown that the sustained

activation of ROCK is sufficient to induce cell cycle progression

and increase cyclin D1 expression in NIH 3T3 fibroblasts [18].

Furthermore, ROCK activation also increases the expression of

cyclin D1 in vascular smooth muscle cells [19]. In this study, the

contribution of both ROCK isoforms in MCL was investigated.

We also explored the possible role of the ROCK gene and protein

expressions in MCL and tested the hypothesis that genetic

variations in the ROCK2 gene may increase the risk of MCL.


Patients

In the present study, tissue samples of 60 patients diagnosed

with MCL between 2006 and 2012, and those of 60 healthy

adults who underwent lymph node biopsy for any reason but

were not diagnosed with any malignant disease and were

reported to have only hyperplasia by the pathology department,

were investigated retrospectively. The study was approved by

the local ethics committee.

Clinical and laboratory information at the date of first diagnosis

was recorded and overall survival was calculated as time from

diagnosis to death or to the date when the patient was seen

for the last time. Patients were identified from the pathological

records and all cases were confirmed by histological evaluation.

All demographic and clinical characteristics as well as prognostic

factors of the study cases were collected from files. The

prognosis of patients was based on the Mantle Cell Lymphoma

International Prognostic Index (MIPI), which is calculated on the

basis of four independent prognostic factors (age, performance

status, serum lactate dehydrogenase level, and leukocyte count).

Immunohistochemistry

Formalin-fixed, paraffin wax-embedded blocks from each

case were selected for immunohistochemical studies using

the antibodies against ROCK1 and ROCK2. Hematoxylin

and eosin-stained lymph node tissue slides were used for

immunohistochemistry. Control tissue sections were made from

the lymph node biopsies of the healthy subjects. Sections of

142



4 µm were cut from paraffin-embedded tissue blocks onto

silane-coated slides. Sections were heated to 60 °C for 20

min prior to deparaffinization with xylene solution. Sections

were then stained using the Bond Polymer Refine Detection

Kit (Bond #DS9800) in an automated slide processing system

(Bond-Max, Leica Microsystems, Buffalo Grove, IL, USA). ROCK1

(rabbit monoclonal, EP786Y, ab45171, Abcam, Cambridge, UK)

and ROCK2 (rabbit polyclonal, ab71598, Abcam, Cambridge, UK)

were used for ROCK1 and ROCK2 immunostaining, respectively.

The percentage of cells staining was evaluated and intensity (–,

+, ++, or +++) was scored from 0 to 3 [20].

DNA Isolation and Genotyping

DNA isolation was done with the paraffin blocks using the

QIAamp DNA FFPE Tissue Kit (Cat. No. 56404). Obtained DNA

was measured with a UV spectrophotometer (Epoch Biotek,

Winooski, VT, USA) and prepared for the study. Various SNPs in

the gene region coding ROCK2 were investigated. Criteria for

the choice of SNPs used were: 1) relatively high minor allele

frequencies in Caucasians; 2) location within the exonic and

intronic sites that could potentially impact ROCK expression and

function; and 3) suitability for the Fluidigm dynamic array chip

designing, i.e. with no high G/C levels. Reference numbers of

SNPs for the ROCK2 gene were rs2290156 in intron 30, rs965665

in intron 3, rs10178332 in intron 3, rs2230774 (Thr431Asn) in

exon 10, rs2230774 (Thr431Ser) in exon 10, rs6755196 in intron

1, and rs726843 in intron 13. Polymorphisms were analyzed in

genomic DNA using the 96.96 Dynamic Array on the BioMark

HD system (Fluidigm, South San Francisco, CA, USA). Digital PCR

Analysis software (Fluidigm, South San Francisco, CA, USA) was

used to process the data after the reaction [21].

Gene Expression

Software Inc., San Diego, CA, USA). For comparisons of the

differences between mean values of two groups, the unpaired

Student t-test was used. The chi-square test for independence

and Fisher exact tests were used for calculation of the significance

of differences in genotype and allele frequencies. The Pearson

test was used to identify the correlations. The Mann-Whitney

U test was used to detect significant differences between

immunohistochemical scores and compare the gene expression

data between groups. All statistical tests and p-values were

two-sided, and p<0.05 was considered statistically significant.

Results

Demographic and clinical characteristics of MCL patients and

controls are outlined in Table 1. There were no statistically

significant differences between patients and control groups in

terms of sex and age distribution. Immunohistochemical study of

the lymph node tissues revealed that ROCK1 and ROCK2 staining

was more marked in the patient group (Figure 1). A widespread

stronger positivity for ROCK1 and ROCK2 staining was observed

in the cytoplasm of the lymph node cells from MCL patients.

The ROCK distribution displayed a similar pattern between

control and MCL sections. There were marked increases in

ROCK1 (1.72±1.08, p=0.0009) and ROCK2 (2.58±0.62, p<0.0001)

staining scores in the lymph nodes of the patient group when

compared to controls (1.07±0.66 for ROCK1 and 1.28±0.69 for

ROCK2; Figure 2). Correlations between the prognostic factors

and ROCK in MCL patients are shown in Table 2. It was found

that there were significant negative correlations between

number of drug therapies and ROCK1 and ROCK2 protein

expressions. However, positive correlation was found between

age and ROCK1 expression. We also noted a positive correlation

Ribonucleic acid (RNA) was extracted from formalin-fixed,

paraffin wax-embedded blocks using the High Pure RNA

Isolation Kit (Cat. No. 03 270 289 001, Roche Diagnostics,

Mannheim, Germany) as described by the manufacturer. The

obtained RNA was prepared for the study by being measured

with UV spectrophotometry. cDNA synthesis was performed

with the Transcriptor First Strand cDNA Synthesis Kit (Roche

Diagnostics, Mannheim, Germany) according to manufacturer’s

protocol. Gene expression analysis was then done using a

BioMark HD device (Fluidigm, South San Francisco, CA, USA)

that utilizes a fluorescent PCR method. Data were analyzed

using the 2-ΔCt method according to the following formula:

ΔC t =C tROCK -C tGAPDH , where C t =threshold cycle [22].


Data were expressed as mean ± standard deviation (SD) or

percentage unless otherwise indicated. Statistical analysis

was performed using GraphPad InStat version 3.05 (GraphPad

Figure 1. Histopathologic images of ROCK staining.

Immunohistochemical staining for lymph node tissues with

ROCK1 in control (a) and in mantle cell lymphoma patients (b),

and ROCK2 staining in control (c) and in mantle cell lymphoma

patients (d). Original magnification 200 x .

143



between ROCK1 and ROCK2 expressions in MCL patients (Table

2). No significant differences were found between MCL patients

and the control group in terms of 7 ROCK2 gene polymorphisms

(Table 3). There was a marked increase in ROCK1 gene expression

in the patient group when compared to controls (p=0.0215).

However, no significant change was observed in ROCK2 gene

expression (p=0.9194; Figure 3).

Discussion

This study provides the first evidence that ROCK1 and ROCK2

protein expressions and ROCK1 gene expression were increased

in MCL patients. However, no marked change in ROCK2 gene

expression was observed. There were also no significant

associations between ROCK2 gene polymorphisms and MCL

cases.

3.0

2.5

2.0

1,5

1.0

0.5

0.0

Figure 2. Comparison of the immunohistochemical scores for

lymph node ROCK1 and ROCK2 staining in healthy controls

(n=60, white bars) and in patients with mantle cell lymphoma

(n=60, black bars). Values are given as mean ± SEM. *p=0.0009

and p<0.0001 values were obtained for ROCK1 and ROCK2,

respectively.

Figure 3. Comparison of the lymph node ROCK1 and ROCK2 gene

messenger ribonucleic acid expressions in healthy controls (white

bars, n=41) and in patients with mantle cell lymphoma (black

bars, n=44). Values are given as mean ± SEM. *p=0.0215 and

p=0.9194 values were obtained for ROCK1 and ROCK2 gene,

respectively.

Information regarding underlying biology and pathogenesis

constantly increases, forming the basis of molecularly targeted

treatment approaches in MCL [23]. Increased protein expressions

of two ROCK isoforms have been found to be associated with

different types of cancer [15,24]. In the present study, elevation

Table 1. Demographic and clinical characteristics of the

study cases.

Cases with

MCL

(n=60)

Controls

(n=60)

p-value

Age (years) a 61.9±10.9 58.6±10.4 0.0910

Sex (n, %)

Male

Female

Stage b (n, %)

42 (70.0)

18 (30.0)

I-II 4 (6.6)

III-IV 56 (93.4)

Chemotherapy protocol (n, %)

R-CHOP

R-HIPERCVAD

R-CVP

R-CEOP

R-FCM

26 (68.4)

6 (15.7)

3 (7.89)

2 (5.26)

1 (2.63)

Average number of drug therapy (n)

R-CHOP

R-HIPERCVAD

R-CVP

R-CEOP

R-FCM

Response to treatment (n)

CR

PR

REFR

RLPS

Average survival (months)

Female

Male

Tissue sample (n, %)

Lymphadenopathy

Bone marrow

Stomach

Tonsil

Nasopharynx

Rectum

Oral mucosa

Orbital mass

5.5

3.2

5.3

3.5

1

15

7

12

20

22.8

21.7

30 (50.0)

13 (21.6)

6 (10.0)

4 (6.6)

3 (5.0)

2 (3.3)

1 (1.6)

1 (1.6)

37 (61.7)

23 (38.3) 0.4413

a Data are mean ± standard deviation. b Staging was carried out according to Ann

Arbor staging system. CR: Complete remission, PR: partial response, REFR: refractory,

RLPS: relapse, LAP: lymphadenopathy, R-CHOP: rituximab, cyclophosphamide,

hydroxydaunorubicin (doxorubicin/adriamycin), oncovin (vincristine), and prednisone,

R-CVP: rituximab, cyclophosphamide, vincristine, and prednisone, R-CEOP:

R-CHOP with etoposide substituted for doxorubicin, R-HIPERCVAD: rituximab,

cyclophosphamide, vincristine, adriamycin (doxorubicin), dexamethasone, alternating

with methotrexate and cytarabine, R-FCM: rituximab, fludarabine, cyclophosphamide,

methotrexate, MCL: mantle cell lymphoma.

144



Table 2. Significant correlations between the prognostic factors and Rho-kinase protein expressions in mantle cell lymphoma

patients.

Prognostic factors Correlation coefficient (r) Coefficient of determination (r 2 ) p-value

Age ↔ ROCK1 0.260 0.067 0.044

Number of drug therapy ↔ ROCK1 -0.394 0.155 0.026

Number of drug therapy ↔ ROCK2 -0.456 0.207 0.009

ROCK1 ↔ ROCK2 0.559 0.312 <0.0001

ROCK: Rho-kinase.

Table 3. Genotype and allele distributions of ROCK2 gene polymorphisms in patients and control groups.

Gene SNP Genotype/allele Control n* MCL patients n* p

ROCK2

rs2290156

GG/GC/CC

G/C

29/25/2

83/29

56 28/22/4

78/30

54 0.9698 + , 0.6719 ‡

0.8703

ROCK2

rs965665

CC/CG/GG

C/G

41/5/5

87/15

51 36/9/1

81/11

46 0.2586 + , 0.2185 ‡

0.6746

ROCK2

rs10178332

AA/AC/CCA/C 45/8/3

98/14

56 44/9/0

97/9

53 1.0000 + , 0.2432 ‡

0.3829

ROCK2

rs2230774 (Thr431Asn)

AA/AC/CC

A/C

15/30/10

60/50

55 14/28/12

56/52

54 1.0000 + , 0.7793 ‡

0.7927

ROCK2

rs2230774 (Thr431Ser)

GG/GC/CC

G/C

40/16/0

96/16

56 39/15/0

93/15

54 1.0000 +

1.0000

ROCK2

rs6755196

GG/GA/AA

G/A

34/17/3

86/23

54 36/16/0

88/16

52 0.9461 + , 0.2397 ‡

0.2940

ROCK2

rs726843

TT/TC/CC

T/C

16/28/12

60/52

56 13/26/15

52/56

54 0.8206 + , 0.5927 ‡

0.5032

*Numbers do not always add up to total numbers because of missing values in the BioMark dynamic array system.

+ Comparison between heterozygous genotype and homozygous wild-type genotype.

‡ Comparison between homozygous variant genotype and homozygous wild-type genotype. ROCK: Rho-kinase, MCL: mantle cell lymphoma.

of the ROCK1 protein expression in MCL patients may be due

to increase in the ROCK1 gene expression. However, we found

an increase in ROCK2 protein, but not gene, expression in MCL

patients, suggesting that other mechanisms are involved in the

ROCK2 protein expression. The underlying mechanism of this

observation is currently unknown, and it may require further

evaluation with other techniques. Lane et al. [25] investigated

the expressions of ROCK1 and ROCK2 in human breast cancer

and showed that expression of ROCK1, at both messenger RNA

(mRNA) and protein levels, is much higher in human breast

tumor tissue compared with normal tissue. Conversely, ROCK2

levels do not seem to vary significantly between normal and

tumor tissue, although a significant decrease was seen in

ROCK2 mRNA levels in patients who died from breast cancer

[25]. ROCK1 is also highly expressed in tumor tissues from

osteosarcoma patients [26]. High expression of ROCK2 protein

has been found to be associated with more aggressive behavior

in hepatocellular carcinomas [27]. Elevated ROCK2 protein

expression levels have also been reported in colon and bladder

cancers and are associated with shorter disease-free survival in

patients with bladder cancer [28,29]. Collectively, these data

may indicate that ROCK is a potential therapeutic target in MCL.

It is known that reactive oxygen species (ROS) can directly

act on the Rho/ROCK signaling pathway [30]. The RhoA/

ROCK pathway may also modulate ROS generation. ROCK is

documented to stimulate expression of NADPH oxidase and

consequent generation of ROS [31]. Continued oxidative stress

can lead to chronic inflammation, which in turn could mediate

cancer [32]. It has been shown that application of the specific

ROCK inhibitors produces suppression of tumor formation,

growth, and metastasis [33,34,35], while specific activation of

ROCK signaling has been shown to lead to increased tumor cell

dissemination and angiogenesis [36]. It was also reported that

ROCK inhibitors inhibited the growth of cancer cells and their

invasion, and increased their sensitivity to chemotherapeutics

[34,37,38]. Taken together, these findings imply that ROCK

inhibitors may be beneficial in targeted cancer treatment.

We have observed a marked positive correlation of ROCK1 protein

expression with age of the patients. However, no correlation

was found between ROCK1 and ROCK2 protein expressions

between overall and disease-free survival. These data may imply

that ROCK has no marked effect on survival in these patients. In

addition, there were significant negative correlations between

145



ROCK1 and ROCK2 expressions and number of drug therapies

in the present study. The underlying reason for this negative

correlation is not known, but these findings may suggest that

short duration of intensive chemotherapy may lead to increased

ROCK1 and ROCK2 expressions.

There are only limited numbers of published studies related to ROCK

polymorphisms in humans. A recent study demonstrated that ROCK2

gene polymorphisms are significantly associated with colorectal

cancer [39] or metastases of breast cancer [40]. However, we have

found no support for a role of the studied variants in the ROCK2

gene in risk of MCL in the present study. This may be due to the

differences in pathogenesis between different types of cancer as

well as the small number of cases in the present study.

Conclusion

In summary, our data strongly suggest that ROCK expressions

may contribute to the development of MCL. This study provides

novel insights into mechanisms of lymphomagenesis. Our

findings may provide an important insight into the future

development or use of potential therapeutic approaches, such

as ROCK inhibitors, for patients with MCL. The results of the

present study may also imply that upregulation of ROCK may

represent a prognostic factor in MCL, and ROCK may be a

potential target for MCL diagnosis and therapy. Further studies

are also required to verify these findings in a larger cohort.

Ethics


local ethics committee, Informed Consent: It was taken.


Surgical and Medical Practices: Didar Yanardağ Açık, Mehmet

Yılmaz, Zeynel A. Sayıner, Salih Subari; Concept: Didar Yanardağ

Açık, Mehmet Yılmaz; Design: Mehmet Yılmaz, Abdullah

T. Demiryürek; Data Collection or Processing: Didar Yanardağ

Açık, Mehmet Yılmaz, İbrahim Sarı, Serdar Öztuzcu, Zeynel

A. Sayıner, Salih Subari, Abdullah T. Demiryürek; Analysis or

Interpretation: Didar Yanardağ Açık, Mehmet Yılmaz; İbrahim

Sarı, Serdar Öztuzcu, Abdullah T. Demiryürek; Literature Search:

Didar Yanardağ Açık, Abdullah T. Demiryürek; Writing: Didar

Yanardağ Açık, Mehmet Yılmaz, Abdullah T. Demiryürek.




included.

Financial Disclosure: This study was funded by a project

(TF.12.38) from Gaziantep University, Turkey.

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147

RESEARCH ARTICLE

DOI: 10.4274/tjh.2015.0094


Prospective Audit of Blood Donor Selection Process in a Tertiary

Care Hospital of a Developing Country

Gelişmekte Olan Bir Ülkede Üçüncü Basamak Bir Hastanede Kan Bağışçı Seçim Sürecinin

İleriye Dönük Denetlenmesi

Naila Raza

Liaquat National Hospital & Medical College, Department of Hematology, Karachi, Pakistan

Abstract

Objective: The aim of this study was to emphasize the significance of

internal audits of the blood donor selection process and documentation

in a resource-limited country by assessing compliance with the

established protocols, and to identify weak areas in the process.

Materials and Methods: This audit reviewed the donor selection

process at the blood bank of Liaquat National Hospital & Medical

College, Karachi, over a 6-month period. Seven variables selected as

performance indicators were graded as very good (%90-100%), good

(80%-89%), satisfactory (70%-79%), or unacceptable (<70%). Blood

bank staff was asked for feedback and suggestions.

Results: Documentation of donor demographics was not within the

acceptable range (documentation rates of 65.14%), donor status

records were satisfactory (77.64%), and donor physical exam records

were graded as good (86.34%). Five performance indicators were

graded as very good (90%-100%).

Conclusion: The audit proved productive in identifying major causes

of irregularities in documentation and in making valuable suggestions

for their rectification.

Keywords: Medical audit, Transfusion medicine, Donor selection

Öz

Amaç: Bu çalışmanın amacı kaynakları kısıtlı bir ülkede kan donörü

seçimi sürecinin hastane içinde denetlenmesinin ve belgelenmesinin

önemini vurgulamak ve sürecin zayıf noktalarının tespitini yapmaktır.

Gereç ve Yöntemler: Bu denetleme ile 6 ay boyunca Liaquat

National Hospital & Medical College, Karachi Kan Bankası’nda donör

seçim süreci gözden geçirdi. Verimliliğini belirlemek için kullanılan 6

değişken şu şekilde derecelendirildi, çok iyi (%90-100), iyi (%80-89),

yeterli (%70-79) ve kabul edilemez (<%70). Kan bankası çalışanlarına

geri bildirimleri ve önerileri soruldu.

Bulgular: Donör bilgilerinin belgelenmesi kabul edilebilir düzeyde

değildi (%65,14), donörlerin durumunun kayıtları yeterliydi (%77,64),

donörlerin fizik muayene kayıtları iyi olarak derecelendirildi (%86,34).

Beş verimlilik belirteci çok iyi olarak derecelendirildi (%90-100).

Sonuç: Denetleme belgelendirme konusundaki düzensizliğin ana

sebeplerini belirlemede ve bunların düzeltilmesinde değerli önerilerde

bulunma konusunda verimli oldu.

Anahtar Sözcükler: Tıbbi denetleme, Transfüzyon tıbbı, Donör seçimi

Introduction

Documentation and record-keeping play integral roles in

transfusion medicine from every step of the vein-to-vein

chain of blood donation to the dispatch of blood components.

Regular medical audits are a part of quality assurance programs

in transfusion medicine and a means of continuous assessment

and improvement of existing systems. For conducting audits

of clinical laboratories, a written set of questions in the form

of a checklist is used, evaluation of which indicates whether

the laboratory is performing its procedures according to its

documented policies and standard operating procedures and

on time. Historically, audits done in blood banks were focused

on clinical uses of blood components to ensure appropriate

use, minimize wastage, and reduce the risk of transfusiontransmissible

diseases. Developing countries like Pakistan

depend heavily on non-remunerated blood donors as only 10%

of blood donations are collected from voluntary donors [1].

Donor deferrals based on pre-donation assessment and workup

acts as a deterrent for future donations, especially among

first-time donors [2]. The World Health Organization (WHO)

calls for a quality system to be put in place for blood donor

selection criteria, staff training, and documentation [3]. A donor

questionnaire is the key tool in donor selection for assessing

donor health and safety and in reducing the risk of transmission

Address for Correspondence/Yazışma Adresi: Naila RAZA, M.D.,

Liaquat National Hospital & Medical College, Department of Hematology, Karachi, Pakistan

Phone : 9221-34557897

E-mail : drnailarahman@yahoo.com

Received/Geliş tarihi: February 24, 2015

Accepted/Kabul tarihi: July 09, 2015

148


Raza N: Audit of Blood Donor Selection Process

of infections. Timely counseling with regular reminders can help

in re-recruiting short-term temporarily deferred donors back

into the donor pool. Prior to these efforts, we have to ensure

that donor screening records are properly maintained. There are

no published data on internal audits done on donor screening

processes in Pakistan.

The objective of our study is to assess compliance with the

established protocols for blood donor selection processes and

documentation, to identify weak areas in these processes, and

to recommend improvements in the system based on feedback

obtained from blood bank staff.


As a part of quality system improvement we planned a

prospective 6-month internal audit of the donor recruitment

process and documentation at the blood bank of a tertiary

care hospital in Karachi, Pakistan, from January to June 2014.

An audit plan was devised and checklists were prepared with

the help of a toolkit developed by the Directorate General of

Health Services, Dhaka WHO, July 2008 [4]. The audit involved

the review of premises and the donor selection process as per

checklists and scrutiny of donor records for documentation. The

audit plan and checklists are shown in Figure 1 and Table 1.

Donor records were grouped into group A (donors deferred

before donation), group B (donors rejected after donation:

seropositive cases), and group C (donors selected for donation:

seronegative cases). We selected documentation of 7 parameters

as performance indicators: donor demographics, donor status,

general physical exam, hemoglobin estimation, informed

consent, reason of deferral, and notification of seropositive

cases. For each group, performance was graded as very good,

good, satisfactory, or unacceptable by maintaining a high level

of scoring documentation rates of 90%-100%, 80%-89%, 70%-

79%, and <70%, respectively. Based on the results, feedback

was obtained from the blood bank staff responsible for

conducting interviews of donors to determine common causes

of nonconformance. A list of recommendations for appropriate

changes in the current system was designed and submitted to

the head of the blood transfusion services of the institute at the

end of this exercise.

the checklist were met. Equipment and materials were being

properly maintained as the department is ISO-9001:2000

certified. Exceptions were absence of privacy for asking

questions related to sexual behavior and lack of written

material for donor self-deferral. Staff members were qualified

and trained. Review of donor records from January 2014 to June

2014 showed that out of 10,041 prospective blood donors, 1027

donors belonged to group A, 496 to group B, and 8518 to group

C. Donor demographic records were inadequately maintained

(documentation rate: 65.14%) as donor identification card

numbers and area of residency were not always documented in

all 3 groups. This was followed by donor status (documentation

rate: 77.64%) and vital statistics (documentation rate: 86.34%)

in that order. See Supplement 1 for review of Total Donor

Screening Forms and Comparison of Documentation rate among

3 groups of Donor Selection Forms.

Among deferred donors, the reason of deferral was mentioned

in all cases but the donor notification rate was 89.51%. The

main reason cited for not documenting the identification

card number was not asking for it due to its nonavailability

at the time of donation, and the area of residency was missed

due to ignorance about the exact zonal divisions of the city.

Irregularities in documentation of donor’s vital statistics were

mainly due to bypassing the set standard operating procedures.

Discussion

This audit gave insight into the existing practices of the donor

selection process at our institute in particular and in developing

countries in general. Random checks were conducted to

evaluate the premises and processes using checklists and

direct observations. Although the overall performance and

documentations were good, some important issues were

highlighted. Lack of privacy for conducting donor interviews

was a concern identified in our study and mentioned by Kumar

The study was conducted after obtaining approval from the

institute’s ethics review committee.

Data analysis was performed using descriptive statistics.

Numerical data are shown as percentages.

Results

In this study, inspection of the donor area was found to be

satisfactory as almost all the prerequisites mentioned in

Figure 1. Audit plan.

149



Table 1. Checklists for donor selection.

Sections Checklist for Donor Room Available Not Available

1. Premises Separate √

Clean

√

Air conditioned

√

Airy

√

Well lit

√

Donor rest room

√

2. Equipment/Material Weighing machine √

Blood pressure and pulse monitor

√

Thermometer

√

Analyzer for hemoglobin estimation

√

Sterile alcohol swabs

√

Gloves

√

Band aid

√

Anticoagulant tubes

√

Disposible syringes

√

Waste bin

√

Blood bags

√

Blood bags sealer

√

Blood bags stripper

√

Blood bags shaker

√

3. Emergency Kit Oxygen cylinder with regulator and mask √

Inj. Adrenaline

√

Inj. Hydrocortisone

√

Inj. Pherimine maleate

√

Inj. Calcium gluconate

√

25% dextrose water √

5% dextrose water 500 mL √

Checklist for Donor Selection Process Yes No

1. Are written sops for donor selection process available? √

2. Are donor selection criteria defined? √

3. Is separate donor interview room available? √

4. Is educational material for self-assessment available? √

5. Is procedure explained to donor? √

6. Are full aseptic measures taken? √

7. Are instructions for postphlebotomy care and possible adverse reactions given? √

8. Is refreshment provided? √

et al. in a similar study from India [5]. Absence of proper

infrastructure and space limitations are common problems

faced by health centers in developing countries.

Review of donor records showed some nonconformance

in donor demographics including irregularities in donor

identification numbers and area of residency in all 3 groups.

The purpose of the former is donor traceability and that of the

latter is use for epidemiological data. Use of mobile numbers for

contacting donors has become the norm as it is much easier and

a quick method for donor notification that can safely replace

identification numbers. However, its documentation rate needs

to be 100%, especially in deferred donors with seropositive

status (group B); in our case, this rate was 94.50%. Area of

residency has also lost credibility as people lack awareness of

exact zonal locations due to formation of new localities and the

constant expansion of the city.

Our study showed a shortcoming as per documentation rate for

donor status (77%). Omission of data regarding donor status

150



Supplement 1. Comparison of documentation rate among 3 groups of donor selection forms and total donor screening forms

reviewed.

Audit of donor selection forms (January-June 2014).

Total Donor Screening Forms Reviewed 10,041

Complete forms 8213 (81.79%)

*Incomplete forms 1828 (18.20%)

Donors deferred before donation

1027

(Group A)



Donors deferred after donation

496

(seronegative) (Group B)



Donors selected for donation

8518

(seropositive) (Group C)



*Computerized national identity card number and/or area of residency excluded

Comparison of documentation rate among 3 groups of donor selection forms.

Performance Group A (n=1027) Group B (n=496) Group C (n=8518)

Indicators

Donor

demographics

Incomplete

Records n

Documentation

Rate %

Incomplete

Records n

Documentation

Rate %

Incomplete

Records n

Documentation

Rate %

ID card number 1027 00.00 496 00.00 8518 00.00 65.14

Area of residency 1027 00.00 496 00.00 8518 00.00

Qualification 75 92.60 2 99.59 274 96.78

Ethnicity 46 95.52 3 99.39 148 98.26

Resident status 30 97.07 15 96.97 20 99.76

Contact number 20 98.05 7 98.58 0 100

Donor status

Voluntary vs. 239 76.72 106 78.62 1909 77.58 77.64

replacement

Vital statistics

General physical 415 59.59 4 99.19 39 99.54 86.11

examination

Hb estimation 0 100 0 100 19 99.77 99.92

Informed consent 0 100 0 100 0 100 100

Deferral reason 0 100 0 100 0 100 100

Donor notification 0 100 52 89.15 N/A N/A 94.57

Cumulative %

can be overcome by using different-colored forms for voluntary

donors or by keeping separate registers. In this way, the focus

can be directed towards voluntary donors with regular reminders

for donations, thus facilitating the donor recruitment program.

Documentation rate of vital signs collectively was good (86%).

Documentation of the remaining 4 indicators was satisfactory.

Feedback from blood bank staff was obtained to determine

the most likely causes for omitted data. Failure to document

identification number and area of residency was due to a silent

understanding among staff about their triviality; hence, this

information was not being collected from donors. Donor status

was not noted mostly due to inattention and the incongruous

location of the question window in the proforma according to

the staff. Documentation rate of vital statistics was selectively

poor in group A (documentation rate: 59.59%). This was

attributed to bypassing of normal standard operating procedures

of conducting a physical exam first, followed by hemoglobin

estimation, by some new staff members due to ignorance of

151



the protocol. Notifying donors about the potential presence

of transfusion-transmissible disease is a major responsibility of

blood banks. In our study, the donor notification documentation

rate was good (94.5%). Failure to inform donors were due to

no response when called, wrong mobile numbers, and failure

to document the mobile number, in that order. Thus, instead

of identity card number, at least two contact phone numbers

should be noted to ensure a 100% donor notification record.

Conclusion and Recommendations

The donor selection process is a vital link in the chain of blood

collection, screening, and transfusion. A detailed audit of this

program showed certain gaps in the documentation process.

The following recommendations are made to minimize chances

of lacunae and further improve the system:

• Privacy must be provided for conducting donor interviews.

• Donor identity card number and area of residency may be

removed from the donor pro forma and replaced by two valid

contact phone numbers.

• Separate registers or color-coded forms can be used for

voluntary blood donors.

• Mini-audits of selected areas must be done apart from the

annual external audits to improve quality.

• Refresher courses for blood bank staff should be conducted

regularly.

• Introduction of electronic record-keeping in blood banks is

vital for easy data retrieval.

Acknowledgment

The author would like to thank the staff of the blood bank of

Liaquat National Hospital & Medical College for their assistance

and cooperation in conducting the audit of the department.

Ethics

Ethics Committee Approval: Ethical Review Committee, Liaquat

National Hospital, Institute for Postgraduate Medical Studies

and Health Sciences, Karachi, Pakistan (App No. 0181-2014

LNH-ERC). Informed Consent: It was taken.

Conflict of Interest: The author of this paper has no conflicts



included.

References

1. National AIDS Control Program, Ministry of Health. National Blood Policy &

Strategic Framework 2008-2012 for Blood Transfusion Services in Pakistan.

Islamabad, Pakistan, Government of Pakistan, 2007. Available online

at http://www.nacp.gov.pk/introduction/ National_Blood_policy_&_

strategic_framework-BT.pdf.

2. Custer B, Chinn A, Hirschler NV, Busch MP, Murphy EL. The consequences

of temporary deferral on future whole blood donation. Transfusion

2007;47:1514-1523.

3. World Health Organization. Blood Donor Selection Guidelines

on Assessing Donor Suitability for Blood Donation. Geneva,

Switzerland, WHO, 2012. Available online at http://apps.who.int/iris/

bitstream/10665/76724/1/9789241548519_eng.pdf?ua=1.

4. Musa SAJM, Hasan MA. Develop Toolkit for Monitoring and Quality

Assurance of Safe Blood Transfusion. Dhaka, Bangladesh, WHO, 2008.

5. Kumar A, Sharma S, Ingole N, Gangane N. An audit of blood bank services.

J Edu Health Promot 2014;3:11.

152

Brief REPORT

DOI: 10.4274/tjh.2015.0335


Regulatory T Cells in Patients with Idiopathic Thrombocytopenic

Purpura

İdiyopatik Trombositopenik Purpura Olgularında Düzenleyici T Hücreler

Alev Akyol Erikçi, Bülent Karagöz, Oğuz Bilgi

Gülhane Military Medical Academy, Haydarpaşa Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey

Abstract

Objective: Immune thrombocytopenic purpura (ITP) is an immunemediated

bleeding disorder in which platelets are opsonized

by autoantibodies and destroyed by an Fc receptor-mediated

phagocytosis by the reticuloendothelial system within the spleen.

Autoimmune processes are also considered in the pathogenesis of this

disorder. CD4+CD25+FoxP3+ regulatory T (Treg) cells and CD8+CD28-

Treg cells have roles in autoimmune diseases. We investigated these

regulatory cells in ITP patients.

Materials and Methods: We included 22 ITP patients and 16

age-matched healthy subjects. CD4+CD25+FoxP3+ Treg cells and

CD8+CD28- cells were investigated by three-color flow cytometry. The

ratios of these cell populations to total lymphocytes were calculated.

Statistical analysis was carried out with the Mann-Whitney U test.

Results: CD4+CD25+ Treg cells were 9.69±3.70% and 12.99±5.58%

in patients with ITP and controls, respectively. CD4+CD25 high FoxP3+

cells were 27.72±19.74% and 27.55±23.98% in ITP patients and

controls, respectively. The percentages of both of these cell types were

not statistically significant when compared to the control group.

Conclusion: We did not find any differences in ratios of

CD4+CD25+FoxP3+ Treg cells or CD8+CD28- T cells in lymphocytes

between patients and healthy subjects. We conclude that these

circulatory cells are not different in ITP, but further studies are needed

to explore the putative roles of these regulatory cells.

Keywords: Idiopathic thrombocytopenic purpura, Regulatory T cells

Öz

Amaç: İmmün trombositopenik purpura (İTP) trombositlerin

otoantikorlar tarafından opsonize edildiği ve retiküloendotelyal sistem

tarafından Fc reseptör aracılı fagositoz ile dalakta yıkıldığı immün

kaynaklı bir kanama bozukluğudur. Bu bozukluğun patogenezinde

otoimmün süreçler de sorumlu tutulmaktadır. CD4+CD25+Foxp3+

regulatuvar T (Treg) hücreleri ve CD8+CD28- Treg hücreler otoimmün

hastalıklarda rol oynamaktadır. Çalışmamızda İTP’li hastalarda bu

regülatuvar hücreleri araştırdık.

Gereç ve Yöntemler: İTP’li 22 hasta ile yaş uyumlu 16 sağlıklı birey

dahil edildi. CD4+CD25+Foxp3+ Treg hücreler ve CD8+CD28- hücreler

üç renkli akım sitometri ile çalışıldı. Bu hücre popülasyonunun tüm

lenfositlere oranı hesaplanmıştır. İstatiktiksel değerlendirmede Mann-

Whitney U testi kullanılmıştır.

Bulgular: CD4+CD25+ Treg hücreler İTP’de ve kontrol grubunda

%9,69±3,70 ve %12,99±5,58 saptandılar. CD4+CD25 yüksek

FoxP3+ hücreler ise İTP’de ve kontrol grubunda %27,72±19,74 ve

%27,55±23,9 olarak saptandı. Her iki hücre tipi de kontrol grubu ile

karşılaştırıldığında istatiktiksel olarak anlamlı bulunmamıştır.

Sonuç: Lenfositlerdeki CD4+CD25+Foxp3+ Treg hücreler ve

CD8+CD28- T hücrelerdeki oranlarında fark bulamadık. Biz

çalışmamızda İTP’de dolaşan regulatuvar hücrelerde fark bulamadık

ama daha geniş kapsamlı çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: İdiopatik trombositopenik purpura, Düzenleyici

T hücreleri

Introduction

Immune thrombocytopenia (ITP) is an autoimmune bleeding

disorder in association with increased platelet destruction and

impaired platelet production. It is mediated by IgG antiplatelet

autoantibodies in which the targets are platelet membrane

glycoproteins (GPs), such as GPIIb/IIIa and GPIb/IX. CD4+CD25+

regulatory T (Treg) cells and CD8+CD28- T lymphocytes have

major roles in self-tolerance. To maintain the immune tolerance

and to prevent autoimmune disease, CD4+CD25+FoxP3+

Treg cells, CD4+ T cells with high expression of CD25, and

transcription factor forkhead box P3 (FoxP3), also referred to

as FoxP3 regulatory T cells, play an important role. Treg cells

account for approximately 5% of circulating CD4+ T cells.

Decreased numbers of Treg cells have been reported in patients

with various autoimmune diseases, including ITP, rheumatoid

arthritis, and systemic lupus erythematosus [1,2,3,4,5].

Address for Correspondence/Yazışma Adresi: Alev AKYOL ERİKÇİ, M.D.,

Gülhane Military Medical Academy, Haydarpaşa Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey

Phone : +90 532 733 03 14

E-mail : aleverikci@yahoo.com



153

Akyol Erikçi A, et al: Regulatory T Cells in Patients with Idiopathic Thrombocytopenic Purpura


In the case of Treg deficiency, peripheral tolerance can fail,

leading to the development of autoimmunity. The purpose of

this study was to evaluate Treg cells in previously untreated

newly diagnosed ITP cases.


Flow Cytometry

Peripheral blood samples were obtained and studied while

still fresh. Flow cytometry was used to count CD4+CD25+

Treg cells and CD8+CD28- suppressive cells. Flow cytometry

was performed on a Becton Dickinson FACSCalibur. Data were

obtained and analyzed using CellQuest software.

Monoclonal Antibodies

Antihuman monoclonal antibodies conjugated with

fluorochromes and appropriate isotype controls were used:

fluorescein isothiocyanate (FITC) conjugated anti-CD28 (BD

Pharmingen Catalog No: 555728), anti-CD4 (Caltag Laboratories

Catalog No: MHCD0401), phycoerythrin-cyanine 5 (PC5)

conjugated anti-CD8 (eBioscience Catalog No: 15-0088), anti-

CD25 (BD Pharmingen Catalog No: 555433), and phycoerythrin

(PE) conjugated anti-FoxP3 (eBioscience Catalog No: 12-4776).

Cell Preparation and Surface Staining

Human peripheral blood mononuclear cells were isolated using

Histopaque (Sigma Catalog No: 1077) gradient centrifugation.

Aliquots of 100 µL were transferred to polypropylene test tubes

(12x75 mm; BD Bioscience Catalog No: 352052) and 20 µL

of conjugated monoclonal antibodies or isotype controls was

added to each tube. Flow cytometric analysis was performed by

BD FACSCalibur after the appropriate staining protocol.

FoxP3 Staining

CD4 and CD25 surface staining was carried out. The CD4+CD25

tube was then washed with cold PBS and resuspended, 1 mL of

freshly prepared fixation/permeabilization working solution was

added, and the tube was incubated at 4 °C for 30-60 min in the

dark and washed twice by adding 2 mL of 1X permeabilization

buffer. Next, 20 µL of PE conjugated antihuman FoxP3

antibody in 1X permeabilization buffer was added and the tube

was incubated at 4 °C for 30 min in the dark. Washing was

repeated twice with 2 mL of 1X permeabilization buffer. After

resuspension, analysis was performed by flow cytometry.

Analysis

CD8+CD28- cell percentages were evaluated using anti-

CD28/anti-CD8 double staining in lymphocyte-gated cells.

CD8+CD28- cells, CD8+CD28+ cells, and the ratio of these cells

were calculated.

Anti-CD4/anti-FoxP3/anti-CD25 triple staining was uved for

CD4+CD25+ Treg cell counts. CD4+CD25 high lymphocytes were

gated and then CD4+CD25 high FoxP3+ cells were calculated in

CD4/FoxP3 histograms.


Statistical analysis was performed using SPSS. The Mann-

Whitney U test was used to investigate immunological

parameters of ITP patients and for comparisons with data of

healthy subjects.

Results

We enrolled 22 previously untreated patients newly diagnosed

with ITP (19 males, 3 females) and 16 age-matched controls

(13 males, 3 females). All of the patients were admitted to our

outpatient clinic. Thrombocytopenia was newly detected and

they had received no previous treatment. The patients were

investigated for possible causes of thrombocytopenia. Viral

serology and other underlying autoimmune diseases were

screened. Demographic findings are illustrated in Table 1. We

performed bone marrow aspiration and biopsy in the relatively

elderly patients (patients numbers 5, 9, and 17). No pathological

findings such as dysplasia were detected. Findings were consistent

Table 1. Patients’ characteristics

Patient number Age Sex Platelets

(x10 9 /L)

1 22 Male 19

2 19 Male 34

3 21 Male 58

4 32 Male 46

5 51 Female 22

6 21 Male 18

7 22 Male 65

8 27 Male 42

9 61 Male 31

10 20 Male 52

11 21 Male 37

12 37 Male 16

13 22 Male 88

14 34 Male 49

15 21 Male 23

16 39 Male 76

17 64 Female 35

18 42 Female 79

19 21 Male 17

20 22 Male 29

21 19 Male 53

22 25 Male 27

154


Akyol Erikçi A, et al: Regulatory T Cells in Patients with Idiopathic Thrombocytopenic Purpura

with ITP, including normal or increased megakaryocytes.

CD4+CD25+ Treg cells and CD4+CD25 high FoxP3+ cells were

calculated in lymphocytes. CD4+CD25+ Treg cells were

9.69±3.70% and 12.99±5.58% in patients with ITP and controls,

respectively. CD4+CD25highFoxP3+ cells were 27.72±19.74%

and 27.55±23.98% in ITP patients and controls, respectively.

Both of these cell counts were not statistically different between

groups.

We also detected no statistically significant difference in

CD8+CD28- suppressor cells between ITP patients and controls

(12.50±9.40% and 11.77±4.64%, respectively).

Discussion

Treg cells suppress effector T cell activation, which leads

to induction of immune tolerance [6]. For this reason it is

assumed that failure of the regulatory T cell system may induce

autoimmunity [7,8,9].

There are increasing numbers of studies demonstrating that

decreased frequency of Treg cells has a role in ITP. Liu et al.

reported that the percentage of Treg cells was significantly

decreased in ITP patients with active disease in which no

remission was achieved [10]. Sakakura et al. reported variations

in Treg amounts according to platelet counts. In patients with

low platelet counts there was no reduction in the percentage

of Treg cells when compared to those with platelet counts over

100,000/µL [11]. In the study by Yu et al., defective circulating

CD25 Treg cells were detected in patients with chronic ITP [12].

However, there are also studies that failed to detect any

differences in Treg frequencies of patients with ITP compared to

healthy controls [13,14].

Similar to our results, Mazzucco et al. detected no significant

difference between Treg cell and platelet counts in patients

with ITP and the control group [15].

In our study we investigated previously untreated newly

diagnosed ITP patients. We detected no significant difference in

Treg cell frequencies in ITP patients and controls. We think that

further studies are needed to explore the putative roles of these

regulatory cells, especially in terms of long-term follow-ups and

response to treatments.

Ethics

Informed Consent: It was taken.


Concept: Alev Akyol Erikçi; Design: Alev Akyol Erikçi; Data

Collection or Processing: Bülent Karagöz, Oğuz Bilgi, Alev Akyol

Erikçi; Analysis or Interpretation: Bülent Karagöz, Literature

Search: Bülent Karagöz, Oğuz Bilgi, Alev Akyol Erikçi; Writing:

Alev Akyol Erikçi, Bülent Karagöz.




included.

Financial Disclosure: Contribution of the Turkish Society of

Hematology.

References

1. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med

2002;346:995-1008.

2. Semple JW, Freedman J. Increased antiplatelet T helper lymphocyte reactivity in

patients with autoimmune thrombocytopenia. Blood 1991;78:2619-2625.

3. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb-IIIa in immune

thrombocytopenic purpura: role in production of anti-platelet autoantibody. J

Clin Invest 1998;102:1393-1402.

4. Ogawara H, Handa H, Morita K, Hayakawa M, Kojima J, Amagai H, Tsumita Y,

Kaneko Y, Tsukamoto N, Nojima Y, Murakami H. High Th1/Th2 ratio in patients

with chronic idiopathic thrombocytopenic purpura. Eur J Haematol 2003;71:283-

288.

5. Semple JW, Milev Y, Cosgrave D, Mody M, Hornstein A, Blanchette V, Freedman

J. Differences in serum cytokine levels in acute and chronic autoimmune

thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet

T-cell reactivity. Blood 1996;87:4245-4254.

6. Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, Sakaguchi

S. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and

suppressive T cells as a key function of the thymus in maintaining immunologic

self-tolerance. J Immunol 1999;162:5317-5326.

7. Nugent DJ. Immune thrombocytopenic purpura of childhood. Hematology

2006;2006:97-103.

8. Cruvinel WM, Mesquita D Jr, Araujo JAP, Salmazi KC, Kallas EG, Andrade LEC.

Natural regulatory T cells in rheumatic diseases. Rev Bras Reumatol 2008;48:342-

355.

9. Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, Fehervari Z, Shimizu J, Takahashi

T, Nomura T. Foxp3+CD25 + CD4 + natural regulatory T cells in dominant selftolerance

and autoimmune disease. Immunol Rev 2006;212:8-27.

10. Liu B, Zhao H, Poon MC, Han Z, Gu D, Xu M, Jia H, Yang R, Han ZC. Abnormality

of CD4 + CD25 + regulatory T cells in idiopathic thrombocytopenic purpura. Eur J

Haematol 2007;78:139-143.

11. Sakakura M, Wada H, Tawara I, Nobori T, Sugiyama T, Sagawa N, Shiku H. Reduced

Cd4 + Cd25 + T cells in patients with idiopathic thrombocytopenic purpura. Thromb

Res 2007;120:187-193.

12. Yu J, Heck S, Patel V, Levan J, Yu Y, Bussel JB, Yazdanbakhsh K. Defective circulating

CD25 regulatory T cells in patients with chronic immune thrombocytopenic

purpura. Blood 2008;112:1325-1328.

13. Andersson PO, Stockelberg D, Jacobsson S, Wadenvik H. A transforming growth

factor-β1-mediated bystander immune suppression could be associated with

remission of chronic idiopathic thrombocytopenic purpura. Ann Hematol

2000;79:507-513.

14. Nishimoto T, Kuwana M. CD4+CD25+Foxp3+ regulatory T cells in the

pathophysiology of immune thrombocytopenia. Semin Hematol 2013;50(Suppl

1):S43-S49.

15. Mazzucco KL, Junior LM, Lemos NE, Wieck A, Pezzi A, Laureano AM, Amorin

B, Valim V, Silla L, Daudt LE, Marostica PJ. Assessment of regulatory T cells in

childhood immune thrombocytopenic purpura. ISRN Hematol 2013;2013:143687.

155

Brief REPORT

DOI: 10.4274/tjh.2015.0206


Serum Zinc Levels in Iron Deficient Women: A Case-Control Study

Demir Eksikliği Anemisi Olan Kadınlarda Serum Çinko Seviyesinin Değerlendirilmesi: Olgu

Kontrol Çalışması

Onur Özhan 1 , Neslihan Erdem 2 , İsmet Aydoğdu 3 , Ali Erkurt 4 , İrfan Kuku 4

1Çukurova Dr. Aşkım Tüfekçi State Hospital, Clinic of Endocrinology, Adana, Turkey

2Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey

3Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa, Turkey

4İnönü University Faculty of Medicine, Department of Hematology, Malatya, Turkey

Abstract

Since similar symptoms and findings can be seen in the deficiencies

of both iron and zinc, we aimed to evaluate the serum zinc levels of

women with iron deficiency anemia (IDA). This study was conducted

with women with iron deficiency and a healthy control group. When

serum zinc levels were compared, they were found to be lower in the

IDA group, which was statistically significant. With the help of these

studies, iron and zinc treatment instead of only iron replacement may

be considered in cases of iron deficiency.

Keywords: Iron, Zinc, Women, Iron deficiency, Anemia

Öz

Demir ve çinko eksikliği benzer belirti ve bulgularla giden hastalıklar

olması sebebiyle, demir eksikliği anemisi (DEA) olan kadınlarda serum

çinko düzeylerinin değerlendirilmesini planladık. Bu çalışma DEA’lı

kadınlar ile sağlıklı kontrol olarak alınan kadınlar üzerinde yapıldı.

Serum çinko düzeyleri karşılaştırıldığında, DEA grubunda istatistiksel

olarak düşük saptandı. Bu çalışmaların yardımıyla sadece demir değil,

demir ve çinkonun beraber tedavisi demir eksikliği olan olgularda

düşünülebilir.

Anahtar Sözcükler: Demir, Çinko, Kadın, Demir eksikliği, Anemi

Introduction

Iron deficiency anemia (IDA) is the most common anemia

around the world and a public health concern in developing

countries that still suffer from malnutrition problems [1,2].

Similar findings and symptoms affecting various systems in the

body may be found both in iron and zinc deficiency; moreover,

coexistence of these deficiencies can exaggerate the symptoms.

However, there are not enough studies about zinc levels in adult

anemic subjects. Therefore, we aimed to evaluate the serum zinc

levels of women with IDA and investigate whether serum zinc

levels in women with iron deficiency were low or not.


Thirty women between 18 and 60 years of age with iron

deficiency who had presented to our outpatient clinics were

enrolled as the patient group and a healthy group consisting

of 30 women with the same age range served as the control

group. The study was conducted in the İnönü University Faculty

of Medicine, Department of Internal Medicine. Women with

history of infection within 1 month, or with chronic diseases

were excluded.

Diagnosis criteria for iron deficiency were hemoglobin below

12 g/dL and serum ferritin level below 20 µg/dL [3]. The normal

values of serum zinc levels were between 70 and 120 µg/dL [4].

Iron and iron-binding capacity were measured by the calorimetric

method with an Olympus OSR6186 (Germany), whereas serum

ferritin levels were measured by nephelometric method with

a 33 Dade Behring (Germany). Complete blood count analysis

was performed with a Beckman Coulter LH 750 analyzer (USA).

Serum zinc levels were measured by atomic absorption method

with the PerkinElmer Analyst 800 (Germany).

Statistics

Results were given as ± standard deviation, and with 95% safety

and distribution. Statistical analysis was conducted with SPSS

and the independent sample t-test.

Results

Thirty healthy women and 30 women with iron deficiency were

included in this study. Ages of the women were between 18

and 60; mean age was 38.4±10.5 years in the IDA group and

39.8±12.5 years in the control group. There was no statistical

difference between the groups (p>0.05).

Address for Correspondence/Yazışma Adresi: Neslihan ERDEM, M.D.,

Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey

Phone : +90 555 729 88 22

E-mail : neslihnerdem@gmail.com


Accepted/Kabul tarihi: November 18, 2015

156


Özhan O, et al: Serum Zinc Levels in Iron Deficiency in Women

Mean hemoglobin level was 10.1±1.4 g/dL in the IDA group

and 14.1±0.5 g/dL in the control group. There was a statistically

significant difference between the groups (p<0.001).

Hematologic parameters of each group are given in Table 1.

Serum iron, ferritin, and transferrin saturation levels, which are

the parameters helping in the diagnosis of IDA, were higher

in the control group compared to the IDA group. There was a

statistically significant difference between the groups (p<0.001).

Iron levels and iron storage parameters are given in Table 2.

When the control and IDA groups were compared, serum zinc

levels were found to be decreased as serum iron levels decreased.

Minimum serum zinc level was 34 µg/dL while the maximum

was 84 µg/dL in the IDA group, and mean serum zinc level was

55.8±10.8.

In the IDA group, serum zinc level was in the normal range

(70-120 µg/dL) in only three of the patients. The remaining 27

patients’ serum zinc levels were below 70 µg/dL. On the other

hand, in the control group, only one individual had a serum

zinc level below 70 µg/dL, while the remaining subjects’ serum

zinc levels were in the normal range. There was a statistically

significant difference between the groups (p<0.0001). Serum

zinc levels of the IDA and control groups are compared in Table 2.

Discussion

Although trace elements are found in minimal quantities, they

have important roles in homeostasis. Two of the most important

trace elements are iron and zinc. IDA is still a serious problem

in Turkey and around the world [1,5,6]. Considering many

etiological factors like low socioeconomic status, malnutrition,

high-fiber diet, pica disorder, parasitic infections, and milk

allergies, it is not a surprise to see zinc deficiency and iron

deficiency at the same time [2,7,8].

Coexistence of iron and zinc deficiency has attracted the

interest of researchers and there have been many studies

about this subject. Furthermore, this situation had led to the

following question: Are there any interactions between these

two elements?

One of the reasons for iron deficiency occurring with zinc

deficiency, other than diet, is the increase in production of

Zn-protoporphyrin and usage of zinc instead of iron in the

protoporphyrin structure [9].

There are also other hypotheses that zinc deficiency can cause

iron deficiency. It has been shown that, in animal studies with

Table 2. Iron levels, zinc levels, and iron storage parameters

of the groups.

IDA group Control p-value

group

Serum iron level (µg/dL)

Distribution

Ferritin (µg/L)

Distribution

Transferrin saturation (%)

Distribution

21.4±11.3*

6-46**

8.01±1.6

2.1-8.4

5.6±3.04

1.6-12.2

99.7±14.9

72-134

96.9±19.02

60-141

32.5±4.2

25.5-43.2

p<0.001

p<0.001

p<0.001

Serum zinc level (µg/dL) 55.8±10.8 80.2±8.6 p<0.001

IDA: Iron deficiency anemia.

*Mean values are given as ± standard deviation.

**Minimum-maximum levels.

Table 1. Hematologic parameters of the iron deficiency anemia and control groups.

IDA group Control group p-value

Hb (g/dL)

Distribution

10.1±1.4*

6.5-12.0**

14.1±0.5

13.1-15.3

p<0.001

Hct (%)

Distribution

WBCs (/mm 3 )

Distribution

Platelets (/mm 3 )

Distribution

MCV (fL)

Distribution

MCH (pg)

Distribution

MCHC (g/dL)

Distribution

31.1±3.8

21.3-36.7

7019.6±2228.2

2200-14,400

318,994.3±98,236.04

94,000-522,000

70.2±6.5

55.9-80

23.1±2.9

15.8-27.8

32.3±1.4

28.3-36.0

42.1±1.6

39.6-45.0

6633.3±1305.5

4100-9000

293,566.7±52,937.6

180,000-369,000

87.1±2.7

80.8-92.8

29.2±1.01

32.8-34.4

33.4±0.4

32.8-34.4

p<0.001

p>0.05

p>0.05

p<0.001

p<0.001

p<0.001

IDA: Iron deficiency anemia, Hb: hemoglobin, WBC: white blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin

concentration.

*Mean values are given as ± standard deviation.

**Minimum-maximum levels.

157

Özhan O, et al: Serum Zinc Levels in Iron Deficiency in Women


mice and rats with zinc deficiency, bone marrow erythrocyte

progenitors and plasma erythropoietin levels are decreased

[10,11,12]. Furthermore, there are also hypotheses that zinc

deficiency can make the erythrocytes vulnerable to oxidative

stress, which can cause anemia [13,14].

In another study, serum zinc levels were measured in children

between 1 and 14 years of age with iron deficiency. The serum

zinc levels were lower in the IDA group than the control group

(p=0.017). There was a statistically significant difference

between the two groups, as in our study. As a result, it has been

suggested that serum zinc levels should be checked in children

with iron deficiency [15].

Serum zinc levels were studied in children with iron deficiency

in Ankara. When zinc deficiency was accepted as levels below

2 SDs of mean levels of the control group, among the 100

anemic patients 23 patients had zinc levels of less than 1 SD

and 19 patients had zinc levels of less than 2 SDs of the mean

levels. These results were compatible with our results. It was also

suggested that zinc deficiency should be evaluated in patients

with IDA, because of the similarity of the symptoms of these

two deficiencies [7].

In Arcasoy’s study in 1985, histopathological changes causing

iron and zinc deficiency in intestinal mucosa were reversed

with zinc treatment and the absorption of zinc and iron were

improved [2].

Because of the lack of studies regarding this subject in adult

women and the high prevalence of IDA in this age group, we

conducted a study with women between 18 and 60 years of age.

We tried to find out whether zinc deficiency coexists with iron

deficiency or not. Although the studies we have mentioned here

mostly involved children, our study has shown similar results.

A study conducted in Vietnam showed that underprivileged

women were at increased risk of insufficient micronutrient

intake due to poor diet quality [16]. The effects of

supplementation in children have also been studied. It was

found that supplementation with iron plus zinc improved serum

zinc and plasma ferritin [17].

Similar findings and symptoms affecting various systems in

the body may be found both in iron and zinc deficiencies;

therefore, the levels of zinc must be carefully evaluated in cases

of iron deficiency. More importantly, as iron deficiency is still

an existing problem in Turkey, further studies investigating the

interactions between these elements must be performed. We

suggest that serum zinc levels should be evaluated in adult

women with IDA, but further studies are needed to evaluate the

benefit of simultaneous zinc and iron treatment instead of only

iron treatment in this age group.

Ethic

This study is ethically approved by İnönü University’s Local

Ethics Committee.


Surgical and Medical Practices: Onur Özhan, İsmet Aydoğdu; Ali

Erkurt, İrfan Kuku; Concept: Onur Özhan, Neslihan Erdem, İsmet

Aydoğdu; Design: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu;

Data Collection or Processing: Onur Özhan, İsmet Aydoğdu;

Analysis or Interpretation: Onur Özhan, İsmet Aydoğdu;

Literature Search: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu;

Writing: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu.




included.

References

1. Berçem İ, İçağasıoğlu D, Cevit Ö, Ergür AT, Berçem G, Gültekin A, Sütçü İ.

The prevalence of iron deficiency and iron deficiency anemia in adolescents.

Türkiye Klinikleri Pediatri Dergisi 1999;8:15-20.

2. Arcasoy A. İnsan sağlığında çinkonun önemi. TÜBİTAK Bilim ve Teknik

Dergisi 1996;12:56 (in Turkish).

3. Türk Hematoloji Derneği. Ulusal Tedavi Kılavuzu 2011 Yetişkinde Demir

Eksikliği. Ankara, Turkey, Türk Hematoloji Derneği, 2011 (in Turkish).

4. Tomita H. Trace Elements in Clinical Medicine: Proceedings of the Second

Meeting of the International Society for Trace Element Research in Humans

(ISTERH). 28 August–1 September 1989, Tokyo, Japan.

5. Gülez P, Kayserili E, Tosun A, Eryılmaz N. Demir eksikliği anemisinde eritrosit

parametrelerinin karşılaştırılması. Klinik Bilimler ve Doktor 1998;4:875-877

(in Turkish).

6. Koç A, Erol Ö, Kösecik M, Vural H, Tatlı MM, Ataş A, Avcı Ş. Şanlıurfa ili 12-

16 yaş arası çocuklarda demir eksikliği araştırması. Klinik Bilimler ve Doktor

1997;3:871-873 (in Turkish).

7. Prasad AS. Zinc deficiency in women, infants and children. J Am Coll Nutr

1996;15:113-120.

8. Prasad AS. Biochemistry of Zinc. New York, NY, USA, Plenum, 1993.

9. Hastka J, Lassere JJ, Schwarzbeck, Hehlmann R. Central role of zinc

protoporphyrin in staging iron deficiency. Clin Chem 1994;40:768-773.

10. King LE, Fraker PJ. Zinc deficiency in mice alters myelopoiesis and

hematopoiesis. J Nutr 2002;132:3301-3307.

11. King LE, Frentzel JW, Mann JJ, Fraker PJ. Chronic zinc deficiency in mice

disrupted T cell lymphopoiesis and erythropoiesis while B cell lymphopoiesis

and myelopoiesis were maintained. J Am Coll Nutr 2005;24:494-502.

12. Konomi A, Yokoi K. Zinc deficiency decreases plasma erythropoietin

concentration in rats. Biol Trace Elem Res 2005;107:289-292.

13. Powell SR. The antioxidant properties of zinc. J Nutr 2000;130:

1447-1454.

14. O’Dell BL. Role of zinc in plasma membrane function. J Nutr 2000;130:1432-

1436.

15. Ece A, Uyanık BS, İşcan A, Ertan P, Yiğitoğlu MR. Increased serum copper

and decreased serum zinc levels in children with iron deficiency anemia.

Biol Trace Elem Res 1997;59:31-39.

16. Nguyen PH, Nguyen H, Gonzalez-Casanova I. Micronutrient intakes among

women of reproductive age in Vietnam. PLoS One 2014;9:e89504.

17. Fallahi E, Kimiagar M, Nazari A, Hasanvand MA, Seifi M. Effect of zinc and

iron supplementation on indicators of iron, zinc and vitamin A status of

primary school children. Pak J Biol Sci 2007;10:1088-1092.

158

CASE REPORT

DOI: 10.4274/tjh.2015.0238


Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal

Masses and Hematuria: A Case Report

Bilateral Renal Kitle ve Hematüri ile Prezente Olmuş Diffüz Büyük B Hücreli Lenfoma Olgusu

Şiyar Erdoğmuş 1 , Serkan Aktürk 1 , Zeynep Kendi Çelebi 1 , Saba Kiremitçi 2 , Gülşah Kaygusuz 2 , Namık Kemal Altınbaş 3 , Evren Üstüner 3 ,

Kenan Keven 1

1Ankara University Faculty of Medicine, Department of Nephrology, Ankara, Turkey

2Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey

3Ankara University Faculty of Medicine, Department of Radiology, Ankara, Turkey

Abstract

Renal involvement is most often seen in conjunction with multisystemic,

disseminated lymphoma either by direct extension from a retroperitoneal

mass or via hematogenous spread. Primary lymphoma of the kidney is

not a common entity and it is a controversial issue on account of the

absence of lymphatic tissues in the normal kidney. In this case report,

we describe a 19-year-old male with hematuria, acute kidney injury,

and bilateral renal masses due to massive lymphomatous infiltration of

the kidneys, which was diagnosed as diffuse large B-cell non-Hodgkin

lymphoma by Tru-Cut biopsy.

Keywords: Acute kidney injury, Hematuria, Lymphoma, Renal biopsy,

Renal masses

Öz

Lenfomada renal tutulum sıklıkla multisistemik olarak, retroperitoneal

kitlenin direkt komşuluğu yoluyla veya hematojen yayılım şeklinde

ortaya çıkar. Böbrekte lenfatik doku yokluğu nedeniyle primer böbrek

lenfoması nadir görülen ve tartışmalı bir durumdur. Bu olguda;

hematüri, akut böbrek hasarı ve böbreklerin masif lenfomatöz

infiltrasyonuna bağlı bilateral renal kitle ile prezente olmuş ve böbrek

biyopsisi ile diffüz büyük B hücreli non-Hodgkin lenfoma tanısı almış

19 yaşında erkek hasta sunulmaktadır.

Anahtar Sözcükler: Akut böbrek hasarı, Hematüri, Lenfoma, Renal

biyopsi, Renal kitle

Introduction

Primary renal lymphoma (PRL) is a very rare disease and a

controversial issue because the kidneys do not normally contain

lymphatic tissue [1,2,3,4,5]. In general, renal lymphoma is most

often seen along with dissemination of systemic disease and

clinically silent. Occasionally, patients present nonspecific

signs and symptoms as well flank pain, weight loss, fever,

hematuria, and palpable mass [6]. Acute renal failure due to

lymphomatous infiltration of the kidney has rarely been reported

[7,8,9,10,11,12,13,14,15]. In this case report, we describe a

19-year-old male who presented with painless hematuria, acute

kidney injury, and bilateral renal masses.

Case Presentation

A 19-year-old male patient was admitted to the Nephrology

Department of Ankara University Faculty of Medicine due to

painless hematuria, bilateral renal masses, and acute kidney

injury for further investigations. He was first evaluated at

another center for hematuria. There was one episode of

hematuria, which had subsided spontaneously. Abdominal

ultrasonography had revealed bilateral diffuse renal masses and

the patient was referred to our center for further examination.

On admission, there was no history of fever, weight loss, night

ssweats and any other health problem. The patient’s physical

examination findings were unremarkable. In particular, there

was no peripheral lymphadenopathy or hepatosplenomegaly.

Also the kidneys were not palpable. Laboratory tests revealed

white blood cell count of 7.7x10 9 /L, hemoglobin of 11.6 g/dL,

platelet count of 315x10 9 /L, serum blood urea nitrogen of 17

mg/dL, serum creatinine of 1.5 mg/dL (normal range: 0.5-0.9),

serum uric acid of 7.6 mg/dL (normal range: 2.4-5.7), serum

lactate dehydrogenase of 1042 U/L (normal range: 125-220), and

serum ferritin of 749 ng/mL (normal range: 11-307). Erythrocyte

sedimentation rate was 52 mm/h and C-reactive protein level

was 27.5 mg/L (normal: <3). Urinalysis showed density of 1010,

Address for Correspondence/Yazışma Adresi: Şiyar ERDOĞMUŞ, M.D.,

Ankara University Faculty of Medicine, Department of Nephrology, Ankara, Turkey

Phone : +90 312 508 21 68

E-mail : si.yar21@hotmail.com

Received/Geliş tarihi: June 12, 2015


159

Erdoğmuş Ş, et al: Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal Masses and Hematuria Turk J Hematol 2016;33:159-162

pH of 5.5, protein of 15 mg/dL, and glucose negative, and urine

microscopy showed 4 leukocytes and 3 erythrocytes per highpower

field. Viral serology tests were negative. C3 and C4 as

well as quantitative immunoglobulin levels were all within

normal limits with negative antinuclear antibody and antineutrophil

cytoplasmic antibody tests. Urinary ultrasonography

demonstrated bilaterally enlarged kidneys without obstruction

(right: 16x8 cm, left: 15.5x8 cm) and numerous solid hypoechoic

nodular cortical masses in both kidneys (largest of 6.5x5.5 cm

in size, numerous variably sized masses) with perirenal and

paraaortic multiple enlarged lymph nodes (largest <2.5 cm in

size).

Contrast-enhanced computerized tomography (CT) scanning of

the abdomen confirmed bilaterally enlarged kidneys, bilateral

variably sized multiple hypodense renal masses, and paraaortic,

paracaval multiple enlarged lymph nodes (Figure 1A). In addition,

the liver was slightly enlarged with normal parenchyma while

the size of the spleen and parenchyma was normal.

Thereafter, 18F-fluorodeoxyglucose positron emission

tomography-computed tomography ( 18 F-FDG PET-CT) was

performed for staging and its role in the differential diagnosis.

It was performed to examine the entire body, revealing

an abnormal accumulation in the thyroid gland, anterior

mediastinum, bilateral hilar, right parasternal lymph node, left

subdiaphragmatic lymph node, bilateral renal cortices, and

multiple abdominal paraaortic, paracaval, and aortocaval lymph

nodes (Figures 1B and 1C).

A percutaneous tru-cut biopsy of the kidney was performed and

histopathological examination showed extensive infiltration of

the renal parenchyma by atypical lymphoid cells (Figures 2A

and 2B). Immunohistochemical studies demonstrated positive

staining of the neoplastic cells for CD20, CD10, bcl-6 (Figures

2C, 2D, 2E, and 2G) and negative results for MUM1 (Figure 2F)

and Bcl-2. The ki-67 proliferation index of neoplastic cells was

80% (Figure 2H). EBER in situ hybridization was negative. A

diagnosis of diffuse large B-cell non-Hodgkin lymphoma (NHL)

was made. To exclude lymphoma involvement of the bone

marrow, the patient underwent bone marrow biopsy and there

was not bone marrow infiltration.

and skin [16,17]. The most common site of genitourinary

involvement is the kidney, usually in patients with intermediate

and high-grade B-cell type NHL or American Burkitt lymphoma.

Additionally, extranodal involvement of lymphoma is seen

in most patients at the time of diagnosis [18,19,20]. Primary

renal NHL is not a common clinical entity and it is a disputed

issue owing to the absence of lymphoid tissue in normal

kidneys. Malbrain et al. [8] suggested the use of some criteria

for the diagnosis of PRL. These include: 1) Renal failure as the

initial presentation, 2) Bilateral enlargement of the kidneys

without obstruction and other organ or nodal involvement, 3)

Diagnosis only made by renal biopsy, 4) Absence of other causes

Figure 1. Computed tomography with intravenous contrast

reveals enlargement of both kidneys with bilateral renal

masses and paraaortic, paracaval lymph nodes (arrows) (A);

18F-fluorodeoxyglucose positron emission tomographycomputed

tomography fusion images showed very intense

diffuse fluorodeoxyglucose uptake in bilaterally enlarged

kidneys (B); maximum intensity projection images of positron

emission tomography-computed tomography scan demonstrated

multifocal increased 18F-fluorodeoxyglucose uptake in the

thyroid, mediastinum, and kidneys (C).

Subsequently, the patient was transferred to the department of

hematology for treatment and chemotherapy regimen as well;

cyclophosphamide, adriamycin, vincristine, prednisolone, and

rituximab (CHOP+R) were started. After a cycle of chemotherapy,

the patient’s renal functions returned to normal.

Discussion and Review of the Literature

Extranodal spread of lymphoma often affects the gastrointestinal

tract, liver, central nervous system, genitourinary tract (e.g.,

kidney, testis, ovary), bone, bone marrow, lungs, breast, thyroid,

Figure 2. Atypical large lymphoid cells infiltrating the renal

interstitium (A, B) (H&E, 65 x , 830 x ), immunohistochemical CD20

(C, D) (37 x , 479 x ), CD10 (E) (506 x ), and BCL6 expression of the

neoplastic cells (G) (333 x ). MUM1 was negative (F) (397 x ). Ki67

immunostaining showed high proliferation index (H) (282 x ).

160


Erdoğmuş Ş, et al: Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal Masses and Hematuria

of renal failure, and 5) Rapid improvement of renal function

after radiotherapy or systemic chemotherapy. Our patient

presented with one episode of hematuria, which had subsided

spontaneously, and bilateral involvement of the kidneys. His

blood tests showed slight renal function impairment (serum

creatinine: 1.5 mg/dL). Furthermore, there were not any causes

of renal failure such as obstructive uropathy, hypercalcemia,

uric acid nephropathy, volume depletion, and nephrotoxic

drugs. The diagnosis of diffuse large B-cell NHL was established

by Tru-Cut biopsy of the kidney. After a cycle of chemotherapy,

his creatinine level returned to normal. The patient had massive

infiltration of the kidneys along with thyroid gland infiltration,

mediastinal involvement, and multiple enlarged lymph nodes in

different sites. Consequently, our patient fulfilled four of the

above criteria, and if we had used these criteria, we could not

have accepted the diagnosis of PRL.

A variety of benign and malignant masses can involve

the kidneys in a bilateral fashion. For example, metastatic

disease, lymphoproliferative disorders, adult polycystic kidney

disease, and angiomyolipoma are more commonly found in a

bilateral fashion compared with transitional cell carcinomas

or oncocytomas [21]. Several radiologic options exist for

the evaluation of renal masses, although CT scan is the most

common imaging modality used for the evaluation of renal

lymphoma. Usually, definitive diagnosis of renal masses is made

by renal biopsy. Urban and Fishman [22] reported that the most

commonly encountered pattern of involvement in patients

with renal lymphoma is multiple renal masses that are mostly

bilateral. Other patterns include renal invasion from contiguous

retroperitoneal tumors, perirenal masses, and diffuse renal

infiltration [18,22,23]. Our patient presented with bilateral renal

enlargement and renal masses in ultrasonography and CT scan.

The patient’s diagnosis was made by ultrasonography-guided

renal biopsy.

Whole-body imaging with 18 F-FDG PET-CT is obligatory to

assess the extent of disease by detecting unexpected extranodal

sites of disease or for exclusion of disease in the presence of

nonspecific extranodal CT findings [24,25]. In the present case,

in addition to the CT findings, involvement of the thyroid gland

and mediastinum was determined by 18 F-FDG PET-CT.

In conclusion, in this case, we present bilateral renal masses

due to massive lymphomatous infiltration of the kidneys, which

was diagnosed as diffuse large B-cell NHL by tru-cut biopsy.

The presence of extrarenal involvement in the thyroid gland and

mediastinal, hilar, subcarinal, and multiple abdominal lymph

nodes made the diagnosis of PRL debatable. Physicians should

be aware of the probability of lymphoma in the differential

diagnosis of renal masses.

Ethics



Concept: Kenan Keven, Şiyar Erdoğmuş; Design: Serkan Aktürk,

Zeynep Kendi Çelebi, Şiyar Erdoğmuş; Data Collection or

Processing: Evren Üstüner, Namık Kemal Altınbaş, Saba Kiremitçi,

Gülşah Kaygusuz, Şiyar Erdoğmuş; Analysis or Interpretation:

Şiyar Erdoğmuş; Literature Search: Şiyar Erdoğmuş; Writing:

Şiyar Erdoğmuş.




included.

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P. Acute renal failure due to bilateral lymphomatous infiltrates. Primary

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Ouzeddoun N. Acute renal failure due to malignant lymphoma infiltration.

Nephrol Ther 2010;6:602-605.

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162

LETTERS TO EDITOR


A Comparison of Healthy Infants and Adults with Respect to

Indirect Microparticle Activity and the Parameters of the Thrombin

Generation Test

Sağlıklı Süt Çocukları ve Erişkinlerin İndirekt Mikropartikül Aktivitesi ve Trombin Jenerasyon

Parametrelerine Göre Karşılaştırılması

Filiz Şimşek Orhon 1 , Nejat Akar 2 , Yonca Eğin 2 , Betül Ulukol 1 , Sevgi Başkan 1

1Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey

2Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Pediatric Molecular Genetics, Ankara, Turkey

To the Editor,

Microparticles express phospholipids and support thrombin

generation, which increases with age [1,2].

In a recently published study, we showed age-dependent

changes in thrombin generation parameters in a healthy infant

population aged 1-24 months [3]. The aim of this present study

was to compare the levels of both indirect microparticle activity

and thrombin generation parameters of healthy infants from our

recent study to those of a healthy adult population. The adult

population consisted of medical students of the Ankara University

School of Medicine. Blood was collected into tubes containing

1 mL of 0.109 M trisodium citrate. For indirect microparticle

activity, plasma samples were studied using the STA-PROCOAG-

PPL Kit (Diagnostica Stago Inc., Asnières sur Seine, France).

Plasma samples were measured using thrombin generation kits,

including a Thrombin Calibrator, PPP-Reagent 5 pM, and the

FluCa-Kit (Diagnostica Stago). Thrombin generation curves were

calculated using Thrombinoscope software (Thrombinoscope BV,

Maastricht, the Netherlands). The following parameters were

derived from the curves: lag time (LT, min), time to initiation

of thrombin generation; endogenous thrombin potential (ETP,

nmol/L/min), area under the thrombin generation curve; peak

thrombin activity (peak, nmol/L); and time to peak thrombin

generated (TTP, min). Statistical analysis was performed using

Statistical Package for the Social Sciences 16.

A total of 58 healthy adults (23 males and 35 females; mean

age: 23.2±0.4 years) were admitted to the study. In our recent

study, 85 healthy infants (51 males and 34 females; mean age:

12.6±8.3 months) were studied. The indirect microparticle

activity in the infant group was significantly lower than that

of the adult group (p<0.001). The ETP and peak levels in the

infant group were significantly lower than those of adults.

Furthermore, the TTP levels of the adult group were lower than

those of infants (p=0.001) (Table 1).

Physiologic concentrations of coagulation proteins gradually

increase after birth [4]. Karlaftis et al. showed that procoagulant

phospholipid activity was increased in neonates and decreased

in children aged 1-16 years [5]. We show that the levels of

indirect microparticle activity are increased in healthy adults

as compared to healthy infants. This may suggest that aging is

correlated to an increase in the indirect microparticle activity,

and also possibly to its procoagulant and proinflammatory

features.

Thrombin generation is influenced by different variables like age,

sex, body mass index, genetic factors, and acquired conditions

[6,7]. In a previous study, the ETP values of children were found

to be lower than those of adults [8]. Positive correlations

were found for age versus thrombin generation parameters

in calibrated automated thrombography in two recent studies

[9,10]. We showed that ETP and peak levels were higher in adults

as compared to infants. Thus, we suggest that ETP and peak

levels, the main parameters of thrombin generation, increase

gradually from infancy to adulthood. As for limitations, our

adult group was not adequate for representing all ages of the

adult population and there was a difference between the groups

Table 1. Data on indirect microparticle activity and thrombin

generation parameters of the study groups.

Healthy

Infants

(n=85)*

Healthy

Adults

(n=58)*

Microparticle release 31.7±7.5 39.7±9.8 0.001

time (s)

Lag time (min) 3.2±0.8 3.1±0.7 0.357

ETP (nmol/L/min) 1363.6±262.2 1691.5±378.1 0.001

Peak (nmol/L) 256.5±79.7 358.4±79.9 0.001

TTP (min) 6.7±1.7 5.4±0.9 0.001

*Values are presented as mean ± standard deviation, **t-test.

ETP: Endogenous thrombin potential, peak: peak thrombin activity, TTP: time to peak

thrombin generated.

p**

163

LETTERS TO EDITOR Turk J Hematol 2016;33:163-166

in terms of sex ratios. However, we may conclude that plasma

from adults may be more procoagulant than that of infants. Our

findings may confirm the presence of a regulation mechanism

in the coagulation parameters throughout the course of life.

Keywords: Infant, Adult, Microparticle, Thrombin

Anahtar Sözcükler: Süt çocuğu, Erişkin, Mikropartikül, Trombin


Study Conception and Design: Nejat Akar, Filiz Şimşek Orhon;

Acquisition and Blood Collection: Filiz Şimşek Orhon, Sevgi

Başkan; Laboratory Analysis: Yonca Eğin; Interpretation of Data:

Nejat Akar, Filiz Şimşek Orhon; Literature Search: Filiz Şimşek

Orhon, Betül Ulukol; Drafting and Writing: Filiz Şimşek Orhon.




included.

Financial Disclosure: This study was supported in part by the

Ankara University Research Fund.

References

1. Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui

A. Endothelial microparticles in diseases. Cell Tissue Res 2009;335:143-151.

2. Brummel-Ziedins KE, Everse SJ, Mann KG, Orfeo T. Modeling thrombin

generation: plasma composition based approach. J Thromb Thrombolysis

2014;37:32-44.

3. Orhon FS, Egin Y, Ulukol B, Baskan S, Akar N. Evaluation of indirect

microparticle activity and parameters of thrombin generation test in

healthy infants. Thromb Res 2014;133:281-284.

4. Kenet G, Krumpel A, Nowak-Gottl U. Bleeding issues in neonates, infants

and young children. Thromb Res 2009;123(Suppl 2):S35-S37.

5. Karlaftis V, Attard C, Summerhayes R, Monagle P, Ignjatovic V. The

microparticle-specific procoagulant phospholipid activity changes with

age. Int J Lab Hem 2014;36:e41-e43.

6. Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011;127(Suppl

3):S21-S25.

7. Butenas S, van’t Veer C, Mann KG. “Normal” thrombin generation. Blood

1999;94:2169-2178.

8. Haidl H, Cimenti C, Leschnik B, Zach D, Muntean W. Age-dependency

of thrombin generation measured by means of calibrated automated

thrombography (CAT). Thromb Haemost 2006;95:772-775.

9. Schneider T, Siegemund T, Siegemund R, Petros S. Thrombin generation

and rotational thromboelastometry in the healthy adult population.

Hamostaseologie 2015;35:181-186.

10. Wu J, Zhao HR, Zhang HY, Ge YL, Qiu S, Zhao J, Song Y, Zhao JZ, Lu SS.

Thrombin generation increasing with age and decreasing with use of

heparin indicated by calibrated automated thrombogram conducted in

Chinese. Biomed Environ Sci 2014;27:378-384.

Address for Correspondence/Yazışma Adresi: Filiz ŞİMŞEK ORHON, M.D.,

Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey

Phone : +90 312 595 72 02

E-mail : simsekfiliz@hotmail.com


Accepted/Kabul tarihi: December 11, 2015

DOI: 10.4274/tjh.2015.0341

Comment: In Response to “Downgraded Lymphoma: B-Chronic

Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell

Lymphoma - De Novo Occurrence or Transformation”

Yorum: Cevap Olarak “Geriletilmiş Lenfoma: Diffüz Büyük B-Hücreli Lenfoma Olduğu Bilinen

Bir Olguda B-Kronik Lenfositik Lösemi - De Novo Oluşum veya Dönüşüm”

Burak Uz, Kadir Acar

Gazi University Faculty of Medicine, Department of Internal Medicine, Division of Adult Hematology, Ankara, Turkey

To the Editor,

We read the letter submitted by Gajendra et al. with deep

interest [1]. The authors described a patient diagnosed with

diffuse large B-cell lymphoma (DLBCL) non-germinal center

B-cell type in 2002 who received 6 cycles of cyclophosphamide,

adriamycin, vincristine, and prednisolone (CHOP) followed by

radiotherapy. He was well for nearly 5 years, but subsequently

his disease locally relapsed. Unfortunately, a planned intensive

salvage regimen could not be given because the patient was lost

to follow-up. In 2010, despite not being given any treatment

modality, he presented with small lymphocytic lymphoma.

Finally, 22 months thereafter, he was diagnosed with Rai stage

IV chronic lymphocytic leukemia and 6 cycles of fludarabine,

164


LETTERS TO EDITOR

cyclophosphamide, and rituximab (FCR) were administered,

resulting in complete remission.

As is known, indolent or low-tumor-burden lymphomas may

transform into aggressive or high-tumor-burden lymphoma

forms in a process called “Richter transformation”. Although

rare, the reverse process may also occur with unknown

mechanisms. At this point, there are two main hypotheses that

can be suggested: initially, there are two existing malignant

clones, and successful eradication of the rapidly proliferating

clone with intensive therapy results in the survival of the less

rapidly growing clone, which may eventually lead to relapsed

disease even many years following the diagnosis [2]; or, less

probable, a separate secondary malignant clone that is distinct

from the initial clone might appear [3].

Previously, two downgraded lymphoma cases were reported

[2,3] after the successful treatment of underlying diffuse

non-Hodgkin lymphoma, 3 and 14 years following the initial

diagnosis. This well-described patient was accepted as having

late-relapsed (~5 years later) DLBCL, which transformed into a

“downgraded lymphoma” without lymphoma-specific therapy.

DLBCL patients generally relapse in the first 2 or 3 years

following treatment. The largest series of patients with DLBCL

who relapsed ≥5 years following diagnosis was reported by a

French group [4]; 3.6% of their cohort had a late relapse and

those patients had some distinct clinical features, including

localized disease (63%), favorable International Prognostic Index

score (82%), and extranodal involvement (65%) at diagnosis. At

the time of relapse, 83% had DLBCL histology, while 17% had

indolent histology. Additionally, having an indolent component

at diagnosis (44.4%) was significantly associated with indolent

histology at relapse. However, nearly all the late-relapsed

patients with initial good-risk disease were treated adequately

with anthracycline-based combined chemotherapy.

Late-relapsed DLBCL patients have poor outcomes; therefore,

they must be treated promptly with rituximab plus chemotherapy

or (if possible) autologous stem cell transplantation [4]. In

the French experience, all late-relapsed patients were heavily

treated and the patients experienced their relapse a median of

7.4 years after diagnosis [4]. As an interesting aside, the present

patient could not be administered any treatment for 3 years

after the confirmation of DLBCL relapse and he presented

with downgraded lymphoma. We could not understand why

the patient’s relapsed high-grade lymphoma resolved without

any treatment attempts. Spontaneous remission of DLBCL is

exceedingly rare, with only a handful of such cases reported

[5,6,7,8]. Given the unexplained clinical course of DLBCL in this

patient, a probable infectious agent or nonprescription usage

of traditional medicinal plants inducing antitumor response by

modulating the immune system against lymphomatous cells

should be sought in his medical history.

Keywords: Diffuse large B-cell lymphoma, Downgraded

lymphoma

Anahtar Sözcükler: Diffüz büyük B-hücreli lenfoma, Geriletilmiş

lenfoma


Concept: Burak Uz; Design: Burak Uz; Data Collection or

Processing: Burak Uz, Kadir Acar; Analysis or Interpretation:

Burak Uz, Kadir Acar; Literature Search: Burak Uz, Kadir Acar;

Writing: Burak Uz.




included.

References

1. Gajendra S, Jha B, Goel S, Sahni T, Dorwal P, Sachdev R. Downgraded

lymphoma: B-chronic lymphocytic leukemia in a known case of diffuse

large B-cell lymphoma-de novo occurrence or transformation. Turk J

Hematol 2015;32:371-372.

2. Kerrigan DP, Foucar K, Dressler L. High-grade non-Hodgkin lymphoma

relapsing as low-grade follicular lymphoma: so-called downgraded

lymphoma. Am J Hematol 1989;30:36-41.

3. Ogata Y, Setoguchi M, Tahara T, Takahashi M. Downgraded non-Hodgkin’s

lymphoma in the neck occurring as a secondary malignancy. ORL J

Otorhinolaryngol Relat Spec 1998;60:295-300.

4. Larouche JF, Berger F, Chassagne-Clement C, Efrench M, Callet-Bauchu

E, Sebban C, Ghesquieres H, Broussais-Guillaumot F, Salles G, Coiffier B.

Lymphoma recurrence 5 years or later following diffuse large B-cell

lymphoma: clinical characteristics and outcome. J Clin Oncol 2010;28:2094-

2100.

5. Mizuno T, Ishigaki M, Nakajima K, Matsue T, Fukushima M, Minato H, Nojima

N, Atsushi S, Ishigami K, Atsumi H, Ito T, Iguchi M, Usuda D, Okamura H,

Urashima S, Asano M, Fukuda A, Izumi Y, Takekoshi N, Kanda T. Spontaneous

remission of Epstein-Barr virus-positive diffuse large B-cell lymphoma of

the elderly. Case Rep Oncol 2013;6:269-274.

6. Buckner TW, Dunphy C, Fedoriw YD, van Deventer HW, Foster MC,

Richards KL, Park SI. Complete spontaneous remission of diffuse large

B-cell lymphoma of the maxillary sinus after concurrent infections. Clin

Lymphoma Myeloma Leuk 2012;12:455-458.

7. Tamás L, Sári E, Répássy G, Szabó P, Bagdi E, Krenács L, Demeter J.

Spontaneous remission in localized diffuse large B-cell lymphoma. Pathol

Oncol Res 2011;17:779-784.

8. Watari J, Saitoh Y, Fujiya M, Nakamura K, Inaba Y, Okamoto K, Tanabe H,

Yasuda A, Miyokawa N, Kohgo Y. Spontaneous remission of primary diffuse

large B-cell gastric lymphoma. J Gastroenterol 2005;40:414-420.

Address for Correspondence/Yazışma Adresi: Burak UZ, M.D.,

Gazi University Faculty of Medicine, Department of Internal Medicine, Division of Adult Hematology, Ankara, Turkey

Phone : +90 312 202 55 79

E-mail : burakuz78@gmail.com; burakuz@yahoo.com

Received/Geliş tarihi: December 30, 2015


DOI: 10.4274/tjh.2015.0452

165

LETTERS TO EDITOR Turk J Hematol 2016;33:163-166

Tumor Necrosis Factor and Splenectomy

Tümör Nekrozis Faktör ve Splenektomi

İrfan Yavaşoğlu

Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey

To the Editor,

The article entitled “Effect of Tumor Necrosis Factor-Alpha

(TNF-α) on Erythropoietin- and Erythropoietin Receptor-

Induced Erythroid Progenitor Cell Proliferation in β-Thalassemia/

Hemoglobin E Patients”, written by Tanyong et al. [1] and

published in a recent issue of your journal, was quite interesting.

Here we would like to emphasize some relevant points.

Splenectomy can increase the release of TNF-α and cell apoptosis

in experimental and clinical studies in different diseases [2,3,4].

Increased serum TNF-α was reported in E/b-Thal patients,

particularly after splenectomy [3,4]. In sickle cell disease

presenting with functional asplenia, increased amounts of

TNF-α, indicative of monocyte activation, and increased serum

C-reactive protein levels were reported [5].

Banyatsuppasin et al. suggested the role of the spleen in

controlling mononuclear phagocytic activity in E/b-Thal patients

[6]. TNF-α play roles as an inducer and effector of monocyte

activation [6]. Additionally, TNF-α returned to normal after 12, 6,

and 3 months of deferiprone treatment [7]. Therefore, chelation

treatment can affect apoptosis independently of splenectomy. It

might be important to know the effect of chelation treatment and

splenectomy on tumor necrosis factor in the study of Tanyong et

al. [1] based on all these investigations stated above [2,3,4,5,6,7].

Keywords: Thalassemia, Tumor necrosis factor, Splenectomy

Anahtar Sözcükler: Talasemi, Tümör nekrozis faktör, Splenektomi

Conflict of Interest: The author of this paper has no conflicts



included.

References

1. Tanyong D, Panichob P, Kheansaard W, Fucharoen S. Effect of tumor necrosis

factor-alpha on erythropoietin- and erythropoietin receptor-induced

erythroid progenitor cell proliferation in β-thalassemia/hemoglobin E

patients. Turk J Hematol 2015;32:304-310.

2. Hiroyoshi T, Tsuchida M, Uchiyama K, Fujikawa K, Komatsu T, Kanaoka

Y, Matsuyama H. Splenectomy protects the kidneys against ischemic

reperfusion injury in the rat. Transpl Immunol 2012;27:8-11.

3. Chuncharunee S, Archararit N, Hathirat P, Udomsubpayakul U, Atichartakarn

V. Levels of serum interleukin-6 and tumor necrosis factor-a in

postsplenectomized thalassemic patients. J Med Assoc Thai 1997;80(Suppl

1):S86-S91.

4. Wanachiwanawin W, Wiener E, Siripanyaphinyo U, Chinprasertsuk S, Mawas

F, Fucharoen S, Wickramasinghe S, Pootrakul P, Visudhiphan S. Serum

levels of tumor necrosis factor-α, interleukin-1, and interferon-γ in β0-

thalassemia/HbE and their clinical significance. J Interferon Cytokine Res

1999;19:105-111.

5. Belcher JD, Marker PH, Weber JP, Hebbel RP. Activated monocytes in sickle

cell disease: potential role in the activation of vascular endothelium and

vaso-occlusion. Blood 2000;96:2451-2459.

6. Banyatsuppasin W, Butthep P, Atichartakarn V, Thakkinstian A, Archararit N,

Pattanapanyasat K, Chuncharunee S. Activation of mononuclear phagocytes

and its relationship to asplenia and phosphatidylserine exposing red blood

cells in hemoglobin E/β-thalassemia patients. Am J Hematol 2011;86:89-92.

7. Del Vecchio GC, Schettini F, Piacente L, De Santis A, Giordano P, De Mattia

D. Effects of deferiprone on immune status and cytokine pattern in

thalassaemia major. Acta Haematol 2002;108:144-149.

Address for Correspondence/Yazışma Adresi: İrfan YAVAŞOĞLU, M.D.,

Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey

Phone : +90 256 212 00 20

E-mail : dr_yavas@yahoo.com

Received/Geliş tarihi: January 23, 2016


DOI: 10.4274/tjh.2016.0040

166

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.2015.0275

Turk J Hematol 2016;33:167

Auer Rod in a Neutrophil in a Nonmalignant Condition

Malign Olmayan Bir Durumda Nötrofilde Görülen Auer Cisimciği

Harish Chandra, Smita Chandra, Vibha Gupta, Divyaa Mahajan

Himalayan Institute of Medical Sciences, Department of Pathology, Dehradun, India

Figure 1. Neutrophil shows an Auer rod at 100 x (Jenner-Giemsa stain), 270x203

mm (72x72 dpi).

Auer rods are normally observed in immature myeloid precursors

including myeloblasts and promyelocytes in cases of acute

myeloid leukemia, while cases have rarely reported Auer rods in

polymorphs in acute myeloid leukemia [1,2].

A 19-year-old female presented with high-grade fever and

abdominal pain for 1 week. Her laboratory investigations

revealed hemoglobin of 65 g/L, red blood cell count of

3.3x1012/L, mean cell volume of 96.2 fL, white blood cell

count of 8.5x109/L, and platelet count of 23x10 9 /L. She was

found to be positive for Salmonella Typhi antigen O in 1:160

dilutions (slide agglutination by Beacon Diagnostics, India). An

interesting finding was observed during her peripheral blood

examination, which showed an Auer rod-like structure within

the cytoplasm of a neutrophil, along with features

of dysmyelopoiesis (Figure 1). Bone marrow

aspiration was done, which was unremarkable and

showed normoblastic maturation.

To the best of our knowledge, no case has been

reported in the literature with Auer rods in a

nonmalignant condition. Therefore, the present

case is being reported, which shows an Auer rod in

a polymorph in a case of typhoid fever.

Keywords: Auer rods, Neutrophil, Typhoid fever

Anahtar Sözcükler: Auer cisimciği, Nötrofil, Tifo

Ethics



Concept: Harish Chandra, Smita Chandra; Design:

Harish Chandra, Smita Chandra; Data Collection or

Processing: Vibha Gupta, Divyaa Mahajan; Analysis

or Interpretation: Harish Chandra, Smita Chandra,

Vibha Gupta; Literature Search: Harish Chandra, Smita Chandra,

Vibha Gupta, Divyaa Mahajan; Writing: Harish Chandra, Smita

Chandra.




included.

References

1. Dawson MA, Whitehead S. Mature neutrophils with multiple Auer rods:

a rarity in normal karyotype acute myeloid leukaemia. Br J Haematol

2007;137:86.

2. Dmitrienko S, Vercauteren S. Auer rods in mature granulocytes of a patient

with mixed lineage leukemia. Blood 2012;119:4348.

Address for Correspondence/Yazışma Adresi: Harish CHANDRA, M.D.,

Himalayan Institute of Medical Sciences,

Department of Pathology, Dehradun, India

E-mail : drharishbudakoti31@yahoo.co.in

Received/Geliş tarihi: July 24, 2015


167


DOI: 10.4274/tjh.2015.0294


Precursor B-Cell Lymphoblastic Lymphoma Presenting as a

Spinal Mass at Initial Diagnosis

İlk Tanı Sırasında Spinal Kitle ile Prezente olan Prekürsör B-Hücreli Lenfoblastik Lenfoma

Oğuzhan Erol 1 , Çiğdem Tokyol 1 , Feyzullah Akyüz 2 , Nuran Ahu Baysal 3 , Mehmet Sezgin Pepeler 4

1Afyon Kocatepe University Faculty of Medicine, Department of Pathology, Afyonkarahisar, Turkey

2Park Hospital, Clinic of Neurosurgery, Afyonkarahisar, Turkey

3Afyonkarahisar Public Hospital, Clinic of Hematology, Afyonkarahisar, Turkey

4Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Figure 1. Lymphoid cells with irregular nuclei, dispersed nuclear

chromatin, prominent nucleoli, and scant cytoplasm (H&E, 400 x ).

Figure 2. Diffuse expression of TdT in tumor cells (200 x ).

An 18-year-old male presented to the emergency department

of our hospital with complaints of bilateral leg numbness and

weakness since about a month. Magnetic resonance imaging

of the spine revealed an extramedullary extradural mass at

the T9-T11 level causing marked spinal cord compression.

Emergent surgery was performed. An epidural mass was

seen after laminectomy and partially removed. Microscopic

examination showed a diffuse infiltration of small- to

medium-sized lymphoid cells with irregular nuclei, dispersed

nuclear chromatin, prominent nucleoli, and scant cytoplasm in

adipose tissue (Figure 1). Immunohistochemical examination

demonstrated that tumor cells stained positively for TdT, CD34,

CD10, CD20, CD79a, Pax-5, CD45, and Bcl-2 (Figure 2). Ki-67

showed immunoreactivity of 80% of tumor cells. Bone marrow

and blood involvements were not detected. These findings led us

to the diagnosis of precursor B-cell lymphoblastic lymphoma. He

was given combination chemotherapy of R-HCVAD (rituximab,

cyclophosphamide, vincristine, doxorubicin, dexamethasone,

cytarabine, mesna, methotrexate). After the second dose of

chemotherapy, complete response was achieved as assessed by

positron emission tomography/computed tomography scan.

The spinal cord is an extremely rare initial site of involvement

for B-cell lymphoblastic lymphoma. To our knowledge, there are

only 3 reported cases in the English literature (Table 1) [1,2,3].

Address for Correspondence/Yazışma Adresi: Çiğdem TOKYOL, M.D.,

Afyon Kocatepe University Faculty of Medicine, Department of Pathology, Afyonkarahisar, Turkey

Phone : +90 272 246 33 04

E-mail : ctokyol@yahoo.com

Received/Geliş tarihi: August 12, 2015


168


Erol O, et al: Spinal Lymphoblastic Lymphoma

Table 1. Cases of isolated primary B-cell lymphoblastic lymphoma of the spine.

Reference Age Location Sex First Manifestations Treatment

Khalid et al. [1] 58 Thoracic spine Female Back pain and numbness S+CT

Esin et al. [2] 29 Thoracic spine Female Acute walking difficulty in pregnancy S+CT

Park et al. [3] 27 Thoracolumbar spine Male Back pain S+CT+RT

Present case 18 Thoracic spine Male Bilateral leg numbness and weakness S+CT

S: Surgery, CT: chemotherapy, RT: radiotherapy.

Lymphoblastic lymphoma should be included in the differential

diagnosis of spinal masses.

Acknowledgment

Presented at the 25th National Congress of Pathology 2015,

Bursa, Turkey.

Keywords: B-cell lymphoblastic lymphoma, Thoracic spine,

Spinal cord compression

Anahtar Sözcükler: B-hücreli lenfoblastik lenfoma, Torasik

vertebra, Spinal kord basısı


Surgical and Medical Practices: Feyzullah Akyüz, Nuran Ahu

Baysal, Mehmet Sezgin Pepeler; Concept: Çiğdem Tokyol;

Design: Çiğdem Tokyol; Data Collection or Processing: Oğuzhan

Erol; Analysis or Interpretation: Çiğdem Tokyol; Literature

Search: Oğuzhan Erol; Writing: Oğuzhan Erol.




included.

References

1. Khalid I, Rival J, Salama ME, Banghar PK, Janakiraman N. Unusual

presentations of hematologic malignancies: Case 2. Precursor B-cell

lymphoblastic lymphoma presenting as spinal cord compression. J Clin

Oncol 2004;22:1331-1333.

2. Esin S, Tarim E, Abali H, Kardes O, Kocer EN, Alkan O. Management

of precursor B-lymphoblastic lymphoma/leukaemia of thoracic spine

in a pregnancy presenting with acute paraplegia. J Obstet Gynaecol

2012;32:485-486.

3. Park DA, Park SG, Kim SW. Solitary lymphoblastic lymphoma of the thoracic

spine. J Korean Neurosurg Soc 2012;52:564-566.

169


DOI: 10.4274/tjh.2014.0470


Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Should

Be Kept in Mind in Children with Febrile Neutropenia, Oral Cavity

Lesions, and Skin Rash

Febril Nötropeni, Oral Kavitede Lezyonlar ve Deri Döküntüsü Olan Çocuklarda Stevens-

Johnson Sendromu/Toksik Epidermal Nekrolizis Akılda Tutulmalıdır

Eda Ataseven, Şebnem Yılmaz Bengoa, Hale Ören

Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Figure 1. Erosions and crusts on the lips and hemorrhagic ulcers

in the oral cavity. A red papular rash spread to the shoulders and

the back.

Figure 2. Regression of the lesions on the lips, oral cavity, and

back.

A 14-year-old boy was diagnosed with acute lymphoblastic

leukemia. Febrile neutropenia developed during induction.

Imipenem and teicoplanin were started because of severe

mucositis. Viral tests and bacterial cultures were unrevealing. On

follow-up, a painful papular rash had appeared and oral mucositis

had become worse (Figure 1). Stevens-Johnson syndrome (SJS)/

toxic epidermal necrolysis (TEN) was suspected. Intravenous

immunoglobulin (IVIG) at 1 g/kg/day and methylprednisolone

at 1 mg/kg/day were started. The lesions regressed in 1 week

(Figure 2). Skin biopsy was consistent with SJS/TEN. Informed

consent was obtained.

SJS and TEN are rare diseases characterized by fever and mucosal

and cutaneous lesions [1]. It is defined as SJS when epidermal

involvement affects less than 10% of the body surface area, as

SJS/TEN overlap when the skin detachment ranges from 10%

to 30%, and as TEN when it involves more than 30% [1,2]. It

may occur after taking a new medication or may rarely have an

infectious origin. Our patient had no predisposing conditions

other than taking chemotherapeutic drugs and antibiotics. The

mortality rate is high in SJS/TEN [1,3]. Rapid withdrawal of

the probable causative drug(s) is important. Use of IVIGs and

corticosteroids is reported as the most commonly used therapy

in childhood [1,4]. Systematic review of adult treatments for SJS

and TEN did not show any benefit of these agents on mortality

rates [3]. Cyclosporine, plasmapheresis, and tumor necrosis

factor-alpha inhibitors have been also reported among other

treatment options [1,2,3,4,5].

Keywords: Acute leukemia, Stevens-Johnson syndrome, Toxic

epidermal necrolysis

Anahtar Sözcükler: Akut lösemi, Stevens-Johnson sendromu,

Toksik epidermal nekrolizis

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 61 41

E-mail : hale.oren@deu.edu.tr

Received/Geliş tarihi: December 08, 2014


170


Ataseven E, et al: Stevens-Johnson/Toxic Epidermal Necrolysis


Concept: Hale Ören, Eda Ataseven; Design: Hale Ören, Eda

Ataseven; Data Collection: Şebnem Yılmaz Bengoa, Eda Ataseven,

Analysis or Interpretation: Hale Ören, Eda Ataseven; Literature

Search: Şebnem Yılmaz Bengoa, Eda Ataseven; Writing: Hale

Ören, Eda Ataseven.




included.

References

1. Ferrandiz-Pulido C, Garcia-Patos V. A review of causes of Stevens-Johnson

syndrome and toxic epidermal necrolysis in children. Arch Dis Child

2013;98:998-1003.

2. Mockenhaupt M. The current understanding of Stevens-Johnson syndrome

and toxic epidermal necrolysis. Expert Rev Clin Immunol 2011;7:803-813.

3. Roujeau J, Bastuji-Garin S. Systematic review of treatments for Stevens-

Johnson syndrome and toxic epidermal necrolysis using the SCORTEN score

as a tool for evaluating mortality. Ther Adv Drug Saf 2011;2:87-94.

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171

Advisory Board of This Issue (June 2016)

Ahmet Muzaffer Demir, Turkey

Ahmet Öztürk, Turkey

Akif Selim Yavuz, Turkey

Anne-Mette Hvas, Denmark

Aurore Keutgens, Belgium

Ayşegül Ünüvar, Turkey

Aytemiz Gürgey, Turkey

Barbara Bain, UK

Bülent Eser, Turkey

Bülent Kantarcıoğlu, Turkey

Cem Ar, Turkey

Cengiz Beyan, Turkey

Davut Albayrak, Turkey

Debra Hoppensteadt, USA

Dunja Mihajlovic, Serbia

Eman Mosad, Egypt

Emre Tekgündüz, Turkey

Eric Berntorp, Sweden

Erol Atalay, Turkey

Fahir Özkalemkaş, Turkey

Fatemah Mohammadi-Nasrabadi, Iran

Fatih Demirkan, Turkey

Fatma Aktepe, Turkey

Fatma Hüsniye Dilek, Turkey

Ferit Avcu, Turkey

Geetha Narayanan, India

Gluseppe Saglio, Italy

Halis Akalın, Turkey

Hayri Özsan, Turkey

Hüseyin Gülen, Turkey

Hyun Kyung Kim, Korea

İbrahim Haznedaroğlu, Turkey

İrfan Yavaşoğlu, Turkey

İsmail Sarı, Turkey

Jawed Fareed, USA

Joachim Deeg, USA

John Bennett, USA

Jun-Sook Ha, Korea

Kaan Kavaklı, Turkey

Kansu Büyükafşar, Turkey

Mahmut Bayık, Turkey

Manuel Olivares, Chile

Muhit Özcan, Turkey

Naci Tiftik, Turkey

Nicola Pavan, Italy

Oral Nevruz, Turkey

Praveen Papareddy, Sweden

Raja Ramachandran, India

Şule Ünal, Turkey

Theodore Tulchinsky, Israel

Türkan Patıroğlu, Turkey

Ulrich Germing, Germany

Usama Gergis, USA

Veysel Sabri Hançer, Turkey

Yehia Abed, Palestine

Ying Ju, China

Yusuf Baran, Turkey

Zahra Sepehri, Iran

BİLİMSEL SEKRETERYA

Kongre Sekreterleri

Prof. Dr. Güner Hayri Özsan (Dokuz Eylül Üniversitesi, İzmir)

E-posta: gensek@thd.org.tr

Doç. Dr. Muhlis Cem Ar (İstanbul Üniversitesi, İstanbul)

E-posta: arassek@thd.org.tr

İletişim Adresi

Turan Güneş Bulv. İlkbahar Mah. 613. Sok. No:8 Çankaya - Ankara

Tel : (312) 490 98 97 • Faks: (312) 490 98 68

E-posta: thdofis@thd.org.tr • Web: www.thd.org.tr

Türk Hematoloji Derneği Merkez İletişim Bilgileri

Mall of İstanbul Rezidans Süleyman Demirel Bulvarı 7 A

Blok No: 26 34306 Başakşehir - İstanbul

Tel: (212) 603 66 55 • Faks: (212) 603 66 35

KONGRE SEKRETERYASI

Serenas Uluslararası

Turizm Kongre Organizasyon A.Ş.

Tel: (312) 440 50 11 • Faks: (312) 441 45 62

E-posta: ulusalhematoloji2016@serenas.com.tr

Web: www.serenas.com.tr

2016-2

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