Turkish Journal of Hematology Volume: 33 - Issue: 3

Volume 33 Issue 3 September 2016 80 TL 

ISSN 1300-7777 

Review Article 

Clinical Interpretation of Genomic Variations 

Müge Sayitoğlu; İstanbul, Turkey 

Research Articles 

The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia 

Jingyi Wang, et al.; Shandong, China 

Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms 

Neslihan Andıç, et al.; Eskişehir, Aydın, Turkey 

Retrospective Study of Incidence and Prognostic Significance of Eosinophilia after Allogeneic Hematopoietic Stem 

Cell Transplantation: Influence of Corticosteroid Therapy 

Wataru Yamamoto, et al.; Yokohama, Japan 

Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression 

Nilgün Işıksaçan, et al.; İstanbul, Turkey 

Finding the Optimal Conditioning Regimen for Relapsed/Refractory Lymphoma Patients Undergoing Autologous 

Hematopoietic Cell Transplantation: A Retrospective Comparison of BEAM and High-Dose ICE 

Onur Esbah, et al.; Ankara, Turkey 

The Changing Epidemiology of Bloodstream Infections and Resistance in Hematopoietic Stem Cell 

Transplantation Recipients 

Mücahit Yemişen, et al.; İstanbul, Turkey 

BK Virus-Hemorrhagic Cystitis Following Allogeneic Stem Cell Transplantation: Clinical Characteristics and Utility 

of Leflunomide Treatment 

Young Hoon Park, et al.; Incheon, Republic of Korea 

A Randomized Study Comparing the Efficacy of Three Hepatitis B Vaccine Induction Regimens in Adult Patients 

with Hematological Malignancies 

Zübeyde Nur Özkurt, et al.; Ankara, Turkey 

Reliability and Validity of the Turkish Version of the PedsQL 3.0 Cancer Module for 2- to 7-Year-Old and the 

PedsQL 4.0 Generic Core Scales for 5- to 7-Year-Old: The Hacettepe University Experience 

Vesile Yıldız Kabak, et al.; Ankara, Turkey 

Cover Picture: 

Mehmet Sönmez 

Trabzon, Turkey 

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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 


Chicago Medical Center University, Chicago, USA 


Karolinska University, Stockholm, Sweden 

Mayo Clinic Saint Marys Hospital, USA 

Heidelberg University, Heidelberg, Germany 

Lille University, Lille, France 

Loyola University, Maywood, USA 

Karolinska University Hospital, Stockholm, Sweden 

Frankfurt University, Frankfurt, Germany 

Colorado Health Sciences University, USA 

University Hospital Cologne, Cologne, Germany 


Albert Ludwigs University, Germany 

University of North Carolina School of Medicine, NC, USA 

Southwestern Medical Center, Texas, USA 

Georgia Health Sciences University, Augusta, USA 

Pavia Medical School University, Pavia, Italy 

Kings College Hospital, London, UK 

Pierre et Marie Curie University, Paris, France 

King’s Hospital, London, UK 

Athens University, Athens, Greece 

Torino University, Torino, Italy 

Wayne State University School of Medicine, Detroit, USA 

Rambam Medical Center, Haifa, Israel 

University of Köln, Germany 

AK St Georg, Hamburg, Germany 

Memorial Şişli Hospital, İstanbul, Turkey 

Santa Creu i Sant Pau Hospital, Barcelona, Spain 

Mayo Clinic, Rochester, Minnesota, USA 

İstanbul Cerrahpaşa University, İstanbul, Turkey 

University of Minnesota, Minnesota, USA 

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

San Martino University, Genoa, Italy 

Language Editor 

Leslie Demir 

Statistic Editor 

Hülya Ellidokuz 

Editorial Office 

İpek Durusu 

Bengü Timoçin 

A-I 

Publishing 

Services 

GALENOS PUBLISHER 

Molla Gürani Mah. Kaçamak Sk. No: 21, 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 

Board of Directors 

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 

15.08.2016 

Cover Picture 

Mehmet Sönmez is currently working at Karadeniz Technical University, 

School of Medicine, Department of Hematology, Trabzon, 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. 

A-II

AIMS AND SCOPE 

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

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

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

periodical encompassing subjects relevant to hematology. 

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

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

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

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

Strengthening the Reporting of Observational Studies in Epidemiology 

(STROBE). 

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

hematological research of the highest scientific quality and clinical relevance. 

Additionally, educational material, reviews on basic developments, editorial 

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

and clinicians covering their experience and comments on hematology 

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

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

reports. Important new findings or data about interesting hematological 

cases may be submitted as a brief report. 

General practitioners interested in hematology and internal medicine 

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

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

Hematology is indexed, as follows: 

- PubMed Medline 

- PubMed Central 

- Science Citation Index Expanded 

- EMBASE 

- Scopus 

- CINAHL 

- Gale/Cengage Learning 

- EBSCO 

- DOAJ 

- ProQuest 

- Index Copernicus 

- Tübitak/Ulakbim Turkish Medical Database 

- Turk Medline 

Impact Factor: 0.827 

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, 


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. 

A-III

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. 

A-IV

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 

A-V

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. 

A-VI

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 

Measurements should be reported using the metric system, according 

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

Guide, New England Journal of Medicine Books, 1992. 

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

edu/~rowlett/units/scales/clinical_data.html. For more details, see http:// 

www.amamanualofstyle.com/oso/public/jama/si_conversion_table.html. 

Example for CBC. 

Hematology component 

SI units 

RBC 6.7-11 x 10 12 /L 

WBC 5.5-19.5 x10 9 /L 

Hemoglobin 

PCV 

MCV 

MCHC 

MCH 

116-168 g/L 

0.31-0.46 L/L 

39-53 fL 

300-360 g/L 

19.5-25 pg 

Platelets 300-700 x 10 9 /L 

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

Abbreviations and Symbols 

Use only standard abbreviations. Avoid abbreviations in the title and 

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

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

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

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

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

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

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

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

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A-VII

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in publication. 

A-VIII

CONTENTS 

Review Article 

172 Clinical Interpretation of Genomic Variations 


Research Articles 

180 The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia 

Jingyi Wang, Jianguo Hao, Na He, Chunyan Ji, Daoxin Ma 

187 Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms 

Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay, İrfan Yavaşoğlu, Vefki Gürhan Kadıköylü, Ali Zahit Bolaman 

196 Retrospective Study of Incidence and Prognostic Significance of Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation: 

Influence of Corticosteroid Therapy 

Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori 

202 Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression 

Nilgün Işıksaçan, Suzan Çınar, Esin Aktaş Çetin, Melih Aktan, Günnur Deniz 

209 Finding the Optimal Conditioning Regimen for Relapsed/Refractory Lymphoma Patients Undergoing Autologous Hematopoietic Cell 

Transplantation: A Retrospective Comparison of BEAM and High-Dose ICE 

Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze Durgun, 

Şerife Kocubaba, Fevzi Altuntaş 

216 The Changing Epidemiology of Bloodstream Infections and Resistance in Hematopoietic Stem Cell Transplantation Recipients 

Mücahit Yemişen, İlker İnanç Balkan, Ayşe Salihoğlu, Ahmet Emre Eşkazan, Bilgül Mete, M. Cem Ar, Şeniz Öngören, Zafer Başlar, 

Reşat Özaras, Neşe Saltoğlu, Ali Mert, Burhan Ferhanoğlu, Recep Öztürk, Fehmi Tabak, Teoman Soysal 

223 BK Virus-Hemorrhagic Cystitis Following Allogeneic Stem Cell Transplantation: Clinical Characteristics and Utility of Leflunomide Treatment 

Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim 

231 A Randomized Study Comparing the Efficacy of Three Hepatitis B Vaccine Induction Regimens in Adult Patients with Hematological Malignancies 

Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı 

236 Reliability and Validity of the Turkish Version of the PedsQL 3.0 Cancer Module for 2- to 7-Year-Old and the PedsQL 4.0 Generic Core 

Scales for 5- to 7-Year-Old: The Hacettepe University Experience 

Vesile Yıldız Kabak, Yavuz Yakut, Mualla Çetin, Tülin Düger 

Brief Reports 

244 Results of Four-Year Rectal Vancomycin-Resistant Enterococci Surveillance in a Pediatric Hematology-Oncology Ward: From 

Colonization to Infection 

Hacer Aktürk, Murat Sütçü, Ayper Somer, Serap Karaman, Manolya Acar, Ayşegül Ünüvar, Sema Anak, Zeynep Karakaş, Aslı Özdemir, 

Kutay Sarsar, Derya Aydın, Nuran Salman 

248 Radiation-Induced Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia 

Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan 

A-IX

251 A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker 

Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç 

Letters to the Editor 

254 A Primary Bone Diffuse Large B-Cell Lymphoma with Ocular Adnexal Involvement 

Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı 

255 Cerebral Sinovenous Thrombosis Mimicking Intracranial Mass 

Derya Özyörük 

256 A Rare Cause of Unexplained Refractory Iron Deficiency Anemia: Unicentric Plasma-Cell Type Castleman’s Disease 

Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden, İbrahim Öner Doğan, Fatih Beşışık 

Images in Hematology 

259 Vaginal Lymphoma: A Possible Cause of Genital Hemorrhage 

Erdoğan Nohuz, Sharif Kullab, Albane Ledoux-Pilon, Cécile Moluçon-Chabrot, Maël Albaut, Luisa De Simone, Xavier Durando 

261 Endothelial Cells, Ankaferd Hemostat, and Estradiol 

Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu 

263 An Unusual Congenital Anomaly in Fanconi Aplastic Anemia: Congenital Lobar Emphysema 

Ali Fettah, Gökçe Pınar Reis, Soner Sertan Kara, Tekin Aksu, Afak Durur Karakaya, Mahmut Subaşı, Atilla Çayır 

A-X

Advisory Board of This Issue (September 2016) 

Agnieszka Bojarska Junak, Poland 

Ahmet Emre Eşkazan, Turkey 

Ahmet Soysal, Turkey 

Akif Selim Yavuz, Turkey 

Alessandro Allegra, Italy 

Anıl Tombak, Turkey 

Ayşegül Ünüvar, Turkey 

Bülent Kantarcıoğlu, Turkey 

Can Balkan, Turkey 

Canan Baydemir, Turkey 

Celalettin Üstün, USA 

Cem Ar, Turkey 

Dae-Young Kim, Korea 

Darko Antic, Serbia 

Dilber Talia İleri, Turkey 

Dilek Sevgi, Turkey 

Dorothy Funk, USA 

Düzgün Özatlı, Turkey 

Eda Tahir Turanlı, Turkey 

Elif Ünal, Turkey 

Eva Posfai, Hungary 

Fortunato Morabito, Italy 

François Vincent, France 

Gökhan Aygün, Turkey 

Güray Saydam, Turkey 

Hakan Özdoğu, Turkey 

Hakim Ouled Haddou, France 

Hüsnü Pullukçu, Turkey 

İbrahim Haznedaroğlu, Turkey 

İbrahim Köker, Turkey 

İhsan Karadoğan, Turkey 

Mahmut Töbü, Turkey 

Mehmet Ali Özcan, Turkey 

Mehmet Sönmez, Turkey 

Nazan Sarper, Turkey 

Nicolaus Kröger, Germany 

Özgür Mehtap, Turkey 

Qing Ma, USA 

Reşat Özaras, Turkey 

Şule Ünal, Turkey 

Takahiko Nakane, Japan 

Takahiro Yamauchi, Japan 

Tunç Fışgın, Turkey 

Türkan Patıroğlu, Turkey 

Umberto Gianelli, Italy 

Varinder Kaur, USA 

Veysel Sabri Hançer, Turkey 

Volkan Hazar, Turkey 

Yener Koç, Turkey 

Yusufhan Yazır, Turkey 

Zühre Kaya, Turkey

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

REVIEW 

DOI: 10.4274/tjh.2016.0149 

Turk J Hematol 2016;33:172-179 

Clinical Interpretation of Genomic Variations 

Genomik Varyasyonların Klinik Yorumlanması 


İstanbul University Faculty of Medicine, Institute of Experimental Medicine, Department of Genetics, İstanbul, Turkey 

Abstract 

Novel high-throughput sequencing technologies generate largescale 

genomic data and are used extensively for disease mapping 

of monogenic and/or complex disorders, personalized treatment, 

and pharmacogenomics. Next-generation sequencing is rapidly 

becoming routine tool for diagnosis and molecular monitoring of 

patients to evaluate therapeutic efficiency. The next-generation 

sequencing platforms generate huge amounts of genetic variation 

data and it remains a challenge to interpret the variations that are 

identified. Such data interpretation needs close collaboration among 

bioinformaticians, clinicians, and geneticists. There are several problems 

that must be addressed, such as the generation of new algorithms for 

mapping and annotation, harmonization of the terminology, correct 

use of nomenclature, reference genomes for different populations, 

rare disease variant databases, and clinical reports. 

Keywords: Genetic variation, Sequencing, Genomic data, Clinical 

interpretation 

Öz 

Yeni dizileme teknolojileri, tek genli ve/veya kompleks kalıtılan 

hastalıklarla ilgili genlerinin haritalanması, kişiye özel tedavi ve 

farmakogenomik alanları için yüksek verimde ve büyük ölçekte genomik 

data üreten teknolojilerdir. Yeni nesil dizileme, hastaların tanı ve tedavi 

yanıtlarını değerlendirecek moleküler izlem süreçlerinde kullanılmak 

üzere, hızlı bir şekilde rutin uygulamada yerini bulmaktadır. Yeni nesil 

dizileme platformları çok büyük ölçekte genetik varyasyon datası 

üretmektedir ve bu varyasyonların klinik olarak anlamlandırılması çok 

zordur. Klinik yorumlamalar, hekimler ile biyoinformatik ve genetik 

uzmanlarının yakın işbirliğine ihtiyaç duymaktadır. Yeni haritalama ve 

hizalama araçlarına duyulan ihtiyaç, terminolojinin harmonizasyonu, 

genetik isimlendirmenin doğru kullanımı, farklı populasyonlar için 

referans genom datasının bulunmaması, nadir hastalıklar için genomik 

veri bankalarının eksikliği ve klinik raporlama, halen aşılması gereken 

sorunlar arasında bulunmaktadır. 

Anahtar Sözcükler: Genetik varyasyon, Dizileme, Genomik data, Klinik 

yorum. 

Introduction 

Next-generation sequencing (NGS) methods provide cheap 

and solid genomic data and are used extensively for de novo 

sequencing, disease mapping, quantifying of expression levels, 

and population genetic studies [1,2,3,4,5]. They can also be 

applied to complex disorders [6], personalized treatment, and 

pharmacogenomics [7,8,9]. The medical genetics field translates 

high-throughput genetic data to clinical settings in order to 

improve diagnostic efficiency and treatment decision-making 

[10,11]. The interpretation of the clinical significance of genomic 

variants in a given patient or in patients’ family members is 

the main challenge of resequencing. In the last decade, several 

diseases and syndromes have been analyzed by NGS, hundreds of 

disease-associated genes have been found, and novel targeted 

therapies have been developed. The most powerful contribution 

of NGS [particularly whole-exome sequencing (WES) and 

whole-genome sequencing (WGS)] is the description of new 

candidate signaling pathways involved in the pathogenesis of a 

clinical condition that will help with prevention, diagnosis, and 

therapeutic opportunities. 

High-throughput sequencing can be implemented within 

different applications including WGS, WES, ribonucleic 

acid sequencing (RNA-seq), or targeted sequencing [12]. 

Commercially available NGS platforms are generally employed 

with similar steps for all these approaches: generation of 

sequencing libraries, sequencing simultaneously in a massively 

parallel fashion, and data analysis [12]. 

Whole-exome sequencing studies have been commonly used to 

identify the responsible gene of a clinical phenotype. The WGS 

approach holds major advantages for the detection of variations 

not only in the exome but also in the noncoding regulatory 

regions for complex and/or multigenic diseases. The analysis of 

WGS data is complicated by the amount of information and 

challenges in elimination of common genetic variations. Wholeexome 

sequencing studies require rough bioinformatics analysis 

Address for Correspondence/Yazışma Adresi: Müge SAYİTOĞLU, M.D., 

İstanbul University Faculty of Medicine, Institute of Experimental Medicine, Department of Genetics, İstanbul, Turkey 

Phone : +90 212 414 22 00-33312 

E-mail : mugeay@istanbul.edu.tr 

Received/Geliş tarihi: April 13, 2016 

Accepted/Kabul tarihi: August 07, 2016 

172


Müge Sayitoğlu. Clinical Interpretation of Genomic Variations 

work and experts and national reference sequences to evaluate 

the population-based genetic variants, along with large budgets. 

Alternatively, WES is relatively cost-efficient and is able to 

discover disease-related rare variants in coding regions and splice 

sites. There are several variations that have been successfully 

identified by WES in monogenic diseases. On the other hand, the 

exome represents less than 1% of the genome and such analysis 

will be excluding noncoding genomic regions such as regulatory 

regions, repetitive sequences, or noncoding RNAs. 

Using gene panels in NGS studies is an alternative option 

that restricts screening to selected genetic regions. Although 

it may simplify the scale of the analysis and interpretation, 

incidental findings still require attention. The most suitable 

NGS approach for routine clinical applications is ampliconbased/targeted 

sequencing. Most genetic disorders have allelic 

and locus heterogeneity, which means that one disease may 

arise from different genetic variations within the same gene 

or different genes. Due to the genetic heterogeneity, it takes 

longer to obtain the genetic test result, which leads to a delay in 

diagnosis. Amplicon-based NGS has provided a major advantage 

for the molecular analysis of heterogeneous genetic disorders, 

including hereditary cancers [13]. 

RNA-seq quantifies the amount of transcripts (all transcribed 

isoforms) and gives a chance to evaluate the whole RNA 

repertoire of a specific cell or tissue. The biggest limitation 

of RNA-seq is the “noncoding RNAs”; most of the genome 

is transcribed but the majority of these transcripts are not 

translated into proteins [14]. 

Accurate Use of the Terminology: Is It a Polymorphism or a 

Disease-Related Variation? 

In common use, a DNA polymorphism is a heritable variation 

that is present in >1% of the population and increasingly 

detected by next-generation resequencing. One of two or 

more alternate forms of a locus (alleles) may result from 

the changes in the nucleotide sequence [single nucleotide 

polymorphisms (SNPs)], deletions, insertions, or other structural 

rearrangements. According to the novel terminology the term 

‘SNP’ is used as single nucleotide variation (SNV) [15]. A genome 

contains repetitive sequences differing in copy numbers (i.e. 

copy number variations) between individuals. Polymorphic 

variations may or may not have phenotypic effects and they 

are valuable tools for genetic mapping studies including linkage 

and association studies of diseases. The vast majority of these 

variations (more than 90%) have been found to be localized 

in the noncoding genomic regions and are possibly involved in 

regulation of gene expression. On the other hand, a mutation is 

defined as a DNA variant detectable in



rely on complex interactions of the chemistry, the hardware, and 

the optical sensors that they use. For example, in the Illumina 

system, the images that are acquired from the instruments are 

prepared and analyzed to determine the base incorporated 

in the complementary strand. In this process, the ordering of 

nucleotides in a template from the noisy signals is referred to 

as base calling [17]. In other words, base calling converts the 

fluorescence signals into actual sequence data with quality 

scores. Base calling accuracy is measured by a Q score (Phred 

quality score), which is a common metric to assess the accuracy 

of a sequencing run. The Q score is defined as the logarithmically 

related base calling error probability (Q=-10 log P/log 10) [18]. 

For example, if Q=40 for a sequencing run, this is equal to the 

probability of an incorrect base call of 1 in 10,000 times, or 

with 99.99% base calling accuracy or a lower Q score of 10 

means, there is the probability of an incorrect call in 1 of 10 

bases. Low Q scores lead to false positive variant calls and need 

resequencing. 

Errors arising from NGS data are generally due to base 

calling and alignment applications. Moreover, low coverage 

sequencing (20× coverage). In association 

studies, sequencing many individuals at a low depth, rather than 

sequencing fewer individuals at a high depth, could maximize 

mapping power. Some of the genomic regions are difficult to 

interpret, such as homopolymer regions (a sequence of identical 

bases, like AAAA or TTTTTTTT), or simple repeats (minisatellitevariable 

number of tandem repeats and microsatellite-short 

tandem repeats). Bioinformaticians use VCF files, “Variant Call 

Format”, to store the gene sequence variations. 

4) Annotation and Prioritization of a Variation 

Several challenges arise for NGS-based diagnostic and research 

efforts in the identification of all genetic variations. Because 

of the increased complexity of data analysis and clinical 

interpretation of the data, it is best to use some universally 

accepted recommendations like those of the American College 

of Medical Genetics and Genomics (ACMG), EuroGentest, and 

the European Society of Human Genetics [25,26,27]. 

The ACMG recommends that both “mutation” and 

“polymorphism” can be replaced by “variant” with the following 

modifiers: pathogenic (P), likely pathogenic (LP), variant of 

uncertain significance (VUS), likely benign (LB), and benign 

(B) [26] (Figure 2). The “likely” term is used to define certainty 

greater than 90% of a variant either being disease-causing or 

benign. 

Pathogenic Variant 

174



in the general population, it is called a “benign” variant. These 

variations are nonpathogenic and have neutral effects. 

American College of Medical Genetics and Genomics suggested 

additional criteria including very strong, strong, and moderate 

support for being pathogenic, and likely pathogenic and likely 

benign variations. 

Nonsense mutations, frame shifts, exonic deletions, and 

promoter variations (very strong) are generally assumed to cause 

loss of function in the genes. These kinds of variations lead to 

reduced or absent gene function and nonsense-mediated decay 

of an altered transcript. These kinds of variations are expected 

to affect the clinical findings. 

Figure 2. Recommended terms for interpretation of clinical 

variants. 

If the previously identified or novel variation has substantial 

evidence that it causes a disease with a known or unknown 

mechanism, is called a “pathogenic” variant. These kinds of 

variations are generally nonsense mutations, frame shift 

variations, or splice site alterations. 

Likely Pathogenic Variant 

If the previously identified or novel variation is consistent 

with the diagnosis, it exists in the conserved genomic region, 

functional studies showed impaired gene product, or the 

function of the gene is known to be associated with a specific 

phenotype, the variation is called a “likely pathogenic” variant. 

Uncertain Significance Variant 

If a variant cannot be classified as pathogenic or benign, it 

is called a “variant of uncertain clinical significance” (VUS). 

It can be a missense variation, an in-frame deletion, or an 

insertion. These kinds of novel variations can cause confusion 

during interpretation and reporting. If there is no other variant 

identified, VUS should be highlighted in the report. 

Likely Benign Variant 

If a variant presents at high frequency in random individuals and 

is not a high penetrant or a disease-causing variant, it is called a 

“likely benign” variant. There is no absolute frequency threshold 

to classify that a variant is likely benign or likely pathogenic. This 

depends on the disease model, clinical characteristics, etc. These 

can be novel or previously reported variations with possible 

neutral effects. Generally, likely benign variants have enough 

evidence that they are not the cause of the disease, and the 

segregation analysis of haplotypes in affected and unaffected 

family members can support this finding. 

Benign Variant 

If a previously reported variation is present at a higher frequency 

Splice site variations may cause exon skipping and shortening or 

inclusion of intronic material due to loss or recreation of donor/ 

acceptor splice sites. These kinds of variations are predicted to 

lead to a null effect that needs additional functional analysis 

(RNA or protein). 

A missense variation is mostly known to be pathogenic; it alters 

the protein function or the nucleotide change and may disrupt 

the splice site. It can be detected by in silico prediction tools 

and then concluded to be a disease-related variation. Missense 

variations should be evaluated with minor allele frequency 

(MAF) values, which refer the second most common allele 

that occurs in a given population. The MAF value provides 

information to differentiate between common and rare variants 

in the population. If the determined missense variation has a 

low MAF value (



known variants that are present in public (dbSNP) and inhouse 

variant databases and published projects such as the 

1000 Genomes Project [28], EXAC, and the Exome Sequencing 

Project (ESP6500) [29] is a very helpful strategy to reduce the 

candidate list of disease-related variations. Population-based 

databases (such as the 1000 Genomes Project or ESP) have been 

created both for large and small local populations [30,31]. They 

are useful to obtain the frequencies of the variations. Disease 

databases mainly contain the variants of a specific disease or 

phenotype [32]. 

There are some limitations to these databases. For example, there 

is no absolute frequency threshold for a given variant, many 

populations are not represented, and there is no information 

about the phenotype. Limited numbers of locus-specific 

databases also exist but those are not available for most genes, 

there are contradictory data between databases, and they may 

not be updated. For correct data interpretation, researchers 

should check the updates of the databases, confirm that HGVS 

nomenclature is being used, and read the relevant publications 

[16,33]. Gene- or disease-oriented biomedical information can 

be found from the OMIM website (Online Mendelian Inheritance 

in Man- http://www.omim.org), in published scientific articles 

(PubMed- http://www.ncbi.nlm.nih.gov/pubmed), and in 

mutation databases (HGMD, Human Gene Mutation Databasehttp://www.hgmd.cf.ac.uk/ac/index.php). 

Clinicians can interpret a variant when it is reported and track 

the genotype-phenotype correlation, population frequency of 

the variation, and clinical assertions. Clinical laboratories should 

increase their collaboration with clinicians to better understand 

the effect of the variation on the phenotype. The ClinVar 

database (http://www.ncbi.nlm.nih.gov/clinvar/) archives 

reports of the relationships among medically important human 

variations and phenotypes. It has access to dbSNP and dbVar 

and includes information about the location of variation and 

phenotypic descriptions included in MedGen (http://www.ncbi. 

nlm.nih.gov/medgen). ClinVar is an interactive tool that can be 

divided into submitter, variation, phenotype, interpretation, 

and evidence. ClinVar represents the interpretation of a single 

allele, compound heterozygotes, haplotypes, and combinations 

of alleles in different genes [34,35]. 

Searching for previously published scientific and medical 

studies is also a valuable tool for the annotation of a detected 

variant. Researchers should be aware of using older versions of 

nomenclature in published reports. The given information about 

the index case, affected family members, and the size of the 

family should be carefully noted to avoid incorrect data. 

4.2. In Silico Functional Prediction 

A variety of algorithms (SIFT, PolyPhen, Provean, CADD, Condel, 

GERP, SNAP, SNPs&Go, PhyloP, and MutationTaster) are used to 

determine the effect of variations and that can be done at the 

nucleotide, amino acid, protein, and transcript/splice variant 

levels (Table 1). Mainly they have been developed to estimate 

the deleterious effect of a variant on a protein. The most 

Table 1. Representative in silico prediction tools and web pages. 

In Silico Prediction Tools 

Missense prediction 

SIFT 

http://sift.jcvi.org 

Mutation Tester 

http://www.mutationtester.org 

Mutation Assessor 

http://mutationassessor.org 

PolyPhen2 

http://genetics.bwh.harvard.edu/pph2 

MutPred 

http://mutpred.mutdb.org 

nsSNP Analyzer 

http://snpanalyzer.uthsc.edu 

Condel 

http://bg.upf.edu/fannsdb/help/condel.html 

CADD 

http://cadd.gs.washington.edu/score 

Provean 

http://provean.jcvi.org/index.php 

Splice site prediction 

Gene Splicer 

http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl.html 

Human Splicing Finder 

http://umd.be/HSF/ 

Net Gene 2 

http://www.cbs.dtu.dk/services/NetGene2 

Conservation prediction 

PhastCons 

http://compgen.bscb.cornell.edu/phast/ 

PhyloP 

http://compgen.bscb.cornell.edu/phast/ 

GERP 

http://mendel.stanford.edu/sidowlab/downloads/gerp/index.html 

176



common use of these tools is to predict the impact of a missense 

variation on a protein and to predict the effect of the variation 

on splicing. The prediction depends on the location, evolutionary 

conservation, amino acid charge, 2D and 3D calculations of the 

effect on protein structure, and biochemical consequences of 

the amino acid substitution. Some of these tools are used for 

the prediction of the effects on splicing and loss or creation of 

the splice sites. As a limitation of in silico tools, variable and 

incompatible interpretation results are derived from different 

algorithms and the use of multiple programs is recommended 

because of the differing performances of the tools [36]. 

4.3. Clinical Interpretation and Reporting of Next- 

Generation Sequencing Results 

Interpretation and reporting of candidate genetic variations 

is the biggest challenge in NGS data analysis and reporting 

processes. Genetic testing based on WGS often results in 

several variations that are not directly clinically actionable. 

The reportable variations should be classified as pathogenic (P), 

likely pathogenic (LP), a variant of uncertain significance (VUS), 

likely benign (LB), or benign (B) as described by the ACMG. 

Misinterpretation of data may be due to annotation errors, 

analytical errors, ethnicity effects (differential MAF values), 

reduced reproducibility in consideration of low-level mutations, 

nomenclature or terminology differences, and variable 

databases. International guidelines and recommendations 

developed to standardize and regulate deep sequencing are 

good references for researchers and clinicians [37]. 

Some general recommendations are summarized below to 

exclude possible incidental findings and ensure correct clinical 

interpretation and reporting of NGS results. 

Data Quality (Base Calling and Mapping) 

- If the Q score is low (Q30 score is lower than 70%) and total 

coverage is lower than 80%, sequencing should be repeated. 

- Different algorithms generate different outputs. Since the 

accuracy of the annotation depends on the success of the 

mapping, it is best to use at least two algorithms for mapping. 

- Previous NGS data generated from the same laboratory (inhouse 

data) are valuable to evaluate and exclude variations that 

arise from technical effects or poor quality of amplicon design. 

In-house laboratory data provide simplified analysis to exclude 

false variants (false positivity). 

- Next-generation sequencing data aim to achieve a high 

diagnostic yield to achieve high coverage in all genomic 

regions covered. If genetic variation is detected by NGS with 

low coverage, resequencing should be repeated, and clear 

communication with the clinician is required if the test results 

cannot be used to exclude a particular clinical diagnosis. 

Reporting 

- Next-generation sequencing results should not be transferred 

to clinical reports and practice without acceptable validation. It 

is essential to confirm the variation from a new DNA sample by 

NGS, Sanger sequencing, or another proper technique to exclude 

false positive results. Validation results should be included in the 

NGS report. 

- All variants should be annotated and reported with regard 

to the gene name; gene symbol; heterozygous, homozygous, 

or compound heterozygous condition; nucleotide changes in 

coding regions; and amino acid changes in proteins according 

to the HGVS [16]. Mutalyzer is useful software to check the 

nomenclature for variations (https://mutalyzer.nl/). Each report 

should include the reference sequence and the use of unique 

nomenclature is critical; “g” represents the genomic sequence, 

“c” represents the coding sequence, “p” represents protein and 

“m” represents mitochondria, and the first translational codon 

(ATG) is the starting point. The universal reference genome 

(hg18, hg19, or hg38) and the latest versions should be used to 

give the correct genomic coordinates and it should cover the 5’ 

and 3′ untranslated regions and promoter regions (http://www. 

ncbi.nlm.nih.gov/refseq/) [38,39]. 

- Reports should state the limitations of each specific NGS test 

regarding the detection of different kinds of mutations. 

- The reference genome, software, and databases (COSMIC, 

ClinVar, dbSNP, etc.) that are used should be specified in the 

report. If the variant was previously identified, the functional 

and clinical significance of the variation should be stated 

referring to the COSMIC database, the HGMD, or a scientific 

publication. 

- For diagnostic purposes, only genes with a known (i.e. 

published and confirmed) relationship between the aberrant 

genotype and the pathology should be included in the analysis. 

The NGS test results should be included with the disease name, 

its targets, the names of the genes tested, their reportable 

ranges, the analytical sensitivity and specificity, and, if possible, 

the diseases not relevant to the clinical phenotype that could be 

caused by mutations in the tested genes [40]. 

- For diagnostic purposes, all pathogenic and likely pathogenic 

variants have to be reported. Whether or not variants of unknown 

significance (VUS) are reported will depend on local practice. 

Researchers should be very cautious if detailed laboratory 

analysis has not been performed and this should be included in 

the report. If no variation has been defined other than a VUS, it 

should be highlighted in the report. In that case, clinicians are 

strongly suggested to discuss the result with a clinical geneticist 

and it is acceptable to request additional analysis (parental 

177



testing, etc.) in order to facilitate the interpretation of the 

result (http://www.acgs.uk.com). The latter has to be clear for 

laboratory scientists, as well as for the referring clinicians [40]. 

Conclusions 

In medical use of genetic discoveries, it is quite important to 

improve the standards of data collection and sharing to define a 

systematic method for the clinical annotation and interpretation 

of genomic and phenotypic variations. Data-sharing platforms 

like the Undiagnosed Diseases Network (UDN- https://www. 

genome.gov) or Matchmaker Exchange Network (http://www. 

matchmakerexchange.org) for researchers of rare diseases and 

clinicians for sharing clinical phenotypes and sequencing data, 

which may allow for identification of other patients with the 

same phenotype, help us to understand the functional relevance 

of the variant that is obtained and reported [41,42]. 

Next-generation sequencing technology is being used as a 

diagnostic tool because of the expanded utility and reduced 

costs. Targeted sequencing offers better running times, costs, 

datasets, and coverage compared to WES or WGS. However, 

there are still many concerns about the application of NGSbased 

diagnostics. The challenges and clinical applications 

of NGS results have been discussed here. These include the 

accumulation and storage of huge amounts of genomic data, 

the need for bioinformatics experts, the need for national 

reference genomes, reimbursement of sequencing costs, and, of 

course, clinical interpretation of novel and VUS results. 

Glossary 

Allele: Alternative form of a given locus. 

Annotation: DNA annotation or genome annotation is the 

identification of the locations of genes and all of the coding 

regions in a genome and determination of their function. 

Frameshift variation: Genetic variation caused by indels 

(insertions or deletions) of a number of nucleotides in DNA. 

Missense variation: A single nucleotide variation that leads 

to amino acid substitution and a codon change. Also called 

nonsynonymous substitution. 

Nonsense variation: A single nucleotide variation that results 

in a premature stop codon, or a nonsense codon in the 

transcribed mRNA, and in a truncated, incomplete, and usually 

nonfunctional protein product. 

Deep sequencing: Indicates that the total number of reads is 

many times larger than the length of the sequence under study. 

Depth: In DNA sequencing refers to the number of times a 

nucleotide is read during the sequencing process. 

Coverage: The average number of reads representing a given 

nucleotide in the reconstructed sequence. 

Whole-genome sequencing (WGS): A laboratory process 

that determines the complete DNA sequence of an organism’s 

genome at a single time. 

Whole-exome sequencing (WES): A technique for sequencing 

all the expressed genes in a genome (known as the exome). 

Amplicon (targeted) sequencing: Amplicon sequencing refers 

to ultradeep sequencing of PCR products for analyzing genetic 

variations. Amplicon sequencing is a highly targeted approach 

for analyzing genetic variation in specific genomic regions. 

Pathogenic: Anything that can produce disease. 

DNA polymorphism: A heritable variation that is present in >1% 

of the population and increasingly detected by next-generation 

resequencing. 

Mutation: DNA variants detectable in



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179

RESEARCH ARTICLE 

DOI: 10.4274/tjh.2015.0220 


The Mutation Profile of Calreticulin in Patients with 

Myeloproliferative Neoplasms and Acute Leukemia 

Miyeloproliferatif Neoplazisi ve Akut Lösemisi Olan Hastalarda Kalretikülin Mutasyon Profili 

Jingyi Wang 1,2 , Jianguo Hao 3 , Na He 1 , Chunyan Ji 1 , Daoxin Ma 1 

1Qilu Hospital of Shandong University, Department of Hematology, Shandong, China 

2Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Department of Hematology, Shandong, China 

3General Hospital of Shandong Stell Group Company, Department of Surgery, Shandong, China 

Abstract 

Objective: Calreticulin (CALR) plays important roles in cell 

proliferation, apoptosis, and immune responses. CALR mutations were 

described recently in Janus kinase 2 gene (JAK2)-negative or MPLnegative 

primary myelofibrosis (PMF) and essential thrombocythemia 

(ET) patients. CALR trails JAK2 as the second most mutated gene in 

myeloproliferative neoplasms (MPNs). However, little is known about 

CALR mutation in Chinese patients with leukemia. In the present 

study, a cohort of 305 Chinese patients with hematopoietic neoplasms 

was screened for CALR mutations, with the aim of uncovering the 

frequency of CALR mutations in leukemia and MPNs. 

Materials and Methods: Polymerase chain reaction and direct 

sequencing were performed to analyze mutations of CALR in 305 

patients with hematopoietic malignancies, including 135 acute 

myeloid leukemia patients, 57 acute lymphoblastic leukemia patients, 

and 113 MPN patients. 

Results: CALR mutations were found in 10.6% (12 of 113) of samples 

from patients with MPNs. CALR mutations were determined in 11.3% 

(6 of 53), 21.7% (5 of 23), and 9.1% (1/11) of patients with ET, PMF, 

and unclassifiable MPN, respectively. 

Conclusion: We showed that MPN patients carrying CALR mutations 

presented with higher platelet counts and lower hemoglobin levels 

compared to those with mutated JAK2. However, all of the leukemia 

patients had negative results for CALR mutations. 

Keywords: Calreticulin mutation, Myeloproliferative neoplasms, 

Leukemia 

Öz 

Amaç: Kalretikülin (CALR) hücre çoğalması, apoptoz ve immün 

yanıtlarda önemli rol oynar. CALR mutasyonları yakın zamanda Janus 

kinaz 2 (JAK2) veya MPL geni negatif primer miyelofibroz (PMF) ve 

esansiyel trombositemi (ET) hastalarında tanımlanmıştır. CALR JAK2’yi 

takiben miyeloproliferatif neoplazilerde (MPN) ikinci sıklıkta görülen 

mutant gendir. Ancak, Çinli lösemi hastalarında CALR mutasyonları 

hakkında bilgi sınırlıdır. Bu çalışmada, hematopoetik neoplazisi olan 

305 Çinli hasta CALR mutasyonları, bu mutasyonların lösemi ve MPN 

hastalarındaki sıklığının ortaya çıkarılması için taranmıştır. 

Gereç ve Yöntemler: Polimeraz zincir reaksiyonu ve direkt dizileme 

yöntemi 135 akut miyeloid lösemi, 57 akut lenfoblastik lösemi ve 113 

MPN olmak üzere toplam 305 hematopoetik malinitesi olan hastada 

CALR mutasyonlarını analiz etmede kullanılmıştır. 

Bulgular: CALR mutasyonu MPN hastalarının %10,6’sında (12/113) 

tespit edilmiştir. Ayrıca bu mutasyonlar ET, PMF ve sınıflandırılamayan 

MPN hastalarında sırasıyla %11,3 (6/53), %21,7 (5/23) ve %9,1 (1/11) 

olarak bulunmuştur. 

Sonuç: CALR mutasyonu taşıyan MPN hastaları JAK2 pozitif olanlara 

göre tanı anında daha yüksek trombosit sayısı ve daha düşük 

hemoglobin düzeylerine sahip olduklarını gösterdik. Ancak, lösemi 

hastalarının tamamında CALR mutasyonları negatif tespit edildi. 

Anahtar Sözcükler: Kalretikülin mutasyonu, Miyeloproliferatif 

neoplazi, Lösemi 

Address for Correspondence/Yazışma Adresi: Daoxin MA, M.D., 

Qilu Hospital of Shandong University, Department of Hematology, Shandong, China 

Phone : +86 531 82169887 

E-mail : daoxinma@sdu.edu.cn 

Received/Geliş tarihi: May 28, 2015 

Accepted/Kabul tarihi: September 02, 2015 

180


Wang J, et al: Calreticulin in Myeloproliferative Neoplasms and Leukemia 

Introduction 

Somatic frameshift mutations in exon 9 of calreticulin (CALR) have 

been identified in a large proportion of JAK2- or MPL-negative 

myeloproliferative neoplasm (MPN) patients, including those 

with primary myelofibrosis (PMF) and essential thrombocythemia 

(ET) [1,2]. The CALR gene, located on chromosome 19p13.3, 

encodes a 48-kDa protein that consists of three domains: the 

amino terminal N-domain (residues 1-180), central proline-rich 

P-domain (residues 181-290), and carboxyl terminal C-domain 

(residues 291-400). The CALR protein is localized primarily in 

the endoplasmic reticulum through its C-terminal KDEL motif 

[3], but it is also found in the cell membrane, cytoplasm, and 

extracellular matrix [4,5]. Functionally, CALR is believed to 

participate in Ca2+ homeostasis as a calcium-binding protein, 

handling misfolded proteins, cell adhesion, immune response to 

cancer, and phagocytosis [4,6,7,8,9,10,11,12,13]. CALR-knockout 

mice are born dead and display impaired cardiac development, 

whereas postnatal overexpression also leads to cardiac defects 

[14,15]. Therefore, CALR regulates key cellular functions like 

proliferation and apoptosis. CALR also plays an important role 

in immune responses [16]. 

Mutations of CALR were found essential for the diagnosis 

and prognosis of MPNs in recent years. All CALR mutations 

seen so far in MPNs mainly involve exon 9 and are somatic 

insertions or deletions. Two mutation variants (type 1 and type 

2) were the most frequent: type 1 (c.1179_1230del) resulted 

from a 52-bp deletion, more frequent in PMF, and type 2 

(c.1234_1235insTTGTC) resulted from a 5-bp TTGTC insertion 

[1]. Andrikovics et al. demonstrated that CALR mutations are 

found in about one-fourth of patients with ET or PMF and are 

associated with distinct clinical characteristics, and another 

study also found that CALR mutations are associated with 

younger age, more severe anemia, higher white blood cell (WBC) 

and platelet counts, lower Dynamic International Prognostic 

Scoring System Plus scores, and better survival compared to 

subjects with JAK2 mutations [17,18]. 

Similar to MPNs, acute leukemia, including acute myeloid 

leukemia (AML) and acute lymphoblastic leukemia (ALL), 

is a group of disorders characterized by abnormal clonal 

proliferation and immune imbalance. To investigate whether 

CALR mutations were present in myeloid neoplasms, Andrikovics 

et al. detected JAK2, CALR, and MPL genes in 289 cases of ET 

and 99 cases of PMF, and they reported that in ET, 154 (53%) 

JAK2V617F mutation-positive, 96 (33%) CALR mutationpositive, 

9 (3%) MPL mutation-positive, and 30 triple-negative 

(11%) cases were identified, while in PMF 56 (57%) JAK2V617F 

mutation-positive, 25 (25%) CALR mutation-positive, 7 (7%) 

MPL mutation-positive, and 11 (11%) triple-negative cases were 

identified [18]. Qiao et al. screened CALR mutations in 104 AML 

patients, 55 chronic myeloid leukemia (CML) patients, 7 chronic 

myelomonocytic leukemia patients, and 8 myelodysplastic 

syndrome (MDS) patients. Although most of these patients 

had negative results, one AML patient was found to harbor a 

CALR mutation (c.1179_1230del) without JAK2V617F or MPL 

W515L/K mutations [19]. 

Unlike AML, ALL is a heterogeneous malignancy caused by the 

clonal proliferation of lymphocytes. However, no data about the 

mutation frequency of CALR in ALL patients have been reported 

to date. Therefore, in the present study, a cohort of 305 Chinese 

patients with hematopoietic neoplasms was screened for CALR 

mutations, with the aim of uncovering the frequency of CALR 

mutations in leukemia and MPNs. The results demonstrate that 

CALR mutation status is an important diagnostic factor in MPN 

patients without JAK2 mutation while it is negative in leukemia 

patients. 

Materials and Methods 

Subjects and Ethics Statement 

Bone marrow or peripheral blood samples from 113 MPN patients 

were collected at Qilu Hospital of Shandong University between 

August 2012 and November 2014, including cases of ET (n=53), 

polycythemia vera (PV; n=20), PMF (n=23), MDS/MPN (n=6), 

and unclassifiable MPN (MPN-U; n=11). We also obtained bone 

marrow samples from 192 patients with other hematopoietic 

neoplasms including AML (n=135) and ALL (n=57). These patients 

were all newly diagnosed before treatment. The characteristics 

of the patients at the time of sampling are presented in Tables 

1 and 2. The patients with AML were treated with standard 

induction chemotherapy (anthracycline and cytarabine). 

The patients with ALL were treated with standard induction 

chemotherapy (vincristine, daunorubicin, L asparaginase, and 

prednisone). Bone marrow mononuclear cells (BMMCs) or 

peripheral blood mononuclear cells (PBMCs) were obtained from 

patients using density-gradient centrifugation with the Ficoll- 

Hypaque technique (Ficoll, Pharmacia LKB Biotechnology Inc., 

Piscataway, NY, USA). The samples were then stored at -80 °C. 

The present study was approved by the Ethics Committee of Qilu 

Hospital, Shandong University (Jinan, China). Written informed 

consent was obtained from all participants for treatment and 

the cryopreservation of bone marrow and peripheral blood 

according to the Declaration of Helsinki. 

Genomic DNA Isolation, Polymerase Chain Reaction 

Amplification, and Sequencing 

Genomic DNA samples from BMMCs or PBMCs of patients were 

extracted using the TIANGEN DNA Extraction Kit (TIANamp 

Genomic DNA Kit, Beijing, China). Oligonucleotide primers 

targeting exon 9 of CALR were used to amplify a 377-bp product: 

forward 5’ - CTG GCA CCA TCT TTG ACA ACT T - 3’, reverse 5’ - 

GGC CTC TCT ACA GCT CGT C - 3’. Polymerase chain reaction 

181

Wang J, et al: Calreticulin in Myeloproliferative Neoplasms and Leukemia Turk J Hematol 2016;33:180-186 

(PCR) was performed in a volume of 25 µL containing 150 ng of 

DNA, 12.5 µL of PCR master mix, 400 nM each of forward and 

reverse primers, and ddH2O. Cycling parameters consisted of an 

initial denaturation at 94 °C for 2 min; 40 cycles of denaturation 

at 94 °C for 15 s, annealing at 56 °C for 30 s, and extension at 

72 °C for 45 s; and a final extension at 72 °C for 1 min. PCR 

products were purified (QIAquick PCR Purification Kit, QIAGEN, 

Valencia, CA, USA) and subjected to bidirectional sequencing. 

Mutations were identified using Mutation Surveyor Software 

(Soft Genetics, LLC, State College, PA, USA). 

JAK2V617F mutation burden was assessed using a quantitative 

PCR-based allelic discrimination assay. Real-time quantitative 

PCR was conducted using an ABI Prism 7500 Real-Time PCR 

System (Applied Biosystems, Foster City, CA, USA) in accordance 

with the manufacturer’s instructions. The primers and probes 

were as follows: JAK2-PCR-Primer-F: AAG CTT TCT CAC AAG CAT 

TTG GTT T, JAK2-PCR-Primer-R: AGA AAG GCA TTA GAA AGC 

CTG TAG TT, MGB probe sequence: JAK2-Probe-WT: VIC- TCT CCA 

CAG ACA CAT AC; JAK2-Probe-V617F: FAM- TCC ACA GAA ACA 

TAC (all the primers and probes were synthesized by Invitrogen, 

USA). The real-time PCR contained, in a final volume of 10 µL, 1 

µL of DNA, 5 µL of Universal PCR Master Mix, 0.4 µL of Primer-F 

and Primer-R, 0.2 µL of Probe-WT, 0.2 µL of Probe-V617F, and 

2.8 µL of distilled water. PCR reaction was done at 50 °C for 2 

min and 95 °C for 15 min, followed by 45 cycles of 95 °C for 

30 s and 62 °C for 1 min. The fluorescence signal was collected 

at 62 °C while ROX Reference Dye was used as a background 

to normalize the fluorescent signal. The cycle threshold (Ct) 

value of VIC or FAM reflects the number of wild-type or mutant 

JAK2V617F gene DNAs, denoted as Ct VIC and Ct FAM. JAK2V617F 

was considered as positive when CtFAM was lower than 38. 

Statistical Analysis 

The Kolmogorov-Smirnov test was performed to test whether 

variables were normally distributed. Then the independentsamples 

t-test was used to compare continuous variables. Chisquare 

or Fisher exact tests were used for dichotomous variables. 

The clinical characteristics of the leukemia and MPN patients, 

including sex, age, WBC count, and other factors, are presented 

in Tables 1 and 2. Statistical analysis was performed using SPSS 

17.0 (SPSS Inc., Chicago, IL, USA). 

Results 

The Profile of CALR Mutations in MPN Patients 

Mutant CALR in MPNs is a result of frameshift mutations, caused 

by exon 9 deletions or insertions; the type 1 variant, a 52-bp 

deletion (c.1179_1230del), and type 2 variant, a 5-bp TTGTC 

insertion (c.1234_1235insTTGTC), constitute more than 80% 

of these mutations. In our study, a total of 10.6% of patients 

(12 of 113) with MPNs were demonstrated to harbor CALR 

mutations. The CALR mutation was found in 11.3% (6 of 53) of 

ET, 21.7% (5 of 23) of PMF, and 9.1% (1/11) of MPN-U patients, 

respectively (Table 3). Moreover, CALR mutations were found in 

24.0% of JAK2V617F-negative ET patients (6 of 25) and 35.7% of 

JAK2V617F-negative PMF patients (5 of 14). No CALR mutation 

was found in patients with PV. For mutation types, a total of 5 

distinct variants of CALR mutation, including 4 deletions and 

1 insertion, were identified (Figure 1). c.1179_1230del, which 

resulted from a 52-bp deletion, and c.1234_1235insTTGTC, 

which resulted from a 5-bp insertion, were the most frequent 

CALR mutations. The two mutations accounted for 50% (6 of 12) 

and 25% (3 of 12) in all cases with mutant CALR, respectively. 

For ET patients, the two mutations were 50% (3 of 6) and 50% 

(3 of 6), respectively. For PMF patients, the two mutations were 

60% (3 of 5) and 0% (0 of 5), respectively. Moreover, we also 

identified other kinds of deletions of CALR genetic variation: 

c.1239_1257del (1/12) and c.1183_1228del (1/12) were found 

Table 1. Clinical characteristics of 192 patients with acute 

leukemia. 

Characteristics AML ALL 

Patients, n 

Age at study entry, years 

Males, n (%) 

Bone marrow blasts at diagnosis, % 

WBC count, x10 3 cells/mm 3 

AML subtype, n (%) 

M2 

M3 

M4 

M5 

135 

42.4±16.1 a 

71 (52.6) 

74.0±0.0 a 

32.1±27.8 a 

16 (11.9) 

40 (29.6) 

29 (21.5) 

50 (37.0) 

57 

37.3±16.5 a 

29 (50.9) 

89.1±13.8 a 

45.1±27.1 a 

a : Data are presented as mean ± standard deviation. WBC: White blood cell, AML: acute 

myeloid leukemia, ALL: acute lymphoblastic leukemia. 

Figure 1. Sequencing results of CALR mutations in patients with 

myeloproliferative neoplasms. 

182



in ET patients, and c.1183_1216del (1/12) was found in a MPN-U 

patient. 

The Profile of CALR Mutations in Leukemia Patients 

To investigate whether CALR mutations were present in other 

hematopoietic neoplasms, we screened 135 patients with AML 

and 57 patients with ALL. However, no CALR exon 9 mutations 

were found in any of these patients. One single nucleotide 

polymorphism (SNP) of CALR, rs143880510 (Figure 2), was found 

in one ALL patient. 

Clinical Features of Patients with CALR Mutations 

All of the 20 PV patients and 6 MDS/MPN patients had wildtype 

CALR. ET patients with mutant CALR had lower WBC counts 

(7.5±4.1×109/L; p


JAK2 exon 12 [23], though much less prevalent in the patients, is 

considered as another robust molecular marker for Ph-negative 

MPNs, and especially for PV patients. 

The mutations in JAK2, MPL, and CALR are driver mutations, and 

they all activate the JAK2 pathway, but additional recurrent 

somatic mutations in several genes (TET2, ASXL1, DNMT3A, CBL, 

LNK, IDH1/2, IKF1, EZH2, TP53, SRSF2), encoding transcriptional 

and epigenetic regulators and signaling proteins, occur in MPNs. 

These additional mutations modulate disease progression and 

can also occur as primary mutations, but it is now convincingly 

demonstrated that MPNs can be initiated from a single 

JAK2V617F hematopoietic stem cell. 

JAK mutations have also emerged in other hematologic diseases, 

and the majority of the pathogenic mutations in JAK2 (also in 

JAK1 and JAK3) localize in or near the pseudokinase domain. 

To date, somatic frameshift mutations in exon 9 of CALR 

have been identified in a large proportion of JAK2- and 

MPL-negative PMF and ET patients. In a study of 617 PMF 

patients by Rumi et al., 399 (64.7%) carried JAK2V617F, 140 

(22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL 

(W515) mutation, and 53 (8.6%) had nonmutated JAK2, 

CALR, and MPL (so-called triple-negative PMF) [24]. Kim et 

al. investigated mutation profiles of CALR, JAK2, and MPL in 

199 Korean patients with MPNs. The overall frequency of 

CALR mutations was 12.6%; it was most frequent in MPN-U 

cases (37.5%), followed by ET (17.7%) and PMF (14.8%). 

CALR mutations were not found in PV or acute panmyelosis 

with myelofibrosis. CALR and JAK2 or MPL mutations were 

mutually exclusive [25]. Wu et al. also found two kinds of 

CALR mutations, c.1179_1230del and c.1234_1235insTTGTC, 

in Chinese patients with MPNs, and female patients showed 

a predisposition to CALR mutation [26]. Li et al. studied 1088 

Chinese patients with MPNs including ET (n=234) and PMF 

(n=50) without JAK2V617F or MPL exon 10 mutations. CALR 

mutation was detected in 53% of subjects with ET and 56% 

of subjects with PMF, and 152 CALR mutations were identified 

clustering into 15 types, including deletions (n=8), insertions 

(n=3), and complex indels (n=4) [27]. 

In our study, mutations in CALR were present in 12 of 113 patients 

with Ph-negative MPNs (10.6%). Mean while, an overwhelming 

majority (75%) of the CALR mutation pattern still lies in 

c.1179_1230del and c.1234_1235insTTGTC. CALR mutations 

were present in 12 of 50 MPN patients without JAK2 mutations 

(24%). Among patients with ET, those with CALR mutations, as 

compared with those with JAK2V617F mutations, presented with 

significantly higher platelet counts and lower hemoglobin levels. 

Whereas the frequency of CALR mutations in MPNs is quite 

consistent in recent studies, it is unclear whether CALR 

mutations occur in up to 8.3% of patients with MDS (10 of 

120 MDS patients) as reported by Nangalia et al. [2], or are 

infrequent in MDS (none of 73) and AML (none of 254) patients 

as reported by Klampfl et al. [1]. This inconsistency could be 

due to the relatively small number of investigated patients. 

Therefore, in addition to MPNs, the mutation profile of CALR 

in other hematopoietic diseases such as leukemia and MDS has 

been given more attention than in the past. CALR mutations 

were identified in 2 of 527 MDS patients (0.38%). None of 328 

patients with MDS were found to have CALR mutations. Two of 

199 patients with AML following MDS had mutated CALR, and 

the frequency of CALR mutations is very low in MDS, supporting 

the use of CALR mutations as a diagnostic marker for ET and PMF 

patients [28]. Recently in a Chinese study, Cui et al. sequenced 

CALR mutations in 14 patients who met the WHO criteria for 

chronic neutrophilic leukemia (CNL) and found that 1 of 14 CNL 

patients had a CALR mutation (c.1154-1155insTTGTC) [29]. 

No CALR mutations were found in 62 patients with ALL [2]. 

However, little attention has been paid to AML and no data 

about the mutation frequency of CALR in Chinese ALL patients 

have been reported until now. Therefore, we screened 135 

AML patients and 57 ALL patients. However, no CALR exon 9 

mutations were found in any of these patients. Only one of the 

leukemia patients was found to have a CALR SNP, rs143880510. 

To date, detection of CALR mutations in peripheral blood has 

been used as a diagnostic tool in the same way that tests for 

JAK2 mutations have simplified and improved the accuracy of 

diagnosis of patients with MPNs. However, in order to develop 

novel therapeutic drugs, further research is needed to explore 

Table 4. Clinical characteristics of essential thrombocythemia and primary myelofibrosis patients with CALR and JAK2 mutation. 

Characteristics ET PMF 

CALR+ JAK2+ p CALR+ JAK2+ p 

Patients, n 

Males, n 

Age, years 

WBC count (x10 9 /L) 

Hemoglobin (g/L) 

Platelets (x10 9 /L) 

6 

2 

44.0±15.1 a 

7.5±4.1 a 

137±34.2 a 

982±24.2 a 28 

11 

56.2±12.9 a 

14±11.0 a 

145±21.4 a 

515±31.6 a 0.784 

0.001 

0.001 

0.002 

0.001 

5 

4 

59.8±3.6 a 

10.42±3.6 a 

66.5±14.1 a 

171±35.2 a 9 

5 

61.9±7.4 a 

15.12±3.6 a 

150±25.7 a 603±56.2 a 0.58 

0.626 

0.612 

0.001 

0.091 

a : Data are presented as mean ± standard deviation. ET: Essential thrombocythemia, PMF: primary myelofibrosis. 

184



the relationship between the pathogenesis of MPNs and the 

function of CALR. 

Conclusion 

In summary, our data from this cohort of Chinese patients with 

MPNs confirmed that CALR mutations were novel molecular 

markers in JAK2V617F-negative MPNs. Patients with the 

c.1179_1230del and c.1234_1235insTTGTC mutations have 

shown distinct clinical characteristics, but further research is 

required to confirm this result. 

Acknowledgment 

This work was supported by grants from the National Natural 

Science Foundation of China (No. 81470319, No. 81170515). 

Ethics 

Ethics Committee Approval: The present study was approved 

by the Ethics Committee of Qilu Hospital, Shandong University 

(Jinan, China); Informed Consent: Written informed consent 

was obtained from all participants for treatment and the 

cryopreservation of bone marrow and peripheral blood 

according to the Declaration of Helsinki. 

Authorship Contributions 

Medical Practices: Jingyi Wang, Jianguo Hao, Na He; Concept: 

Daoxin Ma, Chunyan Ji; Design: Daoxin Ma, Chunyan Ji; Data 

Collection or Processing: Jingyi Wang, Jianguo Hao, Na He, 

Chunyan Ji, Daoxin Ma; Analysis or Interpretation: Daoxin Ma, 

Chunyan Ji; Literature Search: Jingyi Wang, Jianguo Hao, Na He, 

Chunyan Ji, Daoxin Ma; Writing: Jingyi Wang, Jianguo Hao, Na 

He, Chunyan Ji, Daoxin Ma. 

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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DOI: 10.4274/tjh.2015.0041 


Clinical Features of 294 Turkish Patients with Chronic 

Myeloproliferative Neoplasms 

Miyeloproliferatif Hastalığı Olan 294 Türk Hastanın Klinik Verileri 

Neslihan Andıç 1 , Mustafa Ünübol 2 , Eren Yağcı 3 , Olga Meltem Akay 1 , İrfan Yavaşoğlu 2 , Vefki Gürhan Kadıköylü 2 , Ali Zahit Bolaman 2 

1Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey 

2Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey 

3Osmangazi University Faculty of Medicine, Department of Internal Medicine, Eskişehir, Turkey 

Abstract 

Objective: Myeloproliferative neoplasms (MPNs) share common clonal 

stem cells but show significant differences in their clinical courses. 

The aim of this retrospective study was to evaluate thrombotic and 

hemorrhagic complications, JAK2 status, gastrointestinal and cardiac 

changes, treatment modalities, and survival in MPNs in Turkish 

patients. 

Materials and Methods: Medical files of 294 patients [112 essential 

thrombocythemia (ET), 117 polycythemia vera (PV), 46 primary 

myelofibrosis, and 19 unclassified MPN cases] from 2 different 

universities in Turkey were examined. 

Results: Older age, higher leukocyte count at diagnosis, and 

JAK2 mutation positivity were risk factors for thrombosis. Platelet 

count over 1000x10 9 /L was a risk factor for hemorrhagic episodes. 

Hydroxyurea treatment was not related to leukemic transformation. 

Median follow-up time was 50 months (quartiles: 22.2-81.75) in 

these patients. Patients with primary myelofibrosis had the shortest 

survival of 137 months when compared with 179 months for ET and 

231 months for PV. Leukemic transformation, thromboembolic events, 

age over 60 years, and anemia were found to be the factors affecting 

survival. 

Conclusion: Thromboembolic complications are the most important 

preventable risk factors for morbidity and mortality in MPNs. Drug 

management in MPNs is done according to hemoglobin and platelet 

counts. Based on the current study population our results support 

the idea that leukocytosis and JAK2 positivity are more important risk 

factors for thrombosis than hemoglobin and platelet values. 

Keywords: Myeloproliferative neoplasms, Survival, Thrombosis, 

Treatment 

Öz 

Amaç: Miyeloproliferatif hastalıklar (MPH) ortak klonal bir kök 

hücreden köken almalarına karşın klinik seyirleri belirgin farklılıklar 

göstermektedir. Bu retrospektif çalışmanın amacı MPH’lardaki 

trombotik ve hemorajik komplikasyonların, JAK2 mutasyon durumunun, 

gastrointestinal ve kardiyak değişikliklerin, tedavi şekillerinin ve yaşam 

sürelerinin incelenmesidir. 

Gereç ve Yöntemler: Türkiye’nin iki farklı üniversite hastanesinden 

294 hastanın [112 esansiyel trombositemi (ET), 117 polisitemia vera 

(PV), 46 primer miyelofibozis, 19 sınıflanamayan MPH] kayıtları 

incelenmiştir. 

Bulgular: İleri yaş, tanıda yüksek lökosit sayısı JAK2 mutasyon 

pozitifliği tromboz için risk faktörü olarak bulunmuştur. Trombosit 

sayımının 1000x10 9 /L üzerinde olması kanama komplikasyonları 

açısından risk faktörüdür. Hidroksiüre tedavisi lösemik dönüşümle 

ilişkili bulunmamıştır. Bu hastalarda: Medyan takip süresi 50 ay (22,2- 

81,75 çeyrekler) idi. Primer miyelofibrozisli hastalar ET için 179 ay ve 

PV için 231 ay olan yaşam süreleri ile karşılaştırıldığında 137 ay ile 

en kısa yaşam süresine sahip hastalardır. Lösemik transformasyon, 

tromboembolik olaylar, 60 yaş üstü olmak ve anemi yaşam süresinin 

etkileyen faktörler olarak bulunmuştur. 

Sonuç: Tromboembolik komplikasyonlar MPH’da en önemli önlenebilir 

mortalite ve morbidite nedenidir. İlaç düzenlemeleri çoğunlukla 

hemoglobin ve trombosit sayımlarına göre yapılmaktadır. Bu 

çalışmamızdaki hasta popülasyonundan elde ettiğimiz veriler lökositoz 

ve JAK2 pozitifliğinin hemoglobin ve trombosit sayımlarından daha 

önemli risk faktorleri olduğu savını desteklemektedir. 

Anahtar Sözcükler: Miyeloproliferatif hastalıklar, Sağkalım, Tromboz, 

Tedavi 

Address for Correspondence/Yazışma Adresi: Neslihan ANDIÇ, M.D., 

Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey 

Phone : +90 532 518 22 63 

E-mail : neslihandic@yahoo.com 

Received/Geliş tarihi: January 20, 2015 


187

Andıç N, et al: Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms Turk J Hematol 2016;33:187-195 

Introduction 

According to the revised World Health Organization (WHO) 

classification, BCR-ABL-negative chronic myeloproliferative 

disorders are now referred to as myeloproliferative neoplasms 

(MPNs) [1,2]. MPNs share common clonal stem cells and 

phenotypic differences occur due to different molecules 

affecting signal transduction. The JAK2V617F mutation is an 

acquired point mutation causing valine-to-phenylalanine 

substitution at codon 617 on the JAK2 gene. JAK2V617F will 

be referred to as JAK2 mutation in the text. JAK2 mutations 

affecting the JAK-STAT signal transduction pathway are found 

in 90%-95% of patients with polycythemia vera (PV) [3], 50%- 

70% of patients with essential thrombocythemia (ET), and 

40%-50% of patients with primary myelofibrosis (PMF) [4]. 

JAK2 mutations cannot be used in distinguishing one MPN from 

another but are useful in excluding reactive hematocrit and 

platelet elevations and reactive myelofibrosis. Absence of JAK2 

mutations cannot exclude the diagnosis of PV, ET, or PMF. Some 

clinical criteria and bone marrow findings are required for the 

diagnosis of JAK2-negative MPN [2]. 

Prognosis of MPNs is determined by thromboembolic and 

hemorrhagic complications and progression to myelofibrosis 

and acute leukemia. The cumulative rate of nonfatal thrombosis 

in PV is 3.8 events per 100 persons per year, and in ET the rate 

of fatal and nonfatal thrombotic events ranges from 2% to 4% 

of patient years. Primary myelofibrosis seems less susceptible 

for thrombotic events as the cumulative percentage is 2.23% 

per patient years [5]. Age and previous thrombosis are known 

risk factors for future thrombosis in MPNs. Leukocytosis and 

JAK2 mutation are shown to be additional risk factors. Extreme 

thrombocytosis (count over 1000 or 1500x109/L) was found to 

be related to hemorrhagic complications but not thrombosis [5]. 

Other complications like gastrointestinal ulcers and 

echocardiographic changes are also reported. Their importance 

in the course of the disease is only partially understood [6,7]. 

Hydroxyurea and anagrelide are the most commonly used drugs 

in the treatment of MPN. Hydroxyurea was shown to reduce 

the incidence of thrombotic events in several studies, but there 

is some evidence that it may increase the risk of leukemic 

transformation [8]. Anagrelide is effective in reducing platelet 

counts in ET and PV patients who are resistant or intolerant to 

hydroxyurea. Risk increment of leukemia has not been shown 

for this drug [9]. 

The aim of this study is to evaluate thrombotic and hemorrhagic 

complications, JAK2 status, gastrointestinal and cardiac changes, 

treatment modalities, and survival in MPN cases. 


The medical files of patients diagnosed with Philadelphia 

chromosome-negative chronic myeloproliferative disease 

(CMPD) and MPN between 2003 and 2013 were retrospectively 

examined. Two centers in Turkey entered the study: Eskişehir 

Osmangazi University and Adnan Menderes University. 

Diagnoses were made according to PV Study Group and WHO 

recommendations. The WHO criteria were revised in 2005 after 

the discovery of JAK2 mutations. In the revision of WHO criteria 

made in 2008, ‘CMPD’ was changed to ‘MPN’. Patients with 

significant fibrosis in the bone marrow but otherwise clinically 

diagnosed with ET by the primary clinician were placed in the 

category of unclassified MPN and will be referred to here as 

MPN(u) patients. We included MPN(u) patients in the statistical 

analysis done for the whole patient group, like statistics of risk 

factors for thrombosis. On the other hand, we did not include 

MPN(u) in one-to-one comparisons with the three MPN groups 

(ET, PV, and PMF) because we wanted to compare the patients 

with exact diagnoses. 

The study was approved by the local ethics committees of both 

universities. Patients above the age of 16 at the time of diagnosis 

were enrolled in the study. All consecutively admitted patients 

during the mentioned period were taken into consideration. 

Clinical and laboratory parameters were recorded. JAK2 

status and other cytogenetic abnormalities and bone marrow 

findings were evaluated. Treatment modalities, thrombotic and 

hemorrhagic complications, and gastrointestinal and cardiac 

findings were noted. 

Proteins C and S were studied by the Siemens BCSX coagulometric 

method and antithrombin was studied by Siemens BNII 

nephelometric method. Factor V Leiden and prothrombin gene 

mutations were studied with a Roche 480 II LightCycler by 

the real-time polymerase chain reaction (PCR) method. Bone 

marrow samples were cultured in 24-48 h in standard 10 µg/ 

mL colcemid solution without mitogen in order to perform 

conventional bone marrow cytogenetics. Twenty metaphases 

were evaluated. A locus-specific LSI D20S108 (20q12) probe was 

used for fluorescence in situ hybridization (FISH) analysis and 

200 cells (metaphase/interphase) were evaluated. JAK2V617F 

mutations were studied by real-time PCR method with a Roche 

480 II LightCycler using the TIB Molbiol LightMix Kit. Bone 

marrow aspirates and biopsies were evaluated in the pathology 

and hematology departments of both universities. Increases in 

megakaryocytes and grades of reticulin fibrosis were defined 

according to the WHO classification of tumors [10,11]. 


Statistical tests were performed using IBM SPSS 20.0 for 

Windows and p


Andıç N, et al: Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms 

assessed using Kaplan-Meier analysis and the log-rank test 

was used for univariate comparisons. The effect of prognostic 

factors on survival was analyzed by Cox proportional hazards 

regression models. 

Results 

A total of 294 patients’ medical files were eligible for the study; 

143 (48.6%) patients were female and 151 (51.4%) were male. 

Median age was 60 years (quartiles: 48-79), with a minimum of 

16 and maximum of 84 years. Sex and age were not statistically 

different between patient groups. The number of patients 

diagnosed with ET was 112 (38.1%), with PV was 117 (39.8%), 

and with PMF was 46 (15.6%). Nineteen patients who were 

diagnosed with and treated for ET by the primary physician 

were later classified as having MPN(u). These patients had no 

leukoerythroblastosis in peripheral blood and their median 

platelet count was 1146x109/L (quartiles: 844-1416). All of them 

had megakaryocytic proliferation in their bone marrow and had 

neither dysplasia nor prominent granulocytic and erythroid 

proliferation. Median follow-up time was 68 months (quartiles: 

15-81). Six of them had grade 2-3 and 13 of them had grade 

3 reticulin fibrosis in their bone marrow. We could not classify 

these cases as ET or PMF so we classified them as MPN(u). 

The longest follow-up period was 311 months (median: 43 

months; quartiles: 15.7-77.2). In 103 patients there were 

no comorbidities, while 155 patients had hypertension and/ 

or diabetes mellitus, and 53 patients had diseases including 

chronic obstructive pulmonary disease, liver failure, renal 

failure, congestive heart disease, and arrhythmias including 

atrial fibrillation. One patient had lung cancer and another 

had prostate cancer. Eighty-five patients (28%) were smoking 

cigarettes. The clinical and laboratory characteristics of patients 

are summarized in Table 1. Splenomegaly and hepatomegaly 

were significantly more frequent in PMF than in other MPNs. 

Hemoglobin was significantly higher in PV than in other 

groups and significantly lower in PMF than in other groups. 

Platelet count was higher in ET than in other groups. Lactate 

dehydrogenase (LDH), potassium, and uric acid values were 

higher in PMF compared to other MPN subtypes. Median LDH was 

above normal limits in all study groups. Bone marrow findings 

at diagnosis are summarized in Table 2. Megakaryocytes were 

more prominent in ET and reticulin fibrosis was more profound 

in PMF than in other MPNs, as expected. 

It was found that 58.5% (38 patients) of ET patients, 86.2% 

(50 patients) of PV patients, and 70.6% (12 patients) of PMF 

patients were positive for JAK2 mutation. Patients with PV were 

carrying JAK2 mutations significantly more so than patients with 

ET (p


these cerebral arterial occlusions were seen in ET patients (16 out 

of 37). Coronary artery disease was the second most common 

thrombotic complication (32 out of 108). Ten patients had deep 

vein thrombosis (9.3%). Twelve patients (11.1%) had thrombosis 

in an intraabdominal vein. Ten patients had both arterial and 

venous thrombotic attacks and most of them were ET patients 

(8 out of 10). Diagnostic groups, sex, treatment modality, and 

bone marrow findings did not differ between patients with 

or without thrombosis. Older age, higher leukocyte count at 

diagnosis, and JAK2 mutation positivity were risk factors for 

thrombosis after univariate analysis. Data are shown in Table 3. 

Hemorrhagic complications were seen in 34.4% (n=101) 

of patients. Almost all (94%) patients with hemorrhagic 

complications had mucocutaneous or gastrointestinal tract 

bleeds. There was no statistical significance between MPN 

groups regarding the frequency and the source of bleeding. 

Hemoglobin values were significantly lower in patients with 

hemorrhagic episodes than patients without hemorrhage 

(p=0.012). Medians and quartiles were 13.1 g/dL (10.3-17) 

and 14.6 g/dL (12.7-17.1), respectively. Platelets count over 

1000x10 9 /L was a risk factor for hemorrhagic episodes; 30.1% 

(n=59) of patients with platelet count less than or equal to 

1000x10 9 /L had hemorrhagic events, whereas 42.9% (n=42) of 

patients with platelet count over 1000x10 9 /L had hemorrhagic 

episodes (p=0.030). Leukocyte counts, fibrinogen levels, and 

treatment modalities were not statistically different between 

patients with and without hemorrhage. 

Electrocardiogram results were considered as normal in 78.2% 

(n=230) of cases. Atrial fibrillation was present in 7.4% 

(n=22) and signs of ischemia in 8.2% (n=24) of patients. 

Echocardiography was performed in 95 patients. Forty-one 

(43.2%) of them had cardiac valve abnormalities and 10 (10.5%) 

had pulmonary hypertension. 

Upper gastrointestinal endoscopy was performed in 80 patients. 

Gastritis and duodenitis were frequent findings (56 patients, 

70%). Nine patients (11.3%) had ulcers. Nine patients had 

esophageal varices. Helicobacter pylori testing was done in 56 

patients and 53.6% of them were positive. 

Treatment modalities are shown in Table 4. Hydroxyurea was the 

first choice in ET, PV, and PMF cases. Anagrelide was mainly used 

in ET. Patients who were receiving anagrelide treatment were 

significantly younger than the patients receiving hydroxyurea 

treatment [49.5 years (39.7-63) and 60 years (50-69), respectively, 

p0.05). Thirty-six patients (12.2%) were treated with 

phlebotomy alone, and 159 patients (54%) received any kind of 

Table 2. Bone marrow findings at diagnosis. 

PV ET PMF p-value 

Increased number of megakaryocytes (% within MPNs) PV-ET



cytoreductive therapy along with phlebotomy. Seven patients 

developed acute myeloid leukemia during follow-up. Three 

of them had ET, 2 of them had PV, and 2 of them had MPN(u). 

Patients who developed leukemia were not different from others 

by means of sex, megakaryocyte number, or grade of reticulin 

fibrosis in the initial bone marrow. Among 3 patients with excess 

(>5%) blasts in the initial bone marrow, 1 developed leukemia 

and the other 2 were diagnosed with PMF. Receiving previous 

treatment with hydroxyurea was not found to be a risk factor 

for leukemic transformation. Median follow-up time of patients 

receiving hydroxyurea treatment was 50 months (quartiles: 22.2- 

81.75). One patient with ET previously treated with hydroxyurea 

showed transition to myelofibrosis. 

Forty-four patients died during follow-up. Among diseaserelated 

deaths, thromboembolic events were the main cause for 

ET patients and progression of the disease was the main cause 

for PMF patients. 

Factors affecting survival in MPN are shown in Table 5. Overall 

survival of the PMF patients was shorter than in the other 

MPN groups (p


Discussion 

This retrospective study was aimed at evaluating the 

characteristics of MPNs in Turkish patients. Two centers 

contributed to the study and 294 patients were enrolled. 

Median age was 60 years and the female/male ratio was 0.9. 

These two findings were consistent with the literature [12,13]. 

Although there is knowledge in the literature that ET is more 

common in women and PV more common in men, we did 

not find any difference in sex between MPN groups [3,14]. 

Splenomegaly and hepatomegaly were common findings in 

physical examination. They were seen in almost 80% of patients 

with PMF, more commonly than in the other MPN groups. This 

finding is consistent with the literature, but our frequencies 

in PV and ET are higher than those reported in other studies 

[13,14,15]. Even minimal enlargement in the spleen, like 130 mm 

in a male patient, was noted as splenomegaly in this study. This 

could be the reason for higher splenomegaly rates in ET and PV 

patients compared with the literature. Hemoglobin levels were 

higher in PV and platelet levels were higher in ET, and they were 

both lower in PMF, as expected (p



mutation monitoring during follow-up is not recommended 

[9]. Based on our results and knowledge of the literature, 

JAK2 mutation positivity could be another high-risk factor 

for thrombosis along with age, previous thrombosis, and 

leukocytosis. However, regarding the high frequency of positive 

results, we cannot recommend cytotoxic treatment for every 

JAK2 mutation-positive patient. 

A total of 101 (34.4%) patients had hemorrhagic episodes. 

Almost all episodes were mucocutaneous or gastrointestinal. 

This finding is consistent with the literature [13,22]. The only 

risk factor for hemorrhage was platelet count over 1000x109/L. 

Elliot and Tefferi reached the same result [41]. It is thought 

that during extreme thrombocytosis the reduced levels of 

high-molecular-weight von Willebrand (vW) factor causes 

acquired vW disease, which is responsible for bleeding tendency. 

Frequency of hemorrhagic and thrombotic episodes was virtually 

the same among the MPN groups. 

Hydroxyurea was the most commonly used agent in all MPN 

groups. Anagrelide was almost always used in ET cases. Patients 

with ET who were using anagrelide were significantly younger 

than the other ET patients. Our treatment choices correlated 

nicely with the current recommendations. Hydroxyurea is the 

initial choice of treatment because of its proven efficiency, 

especially in reducing thrombotic risk [42,43]. However, 

hydroxyurea is recommended to be used with caution in young 

patients regarding the data showing a risk increment of leukemia 

in long-term usage of hydroxyurea by Kiladjian et al. [8]. In the 

ANAHYDRET study, it was shown that anagrelide is as effective 

as hydroxyurea [44]. Secondary leukemia has not been reported 

with anagrelide treatment yet. Interferon alpha was the least 

commonly used agent in all MPN groups, probably because of 

its parenteral usage and poor tolerability. In our study, we did 

not find any relation between the complications of MPN and 

the treatment methods. 

Survival was longest in PV and shortest in PMF cases. 

Polycythemia vera has a life expectancy of 10 to 20 years 

according to the literature and our finding was consistent with 

this knowledge [45]. Although we found that patients with 

PMF had the shortest survival, their mean overall survival was 

11.4 years, which is longer than 5.5 years as reported in the 

literature [46]. None of the PMF cases transformed to leukemia 

and this could be one of the reasons for the finding above. 

Life expectancy of ET patients ranges from 13 to 22.3 years 

according to the literature [14,47,48]. In our ET patient group, 

mean overall survival was 14.9 years. Although the survival of 

ET patients was shorter than that of PV patients, this was not 

statistically significant. 

Older age, leukocytosis, and low hemoglobin and high platelet 

count were found to be related to survival in ET patients in the 

literature [48,49]. In our study, patients older than 60 years at 

diagnosis had shorter survival than those younger than 60. We 

also found that anemia (defined as hemoglobin of


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195


DOI: 10.4274/tjh.2015.0047 


Retrospective Study of Incidence and Prognostic Significance 

of Eosinophilia after Allogeneic Hematopoietic Stem Cell 

Transplantation: Influence of Corticosteroid Therapy 

Allojenik Hematopoetik Kök Hücre Nakli Sonrası Eozinofilinin Sıklığı ve Prognostik Öneminin 

Değerlendirildiği Geriye Dönük Çalışma: Kortikosteroid Tedavisinin Etkisi 

Wataru Yamamoto 1 , Eriko Ogusa 1 , Kenji Matsumoto 1 , Atsuo Maruta 1 , Yoshiaki Ishigatsubo 2 , Heiwa Kanamori 1 

1Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan 

2Yokohama City University Graduate Faculty of Medicine, Department of Internal Medicine and Clinical Immunology, Yokohama, Japan 

Abstract 

Objective: The clinical significance of eosinophilia after allogeneic 

hematopoietic stem cell transplantation is controversial. This study 

aimed to retrospectively study the impact of eosinophilia on the 

outcome of allogeneic hematopoietic stem cell transplantation by 

taking into account the influence of corticosteroid therapy. 

Materials and Methods: We retrospectively studied 204 patients 

with acute myeloid leukemia, acute lymphoblastic leukemia, and 

myelodysplastic syndrome who underwent allogeneic hematopoietic 

stem cell transplantation from January 2001 to December 2010. 

Results: The median age was 43 years (minimum-maximum: 17- 

65 years). Myeloablative conditioning was used in 153 patients and 

reduced intensity conditioning was employed in 51 patients. Donor 

cells were from bone marrow in 132 patients, peripheral blood in 34, 

and cord blood in 38. Eosinophilia was detected in 71 patients and 

there was no significant predictor of eosinophilia by multivariate 

analysis. There was no relationship between occurrence of eosinophilia 

and the incidence or grade of acute graft-versus-host disease when 

the patients were stratified according to corticosteroid treatment. 

Although eosinophilia was a prognostic factor for 5-year overall 

survival by univariate analysis, it was not a significant indicator by 

multivariate analysis. 

Conclusion: These results suggest that the clinical significance of 

eosinophilia in patients receiving allogeneic hematopoietic stem cell 

transplantation should be assessed with consideration of systemic 

corticosteroid administration. 

Keywords: Eosinophilia, Allogeneic hematopoietic stem cell 

transplantation, Corticosteroid therapy, Prognostic factor, Graftversus-host 

disease 

Öz 

Amaç: Allojenik hematopoetik kök hücre nakli sonrası eozinofilinin 

önemi tartışmalıdır. Bu çalışma kortikosteroid tedavisinin etkisini 

hesaba katarak, eozinofilinin allojenik hematopoetik kök hücre 

naklinin sonuçları üzerine etkisini geriye dönük değerlendirmeyi 

amaçladık. 

Gereç ve Yöntemler: Ocak 2001’den Aralık 2010’a kadar akut myeloid 

lösemi, akut lenfoblastik lösemi ve myelodisplastik sendrom tanısıyla 

allojenik hematopoetik kök hücre nakli olan 204 hastayı geriye dönük 

değerlendirdik. 

Bulgular: Ortanca yaş 43 (aralık: 17-65 yaş) idi. Yüz elli üç hastada 

miyeloablatif, 51 hastada azaltılmış yoğunluklu hazırlama rejimi 

uygulandı. Kök hücre kaynağı 132 hastada kemik iliği, 34 hastada 

periferik kan ve 38 hastada kordon kanıydı. Yetmiş bir hastada 

eozinofili saptandı ve çoklu değişken analizinde eozinofiliyi anlamlı 

olarak öngörecek bir belirteç saptanmadı. Hastalar kortikosteroid 

tedavisine göre gruplandığında eozinofili gelişimi ile akut graftverus-host 

hastalığı sıklığı ya da derecesi arasında bağlantı yoktu. 

Tek değişkenli analizde eozinofili 5 yıllık genel sağkalım açısından 

prognostik bir faktör olmasına karşın, çok değişkenli analizde anlamlı 

bir belirteç değildi. 

Sonuç: Bu sonuçlar allojenik hematopoetik kök hücre nakli olan 

hastalarda eozinofilinin klinik öneminin, sistemik kortikosteroid 

uygulamasını dikkate alarak değerlendirilmesi gerektiğinin 

düşündürmektedir. 

Anahtar Sözcükler: Eozinofili, Allojenik hematopoetik kök hücre 

nakli, Kortikosteroid tedavisi, Prognostik faktör, Graft-versus-host 

hastalığı 

Address for Correspondence/Yazışma Adresi: Wataru YAMAMOTO, M.D., 

Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan 

Phone : 81-45-391-5761 

E-mail : watadesu@yahoo.co.jp 


Accepted/Kabul tarihi: November 18, 2015 

196


Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation 

Introduction 

Results 

Proliferation of eosinophils is induced by stimulation with 

cytokines [1] and eosinophilia occurs in various clinical 

settings. Eosinophilia is often found in patients receiving 

allogeneic hematopoietic stem cell transplantation (allo-HSCT) 

and a relationship between eosinophilia and the outcome 

and/or graft-versus-host disease (GVHD) has been reported 

[2,3,4,5,6,7,8,9,10]. However, the role of corticosteroid (CS) 

therapy should be taken into consideration with regard to 

evaluation of eosinophilia after allo-HSCT, because it is known 

that eosinophilia is influenced by such drugs [11,12]. Therefore, 

we retrospectively studied the impact of eosinophilia on the 

outcome of allo-HSCT by taking into account the influence of 

CS therapy. 


Patients who underwent allo-HSCT for hematologic malignancies 

from January 2001 to December 2010 at the Kanagawa 

Cancer Center were retrospectively investigated. We defined 

eosinophilia as a peripheral blood eosinophil count of >500 

µL on more than one occasion, while systemic steroid therapy 

meant CS administration at more than 0.5 mg/kg/day within 

100 days after allo-HSCT. Standard-risk disease was defined as 

acute myeloid leukemia (AML)/acute lymphoblastic leukemia 

(ALL) in the first or second remission and myelodysplastic 

syndrome (MDS) without leukemic transformation, while highrisk 

disease was defined as all others. Grading of acute GVHD 

was done according to established criteria [13]. 


Statistical analyses were performed with R software (version 

2.11.1; R Development Core Team). Differences between groups 

were analyzed by the Wilcoxon rank sum test or Fisher’s exact 

test, as was appropriate for univariate analysis and logistic 

regression analysis for multivariate analysis. Overall survival 

(OS) was calculated from the date of transplantation to the 

date of death from any cause or the date of last follow-up. 

Non-relapse mortality was defined as death without disease 

relapse or resistance. Time-to-event curves were drawn 

according to the Kaplan-Meier method and the statistical 

significance of differences in survival was assessed by the 

log-rank test. Prognostic factors included age, sex mismatch, 

disease risk, conditioning regimen, GVHD prophylaxis, donor 

type, cytomegalovirus infection, CS therapy, and eosinophilia. 

Either the Cox proportional hazard model or the Fine-Gray 

proportional hazard model was used for analysis. Death without 

relapse was considered to be a competing risk for relapse, relapse 

was a competing risk for non-relapse mortality, and relapse and 

death without GVHD were competing risks for GVHD. 

A total of 204 patients received allo-HSCT for AML, ALL, or MDS. 

The median follow-up period was 5.7 years and patients’ clinical 

characteristics are shown in Table 1. The median age was 43 

years (minimum-maximum: 17-65 years) and there were 102 

patients of each sex. The underlying disease was AML in 126 

patients, ALL in 63, and MDS in 15. Myeloablative conditioning 

was used in 153 patients and reduced intensity conditioning was 

employed in 51 patients. Donor cells were from bone marrow in 

132 patients, peripheral blood in 34, and cord blood in 38. 

Eosinophilia was detected in 71 patients (34.8%). Its appearance 

was associated with total body irradiation (TBI), unrelated donor, 

and CS administration within 100 days after transplantation 

by univariate analysis. However, no significant predictors of 

eosinophilia were identified by multivariate analysis (Table 1). 

Among the 204 patients, 90 patients (44%) received systemic 

CS therapy. The reason for CS treatment was acute GVHD in 

76 patients, engraftment syndrome in 4, interstitial pneumonia 

in 4, organizing pneumonia in 3, disease relapse in 1, diffuse 

alveolar hemorrhage in 1, and vasculitis in 1. The incidence of 

eosinophilia within 100 days after transplantation was higher in 

patients without CS (47/114 patients, 41.2%) than in patients 

with CS (24/90, 26.7%) (p=0.038). Among the 90 patients with 

CS, 11 were first given CS therapy after the appearance of 

eosinophilia. The frequency of eosinophilia was higher among 

patients who were not given CS therapy before eosinophilia 

appeared than among patients who were already receiving CS 

therapy (58/125 patients, 46.4% vs. 13/79, 16.5%, respectively; 

p

Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 

OS between patients with and without eosinophilia among 

those not receiving CS therapy (66.3%, 95% CI: 53.6-82.1 vs. 

57.4%, 95% CI: 46.5-70.8, respectively; p=0.139) (Figure 3A). 

Furthermore, the cumulative incidence of relapse showed no 

significant association with eosinophilia (Figures 1B, 2B, and 

3B). However, non-relapse mortality was significantly higher 

in patients receiving CS therapy (Figures 2C and 3C). According 

to univariate analysis, eosinophilia was a good prognostic 

indicator for 5-year OS (hazard ratio: 0.6, 95% CI: 0.4-0.9; 

p=0.017), but it was not an independent prognostic indicator 

by multivariate analysis (hazard ratio: 0.8, 95% CI: 0.5-1.3; 

p=0.385). Finally, high-risk disease, unrelated donor, and CS 

therapy were adverse prognostic indicators for 5-year OS 

according to multivariate analysis (Table 3). 

Discussion 

In the present study, we investigated the incidence and clinical 

implications of eosinophilia occurring within 100 days after allo- 

HSCT, and we also analyzed the prognostic value of eosinophilia 

in relation to the influence of CS administration. The incidence of 

eosinophilia (34.8%) in our patient cohort was comparable with 

that in previous reports, although the definition of eosinophilia 

varies among studies. It is well known that a decrease of 

eosinophils is caused by CS administration [10,11]; hence, we 

assessed the clinical implications of eosinophilia in relation to 

systemic CS administration. The incidence of eosinophilia was 

significantly lower in patients receiving CS treatment compared 

with that for those without CS treatment in the present 

study, and the same result for patients with acute GVHD has 

already been described [2]. In our study, MDS, use of TBI, and 

transplantation from an unrelated donor were also associated 

with a lower incidence of eosinophilia, but the reasons for these 

associations are unknown. 

The relationship between eosinophilia and acute GVHD after allo- 

HSCT remains controversial. We could not find any association 

between eosinophilia and acute GVHD among patients with or 

without CS therapy in this study. Some previous reports suggested 

that eosinophilia is significantly related to the onset of acute GVHD 

[2,9,10]. However, Aisa et al. reported that the onset of eosinophilia 

within 100 days after allo-HSCT is associated with a lower rate of 

grade II-IV acute GVHD (43% vs. 98%; p



Table 1. Patient characteristics and predictors of eosinophilia within 100 days after transplantation. 

Eosinophilia (+) Eosinophilia (-) Univariate Analysis Multivariate Analysis 

(n=71) (n=133) HR (95% CI) p HR (95% CI) p 

Age. median (range) 43 (17-65) 43 (18-64) 0.541 

Sex 

Male 31 71 1.0 - 

Female 40 62 1.5 (0.8-2.8) 0.240 - - 

Disease 

Acute myelogenous leukemia 45 81 1.0 - 

Acute lymphoblastic leukemia 25 38 1.2 (0.6-2.3) 0.633 - - 

Myelodysplastic syndrome 1 14 0.1 (0.0-0.9) 0.022 - - 

Disease risk at transplantation 

Standard risk 51 77 1.0 - 

High risk 20 56 0.5 (0.3-1.0) 0.068 - - 

Conditioning regimen 

Myeloablative 49 104 1.0 - 

Reduced-intensity 22 29 1.6 (0.8-3.2) 0.175 - - 

Total body irradiation 

No 14 9 1.0 1.0 

Yes 57 124 0.3 (0.1-0.8) 0.009 0.4 (0.2-1.0) 0.058 

GVHD prophylaxis* 

Cyclosporine+sMTX 25 35 1.0 - 

Tacrorimus+sMTX 45 98 0.6 (0.3-1.3) 0.196 - - 

Donor type 

Related 37 49 1.0 1.0 

Unrelated 34 84 0.5 (0.3-1.0) 0.039 0.7 (0.4-1.3) 0.236 

Stem cell source 

Bone marrow 49 83 1.0 - 

Peripheral blood 13 21 1.0 (0.4-2.4) 1.000 - - 

Cord blood 9 29 0.5 (0.2-1.3) 0.174 - - 

HLA disparity* 

Match 51 86 1.0 - 

Mismatch 19 47 0.7 (0.3-1.3) 0.272 - - 

Cytomegarovirus † 

Other 61 122 1.0 - 

Recipient negative and donor positive 6 4 2.9 (0.7-14.9) 0.098 - - 

Sex mismatch 

Other 61 108 1.0 - 

Female to male 10 25 0.7 (0.3-1.7) 0.441 - - 

Acute GVHD 

Grade 0-I 42 70 1.0 - 

Grade II-IV 29 63 0.8 (0.4-1.4) 0.381 - - 

CS administration 100 days after transplantation 

No 47 67 1.0 1.0 

Yes 24 66 0.5 (0.3-1.0) 0.038 0.6 (0.3-1.1) 0.090 

*Data is uncertain in one case. † Data is uncertain in eleven cases. HR: Hazard ratio, CI: confidence interval, GVHD: graft-versus-host disease, sMTX: short-term methotrexate, 

CS: corticosteroid. 

199

Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation Turk J Hematol 2016;33:196-201 

Only one study showed that there was no correlation between 

eosinophilia and the outcome of cord blood transplantation in 

adults [8]. In the present study, eosinophilia was associated with a 

better outcome by univariate analysis, but this was not confirmed 

by multivariate analysis. Since the incidence of eosinophilia 

differs among patients with or without CS treatment, we also 

analyzed its effect on prognosis in patients stratified according 

to systemic CS administration, but there was no significant 

impact of eosinophilia on the outcome in either group. Finally, 

multivariate analysis showed that high-risk disease, unrelated 

donor, and CS therapy were adverse predictors of survival with 

statistical significance. However, there is a limitation in that 

we did not treat eosinophilia and CS administration as timedependent 

covariates in multivariate analyses. 

Table 2. Distribution of patients with acute graft-versus-host disease. 

Acute GVHD Corticosteroid (+) (n=90) Corticosteriod (-) (n = 114) 

Grade 

Eosinophilia (+) 

(n=24) 

0 2 4 

Eosinophilia (-) 

(n=66) 

p 

Eosinophilia (+) 

(n=47) 

21 36 

I 2 6 17 24 

II 13 26 0.709 8 7 

III 6 24 1 0 

IV 1 6 0 0 

GVHD: Graft-versus-host disease. 

Eosinophilia (-) 

(n=67) 

p 

0.424 

Table 3. Prognostic factors for overall survival. 

Variables 

Univariate Analysis 

Multivariate Analysis 

HR (95% CI) p HR (95% CI) p 

Age (years) 



Especially after allo-HSCT, systemic CS administration is done 

in patients who develop various complications, such as acute 

GVHD or pulmonary complications. Imahashi et al. reported that 

eosinophilia has an independent influence on the prognosis 

of patients with acute GVHD receiving CS treatment, but not 

that of patients without CS treatment [2]. Taking our results 

together with these findings raises the possibility that the 

severity of acute GVHD and CS therapy for GVHD may strongly 

influence transplantation outcomes regardless of eosinophilia. 

Furthermore, systemic CS administration is often done for allo- 

HSCT patients with severe complications in addition to acute 

GVHD, and eosinophilia may be suppressed in those patients. 

Our findings about non-relapse mortality in the presence or 

absence of CS treatment support this interpretation. 

In addition to the relationship between eosinophilia and acute 

GVHD, there have been several reports on the pathogenesis of 

eosinophilia in the setting of allo-HSCT. Akhtari et al. reported 

that eosinophilia is observed in patients with eosinophilic 

pulmonary syndrome after allo-HSCT [14], but there were no 

specific causes of eosinophilia in our cohort. 

In conclusion, eosinophilia after allo-HSCT was not related 

to the outcome of transplantation or the incidence of acute 

GVHD in patients with or without systemic CS therapy, although 

this study had some limitations because it was a retrospective 

investigation conducted at a single institution. 

Acknowledgment 

This study was supported by a grant from the Kanagawa Health 

Foundation. 

Ethics 

Ethics Committee Approval: Retrospective study; Informed 

Consent: It was taken. 


Surgical and Medical Practices: Wataru Yamamoto, Heiwa 

Kanamori; Concept: Wataru Yamamoto, Heiwa Kanamori; 

Design: Wataru Yamamoto, Heiwa Kanamori; Data Collection or 

Processing: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, 

Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Analysis or 

Interpretation: Wataru Yamamoto, Heiwa Kanamori; Literature 

Search: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, 

Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Analysis or 

Interpretation: Wataru Yamamoto, Heiwa Kanamori; Literature 

Search: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, 

Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Writing: 

Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo 

Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori. 




included. 

References 

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2. Imahashi N, Miyamura K, Seto A, Watanabe K, Yanagisawa M, Nishiwaki S, 

Shinba M, Yasuda T, Kuwatsuka Y, Terakura S, Kodera Y. Eosinophilia predicts 

better overall survival after acute graft-versus-host-disease. Bone Marrow 

Transplant 2010;45:371-377. 

3. Aisa Y, Mori T, Nakazato T, Shimizu T, Yamazaki R, Ikeda Y, Okamoto S. Blood 

eosinophilia as a marker of favorable outcome after allogeneic stem cell 

transplantation. Transpl Int 2007;20:761-770. 

4. Kim DH, Popradi G, Xu W, Gupta V, Kuruvilla J, Wright J, Messner HA, 

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impact on transplant outcomes afterallogeneic peripheral blood stem cell 

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5. Sato T, Kobayashi R, Nakajima M, Iguchi A, Ariga T. Significance of 

eosinophilia after stem cell transplantation as a possible prognostic marker 

for favorable outcome. Bone Marrow Transplant 2005;36:985-991. 

6. Nakane T, Nakamae H, Hirose A, Nakamae M, Koh H, Hayashi Y, Nishimoto 

M, Umemoto Y, Yoshimura T, Bingo M, Okamura H, Yoshida M, Ichihara H, 

Aimoto M, Terada Y, Nakao Y, Ohsawa M, Hino M. Eosinophilia, regardless of 

degree, is related to better outcomes after allogeneic hematopoietic stem 

cell transplantation. Intern Med 2012;51:851-858. 

7. Ahmad I, Labbé AC, Chagnon M, Busque L, Cohen S, Kiss T, Lachance S, Roy 

DC, Sauvageau G, Roy J. Incidence and prognostic value of eosinophilia 

in chronic graft-versus-host disease after nonmyeloablative hematopoietic 

cell transplantation. Biol Blood Marrow Transplant 2011;17:1673-1678. 

8. Tomonari A, Takahashi S, Ooi J, Tsukada N, Konuma T, Kato S, Kasahara 

S, Iseki T, Tojo A, Asano S. Blood eosinophilia after unrelated cord blood 

transplantation for adults. Bone Marrow Transplant 2008;42:63-65. 

9. Basara N, Kiehl MG, Fauser AA. Eosinophilia indicates the evolution to acute 

graft-versus-host disease. Blood 2002;100:3055. 

10. McNeel D, Rubio MT, Damaj G, Emile JF, Belanger C, Varet B, Brousse N, 

Hermine O, Buzyn A. Hypereosinophilia as a presenting sign of acute 

graft-versus-host disease after allogeneic bone marrow transplantation. 

Transplantation 2002;74:1797-1800. 

11. Walsh GM. Eosinophil apoptosis: mechanisms and clinical relevance in 

asthmatic and allergic inflammation. Br J Haematol 2000;111:61-67. 

12. Schleimer RP. Effects of glucocorticosteroids on inflammatory cells relevant 

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69. 

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14. Akhtari M, Langston AA, Waller EK, Gal AA. Eosinophilic pulmonary 

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201


DOI: 10.4274/tjh.2014.0469 


Cytokine Contents in Chronic Lymphocytic Leukemia: Association 

with ZAP70 Expression 

Kronik Lenfositik Lösemi Sitokin İçeriği: ZAP70 Ekspresyonu ile İlişkisi 

Nilgün Işıksaçan 1 , Suzan Çınar 2 , Esin Aktaş Çetin 2 , Melih Aktan 3 , Günnur Deniz 2 

1Bakırköy Dr. Sadi Konuk Training and Research Hospital, Central Laboratory, İstanbul, Turkey 

2İstanbul University Institute of Experimental Medicine, Department of Immunology, İstanbul, Turkey 

3İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 

Abstract 

Objective: Chronic lymphocytic leukemia (CLL) is a disease that shows 

varying clinical progression, and expression of the protein tyrosine 

kinase ZAP70 has been described as a very valuable prognostic 

factor. Patients with ZAP70 positivity are characterized by worse 

clinical course and significantly shorter progression-free and overall 

survival. In this study, intracytoplasmic interferon gamma (IFN-γ) 

and interleukin-4 (IL-4) content of T, B, and CLL cells in CLL patients 

and their correlations with Rai staging and ZAP70 positivity were 

investigated. 

Materials and Methods: CLL patients newly diagnosed or in followup 

at the İstanbul University İstanbul Medical Faculty Hematology 

Department were included in this study. These patients were classified 

according to Rai staging and ZAP70 expression. IL-4, IFN-γ, and ZAP70 

expressions in peripheral blood T, B, and CLL cells were measured by 

four-color flow cytometry. 

Results: There was a statistically significant correlation between 

advanced disease and ZAP70 positivity. IL-4-secreting T cells were 

significantly increased; however, IFN-γ secretion was significantly 

decreased in CLL patients compared to healthy individuals, whereas 

IL-4-secreting B cells were significantly diminished in contrast to T 

cells. 

Conclusion: These findings suggest damage in the cellular immunity 

and that IL-4 might lead to many complications and may be important 

in disease progression. 

Keywords: ZAP70, Interleukin-4, Interferon gamma, T cells, B cells, 

Chronic lymphocytic leukemia 

Öz 

Amaç: Kronik lenfositik lösemi (KLL) klinik olarak çok farklı seyir 

gösterebilen bir hastalıktır ve tirozin kinaz ZAP70’in ekspresyonu, 

çok değerli bir prognostik faktör olarak tanımlanmıştır. ZAP70 pozitif 

hastalar, kötü klinik seyir, belirgin olarak kısa hastalıksız geçen süre ve 

düşük sağkalım oranı ile karakterizedir. Bu çalışmada KLL hastalarının 

T, B ve KLL hücrelerinde intrasitoplazmik interferon gama (IFN-y) ve 

interlökin-4 (IL-4) içeriği, Rai evrelemesi ve ZAP70 pozitifliği arasındaki 

korelasyon araştırılmıştır. 

Gereç ve Yöntemler: Bu çalışmaya İstanbul Üniversitesi İstanbul Tıp 

Fakültesi Hematoloji Kliniği’nde tanısı yeni konan veya takip edilmekte 

olan KLL hastaları dahil edilmiştir. Bu hastalar Rai evreleme sistemi 

ve ZAP70 ekspresyonlarına göre sınıflandırılmıştır. Periferik kan T, B 

ve KLL hücrelerinin IL-4, IFN-g ve ZAP70 ekspresyonları 4 renkli flow 

sitometri ile ölçülmüştür. 

Bulgular: Hastalığın evresindeki ilerleme ile ZAP70 pozitifliği 

arasında istatistiksel olarak anlamlı bir korelasyon bulunmaktadır. 

KLL hastalarının sağlıklı bireylerle kıyaslandığında IL-4 salgılayan T 

hücreleri sayısı anlamlı olarak artmış, ancak IFN-g salgıları anlamlı 

olarak azalmış, IL-4 salgılayan B hücre sayısı ise T hücrelerinin aksine 

anlamlı olarak azalmıştır. 

Sonuç: Bu bulgular, hücresel immünitede hasarın olabileceğini ve 

IL-4’ün birçok komplikasyonlara yol açarak hastalığın ilerlemesinde 

önemli olabileceğini düşündürtmektedir. 

Anahtar Sözcükler: ZAP70, Interlökin-4, Interferon gama, T hücreleri, 

B hücreleri, Kronik lenfositik lösemi hücreleri 

Address for Correspondence/Yazışma Adresi: Günnur DENİZ, PhD., 

İstanbul University Institute of Experimental Medicine, Department of Immunology, İstanbul, Turkey 

Phone : +90 212 414 20 00 (pbx) 33306 

E-mail : gdeniz@istanbul.edu.tr 

Received/Geliş tarihi: December 06, 2014 

Accepted/Kabul tarihi: April 20, 2015 

202


Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia 

Introduction 

Chronic lymphocytic leukemia (CLL) is a disease that shows 

varying clinical progression. The Rai and Binet classification 

systems are useful to predict treatment requirements and 

survival rates for CLL patients, but current classifications fail 

to distinguish patients who may develop aggressive disease 

[1,2,3,4]. The somatic mutations in the immunoglobulin heavy 

chain variable (IGVH) region are very valuable prognostic factors 

and, in CLL cases, IGVH-mutated patients have a better clinical 

outcome while nonmutated patients have a poorer prognosis 

and impaired response to chemotherapy [5,6,7]. 

The protein tyrosine kinase zeta-associated protein (ZAP70) is a 

protein tyrosine kinase in the T cell receptor signal transduction 

system that can be detected in CLL cells. Studies have pointed 

out a correlation between ZAP70 expression in CLL cells and 

IGVH status, and showed that ZAP70 positive patients have an 

aggressive course, an immediate treatment requirement, longer 

therapy time, and lower survival rates [8,9,10]. Expression of 

ZAP70 has been described as a very valuable prognostic factor 

[2,8]. 

Many researchers have recently examined the cytokine content 

of the T cells in CLL patients and emphasized the significance 

of the T cell activity in the prognosis of the disease [11,12]. 

Interleukin-4 (IL-4) production by T cells has been shown 

to significantly increase in patients with progressive disease 

[11,12]. In light of these data, it was suggested that in CLL, the 

type 1 T cell cytokine profile is converted to the type 2 T cell 

profile in the advanced stages of the disease [13]. The aim of 

this study was to evaluate the expression of ZAP70 changing 

during disease progression, the intracellular interferon gamma 

(IFN-γ) and IL-4 content of T and B lymphocytes and the CLL cell 

subset (CD5 + CD19 + ) in CLL patients and healthy subjects, and 

ZAP70 correlation with cytokine production. 


Study Population 

Twenty-eight patients in follow-up at the İstanbul University 

İstanbul Medical Faculty Hematology Department were included 

in the study. Patients were diagnosed according to the CLL 

diagnosis criteria published in 1996 by a study group supported 

by the National Cancer Institute. Clinical data and follow-up 

files of all the patients included in the study were gathered. All 

patients received written information about the study, including 

ethics committee approval, before the study was initiated. 

Twenty-eight CLL patients (18 males, 10 females) and 10 healthy 

individuals (3 males, 7 females) were involved in this study 

(Table 1). The ZAP70 expression was measured for all CLL cases 

and cytokine levels were measured for 12 CLL cases and the 10 

healthy volunteers. The ages of the patients were between 36 

and 81 (59±11) years, and the mean values were 60±12 years for 

males and 56±7 years for females. 

Peripheral Blood Mononuclear Cell Isolation and Flow Cytometric 

Analyses of Lymphocyte Subsets 

Samples were processed using a whole-blood lysis method 

to analyze lymphocyte subsets. Heparinized blood samples 

were collected from patient and healthy donors and stained 

with anti-CD19-fluorescein isothiocyanate (FITC), anti-CD3- 

allophycocyanin (APC), anti-CD5-TRI-COLOR (TC), anti-CD45- 

FITC, anti-CD14-phycoerythrin (PE), anti-CD23-PE, anti-CD38- 

PE, and PE, FITC, TC, or APC conjugated isotype control (IC) 

monoclonal antibodies (mAbs) (all from Caltag Laboratories, 

Austria) for 30 min at room temperature. Lysis was performed 

with FACS Lysing Solution (BD Biosciences, USA). After washing 

cells with phosphate-buffered saline (PBS), stained cells were 

fixed in 1% paraformaldehyde and the cells were analyzed with 

BD FACSCalibur with CellQuest Software (BD Biosciences). 

For the intracytoplasmic ZAP70 expression, the cells were labeled 

with anti-CD19-FITC and anti-CD5-TC mAbs (Caltag Laboratories) 

for 30 min at room temperature. After the lysing period, cells 

were washed with PBS and were fixed and permeabilized with 

paraformaldehyde/saponin solution (Cytofix&Cytoperm Kit, BD 

Biosciences), and then were stained with PE-conjugated-IgG1 

or PE-labeled ZAP70 (Caltag Laboratories) mAbs for 30 min 

at room temperature and analyzed by flow cytometry. Each 

analysis was performed using at least 5000 cells gated in the 

region of the B cell population, and the cells were analyzed 

with BD FACSCalibur with CellQuest Software (BD Biosciences). 

ZAP70 expression was investigated in CLL patients for positivity; 

20% was used as the cut-off [9]. 

Table 1. Demographic and clinical characteristics of patient 

group. 

Variables 

CLL (n=28) 

Age, years 59±11 

Sex, female, n (%) 10 (35.7) 

Sex, male, n (%) 18 (64.3) 

Rai stage 0-1, n (%) 15 (43.9) 

Rai stage 2-4, n (%) 13 (19.3) 

Zap70 expression, % 23.5 

CD38 expression, % 5.9 

CD23 (n=10), % ≥20 

Values are presented as mean ± standard deviation, median (interquartile range), or 

number or percentage of patients. CLL: Chronic lymphocytic leukemia. Rai staging: 

Stage 0: lymphocytosis, Stage 1: lymphocytosis with lymphadenopathy; Stage 2: 

lymphocytosis with either hepatomegaly or splenomegaly, Stage 3: lymphocytosis 

and anemia (hemoglobin

Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia Turk J Hematol 2016;33:202-208 

CD19+ cells were analyzed for the expression of CD5, CD38, 

and ZAP70 (Figure 1A). Most of the CD19+ B cells expressed 

CD5; however, there was only one patient expressing both CD5 

and CD38 (Figures 1B and 1C, respectively). Expression of CD5 

showed heterogeneity among the patients. Intracytoplasmic 

ZAP70 together with CD5 was expressed in different levels in 

CD19+ B cells (Figures 1D, 1E, and 1F). 

Intracytoplasmic Cytokine Staining 

Peripheral blood mononuclear cells (PBMCs) were separated 

using Ficoll-Hypaque (Sigma Chem. Co., USA) density gradient 

centrifugation, and cells were washed twice in Hank’s 

balanced salt solution. The cells were finally adjusted to a final 

concentration of 1x10 6 cells/mL in complete RPMI-1640 medium 

(Sigma Chem. Co.) supplemented with 10% heat-inactivated 

fetal calf serum, penicillin (100 U/mL), streptomycin (100 mg/ 

mL), gentamicin (50 mg/mL), and 50 µM 2-mercaptoethanol. 

Freshly purified PBMCs were washed and 1x106 cells/mL were 

stimulated for 18 h by a combination of phorbol ester, phorbol- 

12-myristate-13-acetate (50 ng/mL), and Ca2+ ionophore 

(ionomycin, 250 ng/mL) in a 24-well round-bottom plate (all 

from Sigma Chem. Co.). The combination of these two stimuli 

was used to achieve the strongest stimulus for intracytoplasmic 

cytokine secretion. Monensin (Sigma Chem. Co.) was added at 

a final concentration of 1 µM to the cultures in the final 4 h. 

After incubation, PBMCs were washed with PBS solution and 

stained with anti-CD19-PE and anti-CD3-APC mAbs for 30 min 

(Caltag Laboratories) for determining the cytokine secretion 

of T and B cells. Cells were washed with PBS and then fixed 

and permeabilized with paraformaldehyde/saponin solution 

(Cytofix&Cytoperm Kit, BD Biosciences). After washing, the 

cells were stained with FITC conjugated IC (IgG1), anti-IL-4- 

FITC, and anti-IFN-γ-FITC (Caltag Laboratories) mAbs for 30 min 

at room temperature. After washing, cells were resuspended in 

1% paraformaldehyde (Sigma Chem. Co.) and analyzed by flow 

cytometry. Each sample was acquired with a BD FACSCalibur 

(BD Biosciences) and analyzed with the instrument’s operating 

software, CellQuest (BD Biosciences). 


Statistical analysis was performed using a standard nonparametric 

Mann-Whitney U test using SPSS 17.0 for Windows. The results 

are presented as median values and p0.05). Seventeen patients were 

SSC 

Figure 1. Gating strategy of chronic lymphocytic leukemia. Peripheral blood mononuclear cells from chronic lymphocytic leukemia 

patients (n=28) were stained for CD5, CD19, CD38, and ZAP70 mAbs and analyzed by flow cytometry. CD19 + cells were gated versus 

SSC (A). Representative dot-plot analyses for the expression of a patient negative (B) and positive (C) for CD38 with CD5 + in CD19 + 

cells are shown. In the CD19 + B cell population, CD5 + ZAP70 + (D, E) and CD5 + ZAP70- (F) plots from three different patients with chronic 

lymphocytic leukemia are also indicated. The numbers indicate the proportion of cells positive for indicated markers. 

204



positive for ZAP70; 8 of them were Rai 0-1 and 9 of them were 

Rai 2-4. However, 7 patients negative for ZAP70 were Rai 0-1 

and 4 patients for Rai 2-4. When patients are divided into two 

groups according to Rai staging system, the first group includes 

15 patients in stages 0 and 1, and the second group includes 

13 patients in stages 2 and 4. The difference between ZAP70 

expression in the first and second Rai groups was statistically 

significant (p


Discussion 

Chronic lymphocytic leukemia is leukemia of small, mature 

B cells; it mostly affects adults over 65 years of age and it is 

the most common form of lymphoid malignancy. The Rai and 

Binet classification systems are useful to predict treatment 

requirements and survival for CLL patients, but the prognostic 

value of these classifications is limited in early stage cases. 

From 30% to 40% of patients in the early stage may develop 

aggressive disease and die in a short time period [15,16]. 

It has been shown that for ZAP70 positivity, when a cut-off 

value of 20% was used, CLL patients could be classified into 

two groups: those with levels of



the disease. On the other hand, IL-4 can lead to the release of 

the growth factors that are increased in the cytoplasm of CLL 

cells. 


This work was supported by the Scientific Research Projects 

Coordination Unit of İstanbul University, project number 

T697/30062005. It was presented at the 20 th National 

Immunology Congress held in Cyprus in 2009 (best poster 

award, poster reference no. 97). 

Ethics 

Ethics Committee Approval: The study protocol was approved 

by the local ethical committee; Informed Consent: It was taken. 


Concept and Design: Melih Aktan, Günnur Deniz, Nilgün 

Işıksaçan; Data Collection or Processing: Melih Aktan, Nilgün 

Işıksaçan; Analysis: Suzan Çınar, Nilgün Işıksaçan, Esin Aktaş 

Çetin; Interpretation: Suzan Çınar, Nilgün Işıksaçan, Günnur 

Deniz; Literature Search: Nilgün Işıksaçan; Writing: Nilgün 

Işıksaçan, Suzan Çınar, Esin Aktaş Çetin, Melih Aktan, Günnur 

Deniz. 




included. 

Financial Disclosure: This work was supported by the Scientific 

Research Projects Coordination Unit of İstanbul University, 

project number T697/30062005. 

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DOI: 10.4274/tjh.2014.0214 


Finding the Optimal Conditioning Regimen for Relapsed/ 

Refractory Lymphoma Patients Undergoing Autologous 

Hematopoietic Cell Transplantation: A Retrospective Comparison 

of BEAM and High-Dose ICE 

Otolog Hematopoetik Kök Hücre Nakli Yapılan Nüks/Dirençli Lenfoma Hastalarında BEAM ve 

Yüksek Doz ICE Rejimlerinin Geriye Dönük Karşılaştırılması 

Onur Esbah 1 , Emre Tekgündüz 2 , Itır Şirinoğlu Demiriz 2 , Sinem Civriz Bozdağ 2 , Ali Kaya 2 , Ayşegül Tetik 2 , Ömür Kayıkçı 2 , Gamze Durgun 2 , 

Şerife Kocubaba 2 , Fevzi Altuntaş 2 

1Ankara Oncology Hospital, Clinic of Medical Oncology, Ankara, Turkey 

2Ankara Oncology Hospital, Hematology and Stem Cell Transplantation Unit, Ankara, Turkey 

Abstract 

Objective: High-dose chemotherapy followed by autologous 

hematopoietic stem cell transplantation (AHCT) is a well-defined 

treatment modality for relapsed/refractory non-Hodgkin’s lymphoma 

(NHL) and Hodgkin’s lymphoma (HL). Although there are several options 

in terms of conditioning regimens before AHCT, no one treatment is 

accepted as a standard of care. This study aimed to compare different 

conditioning regimens for the treatment of NHL and HL. 

Materials and Methods: Medical records of 62 patients who had 

undergone AHCT following BEAM (BCNU, etoposide, cytarabine, and 

melphalan) and high-dose ICE (hICE; ifosfamide, carboplatin, and 

etoposide) conditioning regimens were analyzed retrospectively and 

compared in terms of efficacy and adverse effects. 

Results: The study included a total of 29 and 33 patients diagnosed 

with relapsed/refractory NHL and HL, respectively. Patients received 

BEAM (n=37) or hICE (n=25) regimens for conditioning. One-year 

overall survival was 73±6% in all patients. One-year overall survival 

was 71±8% and 74±9% in the BEAM and hICE groups, respectively 

(p=0.86). The incidences of nausea/vomiting (grade ≥2) (84% vs. 

44.7%; p=0.04) and mucositis (grade ≥2) (13% vs. 3%; p=0.002) were 

higher in the hICE group compared to the BEAM group. In addition, 

we witnessed significantly more hepatotoxicity of grade ≥2 (40% 

vs. 2.7%; p

Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE Turk J Hematol 2016;33:209-215 

Introduction 

About 50% and 20% of patients presenting with non-Hodgkin’s 

lymphoma (NHL) and Hodgkin’s lymphoma (HL) will not be 

cured after initial combination chemotherapy, respectively 

[1,2]. High-dose chemotherapy combined with autologous 

hematopoietic stem cell transplantation (AHCT) is an accepted 

treatment option for relapsed/refractory chemosensitive NHL/ 

HL patients [3,4]. Predictive markers for post-AHCT outcome 

are chemosensitivity, number of chemotherapy lines before 

AHCT, disease status at the time of AHCT, relevant prognostic 

scores for histological subtypes of lymphoma, and time of 

relapse following first-line therapy (12 

months) [5,6,7,8]. The best conditioning regimen before AHCT 

in patients with relapsed/refractory lymphoma is an undefined 

issue. Commonly used regimens in this scenario are BEAM 

(BCNU, etoposide, cytarabine, and melphalan) [8,9], BEAC 

(BCNU, etoposide, cytarabine, cyclophosphamide) [9], highdose 

ICE (hICE; ifosfamide, carboplatin, and etoposide) [10], 

CMV (cyclophosphamide, melphalan, and etoposide) [11], 

CBV (cyclophosphamide, BCNU, and etoposide), combination 

regimens including total body irradiation (TBI) [12], and 

rituximab or I 131-tositumomab combined with BEAM [13]. 

Few studies were reported comparing conditioning regimens in 

terms of toxicity and efficacy [9,13,14,15,16]. As we are unaware 

of any study comparing hICE and BEAM, we retrospectively 

analyzed our lymphoma patients who had undergone AHCT and 

received either hICE or BEAM regimens as conditioning. 


Patient Characteristics 

The clinical and laboratory records of all consecutive relapsed/ 

refractory HL/NHL patients who were treated with AHCT between 

2010 and 2012 were retrospectively analyzed. We did not use 

any exclusion criteria. All patients gave informed consent for 

all aspects of AHCT and the institutional review board approved 

the study. 

Mobilization Strategy 

We used a step-by-step mobilization strategy. Granulocytecolony 

stimulating factor (G-CSF; filgrastim or lenograstim) at 

a dose of 10 µg/kg/day in two divided doses is our first-line 

mobilization protocol. A progenitor cell yield of 500/mm3). Platelet 

transfusions were given if platelet counts were


Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE 

for the first 2 years, every 6 months for the next 3 years, and 

then annually. 

Definition of Engraftment, Febrile Neutropenia, and Veno- 

Occlusive Disease 

Neutrophil engraftment was defined as the first of 3 consecutive 

days on which the absolute neutrophil count exceeded 500/ 

mm3 without G-CSF support. Platelet engraftment was defined 

as the first day of 7 consecutive days on which platelet count 

exceeded 20,000/mm3 without platelet transfusion [25]. We 

used Infectious Disease Society of America [26] and Seattle [27] 

criteria for defining febrile neutropenia and veno-occlusive 

disease, respectively. 

Management of Febrile Neutropenia 

The details of our protocol can be found elsewhere [28]. 

Briefly, patients with febrile neutropenia who were not 

responding to broad-spectrum antibiotics for 72 h were 

evaluated for opportunistic fungal infections. Patients who had 

hemodynamic instability and/or two consecutive positive serum 

galactomannan assays (ELISA: optical density of ≥0.5) and/or 

thorax computerized tomography findings suggesting invasive 

pulmonary aspergillosis (nodules with/without halo sign, air 

crescent sign, and cavitation) supported by mycological cultures 

received antifungal treatment. Patients with mycological 

evidence of Aspergillus spp. were treated with voriconazole. All 

others received caspofungin. 

Calculation of Direct Treatment Costs of Conditioning Regimens 

Direct drug costs of BEAM and hICE conditioning regimens were 

calculated based on an average patient with a body surface area 

of 1.7 m2 as of October 2013. 


Descriptive statistics are presented as median and minimummaximum. 

Comparisons of continuous variables between the 

two groups were performed using the nonparametric Mann- 

Whitney U test. Proportions were compared using the chisquare 

test. Survival analysis was calculated with Kaplan-Meier 

analysis. A p-value below 0.05 was considered to be statistically 

significant. 

Results 

The demographic and clinical characteristics of the study 

cohort are summarized in Table 1. Fifteen (40%) and 5 (20%) 

patients had primary refractory disease following their firstline 

chemotherapy in the BEAM and hICE groups, respectively 

(p=0.09). While 6 patients in the BEAM group received 

standard-dose ICE (sICE) as rescue before AHCT, no patient in 

the hICE arm was treated with sICE as salvage chemotherapy. 

Fourteen (38%) and 6 (25%) patients of the BEAM and hICE 

arms had refractory disease at AHCT, respectively. Twenty-six, 

28, 8 patients were mobilized with G-CSF alone, G-CSF plus 

chemotherapy, G-CSF plus plerixafor, respectively. There were 

no significant differences in terms of conditioning regimens 

among patient groups (p=0.5 both for HL and NHL patients). 

The treatment arms were also similar according to age, sex, 

stage, previous radiotherapy, chemotherapy history, and disease 

status at AHCT and mobilization protocol. On the other hand, 

the BEAM group had significantly worse performance status 

compared to the hICE arm (p=0.011) (Table 1). 

Mobilization success, engraftment kinetics, and side effect 

profiles of the conditioning regimens are given in Table 2. The 

BEAM and hICE treatment arms were similar in terms of infused 

stem cells, median days with febrile neutropenia, engraftment 

kinetics, and duration of hospitalization. We observed 

significantly more adverse effects (grade ≥2) in terms of nausea/ 

vomiting, mucositis, hepatotoxicity, and nephrotoxicity among 

patients treated with hICE conditioning compared to patients 

who received BEAM. Significantly more patients (n=4; 25%) in 

the hICE group experienced veno-occlusive disease compared 

to the BEAM arm, where no patients developed veno-occlusive 

disease (p=0.01). The treatment arms were comparable according 

to diarrhea rate (p=0.09). 

Relapse rates following BEAM and hICE conditioning regimens 

were 13.5% (5/37) and 32% (8/25) (p=0.07). One patient of 

the BEAM arm died before day 30 following AHCT as a result 

of sepsis. Additionally, two patients (one patient in each arm) 

died before day 100. The reasons for mortality were sepsis/ 

engraftment failure and Cytomegalovirus pneumonia in the 

patients of the BEAM and hICE groups, respectively. Transplantrelated 

mortality for the entire cohort on day 100 was 4.8% 

(BEAM: 5.4%; hICE: 4%; p=0.8). Following AHCT, 5 (13.5%) and 

8 (32%) patients of the BEAM and hICE arms relapsed (p=0.07). 

Three-year disease-free survival (DFS) and overall survival (OS) 

rates were 52±10% and 57±6% in the whole study cohort, 

respectively. There was no difference in terms of 3-year DFS 

rates according to conditioning regimens (BEAM: 63±13%; hICE: 

42±15%; p=0.187) (Figure 1). Three-year OS was 56.8±8% and 

58±10% in the BEAM and hICE groups, respectively (p=0.781) 

(Table 2, Figure 2). 

Direct treatment costs of hICE and BEAM regimens were found 

to be 1721 and 582 euro, respectively. 

Discussion 

Although many different conditioning regimens for relapsed/ 

refractory HL and NHL have been proposed, none of them can 

be considered as a standard of care [8,9,10,11,12]. Different 

types of hICE conditioning regimens were described according 

211


to large ranges of cumulative dosages and administrations of 

drugs [10,29]. To our knowledge, there is no direct comparison 

in the literature of BEAM and hICE chemotherapy regimens 

in patients who have undergone AHCT for relapsed/refractory 

lymphoma. 

In the current study, we observed statistically significant 

differences in terms of toxicity favoring the BEAM regimen 

compared to hICE. It was not surprising that nausea and 

vomiting were more frequent in the hICE arm, as ifosfamide 

and carboplatin have high emetogenic potential. Mucositis is an 

important toxicity of BCNU and etoposide [30,31]. According to 

several studies, BCNU-related mucositis rates were higher when 

the BCNU dose was increased from 450 to 600 mg/m2 [30]. The 

BCNU dose in the BEAM conditioning arm was 300 mg/m2 in 

our study. The total doses of etoposide were 1500 mg/m2 and 

800 mg/m2 in the hICE and BEAM arms, respectively. The higher 

etoposide dose may be responsible for the higher mucositis 

rate observed in the hICE arm. Nephrotoxicity was significantly 

higher in the hICE group (p



Head-to-head comparisons of different conditioning regimens 

in relapsed/refractory lymphoma patients before AHCT are 

scarce. Jo et al. observed superior OS and event-free survival at 

2 years in patients on BEAM compared to BEAC regimens (62.4% 

vs. 32.1% and 62.4% vs. 28.6%, respectively). However, diarrhea 

and mucositis were more frequent in patients of the BEAM arm 

[9]. In their single-center analysis, Jantunen et al. reported similar 

efficacy of BEAM and BEAC conditioning regimens in terms of 

OS and progression-free survival in patients undergoing AHCT 

for NHL, but BEAM was found more toxic to the gastrointestinal 

system [16]. In recent years the BEAM regimen was also 

compared with the CEB (carboplatin, etoposide, and bleomycin) 

regimen with better OS in favor of BEAM [14], but other 

studies reported conflicting results [15]. Salar et al. reported 

Spanish GEL/TAMO registry data including 395 consecutively 

autografted diffuse large B-cell lymphoma (DLBCL) patients. 

Figure 1. Kaplan-Meier plots of disease-free survival following 

autologous hematopoietic stem cell transplantation according 

to conditioning regimens. Three-year disease-free survival rates 

were 63±13% (BEAM) vs. 42±15% (hICE) (p=0.187). 

Figure 2. Kaplan-Meier plots of overall survival following 

autologous hematopoietic stem cell transplantation according 

to conditioning regimens. Three-year overall survival rates were 

56±8% (BEAM) vs. 58±10% (hICE) (p=0.781). 

Table 2. Mobilization yield, engraftment kinetics, efficacy, and toxicity profiles of conditioning regimens. 

BEAM (n=37) hICE (n=25) p 

CD34+ cell counts, median (min-max) 5.9 (2.5-16.7) 5.7 (3.5-11.29) 0.36 

Febrile neutropenic days, median (min-max) 4 (1-10) 3 (1-12) 0.79 

Neutrophil engraftment, days, median (min-max) 11 (8-19) 12 (9-34) 0.24 

Platelet engraftment, days, median (min-max) 12 (8-16) 12 (10-25) 0.24 

Hospitalization days, median (min-max) 24 (12-67) 26 (20-50) 0.53 

Nausea/vomiting, n (grade ≥2) 17 21 0.04 

Diarrhea, n (grade ≥2) 22 20 0.09 

Mucositis, n (grade ≥2) 3 13 0.002 

Nephrotoxicity, n, (grade ≥2) 1 12


The main message of that study was that chemotherapy-only 

conditioning regimens (BEAM, BEAC, or CBV) significantly 

improved 8-year OS compared to TBI + cyclophosphamide [15]. 

Recently, the rituximab-BEAM (R-BEAM) conditioning regimen 

was compared to the I131-tositumomab-BEAM (B-BEAM) in a 

phase III randomized study in relapsed, chemosensitive DLBCL 

patients. Two-year progression-free survival and OS rates were 

comparable, but B-BEAM was found to be more toxic in terms 

of mucositis [13]. Although the observation period of our study 

cohort is limited, 3-year OS rates were similar in the BEAM and 

hICE arms (56±8% vs. 58±10%; p=0.781). There was a trend for 

lower relapse rates following BEAM compared to hICE (13.5% vs. 

32%; p=0.07). There were more patients with primary refractory 

disease (40% vs. 20%) and refractory disease at AHCT (38% vs. 

24%) in the BEAM arm compared to patients receiving hICE 

conditioning. The aforementioned points underline the strong 

antitumor effect of the BEAM regimen compared to hICE. 

Taking the cost and safety advantages of BEAM over hICE in 

addition to similar short-term DFS and OS into account, it seems 

reasonable to suggest that BEAM seems to be a better option 

than hICE for conditioning in relapsed/refractory lymphoma 

patients undergoing AHCT. 

Our study has several limitations that make it difficult to 

draw firm conclusions, such as the limited number of patients, 

retrospective design, and heterogeneous lymphoma subtypes 

of the cohort. As we included patients with HL and various 

pathologic subgroups of NHL (DLBCL, follicular lymphoma, 

mantle cell lymphoma, peripheral T cell lymphoma, and 

anaplastic large cell lymphoma), generalization of our findings 

may not be appropriate for specific patient populations with 

lymphoma. We also had a very limited number of patients with 

each subtype of lymphoma, making disease-specific statistical 

evaluation of hICE and BEAM conditioning regimens impossible. 

Although the BEAM treatment arm included more patients with 

poor performance status, the toxicity profile of BEAM was lower 

compared to ICE. This point again emphasizes that BEAM is a 

safe and effective conditioning regimen even for patients with 

poor performance. 

In conclusion, the current retrospective study showed that 

BEAM seems to be a better option compared to hICE as a 

conditioning regimen in relapsed/refractory lymphoma patients 

before AHCT with similar efficacy but low toxicity. Although 

there was no difference in 3-year DFS and OS, the nausea/ 

vomiting, mucositis, nephrotoxicity, and hepatotoxicity rates 

were significantly higher in the hICE group compared to the 

BEAM group. Prospective studies with homogeneous patient 

populations and incorporating novel agents in the therapeutic 

armamentarium will be very informative in the search for the 

optimal conditioning regimen in specific lymphoma subtypes in 

the future. 

Ethics 

Ethics Committee Approval: Ethical Committee approval has 

been not taken because it is retrospective research; Informed 



Medical Practices: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu 

Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür 

Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş; Concept: 

Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz; Design: Onur 

Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz; Data Collection or 

Processing: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, 

Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze 

Durgun, Şerife Kocubaba, Fevzi Altuntaş; Analysis or Interpretation: 

Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz 

Bozdağ; Literature Search: Onur Esbah, Emre Tekgündüz, Itır 

Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, 

Ömür Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş; 

Writing: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, 

Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze 

Durgun, Şerife Kocubaba, Fevzi Altuntaş. 




included. 

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215


DOI: 10.4274/tjh.2014.0378 


The Changing Epidemiology of Bloodstream Infections and 

Resistance in Hematopoietic Stem Cell Transplantation Recipients 

Hematopoetik Kök Hücre Nakli Alıcılarında Kan Akım Enfeksiyonu ve Direnç 

Epidemiyolojisindeki Değişim 

Mücahit Yemişen1, İlker İnanç Balkan1, Ayşe Salihoğlu2, Ahmet Emre Eşkazan2, Bilgül Mete1, M. Cem Ar2, Şeniz Öngören2, Zafer Başlar2, 

Reşat Özaras1, Neşe Saltoğlu1, Ali Mert1, Burhan Ferhanoğlu3, Recep Öztürk1, Fehmi Tabak1, Teoman Soysal2 

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

2İstanbul University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Heamatology, İstanbul, Turkey 

3Koç University Faculty of Medicine, American Hospital, Clinic of Internal Medicine, Division of Heamatology, İstanbul, Turkey 

Abstract 

Objective: Patients receiving hematopoietic stem cell transplantation 

(HSCT) are exposed to highly immunosuppressive conditions and 

bloodstream infections (BSIs) are one of the most common major 

complications within this period. Our aim, in this study, was to 

evaluate the epidemiology of BSIs in these patients retrospectively. 

Materials and Methods: The epidemiological properties of 312 

patients with HSCT were retrospectively evaluated. 

Results: A total of 312 patients, followed between 2000 and 2011, 

who underwent autologous (62%) and allogeneic (38%) HSCT were 

included in the study. The most common underlying malignancies 

were multiple myeloma (28%) and Hodgkin lymphoma (21.5%). A 

total of 142 (45%) patients developed at least 1 episode of BSI and 

193 separate pathogens were isolated from the blood cultures. There 

was a trend of increase in the numbers of BSIs in 2005-2008 and 

a relative increase in the proportion of gram-positive infections in 

recent years (2009-2011), and central venous catheter-related BSI was 

found to be most common source. Coagulase-negative staphylococci 

(49.2%) and Acinetobacter baumannii (8.8%) were the most common 

pathogens. Extended-spectrum beta-lactamase-producing strains 

were 23% and 22% among Escherichia coli and Klebsiella spp. 

isolates, respectively. Quinolone resistance was detected in 10% of 

Enterobacteriaceae. Resistance to carbapenems was not detected 

in Enterobacteriaceae, while it was seen at 11.1% and 23.5% in 

Pseudomonas and Acinetobacter strains, respectively. 

Conclusion: A shift was detected from gram-negative bacteria to 

gram-positive in the etiology over the years and central lines were the 

most common sources of BSIs. 

Keywords: Hematopoietic stem cell transplantation, Bloodstream 

infection, Epidemiology, Resistance, Central venous catheter 

Öz 

Amaç: Hematopoetik kök hücre transplantasyonu (HKHT) yapılan 

hastaların bağışıklık sistemi ciddi şekilde baskılanmıştır ve kan 

akımı enfeksiyonları (KAE) bu süre içinde karşılaşılan majör 

komplikasyonlardan biridir. Bu çalışmada amacımız, geriye dönük 

olarak bu hastalarda KAE’lerinin epidemiyolojisini değerlendirmektir. 

Gereç ve Yöntemler: HKHT yapılan 312 hastanın epidemiyolojik 

özellikleri retrospektif olarak değerlendirildi. 

Bulgular: 2000 ve 2011 yılları arasında otolog (%62) ve allojeneik 

(%38) HKHT yapılan 312 hasta, çalışmaya dahil edildi. Çalışmaya 

dahil edilen hasta grupları en sık multipl miyelom (%28) ve Hodgkin 

lenfoma (%21,5) tanılı hastalar idi. Yüz kırk iki hastada (%45) en az bir 

kez KAE gelişmiş ve kan kültürlerinden 193 ayrı patojen elde edilmiştir. 

KAE’lerde 2005-2008 yılları arası bir artışın yanında, 2009-20011 

yılları arasında da gram pozitiflerde göreceli bir artış da saptanmış 

ve en sık KAE kaynağı santral venöz kataterler olarak tespit edilmiştir. 

Koagülaz negatif staphylococi (%49,2) ve Acinetobacter baumannii 

(%8,8), kan kültürlerinden en sık elde edilen patojenlerdir. Genişlemiş 

spektrumlu beta laktamaz üretimi Escherichia coli ve Klebsiella 

spp. suşları arasında sırası ile %23 ve %22 idi. Kinolon dirençli 

Enterobacteriaceae oranı %10 olarak tespit edilmiştir. Pseudomonas 

ve Acinetobacter suşlarında karbapenem direni sırasıyla, %11,1 ve 

%23,5 iken, Enterobacteriaceae grubunda karbapenemlere hiç direnç 

saptanmamıştır. 

Sonuç: Yıllar içinde, gram negatif bakterilerden gram pozitiflere 

doğru bir kayma gözlenirken, en sık KAE kaynağı santral kataterler 

olarak saptanmıştır. 

Anahtar Sözcükler: Hematopoetik kök hücre nakli, Kan akımı 

enfeksiyonu, Epidemiyoloji, Direnç, Santral venöz kateter 

Address for Correspondence/Yazışma Adresi: Mücahit YEMİŞEN, M.D., 

İstanbul University Cerrahpaşa Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, 

İstanbul, Turkey 

Phone : +90 212 414 30 95 E-mail: yemisenmucahit@hotmail.com 

Received/Geliş tarihi: September 24, 2014 

Accepted/Kabul tarihi: December 15, 2014 

216


Yemişen M, et al: Epidemiology of Bloodstream Infections 

Introduction 

Bloodstream infection (BSI) is the most common infectious 

problem in patients undergoing hematopoietic stem cell 

transplantations (HSCTs). Depending on the protocol used for 

transplantation and the duration of neutropenia, approximately 

13%-60% of patients develop BSIs, which can result in delays in 

chemotherapies, extension of admission period, and increased 

costs of antimicrobial therapy against target organisms [1,2]. 

The differences in results of these studies are probably due 

to different study designs, study populations, conditioning 

regimens, and prophylactic antibiotic protocols [1]. Beside 

neutropenia, the other risk factors for BSI include age, 

underlying disease, presence of a central catheter, severe graftversus-host 

disease (GVHD), mucositis, and steroid use [1,3,4]. 

The etiology of BSIs has changed and showed different patterns 

in the past years. While gram-negative BSIs among neutropenic 

cancer patients were formerly the leading cause of bacteremia, 

the etiology of BSIs in this patient population has become 

predominantly gram-positive, and especially viridans group 

streptococci and coagulase-negative staphylococci, over the last 

2 decades [5,6]. Besides this shift, resistance rates and patterns 

also started to change and more resistant microorganisms are 

now found as the causes of BSIs. For example, the emergence 

of fluoroquinolone-resistant bacteria, increase in multidrugresistant 

gram-negative bacteria, increase in nosocomial 

methicillin-resistant Staphylococcus aureus infections, and 

emergence of extended-spectrum beta-lactamase (ESBL) 

producers have all been reported in the literature in neutropenic 

patients [3]. Due to the diversity of the causative microorganisms 

of BSIs in patients with HSCT, information about etiology and 

antibiotic susceptibility of BSIs is important to initiate effective 

antibiotic treatment, a parameter that has been shown to be 

closely associated with survival in bacteremic patients [7]. In this 

study, we aimed to assess the etiology and clinical characteristics 

of BSIs in patients with hematological malignancies undergoing 

HSCT over a 12-year period. We also evaluate the risk factors, 

resistance patterns, and sources of BSIs in this group of patients 

as a secondary objective. 


Patients 

A total of 312 patients who underwent autologous and allogeneic 

bone marrow transplantation in the Stem Cell Transplantation 

Unit of the İstanbul University Cerrahpaşa Medical School from 

1 January 2000 to 31 December 2011 were included in the 

study. Data on demographic features of the patients, underlying 

disease, disease status prior to HSCT, HSCT protocols, prophylaxis 

regimens, and emerging resistance profiles of bacteremia were 

retrospectively analyzed. The data of the patients were recorded 

from the initiating day of conditioning until the 100th day after 

transplantation. 

Hematologic Definitions 

All patients were followed in isolated single rooms equipped 

with high-efficiency particulate air filters and underwent 

central venous catheter (CVC) insertion. Conditioning was 

done using standard protocols such as cyclophosphamide alone 

or in combination with total body irradiation for allogeneic 

transplantation and CBV (cyclophosphamide, VP-16, BCNU) or 

BEAM (BCNU, VP-16, cytarabine, melphalan) for autologous 

stem cell transplantation. Almost all allogeneic transplantations 

were done from HLA-identical sibling or matched unrelated 

donors. Neutrophil engraftment was defined as the first of 

3 consecutive days on which the absolute neutrophil count 

remained at or above 500/mm 3 after stem cell infusion. GVHD 

diagnosis and staging were performed according to previously 

established criteria [8,9]. 

Microbiological Definitions 

We obtained at least 2 blood cultures from all febrile neutropenic 

patients and initiated an antipseudomonal antibiotic. Febrile 

neutropenia was investigated and managed according to the 

Infectious Disease Society of America guidelines [10,11]. 

BSI (mono or poly) and catheter-associated BSI were accepted 

according to the established criteria [12,13,14]. Antimicrobial 

susceptibility tests of bacteria obtained from blood cultures 

were evaluated by the disk diffusion method according to 

the current Clinical and Laboratory Standards Institute (CLSI, 

formerly NCCLS) criteria [15]. Intermediate sensitivity or 

resistance results were accepted as resistant. The screening of 

multidrug-resistant phenotypes including methicillin-resistant 

Staphylococcus aureus, ampicillin- and vancomycin-resistant 

enterococci, ESBL production, and carbapenemase production 

was conducted according to CLSI recommendations [16,17]. 

Multidrug resistance was defined as acquired nonsusceptibility 

to at least 1 agent in 3 or more antimicrobial categories; 

extensive drug resistance was defined as nonsusceptibility to 

at least 1 agent in all but 2 or fewer antimicrobial categories, 

and pandrug resistance was defined as nonsusceptibility to all 

agents in all antimicrobial categories [18]. 


The categorical data were compared by chi-square tests, 

and p



Results 

A total of 312 patients were included in the study. The number 

of female patients was 137 (44%) and the mean age was 39 

years (minimum-maximum: 12-73 years). The most common 

underlying conditions of the patients were multiple myeloma in 

87 (28%) and Hodgkin lymphoma in 67 (21.5%). The number of 

patients who underwent autologous and allogeneic HSCT was 

194 (62%) and 118 (38%), respectively. The stem cell source was 

peripheral blood in 295 (94.5%) patients and bone marrow in 

17 (5.5%) patients. The mean time to neutrophil engraftment 

was 14 days and the number of patients having detectable 

cytomegalovirus-DNA was 38 (12.2%). The number of patients 

having acute GVHD equal to or above stage 2 was 36 (11.5%). 

Table 1 shows the characteristics of the patients. 

We obtained a total of 193 microbial isolates from patients’ 

blood cultures; of these 193 isolates, 12 were obtained after 

the conditioning regimen (before infusion of cells), 140 were 

obtained after infusion of cells (before neutrophil engraftment), 

and 41 were obtained after engraftment. Table 2 shows 

the properties of the isolates obtained from blood cultures. 

Gram-positive, gram-negative, and fungal isolates obtained 

from the blood cultures were 112 (58%), 74 (38.3%), and 7 

(3.7%), respectively. A total of 142 (45.5%) of 312 patients 

developed at least 1 episode of BSI. Of these 142 patients, 68 

had autologous and 74 had allogeneic HSCT. In our study, 106 

patients developed 1 episode of BSI, 32 patients had 2 episodes, 

3 patients had 3 episodes, and 1 patient had 4 episodes. The 

numbers of monomicrobial and polymicrobial episodes were 

168 and 14, respectively. The source of BSI was determined as 

CVC-associated for 151 (78.2%) isolates while no source could 

be determined for the remaining isolates. Of those 151 CVCassociated 

isolates, 69.5% were gram-positive bacteria. 

The most frequently isolated gram-positive bacteria were 

coagulase-negative staphylococci with 95 isolates, and then 

Streptococcus spp. with 8, S. aureus with 5, Enterococcus spp. 

with 2, and gram-positive rods with 2 isolates. The numbers 

of gram-negative isolates obtained from blood cultures were 

as follows: Acinetobacter baumannii, 17; Stenotrophomonas 

maltophilia, 14; Escherichia coli, 13; Klebsiella spp., 9; 

Pseudomonas aeruginosa, 9; and other gram-negative bacteria, 

12 isolates. A total of 7 fungal isolates comprised 3 Candida 

parapsilosis, 1 Candida tropicalis, 1 Fusarium spp., and 2 

Candida spp. isolates. 

Between 2000 and 2005, the number of gram-negative isolates 

was greater than the number of gram-positive isolates; after 

2005, gram-positive isolates increased in frequency and became 

the major causative group for BSIs. Figure 1 shows the etiology 

of BSIs (gram-positive, gram-negative, and fungal) according 

to year; there was a trend of increase in the numbers of BSIs 

Figure 1. Evolution of bloodstream infection etiology. 

Table 1. Characteristics of the patients. 

Characteristics n (%) 

Total number of patients 312 

Patients with BSI 142 (45.5%) 

Patients without BSI 170 (54.5%) 

Median age 39 (12-73) 

Sex 

Male 175 (56%) 

Female 137 (44%) 

Underlying disease 

MM 87 (28%) 

HL 67 (21.5%) 

NHL 44 (14%) 

AML 42 (13.5%) 

ALL 39 (12.5%) 

CML 19 (6%) 

Others 14 (4.5%) 

Type of transplantation 

Autologous 194 (62%) 

Allogeneic 118 (38%) 

Graft source 

Peripheral blood 295 (94.5%) 

Bone marrow 17 (5.5%) 

Mean duration of neutrophil engraftment (days) 14 (7-36) 

Comorbid conditions 

DM 13 (4.1%) 

Hepatic 11 (3.5%) 

Cardiac 11 (3.5%) 

Pulmonary 6 (1.9%) 

Solid tumor 4 (1.2%) 

Rheumatic disease 3 (0.9%) 

Crude mortality (in 100 days) 40 (12.8%) 

BSI: Bloodstream infection, MM: multiple myeloma, HL: Hodgkin lymphoma, NHL: 

non-Hodgkin lymphoma, AML: acute myeloid leukemia, ALL: acute lymphoblastic 

leukemia, CML: chronic myeloid leukemia, DM: diabetes mellitus. 

218



in 2005-2008 and also a relative increase in the proportion of 

gram-positive BSIs in more recent years (2009-2011). 

The number of ESBL-producing Enterobacteriaceae isolates was 

6 (20.6%). Among the 13 E. coli isolates, 3 were ESBL-producing 

and 4 were resistant to ciprofloxacin, 5 to aminoglycosides, 

2 to cefepime, and 3 to third-generation cephalosporin and 

piperacillin/tazobactam. For Klebsiella spp., 2 isolates were 

resistant to third-generation cephalosporins, 3 to piperacillin/ 

tazobactam, and 2 to cefepime, and 2 were ESBL-producing. We 

found no resistant isolates for aminoglycosides or ciprofloxacin. 

The proportion of isolates that were ESBL-producing among 

E. coli and Klebsiella spp. was 23% and 22%, respectively. No 

resistance to carbapenems was observed. 

In the P. aeruginosa group, 2 strains were resistant to ceftazidime; 

1 was resistant to piperacillin/tazobactam, cefepime, and 

carbapenems; and no strains were resistant to aminoglycosides or 

ciprofloxacin. Of the 17 A. baumannii strains, 4 were resistant to 

carbapenems; 3 to aminoglycosides, ceftazidime, and cefepime; 2 

to piperacillin/tazobactam; and 1 to ciprofloxacin. All S. maltophilia 

strains were susceptible to trimethoprim/sulfamethoxazole. Among 

all gram-negative strains, the rate of multidrug-resistant bacteria 

was 12.1%, and the rate of extensively drug resistant bacteria was 

8.1%. We did not identify any pandrug resistance in our study. 

Table 3 shows the resistance patterns of the gram-negative 

bacteria obtained from blood cultures. 

Among all gram-positive bacteria, 95 (84.8%) were coagulasenegative 

staphylococci, and only 3 (3.1%) strains were susceptible 

to methicillin. In 5 S. aureus strains, only 1 was resistant 

to methicillin, and the remaining were susceptible. Among 

the 8 Streptococcus strains isolated, 7 were viridans group 

streptococci and 1 was group A beta-hemolytic streptococcus. 

Table 2. Etiology and source of bloodstream infections. 

Bacteria Eng Total 

CVC 

Source 

Unknown 

MRCNS 7 71 14 92 92 0 

MSCNS 0 3 0 3 3 0 

Enterococcus sp. 0 0 2 2 2 0 

Streptococcus sp. 0 8 0 8 2 6 

MRSA 0 1 0 1 1 0 

MSSA 0 3 1 4 3 1 

Other gram-positives 0 2 0 2 2 0 

Escherichia coli 2 9 2 13 6 7 

Klebsiella sp. 0 5 4 9 3 6 

Pseudomonas aeruginosa 0 8 1 9 6 3 

Acinetobacter baumannii 2 10 5 17 11 6 

Stenotrophomonas maltophilia 0 10 4 14 8 6 

Other gram-negatives 0 7 5 12 6 6 

Candida parapsilosis 0 3 0 3 3 0 

Other fungi 1 0 3 4 3 1 

Total 12 (6.2%) 140 (72.5%) 41 (21.3%) 193 151 (78.2%) 42 (21.8%) 

BSI: Bloodstream infection, Eng: engraftment, CVC: central venous catheter, MRCNS: methicillin-resistant coagulase-negative Staphylococcus, MSCNS: methicillin-susceptible 

coagulase-negative Staphylococcus, MRSA: methicillin-resistant Staphylococcus aureus, MSSA: methicillin-sensitive Staphylococcus aureus. 

Table 3. Resistance pattern of gram-negative isolates. 

AK CIP CAZ CTX/ TZP FEP ESBL IPM/MEM MDR XDR PDR Total 

CRO 

Escherichia coli 5 4 3 3 3 2 3 - 3 2 - 13 

Klebsiella sp. - - 2 2 3 2 2 - 1 - - 9 

Pseudomonas aeruginosa - - 2 - 1 1 - 1 - - - 9 

Acinetobacter baumannii 3 1 3 - 2 3 - 4 2 2 - 17 

Others 3 1 3 4 2 3 1 1 - - - 12 

AK: Amikacin, CIP: ciprofloxacin, CAZ: ceftazidime, CTX/CRO: cefotaxime/ceftriaxone, TZP: piperacillin/tazobactam, FEP: cefepime, ESBL: extended-spectrum beta-lactamase, IPM/ 

MEM: imipenem/meropenem, MDR: multidrug-resistant, XDR: extensively drug resistant, PDR: pandrug resistant. 

219



Univariate analysis to determine risk factors for bacteremia 

identified HSCT type, any comorbidity, duration of engraftment 

longer than 10 days, and GVHD grade of 2-4 (p



Our study had some limitations. It was performed retrospectively 

and, due to missing data, some patients had to be excluded. The 

initial empiric antibiotic treatment might have influenced the 

resistance of the bacteria, but we could not account for the 

effect of empiric antibiotic treatment. The impact of the stem 

cell source on bacteremia in allogeneic HSCT recipients could 

not be analyzed since almost all patients received peripheral 

stem cells from fully matched donors. 

In conclusion, BSI in HSCT recipients is still a great problem. 

The global switch from a gram-negative etiology to a grampositive 

one was also observed in our study. In addition to other 

gram-negative bacteria, A. baumannii and S. maltophilia were 

frequent causes of bacteremia but were generally not covered 

by initial empirical therapy. Accordingly, we observed a higher 

rate of mortality due to S. maltophilia bacteremia. It is generally 

difficult to identify the source of bacteremia in HSCT patients. 

In our study, CVCs were the only source suggested and they 

were usually associated with unusual pathogens. However, with 

a dedicated CVC team and the use of a catheter-care bundle, we 

could reduce the rate of catheter-related BSIs. HSCT recipients 

are especially at risk of CVC-related BSIs, which may include 

difficult-to-treat pathogens. 

Ethics 

Ethics Committee Approval: Retrospective study; Informed 



Concept: Mücahit Yemişen, İlker İnanç Balkan; Design: Mücahit 

Yemişen, Ahmet Emre Eşkazan; Data Collection or Processing: 

Mücahit Yemişen, İlker İnanç Balkan, Ayşe Salihoğlu, Ahmet Emre 

Eşkazan, Bilgül Mete, M. Cem Ar, Şeniz Öngören, Zafer Başlar, 

Reşat Özaras, Neşe Saltoğlu, Ali Mert, Burhan Ferhanoğlu, Recep 

Öztürk, Fehmi Tabak, Teoman Soysal; Analysis or Interpretation: 

Mücahit Yemişen, İlker İnanç Balkan, Ayşe Salihoğlu, Ahmet 

Emre Eşkazan, Bilgül Mete, M. Cem Ar, Şeniz Öngören, Zafer 

Başlar, Reşat Özaras, Neşe Saltoğlu, Ali Mert, Burhan Ferhanoğlu, 

Recep Öztürk, Fehmi Tabak, Teoman Soysal; Literature Search: 

Bilgül Mete, Ayşe Salihoğlu; Writing: Mücahit Yemişen. 




included. 

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222


DOI: 10.4274/tjh.2015.0131 


BK Virus-Hemorrhagic Cystitis Following Allogeneic Stem Cell 

Transplantation: Clinical Characteristics and Utility of Leflunomide 

Treatment 

Allojenik Kök Hücre Transplantasyonu Sonrası BK Virüs Hemorajik Sistiti: Klinik Özellikleri 

ve Leflunomid Tedavisinin Etkisi 

Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim 

Inha University Faculty of Medicine and Hospital, Department of Hematology-Oncology, Incheon, Republic of Korea 

Abstract 

Objective: BK virus-hemorrhagic cystitis (BKV-HC) is a potential cause 

of morbidity and mortality in patients having undergone allogeneic 

stem cell transplantation (Allo-SCT). We analyzed the clinical features 

of BKV-HC following Allo-SCT and reported the utility of leflunomide 

therapy for BKV-HC. 

Materials and Methods: From January 2005 to June 2014, among the 

69 patients that underwent Allo-SCT in our institution, the patients 

who experienced BKV-HC were investigated retrospectively. 

Results: HC was observed in 30 patients (43.5%), and among them, 

18 of the cases (26.1%) were identified as BKV-HC. The median age 

of the patients (12 males and 6 females) was 45 years (minimummaximum: 

13-63). Patients received Allo-SCT for acute myeloid 

leukemia (n=11), aplastic anemia (n=4), myelodysplastic syndrome 

(n=2), and non-Hodgkin lymphoma (n=1). The donor types were 

human leukocyte antigen (HLA)-matched sibling donor for six 

patients, HLA-matched unrelated donor for nine, and haploidentical 

familial donor for two. The median onset and duration of BKV-HC 

was on day 21 after transplantation (minimum-maximum: 7-97) 

and 22 days (minimum-maximum: 6-107). Eleven patients (62.1%) 

had grade I-II HC and seven patients (38.9%) had grade III-IV (highgrade) 

HC. Among the seven patients who had high-grade HC, one 

had complete response, one had partial response, and five had 

no response. Among the five nonresponders, one died of BKV-HC 

associated complications. The remaining four patients were treated 

with leflunomide, achieving complete response (n=2) and partial 

response (n=2). The median duration from the start of leflunomide 

therapy to response was 13 days (minimum-maximum: 8-17 days). 

All patients tolerated the leflunomide treatment well, with three 

patients having mild gastrointestinal symptoms, including anorexia 

and abdominal bloating. 

Conclusion: BKV-HC was commonly observed in patients with 

HC following Allo-SCT. In high-grade BKV-HC patients who do not 

respond to supportive care, leflunomide may be a feasible option 

without significant toxicity. 

Keywords: BK virus, Hemorrhagic cystitis, Allogeneic stem cell 

transplantation, Leflunomide 

Öz 

Amaç: BK-virüs hemorajik sistiti (BKV-HS) allojenik kök hücre nakli 

(Allo-KHN) uygulanan hastalarda morbidite ve mortalitenin önemli 

bir nedenidir. Bu çalışmada Allo-KHN sonrası BKV-HS olan olguların 

klinik özellikleri ve leflunomid tedavisinin BKV-HS’deki etkinliği 

araştırılmıştır. 

Öz 

Gereç ve Yöntemler: Kliniğimizde Ocak 2005-Haziran 2014 arası Allo- 

KHN uygulanmış 69 hastada, BKV-HS geçirmiş olanlar retrospektif 

olarak değerlendirildi. 

Bulgular: Otuz hastada (%43,5) HS gözlendi. Bu olguların 18’inde 

(%26,1) BKV-HS’si saptandı. Hastaların (12’si erkek, altısı kadın) 

medyan yaşı 45 (13-63) idi. Hastalara akut miyeloid lösemi (n=11), 

aplastik anemi (n=4), miyelodisplastik sendrom (n=2) ve non-Hodgkin 

lenfoma (n=1) nedeni ile Allo-KHN uygulanmıştı. Altısında insan 

lökosit antijeni (İLA)-uygun kardeş, dokuzunda İLA-uygun akraba 

dışı donör ve ikisinde haplo-identik donör kullanılmıştı. Transplant 

sonrası BKV-HS medyan başlangıç zamanı 21 gün (7-97 gün), medyan 

süresi 22 gün (6-107 gün) idi. On bir olguda (%62,1) derece I-II, yedi 

olguda (%38,9) derece III-IV (yüksek derecede) HS saptandı. Yüksek 

derece HS’li yedi hastanın, birinde tam yanıt, birinde kısmi yanıt elde 

edilirken, beş hastada yanıt alınamadı. Yanıt alınmayan beş hastanın 

birisi BKV-HS ilişkili komplikasyonlardan kaybedildi. Geri kalan dört 

hasta leflunomid ile tedavi edildi. Bu hastaların ikisinde tam yanıt, 

ikisinde kısmi yanıt elde edildi. Leflunomidin başlangıcından itibaren 

medyan yanıt süresi 13 gündü (8-17 gün). Tüm hastalar leflunomidi iyi 

tolere ederken, üç hastada anoreksi ve abdominal gaz şikayetleri dahil 

hafif şiddetli gastrointestinal yan etkiler gözlendi. 

Sonuç: Allo-KHN sonrası izlemde BKV-HS yaygın olarak gözlenmiştir. 

Destek tedavisine yanıt vermeyen yüksek derece BKV-HS’li olgularda 

leflunomid, anlamlı toksisitesi olmaksızın bir seçenek olabilir. 

Anahtar Kelimeler: BK virüs, Hemorajik sistit, Allojenik kök hücre 

transplantasyonu, Leflunomid 

Address for Correspondence/Yazışma Adresi: Chul Soo KIM, M.D., 

Received/Geliş tarihi: March 23, 2015 

Inha University Faculty of Medicine and Hospital, Department of Hematology-Oncology, Incheon, Republic of Korea Accepted/Kabul tarihi: December 21, 2015 

Phone : +82-32-890-2581 

E-mail : cskimmd@inha.ac.kr 

223

Park YH, et al: BK Virus-Hemorrhagic Cystitis 


Introduction 

Hemorrhagic cystitis (HC) is a potential cause of morbidity and 

mortality in patients that have undergone allogeneic stem cell 

transplantation (Allo-SCT) [1,2,3]. Its incidence ranges from 5% 

to 68% of Allo-SCT recipients, with severe-grade hematuria 

in 29%-44% of cases [3,4,5,6,7]. Variable etiologies for the 

development of HC in Allo-SCT recipients include noninfectious 

and infectious causes. As an infectious cause of HC, BK virus- 

HC (BKV-HC) occurs later after transplantation, usually in 

the post-engraftment period [3]. The BKV, a member of the 

family Polyomaviridae, is typically acquired in childhood and 

embedded in urothelial cells of the urinary tract in the latent 

dormant stage [8]. BKV reactivation is commonly associated 

with HC in Allo-SCT settings, occurring in 10% to 25% of 

patients [8]. The clinical symptoms of BKV-HC vary to a great 

extent in Allo-SCT recipients from asymptomatic hematuria to 

massive hemorrhage leading to urinary obstruction and renal 

failure [2,9,10]. Previous studies demonstrated that BKV-HC 

is associated with not only increased morbidity and but also 

increased mortality in Allo-SCT patients [6,7,11,12], and studies 

have also defined potential risk factors for the development of 

BKV-HC [4,5,13,14,15], most of which have not been observed 

consistently in several reports. 

Leflunomide, an immunomodulatory agent with antiviral 

activity, has been found effective against cytomegalovirus 

(CMV), herpes simplex, and BKV based on in vitro data [6,16,17]. 

In renal allografts, leflunomide has been widely used to treat 

biopsy-proven BKV nephropathy [18,19], but it has not been 

well studied in Allo-SCT settings. Only two reports showed 

satisfactory results of leflunomide therapy in the treatment of 

BKV-HC after Allo-SCT [20,21]. 

In this retrospective study, we report the incidence, severity, 

and outcome of clinical BKV-HC in patients who underwent 

Allo-SCT to treat variable hematologic diseases. Furthermore, 

we report high-grade BKV-HC patients who achieved favorable 

response to leflunomide therapy. 


Patients 

A total of 69 patients underwent Allo-SCT in our institution from 

January 2005, when BKV polymerase chain reaction (PCR) testing 

became clinically available, to June 2014. Baseline demographic 

and transplantation data were collected, including age, sex, 

underlying disease, conditioning regimen, stem cell source, donor 

type, prophylaxis to infection and graft-versus-host disease (GVHD), 

time to engraftment, presence and grade of GVHD, and survival at 

last follow-up. Patients received either myeloablative or reducedintensity 

conditioning regimen according to disease status, age, 

or comorbidities. As prophylaxis against HC, hyperhydration 

(intravenous isotonic saline over 3 L/m 2 per day) with forced 

diuresis was used to prevent toxicity of conditioning regimen for 

Allo-SCT. In addition, for patients receiving a preparative regimen 

containing cyclophosphamide (Cy), 2-mercaptoethane sulfonate 

(MESNA) was given prior to the Cy administration and thereafter 

as a continuous infusion until the last dose of Cy. Acute GVHD and 

chronic GVHD were diagnosed and graded according to previously 

published criteria [22,23]. 

Diagnosis of BK Virus-Hemorrhagic Cystitis 

HC was defined as the presence of sustained hematuria and 

urinary symptoms after the beginning of conditioning therapy 

in the absence of gynecological-related bleeding, generalized 

bleeding diathesis, and urinary tract infection. Severity of 

HC was graded according to the following criteria [7]: grade 

0 (no hematuria), grade I (microscopic hematuria), grade II 

(macroscopic hematuria), grade III (macroscopic hematuria 

with presence of blood clots), and grade IV (macroscopic 

hematuria with clots and renal impairment due to urinary 

obstruction). Grades III and IV were defined as high-grade 

HC. The date of onset of HC was defined as the first day of 

symptoms or laboratory evidence appearing after transplant. 

Routine urinalysis was performed at least twice a week during 

hospitalization and thereafter at outpatient visits. For patients 

who had urinary symptoms or gross hematuria, BKV testing by 

a qualitative PCR-based method was performed with the urine 

specimen to identify the presence of virus. 

BK Virus Polymerase Chain Reaction Test 

Each urine sample was submitted to DNA extraction using a 

QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to 

the manufacturer’s instructions. A primer was designed for a 

highly conserved region of gene large T antigen to obtain a 160- 

bp amplicon from the BKV genome using the GeneAmp 2720 

Thermal Cycler (ABI, Foster City, CA, USA). PCR of urine for BKV 

viruria had a sensitivity of 90% and a specificity of 96.5% (cutoff 

value: 160 copies/mL). 

Urine Cytology 

Each urine specimen was processed for cytological evaluation by 

centrifugation. After removal of the supernatant, the sediment 

was examined for clarity. Cytospin slides were fixed in 95% 

alcohol, stained by the Papanicolaou method, and observed for 

the presence of urine decoy cells (characterized by a groundglass 

appearance with an enlarged nucleus, which is occupied by 

a homogeneous basophilic inclusion surrounded by chromatin) 

by a well-trained pathologist. 

Treatment and Criteria of Response 

All patients diagnosed with BKV-HC received supportive 

treatment, including hyperhydration with normal saline, forced 

224



diuresis, urine alkalinization, analgesics, and blood transfusion 

to maintain platelet counts at ≥50x109/L and a hemoglobin 

level of ≥8 g/dL. Foley catheterization and bladder irrigation 

were considered for patients with high-grade HC who had 

no response (NR) to supportive care. Leflunomide therapy 

was indicated as follows: 1) grade III-IV HC; 2) no change or 

worsening in urinary symptoms or grade of hematuria during 

supportive care within 2 weeks; 3) no abnormality in liver 

function tests; 4) absolute neutrophil count of >1.0x109/L; and 

5) no abnormality on chest radiology. For the patients receiving 

leflunomide therapy, leflunomide at 100 mg/day orally was 

used as a loading dose for 5 days, followed by maintenance 

doses of 20 mg/day until resolution of hematuria and urinary 

symptoms. Clinical response was defined as follows: complete 

response (CR), completely improved in symptoms with absence 

of hematuria; partial response (PR), downgrading of severity 

with persistent hematuria; NR, unchanged or worsening urinary 

symptoms or grade of hematuria; refractoriness, NR even after 

about 2 weeks of supportive care. The response was evaluated 

after 20 days of leflunomide treatment. 


The cumulative incidence of BKV-HC was estimated with the 

interval starting at Allo-SCT until the day of the first PCRpositive 

urine sample. The Mann-Whitney test and chi-square 

test were used for comparisons for continuous and categorical 

data, respectively, between the patients with low-grade BKV-HC 

and those with high-grade BKV-HC. Univariate and multivariate 

analyses of risk factors for BKV-HC occurrence were performed 

using the Cox proportional hazard model. SPSS 14.0 (SPSS Inc., 

Chicago, IL, USA) was used for all statistical analyses, and all 

were two-sided. Statistical significance was defined as p



Table 1. Demographic characteristics of the hemorrhagic cystitis patients and posttransplant outcomes. 

Variable BKV-HC (n=18) Non-BKV-HC (n=12) 

Age, years 45 (13-63) 42 (21-66) 

Sex 

Male 

Female 

Diagnosis 

Acute myeloid leukemia 

Aplastic anemia 

Myelodysplastic syndrome 

Non-Hodgkin lymphoma 

Type of donor 

Matched sibling 

Matched unrelated 

Haploidentical familial 

Donor source 

Peripheral blood stem cell 

Bone marrow stem cell 

Conditioning regimen 

Busulfan/fludarabine 

Busulfan/cyclophosphamide 

Fludarabine/melphalan 

Cyclophosphamide/fludarabine 

Cyclophosphamide/total body irradiation 

T-cell depletion 

None 

In vivo 

Engraftment 

Neutrophil engraftment (ANC >0.5x10 9 /L) 

Platelet engraftment (>20x10 9 /L) 

Acute GVHD 

Grade II-IV 

Chronic GVHD 

None 

Limited 

Extensive 

Values are presented as median (minimum-maximum) or number (%). 

HC: Hemorrhagic cystitis, ANC: absolute neutrophil count, GVHD: graft-versus-host disease. 

12 (66.7) 

6 (33.3) 

11 (61.1) 

4 (22.2) 

2 (11.1) 

1 (5.6) 

7 (38.9) 

9 (50.0) 

2 (11.1) 

18 (100) 

0 (0) 

8 (44.4) 

5 (27.8) 

3 (16.7) 

1 (5.6) 

1 (5.6) 

11 (61.1) 

7 (38.9) 

18 (100) 

14 (77.7) 

9 (50.0) 



5 (27.8) 

7 (38.9) 

7 (58.3) 

5 (41.7) 

6 (50) 

2 (16.7) 

3 (25) 

1(8.3) 

8 (66.7) 

3 (25) 

1 (8.3) 

11 (91.7) 

1 (8.3) 

6 (50) 

5 (41.7) 

0 (0) 

1 (8.3) 

0 (0) 

6 (50) 

6 (50) 

11 (91.7) 

9 (75) 

7 (58.3) 

3 (25) 


5 (41.7) 

3 (25) 

Leflunomide Therapy 

Detailed information about the four patients receiving 

leflunomide therapy is summarized in Table 4. Of the four 

patients, three had acute myeloid leukemia and one had highrisk 

myelodysplastic syndrome. All patients had acute GVHD 

before development of BKV-HC. After leflunomide treatment, 

two (50%) patients achieved CR and two (50%) achieved PR. 

One CR patient and one PR patient had a negative PCR for 

BKV, but BKV in urine still remained detectable in one patient 

achieving CR. The median duration from the start of leflunomide 

therapy to response was 13 days (minimum-maximum: 8-17). 

The dose of leflunomide was not reduced for any patients. All 

patients tolerated the leflunomide treatment well, with three 

patients having mild gastrointestinal symptoms, including 

anorexia and abdominal bloating. No significant adverse effects, 

such as hepatotoxicity, skin reactions, diarrhea, bone marrow 

suppression, or pneumonia, were observed during leflunomide 

treatment. There was no recurrence of hematuria in the two 

patients achieving CR after discontinuation of leflunomide 

therapy. Of the patients achieving PR, one died of leukemia 

relapse 16.3 months after Allo-SCT. The remaining three patients 

are still alive without hematuria. 

226



Table 2. Characteristics of BK virus-hemorrhagic cystitis and treatment outcomes. 

Characteristics 

Onset, days after Allo-SCT, median (minimum-maximum) 21 (7-97) 

Duration, days, median (minimum-maximum) 22 (6-107) 

Platelet count at the onset of BKV-HC (x10 9 /L), median (minimum-maximum) 64 (17-260) 

Platelet unit transfusions, median (minimum-maximum) 10 (0-48) 

RBC unit transfusions, median (minimum-maximum) 0 (0-10) 

Serum creatinine at the onset of BKV-HC (mg/dL), median (minimum-maximum) 1.15 (0.52-2.57) 

Severity, n (%) 

I/II 

III/IV 

Concomitant CMV antigenemia, n (%) 9 (50) 

Treatment, n (%) 

Intravenous hyperhydration 

Quinolone antibiotics (ciprofloxacin or levofloxacin) 

Blood transfusion (RBC or platelet) 

Insertion of urinary catheter 

Cystoscopy 

Continuous bladder irrigation with water 

Leflunomide 

Outcome after initial therapy, n (%) 

Complete response 

Partial response 

No response 

Survival, n (%) 

Alive 

Dead 

Relapse/refractory disease 

GVHD 

BKV-HC related renal failure 

BKV-HC: BK virus hemorrhagic cystitis, Allo-SCT: allogeneic stem cell transplantation, RBC: red blood cell, CMV: cytomegalovirus, GVHD: graft-versus-host disease. 

Table 3. Univariate and multivariate analysis for development of BK virus hemorrhagic cystitis. 

5 (27.8)/6 (33.3) 

5 (27.8)/2 (11.1) 

18 (100) 

9 (50.0) 

12 (66.7) 

5 (27.8) 

1 (5.6) 

2 (11.1) 

4 (22.2) 

9 (50.0) 

4 (22.2) 

5 (27.8) 

11 (61.1) 

7 (38.9) 

4 (22.2) 

2 (11.1) 

1 (5.6) 

Characteristics Univariate Multivariate 

HR (95% CI) p-value HR (95% CI) p-value 

Sex 

Male 1.46 (0.98-6.33) 0.097 - - 

Age at transplant 2.11 (1.22-5.38) 0.241 - - 

HLA match 

Mismatched donor 2.23 (1.41-3.56) 0.034 - - 

Conditioning regimen intensity 

Myeloablative conditioning 2.01 (1.10-3.88) 0.092 - - 

Conditioning agents 

Cyclophosphamide-containing regimen 1.98 (0.89-5.31) 0.366 - - 

GVHD prophylaxis 

T-cell depletion 0.75 (0.43-1.24) 0.082 - - 

Acute GVHD 

Grades II-IV 3.52 (1.68-7.83)



Table 4. Clinical characteristics of the patients receiving leflunomide therapy. 

Patient 1 Patient 2 Patient 3 Patient 4 

Age (years)/Sex 40/M 49/M 50/F 52/M 

Diagnosis AML AML MDS AML 

Donor type MSD MSD MUD MSD 

Conditioning Bu/Flu Bu/Flu Bu/Flu Bu/Cy 

GVHD prophylaxis CsA/MTX CsA/MTX CsA/MTX CsA/MTX 

T-cell depletion No No Yes No 

Onset of BKV-HC (days) 24 21 19 23 

Severity of HC Grade III Grade III Grade III Grade III 

Acute GVHD Grade II (skin) Grade II (skin, liver) Grade I (skin) Grade II (skin) 

Chronic GVHD Extensive Extensive Extensive Limited 

CMV antigenemia No Yes No No 

Response after therapy CR PR PR CR 

PCR positivity for BKV after therapy Positive Negative Positive Negative 

Adverse effects Nausea (mild) Nausea (mild) Abdominal bloating None 

Duration to response (days) 8 14 12 17 

Outcome Alive Dead (due to leukemia Alive 

Alive 

relapse) 

M: Male, F: female, AML: acute myeloid leukemia, MDS: myelodysplastic syndrome, MSD: matched sibling donor, MUD: matched unrelated donor, Bu: busulfan, Flu: fludarabine, Cy: 

cyclophosphamide, CsA: cyclosporine, MTX: methotrexate, Allo-SCT: allogeneic stem cell transplantation, BKV-HC: BK virus hemorrhagic cystitis, GVHD: graft-versus-host disease, 

CMV: cytomegalovirus, CR: complete response, PR: partial response, PCR: polymerase chain reaction. 

Discussion 

Our results, with an overall incidence of HC following Allo-SCT 

of 43.5%, are consistent with other studies’ findings, which 

reported frequencies of HC following SCT ranging from 5% to 

68% [3,4,5,6,7]. BKV was identified in 60.0% of cases (18 of 

30 patients) by a qualitative PCR-based assay. A study of 22 

Allo-SCT patients who experienced HC showed that the most 

frequent virus detected was BKV, with an incidence of 54.5% 

of patients, followed by JC virus and CMV [24]. In a study of 

102 children who underwent Allo-SCT for malignancies and 

nonmalignant diseases, HC occurred in 26 patients (25.5%), and 

among them, BKV was identified in 21 (80.8%) patients [25]. 

These findings demonstrated that HC is a frequent complication 

after Allo-SCT and BKV is mainly responsible for HC in Allo-SCT 

recipients. 

Hemorrhagic cystitis after SCT frequently caused prolongation 

of hospitalization and occasionally death [10,24,26]. Gilis et 

al. demonstrated that the median duration of hospitalization 

for Allo-SCT was significantly longer for patients developing 

BKV-HC compared with those without BKV-HC (50 vs. 40 days, 

p



of patients, respectively [21]. In a pilot study with five pediatric 

patients with severe BKV-HC after Allo-SCT, significantly 

shorter duration of BKV-HC (p



20. Wu KH, Weng T, Wu HP, Peng CT, Sheu JN, Chao YH. Effective treatment of 

severe BK virus-associated hemorrhagic cystitis with leflunomide in children 

after hematopoietic stem cell transplantation: a pilot study. Pediatr Infect 

Dis J 2014;33:1193-1195. 

21. Chen XC, Liu T, Li JJ, He C, Meng WT, Huang R. Efficacy and safety of 

leflunomide for the treatment of BK virus-associated hemorrhagic cystitis 

in allogeneic hematopoietic stem cell transplantation recipients. Acta 

Haematol 2013;130:52-56. 

22. Przepiorka D, Anderlini P, Saliba R, Cleary K, Mehra R, Khouri I, Huh YO, 

Giralt S, Braunschweig I, van Besien K, Champlin R. Chronic graft-versushost 

disease after allogeneic blood stem cell transplantation. Blood 

2001;98:1695-1700. 


Thomas ED. 1994 Consensus conference on acute GVHD grading. Bone 


24. Kim SY, Lee JW, Lee KM, Cho BS, Eom KS, Kim YJ, Lee S, Min CK, Kim HJ, 

Cho SG, Kim DW, Min WS, Kim CC. Viruria in adult hemorrhagic cystitis 

patients following allogeneic hematopoietic stem cell transplantation and 

implication of antiviral treatment. Korean J Hematol 2007;42:114-121. 

25. Gorczynska E, Turkiewicz D, Rybka K, Toporski J, Kalwak K, Dyla A, Szczyra 

Z, Chybicka A. Incidence, clinical outcome, and management of virusinduced 

hemorrhagic cystitis in children and adolescents after allogeneic 

hematopoietic cell transplantation. Biol Blood Marrow Transplant 

2005;11:797-804. 

26. Gilis L, Morisset S, Billaud G, Ducastelle-Lepretre S, Labussiere-Wallet H, 

Nicolini FE, Barraco F, Detrait M, Thomas X, Tedone N, Sobh M, Chidiac 

C, Ferry T, Salles G, Michallet M, Ader F; Lyon BK Virus Study Group. High 

burden of BK virus-associated hemorrhagic cystitis in patients undergoing 

allogeneic hematopoietic stem cell transplantation. Bone Marrow 

Transplant 2014;49:664-670. 

27. Vats A, Shapiro R, Singh Randhawa P, Scantlebury V, Tuzuner A, Saxena 

M, Moritz ML, Beattie TJ, Gonwa T, Green MD, Ellis D. Quantitative viral 

load monitoring and cidofovir therapy for the management of BK 

virus-associated nephropathy in children and adults. Transplantation 

2003;75:105-112. 

28. Kadambi PV, Josephson MA, Williams J, Corey L, Jerome KR, Meehan SM, 

Limaye AP. Treatment of refractory BK virus-associated nephropathy with 

cidofovir. Am J Transplant 2003;3:186-191. 

29. Thamboo TP, Jeffery KJ, Friend PJ, Turner GD, Roberts IS. Urine cytology 

screening for polyoma virus infection following renal transplantation: the 

Oxford experience. J Clin Pathol 2007;60:927-930. 

30. Gonzalez-Fraile MI, Canizo C, Caballero D, Hernandez R, Vazquez L, Lopez 

C, Izarra A, Arroyo JL, de la Loma A, Otero MJ, San Miquel JF. Cidofovir 

treatment of human polyomavirus-associated acute haemorrhagic cystitis. 

Transpl Infect Dis 200;3:44-46. 

31. Held TK, Biel SS, Nitsache A, Kurth A, Chen S, Gelderblom HR, Sieqert W. 

Treatment of BK virus-associated hemorrhagic cystitis and simultaneous CMV 

reactivation with cidofovir. Bone Marrow Transplant 2000;26:347-350. 

32. Hatakeyama N, Suzuki N, Kudoh T, Hori T, Mizue N, Tsutsumi H. Successful 

cidofovir treatment of adenovirus-associated hemorrhagic cystitis and 

renal dysfunction after allogenic bone marrow transplant. Pediatr Infect 

Dis J 2003;22:928-929. 

33. Leung AY, Suen CK, Lie AK, Liang RH, Yuen KY, Kwong YL. Quantification 

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230


DOI: 10.4274/tjh.2015.0079 


A Randomized Study Comparing the Efficacy of Three Hepatitis B 

Vaccine Induction Regimens in Adult Patients with Hematological 

Malignancies 

Erişkin Hematolojik Maligniteli Hastalarda Üç Tip Hepatit B Aşılama Rejimini Karşılaştıran 

Randomize Bir Çalışma 

Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı 

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

Abstract 

Objective: Non-responsiveness to hepatitis B virus (HBV) vaccines is 

not rare in hemato-oncological patients due to disease-associated or 

treatment-induced immune suppression. Although different strategies 

have been employed to improve the response rates, to date there is 

not an approved schedule for HBV immunization in patients with 

hematological malignancies. We designed a prospective randomized 

study to evaluate the efficacy of 3 different induction regimens for 

HBV vaccination. 

Materials and Methods: In the standard-dose (SD) group, total 

vaccine dose delivered was 40 µg and patients were vaccinated with 

20 µg at weeks 0 and 4. In the high-dose dose-intensive (HDDI) group, 

total vaccine dose delivered was 80 µg and patients were vaccinated 

with 40 µg at weeks 0 and 4. In the high-dose time-intensive (HDTI) 

group, total vaccine dose delivered was 80 µg and patients were 

vaccinated with 20 µg at weeks 0, 2, 4, and 6. 

Results: In a cohort of 114 patients, 38.6% responded to HBV 

vaccination. The response rate in the SD arm, HDDI arm, and HDTI arm 

was 26.2%, 29.7%, and 44.4%, respectively (p>0.05). Age was the only 

variable identified as having a negative impact on response. 

Conclusion: Short of achieving statistical significance, a higher 

response rate was observed in the HDTI arm. Therefore, this study 

supports a high-dose, time-intensive HBV vaccine induction regimen 

in patients with hematological malignancies who are not on 

chemotherapy. 

Keywords: Hepatitis B, Vaccine, Hematological malignancies 

Amaç: Hematolojik maliniteli hastalarda hastalık veya tedavi ilişkili 

immün baskılanma yüzünden Hepatit B virüsü (HBV) aşısı yanıtsızlığı 

nadir değildir. Aşı yanıtını düzeltecek yöntemler denenmiş olsa da 

hematolojik maligniteli hastalarda kabul görmüş bir protokol henüz 

mevcut değildir. 

Öz 

Öz 

Gereç ve Yöntemler: Üç farklı HBV aşılama rejimini karşılaştıran 

ileriye dönük ve randomize bir çalışma tasarlandı. Standart doz (SD) 

grubuna toplam 40 µg olmak üzere 0. ve 4. haftada 20 µg HBV aşısı 

yapıldı. Yüksek doz-doz yoğun (YDDY) gruba toplam 80 µg HBV aşısını 

0. ve 4. haftada 40 µg uygulandı. Yüksek doz-sık uygulama (YDSU) 

grubuna toplam 80 µg HBV aşısı 0., 2., 4. ve 6. haftalarda 20 µg yapıldı. 

Bulgular: Yüz on dört hastayı içeren bu çalışmada HBV aşısına yanıt 

%38,6 bulundu. Yanıt oranı SD, YDDY ve YDSU kolu için yanıt sırası ile 

%26,2, %29,7 ve %44,7 bulundu (p>0,05). Aşı yanıtı üzerine olumsuz 

etkili tek değişkenin yaş olduğu saptandı. 

Sonuç: İstatistiksel anlamı olmasa da YDSU kolunda HBV aşı yanıtı 

oransal olarak daha yüksek bulundu. Bu çalışmada elde edilen sonuçlar 

hematolojik maligniteli hastalarda yüksek doz ve daha sık uygulanan 

HBV aşılama rejiminin daha etkin olduğunu düşündürmektedir. 

Anahtar Sözcükler: Hepatit B, Aşılama, Hematolojik malignite 

Introduction 

Hepatitis B virus (HBV) infection, the most common chronic viral 

infection in the world, can have serious clinical complications 

ranging from fulminant hepatitis to cirrhosis and hepatocellular 

carcinoma [1]. Vaccination is most effective in preventing HBV 

infection and complications. The complete vaccine series induce 

protective antibody levels in more than 95% of infants, children, 

and young adults. Protection has been estimated to last at least 20 

years and is possibly lifelong [2]. It is generally held that patients 

Address for Correspondence/Yazışma Adresi: Zübeyde Nur ÖZKURT, M.D., 

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

Phone : +90 312 202 63 17 

E-mail : zubeydenurozkurt@yahoo.com 

Received/Geliş tarihi: February 10, 2015 


231

Özkurt ZN, et al: A Step Further for Improving Hepatitis B Vaccine Response 


with hematological malignancies are immunosuppressed, either 

as a result of the underlying hematological malignancy or due 

to treatment with chemotherapy. Immunosuppressive diseases 

like hematological malignancies are a risk factor for nonresponsiveness 

to HBV vaccination. Despite low response rates, 

it is recommended that HBV-naive patients with hematological 

malignancies be immunized against HBV [3]. 

Although a rapid and effective strategy for HBV immunization 

of patients with hematological malignancies is highly desirable, 

to date there is not an approved schedule for these patients. 

Different strategies have been employed to improve the response 

rates in immunologically compromised patients, including HIVinfected 

adult patients, and in patients with chronic kidney 

disease [4,5,6]. We designed a prospective randomized study 

to evaluate the efficacy of 3 different induction regimens for 

HBV immunization in patients with hematological malignancies. 

In this study we aim to compare the results of the 3 different 

induction regimens for HBV vaccine at week 8. The basis of this 

deviation of omitting the consolidation dose at month 6, the 

current standard of care for routine HBV vaccination, was to 

decrease the drop-out rate due to stem cell transplantation, 

progression, relapse, death, frequent infections, and the effect 

of infection, antibiotics, and intravenous immunoglobulin on 

vaccination, and to allow more patients to be recruited into the 

study. 


Patients with hematological malignancies followed by the 

Division of Hematology at Gazi University in Turkey between 

January 2008 and December 2013 were included in the study. 

Inclusion criteria were: 

a. Age >18 years 

b. Eastern Cooperative Oncology Group performance status ≥2, 

c. Negative serology for HBsAg, anti-HBc, and anti-HBs, 

d. Negative serology for hepatitis C and HIV, 

e. Chemotherapy-naive patients, 

f. Patients who achieved remission or stable disease after 

chemotherapy, 

g. In subjects with a history of treatment, a time interval of 

at least 3 months from the last dose of chemotherapy and/or 

radiotherapy. 

Exclusion criteria were: 

a. History of prior HBV vaccination, 

b. Evidence of ongoing systemic infection, 

c. Ongoing immunosuppressive therapy, 

d. History of prior stem cell transplantation, 

e. History of a non-hematological malignancy, except 

adequately treated squamous or basal cell carcinoma of the skin 

or cervical carcinoma in situ. 

Chemotherapy regimens were categorized into 4 groups, 

namely alkylating agent-based, purine analog-based, chemo 

immunotherapy, and acute leukemia type, in an attempt to 

evaluate the effect of various drugs with different modes 

of action on vaccine response. Disease status at the time of 

vaccination was evaluated as untreated, in complete remission, 

or stable disease. 

Before vaccination blood was drawn for complete blood count 

and IgG, IgA, and IgM levels. Absolute CD4, CD8, CD3+ CD56 + , 

and CD4+ CD25 + counts were determined by flow cytometry 

(Becton Dickinson, FACSCalibur). Anti-HBs levels were planned 

to be measured at week 8. Patients who completed the 

vaccine schedule and had an anti-HBs serology at week 8 were 

considered eligible for response evaluation. 

Study Protocol 

All patients participating in the study received a recombinant 

HBV vaccine (Genhevac B, Sanofi Pasteur) in the deltoid region, 

intramuscularly. Patients were randomized (1:1:1 ratio) into 1 of 

the 3 groups below to receive the hepatitis B vaccine. Groups 

were named based on the total vaccine dose delivered and 

vaccination frequency. 

Group 1: Standard dose (SD): Patients were vaccinated with 20 

µg at weeks 0 and 4; total vaccine dose was 40 µg. 

Group 2: High-dose dose-intensive (HDDI): Patients were 

vaccinated with 40 µg at weeks 0 and 4; total vaccine dose was 

80 µg. 

Group 3: High-dose time-intensive (HDTI): Patients were 

vaccinated with 20 µg at weeks 0, 2, 4, and 6; total vaccine dose 

was 80 µg. 

Patients who achieved anti-HBs levels of >10 IU/L were defined 

as vaccine responders. The primary endpoint was seroprotection 

rate at week 8 and the secondary endpoint was comparison of 

antibody levels in 3 different HBV vaccination regimens. The 

study protocol was approved by the Local Ethics Committee 

of Gazi University Faculty of Medicine and written informed 

consent was obtained from all subjects prior to study entry. 


Statistical evaluation was done with SPSS 15. Data were 

described as numbers and percentages or medians and 

minimum-maximum, as appropriate. Chi-square test was used 

for evaluating categorical values, and Kruskal-Wallis and oneway 

ANOVA tests were used for continuous values in patient 

groups. All p-values were 2-sided with statistical significance 

at 0.05 alpha levels. Logistic regression analysis was used for 

multivariate analysis to evaluate the factors affecting the 

vaccination response. 

232



Results 

A total of 124 patients were randomized during the study 

period. Ten patients were excluded from the study due to 

cerebrovascular event before the first dose of the vaccine (1 

patient) or not fulfilling the criteria for response evaluation 

after vaccination (9 patients). Characteristics of the patients are 

summarized in Tables 1 and 2. Significant differences existed 

only in CD4/CD8 ratio between the 3 vaccine groups. 

Table 1. Baseline demographic characteristics of the study 

patients. 

Group 1 Group 2 Group 3 p 

Age 54 (22-78) 57 (25-78) 54 (20-80) 0.83 

Sex (Male/ 20/22 25/12 17/18 0.25 

Female) 

Patient (n) 42 37 35 

CLL 18 16 16 

NHL 8 9 7 

HD 7 5 5 

0.92 

AL 6 4 4 

HCL 1 2 2 

PCD 2 1 1 

Treatment 0.66 

No 13 11 13 

Yes 29 26 22 

Radiotherapy 0.76 

No 38 34 33 

Yes 4 3 2 

DTV (months) 10.5 (0-144) 11 (0-83) 12.5 (0-89) 0.96 

LTTV (months) 6 (3-82) 6 (2-64) 9 (3-89) 0.77 

Values are expressed as medians and minimum-maximum where necessary. 

CLL: Chronic lymphocytic leukemia, NHL: non-Hodgkin lymphoma, HD: Hodgkin’s 

disease, AL: acute leukemia, HCL: hairy cell leukemia, PCD: plasma cell disorder, DTV: 

time from diagnosis to vaccination, LTTV: time from last treatment to vaccination. 

Response Evaluation at Week 8 

At the end of the study, 114 patients were eligible for response 

evaluation. Overall, 44 of 114 patients responded, with a 

response rate of 38.6% at week 8. On the other hand, 61.4% of 

the patients did not respond to HBV vaccination. The response 

rate in the SD arm, HDDI arm, and HDTI arm was 26.2%, 29.7%, 

and 44.4%, respectively (Figure 1). The high response rate in the 

HDTI arm did not translate to statistical significance, possibly 

due to relatively low patient numbers (p>0.05). The median 

antibody concentration (MAC) in the SD arm, HDDI arm, and 

HDTI arm was 46.6 IU/L (12.4-706), 73.95 IU/L (14.7-779), and 

47.4 IU/L (11.6-779), respectively (Figure 2). The differences in 

MAC between groups were not statistically significant. Among 

the variables evaluated, only age had a negative impact on 

response (p



600.0 

500.0 

400.0 

trials in patients with hematological malignancies is very limited 

and mostly confined to pediatric patients with acute leukemia 

[8,9,10,11]. The data supporting HBV vaccination almost 

completely come from general vaccination strategies and no 

evidence-based recommendations for the dose, frequency, and 

timing of HBV vaccination in adult hematological patients are 

available. 

300.0 

200.0 

100.0 

00.0 

1 2 3 

The risk of HBV transmission, just after the diagnosis or 

during the chemotherapy of patients, is high due to frequent 

transfusions and interventions. Immediate vaccination is highly 

desirable; however, disease and chemotherapy may compromise 

antibody response. If the vaccination is postponed until 

after the chemotherapy, disease and chemotherapy-related 

immunosuppression are lessened and probability of response 

increases, but active protection from HBV during the high-risk 

period is missed. 

Figure 2. Median antibody concentration response to 3 different 

regimens at week 8. 

Discussion 

In this study, overall response rate, defined as anti-HBs of 

>10 IU/L at week 8, was 38.6%. The response rate in the SD 

arm, HDDI arm, and HDTI arm was 26.2%, 29.7%, and 44.4%, 

respectively. Short of achieving statistical significance, a higher 

response rate was observed in the HDTI arm. The MAC in the SD 

arm, HDDI arm, and HDTI arm was not statistically significant. 

Among the variables evaluated, only age had a negative impact 

on response. 

HBV reactivation is a common complication in HBsAg-positive 

patients undergoing immunosuppressive anticancer therapy. 

The clinical consequences of HBV reactivation are observed as 

asymptomatic liver function disturbances, liver failure, and delay 

or premature cessation of chemotherapy courses with adverse 

prognostic consequences for the hematological disease. It is 

strongly recommended that all hemato-oncological patients be 

screened for HBV markers and immunization against hepatitis 

B be performed in HBV-naive patients when appropriate [3,7]. 

Conducting HBV vaccination trials in adult patients with 

hematological malignancies is troublesome. Heterogeneity of 

both the underlying hematological conditions and chemotherapy 

regimens, maintenance therapies, relapse of the disease, and 

salvage regimens including high-dose chemotherapy with 

stem cell support make the situation more complex. Therefore, 

recruiting a sufficient number of patients for randomized trials 

and multivariate analysis requires the active collaboration of 

centers, especially in developing countries. 

As a result of these difficulties, the number of HBV vaccination 

Accelerated vaccination strategies could be useful for atrisk 

groups in terms of rapid seroconversion and increasing 

adherence. Studies conducted in high-risk healthy adults, 

drug users, lung transplantation candidates, and HIV-infected 

patients elicited similar or better anti-HBs responses and could 

be advantageous for the short term in this population. However, 

additional studies on long-term protection and effectiveness of 

accelerated schedules are necessary [12,13,14,15]. 

Non-responsiveness to HBV vaccine is not rare in hematooncological 

patients due to disease-associated or treatmentinduced 

immune suppression. There are a number of means to 

augment the immune response to HBV immunization in nonresponders, 

including adding additional doses, doubling the 

vaccine dose, intradermal injection of the vaccine, combination 

with granulocyte-macrophage colony-stimulating factor (GM- 

CSF), and use of new, more immunogenic HBV vaccines [16]. We 

have identified 2 studies on improving serological response to 

HBV vaccine in adult patients with hematological malignancies. 

First, Pullukcu et al. carried out a non-randomized study in 42 

HBV-naive hematology patients during chemotherapy. Patients 

were administered a 20 µg HBV vaccine on days 0, 14, and 28. 

Overall, 23.8% of the patients responded to this accelerated 

schedule during chemotherapy [17]. 

The second study was a randomized one comparing the efficacy 

of a single dose of 40 µg HBV vaccine with one course of 40 µg 

HBV vaccine after 5 µg/kg recombinant GM-CSF injection in 94 

patients with lymphoproliferative disorders (LPDs). Although the 

seroprotection rate was higher in the GM-CSF + HBV vaccine 

group (25.5% vs. 17%), the difference did not reach a significant 

level. In multivariate analysis, age was the only predictor of 

achieving a seroprotective response. The authors concluded that 

in LPDs, the response to HBV vaccine is impaired and GM-CSF 

may enhance the response rate to HBV vaccine [18]. 

234



The available data are far from allowing definite recommendations. 

However, some useful information about the dose, frequency, 

and timing of the vaccination may be collected for the design of 

future studies in adult patients with hematological disease. First 

of all, response to standard HBV vaccination is impaired and 

doubling the vaccine dose per injection does not increase the 

response rate at week 8. On the other hand, frequent antigenic 

stimulation seems to induce better immune response than a 

standard schedule. Thus, an accelerated vaccination schedule is 

feasible in this patient group. 

The durability of the response, even in stable disease conditions, 

is another factor that needs to be taken into consideration. In 

our previous study, some of the patients lost their seroprotective 

anti-HBs responses beginning at the sixth month of the 

vaccination [18]. Therefore, we suggest that a single booster 

dose of vaccine should be given to responders at the sixth month 

of vaccination. Finally, response rate to HBV vaccination during 

chemotherapy is low even though a frequent injection scheme 

is used [17]. Moreover, anti-HBs response may be lost during a 

chemotherapy course [19]. Whether patients who lost anti-HBs 

response during chemotherapy need re-vaccination or a single 

booster dose of vaccine to reinduce antibody production is not 

known. 

The findings from this study suggest a time-intensive approach, 

at 20 µg biweekly of 3-4 doses of HBV vaccine, for the design of 

future studies of adult patients with hematological disease who 

are not on chemotherapy. 

Ethics 

Ethics Committee Approval: The study protocol was approved 

by the Local Ethics Committee of Gazi University Faculty of 

Medicine; Informed Consent: Written informed consent was 

obtained from all subjects prior to study entry. 


Medical Practices: Zübeyde Nur Özkurt, Elif Suyanı, Rauf 

Haznedar, Münci Yağcı; Concept: Zübeyde Nur Özkurt, Münci 

Yağcı; Design: Münci Yağcı, Zübeyde Nur Özkurt; Data Collection 

or Processing: Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, 

Münci Yağcı; Analysis or Interpretation: Zübeyde Nur Özkurt, 

Elif Suyanı, Münci Yağcı; Literature Search: Zübeyde Nur Özkurt, 

Elif Suyanı, Rauf Haznedar, Münci Yağcı; Writing: Zübeyde Nur 

Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı. 

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



included. 

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2063. 

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standard hepatitis B vaccination schedules in high-risk healthy adults: a 

meta-analysis of randomized controlled trials. PLoS One 2015;10:e0133464. 

13. Shah DP, Grimes CZ, Nguyen AT, Lai D, Hwang LY. Long-term effectiveness 

of accelerated hepatitis B vaccination schedule in drug users. Am J Public 

Health 2015;105:36-43. 

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Baden LR,Marty FM, Issa NC. Serologic response to hepatitis B vaccination 

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15. Mena G, Llupià A, García-Basteiro AL, Díez C, León A, García F, Bayas JM. 

Assessing the immunological response to hepatitis B vaccination in HIVinfected 

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16. Chen DS. Hepatitis B vaccination: the key towards elimination and 

eradication of hepatitis B. J Hepatol 2009;50:805-816. 

17. Pullukcu H, Ertem E, Karaca Y, Yamazhan T, Sertoz RY, Altuglu I. Efficacy 

of accelerated hepatitis B vaccination program in patients being actively 

treated for hematologic malignancies. Int J Infect Dis 2008;12:166-170. 

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lymphoproliferative disorders: a prospective randomized study evaluating 

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235


DOI: 10.4274/tjh.2015.0242 


Reliability and Validity of the Turkish Version of the PedsQL 3.0 

Cancer Module for 2- to 7-Year-Old and the PedsQL 4.0 Generic 

Core Scales for 5- to 7-Year-Old: The Hacettepe University 

Experience 

Çocuklar için Yaşam Kalitesi Ölçeği Kanser Modülü Türkçe Versiyonunun 2-7 Yaşları 

Arasındaki Çocuklarda ve Genel Skalası’nın 5-7 Yaşları Arasındaki Çocuklarda Geçerlik ve 

Güvenilirliği: Hacettepe Üniversitesi Deneyimi 

Vesile Yıldız Kabak 1 , Yavuz Yakut 1 , Mualla Çetin 2 , Tülin Düger 1 

1Hacettepe University Faculty of Health Sciences, Department of Physical Therapy and Rehabilitation, Ankara, Turkey 

2Hacettepe University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey 

Abstract 

Objective: The aim of this study was to investigate the reliability and 

validity of the Turkish version of the Pediatric Quality of Life Inventory 

(PedsQL) 3.0 Cancer Module for 2- to 7-year-old and the PedsQL 4.0 

Generic Core Scales for 5- to 7-year-old in childhood cancer. 

Materials and Methods: The PedsQL 3.0 Cancer Module and PedsQL 

4.0 Generic Core Scales were administered to children with cancer 

and their parents at Hacettepe University. Internal consistency was 

determined by using Cronbach’s alpha and test-retest reliability 

was determined by using the intraclass correlation coefficient (ICC). 

Construct validity was assessed by comparing the results of the PedsQL 

3.0 Cancer Module with those of the PedsQL 4.0 Generic Core Scales. 

Results: Cronbach’s alpha of the PedsQL 3.0 Cancer Module varied 

from 0.803 to 0.873 and that of the PedsQL 4.0 Generic Core Scales 

from 0.665 to 0.841. Test-retest ICC values of the PedsQL 3.0 Cancer 

Module varied from 0.877 to 0.949 and those of the PedsQL 4.0 

Generic Core Scales from 0.681 to 0.824. The correlation of the PedsQL 

3.0 Cancer Module with subscale scores of the PedsQL 4.0 Generic 

Core Scales showed that there were excellent to fair correlations 

between the two scales. The relationship between parent proxy-report 

and child self-report of the PedsQL 3.0 Cancer Module had very good 

correlation (r=0.694, p


Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University 

Introduction 

Quality of life (QOL) has been described as a subjective term and 

is defined as a person’s sense of social, emotional, and physical 

well-being and his/her ability to function in ordinary tasks of 

daily living [1,2,3,4]. Therefore, a health-related quality of life 

(HRQOL) instrument should include physical, mental, and social 

health dimensions [5,6]. It is increasingly acknowledged as an 

important health outcome measure in clinical trials and health 

service research and evaluation [7]. 

Disease-specific HRQOL assessment instruments have been 

developed to determine the impact of disease and treatment on 

the quality of patients’ life. However, there are a limited number 

of instruments designed to measure the HRQOL of pediatric 

patients with cancer [8,9,10]. 

The Pediatric Quality of Life Inventory (PedsQL), which has both 

generic and disease-specific modules as well as patient and 

parent versions, is one of the very few instruments that is widely 

used to assess HRQOL among children and adolescents between 

the ages of 2 and 18 years [3,11]. It is brief, can be applied in 5-15 

min, and can be scored easily [12,13]. Evaluation is conducted 

by both children and parents; children aged 5 to 18 years are 

asked to evaluate their own HRQOL (child self-report) and the 

parents of children aged 2 to 18 years are asked to evaluate 

their child’s HRQOL (parent proxy-report) [14]. The PedsQL 4.0 

Generic Core Scales were specifically designed for application 

in both healthy and patient populations. The PedsQL 3.0 Cancer 

Module was designed to measure HRQOL dimensions specific to 

pediatric cancers [7]. This instrument has already been validated 

in English [15], German [16], Chinese [11], Japanese [14], Urdu 

[17], and Portuguese [8]. In Turkey, the validity and reliability of 

the PedsQL 4.0 Generic Core Scales for 8- to 12-year-old and 

13- to 18-year-old children were evaluated, as was the PedsQL 

3.0 Cancer Module for 8- to 12-year-old children [18,19,20]. 

Uneri et al. investigated the reliability and validity of the Turkish 

translation of the PedsQL 4.0 Generic Core Scales for 2- to 

4-year-old and 5- to 7-year-old Turkish children. They found 

that the validity of the parent proxy-reports for the two age 

groups was sufficient, whereas the validity of the child selfreport 

of the 5- to 7-year-old age group was low [12]. 

The aim of this study was to investigate the reliability and 

validity of the Turkish version of the PedsQL 3.0 Cancer Module 

for 2- to 4-year-old and 5- to 7-year olds and the reliability and 

validity of the Turkish version of the PedsQL 4.0 Generic Core 

Scales for 5- to 7-year-old in childhood cancer. 


Patients and Setting 

This study was developed in Turkey. We recruited children with 

cancer and their parents from Hacettepe University Hospital. 

Children between the ages of 2 and 7 who were diagnosed 

with cancer at least 2 months earlier, who agreed to participate 

in the study, and who had good verbal communication were 

included. Children with comorbid disease, major developmental 

disorders, and neurologic problems were excluded from the 

study. The 2- to 4-year-old age group consisted of 43 children 

and their parents. The 5- to 7-year-old age group consisted of 

31 children and their parents. Informed consent was obtained 

from all individual participants included in the study. 

Instruments 

PedsQL 4.0 Generic Core Scales 

The PedsQL 4.0 Generic Core Scales were designed by Varni et al. 

in 1999 as a HRQOL measurement for children and adolescents 

aged between 2 and 18 years [13]. It consists of 23 items: physical 

functioning (8 items), emotional functioning (5 items), social 

functioning (5 items), and school functioning (5 items). Child 

self-reports include 3 age groups: 5-7 years (young children), 

8-12 years (children), and 13-18 years (teens). The parent proxyreport, 

however, includes 4 age groups: 2-4 (toddlers), 5-7, 

8-12, and 13-18. The response scale is a 5-point Likert scale for 

all age groups, except for the 5- to 7-year-old’ version. Items 

are reverse-scored and linearly transformed to a 0-100 scale, 

so that higher scores indicate better HRQOL. The response scale 

of the 5- to 7-year-old version is completed with the help of 

an interviewer and simplified to a 3-point scale (0, 2, and 4 

points). The child answers the items with the help of a visual 

scale (happy, neutral, and sad faces). The PedsQL 4.0 computes 

the scale scores as well as the Psychosocial Health Summary 

Scores by adding the sum of points from the Emotional, Social, 

and School Functioning Subscales and dividing them by the 

total number of items answered [21,22]. 

PedsQL 3.0 Cancer Module 

The PedsQL 3.0 Cancer Module was developed and tested for 

validity and reliability by Varni et al. [3,22,23]. This module 

consists of 27 items: pain and hurt (2 items), nausea (5 items), 

procedural anxiety (3 items), treatment anxiety (3 items), worry 

(3 items), cognitive problems (5 items), perceived physical 

appearance (3 items), and communication (3 items). The format, 

instructions, Likert response options, and scoring method are 

similar to those of the PedsQL 4.0 Generic Core Scales [22]. This 

scale comprises two parallel forms for child and for parent. 

Higher scores indicate better HRQOL [21]. 

Translation and Cross-Cultural Adaptation 

This study was conducted in these phases: 

1. Translation, validation, and reliability of the PedsQL 3.0 

Cancer Module for 2- to 4-year-old parent proxy-report and 

5- to 7-year-old children’s form and parent proxy-report. 

237



2. Translation, validation, and reliability of the PedsQL 4.0 

Generic Core Scales for 5- to 7-year-old children’s form. 

In the translation process, we used the guidelines for crosscultural 

adaptation and we obtained permission for the Turkish 

version from Varni et al. (Mapi Research Trust) [4,24]. Approval of 

the study was obtained from the Ethics Committee of Hacettepe 

University (GO 14/455). 

The original English instruments (PedsQL 4.0 Generic Core Scale 

/5-7 years and PedsQL 3.0 Cancer Module/2-4 and 5-7 years) 

were translated independently into Turkish. Two translations 

from English to Turkish were done by two different and 

independent native Turkish translators. The Turkish translations 

were then compared for inconsistencies. The two translations 

were then retranslated, also blindly and independently, into 

English by two native English speakers. The Turkish version was 

then jointly reviewed by a bilingual team, including the four 

translators, three physical therapists, and a physician, to assess 

the necessity of cultural adaptation. The Turkish version was 

compared with the original English version to detect possible 

errors of interpretation and nuances that might have been 

missed. The final stage of the adaptation process was to test 

the prefinal version. Ten children were tested in this stage. The 

results eliminated the necessity for Turkish cultural adaptation. 


Reliability 

Two common forms of reliability are test-retest reliability and 

internal consistency. For test-retest reliability, the forms were 

applied in 7-day intervals. We used the intraclass correlation 

coefficient (ICC) to evaluate test-retest reliability. The ICC can 

vary from 0.00 to 1.00, where values of 0.60 to 0.80 are regarded 

as evidence of good reliability and those above 0.80 indicate 

excellent reliability [25,26,27]. 

The internal consistency of a scale relates to its homogeneity. 

The coefficient of internal consistency is mainly assessed with 

Cronbach’s alpha. It is suggested that the value of alpha should 

be above 0.80 for acceptance as high internal consistency [28]. 

Validity 

In this study, construct validity was assessed by comparing the 

responses to the PedsQL 3.0 Cancer Module to the results of the 

PedsQL 4.0 Generic Core Scales. Construct validity coefficients (r) 

were accepted as follows: 0.81-1.0 as excellent, 0.61-0.80 very 

good, 0.41-0.60 good, 0.21-0.40 fair, and 0-0.20 poor. Construct 

validity was measured by Pearson’s correlation coefficient [29]. 

All assessments were repeated 7 days later by the physical 

therapist. Means and standard deviations were determined to 

describe the demographic data of the patients. All statistical 

analyses were done with IBM-SPSS 22.0 for Windows. A 

probability value of p



Table 1. Demographic characteristics of the samples. 

2- to 4-year-old (n=43) 5- to 7-year-old (n=31) 

n % n % 

Sex 

Girl 21 51.2 14 45.2 

Boy 22 48.8 17 54.8 

Current treatment status 

On-treatment 8 18.6 3 9.7 

Off-treatment 8 18.6 14 45.2 

Antibiotic treatment 27 62.8 10 32.3 

Respondent parent 

Mother 37 86.0 26 83.9 

Father 4 9.3 3 9.7 

Other 2 4.65 2 6.5 

Education level of the parent 

Illiterate 4 9.3 1 3.2 

Primary school 9 20.9 6 19.4 

Secondary school 2 4.7 3 9.7 

High school 13 30.2 7 22.6 

University 15 34.9 12 38.7 

Mean SD Mean SD 

The age of the children (years) 3.24 0.12 6.27 0.77 

The age of the parents (years) 31.81 0.93 37.0 7.95 

Total duration after diagnosis (months) 11.95 1.65 18.60 18.15 

Body mass index (kg/m 2 ) 16.05 0.4 16.54 3.22 

SD: Standard deviation. 

Table 2. Pediatric Quality of Life Inventory 3.0 Cancer Module and 4.0 Generic Core Scale Scores. 

2- to 4-year-old 5- to 7-year-old 

Parent Proxy-Report Parent Proxy-Report Child Self-Report 

Mean SD Mean SD Mean SD 

PedsQL 3.0 Cancer Module 

Pain and hurt 72.49 21.03 75.29 20.27 76.66 25.37 

Nausea 72.23 21.75 57.43 29.89 67.24 25.05 

Procedural anxiety 38.19 28.52 50.25 29.95 61.36 31.25 

Treatment anxiety 59.12 35.39 76.96 32.57 84.30 28.14 

Worry 70.26 35.00 80.25 31.97 73.85 24.96 

Cognitive problems 71.81 23.08 81.45 21.74 78.83 20.45 

Perceived physical appearance 78.83 25.80 68.80 30.07 72.89 28.07 

Communication 62.90 38.01 64.67 42.55 77.66 27.64 

Total score 64.51 18.27 68.16 22.05 73.33 17.85 

PedsQL 4.0 Generic Core Scale 

Physical functioning 70.98 19.99 68.64 26.86 71.73 20.44 

Emotional functioning 60.47 20.23 70.48 23.03 74.63 17.37 

Social functioning 84.40 18.89 78.53 23.03 82.82 19.10 

School-related problems 67.57 33.50 59.90 30.17 66.55 26.23 

Total score 70.53 15.22 70.32 18.85 73.73 16.08 

SD: Standard deviation, PedsQL: Pediatric Quality of Life Inventory. 

239



Table 3. Pearson’s correlation coefficients between total scores and subscale scores of the scales. 

PedsQL 3.0 Cancer Module Subscale Scores 

PH N PA TA W CP PPA C 

Total scores (5-7 years old) 

Parent proxy-report 0.533* 0.949** 0.709** 0.768** 0.709** 0.512* 0.864** 0.744** 

Child self-report 0.408* 0.829** 0.664** 0.622** 0.701** 0.702** 0.516* 0.816** 


Parent proxy-report 0.393* 0.621** 0.671** 0.744** 0.620** 0.588** 0.746** 0.736** 

Child self-report - - - - - - - - 

PedsQL 4.0 Generic Core Scale Subscale Scores 

PF EF SF SRP 


Parent proxy-report 0.678** 0.761** 0.647** 0.817** 

Child self-report 0.908** 0.748** 0.743** 0.526* 

*p



Table 5. Pearson’s correlation coefficients between subscale scores of the 5- to 7-year-old parent proxy-report of the Pediatric 

Quality of Life Inventory 3.0 Cancer Module and Pediatric Quality of Life Inventory 4.0 Generic Core Scales. 

PedsQL 3.0 Cancer Module, 

5- to 7-year-old 

Physical Functioning 

Emotional 

Functioning 

PedsQL 4.0 Generic Core Scale, 

5- to 7-year-old 

Social Functioning 

School-Related 

Problems 

Pain and hurt 0.455* 0.179 -0.120 0.060 0.296 

Nausea 0.227 0.625** 0.399* 0.583* 0.586* 

Procedural anxiety 0.197 0.418* 0.357 0.578* 0.460* 

Treatment anxiety -0.136 0.739** 0.566* 0.674* 0.468* 

Worry -0.005 0.769** 0.578* 0.701* 0.583* 

Cognitive problems 0.038 0.649** 0.690** 0.706** 0.503* 

Perceived physical appearance 0.103 0.713** 0.536* 0.513* 0.545* 

Communication 0.154 0.604** 0.312 0.627* 0.510* 

Total score 0.179 0.810** 0.562* 0.795** 0.694** 

*p

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University Turk J Hematol 2016;33:236-243 

significant and good (r=0.540, p=0.002). This lower correlation 

and internal consistency of the 5- to 7-year-old age group may 

be related to school functioning. In Turkey, children of this age 

do not go to school yet, and children with chronic diseases 

quit school. Therefore, most of the children in this group had 

difficulties understanding school functioning questions and 

some parents mentioned that their children were too young to 

understand some of the questions. Only 16 parents and children 

answered the school functioning questions, which might be due 

to misunderstanding some questions (for example, forgetting 

things). This indicated that not only are some modifications to 

the school functioning questions for children aged 5-7 years 

necessary, but also that the parent-proxy report and child selfreport 

should be applied together in this age group. 

The PedsQL 3.0 Cancer Module Scales internal consistency 

reliabilities generally exceeded the recommended minimum 

alpha coefficient standard of 0.70 for group comparison for 

child self-report for ages 8-18 years and parent proxy-report 

for ages 2-18 years. For self-report at ages 5-7 years, only the 

procedural anxiety and treatment anxiety scales met the 0.70 

standard and for most scales values were in the range of 0.80 

to 0.90 [4]. 

In our study, Cronbach’s coefficient alpha of the PedsQL 3.0 

Cancer Module for 2- to 7-year-old was from 0.803 to 0.73 

for the parent-proxy reports and the child self-reports (high). 

Test-retest ICC values were 0.877 to 0.949 (excellent) and the 

correlations between total score and subscale scores for 5- to 

7-year-old were higher than those for 2- to 4-year-old. The 

Turkish version of the PedsQL 3.0 Cancer Module in children 

aged 2-7 years can be applied for the Turkish population. 

The intercorrelations between the PedsQL 3.0 Cancer Module 

and PedsQL 4.0 Generic Core Scales parent proxy-reports were 

in the range of moderate to high, except for ‘worry’ and ‘schoolrelated 

problems’ in 2- to 4-year-old, and except for ‘pain and 

hurt’ and ‘physical functioning’ in 5- to 7-year-old. In Turkey, 

many children between 2 and 4 years old do not go to school 

or kindergarten. Therefore, not all parents answered schooling 

questions. At these ages, on the other hand, children do not 

know about their disease and its treatment. In 5- to 7-yearold, 

subscales and their questions of the cancer module do not 

correlate with physical situations. According to the results of 

5- to 7-year-old, while parents correlated physical dysfunctions 

with ‘pain and hurt’, children correlated them with ‘nausea’. In 

this self-report, ‘procedural anxiety’ and ‘perceived physical 

appearance’ do not correlate with Generic Core Scale subscales, 

and the ‘social functioning subscale’ of the Generic Core Scale 

does not correlate with Cancer Module subscales. These low 

correlations in subscales might be due to the small number of 

items that compose the subscales, the low level of schooling in 

these ages, or the absence of physical function subscales in the 

cancer module. Total scale score may be suitable as a summary 

score for the primary analysis of the PedsQL Inventory in clinical 

trials and other group comparisons. 

Parent-child agreement about QOL is controversial in the 

literature [30]. Some studies reported high agreement, whereas 

others reported low agreement [31,32,33]. In our study, there was 

an excellent correlation between the parent-proxy report and 

child self-report for 5- to 7-year-old with the PedsQL 3.0 Cancer 

Module. Although patient self-report is considered the standard 

for measuring perceived HRQOL, it is the parent’s perception of 

the child’s HRQOL that may influence healthcare utilization [34]. 

In clinical practice, there may be circumstances in which the child 

is too young to understand the questions, or too ill and unwilling 

to complete an instrument. Therefore, in cases in which pediatric 

patients are not able to provide self-reports, reliable and valid 

parent proxy-report instruments are needed [35]. 

In conclusion, our results indicated that the Turkish versions of 

the PedsQL 4.0 Generic Core Scales for 5- to 7-year-old and 

the PedsQL 3.0 Cancer Module for 2- to 7-year-old are easy 

to understand, reliable, and valid instruments in the Turkishspeaking 

population. These instruments may be utilized as 

outcome measures in pediatric cancer clinical trials, research, 

and clinical practice for HRQOL outcome assessment. However, 

we suggest that the parent proxy-report and child self-report 

should be used together for 5- to 7-year-old. Further studies 

should focus on testing the responsiveness and reliability of the 

PedsQL in patients who continue or finish treatments and in 

long-term follow-up measurements. 

Ethics 

Ethics Committee Approval: The approval of the Ethic Committee 

of the Hacettepe University was obtained about this study (GO 

14/455); Informed Consent: It was taken. 


Concept: Tülin Düger; Design: Mualla Çetin; Data Collection or 

Processing: Vesile Yıldız Kabak; Analysis or Interpretation: Yavuz 

Yakut, Vesile Yıldız Kabak; Literature Search: Vesile Yıldız Kabak; 

Writing: Tülin Düger. 

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



included. 

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243

BRIEF REPORT 

DOI: 10.4274/tjh.2015.0368 


Results of Four-Year Rectal Vancomycin-Resistant Enterococci 

Surveillance in a Pediatric Hematology-Oncology Ward: From 

Colonization to Infection 

Bir Pediatrik Hematoloji-Onkoloji Ünitesinde Vankomisine Dirençli Enterokok Kolonizasyon 

ve Enfeksiyonu: Dört Yıllık Sürveyans Sonuçları 

Hacer Aktürk 1 , Murat Sütçü 1 , Ayper Somer 1 , Serap Karaman 2 , Manolya Acar 1 , Ayşegül Ünüvar 2 , Sema Anak 2 , Zeynep Karakaş 2 , Aslı 

Özdemir 3 , Kutay Sarsar 4 , Derya Aydın 4 , Nuran Salman 1 

1İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Infectious Diseases, İstanbul, Turkey 

2İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 

3İstanbul University İstanbul Faculty of Medicine, Infection Control Committee, İstanbul, Turkey 

4İstanbul University İstanbul Faculty of Medicine, Department of Clinical Microbiology, İstanbul, Turkey 

Abstract 

Objective: To investigate the clinical impact of vancomycinresistant 

enterococci (VRE) colonization in patients with hematologic 

malignancies and associated risk factors. 

Materials and Methods: Patients colonized and infected with VRE 

were identified from an institutional surveillance database between 

January 2010 and December 2013. A retrospective case-control 

study was performed to identify the risk factors associated with 

development of VRE infection in VRE-colonized patients. 

Results: Fecal VRE colonization was documented in 72 of 229 

children (31.4%). Seven VRE-colonized patients developed subsequent 

systemic VRE infection (9.7%). Types of VRE infections included 

bacteremia (n=5), urinary tract infection (n=1), and meningitis (n=1). 

Enterococcus faecium was isolated in all VRE infections. Multivariate 

analysis revealed severe neutropenia and previous bacteremia with 

another pathogen as independent risk factors for VRE infection 

development in colonized patients [odds ratio (OR): 35.4, confidence 

interval (CI): 1.7-72.3, p=0.02 and OR: 20.6, CI: 1.3-48.6, p=0.03, 

respectively]. No deaths attributable to VRE occurred. 

Conclusion: VRE colonization has important consequences in 

pediatric cancer patients. 

Keywords: Colonization, Infection, Pediatric malignancy, Vancomycinresistant 

enterococci 

Öz 

Amaç: Hematolojik malignitesi olan pediatrik hastalarda vankomisine 

dirençli enterokok (VDE) kolonizasyonunun Öz 

klinik öneminin 

araştırılması ve eşlik eden risk faktörlerinin değerlendirilmesi 

amaçlanmıştır. 

Gereç ve Yöntemler: Ocak 2010-Aralık 2013 tarihleri arasında yapılan 

VDE sürveyans kayıtlarından faydalanılarak VRE ile kolonize ve/veya 

enfekte olmuş hastalar belirlenmiştir. VDE ile kolonize hastalarda VRE 

enfeksiyonu gelişimi ile ilişkili risk faktörlerini belirlemek amacıyla 

retrospektif olgu-kontrol çalışması yapılmıştır. 

Bulgular: Çalışma süresince yatırılan 229 hastanın 72’sinde (%31,4) 

rektal VRE kolonizasyonu saptanmıştır. VRE kolonize hastaların 

yedisinde (%9,7) kolonizasyon sonrası sistemik VRE enfeksiyonu 

gelişmiştir. Beş hastada bakteriyemi (n=5), bir hastada idrar yolu 

enfeksiyonu (n=1) ve bir hastada menenjit (n=1) gözlenmiştir. 

Tüm enfeksiyonlarda ilgili klinik örneklerde Enterococcus faecium 

üretilmiştir. Multivaryant analiz sonucunda, ciddi nötropeni ve başka 

bir patojene bağlı bakteriyemi öyküsü VDE kolonize hastalarda VDE 

enfeksiyonu gelişimi için risk faktörleri olarak bulunmuştur [odds 

ratio (OR): 35,4; güven aralığı (GA): 1,7-72,3; p=0,02 ve OR: 20,6; 

GA: 1,3-48,6; p=0,03; sırasıyla]. VDE enfeksiyonuna bağlı mortalite 

saptanmamıştır. 

Sonuç: Pediatrik kanser hastalarında VDE kolonizasyonunun önemli 

klinik sonuçları olduğu gözlenmiştir. 

Anahtar Sözcükler: Kolonizasyon, Enfeksiyon, Pediatrik malignite, 

Vankomisine dirençli enterokok 

Address for Correspondence/Yazışma Adresi: Serap KARAMAN, M.D., 

İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 

Phone : +90 212 414 20 00 

E-mail : drkaramans@yahoo.com 

Received/Geliş tarihi: November 04, 2015 

Accepted/Kabul tarihi: February 29, 2016 

244


Aktürk H, et al: Vancomycin-Resistant Enterococci Infections in Pediatric Malignancies 

Introduction 

Children with cancer are at high risk of developing systemic 

infections by the microorganisms that colonize their own 

intestinal system [1,2]. Vancomycin-resistant enterococci (VRE) 

are health care-associated opportunistic pathogens. Limited data 

exist on the incidence of subsequent VRE infection development 

among VRE-colonized pediatric cancer patients and associated 

risk factors, which were investigated in this study. 


All patients admitted to the pediatric hematology/oncology 

ward were sampled within 48-72 h after admission and weekly 

thereafter as part of institutional rectal VRE surveillance. An 

infection control nurse assigned by the Hospital Infection 

Control Committee (HICC) prospectively tracked the results of 

rectal surveillance and all health care-associated infections 

occurring in the hematology/oncology ward. VRE-colonized 

and VRE-infected patients were identified from the HICC 

surveillance database retrospectively. Detailed clinical and 

laboratory features of these patients were collected from their 

medical records. The overall rate of VRE colonization and the 

subsequent infection occurrence throughout the study period 

were determined. To identify the risk factors associated with 

VRE infection occurrence in a colonized patient, a retrospective 

case-control study was performed. Patients were defined as VREcolonized 

(VRE-C) when the culture of the rectal swab yielded 

VRE in the absence of any clinical specimens positive for VRE 

[3]. Systemic VRE infection (VRE-I) was defined as isolation of 

VRE from a clinical specimen together with signs and symptoms 

of infection. Statistical analysis was performed with SPSS 21.0 

for Windows. Parameters were compared between groups with 

the chi-square test, Fisher exact test, or Mann-Whitney U test. 

Variables with a p-value of ≤0.1 in univariate analysis were fitted 

to perform logistic regression analysis to identify independent 

risk factors associated with VRE infection occurrence. 

Results 

A total of 229 children were admitted to the hematology/ 

oncology ward. Fecal VRE-C was documented in 72 of these 

patients (31.4%). Excluding eight patients who were transferred 

from the pediatric intensive care unit, 89% of the patients were 

colonized during their stay in the hematology/oncology ward. 

Species determination could be performed in 32 VRE-colonized 

patients: Enterococcus faecium was isolated in 28 patients, 

Enterococcus gallinarum in 2 patients, and nontypeable 

Enterococcus in 2 patients. 

VRE-I was detected in 7 patients, all of whom were previously 

colonized with VRE. The overall rate of VRE-I developing in 

patients with VRE-C was 9.7%. VRE bacteremia was detected 

in five patients (6.9%). Other VRE infections were urinary 

Table 1. Demographic and clinical characteristics of vancomycin-resistant enterococci-colonized patients who developed 

systemic vancomycin-resistant enterococci infection and those who did not. 

VRE-Colonized Patients 

Who Did Not Develop VRE 

Infection 

(n=65) 

VRE-Colonized Patients Who 

Developed VRE Infection 

(n=7) 

Age, months; mean ± SD 77.7±6.1 45.2±12.9 0.16 

Sex, n (%) 

Male 

Female 

Underlying malignancy, n (%) 

ALL 

AML 

Solid tumor 

Duration of time between admission and determination of VRE 

colonization, days; mean ± SD 

38 (58.5) 

27 (41.5) 

33 (50.8) 

15 (23.1) 

17 (26.2) 

2 (28.6) 

5 (71.4) 

2 (28.6) 

3 (42.9) 

2 (28.6) 

p 

0.13 

0.67 

27.8±3.9 25.5±7.5 0.85 

Severe neutropenia (



tract infection in one patient and meningitis in one patient. 

Enterococcus faecium was isolated in all patients with VRE-I. The 

mean duration of time from identification of VRE colonization 

to development of a VRE infection was 32.4±8.6 days (median: 

25 days, range: 10-73 days). 

Univariate analysis of demographic and clinical variables 

associated with development of VRE-I among patients with 

VRE-C is presented in Table 1. Duration of neutropenia was 

significantly longer in patients with VRE-I than in patients with 

VRE-C (12.8±1.4 days vs. 38.5±7.3 days; p=0.016). Similarly, 

total parenteral nutritional support was found to be received 

for a longer time by patients with VRE-I (9.37±0.8 days vs. 

15.33±5.5 days; p=0.04). Antimicrobials used among patients 

are shown in Table 2. Four out of seven patients were receiving 

a glycopeptide antibiotic when a systemic VRE infection was 

diagnosed. Multivariate analysis revealed severe neutropenia 

and history of previous bacteremia with another pathogen as 

independent risk factors for development of VRE infection in a 

VRE-colonized patient with cancer (Table 3). 

All VRE infections were treated with linezolid. No VREattributable 

deaths occurred during systemic VRE infections. 

However, crude mortality was higher in patients who suffered 

from VRE infection than those who did not (2/7 (28.6%) vs. 4/65 

(6.2%), respectively; p=0.04). 

Discussion 

Overall, 31.4% of patients admitted to our hematology/oncology 

ward were colonized with VRE. Colonization rates in other studies 

ranged from 4.7% to 38% [4,5,6,7]. Systemic VRE infections 

develop mostly in VRE-colonized patients [8,9], although some 

contrary cases may exist rarely [5,10]. In this study, about 1 in 

10 VRE-colonized patients developed subsequent systemic VRE 

infection (9.7%). Studies evaluating cancer patients reported a 

range of 13% to 61% rate of progression of VRE colonization to 

VRE bacteremia [6,10,11,12,13]. 

Results of univariate analysis revealed some risk factors 

associated with VRE-I development. One of them was severe 

neutropenia (



Table 3. Multivariate analysis of risk factors for 

development of vancomycin-resistant enterococci infection 

in patients colonized with vancomycin-resistant enterococci. 

Variables p OR (95% CI) 

Severe neutropenia (

BRIEF REPORT 

DOI: 10.4274/tjh.2015.0259 


Radiation-Induced Tumor Lysis Syndrome in Chronic Lymphocytic 

Leukemia 

Kronik Lenfositik Lösemide Radyasyon İlişkili Tümör Lizis Sendromu 

Ali Alkan 1 , Tuğçe Kütük 2 , Ebru Karcı 1 , Arzu Yaşar 1 , Ayşe Hiçsönmez 2 , Güngör Utkan 1 

1Ankara University Faculty of Medicine, Department of Medical Oncology, Ankara, Turkey 

2Ankara University Faculty of Medicine, Department of Radiation Oncology, Ankara, Turkey 

Abstract 

Tumor lysis syndrome (TLS) is an important oncological emergency that 

is usually observed with hematological malignancies and rarely with 

solid tumors. It can be induced either by therapy or spontaneously. 

Radiotherapy-induced TLS has been rarely reported in the literature. 

Here we present a patient with a diagnosis of metastatic prostate 

cancer and chronic lymphocytic leukemia complicated with TLS 

during palliative radiotherapy. 

Keywords: Chronic lymphocytic leukemia, Radiation, Tumor lysis 

syndrome 

Öz 

Tümör lizis sendromu (TLS) sıklıkla hematolojik malignitelerde 

görülen, nadiren solid tümörlerde karşımıza çıkabilen bir onkolojik 

acildir. Tedavi ilişkili olabileceği gibi spontan olarak da ortaya çıkabilir. 

Radyasyon ilişkili TLS literatürde nadiren bildirilmiştir. Burada, kronik 

lenfositik lösemi ve prostat kanseri tanısıyla izlenen hastada palyatif 

radyoterapi ile indüklenen TLS olgusu sunulmaktadır. 

Anahtar Sözcükler: Kronik lenfositik lösemi, Radyasyon, Tümör lizis 

sendromu 

Öz 

Introduction 

Tumor lysis syndrome (TLS) is one of the important 

oncological emergencies in oncology practice, especially in 

lymphoproliferative malignancies. Aggressive solid tumors with 

shorter doubling time can be complicated with TLS. Although it is 

generally triggered by cytotoxic therapy, it can also be observed 

spontaneously in cases of bulky tumors. Radiation as a cause of 

TLS has been rarely reported in the literature [1,2,3]. Here we 

present a patient with 2 primary malignancies experiencing TLS 

during palliative radiotherapy. 

Case Presentation 

A 69-year-old male patient, without any comorbidities, presented 

with fullness in the left axilla. The initial examination showed 

lymphocytosis and lymphadenopathies in the bilateral axillary 

and inguinal region. There was neither hepatosplenomegaly 

nor pathological lymph nodes in the abdominal and thoracic 

cavity. Lymphocytosis and basket cells were seen in the blood 

smear. Excisional biopsy from the left axilla showed infiltration 

by CD5-positive cells. Immunohistochemistry was consistent 

with a diagnosis of chronic lymphocytic leukemia (CLL) 

infiltration. The bone marrow biopsies supported the diagnosis 

with hypercellularity and diffuse interstitial infiltration of small 

atypical lymphoid cells. The patient had been followed with a 

diagnosis of CLL for 2 years. Due to initial stage 1 disease, the 

patient had been periodically followed without medical therapy. 

In a routine outpatient visit, the clinically asymptomatic 

patient was evaluated with lymph node excisional biopsy due 

to progression of the pathological lymph nodes in order to 

exclude Richter’s transformation. In addition, bone marrow 

sampling was performed. Pathology revealed small atypical 

lymphoid cells consistent with an ongoing lymphoproliferative 

disease and infiltration of an adenocarcinoma as a second 

primary malignancy. Further immunohistochemical staining 

was inconclusive for the origin of the tumor. The age of the 

patient, osteoblastic metastatic lesions detected in a bone scan, 

and prostate-specific antigen (PSA) level of 1712 ng/mL led us 

to order a work-up for prostate cancer. Transrectal prostate 

biopsy showed prostate adenocarcinoma with a Gleason score 

of 10. The patient was treated with goserelin (10.8 mg SC, 

every 12 weeks) and bicalutamide (50 mg daily). The analysis 

of bone scans before hormonal therapy showed metastatic 

Address for Correspondence/Yazışma Adresi: Ali ALKAN, M.D., 

Ankara University Faculty of Medicine, Department of Medical Oncology, Ankara, Turkey 

Phone : +90 312 595 73 21 

E-mail : alkanali@yahoo.com 

Received/Geliş tarihi: July 02, 2015 

Accepted/Kabul tarihi: March 23, 2016 

248


Alkan A, et al: Radiation-Induced Tumor Lysis Syndrome 

lesions in the bilateral scapula, thoracic vertebral column, pelvis, 

bilateral humerus, and femur. For skeletal metastatic disease, 

zoledronic acid (4 mg intravenous, every 4 weeks) was started 

and palliative radiotherapy was planned for painful metastatic 

lesions in the thoracic vertebrae. Radiotherapy to T 3-6 and the 

right scapula with a total of 30 Gy divided into 10 fractions 

was planned. Pathological lymph nodes associated with CLL, 

ranging between 1 and 3 cm in diameter in the bilateral 

inguinal areas, axilla, neck, and hilum, were noted for followup. 

On day 7 of radiotherapy the patient complained about mild 

nausea, progressive malaise, and perioral numbness. Physical 

examination was normal except for pathological lymph nodes 

with minimal regression and paleness. The largest lymph node 

in the right axilla had regressed from 3 to 2 cm and the other 

pathological nodes were stable. The hilar fullness in chest 

X-ray was stable. The only medications used were drugs for 

prostate cancer (bicalutamide and goserelin), lansoprazole for 

dyspepsia, and dexamethasone (8 mg), which was planned with 

the radiotherapy. The laboratory evaluation revealed acute 

renal failure complicated with TLS (Table 1). The PSA value was 

stable. The patient was hospitalized and aggressively hydrated 

with 0.9% NaCl at 500 mL/h in the first 6 h of follow-up with 

monitorization of hourly urine output. In the initial evaluation, 

due to hypocalcemia (5.2 mg/dL) with neuromuscular symptoms 

and prolonged QT interval of 0.50 s, the patient was treated with 

calcium gluconate replacement under cardiac monitorization. 

Laboratory results were checked at 4-h intervals. Until we could 

use rasburicase, allopurinol was the initial specific therapy for 

TLS. The dosage was titrated according to glomerular filtration 

rate and a 0.2 mg/kg single dose of rasburicase could be added 

to therapy on day 3 of hospitalization. The patient’s symptoms 

and renal dysfunction progressively improved (Table 1). The 

patient was free of any findings of infection or septicemia. The 

leukocytosis was linked to the dexamethasone therapy, which 

Figure 1. Digitally reconstructed radiograph of the scapula field 

including axillary lymph nodes, supraclavicular lymph nodes, and 

the upper mediastinal lymph node area. 

was planned during radiotherapy for anti-edema prophylaxis. 

After stopping the drug, leukocytosis improved. Analysis of 

the dose-volume histogram showed that during palliative 

radiotherapy to the thoracal vertebrae, mediastinal, axillary, 

and supraclavicular lymph nodes were also affected with 

maximums of 24.8 Gy (mean: 19.8), 34.2 Gy (mean: 20), and 

28.1 Gy (mean: 19.1), respectively. A digitally reconstructed 

radiograph of the scapula field included axillary lymph nodes, 

supraclavicular lymph nodes, and the upper mediastinal lymph 

node area (Figure 1). After improvement of TLS, reanalysis of 

the patient for progression of CLL showed minimal regression 

of the pathological mediastinal and axillary lymph nodes. There 

was no progression in other lymph nodes or new organomegaly. 

With stable clinical findings of CLL and history of lymph node 

resection three months ago without any aggressive form of 

lymphoproliferative disease or Richter transformation, rebiopsy 

was not planned. After six months of follow-up the patient was 

stable for CLL and the PSA level progressively decreased. 

Discussion and Review of the Literature 

TLS is an oncological emergency that results from massive 

tumor lysis due to therapy or spontaneous bursting of tumor 

cells. The release of intracellular electrolytes into circulation 

causes an electrolyte imbalance and, as a result, acute renal 

failure. Intracellular nucleic acid catabolism and as a result 

Table 1. The laboratory parameters before and after 

radiotherapy and after treatment. 

Laboratory 

Parameters 

Before 

Radiotherapy 

Day 6 of 

Radiotherapy 

Leukocytes (x10 9 /L) 219 867 424 

Hemoglobin (g/L) 129 98 82 

Platelets (x10 9 /L) 177 298 113 

After 

Therapy 

BUN (mmol/L) 11.07 27.85 9.12 

Creatinine (µmol/L) 102.5 624.1 159.1 

Sodium (mmol/L) 140 132 143 

Potassium (mmol/L) 3.7 6.1 3.7 

Calcium (mmol/L) 4.4 2.1 3.2 

Phosphorus (mmol/L) 1.36 2.33 1.26 

Albumin (g/L) 34 28 26 

Corrected calcium 4.6 2.5 3.8 

(mmol/L) 

Uric acid (µmol/L) 398.5 868.4 249.8 

ALT (U/L, reference: 17 27 25 

7-40) 

AST (U/L, reference: 28 27 20 

5-40) 

LDH (U/L, reference: 320 732 443 

210-425) 

ALT: Alanine aminotransferase, AST: aspartate aminotransferase, BUN: blood urea 

nitrogen, LDH: lactate dehydrogenase. 

249

Alkan A, et al: Radiation-Induced Tumor Lysis Syndrome 


hyperuricemia further contribute to renal dysfunction. The 

clinical presentation may range from nonspecific malaise and 

nausea to sudden cardiac death. TLS is diagnosed by both 

laboratory and clinical findings. Laboratory TLS is defined as 

the imbalance of two or more electrolytes (hypocalcemia, 

hyperuricemia, hyperphosphatemia, hyperkalemia), whereas 

clinical TLS is defined as laboratory TLS plus one clinical finding 

(increased creatinine, arrhythmia/sudden death, seizure) 

[4,5,6,7]. 

In the literature, radiation has been rarely reported as a cause 

of TLS. Most reported cases are associated with hematological 

pathologies and splenic radiation is the most often related 

scenario [8]. Total body irradiation before allogeneic stem cell 

transplantation has been related to TLS [9]. TLS as a complication 

of radiation in solid tumors has been rarely reported. Palliation 

of bone metastasis from malignant melanoma [10] and 

prostate carcinoma [3] has been discussed. The outcomes of the 

patients were good and there were no mortalities as a result of 

complications. 

After the diagnosis of a second primary tumor, palliative 

radiotherapy was indicated due to bone pain refractory to 

analgesia. The radiotherapy plan for the right scapula and 

thoracal vertebrae involved the right axilla and mediastinum 

incidentally, where pathological lymph nodes had been 

documented. The regression of axillary pathological lymph 

nodes and stable PSA values after radiotherapy led us to a link 

between TLS and the radiated pathological lymph nodes. CLL 

with a leukocyte value of

BRIEF REPORT 

DOI: 10.4274/tjh.2015.0433 


A Case of Superwarfarin Poisoning Due to Repetitive Occupational 

Dermal Rodenticide Exposure in a Worker 

Tekrarlayan Mesleksel Deri Maruziyetine Bağlı Süpervarfarin Zehirlenmesi Gelişen Bir İşçi Olgusu 

Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç 

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

Abstract 

Superwarfarin poisoning is usually due to chronic occult small-dose 

exposures and can easily be misdiagnosed and may lead to serious 

complications. The diagnosis can be confirmed by a concordant 

history and analyses of blood and urine specimens with the liquid 

chromatography with tandem mass spectrometry (LC-MS/MS) 

technique. Several months of continuous treatment with high doses 

of daily oral vitamin K, as well as other supportive measures, are 

warranted, especially when repeated laboratory measurements to help 

predict the treatment period are not available. In this paper, a case 

of superwarfarin poisoning due to chronic repetitive occupational 

dermal exposure to commercial rodenticides is presented. 

Keywords: Superwarfarin, Acquired coagulopathies, Vitamin K 

Öz 

Süpervarfarin zehirlenmesi genellikle, fark edilmeyen küçük dozlarda 

kronik maruziyete bağlı olarak gelişir ve kolaylıkla yanlış tanı 

konarak ciddi komplikasyonlara yol açabilir. Tanı, uyumlu bir hikaye 

varlığında kan ve idrar örneklerinin sıvı kromatografisi ve tandem 

kitle spektrometresi (LC-MS/MS) tekniği ile analiz edilmesi yoluyla 

doğrulanabilir. Özellikle de, tedavi süresine karar verilebilmesi için 

tekrarlanan laboratuvar ölçümlerinin yapılmasının mümkün olmadığı 

durumlarda, aylar boyunca yüksek dozda günlük oral K vitamini 

verilmesi gereklidir. Bu yazıda, ticari rodentisitlerle tekrarlayan 

mesleksel deri maruziyetine bağlı Özolarak süpervarfarin zehirlenmesi 

gelişen bir olgu sunulmaktadır. 

Anahtar Sözcükler: Süpervarfarin, Kazanılmış koagülopatiler, K 

vitamini 

Introduction 

A 40-year-old man without any prior disease was referred to 

our outpatient clinics with a one-month history of recurrent 

epistaxis, ecchymoses, and hemarthroses. Administration of 

fresh frozen plasma (FFP) in the emergency room corrected 

the markedly abnormal international normalized ratio (INR), 

prothrombin time (PT), and activated partial thromboplastin 

time (aPTT) temporarily. His past medical and family histories 

were unremarkable. There were no bleeding episodes following 

surgery or trauma. 

Laboratory analyses repeatedly revealed a sustained deficiency 

of vitamin K-dependent clotting factors with the following 

results: factor II, 26.9%; factor VII, 0.1%; factor IX, 13.3%; factor 

X, 23.5%; factor V, 82%; factor VIII, 131%; INR, 4.04; PT, 48.5 

s; aPTT, 48.3 s; D-dimer, 21 ng/mL; fibrinogen, 5.01 g/L. PT and 

aPTT normalized in the dilution assay, excluding any acquired 

inhibitors. He had a hypochromic microcytic anemia concordant 

with chronic blood loss. The platelet count was 164x10 9 /L. The 

absence of schistocytes in the peripheral blood smear excluded 

disseminated intravascular coagulation. Liver function tests and 

hepatobiliary ultrasound were normal. Endoscopic examinations 

of the gastrointestinal tract revealed normal mucosa. 

After excluding all other possible causes, intoxication with 

superwarfarin remained the diagnosis of exclusion. Blood and 

urine specimens were analyzed at the biochemistry laboratory 

of the Forensic Medicine Institute in Ankara and revealed a high 

blood level of superwarfarin, 32 ng/dL, and a positive urine test. 

Liquid chromatography with tandem mass spectrometry (LC-MS/ 

MS), which allows for the identification, characterization, and 

quantification of chemical compounds in a specimen based on 

their molecular masses and fragmentation patterns [1], was used 

in analyses. However, various special methods do exist for the 

simultaneous analysis of multiple hydroxycoumarin rodenticides 

in a specimen [2]. Our laboratory could quantify the blood level 

of superwarfarin, but could give only a qualitative result for 

the urine specimens. We were not able to identify the specific 

superwarfarin compound in the blood samples, as this requires 

some special techniques and expertise. 

Address for Correspondence/Yazışma Adresi: Zehra NARLI ÖZDEMİR, M.D., 

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

Phone : +90 312 595 70 99 



251

Narlı Özdemir Z, et al: A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker 


Thereafter, the patient, who had been working in a car-washing 

facility, admitted that he was exposed to various brands of 

commercial rodenticides in this facility and did not take the 

necessary precautions (i.e. gloves and masks) while handling and 

scattering the rodenticidal pellets and pastes, in contrast to his 

coworkers, none of whom had similar symptoms. As previously 

reported, long-term repetitive exposure to rodenticides via direct 

skin contact was the probable route of systemic absorption in 

this case [3]. 

Superwarfarins are lipid-soluble long-acting coumarin 

derivatives available since the 1970s as strong rodenticides and 

they cause prolonged anticoagulation both in rats and human 

[4]. The second-generation anticoagulant rodenticides including 

brodifacoum, bromadiolone, difenacoum, difethialone, and 

flocoumafen have longer elimination half-lives and greater 

accumulation and persistence due to a greater affinity to binding 

sites in the liver [5]. Among these compounds, brodifacoum has 

been reported to have the longest plasma and liver elimination 

half-lives, which are 91.7 days and 307.4 days, respectively [5]. 

The common commercial rodenticidal formulations marketed in 

Turkey are in pellet and paste forms and contain 0.005% (w/w) 

brodifacoum, 0.005% (w/w) difenacoum, or 0.0025% (w/w) 

difethialone. Given the extremely dilute concentrations of 

these compounds in the commercial formulations, only a huge 

amount of a single-dose exposure may cause important side 

effects. In concordance, fatalities from a single-dose exposure 

are reported to be very rare [2]. However, regarding the long 

elimination half-lives and lipid solubility of these compounds, 

repetitive exposures to small doses of these formulations may 

result in chronic poisoning. 

Our patient had intermittent episodes of bleeding together 

with prolonged PT approximately every 3-4 weeks after the 

outpatient clinic visit during a 6-month period. In every visit he 

was prescribed FFP and 10 mg of parenteral phytomenadione, 

and he continued taking oral phytomenadione of 10 mg daily 

for 7-10 days. At the end of six months his coagulation tests 

normalized and bleeding episodes disappeared. 

The half-life of superwarfarin can be estimated by repeated 

measurements of blood levels in order to predict the treatment 

period [6]. In our case, the Forensic Medicine Institute laboratory 

was the only available place able to perform these kinds of 

analyses in the area. However, as it is subject to governmental 

regulations, we could not get permission for repeated 

measurements. Due to the presence of chronic exposure to 

more than one product and failure to identify the specific 

superwarfarin molecule with laboratory tests, it was hard to 

predict the outcome in our case. However, considering the long 

wash-out period of our case, we can speculate that the patient 

might have had more exposure to a product containing the 

superwarfarin with the longest half-life, which is brodifacoum. 

As we could not diagnose the patient earlier, he was given 

vitamin K supplementation at lower doses and shorter periods 

than recommended. Supplemental vitamin K doses of up to 100 

mg daily for 12 months have been reported [7]. Our patient was 

a nonobese male, weighing 72 kg and 175 cm in height. In obese 

patients, considering the lipid solubility of these compounds, 

longer periods and higher doses may be required. 

Superwarfarin poisoning due to accidental, occupational, and 

criminal exposures has been reported and should be suspected 

when an acquired bleeding disorder due to persistent deficiency 

of vitamin K-dependent clotting factors and a lack of sustained 

response to treatment with conventional doses of vitamin K 

are present [8]. Rodent infestation and food contamination 

are important sources of superwarfarin poisoning in poor 

socioeconomic settings. Long-term vitamin K supplementation 

is the recommended treatment [6]. Intravenous administration 

of vitamin K is often necessary initially. However, the risk of 

anaphylactic reactions should be considered [9]. 

This paper aims to refresh awareness about this clinical 

scenario that can easily be misdiagnosed and may lead to 

serious complications. Chronic occult small-dose exposures 

might be overlooked and should be meticulously investigated. 

The diagnosis can be confirmed by a concordant history and 

analyses of blood and urine specimens with the LC-MS/MS 

technique in the absence of warfarin therapy. Several months 

of continuous treatment with high doses of daily oral vitamin K, 

as well as other supportive measures, are warranted, especially 

when repeated laboratory measurements to help predict the 

treatment period are not available. 


We are grateful for the exchange of experience provided by Dr. 

Reyhan Diz Küçükkaya during the diagnostic stages of this case. 

Ethics 

Ethics Committee Approval: Not applicable; Informed Consent: 

It was taken. 


Medical Practices: Zehra Narlı Özdemir, Mustafa Merter, 

Mehmet Gündüz, Meral Beksaç; Concept: Zehra Narlı Özdemir, 

Uğur Şahin, Meral Beksaç; Analysis or Interpretation: Zehra 

Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, 

Berna Ateşağaoğlu, Meral Beksaç; Literature Search: Zehra Narlı 

Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna 

Ateşağaoğlu, Meral Beksaç; Writing: Zehra Narlı Özdemir, Uğur 

Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, 

Meral Beksaç. 

252


Narlı Özdemir Z, et al: A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker 




included. 

References 

1. Vogeser M, Parhofer KG. Liquid chromatography tandem-mass spectrometry 

(LC-MS/MS)-technique and applications in endocrinology. Exp Clin 

Endocrinol Diabetes 2007;115:559-570. 

2. Schaff JE, Montgomery MA. An HPLC-HR-MS-MS method for identification 

of anticoagulant rodenticides in blood. J Anal Toxicol 2013;37:321-325. 

3. Voitsekhovskii VV, Pivnik AV, Bitiutskaia LG, Protsko TT. Acquired hemorrhagic 

coagulopathy due to contact with the rodenticide brodifacoum in the 

Nutcracker bait. Ter Arkh 2012;84:66-71. 

4. Altay S, Cakmak HA, Boz GC, Koca S, Velibey Y. Prolonged coagulopathy 

related to coumarin rodenticide in a young patient: superwarfarin 

poisoning. Cardiovasc J Afr 2012;23:9-11. 

5. Vandenbroucke V, Bousquet-Melou A, De Backer P, Croubels S. 

Pharmacokinetics of eight anticoagulant rodenticides in mice after single 

oral administration. J Vet Pharmacol Ther 2008;31:437-445. 

6. Boettcher S, Wacker A, Moerike K, Kopp HG, Jaschonek K, Grobosch T, 

Kanz L, Salih HR. Acquired coagulopathy caused by intoxication with the 

superwarfarin-type anticoagulant rodenticide flocoumafen. Eur J Haematol 

2011;86:173-175. 

7. Weitzel JN, Sadowski JA, Furie BC, Moroose R, Kim H, Mount ME, Murphy 

MJ, Furie B. Surreptitious ingestion of a long-acting vitamin K antagonist/ 

rodenticide, brodifacoum: clinical and metabolic studies of three cases. 

Blood 1990;76:2555-2559. 

8. Poovalingam V, Kenoyer DG, Mahomed R, Rapiti N, Bassa F, Govender P. 

Superwarfarin poisoning-a report of 4 cases. S Afr Med J 2002;92:874-876. 

9. Spahr JE, Maul JS, Rodgers GM. Superwarfarin poisoning: a report of two 

cases and review of the literature. Am J Hematol 2007;82:656-660. 

253

LETTERS TO EDITOR 


A Primary Bone Diffuse Large B-Cell Lymphoma with Ocular 

Adnexal Involvement 

Oküler Adneks Tutulumu Olan Kemiğin Primer Diffüz Büyük B Hücreli Lenfoması 

Rafet Eren 1 , Ceyda Aslan 1 , Cihan Gündoğan 2 , Osman Yokuş 1 , Mehmet Hilmi Doğu 1 , Elif Suyanı 1 

1İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 

2İstanbul Training and Research Hospital, Clinic of Nuclear Medicine, İstanbul, Turkey 

To the Editor, 

Primary bone lymphomas (PBLs) [1,2] and ocular adnexal (OA) 

lymphomas [3,4] are rare types of extranodal lymphomas. 

Coexistence of these two rare entities without lymph node 

infiltration has not been reported previously. 

A 55-year-old man presented with left hip pain without a 

history of trauma. His medical history and physical examination 

did not reveal any remarkable findings. The X-ray radiographs 

of the pelvis and left hip showed multiple lytic lesions. Body 

18fluorodeoxyglucose positron emission tomography/computed 

tomography (18F-FDG PET/CT) demonstrated multiple osteolytic 

bone lesions in the left zygomatic bone, vertebral column, 

bilateral iliac bones, left caput femoris, and trochanter major 

of the femur with increased 18F-FDG uptake (SUVmax: 31) 

(Figure 1a). Tru-Cut biopsy of the caput femoris showed 

atypical lymphoid cells that were pancreatin (-), s-100 (-), 

CD138 (+), CD30 (-), CD20 (+), CD3 (-), CD5 (-), CD10 (-), bcl- 

6 (+), and MUM-1 (+), consistent with diffuse large B-cell 

lymphoma. Laboratory data were as follows: erythrocyte 

sedimentation rate, 37 mm/h; lactate dehydrogenase level, 405 

U/L; hemoglobin, 12 g/dL; white blood cell count, 6.74x109/L; 

platelet count, 250x109/L; normal liver and renal function tests. 

Bone marrow aspirate and core biopsy were normal. A rituximab, 

cyclophosphamide, doxorubicine, vincristine, and prednisolone 

(R-CHOP) regimen was planned. However, prior to the first 

chemotherapy day, the patient was admitted with swelling 

of the eyelids, exophthalmos, proptosis, and vision loss in his 

left eye that developed progressively over 3 days. Magnetic 

resonance imaging (MRI), which was performed two months 

after the initial 18F-FDG PET/CT, showed an orbital mass with 

diameters of 36x21x38 mm eroding the superior wall of the 

orbita and adjacent soft tissue (Figure 1b). After the detection 

of orbital involvement, investigations for central nervous system 

(CNS) involvement were negative. Chemotherapy treatment was 

commenced immediately without doing a biopsy because of the 

patient’s vision loss. After 4 cycles of R-CHOP chemotherapy, 

the patient’s left hip pain, left eye swelling, exophthalmos, and 

proptosis resolved completely, but his vision did not improve. 

Control 18F-FDG PET/CT showed marked regression of bone 

lesions with decreased 18 F-FDG uptake (SUVmax: 4.6). Orbital 

MRI also showed that the mass had regressed to 14x12 mm 

in size. After obtaining this response, 2 cycles of R-CHOP, 

radiotherapy to the left orbita, and two cycles of high-dose 

methotrexate for CNS prophylaxis were planned. 

Considering the extensive lytic bone lesions and recent 

emergence of the OA tumor, the primary site of the disease 

must have been the bones in the presented case. Thus, the 

diagnosis can be categorized as PBL with OA involvement. An 

orbital mass was detected two months after diagnosis by means 

of MRI, but not by 18F-FDG PET/CT performed at diagnosis. It 

would be speculative to claim that such a large mass had arisen 

in a two month period. Taking into account that MRI is the gold 

standard imaging technique in evaluation of OA tumors [3], the 

orbital mass, which was probably small at the beginning, could 

not have been noticed on 18F-FDG PET/CT. This case emphasizes 

that a high suspicion index of OA involvement in PBL cases with 

any symptoms regarding the eyes and prompt assessment of the 

patients with MRI might prevent undesirable consequences. 

Figure 1a. 18 Fluorodeoxyglucose positron emission tomography/ 

computed tomography image of the patient at diagnosis. 

254




Surgical and Medical Practices: Rafet Eren, Elif Suyanı; Concept: 

Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, 

Mehmet Hilmi Doğu, Elif Suyanı; Design: Rafet Eren, Ceyda 

Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, 

Elif Suyanı; Data Collection or Processing: Rafet Eren, Ceyda 

Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, 

Elif Suyanı; Analysis or Interpretation: Rafet Eren, Ceyda Aslan, 

Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; 

Literature Search: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, 

Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; Writing: Rafet 

Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet 

Hilmi Doğu, Elif Suyanı. 

Figure 1b. Magnetic resonance imaging image of orbital adnexal 

mass. 

Keywords: Primary bone lymphoma, Ocular adnexal lymphoma, 

Diffuse large B-cell lymphoma 

Anahtar Sözcükler: Primer kemik lenfoma, Oküler adneks 

lenfoma, Diffüz büyük B hücreli lenfoma 

Ethics 

Ethics Committee Approval: Not applicable; Informed Consent: 

Not applicable. 

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

interest, including specific financial interests, relationships, and/ 

or affiliations relevant to the subject matter or materials included. 

References 

1. Messina C, Christie D, Zucca E, Gospodarowicz M, Ferreri AJ. Primary and 

secondary bone lymphomas. Cancer Treat Rev 2015;41:235-246. 

2. Kitsoulis P, Vlychou M, Papoudou-Bai A, Karatzias G, Charchanti A, Agnantis 

NJ, Bai M. Primary lymphomas of bone. Anticancer Res 2006;26:325-337. 

3. Ponzoni M, Govi S, Licata G, Mappa S, Giordano Resti A, Politi LS, Spagnuolo 

L, Di Cairano E, Doglioni C, Ferreri AJ. A reappraisal of the diagnostic and 

therapeutic management of uncommon histologies of primary ocular 

adnexal lymphoma. Oncologist 2013;18:876-884. 

4. Woolf DK, Ahmed M, Plowman PN. Primary lymphoma of the ocular adnexa 

(orbital lymphoma) and primary intraocular lymphoma. Clin Oncol (R Coll 

Radiol) 2012;24:339-344. 

Address for Correspondence/Yazışma Adresi: Elif SUYANI, M.D., 

İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 

Phone : +90 212 459 63 04 

E-mail : elifsuyani@hotmail.com 



DOI: 10.4274/tjh.2015.0424 

Cerebral Sinovenous Thrombosis Mimicking Intracranial Mass 

İntrakranial Kitleyi Taklit Eden Serebral Sinovenöz Tromboz 

Derya Özyörük 

Ankara Children’s Hematology and Oncology Training and Research Hospital, Ankara, Turkey 


Cerebral sinovenous thrombosis is rare in children [1]. Most 

common signs and symptoms are seizure, lethargy, and headache 

[1,2,3]. We herein report a case diagnosed as cerebral sinovenous 

thrombosis mimicking an intracranial mass and presenting with 

increased intracranial pressure symptoms in an adolescent girl. 

A 16-year-old girl was admitted to our emergency service with 

complaints of headache, vomiting, and confusion. Her past 

medical history was unremarkable. Physical examination revealed 

facial paralysis and motor weakness on the left side. Magnetic 

resonance (MR) imaging disclosed a mass (65x42x55 mm) in the 

right temporal lobe shifting the midline structure from right 

to left (Figure 1). Because of herniation findings, surgery was 

performed immediately. Histopathologic investigation showed 

255



Figure 1. Cranial magnetic resonance images of the patient 

demonstrate a heterogeneous parenchymal lesion with increased 

T2 signal intensity in the right temporal lobe causing compression 

of the third lateral ventricle, basal ganglion, and thalamus with 

sylvian fissure, sulcal effacement, shifting midline structure, and 

significant edema. 

hematoma, gliosis, inflammation, and vasculitis. The laboratory 

examination revealed iron deficiency anemia (white blood cells: 

12x109/L, Hb: 7.2 g/dL, mean corpuscular volume: 49 fL, red cell 

distribution width: 20%, platelets: 570x10 9 /L, ferritin: 4.4 ng/mL). 

Other hematologic tests and coagulation panels were determined 

to be normal. Prothrombotic markers such as protein C, protein 

S, antithrombin III, plasminogen, heparin cofactor II, factor VIII, 

factor XII, lipoprotein (a), fibrinogen, homocysteine, anticardiolipin 

IgG, lupus anticoagulant levels, prothrombin 20210G, and factor 

V Leiden were normal. Tumor markers were negative. Cranial MR 

venography demonstrated stasis and occlusion in the middle distal 

part of the transverse sinus, sigmoid sinus, and internal jugular 

vein. The patient had no signs of nephrotic syndrome, infection, 

or vasculitis. She was diagnosed with transverse sinus thrombosis 

and anticoagulated with low-molecular-weight heparin. Iron 

supplementation was administered for six months. 

Cases of thrombosis presenting with signs of intracranial mass are 

rarely reported in the literature. Kim et al. [4] reported a 54-yearold 

male patient who was operated on for an intracranial tumor; 

however, the final diagnosis was thrombus in the aneurysm of 

the middle cerebral artery. In another report, a 20-month-old 

girl presented with rapidly progressing hemiparesis on the left 

side. Cranial MR revealed an abscess or glial tumor-like lesion 

in the right thalamus; however, pathological investigation 

of the stereotactic biopsy specimen did not show any signs of 

malignancy. On day 14 of admission, control cranial MR showed 

thrombosis in the superior sagittal sinus and transverse sinus [5]. 

The association of iron deficiency anemia with sinus thrombosis 

has been reported previously in children [6]. Although secondary 

thrombocytosis has been implicated in cerebral venous sinus 

thrombosis associated with iron deficiency, two cases with 

normal platelet count have also been described [7]. As not 

all cases of iron-related thrombotic events occur in patients 

with concomitant high platelet counts, other pathogenic 

mechanisms have been proposed. One of these explanations is 

that iron deficiency may contribute to a hypercoagulable state 

by affecting blood flow patterns within the vessels because of 

reduced deformability and increased viscosity of microcytic 

red blood cells. Furthermore, anemic hypoxia secondary to 

iron deficiency has been suggested to precipitate situations of 

increased metabolic stress, in particularly in vulnerable areas 

of the brain supplied by end arteries [8]. In our patient, iron 

deficiency may also have contributed to the development of 

thrombosis. 

In conclusion, cerebral sinovenous thrombosis associated with 

increased intracranial pressure symptoms may mimic intracranial 

masses in children. 

Keywords: Intracranial mass, Cerebral sinovenous thrombosis, 

Increased intracranial pressure 

Anahtar Sözcükler: İntrakranial kitle, Serebral sinovenöz 

tromboz, Artmış intrakranial basınç 

References 

1. Hedlund GL. Cerebral sinovenous thrombosis in pediatric practice. Pediatr 

Radiol 2013;43:173-188. 

2. Suppiej A, Gentilomo C, Saracco P, Sartori S, Agostini M, Bagna R, Bassi B, 

Giordano P, Grassi M, Guzzetta A, Lasagni D, Luciani M, Molinari AC, Palmieri 

A, Putti MC, Ramenghi LA, Rota LL, Sperlì D, Laverda AM, Simioni P. Stroke 

working group of the Italian Registry of Pediatric Thrombosis. Paediatric 

arterial ischaemic stroke and cerebral sinovenous thrombosis. First report 

from the Italian Registry of Pediatric Thrombosis (R. I. T. I., Registro Italiano 

Trombosi Infantili). Thromb Haemost 2015;113:1270-1277. 

3. Ichord RN, Benedict SL, Chan AK, Kirkham FJ, Nowak-Göttl U. International 

Paediatric Stroke Study Group. Paediatric cerebral sinovenous thrombosis: 

findings of the International Paediatric Stroke Study. Arch Dis Child 

2015;100:174-179. 

4. Kim YJ, Jeun SS, Park JH. Thrombosed large middle cerebral artery aneurysm 

mimicking an intra-axial brain tumor: case report and review of literature. 

Brain Tumor Res Treat 2015;3:39-43. 

5. Haug V, Linder-Lucht M, Zieger B, Korinthenberg R, Mall V, Mader I. 

Unilateral venous thalamic infarction in a child mimicking a thalamic 

tumor. J Child Neurol 2009;24:105-109. 

6. Sébire G, Tabarki B, Saunders DE, Leroy I, Liesner R, Saint-Martin C, Husson 

B, Williams AN, Wade A, Kirkham FJ. Cerebral venous sinus thrombosis 

in children: risk factors, presentation, diagnosis and outcome. Brain 

2005;128:477-489. 

7. Kinoshita Y, Taniura S, Shishido H, Nojima T, Kamitani H, Watanebe T. 

Cerebral venous sinus thrombosis associated with iron deficiency: two case 

reports. Neurol Med Chir (Tokyo) 2006;46:589-593. 

8. Franchini M, Targher G, Montagnana M, Lippi G. Iron and thrombosis. Ann 

Hematol 2008;87:167-173. 

Address for Correspondence/Yazışma Adresi: Derya ÖZYÖRÜK, M.D., 

Ankara Children’s Hematology and Oncology Training and Research Hospital, Ankara, Turkey 

Phone : +90 505 633 52 74 

E-mail : dozyoruk@yahoo.com 



DOI: 10.4274/tjh.2016.0038 

256



A Rare Cause of Unexplained Refractory Iron Deficiency Anemia: 

Unicentric Plasma-Cell Type Castleman’s Disease 

Tedaviye Dirençli Demir Eksikliği Anemisinin Nadir Bir Nedeni: Unisentrik Plazma Hücreli 

Tip Castleman Hastalığı 

Sevgi Kalayoğlu Beşışık 1 , İpek Yönal Hindilerden 1 , Fehmi Hindilerden 2 , İbrahim Öner Doğan 3 , Fatih Beşışık 4 

¹İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 

2İstanbul Bakırköy Sadi Konuk Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 

3İstanbul University İstanbul Faculty of Medicine, Department of Pathology, İstanbul, Turkey 

3İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Gastroenterohepatology, İstanbul, Turkey 


Castleman’s disease (CD) is an uncommon benign 

lymphoproliferative disorder characterized by enlargement of 

hyperplastic lymph nodes with abnormal interfollicular vascular 

growth [1]. It is clinically categorized as unicentric disease or 

multicentric disease. Histopathologic variants of CD include 

hyaline-vascular type, plasma-cell (PC) type, and mixed form 

[2]. CD presents with features ranging from asymptomatic 

lymphadenopathy to systemic manifestations such as serious 

infections, anemia, and nerve damage [3]. In CD, hepcidin 

secretion induced by IL-6 is the main cause of anemia [4,5]. 

Anemia is more common in multicentric CD and is rarely reported 

in unicentric CD [3]. Few cases of unicentric PC type CD associated 

with iron deficiency anemia (IDA) have been reported [6,7,8,9]. To 

our knowledge, there is only one previous reported case of adult 

unicentric PC type CD located in the abdomen and presenting 

with IDA [9]. We describe an adult with occult unicentric PC type 

CD of the abdomen presenting with iron-refractory anemia (IRA) 

and achieving dramatic response to curative resection. 

A 47-year-old man was referred with a 5-year history of IRA. 

Blood analysis showed Hb of 8 g/dL, mean corpuscular volume 

of 70 fL, red cell distribution width of 17%, and platelet count 

of 478,000/mm 3 . Biochemical tests were as follows: serum 

ferritin, 371 µg/L; transferrin saturation, 8.6%; and erythrocyte 

sedimentation rate (ESR), 110 mm/h (reference range: 0-20). 

Serum protein electrophoresis revealed polyclonal gammopathy 

with gamma globulin of 2.16 g/dL. The soluble transferrin 

receptor/log10 ferritin index of 2.3 indicated the presence of 

combined IDA and anemia of inflammation (AI) [10]. Underlying 

chronic inflammatory diseases were excluded. Upper and lower 

gastrointestinal endoscopic evaluations and bone marrow 

examination were normal. Positron emission tomographycomputed 

tomography showed a soft tissue mass with diffuse 

fluorodeoxyglucose uptake with an SUVmax of 11.39 and a 

craniocaudal length of 8 cm extending from the hepatogastric 

ligament and with a maximal diameter of 4.4x4.3 cm in the axial 

plane at the portal region. He underwent exploratory laparotomy 

and the mass was completely excised. Histopathological 

examination of the mass revealed greater retention of the 

nodal architecture with increased secondary lymphoid follicles, 

vascularization of germinal centers, and expansion of mantle 

zones. The interfollicular region contained sheets of CD38-positive 

mature-appearing plasma cells and increased postcapillary 

venules. Plasma cells expressed polytypic immunoglobulins, light 

and heavy chains (Figure 1). These findings were compatible with 

a diagnosis of PC type CD. Four months after complete resection, 

laboratory tests had completely normalized (Hb 14 g/dL, mean 

Figure 1. Histopathological examination of the resected 

specimen. Greater retention of the nodal architecture with 

increased secondary lymphoid follicles, and vascularization of 

germinal centers (a: 40 x ), germinal centers fed by prominent 

vessels; lollipop-like appearance (b: 200 x ). The interfollicular 

region contained sheets of mature-appearing plasma cells and 

increased postcapillary venules (c: 400 x ; d: 200 x ). 

257



corpuscular volume 83 fL, and ESR 10 mm/h). He has been free of 

disease for more than 26 months since the resection. 

Previously reported PC type CD patients with abdominal 

involvement and IDA aged between 11 and 29 years [6,7,9], 

making our patient the oldest reported patient. PC type CD with 

abdominal involvement may show an indolent course, often 

leading to great delay in diagnosis. We report an unusual case 

of unicentric PC type CD in which the patient suffered from 

systemic manifestations of anemia for five years. After surgical 

resection, the anemia completely resolved. CD should be included 

in the differential diagnosis of chronic, unexplained inflammation 

associated with combined IDA and AI. 

Keywords: Iron deficiency anemia, Unicentric plasma-cell type, 

Castleman’s disease 

Anahtar Sözcükler: Demir eksikliği anemisi, Unisentrik plazma 

hücreli tip, Castleman hastalığı 

Ethics 

Informed Consent: It was taken. 


Concept: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi 

Hindilerden, İbrahim Öner Doğan, Fatih Beşışık; Design: Sevgi 

Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden; 

Data Collection or Processing: Sevgi Kalayoğlu Beşışık, İpek 

Yönal Hindilerden, Fehmi Hindilerden, İbrahim Öner Doğan, 

Fatih Beşışık; Analysis or Interpretation: İpek Yönal Hindilerden; 

Literature Search: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, 

Fehmi Hindilerden; Draft the Article: Sevgi Kalayoğlu Beşışık, 

İpek Yönal Hindilerden, Fehmi Hindilerden; Revise the Article: 

Sevgi Kalayoğlu Beşışık, İbrahim Öner Doğan, Fatih Beşışık; 

Writing: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi 

Hindilerden, İbrahim Öner Doğan, Fatih Beşışık. 

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

interest, including specific financial interests, relationships, and/ 

or affiliations relevant to the subject matter or materials included. 

References 

1. Castleman B, Iverson L, Menendez VP. Localized mediastinal lymph node 

hyperplasia resembling thymoma. Cancer 1956;9:822-830. 

2. Keller AR, Hochholzer L, Castleman B. Hyaline-vascular and plasma-cell types 

of giant lymph node hyperplasia of the mediastinum and other locations. 

Cancer 1972;29:670-683. 

3. Bjarnason I, Cotes PM, Knowles S, Reid C, Wilkins R, Peters TJ. Giant lymph 

node hyperplasia (Castleman’s disease) of the mesentery. Observations on the 

associated anemia. Gastroenterology 1984;87:216-223. 

4. Yoshizaki K, Matsuda T, Nishimoto N, Kuritani T, Taeho L, Aozasa K, Nakahata 

T, Kawai H, Tagoh H, Komori T. Pathogenic significance of interleukin-6 (IL-6/ 

BSF-2) in Castleman’s disease. Blood 1989;74:1360-1367. 

5. Song SN, Tomosugi N, Kawabata H, Ishikawa T, Nishikawa T, Yoshizaki K. 

Down-regulation of hepcidin resulting from long-term treatment with an 

anti-IL-6 receptor antibody (tocilizumab) improves anemia of inflammation 

in multicentric Castleman disease. Blood 2010;116:3627-3634. 

6. Yin L, Lu XY, Xu F, Li AJ, Wu MC. Unicentric Castleman’s disease presenting with 

growth retardation and iron deficiency anemia. Am J Med Sci 2012;343:426- 

428. 

7. Chandrakasan S, Bakeer N, Mo JQ, Cost C, Quinn CT. Iron-refractory microcytic 

anemia as the presenting feature of unicentric Castleman disease in children. 

J Pediatr 2014;164:928-930. 

8. Suh JH, Hong SH, Jeong SC, Park CB, Choi KB, Shin OR, Choi SY. Anemia 

resolved by thoracoscopic resection of a mediastinal mass: a case report of 

unicentric Castleman’s disease. J Thorac Dis 2015;7:189-193. 

9. Vinzio S, Ciarloni L, Schlienger JL, Rohr S, Méchine A, Goichot B. Isolated 

microcytic anemia disclosing a unicentric Castleman disease: The 

interleukin-6/hepcidin pathway? Eur J Intern Med 2008;19:367-369. 

10. Punnonen K, Irjala K, Rajamäki A. Serum transferrin receptor and its ratio to 

serum ferritin in the diagnosis of iron deficiency. Blood 1997;89:1052-1057. 

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

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

Division of Hematology, İstanbul, Turkey Phone : +90 535 687 59 92 

E-mail : ipekyonal@yahoo.com.tr 

Received/Geliş tarihi: February 28, 2016 


DOI: 10.4274/tjh.2016.0083 

258

IMAGES IN HEMATOLOGY 

DOI: 10.4274/tjh.2015.0112 


Vaginal Lymphoma: A Possible Cause of Genital Hemorrhage 

Vajinal Lenfoma: Olası Bir Genital Kanama Nedeni 

Erdoğan Nohuz 1 , Sharif Kullab 2 , Albane Ledoux-Pilon 3 , Cécile Moluçon-Chabrot 4 , Maël Albaut 1 , Luisa De Simone 1 , Xavier Durando 2 

1General Hospital of Thiers, Clinic of Obstetrics and Gynecology, Thiers, France 

2Centre Jean Perrin, Clinic of Medical Oncology, Clermont-Ferrand, France 

3Estaing University Hospital, Department of Pathology, Clermont-Ferrand, France 

4Estaing University Hospital, Department of Hematology, Clermont-Ferrand, France 

Figure 1. A) Grenz zone (arrows): CD20-positive immunoreactivity in neoplastic cells (25 x ). B: Immunohistochemical analysis of paraffinembedded 

sections of the mass lesion showing tumor cells expressing the CD20 molecule (400 x ). 

A 59-year-old patient complaining of vaginal bleeding and 

puruloid discharge was admitted to our gynecology department. 

Speculum examination showed a vaginal fungating necrotic 

ulcerated mass. There was no palpable lymphadenopathy or 

hepato-splenomegaly on physical examination. Transvaginal 

ultrasound and abdominopelvic computed tomography 

demonstrated a bulky vaginal mass approximately 5x4x3 cm in 

diameter involving the bladder and the rectovaginal septum. 

With the patient’s approval, a punch biopsy was performed and 

failed to establish the diagnosis (small and necrotic samples 

that were not representative of the lesion). 

Histopathological diagnosis was obtained after a second biopsy 

performed under general anesthesia. Immunohistochemistry 

showed that tumor cells were positive for CD20, CD30, MUM1, 

and bcl-6 and were negative for bcl-2, EMA, CD10, and CD30 

(Figure 1). The patient was diagnosed with primary vaginal diffuse 

large-B-cell non-Hodgkin lymphoma (NHL) and underwent 8 

courses of rituximab, cyclophosphamide, doxorubicin, vincristine, 

prednisone immunochemotherapy. Complete remission was 

achieved without any relapse at 18 months’ follow-up. 

Primary vaginal NHL represents less than 1% of genital 

neoplasms [1,2,3]. Early and accurate diagnosis significantly 

influences the prognosis [4,5]. It should be considered in 

differential diagnosis of patients with vaginal bleeding. A deep 

biopsy may be required. 

Address for Correspondence/Yazışma Adresi: Erdoğan NOHUZ, M.D. 

General Hospital of Thiers, Clinic of Obstetrics and Gynecology, Thiers, France 

E-mail : enohuz@yahoo.fr 

Received/Geliş tarihi: March 09, 2015 

Accepted/Kabul tarihi: November 09, 2015 

259

Nohuz E, et al: Vaginal Lymphoma 


Keywords: Non-Hodgkin’s lymphoma, Vaginal B-cell 

lymphoma, Postmenopausal bleeding, Vaginal discharge 

Anahtar Sözcükler: Non-Hodgkin lenfoma, Vajinal B-hücreli 

lenfoma, Postmenopozal kanama, Vajinal akıntı 

Ethics 



Surgical and Medical Practices: Erdoğan Nohuz, Cécile 

Moluçon-Chabrot; Concept: Erdoğan Nohuz, Maël Albaut; 

Design: Erdoğan Nohuz, Sharif Kullab; Data Collection or 

Processing: Erdoğan Nohuz, Albane Ledoux-Pilon, Luisa De 

Simone; Analysis or Interpretation: Erdoğan Nohuz, Xavier 

Durando; Literature Search: Erdoğan Nohuz, Sharif Kullab, 

Albane Ledoux-Pilon, Cécile Moluçon-Chabrot, Maël Albaut, 

Luisa De Simone, Xavier Durando; Writing: Erdoğan Nohuz. 




included. 

References 

1. Kosari F, Daneshbod Y, Parwaresch R, Krams M, Wacker HH. Lymphomas of 

the female genital tract: a study of 186 cases and review of the literature. 

Am J Surg Pathol 2005;29:1512-1520. 

2. Ragupathy K, Bappa L. Primary vaginal non-Hodgkin lymphoma. J Low 

Genit Tract Dis 2013;17:326-329. 

3. Dane C, Dane B, Kalli E, Erginbaş M, Çetin A. Primary uterine lymphoma. 

Turk J Haematol 2004;21:39-43. 

4. Nohuz E, Albaut M, Kullab S, Fattouh M, Tamburro S, Dauplat MM, 

Benoît C, Durando X. What is your diagnosis? J Turk Ger Gynecol Assoc 

2014;15:262-263. 

5. Ameri M, Memarian A, Behtash N, Karimi Zarchi M. The importance of 

re-examination with deep biopsies in diagnosing cervical malignancies 

despite multiple negative pathology reports: A case report. Int J Surg Case 

Rep 2015;14:48-49. 

260


DOI: 10.4274/tjh.2015.0143 


Endothelial Cells, Ankaferd Hemostat, and Estradiol 

Endotel Hücreleri, Ankaferd ve Estradiol 

Yasemin Ardıçoğlu1, Nejat Akar1, İbrahim Haznedaroğlu2 

1TOBB-ETÜ Hospital, Ankara, Turkey 

2Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey 

Figure 1A. A. Human umbilical vein endothelial cells adhered to 

each other, within seconds, just after the application of Ankaferd 

hemostat (5 µL). 

Figure 1B. Reversible vital endothelial cell adherence/aggregation 

in human umbilical vein endothelial cells 24 h after application of 

Ankaferd hemostat (5 µL). 

We previously demonstrated the effects of Ankaferd hemostat 

(AH) on human umbilical vein endothelial cells (HUVECs) in 

Turkish Journal of Hematology [1]. Endothelial cells adhered to 

each other within seconds and critical intracellular transcription 

factors were activated just after the application of AH (5 µL) to 

the HUVECs (Figure 1A). Rapid vital endothelial cell adherence/ 

aggregation is reversible and could be reversed within 24 h 

(Figure 1B). 

We further determined that the cellular effects of AH on HUVECs 

are clinically important in pharmacobiological hemostasis [1,2,3]. 

Endothelial cells are involved in a range of pathophysiological 

processes including hemostasis, inflammation, and angiogenesis 

[4], all of which are directly related to the effects of AH [1,2,3]. 

However, the relevant receptors on the surface of HUVECs and 

the molecules inside the content of AH affecting the endothelial 

cells remain unknown. Since HUVECs express estrogen receptor 

(ER) beta [4] and rapid HUVEC cellular responses to estrogen 

can be mediated by estrogen binding to ER [5], we herein 

aimed to investigate the estradiol content of AH. Estradiol 

concentration is found to be very high in AH (1452.6 pg/mL), 

whereas progesterone level is 6.06 ng/mL. Those results suggest 

novel hypotheses that shall be tested in future investigations 

regarding the interrelationships of vascular endothelial cells, 

hemostasis, and estradiol inside AH. 

Keywords: Endothelium, Ankaferd, Estradiol 

Anahtar Sözcükler: Endotel, Ankaferd, Estradiol 


Concept: İbrahim Haznedaroğlu, Nejat Akar; Design: Yasemin 

Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu; Data Collection 

Address for Correspondence/Yazışma Adresi: İbrahim HAZNEDAROĞLU, M.D., 

Hacettepe University Faculty of Medicine, Department of Hematology, 


E-mail : haznedar@yahoo.com, ichaznedaroglu@gmail.com 

Received/Geliş tarihi: April 02, 2015 

Accepted/Kabul tarihi: May 12, 2015 

261

Ardıçoğlu Y, et al: Endothelial Cells, Ankaferd Hemostat, and Estradiol 


or Processing: Yasemin Ardıçoğlu, Nejat Akar, İbrahim 

Haznedaroğlu; Analysis or Interpretation: Yasemin Ardıçoğlu, 

Nejat Akar, İbrahim Haznedaroğlu; Literature Search: Yasemin 

Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu; Writing: Yasemin 

Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu. 




included. 

References 

1. Yılmaz E, Güleç Ş, Torun D, Haznedaroğlu İC, Akar N. The effects of 

Ankaferd® Blood Stopper on transcription factors in HUVEC and the 

erythrocyte protein profile. Turk J Hematol 2011;28:276-285. 

2. Karabiyik A, Güleç S, Yilmaz E, Haznedaroglu I, Akar N. Reversible proteaseactivated 

receptor 1 downregulation mediated by Ankaferd Blood Stopper 

inducible with lipopolysaccharides inside the human umbilical vein 

endothelial cells. Clin Appl Thromb Hemost 2011;17:165-170. 

3. Karabıyık A, Yılmaz E, Güleç S, Haznedaroğlu I, Akar N. The Dual Diverse 

Dynamic Reversible Effects of Ankaferd Blood Stopper on EPCR and PAI-1 

Inside Vascular Endothelial Cells with and without LPS Challenge. Turk J 

Hematol 2012;29:361-366. 

4. Toth B, Saadat G, Geller A, Scholz C, Schulze S, Friese K, Jeschke U. Human 

umbilical vascular endothelial cells express estrogen receptor beta (ERβ) 

and progesterone receptor A (PR-A), but not ERα and PR-B. Histochem Cell 

Biol 2008;130:399-405. 

5. Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR. Human vascular 

endothelial cells contain membrane binding sites for estradiol, which 

mediate rapid intracellular signaling. Proc Natl Acad Sci U S A 2000;97:5930- 

5935. 

262


DOI: 10.4274/tjh.2015.0384 


An Unusual Congenital Anomaly in Fanconi Aplastic Anemia: 

Congenital Lobar Emphysema 

Fanconi Aplastik Anemisinde Nadir Bir Konjenital Anomali: Konjenital Lober Amfizem 

Ali Fettah 1 , Gökçe Pınar Reis 1 , Soner Sertan Kara 2 , Tekin Aksu 3 , Afak Durur Karakaya 4 , Mahmut Subaşı 5 , Atilla Çayır 6 

1Erzurum Regional Training and Research Hospital, Clinic of Pediatric Hematology, Erzurum, Turkey 

2Erzurum Regional Training and Research Hospital Clinic of Pediatric Infectious Disease, Erzurum, Turkey 

3Dr. Abdurrahman Yurtaslan Oncology Training and Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey 

4Erzurum Regional Training and Research Hospital, Clinic of Radiology, Erzurum, Turkey 

5Erzurum Regional Training and Research Hospital, Clinic of Thoracic Surgery, Erzurum, Turkey 

6Erzurum Regional Training and Research Hospital, Clinic of Endocrinology, Erzurum, Turkey 

Figure 1. Posterior anterior lung radiography imaging of the 

patient. 

Figure 2. Computerized tomography imaging of the patient. 

A 7-year-old girl who presented with epistaxis was examined 

due to pancytopenia. Her medical history revealed that she had 

respiratory distress in the neonatal period. She was born to a 

second-degree consanguineous marriage. Physical examination 

revealed short stature, microcephaly, microphthalmia, and hypo/ 

hyperpigmented lesions on the trunk and extremities. She did 

not have tachypnea, but she had decreased breathing sounds in 

the left lung. A laboratory work-up revealed hemoglobin of 5.4 

g/dL, mean corpuscular volume of 103/fL, leukocyte count of 

2.7x109/L, and thrombocyte count of 11x10 9 /L. A chromosomal 

breakage test with diepoxybutane was compatible with Fanconi 

anemia (FA). Posteroanterior chest X-ray showed hyperinflation 

of the left lung (Figure 1). Chest computed tomography revealed 

emphysematous changes in the upper part of the left lung, 

compatible with congenital lobar emphysema (Figure 2). 

FA is a rare autosomal recessive disorder and presents with 

numerous organ abnormalities, progressive cytopenia, and 

susceptibility to several malignancies [1,2]. Although absent lung 

lobes and abnormal pulmonary drainage have been reported 

[3], congenital lobar emphysema has not been presented as an 

accompanying pathology with FA. It is striking that the patient 

had no prominent respiratory symptoms since the newborn 

period. Congenital lobar emphysema’s association with FA has 

not been reported previously and it could be in coexistence or 

have an association with FA. 

Address for Correspondence/Yazışma Adresi: Ali FETTAH, M.D., 

Erzurum Regional Training and Research Hospital, Clinic of Pediatric Hematology, Erzurum, Turkey 

Phone : +90 505 675 05 86 

E-mail : alifettah@gmail.com 

Received/Geliş tarihi: November 09, 2015 

Accepted/Kabul tarihi: February 08, 2016 

263

Fettah A, et al: Congenital Lobar Emphysema in Fanconi Anemia 


Keywords: Fanconi, Anemia, Congenital lobar emphysema 

Anahtar Sözcükler: Fanconi, Anemi, Konjenital lober amfizem 

Ethics 



Surgical and Medical Practices: Tekin Aksu, Afak Durur Karakaya, 

Mahmut Subaşı, Atilla Çayır; Concept: Ali Fettah, Gökçe Pınar 

Reis; Design: Ali Fettah, Gökçe Pınar Reis; Data Collection or 

Processing: Ali Fettah, Gökçe Pınar Reis, Soner Sertan Kara, 

Tekin Aksu, Afak Durur Karakaya, Mahmut Subaşı, Atilla Çayır; 

Analysis or Interpretation: Ali Fettah, Gökçe Pınar Reis, Soner 

Sertan Kara; Literature Search: Ali Fettah, Gökçe Pınar Reis, 

Soner Sertan Kara; Writing: Ali Fettah, Soner Sertan Kara. 




included. 

References 

1. Unal S, Ozbek N, Kara A, Alikaşifoğlu M, Gümrük F. Five Fanconi anemia 

patients with unusual organ pathologies. Am J Hematol 2004;77:50-54. 

2. Auerbach AD. Fanconi anemia and its diagnosis. Mutat Res 2009;668:4-10. 

3. Bessler M, Mason PJ, Daniel CL, Wilson DB. Inheritedbone marrow failure 

syndromes. In: Nathan DG, Oski FA (eds). Hematology of Infancy and 

Childhood. Philadelphia, Saunders, 2015. 

264

Turkish Journal of Hematology Volume: 33 - Issue: 3

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