v39i2

Volume 39 Issue 2
June 2022
E-ISSN: 1308-5263
Research Articles
Comprehensive Mutation Profile in Acute Myeloid Leukemia Patients with RUNX1-RUNX1T1 or CBFB-MYH11
Fusions
Wei Qin, Xiayu Chen, Hong Jie Shen, Zheng Wang, Xiaohui Cai, Naike Jiang, Haiying Hua; Changzhou, Wuxi,
Soochow, Suzhou, China
Invasive Fungal Infections in Children with Leukemia: Clinical Features and Prognosis
Melike Sezgin Evim, Özlem Tüfekçi, Birol Baytan, Hale Ören, Solmaz Çelebi, Beyza Ener, Kevser Üstün Elmas,
Şebnem Yılmaz, Melek Erdem, Mustafa Kemal Hacımustafaoğlu, Adalet Meral Güneş; Bursa, İzmir, Turkey
Eltrombopag for Treatment of Thrombocytopenia Following Hematopoietic Stem Cell Transplantation
Zeynep Tuğba Güven, Serhat Çelik, Bülent Eser, Mustafa Çetin, Ali Ünal, Leylagül Kaynar; Kayseri, Antalya,
Turkey
Assessment of Bone Marrow Biopsy and Cytogenetic Findings in Patients with Multiple Myeloma
Ahmet Şeyhanlı, Boran Yavuz, Zehra Akşit, Zeynep Yüce, Sermin Özkal, Oğuz Altungöz, Fatih Demirkan,
İnci Alacacıoğlu, Güner Hayri Özsan; Sivas, İzmir, Turkey
Combination of Haploidentical Hematopoietic Stem Cell Transplantation with Umbilical Cord-Derived
Mesenchymal Stem Cells in Patients with Severe Aplastic Anemia: A Retrospective Controlled Study
Xian-Fu Sheng, Hui Li, Li-Li Hong, Hai-Feng Zhuang; Zhejiang, China
Castleman Disease: A Multicenter Case Series from Turkey
Eren Gündüz, Hakkı Onur Kırkızlar, Elif Gülsüm Ümit, Sedanur Karaman Gülsaran, Vildan Özkocaman,
Fahir Özkalemkaş, Ömer Candar, Tugrul Elverdi, Selin Küçükyurt, Semra Paydaş, Özcan Çeneli, Sema Karakuş,
Senem Maral, Ömer Ekinci, Yıldız İpek, Cem Kis, Zeynep Tuğba Güven, Aydan Akdeniz, Tiraje Celkan, Ayşe Hilal
Eroğlu Küçükdiler, Gülsüm Akgün Çağlıyan, Ceyda Özçelik Şengöz, Ayşe Karataş, Tuba Bulduk, Alper Özcan,
Fatma Burcu Belen Apak, Aylin Canbolat, İbrahim Kartal, Hale Ören, Ersin Töret, Gül Nihal Özdemir, Şule Mine
Bakanay Öztürk; Eskişehir, Edirne, Bursa, İstanbul, Adana, Konya, Ankara, Elazığ, Kayseri, Mersin, Aydın, Denizli,
Trabzon, Samsun, İzmir, Turkey
Cover Picture:
Gül Nihal Özdemir, Eren Gündüz
Gaucher Disease for Hematologists
2

Editor-in-Chief
Reyhan Küçükkaya
İstanbul, Turkey
rkucukkaya@hotmail.com
Associate Editors
Cengiz Beyan
Retired Professor
cengizbeyan@hotmail.com
A. Emre Eşkazan
İstanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine,
Department of Internal Medicine, Division of Hematology, İstanbul, Turkey
emre.eskazan@istanbul.edu.tr
Hale Ören
Dokuz Eylül University Faculty of Medicine, Department of Pediatric
Hematology, İzmir, Turkey
hale.oren@deu.edu.tr
Ali İrfan Emre Tekgündüz
Memorial Bahçelievler Hospital, Adult Hematology and BMT Clinic,
İstanbul, Turkey
emretekgunduz@yahoo.com
Şule Ünal
Hacettepe University Faculty of Medicine, Division of Pediatric Hematology,
Ankara, Turkey
suleunal2003@hotmail.com
Ayşegül Ünüvar
İstanbul University, İstanbul School of Medicine, Division of Pediatric
Hematology-Oncology, İstanbul, Turkey
aysegulu@hotmail.com
Assistant Editors
Olga Meltem Akay
Koç University Faculty of Medicine, Department of Hematology,
İstanbul, Turkey
İnci Alacacıoğlu
Dokuz Eylül University, Institute of Oncology, Clinical Oncology Department,
Division of Hematology, İzmir, Turkey
Claudio Cerchione
Scientific Institute of Romagna for the Study and Treatment of Tumors,
Unit of Hematology, Meldola, Italy
Nil Güler
Pamukkale University Faculty of Medicine, Department of Hematology,
Denizli, Turkey
Veysel Sabri Hançer
İstinye University Faculty of Medicine, Department of Medical Biology,
İstanbul, Turkey
Elif Ünal İnce
Ankara University Faculty of Medicine, Department of Pediatric Hematology,
Ankara, Turkey
Zühre Kaya
Gazi University Faculty of Medicine, Department of Hematology, Ankara,
Turkey
Ebru Koca
Başkent University Ankara Hospital, Clinic of Hematology, Ankara, Turkey
Müge Sayitoğlu
İstanbul University, Aziz Sancar Institute of Experimental Medicine,
Department of Genetics, İstanbul, Turkey
Mario Tiribelli
University of Udine, Department of Medical Area, Division of Hematology
and Stem Cell Transplantation, Udine, Italy
International Advisory Board
Nejat Akar (Turkey)
TOBB University of Economics and Technology, Clinic of Child Health and
Diseases (Pediatrics), Ankara, Turkey
Görgün Akpek (USA)
Dignity Health Center San Francisco, California, USA
Serhan Alkan (USA)
Loyola University Medical Center, Department of Pathology, IL, USA
Meral Beksaç (Turkey)
Ankara University Faculty of Medicine, Department of Hematology, Ankara,
Turkey
Koen van Besien (USA)
Weill Cornell Medical College, New York, USA
M. Sıraç Dilber (Sweden)
Karolinska University Hospital Huddinge, Stockholm, Sweden
Ahmet Doğan (USA)
Memorial Sloan Kettering Cancer Center, New York, USA
Peter Dreger (Germany)
Heidelberg University Hospital, Department of Internal Medicine V,
Heidelberg, Germany
Thierry Facon (France)
Lille University, Department of Hematology, Lille, France
Jawed Fareed (USA)
Loyola University Medical Center, Maywood, IL, USA
Burhan Ferhanoğlu (Turkey)
Koç University Faculty of Medicine, Department of Hematology, İstanbul,
Turkey
Gösta Gahrton (Sweden)
Karolinska Institute at Huddinge and Solna, Department of Medicine,
Stockholm, Sweden
Margarita Guenova (Bulgaria)
National Center of Hematology and Transfusiology, Department of
Morphology, Sofia, Bulgaria
A-I
Publishing
Services
GALENOS PUBLISHER
Molla Gürani Mah. Kaçamak Sk. No: 21/1, Fındıkzade, İstanbul, Turkey
Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr

İbrahim Haznedaroğlu (Turkey)
Hacettepe University Faculty of Medicine, Department of Hematology,
Ankara, Turkey
Dieter Hoelzer (Germany)
University Hospital, Department of Internal Medicine II, Hematology and
Oncology, Frankfurt, Germany
Marilyn Manco-Johnson (USA)
Hemophilia & Thrombosis Center, Section of Hematology, Oncology, and
Bone Marrow Transplantation, Department of Pediatrics, University of
Colorado Denver and The Children’s Hospital, Aurora, CO
Andreas Josting (Germany)
German Hodgkin Study Group (GHSG), Cologne, Germany; Protestant
Hospital Lippstadt, Lippstadt, Germany
Emin Kansu (Turkey)
Retired Professor, Hacettepe University Research Center for Stem Cells
(PEDİ-STEM), Senior Consultant
Winfried Kern (Germany)
University of Freiburg, Faculty of Medicine, University of Freiburg,
Department of Medicine II, Division of Infectious Diseases, Medical
Center, Freiburg, Germany
Nigel Key (USA)
University of Minnesota Medical School, Minneapolis, MN, USA
Selami Koçak Toprak (Turkey)
Ankara University School of Medicine, Department of Hematology,
Ankara, Turkey
Korgün Koral (USA)
University of Texas Southwestern Medical Center at Dallas and
Children’s Medical Center Department of Radiology, Texas, USA
Abdullah Kutlar (USA)
Medical College of Georgia, Department of Medicine, Augusta, GA
Luca Malcovati (Italy)
University of Pavia, Fondazione IRCCS Policlinico San Matteo,
Department of Hematology, Pavia, Italy
Robert Marcus (United Kingdom)
King’s College Hospital, London, United Kingdom
Jean Pierre Marie (France)
Laboratoire de Culture et Cinétique Cellulaire, Hôpital Hôtel-Dieu, Paris,
France
Gerassimos A. Pangalis (Greece)
National and Kapodistrian University of Athens, Laikon General Hospital,
Department of Haematology, Athens, Greece
Semra Paydaş (Turkey)
Çukurova University Faculty of Medicine, Department of Oncology,
Adana, Turkey
Santiago Pavlovsky (Argentina)
FUNDALEU (Foundation to Fight Leukemia) and Centro de Investigacion
Clinica A. Ocampo, Buenos Aires, Argentina
Antonio Giulio Piga (Italy)
University of Turin, Department of Clinical and Biological Sciences,
Torino, Italy
Ananda Prasad (USA)
Wayne State University, School of Medicine, Department of Internal
Medicine, Division of Hematology/Oncology, Detroit, Michigan, USA
Jacob M. Rowe (Israel)
Hebrew University of Jerusalem, Jerusalem, Israel
Jens-Ulrich Rüffer (Germany)
German Fatigue Society, Cologne, Germany
Norbert Schmitz (Germany)
Asklepios Hospital Barmbek, Department of Hematology, Oncology and
Stem Cell Transplantation, Hamburg, Germany
Orhan Sezer (Germany)
Charité Comprehensive Cancer Center, Berlin, Germany
Anna Sureda (Spain)
ICO - Hospital Duran i Reynals, Barcelona, Spain
Ayalew Tefferi (USA)
Mayo Clinic, Divisions of Hematology, Rochester, Minnesota, USA
Nüket Tüzüner (Turkey)
Retired Professor
Catherine Verfaillie (Belgium)
Katholieke Universiteit Leuven, Leuven, Belgium
Srdan Verstovsek (USA)
UT MD Anderson Cancer Center, Department of Leukemia, Houston, TX,
USA
Claudio Viscoli (Italy)
IRCCS San Martino-IST, University of Genoa, Clinic of Infectious
Diseases, Genoa, Italy
Past Editors
Erich Frank
Orhan Ulutin
Hamdi Akan
Aytemiz Gürgey
Senior Advisory Board
Yücel Tangün
Osman İlhan
Muhit Özcan
Teoman Soysal
Ahmet Muzaffer Demir
Güner Hayri Özsan
Language Editor
Leslie Demir
Statistic Editor
Hülya Ellidokuz
Editorial Office
İpek Durusu
Bengü Timoçin Efe
A-II

Contact Information
Editorial Correspondence should be addressed to Dr. Reyhan Küçükkaya
E-mail : rkucukkaya@hotmail.com
All Inquiries Should be Addressed to
TURKISH JOURNAL OF HEMATOLOGY
Address : Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.) No: 8 06550 Çankaya, Ankara / Turkey
Phone : +90 312 490 98 97
Fax : +90 312 490 98 68
E-mail : tjh@tjh.com.tr
E-ISSN: 1308-5263
Publishing Manager
Reyhan Küçükkaya
Management Address
Türk Hematoloji Derneği
Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.)
No: 8 06550 Çankaya, Ankara / Turkey
Online Manuscript Submission
http://mc.manuscriptcentral.com/tjh
Web Page
www.tjh.com.tr
Owner on Behalf of the Turkish Society
of Hematology
Muhlis Cem Ar
Publishing House
Molla Gürani Mah. Kaçamak Sk. No: 21,
34093 Fındıkzade, İstanbul / Turkey
Tel: +90 212 621 99 25
Fax: +90 212 621 99 27
E-mail: info@galenos.com.tr
Publisher Certificate Number: 14521
Publication Date
01.06.2022
Cover Picture
Gül Nihal Özdemir, Eren Gündüz
Gaucher Disease for Hematologists
Bone marrow aspirate showing a number of large
macrophages laden with cerebrosides (arrows: Gaucher cells) in a
patient with Gaucher disease.
International scientific journal published quarterly.
The Turkish Journal of Hematology is published by the commercial enterprise
of the Turkish Society of Hematology with Decision Number 6 issued by the
Society on 7 October 2008.
A-III

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.
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
- ProQuest
- Index Copernicus
- Tübitak/Ulakbim Turkish Medical Database
- Turk Medline
- Hinari
- GOALI
- ARDI
- OARE
Impact Factor: 1.831
Digital Archiving Policy
The journal’s Abstracting/Indexing services store essential information about
articles. In addition, some of our journal’s Abstracting/Indexing services
archive metadata about the article and electronic versions of the articles.
In this way, copies of articles are presented to the scientific community
through these systems as an alternative to journals.
Open Access Policy
This journal provides immediate open access to its content on the principle
that making research freely available to the public supports a greater global
exchange of knowledge. Open Access Policy is based on rules of Budapest
Open Access Initiative (BOAI) http://www.budapestopenaccessinitiative.org/.
By “open access” to [peer-reviewed research literature], we mean its free
availability on the public internet, permitting any users to read, download,
copy, distribute, print, search, or link to the full texts of these articles, crawl
them for indexing, pass them as data to software, or use them for any
other lawful purpose, without financial, legal, or technical barriers other
than those inseparable from gaining access to the internet itself. The only
constraint on reproduction and distribution, and the only role for copyright
in this domain, should be to give authors control over the integrity of their
work and the right to be properly acknowledged and cited.
This work is licensed under a Creative Commons Attribution-NonCommercial-
NoDerivatives 4.0 (CC BY-NC-ND) International License.
CC BY-NC-ND: This license allows reusers to copy and distribute the material
in any medium or format in unadapted form only, for noncommercial
purposes only, and only so long as attribution is given to the creator.
CC BY-NC-ND includes the following elements:
BY – Credit must be given to the creator
NC – Only noncommercial uses of the work are permitted
ND – No derivatives or adaptations of the work are permitted
Permission Requests
Permission is required for the use of any work published under a Creative
Commons Attribution-NonCommercial-NoDerivatives 4.0 (CC BY-NC-ND)
International License with commercial purposes (selling, etc.) to protect
the copyright owner and author rights. Republication and reproduction
of images or tables in any published material should be done with proper
citation of source providing authors’ names; article title; journal title; year
(volume) and page of publication; copyright year of the article.
Subscription Information
The Turkish Journal of Hematology is published electronically only as of
2019. Therefore, subscriptions are not necessary. All published volumes are
available in full text free-of-charge online at www.tjh.com.tr.
Address: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613.
Sok.) No: 8 06550 Çankaya, Ankara / Turkey
Telephone: +90 312 490 98 97
Fax: +90 312 490 98 68
Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
Web page: www.tjh.com.tr
E-mail: tjh@tjh.com.tr
Permissions
Requests for permission to reproduce published material should be sent to
the editorial office.
Editor: Professor Dr. Reyhan Küçükkaya
Adress: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613.
Sok.) No: 8 06550 Çankaya, Ankara / Turkey
Telephone: +90 312 490 98 97
Fax: +90 312 490 98 68
Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
Web page: www.tjh.com.tr
E-mail: tjh@tjh.com.tr
A-IV

Publisher
Galenos Yayınevi
Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey
Telephone : +90 212 621 99 25
Fax : +90 212 621 99 27
info@galenos.com.tr
Instructions for Authors
Instructions for authors are published in the journal and at www.tjh.com.tr
Material Disclaimer
Authors are responsible for the manuscripts they publish in The Turkish
Journal of Hematology. The editor, editorial board, and publisher do not
accept any responsibility for published manuscripts.
If you use a table or figure (or some data in a table or figure) from another
source, cite the source directly in the figure or table legend.
Editorial Policy
Following receipt of each manuscript, a checklist is completed by the
Editorial Assistant. The Editorial Assistant checks that each manuscript
contains all required components and adheres to the author guidelines, after
which time it will be forwarded to the Editor in Chief. Following the Editor
in Chief’s evaluation, each manuscript is forwarded to the Associate Editor,
who in turn assigns reviewers. Generally, all manuscripts will be reviewed
by at least three reviewers selected by the Associate Editor, based on their
relevant expertise. The Associate Editor could be assigned as a reviewer
along with the other reviewers. After the reviewing process, all manuscripts
are evaluated in the Editorial Board Meeting.
The Turkish Journal of Hematology’s editors and Editorial Board members
are active researchers. It is possible that they may desire to submit their
manuscripts to the Turkish Journal of Hematology. This may create 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 the
Editor in Chief, who will act independently. In some situations, this process
will be overseen by an outside independent expert in reviewing submissions
from editors.
A-V

TURKISH JOURNAL OF HEMATOLOGY
INSTRUCTIONS FOR AUTHORS
The Turkish Journal of Hematology accepts invited review articles,
research articles, brief reports, letters to the editor, and hematological
images that are relevant to the scope of hematology, on the condition
that they have not been previously published elsewhere. Basic science
manuscripts, such as randomized, cohort, cross-sectional, and casecontrol
studies, are given preference. All manuscripts are subject
to editorial revision to ensure they conform to the style adopted by
the journal. There is a double-blind reviewing system. Review articles
are solicited by the Editor-in-Chief. Authors wishing to submit an
unsolicited review article should contact the Editor-in-Chief prior to
submission in order to screen the proposed topic for relevance and
priority.
The Turkish Journal of Hematology does not charge any article
submission or processing charges.
Manuscripts should be prepared according to ICMJE guidelines (http://
www.icmje.org/). Original manuscripts require a structured abstract.
Label each section of the structured abstract with the appropriate
subheading (Objective, Materials and Methods, Results, and Conclusion).
Letters to the editor do not require an abstract. Research or project
support should be acknowledged as a footnote on the title page.
Technical and other assistance should be provided on the title page.
Submissions and publication are free of charge.
Original Manuscripts
Title Page
Title: The title should provide important information regarding the
manuscript’s content. The title must specify that the study is a cohort
study, cross-sectional study, case-control study, or randomized study (i.e.
Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability
of ferrous succinate tablet formulations without correction for baseline
circadian changes in iron concentration in healthy Chinese male
subjects: A single-dose, randomized, 2-period crossover study. Clin Ther
2011;33:2054-2059).
The title page should include the authors’ names, degrees, and
institutional/professional affiliations and a short title, abbreviations,
keywords, financial disclosure statement, and conflict of interest
statement. If a manuscript includes authors from more than one
institution, each author’s name should be followed by a superscript
number that corresponds to their institution, which is listed separately.
Please provide contact information for the corresponding author,
including name, e-mail address, and telephone and fax numbers.
Important Notice: The title page should be submitted separately.
Running Head: The running head should not be more than 40
characters, including spaces, and should be located at the bottom of
the title page.
Word Count: A word count for the manuscript, excluding abstract,
acknowledgments, figure and table legends, and references, should be
provided and should not exceed 2500 words. The word count for the
abstract should not exceed 300 words.
Conflict of Interest Statement: To prevent potential conflicts of
interest from being overlooked, this statement must be included in each
manuscript. In case there are conflicts of interest, every author should
complete the ICMJE general declaration form, which can be obtained at
http://www.icmje.org/downloads/coi_disclosure.zip
Abstract and Keywords: The second page should include an abstract
that does not exceed 300 words. For manuscripts sent by authors in
Turkey, a title and abstract in Turkish are also required. As most readers
read the abstract first, it is critically important. Moreover, as various
electronic databases integrate only abstracts into their index, important
findings should be presented in the abstract.
Objective: The abstract should state the objective (the purpose of the
study and hypothesis) and summarize the rationale for the study.
Materials and Methods: Important methods should be written
respectively.
Results: Important findings and results should be provided here.
Conclusion: The study’s new and important findings should be
highlighted and interpreted.
Other types of manuscripts, such as reviews, brief reports, and
editorials, will be published according to uniform requirements.
Provide 3-10 keywords below the abstract to assist indexers. Use
terms from the Index Medicus Medical Subject Headings List
(for randomized studies a CONSORT abstract should be provided: http://
www.consort-statement.org).
Introduction: The introduction should include an overview of the
relevant literature presented in summary form (one page), and whatever
remains interesting, unique, problematic, relevant, or unknown about
the topic must be specified. The introduction should conclude with the
rationale for the study, its design, and its objective(s).
Materials and Methods: Clearly describe the selection of observational
or experimental participants, such as patients, laboratory animals, and
controls, including inclusion and exclusion criteria and a description of
the source population. Identify the methods and procedures in sufficient
detail to allow other researchers to reproduce your results. Provide
references to established methods (including statistical methods),
provide references to brief modified methods, and provide the rationale
for using them and an evaluation of their limitations. Identify all drugs
and chemicals used, including generic names, doses, and routes of
administration. The section should include only information that was
available at the time the plan or protocol for the study was devised
A-VI

(https://www.strobe-statement.org/fileadmin/Strobe/uploads/checklists/
STROBE_checklist_v4_combined.pdf).
Statistics: Describe the statistical methods used in enough detail to
enable a knowledgeable reader with access to the original data to verify
the reported results. Statistically important data should be given in the
text, tables, and figures. Provide details about randomization, describe
treatment complications, provide the number of observations, and specify
all computer programs used.
Results: Present your results in logical sequence in the text, tables, and
figures. Do not present all the data provided in the tables and/or figures
in the text; emphasize and/or summarize only important findings, results,
and observations in the text. For clinical studies provide the number of
samples, cases, and controls included in the study. Discrepancies between
the planned number and obtained number of participants should be
explained. Comparisons and statistically important values (i.e. p-value
and confidence interval) should be provided.
Discussion: This section should include a discussion of the data. New and
important findings/results and the conclusions they lead to should be
emphasized. Link the conclusions with the goals of the study, but avoid
unqualified statements and conclusions not completely supported by the
data. Do not repeat the findings/results in detail; important findings/results
should be compared with those of similar studies in the literature, along with
a summarization. In other words, similarities or differences in the obtained
findings/results with those previously reported should be discussed.
Study Limitations: Limitations of the study should be detailed. In
addition, an evaluation of the implications of the obtained findings/
results for future research should be outlined.
Conclusion: The conclusion of the study should be highlighted.
References
Cite references in the text, tables, and figures with numbers in square
brackets. Number references consecutively according to the order in
which they first appear in the text. Journal titles should be abbreviated
according to the style used in Index Medicus (consult List of Journals
Indexed in Index Medicus). Include among the references any paper
accepted, but not yet published, designating the journal followed by “in
press”.
Examples of References:
1. List all authors
Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow
transplantation from unrelated donors for patients with aplastic anemia
after failure of immunosuppressive therapy. Blood 2006;108:1485-1491.
2. Organization as author
Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of
syngeneic bone marrow graft without preconditioning in post-hepatitis
marrow aplasia. Lancet 1977;2:742-744.
3. Book
Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961.
4. Book Chapter
Perutz MF. Molecular anatomy and physiology of hemoglobin. In:
Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin:
Genetics, Pathophysiology, Clinical Management. New York, Cambridge
University Press, 2000.
5. Abstract
Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin)
stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract).
6. Letter to the Editor
Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further
definition of 20q deletion in myeloid leukemia using fluorescence in situ
hybridization. Blood 1994;84:2821-2823.
7. Supplement
Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant
2005;9(Suppl 7):81-86.
Brief Reports
Abstract length: Not to exceed 150 words.
Article length: Not to exceed 1200 words.
Introduction: State the purpose and summarize the rationale for the study.
Materials and Methods: Clearly describe the selection of the observational
or experimental participants. Identify the methods and procedures in
sufficient detail. Provide references to established methods (including
statistical methods), provide references to brief modified methods, and
provide the rationale for their use and an evaluation of their limitations.
Identify all drugs and chemicals used, including generic names, doses, and
routes of administration.
Statistics: Describe the statistical methods used in enough detail to
enable a knowledgeable reader with access to the original data to verify
the reported findings/results. Provide details about randomization,
describe treatment complications, provide the number of observations,
and specify all computer programs used.
Results: Present the findings/results in a logical sequence in the text, tables,
and figures. Do not repeat all the findings/results in the tables and figures in
the text; emphasize and/or summarize only those that are most important.
Discussion: Highlight the new and important findings/results of the
study and the conclusions they lead to. Link the conclusions with the
goals of the study, but avoid unqualified statements and conclusions not
completely supported by your data.
Invited Review Articles
Abstract length: Not to exceed 300 words.
Article length: Not to exceed 4000 words.
Review articles should not include more than 100 references. Reviews should
include a conclusion, in which a new hypothesis or study about the subject
A-VII

may be posited. Do not publish methods for literature search or level of
evidence. Authors who will prepare review articles should already have
published research articles on the relevant subject. The study’s new and
important findings should be highlighted and interpreted in the Conclusion
section. There should be a maximum of two authors for review articles.
Perspectives in Hematology
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A-X

CONTENTS
Research Articles
84 Comprehensive Mutation Profile in Acute Myeloid Leukemia Patients with RUNX1-RUNX1T1 or CBFB-MYH11
Fusions
Wei Qin, Xiayu Chen, Hong Jie Shen, Zheng Wang, Xiaohui Cai, Naike Jiang, Haiying Hua; Changzhou, Wuxi, Soochow, Suzhou, China
94 Invasive Fungal Infections in Children with Leukemia: Clinical Features and Prognosis
Melike Sezgin Evim, Özlem Tüfekçi, Birol Baytan, Hale Ören, Solmaz Çelebi, Beyza Ener, Kevser Üstün Elmas, Şebnem Yılmaz, Melek
Erdem, Mustafa Kemal Hacımustafaoğlu, Adalet Meral Güneş; Bursa, İzmir, Turkey
103 Eltrombopag for Treatment of Thrombocytopenia Following Hematopoietic Stem Cell Transplantation
Zeynep Tuğba Güven, Serhat Çelik, Bülent Eser, Mustafa Çetin, Ali Ünal, Leylagül Kaynar; Kayseri, Antalya, Turkey
109 Assessment of Bone Marrow Biopsy and Cytogenetic Findings in Patients with Multiple Myeloma
Ahmet Şeyhanlı, Boran Yavuz, Zehra Akşit, Zeynep Yüce, Sermin Özkal, Oğuz Altungöz, Fatih Demirkan, İnci Alacacıoğlu,
Güner Hayri Özsan; Sivas, İzmir, Turkey
117 Combination of Haploidentical Hematopoietic Stem Cell Transplantation with Umbilical Cord-Derived
Mesenchymal Stem Cells in Patients with Severe Aplastic Anemia: A Retrospective Controlled Study
Xian-Fu Sheng, Hui Li, Li-Li Hong, Hai-Feng Zhuang; Zhejiang, China
130 Castleman Disease: A Multicenter Case Series from Turkey
Eren Gündüz, Hakkı Onur Kırkızlar, Elif Gülsüm Ümit, Sedanur Karaman Gülsaran, Vildan Özkocaman, Fahir Özkalemkaş,
Ömer Candar, Tugrul Elverdi, Selin Küçükyurt, Semra Paydaş, Özcan Çeneli, Sema Karakuş, Senem Maral, Ömer Ekinci, Yıldız İpek,
Cem Kis, Zeynep Tuğba Güven, Aydan Akdeniz, Tiraje Celkan, Ayşe Hilal Eroğlu Küçükdiler, Gülsüm Akgün Çağlıyan, Ceyda Özçelik
Şengöz, Ayşe Karataş, Tuba Bulduk, Alper Özcan, Fatma Burcu Belen Apak, Aylin Canbolat, İbrahim Kartal, Hale Ören, Ersin Töret,
Gül Nihal Özdemir, Şule Mine Bakanay Öztürk; Eskişehir, Edirne, Bursa, İstanbul, Adana, Konya, Ankara, Elazığ, Kayseri, Mersin,
Aydın, Denizli, Trabzon, Samsun, İzmir, Turkey
Perspective in Hematology
136 Gaucher Disease for Hematologists
Gül Nihal Özdemir, Eren Gündüz; İstanbul, Eskişehir, Turkey
Images in Hematology
140 Gallbladder Involvement of Diffuse Large B-Cell Lymphoma with 18 F-FDG PET/CT
Esra Arslan, Göksel Alçin, Tamer Aksoy, Tevfik Fikret Çermik; İstanbul, Turkey
142 Mast Cell Leukemia in a Patient with Teratoma
Yan Shen, Zhenni Wang, Huijun Lin, Jinlin Liu; Hangzhou, China
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Letters to the Editor
144 Caution Regarding the Difference Between Flower-Like Lymphocytes and Flower-Like Plasma Cells
Jingnan Zhu, Zhang Li, Yong Wang, Jinlin Liu; Hangzhou, Shaoxing, Ningbo, China
146 Does Treatment of Hepatitis C Reduce Inhibitor Titers in Hemophilia A?
Memiş Hilmi Atay, Emine Türkoğlu, Engin Kelkitli; Samsun, Tokat, Turkey
148 Spontaneous Remission in Paroxysmal Nocturnal Hemoglobinuria: An Extremely Rare Case
Özgür Mehtap, Ayfer Gedük; Kocaeli, Turkey
150 Hematopoietic Stem Cell Transplantation to a Patient with Acute Myeloid Leukemia from a Sibling Donor
Positive for SARS-CoV-2 by RT-PCR Test
Ahmet Koç, Ömer Doğru, Nurşah Eker, Burcu Tufan Taş, Rabia Emel Şenay; İstanbul, Turkey
A-XII

RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2022.2021.0641
Turk J Hematol 2022;39:84-93
Comprehensive Mutation Profile in Acute Myeloid Leukemia
Patients with RUNX1-RUNX1T1 or CBFB-MYH11 Fusions
RUNX1-RUNX1T1 veya CBFB-MYH11 Füzyonları Olan Akut Myeloid Lösemili Hastalarda
Detaylı Mutasyon Profili
Wei Qin 1 *, Xiayu Chen 2 *, Hong Jie Shen 3 , Zheng Wang 3,4 , Xiaohui Cai 1 , Naike Jiang 1 , Haiying Hua 2
1Affiliated Changzhou Second Hospital of Nanjing Medical University, Department of Hematology, Changzhou, China
2Affiliated Hospital of Jiangnan University, Department of Hematology, Wuxi, China
3The First Affiliated Hospital of Soochow University, Department of Hematology, Soochow, China
4Suzhou Jsuniwell Medical Laboratory, Suzhou, China
*These authors contributed equally to this work.
Abstract
Objective: This study was undertaken with the aim of better
understanding the genomic landscape of core-binding factor (CBF)
acute myeloid leukemia (AML).
Materials and Methods: We retrospectively analyzed 112 genes that
were detected using next-generation sequencing in 134 patients
with de novo CBF-AML. FLT3-ITD, NPM1, and CEBPA mutations were
detected by DNA-PCR and Sanger sequencing.
Results: In the whole cohort, the most commonly mutated
genes were c-KIT (33.6%) and NRAS (33.6%), followed by FLT3
(18.7%), KRAS (13.4%), RELN (8.2%), and NOTCH1 (8.2%). The
frequencies of mutated genes associated with epigenetic
modification, such as IDH1, IDH2, DNMT3A, and TET2, were low,
being present in 1.5%, 0.7%, 2.2%, and 7.5% of the total
number of patients, respectively. Inv(16)/t(16;16) AML patients
exhibited more mutations of NRAS and KRAS (p=0.001
and 0.0001, respectively) than t(8;21) AML patients. Functionally
mutated genes involved in signaling pathways were observed more
frequently in the inv(16)/t(16;16) AML group (p=0.016), while the
mutations involved in cohesin were found more frequently in
the t(8;21) AML group (p=0.011). Significantly higher white blood cell
counts were found in inv(16)/t(16;16) AML patients with c-KIT (c-KIT mut )
or NRAS (NRAS mut ) mutations compared to the corresponding t(8;21)
AML/c-KIT mut and t(8;21) AML/NRAS mut groups (p=0.001 and 0.009,
respectively).
Conclusion: The mutation profiles of t(8;21) AML patients showed
evident differences from those of patients with inv(16)/t(16;16)
AML. We have provided a comprehensive overview of the mutational
landscape of CBF-AML.
Keywords: Core-binding factor, Acute myeloid leukemia, Mutation,
Next-generation sequencing
Öz
Amaç: Bu çalışma çekirdek bağlama faktörü (ÇBF) akut myeloid
löseminin (AML) genomik durumunu daha iyi anlamak amacıyla
yapılmıştır.
Yöntemler: Yüz otuz dört de novo ÇBF-AML hastasında yeni nesil
dizileme ile tespit edilen 112 geni geriye dönük olarak analiz ettik.
FLT3-ITD, NPM1 ve CEBPA mutasyonları DNA-PCR ve Sanger dizileme
ile tespit edildi.
Bulgular: Bütün kohortta en sık mutasyonlu genler c-KIT (33,6%) ve
NRAS (33,6%) ve ardından FLT3 (%18,7), KRAS (%13,4), RELN (%8,2),
NOTCH1 (%8,2) idi. IDH1, IDH2, DNMT3A ve TET2 gibi epigenetik
modifikasyonla ilişkili mutasyona uğramış genlerin sıklığı düşüktü ve
toplam hastaların sırasıyla %1,5, %0,7, %2,2 ve %7,5’inde mevcuttu.
Inv(16)/t(16;16) AML hastalarında NRAS ve KRAS mutasyonları
t(8;21) AML hastalarına göre daha fazlaydı (sırasıyla; p=0,001;
0,0001). İşlevsel olarak sinyal yolaklarında yer alan mutasyonlu genler
inv(16)/t(16;16) AML grubunda daha çok gözlenirken (p=0,016),
kohezin içinde yer alan mutasyonlar t(8;21) AML grubunda daha
çok bulundu (p=0,011). c-KIT (c-KIT mut ) veya NRAS mutasyonları
(NRAS mut ) olan inv(16)/t(16;16) AML hastalarında karşılığındaki t(8;21)
AML/c-KIT mut ve t(8;21) AML/NRAS mut gruplarına göre beyaz küre sayısı
daha yüksek bulundu (sırasıyla; p=0,001; 0,009,).
Sonuç: t(8;21) AML hastalarının mutasyon profilleri inv(16)/t(16;16)
AML’den belirgin farklılıklar gösterdi. Bu çalışmada ÇBF-AML’nin
mutasyon profili kapsamlı bir biçimde incelenmiştir.
Anahtar Sözcükler: Çekirdek bağlama faktörü, Akut myeloid lösemi,
Mutasyon, Yeni nesil sekanslama
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Naike Jiang, M.D., Affiliated Changzhou Second Hospital of
Nanjing Medical University, Department of Hematology, Changzhou China
Phone : 8613357894897
E-mail : naike.j@163.com ORCID: orcid.org/0000-0003-4495-567X
Received/Geliş tarihi: November 20, 2021
Accepted/Kabul tarihi: April 21, 2022
84

Turk J Hematol 2022;39:84-93
Qin W. et al: Mutation Profile in CBF-AML
Introduction
Cases of acute myeloid leukemia (AML) involving the
core-binding factor (CBF) include AML with t(8;21) and
inv(16)/t(16;16) chromosomal translocations, leading to
the RUNX-RUNX1T1 and CBFB-MYH11 fusions genes,
respectively. Such AML patients account for approximately
25% of pediatric and 15% of adult de novo AML cases [1],
and CBF-AML was recognized as a unique entity in the
2016 World Health Organization classification of myeloid
neoplasms and acute leukemia [2]. Accumulating evidence
has revealed that the t(8;21)(q22;q22) and inv(16)/t(16;16) CBF
rearrangements are associated with favorable outcomes relative
to other cytogenetic subtypes and that allogeneic
hematopoietic stem cell transplantation is not generally
recommended during the first complete remission (CR)
[3,4]. However, relapse occurs in up to 40% of these cases,
indicating clinicopathological heterogeneity within this
AML subset [5,6,7,8]. Further investigation is still needed to
better understand leukemogenesis and disease progression.
Previous findings have demonstrated that expressions of
translocation-encoded AML1 or CBF fusion proteins are
insufficient by themselves to induce a full leukemic phenotype
[9]. Further evidence supporting this model comes from
the fact that mutations in genes activating tyrosine kinase
signaling (including KIT, N/KRAS, and FLT3) are frequent in
both CBF-AML subtypes [6,10]. Nonetheless, data regarding
the prognostic significance of KIT and RAS in CBF-AML
are contradictory. Duployez et al. [11] reported the presence of
additional aberrations in >90% of CBF-AML cases, and mutations
in epigenetic modifications or cohesin genes were associated
with poor prognosis in t(8;21) AML patients with TK pathway
mutations using next-generation sequencing (NGS). Ishikawa
et al. [12] revealed that the c-KIT exon 17 mutation and the
presence of extramedullary tumors in t(8;21) AML patients were
poor prognostic factors for relapse-free survival, as were the loss
of chromosome X or Y and NRAS mutation in patients with
inv(16)/t(16;16). These findings highlight the multiclonality
of CBF-AML and suggest that the prognostic impact may
differ in the context of certain gene mutations between AML
patients with t(8;21) and those with inv(16)/t(16;16).
Comprehensive genetic analysis using NGS may be helpful in
refining our understanding of the prognosis of CBF-AML [11]. To
the best of our knowledge, limited data are available regarding
the impact of companion gene mutations in CBF-AML. To better
characterize this subtype and to better understand the role of
co-mutations in CBF-AML, we performed extensive mutational
analysis by NGS for 134 CBF-AML patients. The clinical value of
co-mutations in CBF-AML patients was also explored.
Materials and Methods
Patients
A total of 134 newly diagnosed de novo CBF-AML patients
were selected from the Affiliated Changzhou Second Hospital
of Nanjing Medical University, Wuxi Third People’s Hospital, and
First Affiliated Hospital of Soochow University from May
2016 to June 2021. The diagnosis of CBF-AML was based on the
2008 definition of the World Health Organization [2]. Eighty AML
patients with t(8;21)/RUNX1-RUNX1T1 and 54 patients
with inv(16)/t(16;16)/CBFB-MYH11 were included in the
analysis. The study was approved by the local ethics committee
and conducted in accordance with the Declaration of Helsinki.
Mutational Analysis by Next-Generation Sequencing
Genomic DNA was extracted from fresh bone marrow or
peripheral blood samples with the QIAamp DNA Mini Kit
(QIAGEN GmbH, Germany) following the manufacturer’s
instructions. The NGS library was prepared using at least 200 ng
of genomic DNA. Massively parallel sequencing was performed
with an Illumina next-generation sequencer and a variant allele
frequency of >3% was used as the threshold for calling singlenucleotide
variants. A high depth of coverage (1000x) was
obtained for 112 genes, including whole coding regions known
to be frequently mutated in hematological malignancies, such
as genes involved in epigenetic regulators, signaling pathways,
transcription factors, spliceosomes, cohesin complex, tumor
suppressors, and chromatin modifiers (Table 1). Searches were
performed in the COSMIC database for altered DNA sequences
deemed to be mutations or variants with IGV software and
were confirmed in the SNP database (dbSNP). Polymerase chain
reaction (PCR) followed by direct Sanger sequencing was used
to detect FLT3-ITD, NPM1 (exon 12), and CEBPA to avoid possible
false-negative results due to limitations of NGS, as previously
described [13,14].
Other Cytogenetic and Molecular Abnormality Screening
Bone marrow (BM) cells were collected and cultured at the
time of the initial diagnosis. The presence of the t(8;21)
or inv(16)/t(16;16) rearrangement was determined by the
conventional G/R banding method. Fluorescence in situ
hybridization (FISH) of interphase nuclei and/or metaphases was
performed for the chimeric genes RUNX1-RUNX1T1 and CBFB-
MYH11. The fusion transcripts were identified by real-time
quantitative PCR (RT-qPCR) method using bone marrow
or peripheral blood samples at diagnosis, as previously described
[15].
Statistical Analysis
CR was defined as <5% blast cells, no Auer rods, and no clusters
of blast cells by bone marrow analysis as well as no evidence
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Turk J Hematol 2022;39:84-93
of extramedullary leukemia. All statistical analyses of the data
were carried out using IBM SPSS Statistics 20.0 (IBM Corp.,
Armonk, NY, USA). The chi-square and Fisher exact tests were
used for comparisons of categorical variables between different
cohorts. The Student t-test was used to analyze continuous
variables with normal distribution and the Mann-Whitney U test
was used for data that did not comply with normal
distribution. Values of p<0.05 were considered statistically
significant.
Results
Patient Characteristics
The 134 CBF-AML patients enrolled in this study
included 65 women and 69 men with a median age of 35.5
years (range: 16.0-73.0 years). The median white blood cell
(WBC) count was 15.3x10 9 /L (range: 0.9 to 156.0x10 9 /L),
median hemoglobin (Hb) level was 81 g/L (range: 39.0
to 124.0 g/L), and median platelet count was 28.0x10 9/ L
(range: 2.0 to 170.0x10 9 /L). Patients with inv(16) or
t(16;16) AML tended to be older than t(8;21) AML patients
(41 vs. 32 years, p=0.048) and also had higher WBC counts
(35.9x10 9 /L vs. 7.8x10 9 /L, p=0.0001). No significant differences
were identified regarding gender, Hb level, or platelet
count between patients with t(8;21) and inv(16)/t(16;16) AML.
By conventional chromosome analysis, 41.0% (55/134) of the
CBF-AML patients were found to have secondary cytogenetic
abnormalities. Additional chromosomal alterations were found
in 45.0% (36/80) and 35.2% (19/54) of the patients with
t(8;21) and inv(16)/t(16;16), respectively. Loss of the X or Y
chromosome was identified as the most common secondary
alteration (33/134, 24.6%), followed by trisomy 22 (13/134,
9.7%). In patients with inv(16)/t(16;16), the most frequently
identified additional cytogenetic alterations were trisomy 22
and trisomy 8, which were found in 20.1% (13/54) and 9.3%
(5/54) of these patients, respectively.
Table 1. The 112 genes analyzed in this study.
Number Gene Number Gene Number Gene Number Gene
1 ABL1 29 DNM2 57 KMT2A 85 SETBP1
2 ACD 30 DNMT3A 58 KRAS 86 SETD2
3 ANKRD26 31 DNMT3B 59 MAPK1 87 SF1
4 ARIDIA 32 ECT2L 60 KMT2D 88 SF3A1
5 ASXL1 33 EED 61 MPL 89 SF3B1
6 ATG2B 34 EP300 62 MYC 90 SH2B3
7 ATM 35 ETNK1 63 MYD88 91 SMC1A
8 B2M 36 EZH2 64 NF1 92 SMC3
9 BCOR 37 FAM46C 65 NOTCH1 93 SRP72
10 BCOR1 38 BRINP3 66 NOTCH2 94 SRSF2
11 BIRC3 39 FAT1 67 NPM1 95 STAG2
12 BRAF 40 FBXW7 68 NRAS 96 STAT3
13 CALR 41 FGFR3 69 PAX5 97 SUZ12
14 CBL 42 FLT3 70 PDS5B 98 ETV6
15 CCND1 43 GATA1 71 PHF6 99 TERC
16 CCND3 44 GATA2 72 PIGA 100 TERT
17 CCR4 45 GATA3 73 PLCG1 101 TET2
18 CD79B 46 JAK1 74 PRKCB 102 TNFAIP3
19 CDC25C 47 JAK2 75 PRPF40B 103 TP53
20 CDKN2A 48 JAK3 76 PRPS1 104 TPMT
21 CEBPA 49 HNRNPK 77 PTEN 105 TRAF3
22 CREBBP 50 ID3 78 PTPN11 106 U2AF1
23 CSF3R 51 IDH1 79 RAD21 107 U2AF2
24 CUX1 52 IDH2 80 RB1 108 WHSC1
25 CXCR4 53 IKZF1 81 RBBP6 109 WT1
26 DDX3X 54 IL7R 82 RELN 110 XPO1
27 DDX41 55 KDM6A 83 RHOA 111 ZRSR2
28 DIS3 56 KIT 84 RUNX1 112 ZMYM3
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Qin W. et al: Mutation Profile in CBF-AML
Trisomies 8 and 22 were more frequently observed in
inv(16)/t(16;16) AML patients (p=0.006 and 0.0001, respectively),
while loss of the X or Y chromosome was more common in
patients with t(8;21) AML (p=0.0001). Del(9q) was not noted in
any inv(16) patients, while trisomies 8 and 22 were not found in
any t(8;21)patients. In all cases for which FISH and/or RT-qPCR
testing was performed, the results were in agreement with the
results of chromosome analysis (data not shown). Both CD19
and CD56 antigen expressions were observed more frequently in
the t(8;21) AML group (both p=0.0001). Clinical and biological
characteristics of the patients are provided in Table 2.
Comparison of Clinical Features and Incidence of
Genetic Mutations Between AML Patients with t(8;21)
and inv(16)/t(16;16)
Among the participating 134 CBF-AML patients, 68 mutated
genes were detected by screening the 112-gene panel. Thirtytwo
of those 68 genes could be classified as transcription
factor, DNA methylation, signaling, spliceosome, cohesin,
or tumor suppressor genes. An average of 3.19 (range: 1-10)
mutations per individual were detected among these CBF-
AML cases. While 22 patients had 1 alteration, 20 had 2, 32 had
3, and 60 had 4 or more. Among all genes sequenced, the most
commonly mutated genes were c-KIT (45/134, 33.6%) and NRAS
(45/134, 33.6%), followed by FLT3 (25/134, 18.7%), KRAS
(18/134, 13.4%), RELN (11/134, 8.2%), NOTCH1 (11/134,
8.2%), TET2 (10/134, 7.5%), and WT1 (10/134, 7.5%). The other
genes had mutation prevalences of <5%. The most frequently
affected functional pathway was the signaling pathway, with
such mutations observed in as many as 86.6% of cases. In
addition, our findings suggest that the frequencies of
mutations in genes associated with epigenetic modification,
such as IDH1, IDH2, DNMT3A, and TET2, are low in
CBF-AML, being identified in 1.5%, 0.7%, 2.2%, and 7.5% of the
total number of participating patients, respectively.
Concomitant gene abnormalities were found in 100% of
the patients with t(8;21) and 100% of the patients with
inv(16)/t(16;16). The patients with inv(16)/t(16;16) AML were
found to have more mutations in the NRAS and KRAS genes
(53.7% vs. 20.0%, p=0.001 and 27.8% vs. 3.8%, p=0.0001,
respectively) compared to t(8;21) AML patients. The distributions
of c-KIT, FLT3, RELN, TET2, FAT1, and NOTCH1 mutations
within these two groups were similar (Table 3). Functionally
mutated genes involved in signaling pathways were
observed more frequently in the inv(16)/t(16;16) AML group
Table 2. Clinical and biological characteristics of the CBF-AML patients at diagnosis.
Variable
Gender
Total
(n=134)
RUNX1-RUNX1T1
(n=80)
CBFB-MYH11
(n=54)
Male, n (%) 69 (51.5%) 42 (52.5%) 27 (50%)
Female, n (%) 65 (48.5%) 38 (47.5%) 27 (50%)
Age (years)
Median (range) 35.5 (16-73) 32 (16-73) 41 (16-63) 0.048
WBC count (x10 9 /L)
Median (range) 15.3 (0.9-156.0) 7.8 (0.9-123) 35.9 (1.6-156) 0.0001
Hb (g/L)
Median (range) 81 (39.0-124.0) 78 (39-120) 86.5 (40-124) 0.169
PLT count (x10 9 /L)
Median (range) 28.0 (2.0-170.0) 32 (2-170) 26 (3-121) 0.725
Secondary cytogenetic abnormalities
Loss of X or Y chromosome, n (%) 33 (24.6%) 32 (40.0%) 1 (1.9%) 0.0001
del(9q), n (%) 4 (3.0%) 4 (5.0%) 0 0.095
Trisomy 8, n (%) 5 (3.7%) 0 5 (9.3%) 0.006
Trisomy 22, n (%) 13 (9.7%) 0 13 (20.1%) 0.0001
Immunophenotyping
CD19 expression, n (n/N, %) 59 (59/116, 50.86%) 58 (58/73, 79.5%) 1 (1/43, 2.33%) 0.0001
NA, n 18 7 11
CD56 expression, n (n/N, %) 51 (51/92, 55.43%) 50 (50/58, 86.21%) 1 (1/34, 2.94%) 0.0001
NA, n 42 22 20
CBF: Core-binding factor; AML: acute myeloid leukemia; WBC: white blood cell; Hb: hemoglobin; PLT: platelet; NA: not available.
p
0.776
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Qin W. et al: Mutation Profile in CBF-AML
Turk J Hematol 2022;39:84-93
(p=0.016), while cohesin mutations were found more frequently
in the t(8;21) AML group (11.3% vs. 0%, p=0.011). All cohesin
mutations were mutually exclusive among each other
(Figure 1). The mutation distribution is provided in detail
in Table 3.
Relationships Between Clinical Characteristics, CR
Rate, and Mutations
We analyzed the clinical characteristics of patients with
mutations in c-KIT, NRAS, KRAS, CSF3R, TET2, and FLT3-ITD. As
listed in Table 4, significantly higher WBC counts were found
in inv(16)/t(16;16) AML patients with c-KIT (c-KIT mut ) or NRAS
(NRAS mut ) mutations than in t(8;21) AML patients with
c-KIT mut and NRAS mut (p=0.001 and 0.009, respectively).
Patients with both inv(16)/t(16;16) AML/TET2 mut and
inv(16)/t(16;16) AML/FLT3-ITD mut also had higher WBC
counts than those with t(8;21) AML/TET2 mut and t(8;21)
AML/FLT3-ITD mut , but these differences did not reach statistical
significance (p=0.088 and 0.067, respectively). No difference
was found between other factors such as age, gender, Hb
level, or platelet count.
This study also aimed to assess the impact of common gene
mutations on the rate of CR after initial induction
therapy. Among the 134 participating patients, relevant data
were available for 128. The overall CR rate among these cases
was 94.53% (121/128). No differences in CR rate were observed
according to mutated genes (KIT, NRAS, KRAS, TET2, FLT3-ITD)
between t(8;21) AML and inv(16)/t(16;16) AML patients. Clinical
characteristics and CR rates of CBF-AML patients with common
mutations are shown in Table 4.
Discussion
In this study, patients with inv(16)/t(16;16) AML had higher WBC
counts than those with t(8;21) AML. Trisomies 8 and 22 were
more frequently observed in inv(16) patients, while loss of the
X or Y chromosome was more common in t(8;21) AML. Patients
with t(8;21) AML also expressed CD19 and CD56 more frequently
than those with inv(16) AML. These findings are consistent with
the conclusions of previous reports [11,16].
Figure 1. Comparisons of genetic mutations between AML patients with t(8;21) and inv(16)/t(16;16).
AML: Acute myleoid leukemia
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Qin W. et al: Mutation Profile in CBF-AML
Table 3. Concomitant gene abnormalities of CBF-AML at diagnosis.
Total
Mutational genes
(n=134)
RUNX1-RUNX1T1
(n=80)
CBFB-MYH11
(n=54)
NPM1 3 (2.2%) 2 (2.5%) 1 (1.9%) 0.804
Signaling pathways, n (%) 116 (86.6%) 64 (80.0%) 52 (96.3%) 0.016
c-KIT, n (%) 45 (33.6%) 28 (35.0%) 17 (31.5%) 0.672
NRAS, n (%) 45 (33.6%) 16 (20.0%) 29 (53.7%) 0.0001
KRAS, n (%) 18 (13.4%) 3 (3.8%) 15 (27.8%) 0.0001
FLT3, n (%) 25 (18.7%) 15 (18.8%) 10 (18.5%) 1
CSF3R, n (%) 8 (6.0%) 7 (8.8%) 1 (1.9%) 0.143
RELN, n (%) 11 (8.2%) 8 (10.0%) 3 (5.6%) 0.524
NOTCH1, n (%) 11 (8.2%) 7 (8.8%) 4 (7.4%) 1
NOTCH2, n (%) 8 (6.0%) 4 (5.0%) 4 (7.4%) 0.714
JAK2, n (%) 8 (6.0%) 6 (7.5%) 2 (3.7%) 0.363
SH2B3, n (%) 6 (4.5%) 6 (7.5%) 0 0.081
PTPN11, n (%) 3 (2.2%) 1 (1.3%) 2 (3.7%) 0.565
Epigenetic regulators, n (%) 15 (11.2%) 10 (12.5%) 5 (9.3%) 0.781
TET2, n (%) 10 (7.5%) 6 (7.5%) 4 (7.4%) 1
IDH1, n (%) 2 (1.5%) 1 (1.3%) 1 (1.9%) 1
IDH2, n (%) 1 (0.7%) 1 (1.3%) 0 1
DNMT3A, n (%) 3 (2.2%) 3 (3.8%) 0 0.273
Transcription factors, n (%) 17 (12.7%) 8 (10.0%) 9 (16.7%) 0.296
ETV6, n (%) 1 (0.7%) 1 (1.3%) 0 1
RUNX1, n (%) 2 (1.5%) 1 (1.3%) 1 (1.9%) 1
GATA2, n (%) 1 (0.7%) 1 (1.3%) 0 1
SETBP1, n (%) 8 (6.0%) 3 (3.8%) 5 (9.3%) 0.267
CEBPA dm , n (%) 7 (5.2%) 3 (3.8%) 4 (7.4%) 0.439
Spliceosomes, n (%) 4 (3.0%) 4 (5.0%) 0 0.148
SRSF2, n (%) 1 (0.7%) 1 (1.3%) 0 1
SF3B1, n (%) 3 (2.2%) 3 (3.8%) 0 0.273
Tumor suppressors, n (%) 14 (10.45%) 9 (11.25%) 5 (9.26%) 0.712
TP53, n (%) 4 (3.0%) 4 (5.0%) 0 0.148
WT1, n (%) 10 (7.5%) 5 (6.3%) 5 (9.3%) 0.516
Cohesin, n (%) 9 (6.7%) 9 (11.3%) 0 0.011
RAD21, n (%) 4 (3.0%) 4 (5.0%) 0 0.148
SMC1A, n (%) 3 (2.2%) 3 (3.8%) 0 0.273
SMC3, n (%) 2 (1.5%) 2 (2.5%) 0 0.515
Chromatin modifiers, n (%) 15 (11.2%) 11 (13.8%) 4 (7.4%) 0.403
ASXL1, n (%) 8 (6.0%) 6 (7.5%) 3 (5.6%) 0.659
KDM6A, n (%) 2 (1.5%) 2 (2.5%) 0 0.515
BCOR, n (%) 4 (3.0%) 3 (3.8%) 1 (1.9%) 0.648
BCORL1, n (%) 1 (0.7%) 1 (1.3%) 0 1
Number of mutated genes
1 22 (16.4%) 14 (17.5%) 8 (14.8%) 0.681
2 20 (14.9%) 10 (12.5%) 10 (18.5%) 0.338
3 32 (23.9%) 18 (22.5%) 14 (25.9%) 0.648
≥4 60 (44.8%) 38 (47.5%) 22 (40.7%) 0.440
Average number (range) 3.19 (1-10) 3.17 (1-8) 3.22 (1-10) 0.873
CBF: Core-binding factor; AML: acute myeloid leukemia.
p
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Turk J Hematol 2022;39:84-93
Table 4. Clinical characteristics of the common co-mutations in patients with RUNX1-RUNX1T1 or CBFB-MYH11 fusions.
Variables
Age, years,
median (range)
Male/female,
n/n
WBC count,
x10 9 /L, median
(range)
Hb, g/L,
median
(range)
PLT count,
x10 9 /L, median
(range)
CR rate, n/N
(%)
c-KIT mutations (n=45)
RUNX1-RUNX1T1 (n=28) 31.5 (16-56) 13/15 10.1 (2.0-49.9) 78 (38-19) 32.5 (11-170) 50.0% (13/26) *
CBFB-MYH11 (n=17) 32 (16-57) 10/7 29 (5.7-137) 93 (63-124) 40 (15-117) 58.8% (10/17)
p 0.566 0.542 0.001 0.081 0.419 0.571
NRAS mutations (n=45)
RUNX1-RUNX1T1 (n=16) 30.5 (16-69) 9/7 18.6 (2.4-123) 80 (57-119) 28 (7-105) 87.5% (14/16)
CBFB-MYH11 (n=29) 35 (18-63) 14/15 43 (5.0-156) 87 (40-122) 25 (3-117) 92.8% (26/28) *
p 0.618 0.758 0.009 0.506 0.585 0.552
KRAS mutations (n=18)
RUNX1-RUNX1T1 (n=3) 41 (16-51) 3/0 30.9 (3.2-49.9) 79 (64-110) 11 (7-58) 100% (3/3)
CBFB-MYH11 (n=15) 36 (19-58) 9/6 32 (1.56-144) 82 (59-110) 40 (15-121) 80% (12/15)
p 0.824 0.515 0.783 0.824 0.138 1.000
CSF3R mutations (n=8)
RUNX1-RUNX1T1 (n=7) 21 (16-32) 2/5 5.1 (0.9-52.4) 63 (59-119) 28 (8-105) 57.1% (4/7)
CBFB-MYH11 (n=1) 56 (56-56) 1/0 34.9 98 (98-98) 14 (14-14) 100% (1/1)
p 0.127 0.375 0.275 0.275 0.275 1.000
TET2 mutations (n=10)
RUNX1-RUNX1T1 (n=6) 36 (29-49) 2/4 4.2 (0.9-74) 89.5 (45-107) 30.5 (2-80) 66.7% (4/6)
CBFB-MYH11 (n=4) 39.5 (26-52) 1/3 28.6 (18.3-65.4) 77.5 (59-98) 22 (22-44) 100% (4/4)
p 1.00 1 0.088 0.593 0.892 0.467
FLT3-ITD mutations (n=10)
RUNX1-RUNX1T1 (n=7) 43 (24-64) 2/5 23.9 (5.6-40) 75 (57-108) 23 (6-45) 42.9% (3/7)
CBFB-MYH11 (n=3) 40 (36-42) 3/0 54 (33-137) 82 (77-85) 40 (20-57) 66.7% (2/3)
p 0.732 0.167 0.067 0.21 0.138 1.000
WBC: White blood cell; HB: hemoglobin; PLT: platelet; CR: complete remission. *: Some patients did not receive any chemotherapy or were only treated with low-dose cytosine
arabinoside.
Both t(8;21) and inv(16)/t(16;16) disrupt the normal functioning
of the heterodimeric transcription factor CBF complex in AML
with relatively similar clinical outcomes. However, the molecular
genetic abnormalities potentially explaining the differences
between these two AML subtypes have not yet been explored in
detail. We performed extensive mutational analysis by NGS for
134 patients with CBF-AML who ranged in age from 16 to 73
years. As expected, additional aberrations were found in 100%
of these CBF-AML cases and the most commonly mutated gene
was c-KIT, as seen in 35.0% of AML cases with t(8;21) and
31.5% of AML cases with inv(16)/t(16;16). This is in accordance
with the previous research conducted by Duployez et al.
[11]. Interestingly, a significantly different spectrum of gene
mutations was demonstrated in AML between patients with
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Turk J Hematol 2022;39:84-93
Qin W. et al: Mutation Profile in CBF-AML
t(8;21) and inv(16)/t(16;16). We noticed that fewer signaling
pathways were involved in cases of t(8;21) in comparison
to inv(16)/t(16;16) (80.0% vs. 96.3%, p=0.016), while patients
with inv(16)/t(16;16) AML exhibited more mutations
in KRAS and NRAS compared to t(8;21) AML patients.
RAS genes encode a family of membrane-associated proteins that
regulate signal transduction upon the ligand binding to a variety
of membrane receptors, and they play important roles in physical
processes including proliferation, differentiation, and apoptosis
[17,18]. Activating point mutations of RAS genes have generally
been accepted as oncogenic events in the tumorigenesis of
numerous malignancies, including hematological malignancies
such as AML [11,19,20]. RAS mutations seem to be particularly
frequent in inv(16)/t(16;16) AML, with a reported incidence
of up to 54% [11]. Duployez et al. [11] and Boissel et
al. [21] reported that NRAS and KRAS mutations were more
common among AML patients with inv(16)/t(16;16) than those
with t(8;21). Further studies showed that RAS mutations had
no effect on overall/disease-free survival, CR, or relapse rates
[22,23,24]. In our cohort, both NRAS and KRAS mutations were
more frequently found among inv(16)/t(16;16)
AML patients than in the t(8;21) AML group, a finding previously
demonstrated among other cohorts [11,21]. These data suggest
that the synergic cooperation between inv(16)/t(16;16) and RAS
mutations may influence the pathophysiology of CBF-AML.
Cohesin is a multimeric protein complex that is involved
in the cohesion of sister chromatids, post-replicative DNA
repair, and transcriptional regulation and it is composed of 4
core subunits: the SMC1A, SMC3, RAD21, and STAG proteins
[25]. Cohesin mutations have been reported in about 6%
of AML patients [26] and fewer than 2% of cases of CBF-
AML [11]. Recent data revealed the identification of cohesin
and chromatin modifier mutations in t(8;21) but not inv(16)
patients [11,27]. In the present study, mutations in genes
encoding members of the cohesin complex were present in
11.3% of the t(8;21) AML patients and none of the inv(16) AML
patients, and all cohesin mutations were mutually exclusive
among each other, which is consistent with previous studies
[26,27]. It is interesting that cohesin gene mutations are more
frequent in patients with RUNX1-mutated AML [28]. Indeed,
Mazumdar et al. [29] demonstrated that cohesin mutations led
to a state of elevated chromatin accessibility and higher levels
of binding at RUNX1 binding sites. These findings suggest links
between alterations in cohesin and the RUNX1-RUNXT1 fusion
oncoprotein. In addition, our study also suggests that the
frequencies of mutations in genes associated with epigenetic
modification (IDH1, IDH2, DNMT3A, and TET2), are low in CBF-
AML, which is in accordance with the findings of Park et al.
[30] and Duployez et al. [11]. These results may support the
idea that mutations involved in epigenetic modification do not
contribute to leukemogenesis in CBF-AML.
Yang et al. [23] showed that AML patients with RAS mutations had
significantly higher WBC counts at diagnosis than those
without mutations (p=0.001). Boissel et al. [21] demonstrated
that CBF-AML patients with c-KIT mutations had a significantly
higher median WBC count at presentation, but this difference
was mainly observed among patients with inv(16), with
the mutations being less common in patients with t(8;21).
Additionally, no gene mutations predicted poor response to
induction in comparisons with patients without mutations for
particular genes (c-KIT, RAS), except for FLT3 [21]. Jahn et al.
[31] found that both DNMT3A and TET2 were associated with
a significantly worse prognosis in univariate analysis. However,
regarding the subgroup with c-KIT, NRAS, FLT3, CSF3R,
and TET2 mutations, WBC count and CR rate were not compared
between inv(16)/t(16;16) AML and t(8;21) AML patients in
previous studies. Our results showed that patients with
inv(16)/t(16;16) had significantly higher WBC counts than
those with t(8;21) in the context of c-KIT or NRAS mutations,
but no significant differences were found for CR rates.
Conclusion
This study has comprehensively analyzed the genetic mutations
of 134 CBF-AML patients to characterize certain crucial genetic
characteristics, compare the mutational profiles of t(8;21)
AML and inv(16)/t(16;16) AML, and establish unique genetic
maps. The major limitations of our study are the absence of
survival data, because these patients received treatment
in different medical institutions, and the fact that the
prognosis of these patients was affected by diverse factors
including physical status, financial situation, and the
different consolidation regimens that were administered.
The molecular mechanisms, exact characteristics, and clinical
implications of these mutations require further study.
Acknowledgments
This work was partly supported by grants from the Young
Scientists Foundation of Changzhou No. 2 People’s Hospital (No.
2019K002), the Science and Technology Project of the Changzhou
Health Committee (No. QN202035), the Science and Technology
Development Fund Project of Nanjing Medical University
(No. NMUB201), the Major Science Project of the Changzhou
Health Commission (No. ZD202018), the Project of Science and
Technology Support for Social Development of the Science and
Technology Bureau of Changzhou (No. CE20205027), and the
Changzhou Sci-Tech Program (No. CJ20210068).
Ethics
Ethics Committee Approval: The study was approved by the
local ethics committee and conducted in accordance with the
Declaration of Helsinki.
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Turk J Hematol 2022;39:84-93
Authorship Contributions
Surgical and Medical Practices: H.H.; Concept: N.J.; Design: W.Q.;
Data Collection or Processing: X.Ch.; Analysis or Interpretation:
H.J.S., , X.Ca.; Literature Search: Z.W.; Writing: N.J.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
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93

RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2021.2021.0203
Turk J Hematol 2022;39:94-102
Invasive Fungal Infections in Children with Leukemia: Clinical
Features and Prognosis
Lösemili Çocuklarda İnvazif Mantar Enfeksiyonları: Klinik Özellikler ve Prognoz
Melike Sezgin Evim 1 , Özlem Tüfekçi 2 , Birol Baytan 1 , Hale Ören 2 , Solmaz Çelebi 3 , Beyza Ener 4 , Kevser Üstün Elmas 5 ,
Şebnem Yılmaz 2 , Melek Erdem 2 , Mustafa Kemal Hacımustafaoğlu 3 , Adalet Meral Güneş 1
1Uludağ University Faculty of Medicine, Department of Pediatric Hematology, Bursa, Turkey
2Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey
3Uludağ University Faculty of Medicine, Department of Pediatric Infection Disease, Bursa, Turkey
4Uludağ University Faculty of Medicine, Department of Medical Microbiology, Bursa, Turkey
5Uludağ University Faculty of Medicine, Department of Pediatrics, Bursa, Turkey
Abstract
Objective: The incidence of invasive fungal infections (IFIs) has
increased due to intensive chemotherapy in childhood leukemia. The
aim of this study was to evaluate the incidence, risk factors, causative
pathogens, and impact on survival of IFIs among pediatric leukemia
patients.
Materials and Methods: The hospital records of 307 children with
acute lymphoblastic leukemia (ALL, n=238), acute myeloid leukemia
(AML, n=51), and relapsed leukemia (n=18) between January 2010 and
December 2015 were retrospectively evaluated.
Results: A total of 1213 febrile neutropenia episodes were recorded
and 127 (10.4%) of them were related to an IFI. Of 307 children, 121
(39.4%) developed IFIs. The mean age was significantly older in the IFI
group compared to children without IFIs (p<0.001). IFIs were defined
as possible, probable, and proven in 73.2%, 11.9%, and 14.9% of
the attacks, respectively. Invasive aspergillosis (81.9%) was the most
frequent infection, followed by invasive candidiasis (13.4%) and rare
fungal diseases (4.8%). The majority of IFI attacks in both ALL and
AML occurred during the induction phase. In total, the death rate was
24% and the IFI-related mortality rate was 18%. The mortality rate
among children with IFIs was found to be significantly higher than
that of children without IFIs (p<0.001). Overall and event-free survival
rates at 5 years were also found to be significantly lower in the IFI
group (p<0.001). Relapse (odds ratio: 8.49) was the most effective risk
factor for mortality, followed by developing an IFI episode (odds ratio:
3.2) and AML (odds ratio: 2.33) according to multivariate regression
analysis.
Conclusion: Our data showed that IFIs were more common in older
children. Although proven and probable IFI episodes were more
frequently diagnosed in cases of relapse and AML, children with ALL
and AML had similar frequencies of experiencing at least one episode
Öz
Amaç: Çocukluk çağı lösemisinde yoğun kemoterapi nedeniyle invaziv
mantar enfeksiyonlarının (IFI) insidansı artmıştır. Bu çalışmanın amacı,
pediatrik lösemi hastalarında IFI’nın insidansını, risk faktörlerini,
nedensel patojenleri ve sağkalım üzerindeki etkisini değerlendirmektir.
Gereç ve Yöntemler: Ocak 2010 ile Aralık 2015 tarihleri arasında akut
lenfoblastik lösemili (ALL, n=238), akut myeloid lösemili (AML, n=51)
ve relaps lösemili (n=18) 307 çocukların hastane kayıtları geriye dönük
olarak değerlendirildi.
Bulgular: Toplam 1213 febril nötropeni atağı kaydedildi ve bunların
127’si (%10,4) IFI ile ilgiliydi. Bu 307 çocuğun 121’i (%39,4) IFI
geliştirdi. Ortalama yaş, IFI grubunda IFI olmayan çocuklara kıyasla
anlamlı olarak daha büyük bulundu (p<0,001). IFI, atakları sırasıyla;
%73,2, %11,9 ve %14,9’unda olası, yüksek olası ve kanıtlanmış olarak
tanımlandı. İnvazif aspergilloz (%81,9) en sık görülen enfeksiyondu,
bunu invaziv kandidiyazis (%13,4) ve nadir mantar hastalıkları (%4,8)
izledi. Hem ALL hem de AML’deki IFI ataklarının çoğu indüksiyon
aşamasında görüldü. Toplamda ölüm oranı %24 ve IFI bağlantılı ölüm
oranı %18 olarak bulundu. IFI’lı çocuklarda ölüm oranı, IFI olmayan
çocuklarda anlamlı olarak daha yüksek bulundu (p<0,001). Beş yılda
genel ve olaysız sağkalım, IFI grubunda önemli ölçüde daha düşük
bulundu (p<0,001). Relaps (odds oranı, 8,49), mortalite üzerinde en
etkili risk faktörü oldu ve bunu, çok değişkenli regresyon analizi ile
bir IFI epizodu geçirmek (odds oranı, 3,2) ve AML olmak (odds oranı,
2,33) izledi.
Sonuç: Çalışmamız, IFI’nın büyük yaştaki çocuklarda daha yaygın
olduğunu gösterdi. Kanıtlanmış ve olası IFI epizodlarının nüks ve
AML’de daha sık saptanmasına karşın, ALL ve AML’li çocukların da
benzer sıklıkta en az bir IFI atağı geçirdiği görüldü. Nadir mantar
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Melike Sezgin Evim, M.D., Uludağ University Faculty of Medicine,
Department of Pediatric Hematology, Bursa, Turkey
E-mail : melikevim@yahoo.com ORCID: orcid.org/0000-0002-4792-269X
Received/Geliş tarihi: March 25, 2021
Accepted/Kabul tarihi: November 17, 2021
94

Turk J Hematol 2022;39:94-102
Sezgin Evim M. et al: Fungal Infections in Leukemic Children
Abstract
of IFI. Rare fungal diseases were also identified as a major problem.
Despite success in treatment, IFIs increased the rate of mortality in
children with acute leukemia.
Keywords: Fungal infections, Pediatric leukemia, Acute lymphoblastic
leukemia, Acute myeloid leukemia, Febrile neutropenia
Öz
hastalıkları önemli bir sorun olarak tanımlandı. Tedavideki başarıya
rağmen, IFI’nın akut lösemili çocuklarda ölüm oranını artırdığı
saptandı.
Anahtar Sözcükler: Mantar enfeksiyonları, Pediatrik lösemi, Akut
lenfoblastik lösemi, Akut myeloid lösemi, Febril nötropeni
Introduction
The overall survival (OS) rate of pediatric acute leukemia has
improved over the years, reaching 90% for acute lymphoblastic
leukemia (ALL) and 70% for acute myeloid leukemia (AML) [1,2].
However, invasive fungal infections (IFIs) are still a significant
cause of mortality and morbidity in children with hematologic
malignancies [3]. It is hard to estimate the true incidence of
IFIs, but it has been increasing in recent years due to advances
in diagnostic methods, the use of intensive chemotherapy,
prolonged neutropenia, and the increased use of central
catheters [3,4]. Although new antifungals are available, IFIrelated
mortality still remains high, reported to be between 20%
and 70% in various studies [4,5,6,7,8].
The aim of this study was to evaluate the incidence, risk factors,
causative pathogens, and impact of IFIs on survival among
pediatric acute leukemia patients treated with Berlin-Frankfurt-
Munich (BFM) protocols in two major pediatric hematology
centers of Turkey.
Materials and Methods
The medical records of acute leukemia patients diagnosed
between 2010 and 2015 in the pediatric hematology
departments of Uludağ and Dokuz Eylül University Hospitals
were retrospectively evaluated. The study included only children
with ALL and AML, excluding hematopoietic stem cell recipients.
De nova ALL and AML patients were treated with BFM-based
protocols (ALL IC-BFM 2009 and AML-BFM 2004 and 2012).
ALL-REZ BFM 2002 was used for relapsed ALL and cases of
relapsed AML were treated according to AML-REZ BFM 2001/01.
A total of 307 patients’ data were eligible for evaluation. Febrile
neutropenia attacks were analyzed for defining and classifying
IFIs according to the European Organization for the Research
and Treatment of Cancer/Mycoses Study Group (EORTC/MSG)
classification system [9]. Based on this classification, probable
IFI requires the presence of a host factor, a clinical criterion,
and a mycological criterion. Cases that meet the criteria for
a host factor and the clinical criterion without a mycological
criterion are considered as possible IFI. Proven IFI is defined as
fungus detected by either histological analysis or culturing of a
specimen of tissue taken from a site of disease.
The patients were hospitalized in single rooms without highefficiency
particulate air filtered systems. Data including age,
gender, leukemia type, risk groups, treatment phase, duration of
neutropenia, and steroid use prior to the diagnosis of IFI were
collected from patients’ files. Neutropenia was defined as an
absolute neutrophil count (ANC) below 500/mm 3 and severe
neutropenia was defined as an ANC below 100/mm 3 . Bacterial
and fungal surveillances were performed for peripheral blood,
central venous catheters, throat, urine, and stool. Tissue biopsy
from the affected site for mycological examination was
evaluated when available. Chest high-resolution computed
tomography (HRCT) and abdominal ultrasound were performed
within the first week of persistent fever in spite of proper
antimicrobial treatment. In the presence of a localized finding
or persistent, prolonged fever, sinonasal and cranial imaging
(magnetic resonance imaging or computed tomography scan)
were also performed. Serum galactomannan (GM) levels were
measured twice weekly during febrile episodes using the Platelia
Aspergillus Enzyme Immunoassay Test (Bio-Rad). Samples with
an index greater than 0.5 in two consecutive measurements
were considered positive. The use of antifungal drugs for either
prophylaxis or treatment was recorded. The survival rates and
the risk factors affecting mortality in children with IFIs and
without IFIs were compared. The study was approved by the
relevant ethics committee on October 4, 2016 (decision number:
2016-17/12).
Statistical Analysis
All statistical analyses were carried out using IBM SPSS Statistics
23.0 (IBM Corp.). Descriptive data were presented as median and
minimum-maximum. Variables with non-normal distribution
were analyzed using the Mann-Whitney U and chi-square tests.
Risk factors for mortality were assessed using univariate and
multivariate logistic regression analysis. Values of p<0.05 were
considered statistically significant. Analyses for event-free
survival (EFS) and OS rates were performed according to the
Kaplan-Meier method, and survival curves were compared with
the log-rank test.
Results
The data of a total of 307 pediatric patients with acute leukemia
were retrospectively screened. Of these, 289 cases were de novo
95

Sezgin Evim M. et al: Fungal Infections in Leukemic Children
Turk J Hematol 2022;39:94-102
acute leukemia (ALL=238, AML=51) and the rest (n=18) were
relapsed cases. In total, 1213 febrile neutropenia episodes were
recorded and 127 (10.4%) of them were related to IFIs according
to the EORTC/MSG criteria. IFI developed in 121 children
(39.4%). The median age of the whole cohort was 78 (1.7-215.7)
months and it was found to be significantly higher in the IFI
group (n=121) compared to the children without IFIs (n=186)
(96 months vs. 58 months; p<0.001). The male-to-female ratio
was 1.47 (n=183/124). No significant difference was determined
in terms of gender (p>0.05).
Classification of IFIs
The distributions of patients and episodes of IFI are shown in
Table 1. According to the EORTC/MSG criteria, the episodes
were classified as proven (14.9%; n=19/127), probable (11.9%;
n=15/127), or possible (73.2%; n=93/127). In total, 35.2%
(n=84/238) of ALL, 39% (n=20/51) of AML, and 94.4% (n=17/18)
of relapsed patients experienced at least one episode of IFI. The
incidences of proven and probable IFI episodes were evaluated
together and found to be significantly higher in the relapsed
leukemia group compared to the de novo ALL and AML patients
(p<0.001).
Neutropenia was present in 82% (n=104/127) and severe
neutropenia was demonstrated in 73% (n=76/104) of all IFI
episodes. The median duration of neutropenia was 20 (1-78)
days. The group with proven or probable IFI together had a
significantly longer median duration of neutropenia than the
possible IFI cases (30 days vs. 16 days; p=0.002). The frequency
of IFI attacks in the ALL group was higher during induction
therapy (n=55/87), followed by high-risk blocks (n=18/87) and
the consolidation phase (n=14/87). In AML cases, IFIs usually
occurred during induction (72.7%, n=16/22).
Mycologic agents isolated during the episodes are given in
Table 2. The majority of IFI episodes were related to invasive
aspergillosis (IA) (81.9%), followed by invasive candidiasis (IC)
(13.4%) and rare fungal infections (4.8%). The features of rare
fungal infections are given in Table 3.
Generally, children with ALL in the high-risk group received
fluconazole prophylaxis while AML and relapse patients had
posaconazole or itraconazole. Access to posaconazole is
restricted over 13 years of age in Turkey. For younger children,
it could only be administered with the special approval of the
Turkish Ministry of Health. In the IFI group, 40% of the episodes
occurred under antifungal prophylaxis; the rates of IFI attacks
in ALL, AML, and relapsed leukemia cases were 30% (n=26/87),
50% (n=11/22), and 77% (n=14/18), respectively. Children with
proven or probable episodes were under antifungal prophylaxis
at the time of 21% (n=4/19) and 80% (n=12/15) of the episodes,
respectively. The most commonly used antifungal agents for
prophylaxis were fluconazole (42.9%), itraconazole suspension
(33.3%), and posaconazole (17.2%). Secondary antifungal
prophylaxis was administered for 63% (n=80/127) of the IFI
episodes and the major preferred agent was voriconazole (46%).
For the treatment of IFI episodes, voriconazole (38%, n=49/127),
caspofungin (22.8%, n=29/127), or liposomal amphotericin B
(11%, n=14/127) was given as a single agent. Combination
therapy was administered in 27% (n=35/127) of the attacks
when the disease was not taken under control by a single
agent. Caspofungin and voriconazole (n=13/35) were the most
commonly preferred combination therapy.
Outcome
In the whole cohort, the 5-year OS and EFS rates were found to
be 83.6% and 79% for ALL, 68.7% and 60.8% for AML, and 22%
and 17.6% for relapsed leukemia cases. In total, the mortality
rate was 24% (n=72/307). It was found to be significantly
Table 1. Distribution of patients and episodes of invasive fungal infections according to diagnosis.
Total, n ALL, % (n) AML, % (n)
Relapsed
leukemia, % (n)
Total patients 307 77.6% (238/307) 16.6% (51/307) 5.8% (18/307)
Patients without IFI 60.6% (186/307) 82.7% (154/186) 16.6% (31/186) 0.7% (1/186)
Patients with IFI 39.4% (121/307) 69.4% (84/121) 16.5% (20/121) 14.1% (17/121)
Total IFI episodes 127 68.5% (87/127) 17.3% (22/127) 14.2% (18/127)
Possible 73.2% (93/127) 80.5% (70/87) 68.2% (15/22) 44.5% (8/18)
Probable 11.9% (15/127) 8% (7/87) 9.1% (2/22) 33.3% (6/18)
Proven 14.9% (19/127) 11.5% (10/87) 22.7% (5/22) 22.2% (4/18)
Proven + probable 28.8% (34/127) 19.5% (17/87) 31.8% (7/22) 55.5% (10/18)*
ALL: Acute lymphoblastic leukemia, AML: acute myeloid leukemia, IFI: invasive fungal infection, *: p<0.001.
96

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Sezgin Evim M. et al: Fungal Infections in Leukemic Children
higher in the IFI group (34%, n=41/121) compared to the non-
IFI group (16.6%, n=31/186) (p<0.001). IFI-related mortality was
observed in 18% (n=22/121) of cases (Figure 1). The mortality
rate in children with IFIs increased to 52.9% (n=18/34) when
possible IFI attacks were excluded. Five-year EFS and OS rates
were found to be significantly lower in the IFI group than in
cases without IFIs (EFS: 79.6% vs. 62.7%, OS: 83.9% vs. 67.5%)
(p<0.001) (Figure 2). Both EFS and OFS at 5 years in the IFI group
were found to be even lower when possible IFI attacks were
removed from the analysis (47.1%).
Table 2. Classification according to the diagnosis of invasive fungal infection.
Diagnosis of IFI
Episodes of IFI
(n=127)
Proven IFI,
14.9% (n=19)
Probable IFI,
11.9% (n=15)
Possible IFI,
73.2% (n=93)
Invasive aspergillosis 81.9% (n=104) 3 15 86
IPA 76.3% (n=97) - 15 82
SNIA 4.8% (n=6) 3* - 3
CNSIA 0.8% (n=1) - - 1
Invasive candidiasis 13.4% (n=17) 10** - 7
HSC 7.8% (n=10) 3 - 7
Candidemia 6.3% (n=7) 7 - -
Rare fungal inf. 4.8% (n=6)*** 6 - -
Mucorales (n=3) 3 - -
Fusarium (n=1) 1 - -
Dematiaceous (n=1) 1 - -
Alternaria (n=1) 1 - -
IFI: Invasive fungal infection, IPA: invasive pulmonary aspergillosis, SNIA: sinonasal invasive aspergillosis, CNSIA: central nervous system invasive aspergillosis, HSC: hepatosplenic
candidiasis.
*: Biopsy proven from sinonasal cavity: A. flavus (n=3).
**: Isolated from blood cultures: C. parapsilosis (n=6), C. albicans (n=3), C. guilliermondii (n=1).
***: Biopsy proven from sinonasal cavity (n=5), left maxillectomy (n=1).
Table 3. Features of rare fungal infections.
Rare fungal
infection
Diagnosis
Age,
months
Primary
prophylaxis
Biopsy region Treatment Status
Mucorales Relapsed AML 40 Posaconazole Left maxillectomy
Mucorales ALL-HRG 204 No Sinonasal cavity
Mucorales ALL-HRG 40 No Sinonasal cavity
Fusarium Relapsed ALL 196 Posaconazole Sinonasal cavity
Liposomal
amphotericin B
Liposomal
amphotericin B
Liposomal
amphotericin B
Liposomal
amphotericin B
Death due to
resistant disease
Death due to IFI
Death due to IFI
Death due to IFI
Dematiaceous AML 52 No Sinonasal cavity Caspofungin Alive
Alternaria ALL 182 No Sinonasal cavity
ALL: Acute lymphoblastic leukemia, AML: acute myeloid leukemia, HRG: high-risk group, IFI: invasive fungal infection.
Liposomal
amphotericin B
Alive
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Turk J Hematol 2022;39:94-102
Factors Affecting Mortality
For 307 children, the risk factors affecting mortality were
evaluated by univariate regression analysis. This revealed
that high-risk ALL (n=30) and AML (n=51), relapse (n=18),
neutropenia longer than 10 days (n=67), development of an
IFI episode, and presence of proven or probable IFI significantly
increased the rate of mortality (p<0.001). Gender and age at
diagnosis did not have any effect (p>0.05).
Independent risk factors for mortality were determined by
multivariate regression analysis (Table 4). Relapse (odds ratio:
8.49) was the most effective risk factor for mortality, followed
by development of an IFI episode (odds ratio: 3.2) and AML
(odds ratio: 2.33).
Discussion
We investigated a large sample of IFI data in children with
acute leukemia, which provided a valuable assessment of IFI
Figure 1. The rate and causes of mortality.
Figure 2. a). Event-free survival, b) Overall survival.
IFI: Invasive fungal infection
Table 4. Factors affecting mortality according to multivariate regression analysis.
Β p Odds ratio 95% CI
Relapse 2.14 <0.001 8.49 3.98-18.1
IFI episode 1.18 0.001 3.2 1.58-6.75
Leukemia type (AML) 0.84 0.02 2.33 1.1-4.92
AML: Acute myeloid leukemia, IFI: invasive fungal infection, CI: confidence interval.
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Turk J Hematol 2022;39:94-102
Sezgin Evim M. et al: Fungal Infections in Leukemic Children
epidemiology and outcomes in our country. In the current
study, the incidence of IFI episodes was found to be 10.4%.
The incidence in cases of hematologic malignancies ranged
between 1.7% and 35.4% in various studies [4,9,10,11,12].
However, it may increase further in autopsy findings [13]. The
wide range among studies could be explained by differences
in study populations, hospital conditions, usage of prophylactic
antifungal agents, and the criteria used in defining IFI. The
current study included only children with acute leukemia, and
IFI attacks were defined according to EORTC/MSG criteria. The
majority of attacks were in the group with possible IFIs (73.2%),
since the use of invasive techniques such as bronchoalveolar
lavage (BAL) was limited. The classification was mainly based
on host factors, clinical criteria, imaging techniques, and serum
GM levels. However, some of the cases within the possible group
might be confirmed as Mucorales or other fungal diseases if
histopathologic diagnosis methods are available, similarly to a
case reported in the literature [14]. Among the present study
group, proven and probable attacks were determined in 14.9%
and 11.9% of the cases, respectively. Proven and probable IFI
incidences in recent studies with BFM groups were reported
to be lower than 10% [15,16]. Unlike our study, those two
studies included not only hematologic malignancies but
also hematopoietic stem cell transplantation recipients and
antifungal prophylaxis was administered to the majority of
them.
The mean age in the IFI group was significantly higher compared
to children in the non-IFI group. The incidence of IFIs is reported
to increase with age [4,7,17]. This finding could be attributed
to the fact that colonization is less frequent in the younger
population due to less contact with fungal spores and the lower
frequency of unfavorable genetic mutations [1,18].
We found that children with ALL and AML had a similar
frequency of experiencing at least one episode of IFI. This finding
is contrary to the previous data in the literature suggesting that
the frequency of IFI episodes in AML is higher than that in ALL
[4,19]. The prevalence of IFI in ALL is reported to be between
4% and 35% depending on the era, chemotherapy protocol, risk
categories, and antifungal prophylaxis [6,7,12,17,20,21,22]. In
the current study, only 30% of children with ALL were receiving
antifungal prophylaxis and the majority of them developed IFI
during induction therapy or high-risk blocks, which are typically
associated with severe neutropenia and high-dose steroids.
Prolonged and severe neutropenia was defined as a determinant
for IFI [3,4]. In our study, proven and probable IFI attacks also
had a significantly longer mean duration of neutropenia than
possible IFI attacks (p=0.002). Similarly, reports from different
countries showed that the majority of IFI attacks in both ALL
and AML cases occurred during the induction phase and with
more intensive chemotherapy protocols [12,20,23,24,25].
In developing countries, ALL patients receiving intensive
chemotherapy phases may benefit from antifungal prophylaxis
for decreasing IFI episodes in these patients.
The most common cause of IFI episodes in our study was IA
(81.9%), and the majority were cases of invasive pulmonary
aspergillosis (IPA) (76.3%). Aspergillus flavus was isolated
from the sinonasal cavity in 3 out of 8 biopsy materials. After
excluding possible IFI episodes, the proven and probable
incidence of IA was 17%. The incidences of IA and IPA have
been increasing in patients with pediatric leukemia since 2000
with the use of new diagnostic methods [4,26]. Crassard et al.
[5] showed that BAL results were suggestive of IPA in 92% of
children with pediatric leukemia. However, invasive procedures
like BAL and biopsy are generally avoided in children due to
thrombocytopenia and the risk of bleeding. Instead of biopsy,
the combination of chest HRCT and serum GM level is more
widely used for non-invasive diagnosis and it is also the
preferred approach in Turkey. However, GM levels may not be
elevated in localized IA, especially under antifungal prophylaxis
or empiric antifungal treatment [27,28].
The frequency of IC in total IFI episodes was found to be 13.4%.
However, it increased to 52.6% (n=10/19) among the proven IFI
episodes. In our series, the incidence of hepatosplenic candidiasis
(HSC) was determined as 7.8% and Candida spp. were isolated
from blood cultures in 30% of those cases. Celkan et al. [29],
in a multicentric study from Turkey, recently reported that 8
out of 40 (22.5%) children with HSC had Candida spp. and
only one case was C. albicans. In the current study, the most
commonly isolated pathogen was also non-albicans, namely C.
parapsilosis (Table 2). It appears that C. albicans, which used to
be the most commonly reported pathogen, has been replaced
by C. parapsilosis and other non-albicans species [6,12,19,30].
The current data showed a high rate of rare fungal infections,
which were detected in 6 of 127 IFI episodes (4.8%). However,
this incidence was increased to 31.5% (n=6/19) among proven
attacks. Epidemiological data for children regarding rare fungal
infections are limited [14,31,32,33,34]. Three out of 6 children
had mucormycosis and the others had fusarium, dematiaceous,
and alternaria involvements. The largest registry for invasive
mucormycosis in children showed that its frequency was
higher in cases of hematologic malignancies compared to
other malignancies and a variety of other disorders [31,32,33].
A retrospective study from Italy also reported that underlying
hematologic malignancies, particularly acute leukemia and
lymphoma, are risk factors for developing mucormycosis [34].
Two patients with mucormycosis and one with fusarium died
despite effective antifungal treatment combined with surgical
resection. The mortality rates for both mucormycosis and
fusarium are reported to be high [31,33]. Limited data related
to dematiaceous and alternaria cases in children with leukemia
are available in the literature [35,36]. In our study, these cases
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Turk J Hematol 2022;39:94-102
were successfully cured. The most important prognostic factor
is early clinical suspicion, timely aggressive systemic antifungal
treatment, and surgical procedures.
In the present study, the mortality rate for the whole cohort
was 24% and the rate of deaths attributable to IFI was found to
be 18%. IFI-related mortality in cases of hematologic disorders
ranged from 5% to 14% in Turkey [10,12,37]. It is also reported
to be as high as 21% to 48% in children with leukemia [7,21,38].
The current study also separately analyzed the mortality rates
for IA, IC, and rare fungal infections, which were found to be
16%, 17%, and 50%, respectively. The high mortality rate in the
rare fungal infection subgroup still remains an important issue
[19,31]. Although mortality rates due to IFIs were reported high
in the 1990s, they significantly decreased with the development
of early diagnostic tools and new antifungal agents [3,5,26,39].
Our data showed that OS and EFS were significantly lower in
children with IFIs than those without IFIs. Multivariate regression
analyses revealed that independent risk factors affecting
mortality were recurrent disease, development of an IFI episode,
and AML, which increased the mortality rate 8.4, 3.2, and 2.3
times, respectively. Data on the impact of IFIs on survival rates
in children with hematologic malignancies are rare. There are
three major studies reported regarding this issue. Two of them
did not find any differences in survival rates [40,41]. However,
those studies included small numbers of children. Similar to
our findings, Kobayashi et al. [7,42] found significantly lower
survival in patients with IFIs compared to those without IFI in a
patient population including hematologic malignancies, other
malignant diseases, and aplastic anemia.
Study Limitations
The data were retrospectively collected. The classification
was mainly based on host factors, clinical criteria, imaging
techniques, and GM levels. BAL was not administered for any of
the patients. Therefore, most of the attacks were in the “possible”
group. However, it may have been seen that these cases were
caused by other fungal pathogens if invasive techniques were
available.
Conclusion
Proven and probable IFI episodes concurrently occurred in ALL,
AML, and relapsed cases at rates of 19.5%, 31.8%, and 55.5%,
respectively. Although the majority of attacks were related to
IA, rare fungal infections were also isolated in almost 5% of
patients. Prolonged severe neutropenia was one of the major
risk factors for IFIs. Multivariate regression analysis showed
that IFIs significantly increased mortality, and the survival rates
were significantly lower in patients with IFIs. Our study clearly
shows that IFIs are poor prognostic factors in children with
hematologic malignancies.
Ethics
Ethics Committee Approval: The study was approved by the
relevant ethics committee on October 4, 2016 (decision number
2016-17/12).
Informed Consent: Obtained.
Authorship Contributions
Concept: M.S.E., H.Ö., A.M.G., B.B., Ş.Y.; Data collection or
processing: Ö.T., B.B., K.Ü.E., B.E., S.Ç., Ş.Y., M.E., M.K.H.; Analysis
or interpretation: M.S.E., Ö.T., H.Ö., A.M.G., B.E., K.Ü.E., S.Ç.,
M.K.H.; Literature review: M.S.E., H.Ö., K.Ü.E., A.M.G.; Writing:
M.S.E., H.Ö., A.M.G.
Conflicts of Interest: No conflicts of interest were declared by
the authors.
Financial Disclosure: The authors declared that this study
received no financial support.
Acknowledgments: The authors declared that they had no
financial, consulting, or personal relationships with other
people or organizations. This study was presented orally at the
44 th National Hematology Congress.
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102

RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2022.2021.0675
Turk J Hematol 2022;39:103-108
Eltrombopag for Treatment of Thrombocytopenia Following
Hematopoietic Stem Cell Transplantation
Hematopoetik Kök Hücre Nakli Sonrası Trombositopeni Tedavisinde Eltrombopag Kullanımı
Zeynep Tuğba Güven 1 , Serhat Çelik 1 , Bülent Eser 2 , Mustafa Çetin 1 , Ali Ünal 1 , Leylagül Kaynar 1
1Erciyes University Faculty of Medicine, Department of Hematology, Kayseri, Turkey
2Medical Park Hospital, Antalya, Turkey
Abstract
Objective: This study aimed to evaluate the efficacy and safety of
eltrombopag (ELT) in the treatment of thrombocytopenia following
hematopoietic stem cell transplantation (HSCT).
Materials and Methods: Forty-eight patients treated with ELT for
thrombocytopenia after allogeneic or autologous transplantation at
the Erciyes University Bone Marrow Transplantation Center between
July 2017 and July 2021 were evaluated retrospectively.
Results: Forty-eight HSCT recipients were included in this study.
Thirty (62.5%) patients were evaluated as having experienced delayed
platelet recovery (DPR) and 18 (37.5%) patients as having experienced
secondary failure of platelet recovery (SFPR). The median platelet
count before ELT treatment was 13x10 9 /L (range: 3-20x10 9 /L). Twentythree
patients responded to treatment and the cumulative incidence
of successful platelet recovery was 48%. Patients with both DPR
and SFPR responded, but patients with DPR had a higher response
rate (50% vs. 44%). The median platelet count of the 23 responding
patients was 12x10 9 /L (5-19x10 9 /L) before treatment and 68x10 9 /L
(52-266x10 9 /L) after treatment (p<0.0001). While the number of bone
marrow megakaryocytes before treatment was adequate in 22 (46%)
cases, it was decreased in 26 (54%) cases. Patients with adequate bone
marrow megakaryocytes had a better response rate than those without
(77% vs. 23%, p<0.0001). The group with adequate megakaryocytes
responded to treatment at a median of 33 days (range: 9-174 days).
Patients with decreased megakaryocytes responded at a median of
55 days (30-164 days) (p=0.002). No drug-related side effects were
observed in any patients.
Conclusion: This real-life experience demonstrates that ELT is an
effective and safe treatment option for thrombocytopenia after HSCT.
The adequacy of bone marrow megakaryocytes before ELT treatment
was an important factor affecting response to treatment.
Keywords: Eltrombopag, Thrombocytopenia, Hematopoietic stem cell
transplantation, Platelet recovery
Öz
Amaç: Transplantasyon sonrası trombositopeni tedavisinde
eltrombopagın (ELT) etkinlik ve güvenliğini değerlendirmeyi amaçladık.
Gereç ve Yöntemler: Erciyes Üniversitesi Kemik İliği Nakil Merkezi’nde
Temmuz 2017-Temmuz 2021 tarihleri arasında allojenik veya otolog
transplantasyon sonrası trombositopeni nedeniyle ELT ile tedavi edilen
48 hasta retrospektif olarak değerlendirildi.
Bulgular: Bu çalışmaya 48 hematopoietik kök hücre nakli (HKHN)
alıcısı dahil edildi. Otuz (%62,5) hasta gecikmiş trombosit iyileşmesi
(DPR) ve 18 (%37,5) hasta ikincil trombosit iyileşme başarısızlığı (SFPR)
olarak değerlendirildi. ELT tedavisinden önce medyan trombosit sayısı
13x10 9 /L (aralık, 3-20x10 9 /L) idi. Yirmi üç hasta tedaviye yanıt verdi ve
başarılı trombosit iyileşmesinin kümülatif insidansı %48 idi. Hem DPR
hem de SFPR’li hastalar yanıt verdi, ancak DPR’li hastalarda yanıt oranı
daha yüksekti (%50’ye karşı %44). Yanıt veren 23 hastanın medyan
trombosit sayısı tedaviden önce ve sonra 12 (5-19)x10 9 /L ve 68 (52-
266)x10 9 /L idi (p<0,0001). Tedavi öncesi kemik iliği megakaryosit sayısı
22 (%46) hastada yeterli iken 26 (%54) hastada azalmıştı. Yeterli
kemik iliği megakaryositleri olan hastalar, azalmış olanlara göre daha
iyi bir yanıt oranına sahipti (%77’ye karşı %23, p<0,0001). Yeterli
megakaryositleri olan grup, tedaviye medyan 33 (aralık, 9-174) günde
yanıt verdi. Megakaryositleri azalmış hastalar, ortalama 55 (aralık, 30-
164) gün yanıt verdi (p=0,002). Hiçbir hastada ilaca bağlı yan etki
gözlenmedi.
Sonuç: Bu gerçek yaşam deneyimi, ELT’nin HKHN sonrası
trombositopeni için etkili ve güvenli bir tedavi seçeneği olduğunu
göstermektedir. ELT tedavisi öncesi kemik iliği megakaryositlerinin
yeterliliği tedaviye yanıtı etkileyen önemli bir faktördü.
Anahtar Sözcükler: Eltrombopag, Trombositopeni, Hematopoietik
kök hücre nakli, Trombosit iyileşmesi
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Zeynep Tuğba Güven, M.D., Erciyes University Faculty of Medicine,
Department of Hematology, Kayseri, Turkey
E-mail : drztkarabulutguven@gmail.com ORCID: orcid.org/0000-0003-1600-9731
Received/Geliş tarihi: December 8, 2021
Accepted/Kabul tarihi: March 11, 2022
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Güven Z.T. et al: Eltrombopag for Post-transplant Thrombocytopenia
Turk J Hematol 2022;39:103-108
Introduction
Hematopoietic stem cell transplantation (HSCT) is an effective
treatment method used in the treatment of many malignant
and non-malignant diseases today. After transplantation,
engraftment is expected within weeks and blood parameters are
anticipated to return to normal levels; however, some patients
may suffer from cytopenia. The most frequently observed
cytopenia following allogeneic and autologous transplantation
is thrombocytopenia and it can be seen in up to 40% of patients
after transplantation [1,2,3]. Many underlying causes including
infections, side effects of drugs used, disease recurrence,
alloantibodies, insufficient graft function, and microangiopathy
can be identified [3,4,5]. Persistent thrombocytopenia after
HSCT is an important cause of mortality and morbidity
[1,2,6]. Thrombocytopenia may be severe in some patients and
may continue for a long time. However, there is no standard
approach for the treatment of these patients. Although it is
only a temporary fix, transfusion support is used often to treat
bleeding in such cases.
Eltrombopag (ELT) and romiplostim are thrombopoietin receptor
agonists (rhTPO) that increase platelet production and are used
in the treatment of idiopathic (immune) thrombocytopenic
purpura in adults and children [7,8,9,10]. In the literature,
it has been reported that ELT provides convenience with oral
use, and it is also used in the treatment of aplastic anemia,
low-risk myelodysplastic syndrome, and chemotherapyassociated
thrombocytopenia [11,12].
There are various reports on the use of ELT in the treatment
of post-transplant thrombocytopenia [13,14,15,16]. In this
study, we aim to share our experiences with ELT treatment in
48 patients with delayed platelet recovery (DPR) or secondary
failure of platelet recovery (SFPR) following HSCT.
Materials and Methods
Patients
The medical records of 634 patients who underwent HSCT at the
Erciyes University Bone Marrow Transplantation Center between
July 2017 and July 2021 were reviewed retrospectively. We
identified 48 patients treated with ELT for thrombocytopenia
after allogeneic or autologous transplantation. Successful
neutrophil engraftment was achieved in all cases. Patients
were classified into two groups as having experienced DPR or
SFPR. Patients with primary graft failure were not included in
the study and all patients were in remission. Signed written
consent forms were obtained from all participating patients
and the study was approved by the Ethics Committee of Erciyes
University (2020/156).
Definitions
Neutrophil engraftment was defined as an absolute neutrophil
count of ≥0.5x10 9 /L for 2 consecutive days. Platelet engraftment
was defined as a platelet count of ≥20x10 9 /L without transfusion
for 3 consecutive days. DPR was defined as the absence of
platelet engraftment on the 35 th day after transplantation
despite the occurrence of neutrophil engraftment [17]. SFPR
was defined as a platelet count of ≤20x10 9 /L after platelet
engraftment for more than 7 days [2,18]. Patients were excluded
if they had secondary causes of thrombocytopenia such as
drug-induced thrombocytopenia or viral infection-associated
thrombocytopenia. Liver function test results were normal for
all patients.
Bone marrow aspiration and biopsy were performed to evaluate
bone marrow cellularity just before the ELT treatment was
started. Megakaryocyte count was evaluated by bone marrow
aspirate smear and a count of ≥8/mm 2 was considered adequate
[13]. Platelet recovery after ELT was defined as a platelet
count above 50x10 9 /L for at least 7 days without the need for
transfusion.
Eltrombopag Treatment
While the initial dose of ELT was typically 25 mg or 50 mg, the dose
was increased by 25 mg per week based on the response status
and the maximum dose reached was 150 mg. The drug doses
given to the patients were adjusted according to their weekly
blood counts. When the platelet count exceeded 100x10 9 /L, the
dose was reduced by 25 mg weekly. Platelet transfusions were
performed according to institutional guidelines. Side effects
were graded according to Version 4.0 of the National Cancer
Institute Common Toxicity Criteria.
The efficacy of the treatment was determined by platelet count.
The primary outcome was a platelet count of ≥50x10 9 /L for 7
consecutive days without transfusion. Secondary outcomes
were the effect of bone marrow megakaryocyte count before
ELT treatment on platelet recovery and treatment-related side
effects.
Statistical Analysis
Continuous data conforming to normal distribution were
expressed as mean ± standard deviation, while continuous data
not conforming to normal distribution were expressed as median
and minimum-maximum and categorical data were expressed
as percentages (%). Categorical data were compared using
chi-square tests. The cumulative incidence of platelet recovery
was analyzed using Cox regression. Days without transfusion
from initiation of ELT to a platelet count of >50x10 9 /L were
compared between groups using the Wald chi-square test. Data
were analyzed with IBM SPSS Statistics 22.0 for Windows (IBM
Corp., Armonk, NY, USA). Values of p<0.05 were considered
significant.
Results
The clinical characteristics of the patients are summarized in
Table 1. Forty-eight HSCT recipients were included in this study.
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Güven Z.T. et al: Eltrombopag for Post-transplant Thrombocytopenia
Thirty (62.5%) patients were evaluated as having experienced
DPR and 18 (37.5%) patients as having experienced SFPR. There
were 34 male patients, and the median age was 53 years (range:
21-69 years). Allogeneic stem cell transplantation was performed
for 28 (58%) patients and 18 of those patients were diagnosed
with acute myeloid leukemia. Myeloablative conditioning
regimens were used for all patients undergoing allogeneic
transplantation. Twenty-four patients of the allogeneic
transplantation group had a sibling HLA-matched donor, while
4 patients had a haploidentical donor. Peripheral blood was used
as a stem cell source in all cases. Cyclosporine and methotrexate
were used most frequently for graft-versus-host disease (GVHD)
prophylaxis, while post-transplant cyclophosphamide was
preferred in cases of haploidentical transplantations. Autologous
stem cell transplantation was performed for 20 (42%) patients
and 12 of those patients were diagnosed with non-Hodgkin
lymphoma. Neutrophil engraftment occurred in all patients
at a median of 17 days (range: 8-24 days). Grade II-IV GVHD
developed in 6 (12.5%) patients before ELT treatment. None of
the patients had a previous history of using rhTPO. Bone marrow
biopsy was performed for all patients before ELT treatment.
were 38% among patients with DPR and 50% among those
with SFPR for allogeneic transplantation, respectively. The
cumulative incidences of platelet recovery were 64% among
patients with DPR and 33% among those with SFPR for
autologous transplantation. Response rates were 55% and 43%
among patients who underwent autologous and allogeneic
transplantation, respectively. Patients with both DPR and
SFPR responded, but patients with DPR had a higher response
rate (50% vs. 44%). The median time to platelet recovery was
35 days (range: 9-174 days) and these patients had received
ELT for a median of 77 days (15-293 days). No recurrence of
thrombocytopenia was observed in any of these cases while ELT
was reduced. The median platelet count of the 23 responding
Outcomes of ELT treatment are listed in Table 2. ELT was started
at a median of 57 days after HSCT (range: 36-513 days). The
median platelet count before ELT treatment was 13x10 9 /L
(range: 3-20x10 9 /L). The starting dose of ELT was 50 mg per day
for most patients, with a maximum dose of 150 mg per day.
Twenty-three patients responded to treatment and the
cumulative incidence of successful platelet recovery was 48%
(Figure 1). The cumulative incidences of platelet recovery
Figure 1.Median platelet count before and after eltrombopag
treatment. The analysis included 23 responding patients
(p<0.0001).
Table 1. Clinical characteristics of the patients.
Characteristic Type of thrombocytopenia, n (%) All, n (%)
DPR (n=30) SFPR (n=18) (n=48)
Age, years, median (range) 54.5 (21-69) 50.5 (31-63) 53 (21-69)
Sex
Male
Female
Type of HSCT
Allo-HSCT
Auto-HSCT
Disease
AML
ALL
MM
HL
NHL
23 (77)
7 (23)
16 (53)
14 (47)
9 (30)
5 (17)
6 (20)
3 (10)
7 (23)
11 (61)
7 (39)
12 (67)
6 (33)
9 (50)
2 (11)
2 (11)
-
5 (28)
34 (71)
14 (29)
28 (58)
20 (42)
18 (37.5)
7 (14.5)
8 (17)
3 (6)
12 (25)
Neutrophil engraftment, day, median (range) 17 (10-24) 16 (8-23) 17 (8-24)
Grade II-IV GVHD 2 (7) 4 (22) 6 (12.5)
DPR: Delayed platelet recovery; SFPR: secondary failure of platelet recovery; HSCT: hematopoietic stem cell transplantation; Allo-HSCT: allogeneic HSCT; Auto-HSCT: autologous
HSCT; AML: acute myeloblastic leukemia; ALL: acute lymphoblastic leukemia; MM: multiple myeloma; HL: Hodgkin lymphoma; NHL: non-Hodgkin lymphoma; GVHD: graft-versushost
disease.
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Güven Z.T. et al: Eltrombopag for Post-transplant Thrombocytopenia
Turk J Hematol 2022;39:103-108
patients was 12x10 9 /L (range: 5-19x10 9 /L) before treatment and
68x10 9 /L (52-266x10 9 /L) after treatment (p<0.0001; Figure 2).
Among the 25 non-responders, ELT was discontinued in 11 cases
due to death. It was continued for a median of 77 days among
non-responder patients (range: 10-232 days).
While the number of bone marrow megakaryocytes before
treatment was adequate in 22 (46%) cases, it was decreased
in 26 (54%) cases. Patients with adequate bone marrow
megakaryocytes had a better response rate than those without
(77% vs. 23%). This finding was significant (p<0.0001). The
group with adequate megakaryocytes responded to treatment
Figure 2. Cumulative incidence of platelet recovery (48%).
at a median of 33 days (range: 9-174 days), while patients with
decreased megakaryocytes responded at a median of 55 days
(30-164 days) (p=0.002; Table 3).
The treatment response was permanent in all patients and no
platelet count below 50x10 9 /L was observed even after ELT was
discontinued. The median follow-up time after discontinuation
of ELT treatment was calculated as 365 days (range: 61-921 days).
No drug-related side effects were observed in any patients. The
patients were fully compliant with treatment and none of the
patients had to discontinue the drug due to side effects.
Discussion
In this study, we have presented our single-center experience
with 48 patients treated with ELT for primary and secondary
platelet failure following HSCT. The cumulative incidence of
platelet recovery after treatment with ELT was 48%. These
patients, who needed continuous platelet transfusion, became
transfusion-free in a median of 35 days. Furthermore, after
discontinuation of ELT, platelet counts were maintained
permanently in all responding patients.
The use of ELT is effective in thrombocytopenia that develops
after autologous and allogeneic transplantation [13,19,20]. In
our study, response to ELT was observed in both the autologous
transplant and allogenic transplant groups (55% vs. 43%). In
Table 2. Outcomes of eltrombopag treatment.
Characteristics
Outcomes
Duration from transplantation to eltrombopag treatment, median (range), days 57 (36-513)
Starting dose of eltrombopag
25 mg daily, n (%)
50 mg daily, n (%)
Maximum dose of eltrombopag
75 mg daily, n (%)
100 mg daily, n (%)
125 mg daily, n (%)
150 mg daily, n (%)
6 (12.5)
42 (87.5)
3 (6)
17 (36)
4 (8)
24 (50)
Table 3. Bone marrow megakaryocytes of patients.
Achievement of platelet response
Yes, n (%)
No, n (%)
23 (48)
25 (52)
Days from starting eltrombopag to platelet response, median (range), days 35 (9-174)
Duration of treatment among patients with platelet response, median (range), days 77 (15-293)
Response
Adequate megakaryocytes
(n=22)
Positive response, n (%) 17 (77) 6 (23)
No response, n (%) 5 (23) 20 (77)
Days from starting eltrombopag to response,
median (range), days
Decreased megakaryocytes
(n=26)
p
<0.0001
33 (9-174) 55 (30-164) 0.002
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Turk J Hematol 2022;39:103-108
Güven Z.T. et al: Eltrombopag for Post-transplant Thrombocytopenia
their study, Yuan et al. [21] used ELT therapy for 13 patients
with primary and secondary platelet failure. Response was
observed in 50% of the patients with primary failure and 71%
of those with secondary failure. In another study involving two
groups of patients who underwent autologous and allogeneic
transplantation, the response to ELT after transplantation was
100% and 61%, respectively. Hence, it was effective in cases
of both primary and secondary platelet failure [22]. In our
study, it was similarly observed that ELT was effective in both
primary and secondary platelet failure. There was more platelet
recovery in the group with primary failure (50% vs. 44%), but
that finding was not statistically significant.
Tanaka et al. [13] administered ELT for the treatment of
thrombocytopenia after allogeneic transplantation. Both
groups of patients with prolonged thrombocytopenia and
platelet recovery failure had positive results. Platelet recovery
was in line with the amount of bone marrow megakaryocytes
before treatment [13]. Similarly, patients with adequate
megakaryocyte counts prior to initiation of ELT therapy had
a higher platelet recovery rate than patients with decreased
megakaryocytes in the present study (77% vs. 23%, p<0.0001).
Based on these results, it can be thought that the number of
bone marrow megakaryocytes before treatment is a determining
factor in the response to rhTPO agonists. Patients with adequate
megakaryocytes responded to treatment in a shorter time than
patients with inadequate megakaryocytes (33 days vs. 55 days,
p=0.002). A normal megakaryocyte count allows the platelet
count to improve faster. In a study involving 13 patients,
ELT was used for thrombocytopenia following allogeneic
transplantation. The overall response rate was 62%. When
evaluated in terms of bone marrow reserve, the ELT response was
similar and sufficient in the group with lower megakaryocyte
counts [21]. It remains to be determined whether higher doses
of rhTPO agonists improve platelet recovery in patients with
reduced megakaryocyte counts. We used a maximum dose of
150 mg of ELT for our patients.
Study Limitations
Our study has several limitations; it was a retrospective study
and it included a small number of patients. In addition, the
patient group was heterogeneous. Although we investigated
other causes of thrombocytopenia for all patients, it was not
always possible to exclude them completely. However, we think
that our study nevertheless supports the safety and efficacy of
ELT in the treatment of thrombocytopenia following HSCT.
Conclusion
The results that we have presented here demonstrate that ELT
is an effective and safe treatment option for thrombocytopenia
following HSCT. However, prospective randomized studies are
needed to demonstrate the efficacy and optimal dose of ELT
in the treatment of post-transplant thrombocytopenia. Patients
with normal megakaryocyte counts responded better and
faster to ELT. Adequacy of bone marrow megakaryocytes before
ELT treatment was an important factor affecting treatment
response.
Ethics
Ethics Committee Approval: Signed written consent forms
were obtained from all participating patients and the study
was approved by the Ethics Committee of Erciyes University
(2020/156).
Informed Consent: Signed written consent forms were obtained.
Authorship Contributions
Surgical and Medical Practices: Z.T.G., S.Ç., B.E., M.Ç., A.Ü., L.K.;
Concept: Z.T.G., L.K., Design: Z.T.G., L.K.; Data Collection or
Processing: Z.T.G., S.Ç., B.E.; Analysis or Interpretation: Z.T.G.,
S.Ç., B.E., M.Ç.; Literature Search: Z.T.G., S.Ç., B.E., M.Ç., A.Ü., L.K.;
Writing: Z.T.G., S.Ç.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
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108

RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2021.2021.0325
Turk J Hematol 2022;39:109-116
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Patients with Multiple Myeloma
Multipl Myelomlu Hastalarda Kemik İliği Biyopsisi ve Sitogenetik Bulguların
Değerlendirilmesi
Ahmet Şeyhanlı 1 , Boran Yavuz 2 , Zehra Akşit 3 , Zeynep Yüce 4 , Sermin Özkal 5 , Oğuz Altungöz 4 , Fatih Demirkan 2 ,
İnci Alacacıoğlu 2 , Güner Hayri Özsan 2
1Sivas Numune Hospital, Clinic of Hematology, Sivas, Turkey
2Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey
3Dokuz Eylül University Faculty of Medicine, Department of Internal Medicine, İzmir, Turkey
4Dokuz Eylül University Faculty of Medicine, Department of Medical Biology, İzmir, Turkey
5Dokuz Eylül University Faculty of Medicine, Department of Pathology, İzmir, Turkey
Abstract
Objective: Multiple myeloma (MM) is a malignant condition
characterized by the accumulation of malignant plasma cells.
Although MM remains incurable, the survival of MM patients has
improved considerably due to the application of autologous stem cell
transplantation, novel agents, and advanced treatment strategies. This
study aimed to determine the cytogenetic characterization and bone
marrow (BM) features of Turkish patients with MM.
Materials and Methods: Eighty-five MM patients were admitted to
Dokuz Eylül University Hospital in Turkey. BM samples of these MM
patients were subjected to cytogenetic analyses at diagnosis and
during therapy as a part of therapeutical and clinical evaluation.
A complete cytogenetic study was performed using the G-banding
technique. Fluorescence in situ hybridization (FISH) analysis was
performed using cytoplasmic immunoglobulin. The degree of BM
fibrosis was determined using reticulin histochemical staining. We
determined the percentage of BM plasma cells based on the extent
of CD38 staining.
Results: Eighty-five MM patients were retrospectively identified
between 2015 and 2021. The median age was 63 (38-90) years. Of
the 85 patients, 60 (70.6%) were male and 25 (29.4%) were female.
Seventy-two (84.7%) cases had BM fibrosis at the time of diagnosis.
The most common was grade 2 fibrosis, recorded in 35 cases (41.2%).
About 72.9% of the patients showed more than 50% plasma cells.
FISH analysis indicated the presence of abnormal chromosomes in
37% (32/85) of the patients. The most frequent abnormality was
Immunoglobulin heavy-chain (IGH) translocation (21.3%).
Conclusion: Subgroup analysis of IGH mutations is crucial in the
identification of high-risk MM patients. We believe that our study
will contribute to the determination of BM biopsy and cytogenetic
features of MM patients in our country.
Keywords: Multiple myeloma, Bone marrow biopsy, Cytogenetic
analysis, Fluorescence in situ hybridization
Öz
Amaç: Multipl myelom (MM), malign plazma hücrelerinin birikmesi
ile karakterize malign bir durumdur. MM tam kür sağlamanan hastalık
olmasa da, uygulanan otolog kök hücre nakli ve yeni ilaçlar ve yeni
tedavi stratejileri nedeniyle MM hastalarının sağkalımı önemli
ölçüde gelişmiştir. Bu çalışmada MM’li Türk hastalarda sitogenetik
karakterizasyon ve kemik iliği (Kİ) özelliklerinin belirlenmesi
amaçlanmıştır.
Gereç ve Yöntemler: Türkiye’deki Dokuz Eylül Üniversite
Hastanesi’ndeki 85 MM hastasını kaydettik. Bu MM hastalarının kemik
iliği örneklerinde, tedavi ve klinik değerlendirmenin bir parçası olarak
tanı anında ve tedavi süresince sitogenetik analiz yapıldı. G-bantlama
tekniği kullanılarak tam bir sitogenetik çalışma gerçekleştirildi.
Fazlar arası Floresan in situ hibridizasyon (FISH) analizi, sitoplazmik
immünoglobulin ile yapıldı. Kemik iliği fibrozunun derecesi, retikülinin
histokimyasal boyası kullanılarak belirlendi. Kİ plazma hücrelerinin
yüzdesi, CD38 boyamasınına göre belirlendi.
Bulgular: 2015 ve 2021 yılları arasında geriye dönük olarak 85 MM
hastası belirlendi. Ortanca yaş 63 (38-90) yıldı. Seksen beş hastanın
60’ı (%70,6) erkek, 25’i (%29,4) kadındı. Tanı anında kemik iliği
fibrozu 72 olguda (%84,7) mevcuttu. En sık 35 hastada (%41,2) derece
2 fibrozis görüldü. Hastaların %72,9’unda plazma hücre yüzdeleri
%50’den fazlaydı. FISH analizi sonuçlarında kromozom anomalilerinin
varlığı %37 (32/85) oranındadır. En sık kromozom görülen anormallik
IGH translokasyonuydu (%21,3).
Sonuç: IGH mutasyonlarının alt grup analizi, yüksek riskli MM
hastalarının belirlenmesinde kritik öneme sahiptir. Çalışmamızın
ülkemizdeki MM hastalarının Kİ biyopsisi ve sitogenetik özelliklerinin
belirlenmesine katkı sağlayacağına inanıyoruz.
Anahtar Sözcükler: Multipl myelom, Kemik iliği biyopsisi, Sitogenetik
analiz, Floresan in situ hibridizasyon
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Ahmet Şeyhanlı, M.D., Sivas Numune Hospital, Clinic of Hematology,
Sivas, Turkey
E-mail : ahmet8563@yahoo.com ORCID: orcid.org/0000-0001-6082-2995
Received/Geliş tarihi: May 22, 2021
Accepted/Kabul tarihi: November 25, 2021
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Şeyhanlı A. et al: Genetic and Bone Marrow Features in Multiple Myeloma
Turk J Hematol 2022;39:109-116
Introduction
Multiple myeloma (MM) is a malignant condition characterized
by the accumulation of malignant plasma cells. It is the second
most common hematological malignancy that develops in
the bone marrow (BM) [1]. Although MM remains incurable,
the survival of MM patients has improved considerably due
to the application of autologous stem cell transplantation
(ASCT), novel agents, and advanced treatment strategies.
However, this development has not been uniform. The existing
heterogeneity is dependent on patient-specific factors such
as age, comorbidities, and disease-related factors, including
cytogenetic and molecular features, BM fibrosis (BMF), and the
plasma cells in the BM. Several risk classifications have been
made for personalized therapeutic approaches. A clinical staging
system for MM was first developed by Durie and Salmon [2]
in 1975. The International Staging System (ISS) score was then
defined by Greipp et al. [3] in 2005 based on two parameters:
serum β2-microglobulin level and serum albumin. Avet-Loiseau
et al. [4] and Cavo-Rosinol et al. [5] combined cytogenetics with
the ISS to improve risk stratification. Several techniques are
available to detect genetic abnormalities in MM. Karyotyping is
applied to detect cytogenetic abnormalities and abnormalities
dependent on the proliferative index of malignant plasma
cells but provides limited information in vitro due to the low
proliferative ability of malignant plasma cells [6]. Kishimoto et al.
[7]reported that abnormal karyotypes are seen in approximately
30%-50% of MM cases. The gold-standard approach for the
detection of genomic abnormalities in MM is fluorescence in
situ hybridization (FISH), which has been implemented and
validated by various cytogenetic laboratories around the globe
[8]. The identification of high-risk subtypes of myeloma is vital.
According to previous works, high-risk MM patients showed
a 20%-50% reduction in overall survival (OS) compared to
standard-risk patient groups when undergoing induction with
conventional chemotherapy followed by ASCT. The present
study aimed to determine the cytogenetic characters and BM
features of Turkish patients with MM.
Materials and Methods
From January 2015 to January 2021, 85 MM patients were
admitted to Dokuz Eylül University Hospital in Turkey. BM samples
of these MM patients were subjected to cytogenetic analyses
at diagnosis and during therapy as a part of therapeutical and
clinical evaluation. Conventional cytogenetic analysis was
performed for all samples at presentation and after a short-term
culture period of 24/72 h. Cells were treated with colcemid and
harvested for 20 min. Karyotypes were then analyzed using the
standard G-banding technique. A minimum of 20 metaphases
per case were analyzed. The karyotypes were reported according
to the 2016 International System for Human Cytogenetic
Nomenclature. An interphase FISH study was performed on BM
samples according to the relevant manufacturer’s hybridization
and post-hybridization stringency conditions with minimal
modifications. Dual-color CytoCell FISH probes specific to loci
13q14.3 (LPH006), CKS1B (1q21-22)/CDKN2C (1p32.3) (LPH039),
IGH (14q32.33) (Break-apart, LPH 014), P53 (17p13.1)/ATM
(11q32) (LPH 052), and MLL (11q23.3) (Break-apart, LPH 013)
were used. Threshold values of copy number gain, deletion,
and break-apart positivity were 7%, 7%, and 10%, respectively.
FISH slides were analyzed with a motorized Olympus BX61
fluorescence microscope equipped with 4′,6-diamidino-
2-phenylindole, fluorescein isothiocyanate, rhodamine, dual,
and triple band-pass filters (Chroma, Bellows Falls, VT, USA). Two
independent observers analyzed the slides blindly, enumerating
at least 200 optimally hybridized nuclei. The degree of BMF
was detected using reticulin histochemical staining. The level
of fibrosis as determined by a pathologist was scored according
to the guidelines of the World Health Organization [grade 0:
no fibrosis, grade 1 (mild): low (fine reticulin network), grade 2
(moderate): intermediate (multifocal or diffuse non-confluent
fibrosis), grade 3 (severe): high (marked and diffuse fibrosis)].
The percentage of BM plasma cells was determined based on
the extent of CD38 staining and categorized as Group 1 (<20%),
Group 2 (20%-50%), or Group 3 (>50% plasma cells). The ISS
was used based on the combination of serum β2-microglobulin
and serum albumin.
Statistical Analysis
IBM SPSS Statistics 24.0 for Windows (IBM Corp., Armonk, NY,
USA) was used for statistical analysis. Numerical variables are
presented as median or mean, while categorical variables are
given as number (n) and percentage (%). The OS curves were
estimated using the Kaplan-Meier method whereby OS was
defined as the time from the diagnosis of MM to death from any
cause. The confidence interval was determined as 95% in the
analyses and values of p<0.05 were considered to be statistically
significant.
Results
Eighty-five MM patients were retrospectively identified between
2015 and 2021. The median age was 63 (38-90) years and 56.5%
of the patients were under 65 years old. Of the 85 patients, 60
(70.6%) were male and 25 (29.4%) were female. Bone lesions
were detected in 57.6% of these cases, anemia in 42.4%,
impaired renal function in 27.1%, and hypercalcemia in 18.8%.
Most patients were in ISS stage III. More than half of the patients
received proteasome inhibitor-based therapy for induction.
Information about the induction therapy of five patients
could not be obtained. Further details about the demographic,
laboratory, and clinical characteristics are presented in Table 1.
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Şeyhanlı A. et al: Genetic and Bone Marrow Features in Multiple Myeloma
Table 1. Demographic, laboratory, and clinical characteristics
of multiple myeloma (MM) patients.
Characteristics n (%)
Age, median, years (range) 63 years (38-90)
<65 years 48 (56.5)
≥65 years 37 (43.5)
Sex
Female 25 (29.4)
Male 60 (70.6)
Immunoglobulin (Ig) isotype
IgG 48 (56.5)
IgA 13 (15.3)
Light chain MM 24 (28.2)
Type of light chain
Kappa 54 (63.5)
Lambda 31 (26.5)
ISS stage
I 21 (24.7)
II 23 (27.1)
III 41 (48.2)
Anemia
Hemoglobin >10 g/dL 49 (57.6)
Hemoglobin <10 g/dL 36 (42.4)
Renal impairment
Creatinine ≤2 mg/dL 62 (72.9)
Creatinine >2 mg/dL 23 (27.1)
Hypercalcemia
Serum calcium <11 mg/dL 69 (81.2)
Serum calcium >11 mg/dL 16 (18.8)
Bone lesions
Absent 36 (42.4)
Present 49 (57.6)
High ESR level (>15 mm/h)
Absent 26 (30.5)
Present 52 (61.1)
Not available 7 (8.4)
High LDH level (>250 U/L)
Absent 60 (70.6)
Present 25 (29.4)
High ferritin level (>335 ng/mL)
Absent 51 (60.0)
Present 17 (20.0)
Not available 17 (20.0)
Treatment type used for induction
VAD 4 (4.70)
Proteasome inhibitor 54 (63.5)
Immunomodulatory drug 3 (3.52)
Both proteasome inhibitor and
immunomodulatory drug
19 (22.3)
Not available 5 (5.88)
Total 85 (100)
ISS: International Staging System; ESR: erythrocyte sedimentation rate; LDH: lactate
dehydrogenase; VAD: vincristine, doxorubicin, and dexamethasone.
BMF was observed at the time of diagnosis in 72 cases (84.7%).
The most common finding was grade 2 fibrosis in 35 patients
(41.2%). In 72.9% of the patients, a plasma cell percentage of
over 50% was observed, and the most common type of plasma
cell infiltration was the diffuse involvement pattern (50.6%).
Based on FISH analysis, 37% (32/85) of cases showed the presence
of chromosomal anomalies. The most frequent abnormality was
the IGH translocation (21.3%). Based on karyotype analysis,
54.4% (37/68) of the patients had normal karyotypes while
5.8% (4/68) had abnormal karyotypes. Table 2 presents plasma
cell percentages, plasma cell infiltration patterns, and fibrosis
results from BM biopsy samples. Detailed cytogenetic results of
these MM patients are presented in Table 3.
In this study, the median OS of the entire population was 29.4
months (Figure 1a). Del(17p13)-negative patients had longer OS
compared to del(17p13)-positive patients (32.4 months vs. 11.1
months, p=0.015) (Figure 1b).
IGH-negative and del(13q)-negative patients had longer OS
compared to IGH-positive and del(13q)-positive patients.
However, this was not statistically significant (p=0.594 and
p= 0.094, respectively). A lower percentage of plasma cells in
the BM was associated with better OS than higher plasma cell
percentages. The median OS was 42.6 months in patients with
0%-19% plasma cells, 33.5 months in patients with 20%-49%
plasma cells, and 28.4 months in patients with 50%-100%
plasma cells. However, this observation was not statistically
significant (p=0.969). The results of multivariate analysis
adjusting for the del(17p13) mutation, IGH mutation, del(13)
mutation, BMF, plasma cell infiltration pattern, and plasma cell
percentage are shown in Table 4.
Table 2. Analysis of plasma cell percentage, plasma cell
infiltration pattern, and fibrosis in bone marrow biopsy
samples from patients with multiple myeloma at diagnosis.
Bone marrow plasma cell percentage (%) n (%)
0-19 5 (5.9)
20-49 62 (72.9)
50-100
Pattern of plasma cell infiltration 33 (38.8)
Interstitial 9 (10.6)
Nodular
Diffuse 43 (50.6)
Bone marrow fibrosis
MF-0 13 (15.3)
MF-1 35 (41.2)
MF-2 9 (10.6)
MF-3
Total 85 (100)
MF: Marrow fibrosis.
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Turk J Hematol 2022;39:109-116
Figure 1. a) Kaplan-Meier curve of overall survival (OS) for the entire study cohort (n=85). The median OS was 29.4 months. b) Del(17p13)-
negative patients had longer OS compared to del(17p13)-positive patients (32.4 months vs. 11.1 months, p=0.015).
Table 3. Detailed cytogenetic results of multiple myeloma patients.
Characteristics Information available, n Positive patients, n (%)
Del(17p13) mutation 85 6 (7.1)
Del(13q14) mutation 49 9 (18.4)
IGH translocations 80 17 (21.3)
CKS1B mutation 1 0 (0.00)
MLL mutation 15 0 (0.00)
Karyotyping 68
Normal 37 (54.4)
Hyperdiploid 2 (2.9)
Non-hyperdiploid 2 (2.9)
Non-diagnostic 27 (39.7)
Discussion
MM remains an incurable malignancy with varying clinical and
prognostic differences depending on various factors including
age, comorbidity, cytogenetic and genomic features, and tumor
microenvironment characteristics in the bone marrow. The
disease incidence ranges from 5 to 7 per 100,000 and increases
with age. The median age at diagnosis of MM is 69 years. Fewer
than 14% of patients are younger than 55 years. MM is seen
more often in men and African Americans. The etiological
reasons behind these differences are not sufficiently understood.
Survival among patients with MM ranges from 1 year to more
than 10 years. Median survival in unselected patients with MM
is 3 years. The 5-year relative survival rate is 46.6% [9]. In our
study, the median age was 63 and male patients constituted
70.6% of the total. These findings are slightly different from
those published in the literature, possibly due to environmental
and genetic factors.
Understanding the alterations in the BM microenvironment
and the molecular pathways related to these changes is
crucial for the further improvement of MM treatment and
the outcomes of these patients. Sailer et al. [10] demonstrated
that characteristics of the tumor microenvironment such as
plasma cell differentiation, volume of plasma cell infiltration,
and infiltration pattern have predictive value. However, the
prognostic efficacy of BMF at the time of diagnosis is still
controversial [11,12]. Babarović et al. [13] reported that the
5-year survival rates of patients with increased BMF subjected
to post-treatment BM biopsy were significantly shorter than
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Şeyhanlı A. et al: Genetic and Bone Marrow Features in Multiple Myeloma
Table 4. Multivariable Cox regression analysis for overall survival (OS).
Characteristics Median OS (months) Hazard ratio 95% CI p
Del(17p13) 0.015
Del(17p13)-negative 32.4 1
Del(17p13)-positive 11.1 3.45 1.18-10.0
IGH translocations 0.594
IGH-negative 28.9 1
IGH-positive 22.8 1.35 0.44-4.11
Del(13q) mutation 0.094
Del(13q)-negative 28.4 1
Del(13q)-positive 23.3 1.88 0.89-3.96
Bone marrow fibrosis 0.938
MF-0 34.4 1
MF-1 33.0 0.90 0.35-2.29
MF-2 27.4 1.15 0.46-2.87
MF-3 33.4 1.08 0.31-3.72
Pattern of plasma cell infiltration 0.466
Interstitial 33.4 1
Nodular 23.3 1.80 0.69-473
Diffuse 28.4 1.12 0.55-2.27
Bone marrow plasma cell percentage (%)
0-19 42.6 1 0.969
20-49 33.5 0.86 0.23-3.31
50-100 28.4 0.97 0.29-3.26
CI: Confidence interval; MF: marrow fibrosis; IGH: immunoglobulin heavy chain.
those of patients without fibrosis. Thus, the assessment of BMF
was concluded to have prognostic value in the follow-up of MM
patients [13]. A recent study by Paul et al. [14] showed that the
median progression-free survival in patients without BMF was
30.2 months, while in those with BMF, it was 21 months. On
the other hand, median OS was 61.2 months and 45.1 months
among patients without and with BMF, respectively. These
findings were statistically significant [14]. Our results suggested
that the pattern of plasma cell infiltration and the degree of
fibrosis at diagnosis did not significantly influence survival
times, possibly due to the relatively small groups in this study.
Several predictive models like the Durie and Salmon [2] staging
system and the ISS have been used to categorize MM patients
into risk groups. The Durie and Salmon [2] staging system was
the first such system devised for MM and it has been widely
used for several decades. Later proposed by Greipp et al. [3], the
ISS became more widely used. The ISS is based on the prognostic
factors of serum β2-microglobulin and serum albumin. For
ISS stages I, II, and III, the median survival of MM patients
was determined to be 62 months, 44 months, and 29 months,
respectively [3]. A major limitation of the ISS classification is
that it does not consider patient-specific factors. Approximately
half of our patients were in ISS stage III and their median OS was
28.4 months. Our results were thus consistent with the study
of Greipp et al. [3]. Chromosomal abnormalities detected by
FISH were combined with the ISS to improve risk stratification
[4,5]. Because karyotypes are highly sensitive in the detection
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Turk J Hematol 2022;39:109-116
of numerical chromosomal abnormalities, patients can be
classified into specific categories such as normal, hyperdiploid,
or non-hyperdiploid (including hypodiploid, pseudodiploid,
and near-tetraploid) karyotypes [15]. Among non-hyperdiploid
cases of MM, the hypodiploid subtype is associated with
poor prognosis [16,17]. In our study, abnormal karyotypes
were detected in 4 of 68 (5.8%) patients. Among these cases,
non-hyperdiploid abnormalities were observed in two patients.
However, chromosomal abnormalities could not be evaluated in
39.7% of the total cases because of the absence of metaphase
cells. FISH does not require metaphase cells before analysis
and it is accepted as the gold-standard test for the detection
of genomic abnormalities in MM, being applied and verified
by cytogenetics laboratories around the globe [8]. Further
studies are underway for identifying the optimal risk-based
classification and treatment for MM and especially for the
identification of high-risk MM patients. The International
Myeloma Working Group provided a consensus decision stating
that FISH panel testing should be performed for at least t(4;14),
t(14;16), and del(17p13) to define patients with high-risk disease
[18]. In addition to that recommendation, Boyd et al. [19]
suggested that FISH panel tests for +1q21 and t(14;20) should
be performed in MM risk classification. Deletion of chromosome
17p13 increases the immortalization of tumor cells by inhibiting
apoptosis [20]. Del(17p13) is observed in about 10% of MM
cases and has been associated with impaired OS [21,22,23,24].
In our study, the incidence rate of the del(17p13) mutation was
7.1% and del(17p13)-negative patients had significantly longer
OS compared to del(17p13)-positive patients (32.4 months vs.
11.1 months, respectively). Previously, del(13q) was in the poor
prognosis group, but it is now excluded from risk stratification
as it was detected in almost 50% of myeloma patients and is
thought to be secondary to the close association with t(4;14),
del(17p) [19,25,26]. In this study, del(13q) was detected in 9 of
49 cases (18.4%) and a significant adverse effect of del(13q) on
OS was not observed (p=0.094).
Chromosome 14q32 translocations (heavy chain IGH
translocations) in MM also increase dysregulated proliferation
and inhibit differentiation. Bergsagel and Kuehl [27] reported an
incidence of IGH translocations of 60%-65% in intramedullary
MM. However, in our study, the incidence of IGH mutations
was lower (21.3%). Over 20 different IGH translocations
have been revealed, among which IGH translocations t(4;14),
t(14;16), and t(14;20) are associated with poor prognosis in MM
patients [28]. The use of proteasome inhibitor-based induction
is recommended to improve the prognosis of patients with
t (4;14) but not those with del(17p13) [29]. Unfortunately, our
study does not include subgroup analysis of IGH mutations
that involve several recurrent translocations. Recently, gene
expression profiling methods, such as sequencing comparative
genomic hybridization (CGH), single nucleotide polymorphism
sequencing CGH, gene expression profiling, and RNA sequencing
have been introduced to identify patients with particularly
high-risk MM [30,31,32]. However, this is not feasible in routine
practice due to the high costs.
Study Limitations
The present study has a few limitations. It was a retrospective
study based on single-center data. Possibly because of the small
sample size, we did not observe a significant adverse effect of
IGH mutation on OS. Since our study did not include subgroups
of IGH mutations, high-risk patients could not be determined
clearly.
Conclusion
To the best of our knowledge, this study is the first of its
kind conducted in Turkey to include genetic results. This is
especially crucial in the identification of high-risk myeloma
subtypes. The subgroup analysis of IGH mutations is critical in
identifying high-risk MM patients. IGH subgroup analyses have
been conducted recently in our center. The present study will
contribute to determining BM biopsy and cytogenetic features
of MM patients in our country.
Ethics
Ethics Committee Approval: This study was approved by the
Ethics Committee of Dokuz Eylül University (2021/10-25).
Informed Consent: Retrospective study.
Authorship Contributions
Concept: A.Ş., B.Y., Z.A., Z.Y., S.Ö., O.A., F.D., İ.A., G.H.Ö.; Design:
A.Ş., B.Y., Z.A., Z.Y., S.Ö., O.A., F.D., İ.A., G.H.Ö.; Data Collection or
Processing: A.Ş., B.Y., Z.A., Z.Y., S.Ö., O.A., F.D., İ.A., G.H.Ö.; Analysis
or Interpretation: A.Ş., B.Y., Z.A., Z.Y., S.Ö., O.A., F.D., İ.A., G.H.Ö.;
Literature Search: A.Ş., B.Y., Z.A., Z.Y., S.Ö., O.A., F.D., İ.A., G.H.Ö.;
Writing: A.Ş., B.Y., Z.A., Z.Y., S.Ö., O.A., F.D., İ.A., G.H.Ö.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
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116

RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2022.2022.0084
Turk J Hematol 2022;39:117-129
Combination of Haploidentical Hematopoietic Stem Cell
Transplantation with Umbilical Cord-Derived Mesenchymal Stem
Cells in Patients with Severe Aplastic Anemia: A Retrospective
Controlled Study
Şiddetli Aplastik Anemisi Olan Hastaların Göbek Kordonu Kaynaklı Mezenkimal Kök
Hücreleri ile Haplo Uyumlu Hematopoetik Kök Hücre Naklinin Kombinasyonu: Geriye Dönük,
Kontrollü Bir Çalışma
Xian-Fu Sheng*, Hui Li*, Li-Li Hong, Hai-Feng Zhuang
Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China
*These authors contributed equally to this work.
Abstract
Objective: We retrospectively compared the outcomes of patients
with severe aplastic anemia (SAA) who received haploidentical
hematopoietic stem cell transplantation (haplo-HSCT) combined or
not combined with umbilical cord-derived mesenchymal stem cells
(UC-MSCs).
Materials and Methods: A total of 101 patients with SAA were
enrolled in this study and treated with haplo-HSCT plus UC-MSC
infusion (MSC group, n=47) or haplo-HSCT alone (non-MSC group,
n=54).
Results: The median time to neutrophil engraftment in the MSC
and non-MSC group was 11 (range: 8-19) and 12 (range: 8-23) days,
respectively (p=0.049), with a respective cumulative incidence (CI) of
97.82% and 97.96% (p=0.101). Compared to the non-MSC group, the
MSC group had a lower CI of chronic graft-versus-host disease (GVHD)
(8.60±0.25% vs. 24.57±0.48%, p=0.048), but similar rates of grades II-
IV acute GVHD (23.40±0.39% vs. 24.49±0.39%, p=0.849), grades III-IV
acute GVHD (8.51±0.17% vs. 10.20±0.19%, p=0.765), and moderatesevere
chronic GVHD (2.38±0.06% vs. 7.45±0.18%, p=0.352) were
observed. The estimated 5-year overall survival (OS) rates were
78.3±6.1% and 70.1±6.3% (p=0.292) while the estimated 5-year
GVHD-free, failure-free survival (GFFS) rates were 76.6±6.2% and
56.7±6.9% (p=0.045) in the MSC and non-MSC groups, respectively.
Conclusion: In multivariate analysis, graft failure was the only adverse
predictor for OS. Meanwhile, graft failure, grades III-IV acute GVHD,
and moderate-severe chronic GVHD could predict worse GFFS. Our
results indicated that haplo-HSCT combined with UC-MSCs infusion
was an effective and safe option for SAA patients.
Keywords: Severe aplastic anemia, Haploidentical, Hematopoietic
stem cell transplantation, Mesenchymal stem cells
Öz
Amaç: Göbek kordonundan elde edilen mezenkimal kök hücrelerin (UK-
MKH’ler) infüzyonu ile kombine edilmiş ve edilmemiş, haploidentik
hematopoietik kök hücre nakli (haplo-HKHN) yapılan şiddetli aplastik
anemili (ŞAA) hastalarının sonuçları geriye dönük olarak karşılaştırıldı.
Gereç ve Yöntemler: Bu çalışmaya ŞAA’lı toplam 101 hasta alındı
ve hastalar haplo-HSCT ile birlikte UK-MKH infüzyonu (MKH grubu,
N=47) ve tek başına haplo-HKHN (MKH olmayan grup, N=54) şeklinde
iki gruba ayrıldı.
Bulgular: MKH alan ve almayan gruplarda nötrofil engraftmanı için
medyan süreler sırasıyla, 11 (aralık, 8-19) ve 12 (aralık, 8-23) gün
(p=0,049) ve kümülatif insidans (CI) sırası ile %97,82 ve %97,96
(p=0,101) bulundu. MKH almayan grupla karşılaştırıldığında, MKH
alan grupta daha düşük kronik graft-versus-host hastalığı (cGVHD)
CI saptandı (%8,60±0,25’e karşı %24,57±0,48, p=0,048), ancak II-
IV akut GVHD (aGVHD) (%23,40±0,39-%24,49±0,39, p=0,849), III-
IV aGVHD (%8,51±0,17 ve %10,20±0,19, p=0,765) ve orta-şiddetli
cGVHD (%2,38±0,06 vs. %7,45±0,18, p=0,352) gruplarında fark
görülmedi. MKH alan ve MKH almayan gruplarda tahmini beş yıllık
genel sağkalım oranları %78,3±6,1 ve %70,1±6,3 (p=0,292) iken,
GVHD’siz, hastalıksız sağkalım (GFFS) oranları sırasıyla, %76,6 ±%6,2
ve %56,7±6,9 (p=0,045) idi.
Sonuç: Çok değişkenli analizde greft yetmezliği, OS için tek olumsuz
gösterge idi. Bu arada, greft yetmezliği, III-IV aGVHD ve orta-şiddetli
cGVHD arasında daha kötü GFFS’yi öngörebilir. Sonuçlarımız, UC-MKH
infüzyonu ile birlikte haplo-HKHN’nin ŞAA hastaları için etkili ve
güvenli bir seçenek olduğunu gösterdi.
Anahtar Sözcükler: Şiddetli aplastik anemi, Haplo uyumlu,
Hematopoetik kök hücre nakli, Mezenkimal kök hücre
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Hai-Feng Zhuang, M.D., Zhejiang Chinese Medical University,
Hangzhou, Zhejiang, China
Phone : +86 13858159652
E-mail : zhuanghaifeng5@163.com ORCID: orcid.org/0000-0003-2802-7910
Received/Geliş tarihi: March 2, 2022
Accepted/Kabul tarihi: April 21, 2022
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Turk J Hematol 2022;39:117-129
Introduction
Severe aplastic anemia (SAA) is a rare, life-threatening bone
marrow (BM) failure syndrome characterized by pancytopenia
and hypoplastic bone marrow. The incidence is 2-2.4 per million
per year in Europe but higher in China, affecting 7.4 patients
per million [1].
Although hematopoietic stem cell transplantation (HSCT) from
a human leukocyte antigen (HLA)-matched related donor (MRD)
is the first choice for SAA patients who are <40 years old, it
is difficult to search for HLA-matched siblings in China [2].
Immunosuppressive therapy (IST) with antithymocyte globulin
(ATG) and cyclosporin A (CsA) is recommended as the first-line
treatment for SAA patients who lack an appropriate MRD and
those aged >40 years [3]. However, most patients cannot be
cured by IST. Previous studies showed that response rates were
in the range of 40%-60% and 25% of responders subsequently
relapsed [4,5,6]. Therefore, the treatment of SAA patients
who lack sibling donors or fail to respond to IST is extremely
challenging.
Stem cells from haploidentical family donors have become the
most common source of HSCT in China due to the advantage of
immediate availability for almost any patient [7]. Haploidentical
HSCT (haplo-HSCT) has greatly developed in recent years. With
improvements in donor selection, conditioning regimens, and
graft-versus-host disease (GVHD) prophylaxis, studies comparing
haplo-HSCT with MRD-HSCT in young patients have revealed
limited differences in overall survival (OS) [6,8,9]. However, the
higher incidence of graft failure (GF) and GVHD has limited the
clinical application of haplo-HSCT for SAA patients [6,10,11,12].
Mesenchymal stem cells (MSCs) have been shown to support
hematopoiesis and display potent immunomodulatory
properties for treating GVHD after HSCT [13,14]. It was reported
that human umbilical cord-derived MSCs (UC-MSCs) have
higher activity levels of proliferation and differentiation in
comparison with bone marrow-derived MSCs (BM-MSCs) [11].
Thus, UC-MSCs are highly promising for use in haplo-HSCT. In
recent years, some studies have reported the co-transplantation
of haplo-HSCT and UC-MSCs with favorable outcomes in cases
of SAA [11,12,15,16]. To the best of our knowledge, there are
still no head-to-head studies exploring the efficiency and
safety of haplo-HSCT with and without UC-MSCs. In the present
study, we report the outcomes of haplo-HSCT with or without
third-party UC-MSCs for the treatment of SAA patients in our
centers from June 2014 to June 2021.
Materials and Methods
Patients
Between June 2014 and June 2021, 101 consecutive SAA
patients who underwent haplo-HSCT in our transplant unit
were enrolled in this study. Informed consent was obtained from
all patients or their parents and the study was approved by the
Ethics Committee of the First Affiliated Hospital of Zhejiang
Chinese Medical University. All patients met the following
inclusion criteria: (1) diagnosed with SAA or very severe aplastic
anemiaSAA according to the guidelines of the International
Aplastic Anemia Study Group; (2) no HLA-identical sibling
donor; (3) no serious infectious disease or acute hemorrhaging;
(4) absence of severe liver, renal, lung, and heart diseases;
(5) Eastern Cooperative Oncology Group score of 0-2 points.
HLA compatibility was determined by high-resolution DNA
techniques for the HLA-A, B, C, DRB1, and DQB1 loci. Donors
were ranked on the basis of HLA match, age (younger preferred),
gender (same preferred), and health status (better preferred).
Conditioning Regimen
Patients who underwent haplo-HSCT were placed on regimens
including fludarabine (Flu)/cyclophosphamide (Cy)/ATG or
Bu/Cy/ATG. Patients with acute SAA received the following
regimen: intravenous fFlu at 30 mg/m 2 /day on days -5 and
-2; intravenous c (Cy) at 50 mg/kg/day on days -5 to -2; and
intravenous ATG (rabbit, Genzyme Polyclonals SAS, Lyon, France)
at 2.5 mg/kg/day on days -5 to -2. For patients with chronic
SAA (SAA-II), or, in other words, patients who had progressed to
SAA from non-SAA [17], the same protocol for Cy and ATG was
applied as above with the addition of busulfan (Bu) at 3.2 mg/
kg/day from day -7 to -6.
All patients underwent testing for donor-specific anti-HLA
antibodies (DSA) before transplantation. If the DSA results were
positive (MFI >5000), the patient received rituximab (once a
week, 4 times) and plasmapheresis (2-3 times). The DSA results
were then reviewed again and transplantation occurred if the
value dropped below 5000. Among the 101 patients enrolled in
this study, 5 patients had positive DSA results. After treatment,
all of these patients had negative DSA results.
Stem Cell Collection and Infusion
All donors were injected with granulocyte colony-stimulating
factor (G-CSF) subcutaneously at 10 µg/kg/day for 4-5
continuous days. BM grafts were collected by BM aspiration in
an operating room on day +1. The target volume was 10 to 20
mL/kg of the recipient’s weight. In cases of ABO incompatibility,
red blood cells were removed by sedimentation with Hespan.
Peripheral blood stem cells (PBSCs) were collected with a COBE
Spectra device (Gambro BCT, Lakewood, CO, USA) on day +2. The
target mononuclear cell) count from BM and peripheral blood
was ≥5x10 8 /kg and the target CD34 + cell count was ≥2x10 6 /kg
of the recipient’s weight. An additional collection of PBSCs was
performed on day +3 if the initial cell numbers were insufficient.
UC-MSCs were purchased from the Shanghai Cord Blood Bank.
A total of 1x10 6 /kg UC-MSCs was infused 4 h before the infusion
of the graft on day +1.
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Sheng X-F. et al: Combination of Haplo-HSCT with UC-MSCs
GVHD Prophylaxis
In the protocol applied for these patients, prophylaxis for acute
GVHD (aGVHD) included CsA, mycophenolate mofetil (MMF),
and short-term methotrexate (MTX). CsA was administrated
intravenously twice daily at a dose of 1.5 mg/kg/day from day
-7 until bowel function returned to normal, at which time
patients received oral CsA. A target trough blood concentration
of 200-300 ng/mL was maintained for ≥1 year after
haplo-HSCT. CsA was gradually tapered thereafter and was
withdrawn completely in the following 2-3 months. MMF was
given orally at 500 mg every 12 h (250 mg for children) from
day -7 to +30 and subsequently at 250 mg from day +30 to
+90. MTX was administrated intravenously at a dose of 15
mg/m2 on day +1 and 10 mg/m2 on days +3, +6, and +11. If
GVHD occurred in any organ, CsA and MMF were continued and
adjusted to therapeutic concentrations.
Definitions and Post-transplantation Evaluations
Neutrophil engraftment was defined as occurring on the first
of 3 consecutive days with an absolute neutrophil count of
>0.5x10 9 /L. Platelet engraftment was defined as occurring on the
first day of a platelet count of >20x10 9 /L without transfusion
for 7 consecutive days. Short-tandem repeat polymerase chain
reaction of peripheral whole blood was performed monthly to
assess hematopoietic chimerism from the time of neutrophil
recovery.
Primary GF (PGF) was defined as the failure to achieve
neutrophil engraftment before day +28 after transplantation.
Secondary GF was defined as graft loss after initial engraftment,
with the recipient experiencing pancytopenia and hypocellular
BM without moderate to severe aGVHD [15]. Delayed platelet
recovery was defined as platelet engraftment achieved after day
+28. aGVHD was defined according to the criteria proposed by
the 1994 Consensus Conference on aGVHD Grading [18], while
chronic GVHD (cGVHD) was defined according to the National
Institutes of Health Consensus Conference on cGVHD [19].
Diagnoses of cytomegalovirus (CMV) infection, CMV pneumonia,
Epstein-Barr virus (EBV) infection, and EBV-associated posttransplant
lymphoproliferative disorders (PTLDs) were based on
standard clinical criteria.
OS was defined as the time from the date of haplo-HSCT to the
date of death or last follow-up. GVHD-free, failure-free survival
(GFFS) was defined as survival without grades III-IV aGVHD,
moderate-severe cGVHD, or HSCT failure. Death, primary or
secondary GF, and relapse were considered as HSCT failures.
Supportive Care
All patients were admitted to class 100 laminar flow clean
wards after a skin-care bath. Patients received oral antibiotics
(levofloxacin for adults, gentamycin for children) for
gastrointestinal decontamination before transplantation.
Fluconazole, acyclovir, and trimethoprim/sulfamethoxazole
were administered to prevent fungal, viral, and Pneumocystis
pneumonia infection, respectively. Heparin and prostaglandin E1
were administrated to prevent veno-occlusive disease. Human
immunoglobulin was administered intravenously at a dose of 10
g once a week from day 0 to day +100 after the transplantation.
Recombinant human G-CSF was given at a dose of 5 µg/kg/day
from day +3 until neutrophil recovery. Irradiated erythrocytes
and platelets were given to maintain a hemoglobin level of >60
g/L and a platelet count of >20x10 9 /L.
Statistical Analysis
The date of last follow-up for all surviving patients was December
31, 2021. Patient baseline characteristics were compared by
chi-square test for categorical variables and by Mann-Whitney U
and Kruskal-Wallis tests for continuous data. OS and GFFS were
calculated using the Kaplan-Meier method and the groups were
compared using the log-rank test. The cumulative incidence
(CI) method was used to calculate the incidence of aGVHD and
cGVHD with death and GF as competing risks according to the
competing risk model. We used univariate and multivariate
analyses to determine whether any of the selected factors were
predictive of OS and GFFS. In multivariate analysis, all factors
with values of p<0.1 in univariate analysis were included in the
Cox regression model. Values of p<0.05 were considered to be
significant. SPSS 19.0 (IBM Corp., Armonk, NY, USA) was used
for statistical analyses.
Results
Characteristics of Patients and Donors
SAA patients undergoing haplo-HSCT with UC-MSC infusion
(MSC group, n=47) or without UC-MSC infusion (non-MSC
group, n=54) were enrolled in this study. Characteristics of the
patients and their donors were compared between groups as
shown in Table 1. The two cohorts were similar with regard to
age distribution, sex ratio, disease severity, interval between
diagnosis and transplantation, and previous treatments. No
differences were found in the baseline characteristics of donors
and grafts between the two groups.
Engraftment
Forty-seven patients (100%) in the MSC group and 49 patients
(90.74%) in the non-MSC group survived for 28 days. The five
patients who experienced early mortality before engraftment
were not considered in the subsequent analysis. One patient
in each group experienced PGF. Two patients died suddenly
before a second HSCT could be performed. The CIs of 28-day
neutrophil engraftment were 97.82±0.06% and 97.96±0.06%
in the MSC and non-MSC groups, respectively (p=0.101).
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Turk J Hematol 2022;39:117-129
Table 1. Characteristics of patients with severe anaplastic anemia, their donors, and grafts.
Variable MSC group (n=47) Non-MSC group (n=54) p
Median age, years (range)
≤20 years, n (%)
21-39 years, n (%)
≥40 years, n (%)
Sex, n (%)
Male
Female
Disease severity, n (%)
SAA
VSAA
SAA-PNH
25 (8-56)
15 (31.9)
26 (55.3)
6 (12.8)
24 (51.1)
23 (48.9)
23(48.9)
21 (44.7)
3 (6.4)
33 (9-55)
14 (25.9)
24 (44.4)
22 (21.8)
33 (61.1)
21 (38.9)
35 (64.8)
17 (31.5)
2 (3.7)
Median time from diagnosis to HSCT, months (range) 3 (1-300) 7 (1-120) 0.502
Previous treatment, n (%)
CsA ± Andriol
ATG/ALG ± CsA ± Andriol
Supportive care
34 (72.3)
7 (14.9)
6 (12.8)
43 (79.6)
2 (3.7)
9 (16.7)
0.145
Donor-recipient sex match, n (%)
Male-male
Male-female
Female-male
Female-female
15 (31.9)
11 (23.4)
9 (19.1)
12 (25.5)
18 (33.3)
7 (13.0)
15 (27.8)
14 (25.9)
Donor-recipient relationship, n (%) 0.574
Mother-child 5 (10.6) 5 (9.3)
Father-child 12 (25.5) 10 (18.5)
Child-mother 6 (12.8) 10 (18.5)
Child-father
Siblings
5 (10.6)
19 (40.4)
11 (20.4)
18 (33.3)
HLA mismatched, n (%) 0.950
Haplo 5 locus mismatched
Haplo 4 locus mismatched
Haplo 3 locus mismatched
Haplo 2 locus mismatched
Haplo 1 locus mismatched
ABO blood types match, n (%)
Matched
Major unmatched
Minor unmatched
Major and minor unmatched
Conditioning regimen, n (%)
Flu/Cy/ATG
Bu/Cy/ATG
Graft type, n (%)
BM + PB
PB
Stem cells
MNCs, x10 8 /kg, median (range)
CD34 + , x10 6 /kg, median (range)
30 (63.8)
8 (17.0)
7 (14.9)
1 (2.1)
1 (2.1)
27 (57.4)
6 (12.8)
10 (21.3)
4 (8.5)
35 (74.5)
12 (25.5)
47 (100.0)
0
8.68 (2.49-21.80)
4.90 (1.92-12.70)
31 (57.4)
12 (22.2)
9 (16.7)
1 (1.9)
1 (1.9)
33 (61.1)
9 (16.7)
8 (14.8)
4 (7.4)
45 (83.3)
9 (16.7)
52 (96.3)
2 (3.7)
7.33 (2.35-26.00)
5.21 (1.98-16.86)
MSCs: Mesenchymal stem cells; SAA: severe aplastic anemia; VSAA: very severe aplastic anemia; PNH: paroxysmal nocturnal hemoglobinuria; HSCT: hematopoietic stem cell
transplantation; CsA: cyclosporin A; ATG: antithymocyte globulin; ALG: antilymphocyte globulin; HLA: human leukocyte antigen; Haplo: haploidentical; Flu: fludarabine; Cy:
cyclophosphamide; Bu: busulfan; BM: bone marrow; PB: peripheral blood; MNCs: mononuclear cells. Values of p<0.05 were considered to be significant.
0.134
0.123
0.310
0.256
0.505
0.815
0.273
0.497
0.251
0.817
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The median times to neutrophil engraftment were 11
(range: 8-19) and 12 (range: 8-23) days, respectively (p=0.049).
Forty-three and 48 patients achieved platelet engraftment in
the MSC and non-MSC groups, respectively, with respective
CIs of platelet engraftment of 91.49±0.19% and 91.84±0.16%
(p=0.345). The median times to platelet engraftment were 12
(range: 7-43) and 14 (range: 6-70) days, respectively (p=0.047).
In the MSC group, 2 patients (4.26%) experienced secondary
GF and 3 patients (6.38%) had delayed platelet recovery. In the
non-MSC group, 3 (6.12%) and 6 (12.24%) patients experienced
secondary GF and delayed platelet recovery, respectively. The
patients who experienced secondary GF subsequently died due
to infection or cerebral hemorrhage. No significant differences
were observed between groups in terms of PGF, secondary GF, or
delayed platelet recovery (p>0.05, Table 2).
GVHD
Table 2 presents the incidence and severity of GVHD for both
groups. Patients with primary engraftment were included in
the aGVHD analysis. In the MSC group, 17 patients experienced
aGVHD after HSCT. These included 6 patients with grade I, 7
with grade II, 3 with grade III, and 1 with grade IV aGVHD. Of
the 49 patients in the non-MSC groups, 21 experienced aGVHD
after HSCT, including 9 with grade I, 7 with grade II, 4 with
Table 2. Comparison of clinical outcomes between the groups after haploidentical hematopoietic stem cell transplantation.
Variable MSC group (n=47) Non-MSC group (n=54) p
Patients surviving longer than 30 days, n (%) 47 (100) 49 (90.7) 0.059
Engraftment
Median myeloid recovery, days (range) 11 (8-19) 12 (8-23) 0.049
Median platelet recovery, days (range) 12 (7-43) 14 (6-70) 0.047
Primary graft failure, n (%) 1 (2.1) 1 (2.0) 1.000
Secondary graft failure, n (%) 2 (4.3) 3 (6.1) 1.000
Delayed platelet recovery, n (%) 3 (6.4) 6 (12.2) 0.526
Acute GVHD n=47 n=49
None, n (%) 30 (63.8) 28 (57.1) 0.503
II-IV, n (%) 11 (23.4) 12 (24.5) 0.901
III-IV, n (%) 4 (8.7) 5 (10.2) 1.000
Patients surviving longer than 100 days, n (%) 42 (89.4) 44 (81.5) 0.267
Chronic GVHD n=42 n=44
None, n (%) 38 (90.5) 34 (77.3) 0.097
Mild, n (%) 2 (4.8) 7 (15.9) 0.182
Moderate to severe, n (%) 2 (4.8) 3 (6.8) 1.000
Infection, n (%) n=47 n=54
Viremia
CMV 14 (29.8) 15 (27.8) 0.824
EBV 31 (66.0) 30 (55.6) 0.286
EBV-associated PTLD 3 (6.4) 4 (7.4) 1.000
Pulmonary infections 14 (29.8) 16 (29.6) 0.986
Septicemia 7 (14.9) 11 (20.4) 0.473
Herpes zoster 2 (4.3) 10 (18.5) 0.027
Causes of death, n (%) n=47 n=54
Infection 4 (8.5) 5 (9.3) 1.000
RRTs 0 3 (5.6) 0.246
GF 2 (4.3) 3 (5.6) 1.000
GVHD 3 (6.4) 2 (3.7) 0.873
TMA 1 (2.1) 0 0.465
Leukoencephalopathy syndrome 0 1 (1.9) 1.000
Median follow-up time among living patients, months (range) 30.8 (6.5-91.0) 28.1 (6.8-84.5) 0.427
MSCs: Mesenchymal stem cells; GVHD: graft-versus-host disease; CMV: cytomegalovirus; EBV: Epstein-Barr virus; PTLD: post-transplant lymphoproliferative disorder; RRTs: regimenrelated
toxicities; GF: graft failure; TMA: thrombotic microangiopathy. Values of p<0.05 were considered to be significant.
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grade III, and 1 with grade IV. At 100 days after transplantation,
the CIs of grade II-IV aGVHD for the MSC and non-MSC groups
were 23.40±0.39% and 24.49±0.39%, respectively (p=0.849,
Figure 1a). The CIs of grade III-IV aGVHD in the MSC and
non-MSC groups were 8.51±0.17% and 10.20±0.19%,
respectively (p=0.765, Figure 1b).
Forty-two patients in the MSC group and 44 in the non-MSC
group with survival longer than 100 days after transplantation
were assessed for cGVHD. In the MSC group, 2 patients had mild
cGVHD, 2 had moderate cGVHD, and none had severe cGVHD. In
the non-MSC group, 7 patients had mild cGVHD, 2 had moderate
cGVHD, and 1 had severe cGVHD. Patients in the MSC group
had a lower CI of cGVHD than the non-MSC group (8.60±0.25%
vs. 24.57±0.48%, p=0.048, Figure 1c). However, there was no
significant difference in the CIs of moderate-severe cGVHD
between the two groups (2.38±0.06% vs. 7.45±0.18%, p=0.352,
Figure 1d).
Infectious Complications
The most common infections after transplantation were the
reactivation of CMV and EBV, pulmonary infections, septicemia,
and herpes zoster (Table 2).
Fourteen of the 47 patients (29.8%) in the MSC group
experienced CMV reactivation following transplantation as
detected by DNA testing. In the non-MSC group, 15 of 54
patients (27.8%) experienced CMV reactivation. There was no
Figure 1. Cumulative incidences of graft-versus-host disease (GVHD) for the two groups. a) Cumulative incidences of grade II-IV acute
GVHD (aGVHD): mesenchymal stem cell (MSC) group 23.40±0.39% vs. non-MSC group 24.49±0.39% (p=0.849). b) Cumulative incidences
of grade III-IV aGVHD: MSC group 8.51±0.17% vs. non-MSC group 10.20±0.19% (p=0.765). c) Cumulative incidences of chronic GVHD
(cGVHD): MSC group 8.60±0.25% vs. non-MSC group 24.57±0.48% (p=0.048). d) Cumulative incidences of moderate-severe cGVHD:
MSC group 2.38±0.06% vs. non-MSC group 7.45±0.18% (p=0.352).
HSCT: Hematopoietic stem cell transplantation.
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significant difference in CMV reactivation between the two
groups (p=0.824, Table 2). Viral infections were treated with
foscarnet or ganciclovir and gamma globulin. In the non-MSC
group, one patient developed CMV retinitis and recovered with
an infusion of CMV-specific cytotoxic T lymphocytes. Other
patients who did not develop CMV disease were mostly cured
after antiviral treatment.
As shown in Table 2, 31 patients in the MSC group (66.0%)
and 30 patients in the non-MSC group (55.6%) experienced
EBV reactivation (p=0.286). Nineteen patients (10 in the MSC
group and 9 in the non-MSC group) who were not successfully
treated with antiviral drugs were treated with rituximab. EBV
copy numbers decreased to the normal range in most cases.
However, one patient in the non-MSC group only recovered with
EBV-specific cytotoxic T lymphocytes. Three patients (6.4%)
and 4 patients (7.4%) progressed to EBV-associated PTLD,
respectively, in the MSC and non-MSC groups (p=1.000). All
patients with PTLD received rituximab treatment, and some
patients received it in combination with chemotherapy. Three
patients with PTLD in the non-MSC group died of complications
resulting from immunochemotherapy on day +274, day +205,
and day +134, respectively. Others recovered from PTLD with the
regression of enlarged lymph nodes and EBV copy numbers that
declined to normal values.
In the MSC group, 14 patients (29.8%) experienced pulmonary
infections, 7 patients (14.9%) experienced septicemia, and
2 patients (4.3%) experienced herpes zoster. In the non-MSC
group, 16 patients (29.6%) experienced pulmonary infections,
11 patients (20.4%) experienced septicemia, and 10 patients
(18.5%) experienced herpes zoster. The incidence of herpes
zoster was higher in the non-MSC group than the MSC group
(p=0.027), but there was no significant difference in the
incidence of pneumonia or sepsis between the groups (p>0.05,
Table 2).
3 cases, GVHD in 2 cases (1 each of severe aGVHD and cGVHD),
and leukoencephalopathy syndrome in 1 case.
Survival
The estimated 5-year OS rates in the MSC and non-MSC groups
were 78.3±6.1% and 70.1±6.3%, respectively, and the difference
was not significant (p=0.292, Figure 3a). However, the estimated
5-year GFFS rate in the MSC group was significantly higher
than that of the non-MSC group (76.6±6.2% vs. 56.7±6.9%,
respectively, p=0.045) (Figure 3b). In univariate analysis, age,
disease severity, donor-recipient relationship, graft type, and GF
significantly predicted OS and GFFS (Tables 3 and 4). Meanwhile,
grade II-IV aGVHD, grade III-IV aGVHD, and moderate-severe
cGVHD also predicted worse GFFS (Table 4). In multivariate
Cox regression analysis, only GF was found to be predictive
of worse OS (p=0.000, Table 3), while GF, grade III-IV aGVHD,
and moderate-severe cGVHD were significantly associated with
lower GFFS (p<0.05, Table 4).
Discussion
Haplo-HSCT is usually considered as a salvage treatment option
for SAA patients after failed IST. In recent years, some studies
have evaluated haplo-HSCT as an upfront therapy for newly
diagnosed SAA patients [6,20,21]. The currently available data
reflect a favorable survival outcome of haplo-HSCT in China.
However, higher incidences of GF and GVHD have limited its
clinical applications [7]. Numerous trials have been undertaken
to overcome these challenges, including the co-transplantation
of MSCs [13,22,23,24]. MSCs possess the capacity to differentiate
into various types of cells, modulate immune response, and
Transplantation-Related Mortality
During a median follow-up period of 29.5 (range: 6.5-91)
months, 10 and 14 patients died of transplantation-related
mortality (TRM) in the MSC and non-MSC groups, respectively,
with a median time to death of 91 (range: 31-270) and 76
(range: 1-274) days (p=0.334). The CIs of TRM in the MSC and
non-MSC groups were 21.7±6.1% and 27.2±6.2% (p=0.424,
Figure 2). Severe infection was the primary cause of death in
both groups (Table 2). In the MSC group, 4 patients died of
infection (3 pulmonary infections and 1 case of septicemia), 3
died of GVHD (1 severe aGVHD and 2 severe cGVHD), 2 of GF
(1 PGF and 1 secondary GF), and 1 of thrombotic microangiopathy.
In the non-MSC group, the causes of TRM included infection in
5 cases (4 pulmonary infections and 1 case of septicemia), GF in
3 cases (1 PGF and 2 secondary GF), regimen-related toxicities in
Figure 2. Cumulative incidences of transplantation-related
mortality (TRM) during follow-up in the mesenchymal stem
cell (MSC) group and non-MSC group were 21.7±6.1% and
27.2±6.2%, respectively (p=0.424).
HSCT: Hematopoietic stem cell transplantation.
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Figure 3. Overall survival and graft-versus-host disease (GVHD)-free, failure-free survival of the two groups. a) Comparison of estimated
5-year overall survival between mesenchymal stem cell (MSC) group and non-MSC group (78.3±6.1% vs. 70.1±6.3%, p=0.292). b)
Comparison of estimated 5-year GVHD-free, failure-free survival between MSC group and non-MSC group (76.6±6.2% vs. 56.7±6.9%,
p=0.045).
HSCT: Hematopoietic stem cell transplantation.
support hematopoiesis. They are currently widely used in cases
of hematological disease, and especially in treating aplastic
anemia and GF, promoting HSC engraftment, and preventing
and suppressing GVHD [25]. Given the encouraging results,
some researchers have tried co-transplanting MSCs with HSCs
in haplo-HSCT for SAA patients [9,12,13,14,26,27]. Due to the
rarity of SAA, however, there are still no trials directly evaluating
the role of MSCs in haplo-HSCT for SAA patients. To the best
of our knowledge, we present the first prospective, head-tohead
study comparing the efficacy and safety of haplo-HSCT
combined with or not combined with MSC infusion. Our results
have demonstrated that co-transplantation of MSC could
shorten the engraftment time, reduce the incidence of cGVHD,
and extended the GFFS.
GF is always a major risk of HSCT in cases of SAA, occurring
more frequently in these cases than in other malignant diseases.
In earlier years, the GF rate was as high as 70% [28]. In recent
years, the GF rate has significantly fallen to less than 10% due
to improved conditioning regimens, the use of G-CSF-mobilized
grafts, and cell therapy strategies such as cord-blood units
and MSC infusions [27,29,30,31,32]. MSCs have been shown to
support hematopoiesis in vitro and in vivo [25]. Some researchers
combined haplo-HSCT with MSC infusions in a series of singlearm
trials, and the promising results included engraftment rates
ranging from 93.2% to 98.9% [9,11,12,13,14,15]. Although the
current incidence of engraftment appears to be higher than
the historically reported values, there are no head-to-head
studies to date to confirm this. Our results showed no statistical
differences in the CIs of neutrophil engraftment (97.82±0.06%
vs. 97.96±0.06%, p=0.101) or platelet engraftment
(91.49±0.19% vs. 91.84±0.16%, p=0.345) in the MSC and non-
124
MSC groups. However, patients in the MSC group experienced
faster neutrophil engraftment (11 (range: 8-19) vs. 12 (range:
8-23) days, p=0.049) and platelet engraftment (12 (range: 7-43)
vs. 14 (range: 6-70) days, p=0.047) compared to those in the
non-MSC group. Our results thus indicate that co-transplantation
of MSCs could enhance engraftment in HSCT.
GVHD is another major challenge in haplo-HSCT for SAA. MSC
applications in the field of GVHD treatment have achieved great
success due to the immunoregulatory properties of these cells
[33,34]. However, the efficacy of MSCs for GVHD prophylaxis
varies in different reports [25,35]. In a phase II study, 37 patients
with hematological malignant diseases were randomly divided
into a standard GVHD prophylaxis group (Group 1) and a group
receiving standard GVHD prophylaxis combined with MSCs
(Group 2). Results showed that the incidence of grade II-IV
aGVHD was lower in Group 2 compared to Group 1 (5.3% vs.
38.9%, p=0.002) [36]. There have been no controlled studies
to date to confirm the efficacy of MSCs for GVHD prophylaxis
in SAA patients, although some studies reported that cotransplantation
of haplo-HSCs and MSCs in SAA patients was
safe and effective [12,13,14,15]. The reported CIs of grade II-IV
aGVHD, grade III-IV aGVHD, and cGVHD were previously found
to be approximately 23.5%-29.3%, 4.9%-11.4%, and 18.2%-
26.8%, respectively [12,13,14,15]. These results did not appear
to differ significantly from the historical findings. On the other
hand, a meta-analysis concluded that MSCs may make little
or no difference for the risk of GVHD compared to treatment
without MSCs in haplo-HSCT for SAA patients [16]. Our results
showed that the CI of cGVHD in the MSC group was significantly
lower compared to that of the non-MSC group (8.60±0.25%
vs. 24.57±0.48%, p=0.048). However, there were no statistical

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Table 3. Univariate and multivariate analysis of factors associated with overall survival.
Variable
Age, years
Univariate
OS
Multivariate
HR (95% CI) p HR (95% CI) p
21-39 vs. ≤20 0.544 (0.235-1.285) 0.011 0.296 (0.052-1.671) 0.168
≥40 vs. ≤20 0.185 (0.050-0.684) 0.155 0.674 (0.216-2.106) 0.497
Gender
Male vs. female 1.331 (0.616-2.873) 0.467
Disease severity
VSAA vs. SAA 0.275 (0.078-0.966) 0.044 0.443 (0.100-1.958) 0.283
SAA-PNH vs. SAA 0.346 (0.095-1.258) 0.107 0.418 (0.103-1.691) 0.221
Time from diagnosis to HSCT
≤2 vs. > 2 months 0.767 (0.355-1.654) 0.499
Previous treatment
ATG/ALG vs. CsA 0.843 (0.287-2.479) 0.757
Supportive care vs. CsA 1.280 (0.286-5.719) 0.747
Donor-recipient relationship
Mother-child vs. siblings 0.269 (0.082-0.883) 0.030 0.580 (0.157-2.149) 0.415
Father-child vs. siblings 0.684 (0.171-2.735) 0.591 2.246 (0.448-11.262) 0.325
Child-mother vs. siblings 0.381 (0.107-1.350) 0.135 0.933 (0.214-4.064) 0.927
Child-father vs. siblings 1.186 (0.411-3.420) 0.752 1.042 (0.314-3.455) 0.946
Graft type
PB vs. BM + PB 0.168 (0.040-0.715) 0.016 0.165 (0.025-1.094) 0.062
Treatment group
MSC vs. non-MSC 1.524 (0.691-3.359) 0.296
GF
Yes vs. no 0.073 (0.027-0.197) 0.000 0.080 (0.027-0.237) 0.000
Grade II-IV aGVHD
Yes vs. no 0.831 (0.349-1.978) 0.676
Grade III-IV aGVHD
Yes vs. no 0.491 (0.169-1.426) 0.191
Moderate-severe cGVHD
Yes vs. no 0.712 (0.168-3.014) 0.645
SAA: Severe aplastic anemia; VSAA: very severe aplastic anemia; PNH: paroxysmal nocturnal hemoglobinuria; HSCT: hematopoietic stem cell transplantation; ATG: antithymocyte
globulin; ALG: antilymphocyte globulin; CsA: cyclosporin A; BM: bone marrow; PB: peripheral blood; MSC: mesenchymal stem cell; aGVHD: acute graft-versus-host disease; cGVHD:
chronic GVHD; OS: overall survival; HR: hazard ratio; CI: confidence interval. Values of p<0.05 were considered to be significant.
differences in the CIs of grade II-IV aGVHD, grade III-IV aGVHD,
or moderate-severe cGVHD between the two groups. Another
phase II multicenter, randomized, double-blind controlled
study similarly demonstrated that prophylactic infusion of
MSCs after haplo-HSCT may reduce the incidence of cGVHD
in hematological malignant diseases (27.4% vs. 49%, p=0.021)
[37]. Our data and previous studies thus suggest that MSCs cotransplanted
during HSCT decrease the incidence of cGVHD to
some extent.
Infection is another major obstacle to survival, and particularly
lethal viral infections. Accordingly, another issue of concern
is whether MSCs increase the incidence of infection. In the
past, some studies suggested that MSCs did increase the risk
of infection by suppressing T-cell response and increasing the
secretion of some proinflammatory cytokines [38,39]. However,
our results showed that the incidences of all types of infections
did not increase after MSC administration. The incidence of
CMV and EBV reactivation in the MSC group was 29.8% and
66.6%, respectively, and these values were consistent with some
recent studies [14,40]. However, other studies reported a higher
risk of CMV reactivation (51.7% to 65.9%) and a lower risk
of EBV reactivation (22.7% to 31.8%) [2,12,13], which seems
to be contrary to our results. This may be related to different
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Table 4. Univariate and multivariate analysis of factors associated with graft-versus-host disease-free, failure-free survival.
Variable
Age, years
Univariate
GFFS
Multivariate
HR (95% CI) p HR (95% CI) p
21-39 vs. ≤20 0.152 (0.049-0.467) 0.001 0.634 (0.133-3.026) 0.567
≥40 vs. ≤20 0.387 (0.187-0.799) 0.010 1.033 (0.363-2.936) 0.951
Gender
Male vs. female 1.291 (0.659-2.530) 0.457
Disease severity
VSAA vs. SAA 0.351 (0.102-1.208) 0.097 0.461 (0.099-2.137) 0.322
SAA-PNH vs. SAA 0.577 (0.167-1.994) 0.384 0.500 (0.116-2.164) 0.354
Time from diagnosis to HSCT
≤2 vs. >2 months 0.633 (0.323-1.241) 0.183
Previous treatment
ATG/ALG vs. CsA 0.866 (0.331-2.263) 0.769
Supportive care vs. CsA 1.331 (0.357-4.958) 0.670
Donor-recipient relationship
Mother-child vs. siblings 0.228 (0.085-0.612) 0.003 0.403 (0.123-1.320) 0.133
Father-child vs. siblings 0.537 (0.165-1.745) 0.301 0.594 (0.120-2.941) 0.523
Child-mother vs. siblings 0.220 (0.068-0.716) 0.012 0.649 (0.149-2.830) 0.565
Child-father vs. siblings 0.949 (0.385-2.335) 0.909 1.495 (0.487-4.583) 0.482
Graft type
PB vs. BM + PB 0.222 (0.053-0.929) 0.039 0.504 (0.086-2.940) 0.446
Treatment group
MSC vs. non-MSC 2.053 (1.001-4.212) 0.050 1.694 (0.731-3.925) 0.219
GF
Yes vs. no 0.125 (0.051-0.311) 0.000 0.049 (0.015-0.162) 0.000
Grade II-IV aGVHD
Yes vs. no 0.447 (0.221-0.906) 0.025 1.022 (0.254-4.110) 0.975
Grade III-IV aGVHD
Yes vs. no 0.064 (0.025-0.161) 0.000 0.023 (0.004-0.137) 0.000
Moderate-severe cGVHD
Yes vs. no 0.242 (0.093-0.629) 0.004 0.128 (0.039-0.414) 0.001
SAA: Severe aplastic anemia; VSAA: very severe aplastic anemia; PNH: paroxysmal nocturnal hemoglobinuria; HSCT: hematopoietic stem cell transplantation; ATG: antithymocyte
globulin; ALG: antilymphocyte globulin; CsA: cyclosporin A; BM: bone marrow; PB: peripheral blood; MSC: mesenchymal stem cell; aGVHD: acute graft-versus-host disease; cGVHD:
chronic graft-versus-host disease; GFFS: graft-versus-host disease-free, failure-free survival; HR: hazard ratio; CI: confidence interval. Values of p<0.05 were considered to be
significant.
conditioning regimens and protocols for GVHD prophylaxis.
No significant differences were observed between the MSC
and non-MSC groups in the present study in terms of EBVassociated
PTLD, pulmonary infections, septicemia, and other
non-infection complications. Thus, our data indicate that MSC
applications are safe.
The rate of TRM in the MSC group was 21.7%, comparable to
the rate observed in the non-MSC group (27.2%, p=0.424).
Infection, GF, and GVHD were the most common causes of
death. We also observed comparable rates of OS between
the two groups (78.3±6.1% vs. 70.1±6.3%, p=0.292), with GF
being the main adverse factor. These findings were consistent
with previous reports [12,31]. Surprisingly, we found that
the estimated 5-year GFFS was significantly improved in the
MSC group compared to the non-MSC group (76.6±6.2% vs.
56.7±6.9%, p=0.045). Possible explanations for this finding
include the MSCs promoting faster engraftment to reduce
the chance of infection or MSCs reducing the risk of cGVHD
to improve the quality of life. GF, grade III-IV aGVHD, and
moderate-severe cGVHD were the factors that predicted worse
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GFFS. These findings suggest the need to reduce GF and GVHD,
which may improve survival outcomes. Co-transplantation of
MSCs during HSCT is a good attempt in this direction, although
some results were not satisfactory.
Our study has several limitations. First, certain shortcomings are
inherent in retrospective, single-center studies. A prospective,
multicenter, head-to-head clinical trial should be performed
to confirm our results. Second, other factors that may affect
engraftment and GVHD, such as variables of the patient
populations and conditioning regimens, should be controlled in
future studies. Third, many factors may influence the effects
of MSC treatment, such as the source of the MSCs (BM-MSCs
versus UC-MSCs), the frequency of MSC infusions (once,
twice, or more), therapeutic schedules, and the timing of MSC
administrations. All of these factors require more studies to
explore their impacts on efficacy. Despite these limitations,
however, our study offers remarkable comparative evidence
of the value of the prophylactic use of MSCs in haplo-HSCT
treatment for SAA patients.
Conclusion
The work presented here is the first head-to-head, retrospective
study to date to explore the efficacy and safety of the cotransplantation
of UC-MSCs during haplo-HSCT for the
treatment of patients with SAA. The encouraging results
suggest that infusion of MSCs after haplo-HSCT can promote
engraftment, reduce the incidence of cGVHD, and improve
GFFS. The prophylactic use of MSCs combined with haplo-HSCT
was found to be an effective and safe method for the treatment
of patients with SAA.
Acknowledgments: This work was supported by the Natural
Science Foundation of Zhejiang Province (No. LY19H290003), the
Zhejiang Provincial Medical and Health Science and Technology
Project (Nos. 2020KY196, 2018277310), the Foundation of
Zhejiang Province Chinese Medicine Science and Technology
Planes (Nos. 2017ZB030, 2020ZA044), and the Key Project of
the 2017 School Research Fund of Zhejiang Chinese Medical
University (No. 2017ZZ02).
Ethics
Ethics Committee Approval: This study was approved by the
Ethics Committee of the First Affiliated Hospital of Zhejiang
Chinese Medical University.
Informed Consent: All patients signed informed consent forms
in accordance with the Declaration of Helsinki.
Authorship Contributions
Surgical and Medical Practices: H-F.Z., X-F.S., H.L., L-L.H.;
Concept: H-F.Z.; Design: H-F.Z.; Data Collection or Processing:
H-F.Z., X-F.S., H.L., L-L.H.; Analysis or Interpretation: X-F.S., H.L.,
L-L.H.; Writing: X-F.S., H.L., L-L.H.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
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129

RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2022.2021.0670
Turk J Hematol 2022;39:130-135
Castleman Disease: A Multicenter Case Series from Turkey
Castleman Hastalığı: Türkiye’den Çok Merkezli Bir Olgu Serisi
Eren Gündüz 1 , Hakkı Onur Kırkızlar 2 , Elif Gülsüm Ümit 2 , Sedanur Karaman Gülsaran 2 , Vildan Özkocaman 3 ,
Fahir Özkalemkaş 3 , Ömer Candar 3 , Tugrul Elverdi 4 , Selin Küçükyurt 4 , Semra Paydaş 5 , Özcan Çeneli 6 , Sema Karakuş 7 ,
Senem Maral 8 , Ömer Ekinci 9 , Yıldız İpek 10 , Cem Kis 11 , Zeynep Tuğba Güven 12 , Aydan Akdeniz 13 , Tiraje Celkan 14 ,
Ayşe Hilal Eroğlu Küçükdiler 15 , Gülsüm Akgün Çağlıyan 16 , Ceyda Özçelik Şengöz 17 , Ayşe Karataş 18 , Tuba Bulduk 19 ,
Alper Özcan 12 , Fatma Burcu Belen Apak 7 , Aylin Canbolat 20 , İbrahim Kartal 21 , Hale Ören 22 , Ersin Töret 1 ,
Gül Nihal Özdemir 14 , Şule Mine Bakanay Öztürk 23
1Eskişehir Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey
2Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey
3Uludağ University Faculty of Medicine, Department of Hematology, Bursa, Turkey
4İstanbul University-Cerrahpaşa Cerrahpaşa Faculty of Medicine, Department of Hematology, İstanbul, Turkey
5Çukurova University Faculty of Medicine, Department of Hematology, Adana, Turkey
6Necmettin Erbakan University Meram Faculty of Medicine, Department of Hematology, Konya, Turkey
7Ankara Başkent University Faculty of Medicine, Department of Hematology, Ankara, Turkey
8University of Health Sciences Turkey Ankara Dışkapı Yıldırım Beyazıt Training and Research Hospital, Clinic of Hematology, Ankara, Turkey
9Fırat University Faculty of Medicine, Department of Hematology, Elazığ, Turkey
10Kartal Dr. Lütfi Kırdar City Hospital, Clinic of Hematology, İstanbul, Turkey
11Adana Başkent University Faculty of Medicine, Department of Hematology, Adana, Turkey
12Erciyes University Faculty of Medicine, Department of Hematology, Kayseri, Turkey
13Mersin University Faculty of Medicine, Department of Hematology, Mersin, Turkey
14İstinye University Faculty of Medicine, Department of Pediatric Hematology, İstanbul, Turkey
15Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey
16Pamukkale University Faculty of Medicine, Department of Hematology, Denizli, Turkey
17Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey
18Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey
19University of Health Sciences Turkey Gülhane Training and Research Hospital, Clinic of Hematology, Ankara, Turkey
20İstanbul Medeniyet University Faculty of Medicine, Department of Pediatric Hematology, İstanbul, Turkey
21Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Hematology, Samsun, Turkey
22Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey
23Ankara Yıldırım Beyazıt University Faculty of Medicine, Department of Hematology, Ankara, Turkey
Abstract
Objective: Castleman disease (CD) is a rare disease also known as
angiofollicular lymph node hyperplasia. The two main histological
subtypes are the hyaline vascular and plasma cell variants. It is further
classified as unicentric CD (UCD) or multicentric CD (MCD) according
to the anatomical distribution of the disease and the number of lymph
nodes involved. The aim of this multicenter study was to evaluate all
cases of CD identified to date in Turkey to set up a national registry to
improve the early recognition, treatment, and follow-up of CD.
Öz
Amaç: Anjiyofolliküler lenf nodu hiperplazisi olarak da bilinen
Castleman hastalığı (CH), nadir bir hastalık olup başlıca hiyalin vasküler
ve plazma hücreli olmak üzere 2 histolojik alt tipi vardır. Hastalığın
anatomik yayılımı ve tutulan lenf nodu bölgelerinin sayısına göre
unisentrik (UCH) ya da multisentrik (MCH) olarak sınıflandırılır. Bu
çok merkezli çalışmanın amacı bugüne kadar Türkiye’de tanımlanan
tüm CH olgularını tanımlamak, ulusal bir veri tabanı oluşturarak CH’de
erken tanı, tedavi ve takip sürecine katkı sağlamaktır.
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Eren Gündüz, M.D., Eskişehir Osmangazi University Faculty of Medicine, Received/Geliş tarihi: December 6, 2021
Department of Hematology, Eskişehir, Turkey
Accepted/Kabul tarihi: February 7, 2022
E-mail : erengunduz26@gmail.com ORCID: orcid.org/0000-0002-7660-4092
130

Turk J Hematol 2022;39:130-135
Gündüz E. et al: Case Series of Castleman Disease
Abstract
Materials and Methods: Both adult (n=130) and pediatric (n=10)
patients with lymph node or involved field biopsy results reported as
CD were included in the study. Patients’ demographic information,
clinical and laboratory characteristics, imaging study results, treatment
strategies, and clinical outcomes were evaluated retrospectively.
Results: A total of 140 patients (69 male and 71 female) with a
diagnosis of UCD (n=73) or MCD (n=67) were included. The mean
age was 39 years in the UCD group and 47 years in the MCD group.
Female patients were more common in the UCD group. The most
common histological subtype was hyaline vascular for both UCD
and MCD patients. Asymptomatic patients were more common in
the UCD group. Anemia, elevations of acute phase reactants, and
hypoalbuminemia were more common in the MCD group. The most
commonly used treatment strategies for UCD were surgical excision,
rituximab, and radiotherapy, respectively. All UCD patients were alive
at a median of 19.5 months of follow-up. The most commonly used
treatment strategies for MCD were methyl prednisolone, R-CHOP,
R-CVP, and rituximab. Thirteen MCD patients had died at a median of
34 months of follow-up.
Conclusion: This study is important in presenting the patient
characteristics and treatment strategies for CD from Turkey, with the
potential of increasing awareness about CD. Treatment data may help
in making decisions, particularly in countries that do not have access
to siltuximab. However, larger prospective studies are needed to make
definitive conclusions.
Keywords: Castleman disease, Unicentric, Multicentric, Treatment
Öz
Gereç ve Yöntemler: Çalışmaya lenf nodu ya da tutulan alandan
yapılmış biyopsi sonucu CH olarak rapor edilen hem erişkin
(n=130) hem de pediyatrik (n=10) hastalar dahil edildi. Hastaların
demografik bilgileri, klinik ve laboratuvar özellikleri, görüntüleme
bulguları, aldıkları tedaviler ve tedavi sonuçları geriye dönük olarak
değerlendirildi.
Bulgular: Dahil edilen 140 hastanın 69’u kadın, 71’i erkekti. Yetmiş
üç hasta UCH, 67 hasta MCH olarak sınıflandırılmıştı. Yaş ortalaması
UCH’de 39, MCH’de 47 yıl idi. Kadın hastalar UCH’de daha fazlaydı.
Hem UCH hem de MCH için en sık histolojik alt tip hiyalin vaskülerdi.
Asemptomatik hastalar UCH’de daha fazlaydı. Anemi, akut faz
reaktanı yüksekliği ve hipoalbuminemi MCH’de daha sıktı. UCH’de
en sık uygulanan tedaviler sırasıyla cerrahi eksizyon, rituksimab
ve radyoterapiydi. Median 19,5 aylık takipte tüm UCH’li hastalar
hayattaydı. MCH’de 1. basamak tedaviler metil prednizolon, R-CHOP,
R-CVP ve rituksimab idi. Median 34 aylık takipte 13 MCH’li hasta
kaybedilmişti.
Sonuç: Çalışmamız Türkiye’deki CH hastalarının özellikleri ve tedavi
yaklaşımlarını yansıtması açısından önemli olup hastalıkla ilgili
farkındalığın arttırılması potansiyeline sahiptir. Tedavi verileri özellikle
ülkemizde olduğu gibi siltuksimaba ulaşımı zor olan ülkelerde tedavi
seçimi konusunda fikir verebilir. Kesin sonuçlar çıkarmak için büyük
ölçekli prospektif çalışmalara ihtiyaç vardır.
Anahtar Sözcükler: Castleman hastalığı, Unisentrik, Multisentrik,
Tedavi
Introduction
Castleman disease (CD), also known as angiofollicular lymph
node hyperplasia or giant lymph node hyperplasia, was first
described in 1954 [1,2,3,4]. The two main histological subtypes
are the hyaline vascular and plasma cell variants, and a mixed
variant is seen occasionally [5]. It is further classified as
unicentric CD (UCD) or multicentric CD (MCD) according to the
anatomical distribution of the disease and the number of lymph
nodes involved [6,7]. The estimated incidence is approximately
25 cases per million person-years, which represents fewer than
5200 cases per year in the United States [8,9].
Classically, MCD presents with lymphadenopathy affecting
multiple lymph node stations and is associated with systemic
symptoms such as fever, weight loss, and fatigue, driven by
interleukin-6 and other cytokines. MCD has been subclassified
into human herpesvirus-8 (HHV-8)-associated MCD;
polyneuropathy, organomegaly, endocrinopathy, monoclonal
plasma cell disorder, skin changes (POEMS)-associated MCD; and
idiopathic MCD (iMCD). UCD, on the other hand, involves a single
enlarged lymph node or multiple enlarged lymph nodes within
a single lymph node region. The diagnosis of UCD is frequently
incidental and the lymphadenopathy is often asymptomatic.
However, some patients present with symptoms resulting from
compression of vital structures (e.g., the airways, neurovascular
bundles, or ureters), whereas others will experience iMCDlike
inflammatory syndromes. UCD is virtually always HHV-8-
negative, but rare positive cases have been reported [6].
The aim of this multicenter study is to evaluate all cases of
CD identified to date in Turkey to set up a national registry to
improve the early recognition, treatment, and follow-up of this
disease.
Materials and Methods
We included all patients with a diagnosis of CD based on the
histopathological analysis of a lymph node or other affected area.
Information about the patients was collected retrospectively
and included patients’ demographic information, treatment
strategy, clinical outcome, and the results of laboratory and
imaging studies. Data were collected between April 2018 and
August 2020 and the dates of diagnoses were between 2002
and 2020.
The study was approved by the local ethics committee of
Eskişehir Osmangazi University.
Statistical Analysis
We stratified our patient population based on centricity and
compared baseline clinical characteristics. Categorical variables
131

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Turk J Hematol 2022;39:130-135
are reported as counts and percentages; parametric continuous
variables are reported as means and standard deviations.
Nonparametric continuous variables are reported as medians
and interquartile ranges. To determine differences between
cohorts for categorical variables, we used chi-square and
Fisher exact tests. To determine differences for nonparametric
continuous variables, we used the Mann-Whitney U test. Finally,
to determine differences for parametric continuous variables,
we used the Student t-test. We also constructed Kaplan-Meier
survival curves for patients with UCD and MCD. All p-values are
two-tailed with a significance level of 0.05 reflecting statistical
significance. We performed the statistical analysis using IBM
SPSS Statistics 21.0 for Windows (IBM Corp., Armonk, NY, USA).
Results
A total of 140 patients (69 male and 71 female) from 21 centers
with a diagnosis of UCD (n=73) or MCD (n=67) were included
in the study. Patients were younger and female patients were
more common in the UCD group than the MCD group. The most
common histological type by lymph node biopsy was hyaline
vascular. Symptoms at diagnosis were similar between the
two groups except for fatigue, anorexia, fever, diarrhea, and
affected inguinal lymph nodes. Asymptomatic patients were
more common in the UCD group.
Physical examinations revealed significant differences between
the presence of hepatomegaly, splenomegaly, and affected
submandibular, axillary, and inguinal lymph nodes in the UCD
and MCD groups. Affected lymph nodes in the UCD group
were most frequently found in the submandibular region (n=5,
17.2%) (Table 1).
Serum C-reactive protein (CRP) level was increased in 15 (36.6%)
patients and elevated immunoglobulin (Ig) G was observed in 6
(18.8%) patients.
Imaging studies performed for patients with UCD included neck
computed tomography (CT) for 39 (56.5%), thorax CT for 43
(50%), abdominopelvic CT for 38 (46.9%), and positron emission
tomography (PET)/CT for 31 (44.3%) patients. Lymphadenopathy
was observed in the cervical area for 19 (44.2%), intrathoracic
area for 8 (20.5%), and abdominopelvic area for 14 (28.6%)
patients (Table 2).
Table 1. Clinical characteristics of the patients.
132
Unicentric (n=73) Multicentric (n=67) p
Age at diagnosis (years) 38.95±16.06 48.61±19.45 0.002
Sex ratio (female/male) 45/28 26/41 0.009
Histological subtype
Hyaline vascular
Plasma cell
Mixed
Unknown
Most frequent symptoms at diagnosis
Fatigue
Anorexia
Fever
Weight loss
Sweating
Cough
Diarrhea
Cervical lymph node involvement
Axillary lymph node involvement
Inguinal lymph node involvement
Abdominal pain
Physical examination
Hepatomegaly
Splenomegaly
Submandibular lymph node involvement
Submental lymph node involvement
Supraclavicular lymph node involvement
Axillary lymph node involvement
Inguinal lymph node involvement
53 (72.6%)
11 (15.1%)
5 (6.8%)
4 (5.5%)
15 (35.7%)
4 (22.2%)
4 (21.1%)
11 (40.7%)
6 (40%)
6 (37.5%)
0 (0%)
22 (53.7%)
6 (31.6%)
2 (16.7%)
19 (66.7%)
7 (25.9%)
3 (9.7%)
5 (17.2%)
1 (14.3%)
4 (26.7%)
7 (15.9%)
3 (7.9%)
26 (37.9%)
22 (33.3%)
5 (7.6%)
14 (21.2%)
27 (64.3%)
14 (77.8%)
15 (78.9%)
16 (59.3%)
9 (60%)
10 (62.5%)
4 (100%)
19 (46.3%)
13 (68.4%)
10 (83.3%)
5 (33.3%)
20 (74.1%)
28 (90.3%)
24 (82.8%)
6 (85.7%)
11 (73.3%)
37 (84.1%)
35 (92.1%)
<0.001
<0.001
<0.001
<0.001
0.015
0.012
0.007
0.266
0.451
0.311
0.048
1.000
0.085
0.021
0.359
0.004
0.012
<0.001
0.053
0.064
<0.001
<0.001

Turk J Hematol 2022;39:130-135
Gündüz E. et al: Case Series of Castleman Disease
The most common first-line treatment in cases of UCD was
surgical excision, followed by rituximab and radiotherapy.
Thirty-nine patients were followed without treatment (Table
3). Twenty-eight (87.5%) patients were in complete remission
and 3 (9.4%) patients were in partial remission after first-line
treatment. Response to first-line treatment was not evaluated
for 2 (6.3%) patients. Only 3 patients needed a second-line
treatment and their treatment responses were complete
remission. Second-line therapies were radiotherapy (n=1),
cyclosporine (n=1), and chemoimmunotherapy (R-ESHAP) (n=1).
At the last evaluation after a median follow-up of 19.5 (range:
7-52.5) months, all patients with UCD were alive (Figure 1).
The most common histological type of CD by lymph node biopsy
was also the hyaline vascular type in cases of MCD. Coronary
artery disease, chronic renal failure, and solid malignancy were
more common in the MCD group, possibly due to the older
mean age of this group. Kaposi sarcoma was reported for two
MCD patients.
The most common symptoms and physical examination findings
were reported as fever (n=15, 78.8%), affected inguinal
lymph nodes (n=10, 83.3%), hepatomegaly (n=20, 74.1%),
splenomegaly (n=28, 90.3%), arthralgia (n=2, 40%), abdominal
pain (n=5, 33.3%), fatigue (n=27, 33.3%), and diarrhea (n=4,
100%). Serum CRP levels were increased in 26 (63.4%) cases.
Elevated IgG levels were detected in 26 (81.3%) patients.
Anemia, thrombocytopenia, elevated erythrocyte sedimentation
rate, and hypoalbuminemia were more common in the MCD
group than the UCD group.
Imaging studies performed for MCD patients included neck
CT for 30 (43.5%), thorax CT for 43 (50%), abdominopelvic CT
for 43 (53.1%), and PET/CT for 39 (55.7%). Lymphadenopathy
was identified in the cervical area for 24 (55.8%) patients, the
intrathoracic area for 31 (79.5%), and the abdominopelvic area
for 35 (71.4%). Most of the affected lymph nodes identified
by thorax and abdominopelvic CT were smaller than 5 cm in
diameter.
SUV max
values in PET/CT were similar between the two groups
and most commonly below 6. However, PET/CT positivity was
more common in the MCD group.
Follow up (months)
Figure 1. Survival curves of patients.
Type of involvement
1: Unicentric Castleman disease, 2: Multicentric Castleman disease.
Table 2. Laboratory characteristics of the patients.
Laboratory findings
Anemia
Lymphopenia
Thrombocytopenia
Elevated ESR
Elevated CRP
Elevated β2 microglobulin
Hypoalbuminemia
Elevated IgG
Elevated IgA
HHV-8 positivity
HIV positivity
Imaging studies
Lymph node in neck CT
Lymph node in thorax CT
Lymph node in abdominopelvic CT
Activity in PET/CT
Unicentric (n=73) Multicentric (n=67) p
19 (34.5%)
8 (50%)
2 (13.3%)
9 (19.1%)
15 (36.6%)
11 (25.6%)
3 (9.1%)
6 (18.8%)
1 (5%)
0 (0%)
0 (0%)
19 (44.2%)
8 (20.5%)
14 (28.6%)
22 (36.1%)
36 (65.5%)
8 (50%)
13 (80.9%)
38 (80.9%)
26 (63.4%)
32 (74.4%)
30 (90.9%)
26 (81.3%)
19 (95%)
13 (100%)
0 (0%)
24 (55.8%)
31 (79.5%)
35 (71.4%)
39 (63.9%)
0.002
0.884
0.003
<0.001
0.048
<0.001
<0.001
<0.001
<0.001
<0.001
1.000
0.012
<0.001
<0.001
<0.001
ESR: Erythrocyte sedimentation rate; CRP: C-reactive protein; IgG: immunoglobulin G; IgA: immunoglobulin A; HHV-8: human herpesvirus-8; HIV: human immunodeficiency virus; CT:
computed tomography; PET: positron emission tomography.
133

Gündüz E. et al: Case Series of Castleman Disease
Turk J Hematol 2022;39:130-135
Patients in the MCD group received methyl prednisolone (n=4,
10.5%) R-CHOP (n=5, 13.2%), R-CVP (n=3, 7.9%), CVP (n=4,
10.5%), CHOP (n=3, 7.9%), and rituximab (n=16, 42.1%) as
first-line treatments (Table 2). Thirteen (37.1%) patients were
in complete remission and 13 (37.1%) patients were in partial
remission after first-line treatment. Five (14.3%) patients had
progressive and 4 (11.4%) patients had stable disease. Response
to first-line treatment was not evaluated for 3 patients in this
group.
More patients in the MCD group needed second-line treatment
compared to the UCD group (18.8% vs. 81.3%, p=0.021).
Second-line treatments included combined chemotherapy (CVP,
CHOP; n=5), chemoimmunotherapy (R-CVP, R-etoposide; n=3),
rituximab (n=1), lenalidomide (n=1), tocilizumab (n=1), and
surgical excision (n=1). Treatment responses in these cases were
progressive disease (n=1), complete remission (n=3), partial
remission (n=6), and stable disease (n=4). Two MCD patients
needed third-line (chemoimmunotherapy, tocilizumab) and 2
patients needed fourth-line (lenalidomide, methyl prednisolone)
treatment. One patient underwent autologous hematopoietic
stem cell transplantation as a fifth-line treatment and had
stable disease at the time of evaluation.
At the last evaluation after a median follow-up of 34 (range:
10-59) months, 13 (34.2%) patients with MCD had died (Figure
1).
Discussion
In this study, our aim was to review the largest cohort and set up
a national registry for a rare disease in Turkey. Another aim of
the study was to increase the awareness about CD and prevent
diagnostic delays.
UCD is reported in approximately 75% of cases of CD [10,11].
In our study, UCD patients accounted for 52% of all patients.
The plasmablastic subtype is different from the three main
histological types and is observed in HHV-8-positive patients
[12]. The plasmablastic subtype was not reported among our
patients, but the subtypes of several patients were not known.
Although Pribyl et al. [5] reported a marginal female
predominance among patients with UCD, CD generally affects
both sexes equally [1,13]. We found a significant female
predominance among our patients with UCD. Patients with
UCD tend to present in the second and fourth decades of life,
being significantly younger than those with MCD, the latter
of which has peak incidence in the sixth and seventh decades
of life [7,13]. Our patients with MCD were older than the UCD
patients but generally younger than MCD patients reported in
the literature.
Systemic symptoms and laboratory abnormalities are more
commonly reported in cases of MCD in the literature [2]. Our
MCD patients also had more systemic symptoms and laboratory
abnormalities than those with UCD.
Viral infections are postulated to play a role in the pathogenesis
of CD; in particular, an association of HHV-8 with MCD is
reported [14,15,16,17]. In a meta-analysis conducted by Talat
et al. [2], HHV-8 was reported to be positive in 46 of 49 (93.9%)
patients with MCD. HHV-8 was evaluated for 39 of our MCD
patients and was found to be positive in 13 (33.3%) cases.
Coinfections of HHV-8 and HIV are also commonly observed
[14], but none of our patients were HIV-positive.
Upon histopathological examination, UCD predominantly
consists of the hyaline vascular variant (90%), while in MCD the
plasmacytoid variant is most commonly observed [16,18,19]. The
hyaline vascular variant was the most common histopathological
subtype in both our UCD and MCD groups.
PET/CT is the suggested imaging method if it can be performed
[20]. Maximum SUV max
values are reported as ranging from 3
to 8 in cases of CD and lymphoma should be suspected in the
differential diagnosis if the value is above 8 [21,22]. In our study,
PET/CT SUV max
values were between 2.5 and 5.
MCD is a rare disease and no prospective randomized controlled
trials have been performed. Therefore, treatment strategies
are heterogeneous, particularly in cases of iMCD [23]. This
heterogeneity was also observed in our study and our data were
Table 3. First-line treatments of patients.
First-line treatment
R-CHOP
Rituximab
R-CVP
Surgical excision
Methyl prednisolone
CVP
CHOP
Radiotherapy
Unicentric (n=34)
1 (2.9%)
2 (5.9%)
0 (0%)
28 (82.4%)
1 (2.9%)
0 (0%)
0 (0%)
2 (5.9%)
Multicentric (n=38)
5 (13.2%)
16 (42.1%)
3 (7.9%)
2 (5.3%)
4 (10.5%)
4 (10.5%)
3 (7.9%)
1 (2.6%)
R: Rituximab; CHOP: cyclophosphamide, doxorubicin, vincristine, methyl prednisolone; CVP: cyclophosphamide, vincristine, methyl prednisolone.
134

Turk J Hematol 2022;39:130-135
Gündüz E. et al: Case Series of Castleman Disease
not sufficient for definitive conclusions. Although siltuximab is
commonly suggested as a first-line treatment, no experiences
with siltuximab were reported in our study because we are
unable to access this drug due to reimbursement obstacles in
Turkey. Central pathological revision was not performed and
this may be another limitation of our study.
Conclusion
We have reported the results of a multicenter retrospective study
of patients with CD, which is a rare disease. The data reported
here are important as they represent the patient characteristics
and treatment strategies from Turkey and have the potential of
increasing awareness about CD. Such treatment data may also
help in making decisions, particularly in countries that do not
have access to siltuximab. However, larger prospective studies
are needed to draw definitive conclusions regarding optimal
treatment options.
Acknowledgment: We would like to thank Associate Professor
Cengiz Bal from the Eskişehir Osmangazi University Faculty of
Medicine’s Department of Biostatistics for statistical analysis.
Ethics
Ethics Committee Approval: The study was approved by the
local ethics committee of Eskişehir Osmangazi University.
Informed Consent: Retrospective study.
Authorship Contributions
Surgical and Medical Practices: E.Ö., H.O.K., E.G.Ü., S.K.G., V.Ö.,
F.Ö., Ö.C., T.E., S.Kü., S.P., Ö.Ç., S.Ka., S.M., Ö.E., Y.İ., C.K., Z.T.G.,
A.A., T.C., A.E.H.K., G.A.Ç., C.Ö.Ş., A.K., T.B., A.Ö., F.B.B.A., A.C.,
İ.K., H.Ö., E.T., G.N.Ö., Ş.M.B.Ö.; Concept: E.G., S.Kü., G.N.Ö.,
Ş.M.B.Ö.; Design: E.G., S.Kü., G.N.Ö., Ş.M.B.Ö.; Data Collection
or Processing: E.G.; Analysis or Interpretation: E.G.; Literature
Search: E.G.; Writing: E.G.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The database project of this study was
supported by the Turkish Society of Hematology.
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135

PERSPECTIVE
DOI: 10.4274/tjh.galenos.2021.2021.0683
Turk J Hematol 2022;39:136-139
Gaucher Disease for Hematologists
Hematologlar için Gaucher Hastalığı
Gül Nihal Özdemir 1 , Eren Gündüz 2
1İstinye University Faculty of Medicine, Department of Pediatric Hematology Oncology, İstanbul, Turkey
2Eskisehir Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey
Abstract
Gaucher disease (GD) is a rare hereditary lysosomal storage disease
that arises due to deficiency of glucocerebrosidase. Early diagnosis
is very important for starting proper treatment and preventing
complications. Splenomegaly, anemia, and thrombocytopenia are the
most common findings in GD and so most patients are initially referred
to hematologists. The Turkish Society of Hematology established its
Rare Hematological Diseases Subcommittee in 2015. One of the main
topics of this subcommittee was to increase and improve awareness
and education of rare diseases among hematologists in Turkey.
This review presents GD with an overview of its clinical features,
pathophysiology, and treatment options for hematologists.
Keywords: Gaucher disease, Anemia, Thrombocytopenia, Splenomegaly
Öz
Gaucher hastalığı (GH) nadir görülen kalıtsal bir lizozomal depo
hastalığıdır. Gaucher hastaları sıklıkla geç tanı alırlar. Erken teşhis;
uygun tedaviye başlamak, komplikasyonları ve hastalığın ilerlemesini
önlemek için önemlidir. Splenomegali, anemi ve trombositopeni
GH’de en sık görülen bulgulardır, bu nedenle çoğu hasta başlangıçta
hematologlara sevk edilir. Türk Hematoloji Derneği 2015 yılında “Nadir
hematolojik hastalıklar alt komitesini” kurmuştur. Alt komitenin ana
amaçlarında biri Türkiye’deki hematologların nadir görülen hastalıklar
konusunda bilinçlendirilmesi ve eğitimlerinin artırılması olmuştur. Bu
derleme, hematologlar için GH’nin klinik özellikleri, patofizyolojisi ve
tedavi seçeneklerine bir bakış sunmaktadır.
Anahtar Sözcükler: Gaucher hastalığı, Anemi, Trombositopeni,
Splenomegali
Introduction
Gaucher disease (GD) is a rare hereditary lysosomal storage
disease [1]. It occurs due to deficiency of the lysosomal enzyme
glucocerebrosidase and the results of several compounds
related to the substrate accumulating in cell lysosomes. The
glucocerebrosidase 1 (GBA1) gene encoding glucocerebrosidase
is located on chromosome 1. This disease has traditionally
been classified into subtypes according to clinical findings,
course, and the patient’s ethnic origin. Type 1 GD (GD1, adult
type) is the most prevalent form. Visceral findings such as
hepatosplenomegaly, cytopenia, and bone disease are observed.
While cases of GD1 lack involvement of the central nervous
system, recent studies have shown that Parkinson disease and
peripheral neuropathy are more common in these cases [2]. Type
2 GD (GD2, infantile) is the most severe form and begins within
the first 6 months of life with a life expectancy of <2 years [3].
In addition to enlargement of the spleen and liver, progressive
neurological findings are observed. In type 3 GD (GD3, juvenile),
patients have both visceral and neurological findings with
longer survival. Recently, however, this classification according
to subtypes is being abandoned. The three subtypes are now
thought to be continuations of each other within the spectrum
of the same disease, rather than disorders with different
phenotypes.
Patients with GD often have delays in diagnosis of up to 10
years [4]. Early diagnosis is important for starting proper
treatment and preventing complications as well as disease
progression. Splenomegaly, anemia, and thrombocytopenia are
the most common findings in GD so most patients are initially
referred to hematologists with a differential diagnosis of
leukemia, lymphoma, or immune thrombocytopenia [5,6]. The
Turkish Society of Hematology established a Rare Hematological
Diseases Subcommittee in 2015. One of the main tasks of the
subcommittee was to increase and improve awareness and
education of rare diseases among hematologists in Turkey and
GD was selected as one of the target diseases within this project.
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Gül Nihal Özdemir, M.D., İstinye University Faculty of Medicine,
Department of Pediatric Hematology Oncology, İstanbul, Turkey
E-mail : gul.ozdemir@istinye.edu.tr ORCID: orcid.org/0000-0002-3204-4353
Received/Geliş tarihi: February 6, 2022
Accepted/Kabul tarihi: April 18, 2022
136

Turk J Hematol 2022;39:136-139
Özdemir G.N. and Gündüz E.: Gaucher Disease
Weekend courses, online educational meetings, and guidelines
for the diagnosis and treatment of GD were organized. This
review presents GD with an overview of its clinical features,
pathophysiology, and treatment options for hematologists.
Clinical Findings
There is a distinct phenotypic diversity in GD that cannot be
fully explained by the genotype [7,8,9]. The clinical findings
are shown in Table 1 [7]. The visceral organs, bone marrow
(Figure 1), and bones are affected in almost all cases.
The most common finding is splenomegaly [10]. Isolated
thrombocytopenia alone is the most common cytopenia.
Anemia and rarely leukopenia may be seen. Bone findings
may present as diffuse bone pain and pain attacks associated
with osteonecrosis. Osteolytic lesions, pathological fractures,
and compression fractures are more common in patients who
have undergone splenectomy. Many patients have growth
retardation and delayed puberty [11]. Interstitial lung disease
may be detected in rare cases [12].
Diagnosis
Laboratory and radiological findings are summarized in
Table 2 and Table 3. Serum angiotensin-converting enzyme
and particularly its tartrate-resistant isoenzymes may
be increased [13]. Levels of chitotriosidase, an enzyme
secreted from lipid-laden macrophages, are elevated [14].
Hyperferritinemia is frequently encountered in GD [15].
Lyso-GL1 (glucosylsphingosine), which is a downstream metabolic
product of glucosylceramide, has been identified as a promising
biomarker for the diagnosis and monitoring of patients with GD
in recent years [16]. The definitive diagnosis of GD is made with
glucocerebrosidase enzyme detection and genetic mutation
analysis. Glucocerebrosidase can be examined in peripheral
leukocytes or skin fibroblasts. For this, it is necessary to take a
dry blood sample on filter papers [17]. Genetic analysis provides
additional confirmation of the diagnosis and is also helpful for
genetic counseling and the detection of carriers [18]. GBA1 is
the only gene known to mutate in GD and the most common
Table 1. Signs and symptoms of Gaucher disease.
Splenomegaly 85%
Hepatomegaly 63%
Thrombocytopenia 68%
Anemia 34%
Bleeding
Osteopenia 55%
Bone pain 33%
Pathological fractures 7%
Bone crises 7%
Growth retardation 36%
Frequent (no percentage reported)
mutation is the N370S mutation [7]. Lipid-loaded macrophages
can be seen in the bone marrow in GD, but that is not a specific
finding. Pseudo-Gaucher cells can be seen in other diseases [20].
Bone marrow aspiration/biopsy is not required for the definitive
diagnosis but may be performed to rule out other diseases.
Treatment
The goals of treatment are the elimination of symptoms,
prevention of complications, and improvement of quality
of life [21]. Due to the heterogeneity of the disease and the
uncertainty of disease progression, the management should
be individualized. Enzyme replacement therapy ameliorates
most of the manifestations of GD1 and improves quality of
life [22]. Treatment is not recommended for GD2 patients as it
does not stop the clinical course. Enzyme replacement therapy
may be beneficial for GD3 patients who have chronic visceral
manifestations. Indications for starting treatment in cases of
GD1 are considered according to the severity of the disease
at the initial evaluation or according to the progression of
Table 2. Laboratory findings in cases of Gaucher disease.
• Cytopenia
* Anemia
* Thrombocytopenia
* Leukopenia
* Bicytopenia/pancytopenia
• Coagulation disorders
• Elevated liver enzymes
• Increase in serum ACE level (especially tartrate-resistant
isoenzymes)
• Increase in acid phosphatase activity
• Hyperferritinemia
• Increase in chitotriosidase
• Poly- and monoclonal gammopathy
• Lipid-loaded macrophages in tissues (bone marrow, liver, spleen)
Table 3. Radiological findings in cases of Gaucher disease.
• Bone radiography
° Erlenmeyer flask deformity
° Bone fractures and lytic lesions
• Bone magnetic resonance imaging (MRI)
° Bone marrow involvement
° Bone infarcts
° Osteonecrosis
• Dual-energy X-ray absorptiometry (DEXA)
° Osteopenia
• Abdominal ultrasonography (USG)
° Hepatomegaly
° Splenomegaly
• Echocardiography
° Pulmonary hypertension
• Chest X-ray/thorax computed tomography (CT)
° Lung involvement
137

Özdemir G.N. and Gündüz E.: Gaucher Disease
Turk J Hematol 2022;39:136-139
the disease. The severity of the disease can be evaluated with
scoring systems [23]. Enzyme replacement therapy for GD1
may include imiglucerase, velaglucerase alfa, and taliglucerase
alfa [24,25,26]. There is no consensus on the optimal dose or
frequency in the administration of recombinant enzymes.
The recommended dose for imiglucerase is 15-60 units of
enzyme/kg every 2 weeks intravenously. The ideal dose has
been set at 60 units/kg in most studies, but good response
was also shown at lower doses [27]. Treatment is life-long and
interruptions are not recommended. A small percentage of
patients may develop antibodies (15%) [28]. Glucosylceramide
synthase inhibitors (miglustat and eliglustat) are used for
substrate reduction therapy [29,30]. They reduce the amount of
substrate and prevent the symptoms that develop accordingly.
Miglustat is approved for patients with mild to moderate GD1
who cannot undergo enzyme therapy and also for the small
group of patients for whom enzyme therapy is unsuitable due
to adverse events or venous access problems [31]. The role of
splenectomy has decreased with the availability of enzyme
replacement therapy. Some studies have shown that total
splenectomy worsens bone findings in GD [32]. Bone marrow
transplantation offer the potential of cure, but no clinical trials
to date have assessed its safety and efficacy in comparison to
enzyme replacement therapy or substrate reduction therapy
[33].
Conclusion
Gaucher disease is a rare but treatable metabolic disease. High
levels of suspicion are necessary for early diagnosis as this disease
may present with different clinical findings. Early treatment will
be beneficial in preventing irreversible complications.
Authorship Contributions
Concept: G.N.Ö.; Design: G.N.Ö., E.G.; Literature Search: E.G.;
Writing: G.N.Ö.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
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patient with Gaucher disease.
6. Linari S, Castaman G. Hematological manifestations and complications of
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138

Turk J Hematol 2022;39:136-139
Özdemir G.N. and Gündüz E.: Gaucher Disease
Registry: Demographics and disease characteristics of 1698 patients with
Gaucher disease. Arch Intern Med 2000;160:2835-2843.
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Disease Registry. Mutation analysis and genotype/phenotype relationships
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10. Motta I, Filocamo M, Poggiali E, Stroppiano M, Dragani A, Consonni
D, Barcellini W, Gaidano G, Facchini L, Specchia G, Cappellini MD;
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study for early diagnosis of Gaucher disease in patients with splenomegaly
and/or thrombocytopenia. Eur J Haematol 2016;96:352-359.
11. Baris HN, Cohen IJ, Mistry PK. Gaucher disease: the metabolic defect,
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12. Miller A, Brown LK, Pastores GM, Desnick RJ. Pulmonary involvement in
type 1 Gaucher disease: functional and exercise findings in patients with
and without clinical interstitial lung disease. Clin Genet 2003;63:368-376.
13. Danilov SM, Tikhomirova VE, Metzger R, Naperova IA, Bukina TM, Goker-
Alpan O, Tayebi N, Gayfullin NM, Schwartz DE, Samokhodskaya LM, Kost
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14. van Dussen L, Hendriks EJ, Groener JE, Boot RG, Hollak CE, Aerts JM. Value
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15. Regenboog M, van Kuilenburg AB, Verheij J, Swinkels DW, Hollak CE.
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17. Herrera D, Monaga M, Campos D, Pampín Y, González EC, Lavaut K.
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glucocerebrosidase gene. Am J Hum Genet 1990;47:275-278.
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question. Turk J Hematol 2014;31:428-429.
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GA, Mistry PK, Tylki-Szymańska A. Therapeutic goals in the treatment of
Gaucher disease. Semin Hematol 2004;41(4 Suppl 5):4-14.
22. Charrow J, Scott CR. Long-term treatment outcomes in Gaucher disease.
Am J Hematol 2015;90(Suppl 1):S19-24.
23. Di Rocco M, Giona F, Carubbi F, Linari S, Minichilli F, Brady RO, Mariani
G, Cappellini MD. A new severity score index for phenotypic classification
and evaluation of responses to treatment in type I Gaucher disease.
Haematologica 2008;93:1211-1218.
24. Serratrice C, Carballo S, Serratrice J, Stirnemann J. Imiglucerase in the
management of Gaucher disease type 1: an evidence-based review of its
place in therapy. Core Evid 2016;11:37-47.
25. Smith L, Rhead W, Charrow J, Shankar SP, Bavdekar A, Longo N, Mardach
R, Harmatz P, Hangartner T, Lee HM, Crombez E, Pastores GM. Long-term
velaglucerase alfa treatment in children with Gaucher disease type 1 naïve
to enzyme replacement therapy or previously treated with imiglucerase.
Mol Genet Metab 2016;117:164-171.
26. Zimran A, Elstein D. Management of Gaucher disease: enzyme replacement
therapy. Pediatr Endocrinol Rev 2014;12(Suppl 1):82-87.
27. de Fost M, Hollak CE, Groener JE, Aerts JM, Maas M, Poll LW, Wiersma MG,
Häussinger D, Brett S, Brill N, vom Dahl S. Superior effects of high-dose
enzyme replacement therapy in type 1 Gaucher disease on bone marrow
involvement and chitotriosidase levels: a 2-center retrospective analysis.
Blood 2006;108:830-835.
28. Starzyk K, Richards S, Yee J, Smith SE, Kingma W. The long-term international
safety experience of imiglucerase therapy for Gaucher disease. Mol Genet
Metab 2007;90:157-163.
29. Van Rossum A, Holsopple M. Enzyme replacement or substrate reduction?
A review of Gaucher disease treatment options. Hosp Pharm 2016;51:553-
563.
30. Shemesh E, Deroma L, Bembi B, Deegan P, Hollak C, Weinreb NJ, Cox TM.
Enzyme replacement and substrate reduction therapy for Gaucher disease.
Cochrane Database Syst Rev 2015;2015:CD010324.
31. Heitner R, Elstein D, Aerts J, van Weely S, Zimran A. Low-dose
N-butyldeoxynojirimycin (OGT 918) for type I Gaucher disease. Blood Cells
Mol Dis 2002;28:127-133.
32. Fleshner PR, Aufses AH Jr, Grabowski GA, Elias R. A 27-year experience with
splenectomy for Gaucher’s disease. Am J Surg 1991;161:69-75.
33. Somaraju UR, Tadepalli K. Hematopoietic stem cell transplantation for
Gaucher disease. Cochrane Database Syst Rev 2017;10:CD006974.
139

IMAGES IN HEMATOLOGY
DOI: 10.4274/tjh.galenos.2020.2020.0693
Turk J Hematol 2022;39:140-141
Gallbladder Involvement of Diffuse Large B-Cell Lymphoma with
18
F-FDG PET/CT
18
F-FDG PET/CT Görüntüleme ile Diffüz Büyük B Hücreli Lenfomanın Safra Kesesi Tutulumu
Esra Arslan, Göksel Alçin, Tamer Aksoy, Tevfik Fikret Çermik
University of Health Sciences Turkey, İstanbul Training and Research Hospital, Clinic of Nuclear Medicine, İstanbul, Turkey
Figure 1. Involvement of multiple lymph nodes with high
radiopharmaceutical uptake in supra- and infradiaphragmatic
lymphatic stations.
CT: Computed tomography, PET: positron emission tomography, F: fusion,
MIP: maximum intensity projection.
Figure 2. Increased 18 F-FDG uptake was observed in newly
developed metastatic nodes in addition to previous lesions in
the supra/infradiaphragmatic regions on posttreatment PET/CT.
Additionally, focal 18 F-FDG uptake (dashed arrows) in the upper
outer quadrant of the right breast and unexpected gallbladder
(GB) wall involvement (arrows) were observed.
CT: Computed tomography, PET: positron emission tomography, F: fusion,
MIP: maximum intensity projection.
18
F-Fluoro-2-deoxy-glucose positron emission tomography/
computed tomography ( 18 F-FDG PET/CT) is widely used in staging,
restaging, and evaluation of treatment response in patients with
lymphoma. In this case report, a 59-year-old woman diagnosed
with diffuse large B-cell lymphoma (DLBCL) with gallbladder
involvement is presented. Immunohistochemical analysis
showed strongly positive CD79a, bcl-6, MUM1, bcl-2, and CD43;
weakly positive CD20 and CD5 staining; negative c-myc; and
Ki-67 of 85%. Interim 18 F-FDG PET/CT showed involvement of
multiple lymph nodes with high radiopharmaceutical uptake in
supra- and infradiaphragmatic lymphatic stations (Figure 1). On
the other hand, increased 18 F-FDG uptake was observed in newly
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Esra Arslan, M.D., University of Health Sciences Turkey, İstanbul
Training and Research Hospital, Clinic of Nuclear Medicine, İstanbul, Turkey
E-mail : dresraarslan@gmail.com ORCID: orcid.org/0000-0002-9222-8883
Received/Geliş tarihi: November 19, 2020
Accepted/Kabul tarihi: December 28, 2020
140

Turk J Hematol 2022;39:140-141
Arslan E. et al: Gallbladder Involvement of Diffuse Large B-Cell Lymphoma
developed metastatic nodes in addition to previous lesions in
the supra/infradiaphragmatic regions on posttreatment PET/CT.
Additionally, focal 18 F-FDG uptake (dashed arrows) in the upper
outer quadrant of the right breast and unexpected gallbladder
(GB) wall involvement (arrows) were observed (Figure 2).
18
F-FDG PET/CT is a diagnostic method for detecting metastatic
lesions in GB [1,2,3]. Although the involvement of the breasts
and thyroid in non-Hodgkin’s lymphoma is frequently reported,
involvement of the GB is extremely rare [4,5,6]. Al-Katib
et al. [7] detected extranodal involvement of the GB in an
83-year-old male with intravascular large B-cell lymphoma. Bai
et al. [8] reported increased 18 F-FDG uptake in the GB without
luminal pathology in a 15-year-old girl with Hodgkin’s disease.
In our case, 18 F-FDG PET/CT imaging revealed DLBCL-related
involvement in the GB. PET/CT is a useful tool for demonstrating
unexpected organ involvements, such as in the GB.
18
Keywords: F-FDG PET/CT, Gallbladder,
lymphoma
Non-Hodgkin’s
Anahtar Sözcükler: 18 F-FDG PET/BT, Safra kesesi, Non-Hodgkin
lenfoma
Ethics
Informed Consent: The patient provided verbal and written
consent for the use of the medical findings for research purposes.
Authorship Contributions
Surgical and Medical Practices: T.A.; Concept: E.A.,
T.F.Ç., Design: E.A.; Data Collection or Processing: G.A.;
Analysis or Interpretation: T.A., T.F.Ç.; Literature Search: G.A.;
Writing: E.A.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Chung PH, Srinivasan R, Linehan WM, Pinto PA, Bratslavsky G. Renal cell
carcinoma with metastases to the gallbladder: four cases from the National
Cancer Institute (NCI) and review of the literature. Urol Oncol 2012;30:476-
481.
2. Shaikh F, Awan O, Khan SA. 18F-FDG PET/CT imaging of gallbladder
adenocarcinoma-a pictorial review. Cureus 2015;7:e298.
3. Kursat O, Engin A, Nuri A, Seref K, Erkan O. Watch out for the unexpected:
sole gallbladder metastasis in a patient with malignant melanoma striked
by FDG-PET. J Nucl Med Radiat Ther 2015;6:2.
4. Dravid NV, Ningurkar NU, Nikumbh DB, Gadre AS. Extranodal primary non
hodgkin lymphoma of breast: multimodal approach to diagnosis. Indian J
Pathol Oncol 2018;5:349-351.
5. Binesh F, Akhavan A, Navabii H. Extranodal marginal zone B cell lymphoma
of MALT type with extensive plasma cell differentiation in a man with
Hashimoto’s thyroiditis. BMJ Case Rep 2011;2011:bcr0520114277.
6. Pezzuto R, Di Mauro D, Bonomo L, Patel A, Ricciardi E, Attanasio A, Manzelli
A. An unusual case of primary extranodal lymphoma of the gallbladder.
Hematol Rep 2017;9:6972
7. Al-Katib S, Colvin R, Sokhandon F. Intravascular large B-cell lymphoma
presenting with diffuse gallbladder wall thickening: a case report and
literature review. Case Rep Radiol 2018;2018:2494207.
8. Bai X, Wang X, Zhuang H. FDG accumulation in the lumen of the gallbladder
without related pathology. Clin Nucl Med 2018;43:383-385.
141

IMAGES IN HEMATOLOGY
DOI: 10.4274/tjh.galenos.2022.2021.0530
Turk J Hematol 2022;39:142-143
Mast Cell Leukemia in a Patient with Teratoma
Teratomlu Bir Hastada Mast Hücreli Lösemi
Yan Shen*, Zhenni Wang*, Huijun Lin*, Jinlin Liu
Laboratory Medicine Center, Department of Clinical Laboratory, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou
Medical College), Hangzhou, China
*These authors contributed equally to this work.
Figure 1. Blood smear showed peripheral blood involvement (40%) by promyelocyte-like leukemia cells characterized by a spindle shape
with packed and polar granule aggregates (A), dandelion-like circulating round cells (B), and prominent vacuoles in the cytoplasm (C).
Bone marrow aspirate smear showed clusters of cells resembling metastatic cells (30%) (D, E). Toluidine blue staining of the bone marrow
revealed the cytoplasm to be chemoreactive for mast cells (F).
A 29-year-old man was admitted to the hospital with the
complaints of left upper chest pain, asthenia, melena, and red
but not itchy rash across the whole body. Initial examination
for thrombocytopenia (38x10 9 /L), normocytic anemia (8.2 g/dL),
and white blood cell count (15.6x10 9 /L) yielded a diagnosis
of acute promyelocyte leukemia (APL). Physical examination
revealed hepatosplenomegaly.
A blood smear showed peripheral blood involvement (40%) by
promyelocyte-like leukemia cells (Figures 1A-1C). These cells
were characterized by a spindle shape with packed and polar
granule aggregates (Figure 1A), dandelion-like circulating round
cells (Figure 1B), and prominent vacuoles in the cytoplasm
(Figure 1C). A bone marrow aspirate smear showed clusters
of cells resembling metastatic cells (30%) (Figures 1D and 1E).
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Jinlin Liu, M.D., Ph.D., Zhejiang Provincial People’s Hospital,
Hangzhou Medical College, Cytology Unit, Department of Clinical Laboratory, Hangzhou, China
Phone : 0086-18768137609
E-mail : liujinlinhz@163.com ORCID: orcid.org/0000-0003-0502-4813
Received/Geliş tarihi: September 15, 2021
Accepted/Kabul tarihi: February 22, 2022
142

Turk J Hematol 2022;39:142-143
Shen Y. et al: Mast Cell Leukemia in a Patient with Teratoma
Additionally, chromosome analysis revealed a normal karyotype.
Considering that the patient had a history of malignant
mediastinal teratoma, teratoma metastasis to the bone marrow
was suspected. However, the flow cytometry for these abnormal
cells was positive for CD117, CD33, CD13, CD11c, CD4, CD38,
CD22, CD25, and CD2. Furthermore, toluidine blue staining of
the bone marrow revealed the cytoplasm to be chemoreactive
for mast cells (Figure 1F). Together with the KIT D816V mutation
identified in this patient, the diagnosis of mast cell leukemia
(MCL) was made according to the 2016 systemic mastocytosis
criteria of the World Health Organization. Thus, this patient was
treated with dasatinib, lucitanib, and dexamethasone, and later
with midostaurin, but no improvement was achieved and he
died two months later.
MCL is an aggressive form of systemic mastocytosis. Increasing
promyelocyte-like blasts in the peripheral blood were initially
suggestive of APL, and clusters of blast cells in the bone marrow
were suggestive of the metastasis of teratoma, factors that
could lead to the incorrect diagnosis of MCL. However, as
atypical features were of relevance for other hematological
malignancies, toluidine blue staining, immunostaining, and
KIT D816V mutation were evaluated to further support the
diagnosis of MCL.
Keywords: Mast cell leukemia, Teratoma
Anahtar Sözcükler: Mast hücreli lösemi, Teratom
Acknowledgments
The authors appreciate the support of the members of the
Department of Clinical Laboratory, Zhejiang Provincial People’s
Hospital.
Ethics
Informed Consent: This study did not involve personal
information; only laboratory data were reported. Patient
consent was therefore waived, and this waiver was approved by
the Ethics Committee of Zhejiang Provincial People’s Hospital.
Authorship Contributions
Data Collection or Processing: Y.S., Z.W., H.L.; Writing: J.L.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: This work was supported by the General
Research Program of the Zhejiang Provincial Department of
Health (no. 2018KY249).
143

LETTERS TO THE EDITOR
Turk J Hematol 2022;39:144-151
Caution Regarding the Difference Between Flower-Like
Lymphocytes and Flower-Like Plasma Cells
Çiçek Gibi Lenfositler ile Çiçek Gibi Plazma Hücreleri Arasındaki Farka Dikkat!
Jingnan Zhu 1,2 , Zhang Li 3 , Yong Wang 2 , Jinlin Liu 4
1Zhejiang Chinese Medical University, College of Medical Technology, Hangzhou, China
2Shangyu People’s Hospital of Shaoxing, Department of Clinical Laboratory, Shaoxing, China
3People’s Hospital of Zhenhai, Department of Clinical Laboratory, Ningbo, China
4Zhejiang Provincial People’s Hospital, Hangzhou Medical College, Department of Clinical Laboratory, Hangzhou, China
To the Editor,
We attentively read the paper by Sall et al. [1] recently
published in Turkish Journal of Hematology. These authors
reported a case of multiple myeloma showing abnormal
plasma cells with flower-shaped nuclear features. These cells
with flower-shaped nuclei have also been reported in cases
of plasma cell leukemia in previous studies [2,3,4,5]. The
authors described the morphological features of flower-like
cells in order to help pathologists identify them for diagnostic
purposes. However, the flower-like plasma cells with condensed
chromatin presented in Figure 1B of the original study [1]
look like flower-like lymphocytes. Furthermore, these cells
had not been confirmed by immunocytochemical staining.
CD20 or CD138 immunocytochemistry could have been used
to confirm whether these cells were flower-like plasma cells or
flower-like lymphocytes, as previously reported [6]. Therefore,
the differential diagnosis between flower-like lymphocytes and
flower-like plasma cells should be approached with caution.
As we know, the presence of flower-like lymphocytes is a wellknown
morphological feature in the peripheral blood of patients
with adult T-cell leukemia/lymphoma (ATLL) [7]. However,
evaluation of flower-like lymphocytes must be performed
carefully because these cells appear not only in ATLL patients but
also in patients with various other diseases entailing reactive or
neoplastic lesions, or even in healthy individuals [6,8].
In our experience, these flower-like lymphocytes are frequently
found in the peripheral blood in routine daily blood examination.
We can consider the daily peripheral blood of a hepatitis B
and an anemia patient for example. Flower-like lymphocytes
are clearly visible in the peripheral blood (Figures 1A and 1B).
Figure 1. Peripheral blood smears of a hepatitis B patient (A) and an anemia patient (B) reveal flower-like lymphocytes. These flower-like
lymphocytes clearly have flower-like nuclei (Wright-Giemsa staining, 1000 x ).
144

Turk J Hematol 2022;39:144-151
LETTERS TO THE EDITOR
Therefore, when encountering these flower-like cells in blood
smears, flower-like lymphocytes should also be considered for
the differential diagnosis of flower-like plasma cells.
Keywords: Flower-like lymphocyte, Flower-like plasma cell,
Difference
Anahtar Sözcükler: Çiçek benzeri lenfosit, Çiçek benzeri plazma
hücresi, Fark
Acknowledgment
The authors appreciate the encouragement and support of
the members of the cytology unit of the Clinical Laboratory
Department, Zhejiang Provincial People’s Hospital.
Ethics
Authorship Contributions
Surgical and Medical Practices: J.Z., Concept: J.L.; Design: Y.W.;
Data Collection or Processing: J.Z.; Analysis or Interpretation:
J.Z.; Literature Search: Z.L.; Writing: J.L.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Sall A, Seck M, Samb D, Faye B, Gadji M, Diop S, Touré AO. Flower-like
plasma cell nuclei in multiple myeloma. Turk J Hematol 2021;38:153-154.
2. Gajendra S. Flower-like plasma cell: a comment. Turk J Hematol
2021;38:228-229.
3. De Miguel Sánchez C, Robles de Castro D, Pisón Herrero C, Pérez Persona
E, Salcedo Cuesta L, Guinea de Castro JM. Primary plasma cell leukaemia
presenting with flower-shaped nuclei. Br J Haematol 2021;193:689.
4. Shibusawa M. Plasma cell leukaemia presenting as flower-shaped plasma
cells mimicking adult T-cell leukaemia or lymphoma. Lancet Haematol
2020;7:e270.
5. Delhommeau F, Huguet S, Gachet J, van den Akker J, Lagrange M. Primary
plasma cell leukemia mimicking an adult T-cell leukemia-lymphoma: a case
report. Acta Cytol 2010;54:187-189.
6. Kobayashi M, Horikawa M, Uehara T, Honda T, Kobayashi T, Sakai H. Flowerlike/clover
leaf lymphocytes appear in various diseases: cerebrospinal fluid
cytology case with review of the literature. Diagn Cytopathol 2015;43:88-
90.
7. Dahmoush L, Hijazi Y, Barnes E, Stetler-Stevenson M, Abati A. Adult T-cell
leukemia/lymphoma: a cytopathologic, immunocytochemical, and flow
cytometric study. Cancer 2002;96:110-116.
8. Yaghmour G, Thind R, Kryvenko ON, Ayyad H, Chen A, Kuriakose P. All that
glitters is not gold: differential diagnosis of clover-leaf lymphocytes. Int J
Hematol 2013;97:679-680.
To the Editor,
We thank Zhu et al. for their interest in our article.
We agree with their observations regarding lymphocytes.
However, in our patient, these flower-like plasma cells were
found only in the marrow. Cytochemistry was not performed,
but all relevant cytological evidence was visible on the smear:
for example, rouleaux, plasma cells, and nuclear budding. Upon
immunophenotyping, a homogeneous population of tumor
plasma cells was found. HBV and HTLV1 serologies were also
evaluated and were negative.
In the absence of clinical signs such as bone pain and
spontaneous fractures and in light of the monoclonal gammaglobulin
peak, cytologic immunophenotypic evidence, and
serological negativity, we agree that flower-like lymphocytes
could be considered in the differential diagnosis.
Sincerely,
Abibatou Sall, Moussa Seck, Diama Samb, Blaise Faye, Macoura
Gadji, Saliou Diop, Awa Oumar Touré
©Copyright 2021 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Yong Wang, M.D., Shangyu People’s Hospital of Shaoxing,
Department of Clinical Laboratory, Shaoxing, China / Jinlin Liu, M.D., Ph.D., Zhejiang Provincial People’s
Hospital, Hangzhou Medical College, Department of Clinical Laboratory, Hangzhou, China
E-mail : 291615783@qq.com/liujinlinhz@163.com ORCID: orcid.org/0000-0001-8530-3809/
Received/Geliş tarihi: March 2, 2022
Accepted/Kabul tarihi: March 17, 2022
DOI: 10.4274/tjh.galenos.2022.2022.0087
145

LETTERS TO THE EDITOR
Turk J Hematol 2022;39:144-151
Does Treatment of Hepatitis C Reduce Inhibitor Titers in
Hemophilia A?
Hepatit C Tedavisi Hemofili A’da İnhibitör Titrelerini Azaltır mı?
Memiş Hilmi Atay 1 , Emine Türkoğlu 2 , Engin Kelkitli 1
1Ondokuz Mayıs University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Samsun, Turkey
2Tokat Gaziosmanpaşa University Faculty of Medicine, Department of Infectious Diseases, Tokat, Turkey
To the Editor,
Hemophilia A is an X-linked recessive disorder characterized by
a deficiency of factor VIII in plasma [1]. It has been reported
that approximately 30% of patients who receive recombinant or
plasma-derived factor VIII prophylaxis may develop neutralizing
antibodies or inhibitors [2]. Bleeding episodes in patients with
low-responding inhibitors are treated with factor VIII, whereas
bleeding episodes in those with high-responding inhibitors are
treated with bypassing agents, such as recombinant activated
factor VIIa (rFVIIa) [3]. Here we discuss the case of a patient
with factor VIII inhibitors whose need for factor replacement
decreased with the treatment of chronic hepatitis C.
A 47-year-old male patient underwent surgery due to a fracture
of the right elbow. He had factor VIII deficiency and was first
diagnosed at the age of 10 with a factor VIII level of 4 IU. He
was treated with factor replacement due to bleeding episodes.
He did not have major bleeding episodes or major surgery.
The preoperative factor FVIII level was reported as 1.2 IU/mL.
Inhibitors could not be studied. Before the surgery, he was
given factor VIII concentrate loading of 3000 units and 2x1500
units (50 U/kg) for one week. On the seventh postoperative
day, the levels of factor VIII and inhibitor were assessed due to
ongoing bleeding from the operation site. Factor VIII was found
to be 2 IU/mL and the inhibitor to be 19.2 BU/mL. No factor
VIII inhibitor had been detected in the patient previously. The
treatment was switched to a bypassing agent, rFVIIa, at 5400 µg
(90 µg/kg/dose) with 3 doses to be administered every 3 hours.
Once the bleeding was under control, the rFVIIa treatment was
administered with decreasing frequency. The patient was HCVpositive
with HCV RNA of 550,000 units of copies. He tested
negative for hepatitis B surface antigens and HIV antibodies. The
patient, who had HCV genotype 2, was started on direct-acting
antiviral (DAA) drug treatment with glecaprevir/pibrentasvir at
1x3 tablets daily. After starting DAA treatment, the patient’s
need for factor rFVIIa decreased (Figure 1). As a result, the dose
frequency was decreased and treatment with rFVIIa continued
twice a week. After 8 weeks, HCV RNA was not detected. The
level of factor VIII inhibitor was detected as 1.2 BU/mL.
It has been found that patients who are HCV-positive with factor
VIII inhibitors have a complex immune profile characterized
by higher levels of pro-inflammatory and anti-inflammatory
cytokines compared to those who are not [4,5]. The cure rate of
chronic hepatitis C has reached 90% with DAA therapy [6]. Triple
treatment with interferon, telaprevir, and ribavirin has been
reported to result in a decrease in the level of inhibitors and the
need for recombinant factors in patients with hemophilia A [7].
We believe that DAA treatment reduces the need for rFVIIa by
lowering inhibitor levels through both suppression of hepatitis
C and modulation of cytokines.
Figure 1. Graph demonstrating inhibitor levels and factor VIIa dosage during direct-acting antiviral drug treatment.
146

Turk J Hematol 2022;39:144-151
LETTERS TO THE EDITOR
To the best of our knowledge, this case study is the first to report
reduced inhibitor levels and a decreased need for rFVIIa after
DAA treatment. This treatment may reduce inhibitor titers. The
treatment of hepatitis C may also play a role. We hope that our
findings will facilitate further studies involving larger numbers
of patients.
Keywords: FVIII deficiency, Hepatitis C, Inhibitor
Anahtar Sözcükler: FVIII eksikliği, Hepatit C, İnhibitör
Ethics
Informed Consent: Received.
Authorship Contributions
Surgical and Medical Practices: M.H.A., E.T., E.K.; Concept:
M.H.A.; Design: M.H.A.; Data Collection or Processing: M.H.A.;
Analysis or Interpretation: M.H.A., E.T., E.K.; Literature Search:
M.H.A., E.T., E.K.; Writing: M.H.A., E.T., E.K.
Conflict of Interest: The authors of this paper have no conflicts
of interest, including specific financial interests, relationships,
and/or affiliations relevant to the subject matter or materials
included.
References
1. Ljung RCR. How I manage patients with inherited haemophilia A and B and
factor inhibitors. Br J Haematol 2018;180:501-510.
2. Wight J, Paisley S. The epidemiology of inhibitors in haemophilia A: a
systematic review. Haemophilia 2003;9:418-435.
3. Srivastava A, Santagostino E, Dougall A, Kitchen S, Sutherland M, Pipe
SW, Carcao M, Mahlangu J, Ragni MV, Windyga J, Llinás A, Goddard NJ,
Mohan R, Poonnoose PM, Feldman BM, Lewis SZ, van den Berg HM, Pierce
GF; WFH Guidelines for the Management of Hemophilia Panelists and Co-
Authors. WFH guidelines for the management of hemophilia, 3rd edition.
Haemophilia 2020;26:1-158.
4. Martins ML, Chaves DG, da Silva-Malta MCF, Bolina-Santos E, Barbosa-
Stancioli EF, Carmo RA. Hepatitis C and history of FVIII inhibitor
development in a long-term cohort of Brazilian patients with haemophilia
A. Haemophilia 2020;26:130-133.
5. Bolina-Santos E, Chaves DG, da Silva-Malta MCF, Carmo RA, Barbosa-
Stancioli EF, Lobato Martins M. HCV infection in hemophilia A patients
is associated with altered cytokines and chemokines profile and might
modulate the levels of FVIII inhibitor. J Med Virol 2022;94:683-691.
6. Holmes JA, Thompson AJ. Interferon-free combination therapies for the
treatment of hepatitis C: current insights. Hepat Med 2015;7:51-70.
7. Singh G, Sass R, Alamiry R, Zein N, Alkhouri N. Hepatitis C treatment
with triple therapy in a patient with hemophilia A. World J Clin Cases
2013;16:106-107.
Financial Disclosure: The authors declared that this study
received no financial support.
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Memiş Hilmi Atay, M.D., Ondokuz Mayıs University Faculty of
Medicine, Department of Internal Medicine, Division of Hematology, Samsun, Turkey
Phone : +90 530 468 15 59
E-mail : draatay@gmail.com ORCID: orcid.org/0000-0001-9666-6955
Received/Geliş tarihi: March 2, 2022
Accepted/Kabul tarihi: April 8, 2022
DOI: 10.4274/tjh.galenos.2022.2022.0085
147

LETTERS TO THE EDITOR
Turk J Hematol 2022;39:144-151
Spontaneous Remission in Paroxysmal Nocturnal Hemoglobinuria:
An Extremely Rare Case
Paroksismal Noktürnal Hemoglobinüride Spontan Remisyon: Çok Nadir Bir Olgu
Özgür Mehtap,
Ayfer Gedük
Kocaeli University Faculty of Medicine, Department of Hematology, Kocaeli, Turkey
To the Editor,
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired
clonal disease with the main clinical manifestations of
hemolytic anemia, bone marrow failure, and thrombophilia [1].
While it was previously reported that spontaneous remission
develops in 15%-30% of patients with PNH, this rate is as low
as 3% in recent studies [2,3,4,5]. This inconsistency between
results is probably due to the differences in the definitions of
“spontaneous remission” used in these studies and the analysis
methods applied. Herein, we present a rare case of aplastic
anemia/PNH overlap syndrome where the patient achieved
remission under immunosuppressive and anticomplement
therapy.
In 2012, a 24-year-old female patient was admitted to our
hospital due to severe fatigue. Pancytopenia was detected in
the complete blood count. Biochemical analysis was normal,
except for a slight increase in lactate dehydrogenase and a low
haptoglobin level. Bone marrow biopsy revealed decreased bone
marrow cellularity (40%) and relative erythroid hyperplasia. A
flow cytometric evaluation revealed a PNH granulocyte clone
size of 56.3%. The patient was started on eculizumab treatment,
which continued for 6 years. PNH clone percentages and clinical
parameters over the years are shown in Table 1. The PNH
clone size gradually decreased from 56.3% to 12.96% under
eculizumab treatment. Repeated bone marrow biopsy revealed
Table 1. Paroxysmal nocturnal hemoglobinuria clone percentages and clinical parameters over the years.
Type II erythrocytes, % Type III erythrocytes, % Total erythrocytes, % Monocytes, % Granulocytes, %
2012 2.75 11.63 14.38 48 56.3
2013 1.18 19.5 21.3 51.6 55
2014 1 13.9 14.9 40.3 46.5
2015 0.75 13 13.75 42.4 45
2016 0.28 12.56 12.84 48.5 42.6
2017 0.10 8.9 9 24.5 31.76
2018 10.95 10.53 21.48 11.05 12.96
2019 1.81 0.11 1.92 4.52 4.86
2020 1.68 0.04 1.72 4.26 3.58
2021 0.1 1.1 1.2 1.39 0.72
Neutrophils, /mm 3 Hemoglobin, g/dL Platelets, /mm 3 LDH, NR: 135-225 IU/L
2012 950 8.9 44,000 297 8
2013 1380 9.26 90,300 123 77
2014 1340 9.6 107,000 144 63
2015 4750 11.2 90,300 129 97
2016 1730 13.3 89,800 152 93
2017 1513 10.8 82,900 146 81
2018 695 10.67 48,700 180 138
2019 1634 11.13 95,000 150 99
2020 1896 11.89 102,000 187 118
2021 2119 13.69 123,700 171 106
LDH: Lactate dehydrogenase; NR: normal range.
Haptoglobin,
NR: 30-200 mg/dL
148

Turk J Hematol 2022;39:144-151
LETTERS TO THE EDITOR
a decrease in cellularity to 35% and, due to the tendency of
a decreasing platelet count, cyclosporine was added to the
treatment at 200 mg/day. After 1 year of combined treatment,
the pancytopenia was resolved, PNH clone size had decreased
to 4.86%, and follow-up bone marrow biopsy showed an
increase in cellularity (65%). With these results, eculizumab
and cyclosporine treatment was discontinued in 2019. The PNH
clone size of the patient, who is still asymptomatic 27 months
after the cessation of treatment, continues to shrink. During the
entire clinical course, the patient did not need transfusions and
no complications related to PNH developed.
The disappearance of the PNH clone may be due to various
causes, such as complete recovery, deepening of bone
marrow aplasia, or transformation to leukemia. However, the
underlying mechanisms and the reasons for complete recovery
are unknown. One of the hypotheses put forward is that
clones of cells affected by PNH have a limited lifespan like
normal somatic cells [3]. There are also cases in the literature
that draw attention to the relationship between pathological
PNH clones and bone marrow environmental conditions [6,7].
Recent studies have shown that PNH is a multiclonal disease
and hosts additional somatic mutations that result in a complex
hierarchical clonal architecture similar to that observed in
myeloid neoplasms. Thus, it has been suggested that remission
of PNH may occur through the emergence of a new dominant
clone carrying multiple somatic mutations rather than
restoration of normal hematopoiesis [5,8,9]. As a result, the
highly variable clinical spectrum of PNH is also reflected in cases
of remission. Understanding the underlying pathophysiology
of this extremely rare condition will form the basis for the
development of curative treatments for the disease.
Keywords: Aplastic anemia, Paroxysmal nocturnal
hemoglobinuria, Eculizumab, Cyclosporine
Anahtar kelimeler: Aplastik anemi, Paroksismal noktürnal
hemoglobinüri, Ekulizumab, Siklosporin
Analysis or Interpretation: Ö.M, A.G.; Literature Search: Ö.M,
A.G.; Writing: Ö.M, A.G.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Parker C, Omine M, Richards S, Nishimura J, Bessler M, Ware R, Hillmen
P, Luzzatto L, Young N, Kinoshita T, Rosse W, Socié G; International PNH
Interest Group. Diagnosis and management of paroxysmal nocturnal
hemoglobinuria. Blood 2005;106:3699-3709.
2. Muñoz-Linares C, Ojeda E, Forés R, Pastrana M, Cabero M, Morillo D, Bautista
G, Baños I, Monteserín C, Bravo P, Jaro E, Cedena T, Steegmann JL, Villegas A,
Cabrera JR. Paroxysmal nocturnal hemoglobinuria: a single Spanish center’s
experience over the last 40 yr. Eur J Haematol 2014;93:309-319.
3. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of
paroxysmal nocturnal hemoglobinuria. N Engl J Med 1995;333:1253-1258.
4. Korkama ES, Armstrong AE, Jarva H, Meri S. Spontaneous remission in
paroxysmal nocturnal hemoglobinuria-return to health or transition into
malignancy? Front Immunol 2018;9:1749.
5. Gurnari C, Pagliuca S, Kewan T, Bahaj W, Mori M, Patel BJ, Visconte V,
Maciejewski JP. Is nature truly healing itself? Spontaneous remissions in
paroxysmal nocturnal hemoglobinuria. Blood Cancer J 2021;11:187.
6. Pulini S, Marando L, Natale A, Pascariello C, Catinella V, Del Vecchio L,
Risitano AM, Fioritoni G. Paroxysmal nocturnal hemoglobinuria after
autologous stem cell transplantation: extinction of the clone during
treatment with eculizumab - pathophysiological implications of a unique
clinical case. Acta Haematol 2011;126:103-109.
7. Hakim F, Childs R, Balow J, Cowan K, Zujewski J, Gress R. Development
of paroxysmal nocturnal hemoglobinuria after chemotherapy. Blood
1996;88:4725-4726.
8. Shen W, Clemente MJ, Hosono N, Yoshida K, Przychodzen B, Yoshizato
T, Shiraishi Y, Miyano S, Ogawa S, Maciejewski JP, Makishima H. Deep
sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal
hemoglobinuria. J Clin Invest 2014;124:4529-4538.
9. Babushok DV, Stanley N, Xie HM, Huang H, Bagg A, Olson TS, Bessler
M. Clonal replacement underlies spontaneous remission in paroxysmal
nocturnal haemoglobinuria. Br J Haematol 2017;176:487-490.
Authorship Contributions
Surgical and Medical Practices: Ö.M, A.G.; Concept: Ö.M, A.G.;
Design: Ö.M, A.G.; Data Collection or Processing: Ö.M, A.G.;
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Ayfer Gedük, M.D., Kocaeli University Faculty of Medicine,
Department of Hematology, Kocaeli, Turkey
E-mail : ayfergeduk@hotmail.com ORCID: orcid.org/0000-0002-5603-1178
Received/Geliş tarihi: December 7, 2021
Accepted/Kabul tarihi: January 27, 2022
DOI: 10.4274/tjh.galenos.2022.2021.0672
149

LETTERS TO THE EDITOR
Turk J Hematol 2022;39:144-151
Hematopoietic Stem Cell Transplantation to a Patient with Acute
Myeloid Leukemia from a Sibling Donor Positive for SARS-CoV-2
by RT-PCR Test
SARS-CoV-2 RT-PCR Testi Pozitif Kardeş Donörden Akut Myeloid Lösemili Bir Hastaya
Hematopoetik Kök Hücre Nakli
Ahmet Koç 1,2 , Ömer Doğru 1,2 , Nurşah Eker 1,2 , Burcu Tufan Taş 1,2 , Rabia Emel Şenay 1,2
1Marmara University Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey
2Marmara University Training and Research Hospital, Pediatric Bone Marrow Transplant Unit, İstanbul, Turkey
To the Editor,
Hematopoietic stem cell transplantation (HSCT) is the preferred
treatment modality in cases of high-risk pediatric acute myeloid
leukemia (AML). However, due to the ongoing coronavirus
disease-2019 (COVID-19) pandemic, there is a risk that stem cell
donors may be positive for SARS-CoV-2 by reverse transcription
polymerase chain reaction (RT-PCR) and that the ability to find
suitable donors will be impacted [1,2]. In addition, it has been
reported that the virus can be found in the blood of patients
infected with SARS-CoV-2 [3]. Stem cell collection is not
recommended by the European Society for Blood and Marrow
Transplantation or other international organizations when the
donor’s RT-PCR test is positive for SARS-CoV-2 [1,2]. However,
in the literature, cases of HSCT performed with SARS-CoV-2-
positive donors due to necessity have been reported for a small
number of patients [4,5,6]. In this letter, we present the case of
a patient with high-risk AML who underwent HSCT from a fully
HLA-matched 22-year-old sibling with SARS-CoV-2 positivity
by RT-PCR test. Considering that the COVID-19 pandemic is
still ongoing with the emergence of new mutations, we wanted
to share this experience with colleagues who may encounter
similar situations.
A 15-year-old girl with a diagnosis of AML-M4 was included
in the high-risk AML group due to monosomy-7 positivity. She
received the first four blocks of the AML-BFM 2019 treatment
protocol (cytarabine, idarubicin, and etoposide in the first
treatment block; high-dose cytarabine and mitoxantrone
in the second treatment block; cytarabine and idarubicin
in the third treatment block; and high-dose cytarabine and
mitoxantrone in the fourth treatment block) and good response
was obtained. Before transplantation, her bone marrow was in
complete remission morphologically. Minimal residual disease
as measured by flowcytometry was below 1%. Monosomy-7
disappeared after the first block of induction therapy and
was also negative before transplantation. A nasopharyngeal
swab from the donor before conditioning began was
negative for SARS-CoV-2 by RT-PCR. Intravenous busulfan
(3.2 mg/kg/day, days -7 to -4), fludarabine (30 mg/m 2 /day,
days -7 to -3), and melphalan (140 mg /m 2 /day, day -1) were
administered as myeloablative conditioning. However, the
donor’s RT-PCR on the day before the transplant was positive for
SARS-CoV-2. The donor was asymptomatic and did not receive
any medication for COVID-19. There was no chance of finding
another donor for the patient at this stage. The transplant was
delayed 1 day and the donor’s SARS-CoV-2 test performed 2 days
after the previous test was again found to be positive. Since the
patient had serious infectious conditions during AML treatment,
it was decided to proceed with the transplant considering that
the risk of serious infection would increase significantly if the
transplantation was not performed at this time and the bone
marrow remained aplastic.
Following all personal protection rules issued by all relevant
health institutions, CD34+ peripheral blood stem cells were
collected from the donor by apheresis and were transfused
to the patient on the same day without any manipulation.
The patient and her accompanying mother wore appropriate
personal protective equipment during the transplantation
and did not develop any fever or other COVID-related
symptoms. Cyclosporine A (3 mg/kg/day, intravenous) and
methotrexate (10 mg/kg/day, on days 1, 3, and 6) were used for
graft-versus-host disease prophylaxis. Ciprofloxacin,
voriconazole, and acyclovir were used for infection prophylaxis.
Neutrophil engraftment occurred on day 14 and platelet
engraftment on day 16. The patient underwent a weekly SARS-
CoV-2 test after transplantation and all results were negative.
Bone marrow examination showed 100% donor chimerism at
the end of the fourth week. The patient is currently alive and
healthy in the fifth month after transplantation.
150

Turk J Hematol 2022;39:144-151
LETTERS TO THE EDITOR
In conclusion, this case supports the reports in the literature
that hematopoietic stem cells from asymptomatic SARS-CoV-2-
positive donors may be safe.
Keywords: Acute myeloid leukemia, COVID-19, Hematopoietic
stem cell transplantation
Anahtar Sözcükler: Akut myeloid lösemi, COVID-19,
Hematopoietik kök hücre nakli
Informed Consent: Obtained.
Authorship Contributions
Surgical and Medical Practices: A.K., Ö.D., N.E., B.T.T., R.E.Ş.;
Concept: A.K., Ö.D., N.E., B.T.T., R.E.Ş.; Design: A.K., Ö.D., N.E.,
B.T.T., R.E.Ş.; Data Collection or Processing: A.K., Ö.D., N.E., B.T.T.,
R.E.Ş.; Analysis or Interpretation: A.K., Ö.D., N.E., B.T.T., R.E.Ş.;
Literature Search: A.K.; Writing: A.K., Ö.D., N.E., B.T.T., R.E.Ş.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
References
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S, Duarte R, Dolstra H, Lankester AC, Mohty M, Montoto S, Murray J, de
Latour RG, Snowden JA, Yakoub-Agha I, Verhoeven B, Kröger N, Styczynski
J, for the European Society for Blood and Marrow Transplantation. The
challenge of COVID-19 and hematopoietic cell transplantation; EBMT
recommendations for management of hematopoietic cell transplant
recipients, their donors, and patients undergoing CAR T-cell therapy. Bone
Marrow Transplantation 2020;55:2071-2076.
2. Waghmare A, Abidi MZ, Boeckh M, Chemaly RF, Dadwal S, Boghdadly Z,
Kamboj M, Papanicolaou GA, Pergam SA, Shahid Z. Guidelines for COVID-19
management in hematopoietic cell transplantation and cellular therapy
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safety. Transfus Med Rev 2020;34:75-80.
4. Anurathapan U, Apiwattanakul N, Pakakasama S, Pongphitcha P,
Thitithanyanont A, Pasomsub E, Hongeng S. Hematopoietic stem cell
transplantation from an infected SARS-CoV2 donor sibling. Bone Marrow
Transplant 2020;55:2359-2360.
5. Lázaro Del Campo P, de Paz Arias R, Ramírez López A, de la Cruz Benito B,
Humala Barbier K, Sánchez Vadillo I, López de la Guía A, de Soto Álvarez T,
Jiménez Yuste V, Canales Albendea M. No transmission of SARS-CoV-2 in
a patient undergoing allogeneic hematopoietic cell transplantation from
a matched-related donor with unknown COVID-19. Transfus Apher Sci
2020;59:102921.
6. Leclerc M, Fourati S, Menouche D, Challine D, Maury S. Allogeneic
haematopoietic stem cell transplantation from SARS-CoV-2 positive
donors. Lancet Haematol 2021;8:e167-169.
©Copyright 2022 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Ahmet Koç, Ph.D. Marmara University Faculty of Medicine,
Department of Pediatric Hematology and Oncology, İstanbul, Turkey
E-mail : ahmetkoc1@hotmail.com ORCID: orcid.org/0000-0001-7940-2640
Received/Geliş tarihi: March 2, 2022
Accepted/Kabul tarihi: April 26, 2022
DOI: 10.4274/tjh.galenos.2022.2022.0086
151

Advisory Board of This Issue (June 2022)
Ahmet Emre Eşkazan, Turkey
Ahmet Muzaffer Demir, Turkey
Akif Selim Yavuz, Turkey
Ana Planinc-Peraica, Croatia
Atilla Özkan, Turkey
Burhan Ferhanoğlu, Turkey
Elif Gülsüm Ümit, Turkey
Emin Kaya, Turkey
Emine Zengin, Turkey
Emre Tekgündüz, Turkey
Esra Yıldızhan, Turkey
Fergün Yılmaz, Turkey
Hale Ören, Turkey
Hasan Hashem, Jordan
İnci Alacacıoğlu, Turkey
İrfan Yavaşoğlu, Turkey
Lenka Besse, Switzerland
Massimo Martino, Italy
Mehmet Can Uğur, Turkey
Müge Sayitoğlu, Turkey
Mustafa Baydar, Turkey
Nadim Mahmud, USA
Neslihan Andıç, Turkey
Nükhet Tüzüner, Turkey
Ömür Gökmen Sevindik, Turkey
Ömür Kayıkçı, Turkey
Rejin Kebudi, Turkey
Rouslan Kotchetkov, Canada
Sinan Demircioğlu, Turkey
Stephanie Poulain, France
Tekin Aksu, Turkey
Theoni Kanellopoulou, Greece
Tuba Karapınar, Turkey
Tuğrul Elverdi, Turkey
Tülin Tiraje Celkan, Turkey
Ümit Yavuz Malkan, Turkey