tjh-v38i4

Volume 38 Issue 4

December 2021

E-ISSN: 1308-5263

Research Articles

Clinical Significance of TP53 Abnormalities in Newly Diagnosed Multiple Myeloma

Fang Ye, Tongtong Wang, Aijun Liu, Yanchen Li, Ningning Li, Huan Wang, Wenming Chen; Beijing, China

Generation of Induced Pluripotent Stem Cells from Patients with Multiple Myeloma

İrem Yılmaz Başaran, Erdal Karaöz; Eskişehir, İstanbul, Turkey

LncRNA-DUXAP8 Regulation of the Wnt/β-Catenin Signaling Pathway to Inhibit Glycolysis and Induced

Apoptosis in Acute Myeloid Leukemia

Hong Zhai, Junting Zhao, Juan Pu, Pan Zhao, Jin Wei; Nanchong, China

Efficacy and Safety of Ibrutinib Therapy in Patients with Chronic Lymphocytic Leukemia: Retrospective Analysis

of Real-Life Data

Anıl Tombak, Funda Pepedil Tanrıkulu, Salih Sertaç Durusoy, Hüseyin Derya Dinçyürek, Emin Kaya,

Elif Gülsüm Ümit, İrfan Yavaşoğlu, Özgür Mehtap, Burak Deveci, Mehmet Ali Özcan, Hatice Terzi, Müfide Okay,

Nilgün Sayınalp, Mehmet Yılmaz, Vahap Okan, Alperen Kızıklı, Ömer Özcan, Güven Çetin, Sinan Demircioğlu,

İsmet Aydoğdu, Güray Saydam, Eren Arslan Davulcu, Gül İlhan, Mehmet Ali Uçar, Gülsüm Özet, Seval Akpınar,

Burhan Turgut, İlhami Berber, Erdal Kurtoğlu, Mehmet Sönmez, Derya Selim Batur, Rahşan Yıldırım,

Vildan Özkocamaz, Ahmet Kürşad Güneş, Birsen Sahip, Şehmus Ertop, Olga Meltem Akay, Abdülkadir Baştürk,

Mehmet Hilmi Doğu, Aydan Akdeniz, Ali Ünal, Ahmet Seyhanlı, Emel Gürkan, Demet Çekdemir,

Burhan Ferhanoğlu; Mersin, Adana, Gaziantep, Malatya, Edirne, Aydın, Kocaeli, Antalya, İzmir, Sivas, Ankara,

İstanbul, Konya, Manisa, Hatay, Tekirdağ, Trabzon, Erzurum, Bursa, Şanlıurfa, Zonguldak, Kayseri, Turkey

Pre-Conditioning Serum Uric Acid as a Risk Factor for Sinusoidal Obstruction Syndrome of the Liver in Children

Undergoing Hematopoietic Stem Cell Transplantation

Fatma Visal Okur, Murat Karapapak, Khaled Warasnhe, Umut Ece Arslan, Barış Kuşkonmaz, Duygu Çetinkaya;

Ankara, Turkey

Thrombolysis with Systemic Recombinant Tissue Plasminogen Activator in Children: A Multicenter Retrospective

Study

Emine Zengin, Nazan Sarper, Arzu Yazal Erdem, Işık Odaman Al, Melike Sezgin Evim, Neşe Yaralı, Burcu Belen,

Arzu Akçay, Ayşen Türedi Yıldırım, Tuba Hilkay Karapınar, Adalet Meral Güneş, Sema Aylan Gelen, Hale Ören,

Lale Olcay, Birol Baytan, Hüseyin Gülen, Gülyüz Öztürk, Mehmet Fatih Orhan, Yeşim Oymak, Sibel Akpınar,

Özlem Tüfekçi, Meryem Albayrak, Burçak Tatlı Güneş, Aylin Canpolat, Namık Özbek; Kocaeli, Ankara, İzmir, Bursa,

İstanbul, Manisa, Sakarya, Kırıkkale, Turkey

Assessment of Long-Term Hematologic Effects in Differentiated Thyroid Cancer Patients Treated with Radioactive

Iodine

Bircan Sönmez, Özlen Bektaş, Nergiz Erkut, Mehmet Sönmez; Trabzon, Turkey

Cover Picture:

Nabhajit Mallik, Man Updesh Singh

Sachdeva

Bone Marrow Oxalosis: Crystal

Flowers in the Bone Marrow Garden

4

Editor-in-Chief

Reyhan Küçükkaya

İstanbul, Turkey

rkucukkaya@hotmail.com

Associate Editors

A. Emre Eşkazan

İstanbul University-Cerrahpaşa, İstanbul, Turkey

emre.eskazan@istanbul.edu.tr

Ali İrfan Emre Tekgündüz

Memorial Bahçelievler Hospital, İstanbul, Turkey

emretekgunduz@yahoo.com

Ayşegül Ünüvar

İstanbul University, İstanbul, Turkey

aysegulu@hotmail.com

Cengiz Beyan

Ankara, Turkey

cengizbeyan@hotmail.com

Hale Ören

Dokuz Eylül University, İzmir, Turkey

hale.oren@deu.edu.tr

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey

haznedar@yahoo.com

Selami Koçak Toprak

Ankara University, Ankara, Turkey

sktoprak@yahoo.com

Semra Paydaş

Çukurova University, Adana, Turkey

sepay@cu.edu.tr

Şule Ünal


suleunal2003@hotmail.com

Assistant Editors

Claudio Cerchione

University of Naples Federico II Napoli,

Campania, Italy

Ebru Koca

Başkent University Ankara Hospital,

Clinic of Hematology, Ankara, Turkey

Elif Ünal İnce


İnci Alacacıoğlu

Dokuz Eylül University, İzmir, Turkey

Mario Tiribelli

University of Udine, Udine, Italy

Müge Sayitoğlu

İstanbul University, İstanbul, Turkey

Nil Güler

Ondokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay

Koç University, İstanbul, Turkey

Veysel Sabri Hançer

İstinye University, İstanbul, Turkey

Zühre Kaya

Gazi University, Ankara, Turkey

International Review Board

Nejat Akar

TOBB University of Economics and Technology Hospital, Ankara, Turkey

Görgün Akpek

Maryland School of Medicine, Baltimore, USA

Serhan Alkan

Cedars-Sinai Medical Center, Los Angeles, USA

Çiğdem Altay

Ankara, Turkey

Meral Beksaç


Koen van Besien

Weill Cornell Medicine, New York, USA

M. Sıraç Dilber Karolinska University, Stockholm, Sweden

Ahmet Doğan

Memorial Sloan Kettering Cancer Center, New York, USA

Peter Dreger

Heidelberg University, Heidelberg, Germany

Thierry Facon

Lille University, Lille, France

Jawed Fareed

Loyola University, Maywood, USA

Burhan Ferhanoğlu

Koç University, İstanbul, Turkey

Gösta Gahrton

Karolinska University Hospital, Stockholm, Sweden

Dieter Hoelzer

Frankfurt University, Frankfurt, Germany

Andreas Josting

University Hospital Cologne, Cologne, Germany

Emin Kansu


Winfried Kern

Albert Ludwigs University, Freiburg im Breisgau, Germany

Nigel Key

University of North Carolina School of Medicine, NC, USA

Korgün Koral

Southwestern Medical Center, Texas, USA

Abdullah Kutlar

Medical College of Georgia at Augusta University, Augusta, USA

Luca Malcovati

Pavia Medical School University, Pavia, Italy

Marilyn Manco-Johnson University of Colorado Anschutz Medical Campus, Aurora, USA

Robert Marcus

King’s College Hospital, London, UK

Jean Pierre Marie

Pierre et Marie Curie University, Paris, France

Ghulam Mufti

King’s Hospital, London, UK

Gerassimos A. Pangalis Athens University, Athens, Greece

Antonio Piga

Torino University, Torino, Italy

Ananda Prasad

Wayne State University School of Medicine, Detroit, USA

Jacob M. Rowe

Hebrew University of Jerusalem, Jerusalem, Israel

Jens-Ulrich Rüffer

University of Köln, Köln, Germany

Norbert Schmitz

AK St Georg, Hamburg, Germany

Orhan Sezer

Charité Comprehensive Cancer Center, Berlin, Germany

Anna Sureda

Santa Creu i Sant Pau Hospital, Barcelona, Spain

Ayalew Tefferi

Mayo Clinic, Rochester, Minnesota, USA

Nükhet Tüzüner

İstanbul Cerrahpaşa University, İstanbul, Turkey

Catherine Verfaillie

Katholieke Universiteit Leuven, Leuven, Belgium

Srdan Verstovsek

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

Claudio Viscoli

San Martino University, 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-I

Publishing

Services

GALENOS PUBLISHER

Molla Gürani Mah. Kaçamak Sk. No: 21/1, Fındıkzade, İstanbul, Turkey

Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr

Contact Information

Editorial Correspondence should be addressed to Dr. Reyhan Küçükkaya

E-mail : rkucukkaya@hotmail.com

All Inquiries Should be Addressed to

TURKISH JOURNAL OF HEMATOLOGY

Address : Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.) No: 8 06550 Çankaya, Ankara / Turkey

Phone : +90 312 490 98 97

Fax : +90 312 490 98 68

E-mail : 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

International scientific journal published quarterly.

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

02.12.2021

Cover Picture

Nabhajit Mallik, Man Updesh Singh Sachdeva

Bone Marrow Oxalosis: Crystal Flowers in the Bone Marrow

Garden

Bilateral bone biopsy core showed replacement of bone marrow

with extensive interstitial and paratrabecular deposition of crystals

accompanied by fibrosis (a, 100 x ; b and c, 400 x ). Crystals were

translucent and rod-shaped, arranged in a rosette-like pattern, and

birefringent under polarized light, consistent with calcium oxalate

crystals (d and e, 100 x ; f, 400 x ).

The Turkish Journal of Hematology is published by the commercial enterprise

of the Turkish Society of Hematology with Decision Number 6 issued by the

Society on 7 October 2008.

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AIMS AND SCOPE

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

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

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

periodical encompassing subjects relevant to hematology.

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

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

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

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

Strengthening the Reporting of Observational Studies in Epidemiology

(STROBE).

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

hematological research of the highest scientific quality and clinical relevance.

Additionally, educational material, reviews on basic developments, editorial

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

and clinicians covering their experience and comments on hematology

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

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

reports. Important new findings or data about interesting hematological

cases may be submitted as a brief report.

General practitioners interested in hematology and internal medicine

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

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

Hematology is indexed, as follows:

- PubMed Medline

- PubMed Central

- Science Citation Index Expanded

- EMBASE

- Scopus

- CINAHL

- Gale/Cengage Learning

- EBSCO

- ProQuest

- Index Copernicus

- Tübitak/Ulakbim Turkish Medical Database

- Turk Medline

- Hinari

- GOALI

- ARDI

- OARE

Impact Factor: 1.831

Open Access Policy

Turkish Journal of Hematology is an Open Access journal. This journal

provides immediate open access to its content on the principle that making

research freely available to the public supports a greater global exchange of

knowledge.

Open Access Policy is based on the rules of the Budapest Open Access

Initiative (BOAI) http://www.budapestopenaccessinitiative.org/.

Subscription Information

The Turkish Journal of Hematology is published electronically only as of

2019. Therefore, subscriptions are not necessary. All published volumes are

available in full text free-of-charge online at www.tjh.com.tr.

Address: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613.

Sok.) No: 8 06550 Çankaya, Ankara / Turkey

Telephone: +90 312 490 98 97

Fax: +90 312 490 98 68

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

Web page: www.tjh.com.tr

E-mail: 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

Publisher

Galenos Yayınevi

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

Telephone : +90 212 621 99 25

Fax : +90 212 621 99 27

info@galenos.com.tr

Instructions for Authors

Instructions for authors are published in the journal and at www.tjh.com.tr

Material Disclaimer

Authors are responsible for the manuscripts they publish in The Turkish

Journal of Hematology. The editor, editorial board, and publisher do not

accept any responsibility for published manuscripts.

If you use a table or figure (or some data in a table or figure) from another

source, cite the source directly in the figure or table legend.

Editorial Policy

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

Assistant. The Editorial Assistant checks that each manuscript contains all

required components and adheres to the author guidelines, after which time

it will be forwarded to the Editor in Chief. Following the Editor in Chief’s

evaluation, each manuscript is forwarded to the Associate Editor, who in

turn assigns reviewers. Generally, all manuscripts will be reviewed by at least

three reviewers selected by the Associate Editor, based on their relevant

expertise. Associate editor could be assigned as a reviewer along with the

reviewers. After the reviewing process, all manuscripts are evaluated in the

Editorial Board Meeting.

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

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

to the Turkish Journal of Hematology. This may be creating a conflict of

interest. These manuscripts will not be evaluated by the submitting editor(s).

The review process will be managed and decisions made by editor-in-chief

who will act independently. In some situation, this process will be overseen

by an outside independent expert in reviewing submissions from editors.

A-III

TURKISH JOURNAL OF HEMATOLOGY

INSTRUCTIONS FOR AUTHORS

The Turkish Journal of Hematology accepts invited review articles,

research articles, brief reports, letters to the editor, and hematological

images that are relevant to the scope of hematology, on the condition

that they have not been previously published elsewhere. Basic science

manuscripts, such as randomized, cohort, cross-sectional, and 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.

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-IV

(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



Review articles should not include more than 100 references. Reviews

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

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

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

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

A-V

important findings should be highlighted and interpreted in the Conclusion

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

Perspectives in Hematology

“Perspectives” are articles discussing significant topics relevant to

hematology. They are more personal than a Review Article. Authors

wishing to submit a Perspective in Hematology article should contact

the Editor in Chief prior to submission in order to screen the proposed

topic for relevance and priority. Articles submitted for “Perspectives

in Hematology” must advance the hot subjects of experimental and/

or clinical hematology beyond the articles previously published or in

press in TJH. Perspective papers should meet the restrictive criteria of

TJH regarding unique scientific and/or educational value, which will

impact and enhance clinical hematology practice or the diagnostic

understanding of blood diseases. Priority will be assigned to such

manuscripts based upon the prominence, significance, and timeliness of

the content. The submitting author must already be an expert with a

recognized significant published scientific experience in the specific field

related to the “Perspectives” article.



References: Should not include more than 50 references

Images in Hematology


Authors can submit for consideration illustrations or photos that are

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

of explanatory text and references. Images in Hematology can include

no more than 200 words of text, 5 references, and 3 figures or tables.

No abstract, discussion, or conclusion is required, but please include a

brief title.

Letters to the Editor


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

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

title. The total number is usually limited to a maximum of five authors

for a letter to the editor.

Tables

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

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

Give each column a short or abbreviated heading. Write explanatory

statistical measures of variation, such as standard deviation or standard

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

Figures

Figures should be professionally drawn and/or photographed. Authors

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

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

Each figure should be accompanied by a legend that does not exceed

50 words. Use abbreviations only if they have been introduced in the

text. Authors are also required to provide the level of magnification for

histological slides. Explain the internal scale and identify the staining

method used. Figures should be submitted as separate files, not in

the text file. High-resolution image files are not preferred for initial

submission as the file sizes may be too large. The total file size of the

PDF for peer review should not exceed 5 MB.

Authorship

Each author should have participated sufficiently in the work to assume

public responsibility for the content. Any portion of a manuscript that is

critical to its main conclusions must be the responsibility of at least one

author.

Contributor’s Statement

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

statement should contain substantial contributions to idea and design,

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

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

qualify should be listed. Each author should have participated sufficiently

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

Acknowledgments

Acknowledge support received from individuals, organizations, grants,

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

product or potential product partially or wholly supported by corporate

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

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

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

specific granting institutions’ names and grant numbers provided when

applicable.

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

other associations that might pose a conflict of interest in connection

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A-VI

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A-VIII

CONTENTS

Research Articles

246 Clinical Significance of TP53 Abnormalities in Newly Diagnosed Multiple Myeloma

Fang Ye, Tongtong Wang, Aijun Liu, Yanchen Li, Ningning Li, Huan Wang, Wenming Chen; Beijing, China

254 Generation of Induced Pluripotent Stem Cells from Patients with Multiple Myeloma

İrem Yılmaz Başaran, Erdal Karaöz; Eskişehir, İstanbul, Turkey

264 LncRNA-DUXAP8 Regulation of the Wnt/β-Catenin Signaling Pathway to Inhibit Glycolysis and Induced

Apoptosis in Acute Myeloid Leukemia

Hong Zhai, Junting Zhao, Juan Pu, Pan Zhao, Jin Wei; Nanchong, China

273 Efficacy and Safety of Ibrutinib Therapy in Patients with Chronic Lymphocytic Leukemia: Retrospective

Analysis of Real-Life Data

Anıl Tombak, Funda Pepedil Tanrıkulu, Salih Sertaç Durusoy, Hüseyin Derya Dinçyürek, Emin Kaya, Elif Gülsüm Ümit, İrfan Yavaşoğlu,

Özgür Mehtap, Burak Deveci, Mehmet Ali Özcan, Hatice Terzi, Müfide Okay, Nilgün Sayınalp, Mehmet Yılmaz, Vahap Okan, Alperen Kızıklı,

Ömer Özcan, Güven Çetin, Sinan Demircioğlu, İsmet Aydoğdu, Güray Saydam, Eren Arslan Davulcu, Gül İlhan, Mehmet Ali Uçar, Gülsüm Özet,

Seval Akpınar, Burhan Turgut, İlhami Berber, Erdal Kurtoğlu, Mehmet Sönmez, Derya Selim Batur, Rahşan Yıldırım, Vildan Özkocamaz,

Ahmet Kürşad Güneş, Birsen Sahip, Şehmus Ertop, Olga Meltem Akay, Abdülkadir Baştürk, Mehmet Hilmi Doğu, Aydan Akdeniz, Ali Ünal,

Ahmet Seyhanlı, Emel Gürkan, Demet Çekdemir, Burhan Ferhanoğlu; Mersin, Adana, Gaziantep, Malatya, Edirne, Aydın, Kocaeli, Antalya, İzmir,

Sivas, Ankara, İstanbul, Konya, Manisa, Hatay, Tekirdağ, Trabzon, Erzurum, Bursa, Şanlıurfa, Zonguldak, Kayseri, Turkey

286 Pre-Conditioning Serum Uric Acid as a Risk Factor for Sinusoidal Obstruction Syndrome of the Liver in

Children Undergoing Hematopoietic Stem Cell Transplantation

Fatma Visal Okur, Murat Karapapak, Khaled Warasnhe, Umut Ece Arslan, Barış Kuşkonmaz, Duygu Çetinkaya; Ankara, Turkey

294 Thrombolysis with Systemic Recombinant Tissue Plasminogen Activator in Children: A Multicenter

Retrospective Study

Emine Zengin, Nazan Sarper, Arzu Yazal Erdem, Işık Odaman Al, Melike Sezgin Evim, Neşe Yaralı, Burcu Belen, Arzu Akçay,

Ayşen Türedi Yıldırım, Tuba Hilkay Karapınar, Adalet Meral Güneş, Sema Aylan Gelen, Hale Ören, Lale Olcay, Birol Baytan, Hüseyin Gülen,

Gülyüz Öztürk, Mehmet Fatih Orhan, Yeşim Oymak, Sibel Akpınar, Özlem Tüfekçi, Meryem Albayrak, Burçak Tatlı Güneş, Aylin Canpolat,

Namık Özbek; Kocaeli, Ankara, İzmir, Bursa, İstanbul, Manisa, Sakarya, Kırıkkale, Turkey

306 Assessment of Long-Term Hematologic Effects in Differentiated Thyroid Cancer Patients Treated with

Radioactive Iodine

Bircan Sönmez, Özlen Bektaş, Nergiz Erkut, Mehmet Sönmez; Trabzon, Turkey

Perspective

314 A Rare Lymphoproliferative Disease: Castleman Disease

Eren Gündüz, Nihal Özdemir, Şule Mine Bakanay, Sema Karakuş; Eskişehir, İstanbul, Ankara, Turkey

Brief Report

321 Convalescent Plasma Reduces Endogenous Antibody Response in COVID-19: A Retrospective

Cross-Sectional Study

Ahmet Omma, Abdulsamet Erden, Serdar Can Güven, İhsan Ateş, Orhan Küçükşahin; Ankara, Turkey

A-IX

Images in Hematology

325 Simultaneous Presentation of Hairy Cell Leukemia and Acute Lymphoblastic Leukemia

Mingyong Li, Yuan He, Kang Jiang, Juan Zhang; Chengdu, Ya’an, China

327 Bone Marrow Oxalosis: Crystal Flowers in the Bone Marrow Garden

Nabhajit Mallik, Man Updesh Singh Sachdeva; Chandigarh, India

Letters to the Editor

329 Hematological Findings and Clinical Severity in Pediatric Patients with COVID-19

Pathum Sookaromdee, Viroj Wiwanitkit; Bangkok, Thailand; Pune, India

331 Hematological Malignancy Patients, COVID-19, and Favipiravir

Rujittika Mungmunpuntipantip, Viroj Wiwanitkit; Bangkok, Thailand; Pune, India

333 A Peculiar Disease in a Young Woman Wanting to Get Pregnant

Tülin Tiraje Celkan, Şeyma Fenercioğlu, Ayşe Gonca Kaçar; İstanbul, Turkey

335 Pulmonary Embolism Secondary to Intravenous Immunoglobulin in a Child with Leukemia

Işıl Seren Oğuz, Zühre Kaya, Serap Kirkiz, Ülker Koçak; Ankara, Turkey

337 Severe Lymphocytosis in a Case of Diffuse Large B-Cell Lymphoma Treated by Ibrutinib

Semra Paydaş, Ertuğrul Bayram, Mehmet Türker, Turan Özer; Adana, Turkey

338 Leg Ulcers Associated with Anagrelide

Tuba Oskay, Mehmet Özen; Ankara, Turkey

341 Acute Basophilic Leukemia Arising from Chronic Myeloid Leukemia with Isolated Thrombocytosis

Yun Zhang, Xiaosu Kang, Xiliang Chen, Ting Li; Jinan, Yantai, Beijing, China

344 Treatment with Venetoclax for Chronic Lymphocytic Leukemia with the Highest Known White Blood

Cell Count: Safe and Effective

Mehmet Sönmez, Merve Kestane, Osman Akıdan, Nergiz Erkut, Özlen Bektaş; Trabzon, Turkey

38 th Volume Index

Author Index 2021

Subject Index 2021

A-X

Ye F. et al: TP53 Abnormalities in Multiple Myeloma

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2021.0064

Turk J Hematol 2021;38:246-253

Clinical Significance of TP53 Abnormalities in Newly Diagnosed

Multiple Myeloma

Yeni Tanı Multipl Myelomda TP53 Anormalliklerinin Klinik Önemi

Fang Ye 1 , Tongtong Wang 2 , Aijun Liu 2 , Yanchen Li 2 , Ningning Li 1 , Huan Wang 1 , Wenming Chen 2

1Chuiyangliu Hospital Affiliated to Tsinghua University, Department of Hematology, Beijing, China

2Capital Medical University Beijing Chaoyang Hospital, Department of Hematology, Beijing, China

Abstract

Objective: This study aimed to identify the clinical significance of

TP53 and common cytogenetic abnormalities.

Materials and Methods: A total of 114 patients with newly diagnosed

multiple myeloma (MM) and TP53 abnormalities were selected from

two large patient cohorts of collaborating hospitals from 2010 to 2017.

The characteristics and outcomes of these patients were analyzed.

TP53 and other common mutations in MM patients were quantified

by fluorescence in situ hybridization. Kaplan-Meier curves and logrank

tests were applied for survival analysis. A Cox proportional hazard

model for covariate analysis was used to determine the prognostic

factors.

Results: By extensive data analysis, we found that TP53 amplification

is a strong positive predictor for complete response (CR) to therapy

and positively correlated with patient survival. The number of

simultaneous genomic abnormalities with TP53 mutation has a

modest impact on patient survival. Among these mutations, 1q21

amplification is associated with decreased CR (odds ratio: 4.209)

and FGFR3 levels are positively correlated with progression-free and

overall survival.

Conclusion: TP53 abnormalities at the diagnosis of MM are of great

clinical significance in predicting patient response to therapy and

survival. Furthermore, 1q21 and FGFR3 mutations could potentially

be used in combination with TP53 status to better predict patient

survival and guide the selection of high-risk patients to advance

patient treatment strategies.

Keywords: TP53, Multiple myeloma, Genomic abnormality

Öz

Amaç: Bu çalışma, TP53’ün klinik önemini ve yaygın sitogenetik

anormallikleri belirlemeyi amaçladı.

Gereç ve Yöntemler: 2010 ile 2017 yılları arasında işbirliği yapan

hastanelerin iki büyük hasta grubundan yeni teşhis edilmiş multipl

miyelom (MM) ve TP53 anormallikleri olan toplam 114 hasta seçildi.

Bu hastaların özellikleri ve sonuçları analiz edildi. MM hastalarında

TP53 ve diğer yaygın mutasyonlar, floresan in situ hibridizasyon ile

ölçülmüştür. Hayatta kalma analizi için Kaplan-Meier eğrileri ve logrank

testleri uygulandı. Prognostik faktörleri belirlemek amacı ile

ortak değişken analizi için bir Cox orantılı tehlike modeli kullanıldı.

Bulgular: Kapsamlı veri analizi ile, TP53 amplifikasyonunun tedaviye

tam yanıt (CR) için güçlü bir pozitif öngörücü olduğunu ve hastanın

sağkalımı ile pozitif korelasyon gösterdiğini bulduk. TP53 mutasyonu

ile eşzamanlı genomik anormalliklerin sayısı, hastanın sağkalımı

üzerinde sınırlı bir etkiye sahiptir. Bu mutasyonlar arasında, 1q21

amplifikasyonu, azalmış CR (olasılık oranı: 4.209) ile ilişkilidir ve FGFR3

seviyeleri, progresyonsuz ve genel sağkalım ile pozitif olarak ilişkilidir.

Sonuç: MM tanısındaki TP53 anormallikleri, hastanın tedaviye yanıtını

ve sağkalımı öngörmede büyük klinik öneme sahiptir. Ayrıca, 1q21 ve

FGFR3 mutasyonları, hasta sağkalımını daha iyi tahmin etmek ve hasta

tedavi stratejilerini geliştirmek için yüksek riskli hastaların seçimine

rehberlik etmek amacı ile potansiyel olarak TP53 durumu ile kombine

halde kullanılabilir.

Anahtar Sözcükler: TP53, Multipl myelom, Genomik anormallik

Introduction

Multiple myeloma (MM) is a hematologic malignancy caused

by the proliferation of plasma cells in the bone marrow. It

accounts for approximately 10% of all hematologic

malignancies and 1% of all cancers [1,2]. The tumor plasma

cells infiltrate bone marrow and other organs, which leads to

lethal immune deficiency and organ damage [3,4,5]. Worldwide

studies indicate that the incidence of MM has increased by 126%

globally and the 5-year survival rate is only about 50% [6].

One major factor that contributes to the low survival rate is

that MM is a highly heterogeneous disease, characterized by

numerous genetic alterations [7]. Chromosome gains and losses,

©Copyright 2021 by Turkish Society of Hematology

Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Wenming Chen, M.D., Capital Medical University Beijing Chaoyang

Hospital, Department of Hematology, Beijing, China

Phone : +86-13910107759

E-mail : cebqai@163.com ORCID: orcid.org/0000-0003-1788-2460

Received/Geliş tarihi: January 22, 2021

Accepted/Kabul tarihi: April 29, 2021

246



immunglobulin H translocations, and mutations of specific

genes are often found in MM patients [7,8]. Genetic alterations

are categorized as primary or secondary changes based on when

the changes are observed during disease progression [9,10].

Cytogenetic abnormalities play a very important role in the

survival of MM patients. For example, as determined by

fluorescence in situ hybridization (FISH) detection, gain (1)(q21),

del(17)(p13), and t(4;14)(p16;q32) in MM patients are correlated

with shorter overall survival (OS) [11,12]. The fact that the type

and quantity of genomic abnormalities are directly linked to MM

patients’ survival time and response to treatment suggests that

an investigation of the role of mutations in predicting patient

response and survival is of great clinical significance in MM

patient management [13].

Mapped to the position of chromosome 17p13, the TP53 gene

encodes the p53 protein and regulates the cell cycle. Since

the discovery of the p53 protein, its role in cancer has been

intensively investigated. p53 is an important tumor suppressor

due to its critical role in inducing cell cycle arrest and apoptosis

in response to cellular stress signals [14].

In MM patients, the major abnormalities of the TP53 gene are

mutation and deletion (due to deletion of the 17p13 region).

These abnormalities of the TP53 gene rarely occur at diagnosis;

they increase in late-stage patients, suggesting the essential

role of the TP53 gene in disease progression [15,16]. Many

clinical reports have shown a strong association between a loss

of TP53 and poor prognosis in MM patients [16,17,18,19].

However, due to the heterogeneity of MM and the limited

number of cases, the function of TP53 at diagnosis as a

biomarker in different backgrounds of the major molecular

cytogenetic abnormalities of MM is not well studied. Here, we

provide an intensive retrospective analysis of a large cohort

of newly diagnosed MM patients to identify the clinical

significance of TP53 and common cytogenetic abnormalities. We

compare TP53 loss and amplification together with common

genes dysregulated in MM patients, including chromosome

1q21 amplification, translocation of 4p16.3 (fibroblast growth

factor receptor 3, FGFR3), and translocation of 16q23 (MAF)

to chromosome 14q32. Investigation of the risk factors of MM

relapse/progression will bring insight into the development

of adaptive methods for better treatment of MM patients.

Materials and Methods

Patients

A total of 1046 newly diagnosed MM patients were enrolled

from Beijing Chao-Yang Hospital, the Multiple Myeloma

Research Center of Beijing, and Chuiyangliu Hospital Affiliated

to Tsinghua University from January 2010 to December 2017.

FISH was used to characterize the genetic abnormalities [TP53,

1q21, 14q32/11q13 (CCND1 (cyclin D1 gene)], 14q32/4p16.3

(FGFR3), 14q32/16q23 (MAF) of these patients and diagnostic

criteria were based on those of the International Myeloma

Working Group [20]. Detailed criteria for FISH positivity are

provided in Table 1. Basic patient information including age,

gender, habits, baseline health, and comorbid diseases and

clinical parameters including OS and chemotherapy response

were recorded. The patient selection criterion was a primary

diagnosis with TP53 abnormality. Patients were excluded if

they had refractory/relapsed MM. The study was approved

by the ethics committee of our hospital. All patients gave

written informed consent.

FISH

FISH was performed on interphase cells. CD138-expressing

plasma cells were purified and then FISH was performed as

previously described [21] using probes purchased

from Beijing Hightrust Diagnostic Company Limited. Targets

detected by FISH and thresholds are included in Table 1. At

least 200 plasma cells were scored to determine the prevalence

of each genetic abnormality.

Statistical Analysis

The primary endpoint of this study was correlated with survival

from the time of diagnosis. Progression-free survival (PFS) and OS

were evaluated according to the international uniform response

criteria [22]. PFS was calculated from the time of diagnosis to

the date of death, progression, or last follow-up. OS was defined

as the duration from the time of diagnosis to the date of death

or last follow-up. Descriptive statistics such as mean, standard

deviation, median, and range were used for continuous

variables while frequency counts and percentages were used

for categorical variables. An independent sample t-test was

employed to evaluate the associations between genetic

abnormalities and biological parameters. The chi-square test or

two-sided Fisher exact test was performed to make comparisons

of categorical variables among groups. The Kaplan-Meier

method was employed to plot survival curves, with a log-rank

test to assess the differences. A Cox proportional hazard model

for covariate analysis was used to determine the prognostic

factors for PFS. All statistical analyses were performed using

SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The results were

considered significant at p<0.05.

Table 1. Summary of FISH positivity thresholds.

Probe

Test site

1q21 1q21 6.87

TP53 17p13.1 6.09

IGH/MAF 14q32/16q23 0.77

IGH/FGFR3 14q32/4p16.3 1.11

IGH/CCND1 14q32/11q13 4.85

Positive threshold

% cells tested positive

247



Results

The median follow-up time for the entire population of MM

patients was 32 months (range: 1-192 months). Among the

1046 newly diagnosed MM cases, TP53 abnormalities were

found in 153 cases, and 114 of those 153 cases (64 male

patients, 50 female patients) were followed and included in

the analysis, with a mean age of 59.4±10.3 years (Table 2).

Among those 114 patients, 23 cases were stage I, 27 cases were

stage II, and 64 cases were stage III at the time of diagnosis

based on the International Staging System (ISS) (Table 2). Due

to the significant effect of extramedullary disease (EMD) on

survival rate reduction [23], patients’ EMD statuses at

diagnosis were recorded. Most patients (86.84%) had no EMD

at diagnosis (Table 2). Other medical history (hypertension,

diabetes, heart disease, etc.), lifestyle factors (smoking and

alcohol consumption), and clinical characteristics (neutrophils,

platelet count, hemoglobin level, creatinine level, etc.) that may

affect or reflect disease progression are provided in Tables 2

and 3. Patients mainly received autologous hematopoietic cell

transplantation and/or standard chemotherapies, including but

not limited to bortezomib combined with dexamethasone (PD)

or three-drug combinations of PD with liposomal doxorubicin

or thalidomide (Table 2).

In our analysis, the OS of patients was mainly affected by age

and chemotherapy. Younger age (<60 years old) correlated

with increased OS rate compared to older patients (≥60 years

old) (median survival: 72 months vs. 39 months, p=0.038)

(Figure 1A). Chemotherapy increased the median survival time

from 28 months to 77 months (p=0.029) (Figure 1B). However,

the other major therapy received by our patients, autologous

hematopoietic cell transplantation therapy, did not further

improve patient survival rate (p=0.428; data not shown). Other

Table 2. Summary of patients’ general information.

Gender

Age

n (%)

Male 64 (56.14)

Female 50 (43.86)

Age <60 54 (47.37)

Age ≥60 60 (52.63)

Durie-Salmon System I-II 14 (12.39)

International Staging System

Eastern Cooperative Oncology Group

performance status

Smoking

Alcohol consumption

Hypertension

Diabetes

Heart disease/arteriovenous thrombosis

Chemotherapy

Autologous hematopoietic cell

transplantation

EMD

EMD: Extramedullary disease.

III 99 (87.61)

I 23 (20.18)

II 27 (23.68)

III 64 (56.14)

0 55 (48.25)

≥1 59 (51.75)

No 81 (71.05)

Yes 33 (28.95)

No 90 (78.95)

Yes 24 (21.05)

No 71 (62.28)

Yes 43 (37.72)

No 97 (85.09)

Yes 17 (14.91)

No 103 (90.35)

Yes 11 (9.65)

No 18 (15.79)

Yes 96 (84.21)

No 94 (82.46)

Yes 20 (17.54)

No 99 (86.84)

Yes 15 (13.16)

Figure 1. Factors that influence patient survival. Log-rank analysis of (A) age and (B) chemotherapy on patients’ overall survival. Patient

numbers are indicated on the charts. Age groups are separated based on the mean age in our patient cohorts.

248



factors including gender, ISS, Eastern Cooperative Oncology

Group score, smoking, and alcohol consumption did not

have a significant correlation with PFS or OS rates (data not

shown).

Among the 114 patients with TP53 abnormalities, 54 showed

TP53 amplification and 60 showed TP53 deletion. Compared

to patients with TP53 deletion, those with TP53 amplification

had a higher probability of achieving complete response (Table

4; p=0.008) and had modest PFS and OS advantages (Figures

2A and 2B). When 3-year survival time was used as the cutoff

in analysis, the patients who survived had a higher percentage

of TP53 amplification than patients who died (mean: 61.4% vs.

40.27%; p=0.034). The PFS and OS rates of patients with more

than 51.25% TP53 amplification (value calculated by receiver

operating characteristic curve analysis; data not shown) trended

more highly than those of patients with less TP53 amplification

(Figures 3A and 3B). Together, these data suggest that

TP53 amplification plays a positive role in patient survival.

The genes and chromosomes that are commonly dysregulated in

MM patients were also tested in these 114 patients [chromosome

1q21 amplification, 4p16.3 (FGFR3), 16q23 (MAF), IgH

translocations, abnormal chromosome counts] to show the

potential effects of these common genetic dysregulations in the

background of TP53 abnormality. The genomic changes in these

114 patients are summarized in Table 5.

Overall, our data indicate that patients with four or more types

of mutations in the list have PFS rates similar to those of patients

with fewer than four types of mutation (data not shown).

However, their OS rates trend more highly compared to patients

Table 3. Summary of patients’ clinical features.

Abb. Feature n SD Min Max Median

NE Neutrophils (10 9 /L) 114 5.95 0.49 63.2 3.13

HGB Hemoglobin (g/L) 114 24.2 50 152 90.8

PLT Platelet (10 9 /L) 114 89.07 20 724 165

ALB Albumin (g/L) 114 6.75 17.6 48 34.55

CR Creatinine (µmol/L) 114 182.09 30 1004.9 77.35

LDH Lactate dehydrogenase (U/L) 114 119.95 68 971 163.5

CA Calcium (mmol/L) 114 1.78 1.54 20.8 2.19

BTA β2-microglobulin (mg/L) 112* 9.73 1.42 73.5 4.34

BNP B-type natriuretic peptide 113* 3659.42 5 35000 149.9

LVEF Left ventricular ejection fraction 114 6.38 45 82 69

JXBP Plasma cell % in bone marrow 114 21.48 1 93.5 36.25

*: Missing values, min: minimum, max: maximum.

Figure 2. TP53 level affects patient survival. Log-rank analysis of the effect of TP53 amplification and deletion on (A) progression-free

survival (PFS) and (B) overall survival (OS) of the patients.

249



with lower mutation burden (Figure 4A). When OS analysis was

performed for patients separated by TP53 status, though, no

significant difference was found between patients with four

or more types of mutations and patients with fewer than four

types of mutations, potentially due to the low patient number in

each group (Figures 4B and 4C). The effects of individual genetic

abnormalities in the background of TP53 abnormality on OS

were also tested. In our patient cohorts with TP53 abnormality,

of the five genetic abnormalities (1q21, FGFR3, MAF, IgH

translocations, and chromosome number changes), 1q21

amplification predicted the decreased probability of complete

response (Table 4; odds ratio: 4.209), and the type of FGFR3

mutation was critical in predicting patients’ PFS and OS. FGFR3

amplification yielded a fivefold increase in median survival

time compared to FGFR3 deletion (100 months vs. 19 months)

and a twofold increase compared to patients with normal FGFR3

(100 months vs. 41 months) (Figure 4D). We further analyzed

median survival times for patients with FGFR3 amplification

and normal FGFR3 as separated by their TP53 statuses. Patients

with FGFR3 amplification still had significantly longer median

Table 4. Risk factors involved in complete response to therapies.

Risk factors OR p

Age Unit =1 1.045 (0.997-1.096) 0.068

DS I-I vs. III 0.181 (0.044-0.737) 0.017

Chemotherapy No vs. Yes 12.597 (1.319-120.317) 0.028

TP53

1q21

Amplification vs.

Deletion

Amplification vs.

Deletion

OR: Odds ratio; DS: Durie-Salmon System.

0.225 (0.075-0.677) 0.008

4.209 (1.258-14.076) 0.020

survival time in the background of TP53 amplification (Figure

4E), but not in cases of TP53 loss (Figure 4F).

These data suggest that TP53 status in combination with

common mutations in MM could potentially be used to predict

patient survival at the time of disease diagnosis.

Discussion

TP53 is a critical tumor suppressor and reported to correlate

with MM disease progression. However, TP53 mutation is a rare

Table 5. Summary of patients’ genetic abnormalities.

TP53

1q21

MAF

FGFR3

IgH

Chromosome

IgH: Immunglobulin H.

n (%)

Amplification 54 (47.37)

Deletion 60 (52.63)


Deletion 1 (0.88)

Normal 29 (25.44)


Deletion 24 (21.05)

Normal 61 (53.51)


Deletion 6 (5.26)

Normal 76 (66.67)


Deletion 15 (13.16)

Normal 74 (64.91)

46, XY/XX 95 (83.33)

Other 19 (16.67)

Figure 3. TP53 amplification predicts better patient survival. Log-rank analysis of the effect of the level of TP53 amplification on (A)

progression-free survival (PFS) and (B) overall survival (OS) of the patients. The cutoff threshold of TP53 amplification is based on

receiver operating characteristic curve analysis.

250



Figure 4. The ability of common mutations found in MM patients to predict patient survival among patients with TP53 abnormalities.

(A) Correlation between number of genetic abnormalities and patient OS. (B) Correlation between number of genetic abnormalities and

patient OS in the background of TP53 amplification. (C) Correlation between number of genetic abnormalities and patient OS in the

background of TP53 deletion. (D) FGFR3 level in predicting median patient survival time. (E) FGFR3 status in predicting median patient

survival time in the background of TP53 amplification. (F) FGFR3 status in predicting median patient survival time in the background

of TP53 loss.

MM: Multiple myeloma.

occurrence at diagnosis, being seen in only about 3% of newly

diagnosed patients. The large patient cohorts in our hospitals

provided an opportunity for us to study TP53 mutation in earlystage

MM patients, which brings insight into the clinical

significance of TP53 in newly diagnosed MM patients and also

disease progression.

In 114 newly diagnosed MM patients with TP53 abnormalities,

we found that patient age and stage of the disease were the

strongest predicting factors for patient PFS and OS, with older

age and later stages indicative of worse prognosis, consistent

with reports from other groups [24,25]. Patients’ lifestyles

(smoking, etc.) and preexisting conditions (heart diseases,

etc.) did not have strong effects on patient survival.

TP53 deletion is more commonly found in MM patients.

In the present study we also reported a group of patients

with TP53 amplification, which was associated with increased

PFS and OS. The mechanism of TP53 amplification is unknown,

but it could potentially be caused by the compensating

of non-functional p53 protein. Among the patients

with TP53 mutations, nearly half showed TP53 amplification,

and TP53 amplification was a strong predictor for a complete

response to therapy. Furthermore, the level of TP53 amplification

(≥51.25%) also showed a trend of positive correlation with

patient survival rate. These data indicate that TP53, as a tumor

suppressor, plays an important role in MM patient prognosis;

patients with TP53 deletion at an earlier stage and patients of

older ages will potentially have a decreased chance of reaching

complete response when treated with standard chemotherapy

and autologous hematopoietic cell transplantation therapy.

More advanced and intensive therapeutic strategies are

potentially needed for these patients.

As common mutations in MM patients, 1q21 and FGFR3 levels

were good predictors of patient’s therapy responses and OS in our

cohorts. Copy number gain of chromosome 1q21 is among the

251



most commonly reported genetic abnormalities in MM patients.

The predictive role of 1q21 amplification in MM patients in

terms of chemotherapy response and patient survival, however,

is controversial. Studies have shown that 1q21 amplification

strongly correlates with bortezomib resistance, but others

showed no response prediction or survival benefit for patients

with 1q21 amplification [26,27,28]. Our data indicate that in

patients with TP53 abnormalities, 1q21 amplification is a strong

predictor for worse response to chemotherapy, suggesting that

the study of 1q21’s role in the context of TP53 mutation is of

great clinical importance.

On the other hand, the t(4;14) translocation is associated with

upregulation of FGFR3 amplification, which has been shown

to correlate with poor patient survival [29,30]. Interestingly,

in contradiction with other studies, we found that in newly

diagnosed MM patients with TP53 mutation, FGFR3 levels had

a strong positive correlation with patient PFS and OS. Patients

with FGFR3 amplification had a nearly twofold increase in

median survival time compared to patients with normal

FGFR3 levels. These data suggest that FGFR3 level is a critical

prognosis indicator and a potential therapeutic target in MM

patients with TP53 mutation.

Study Limitations

One limitation of our study is that the patient number is

small, due to the fact that TP53 mutation is rarely present at

diagnosis. Data analysis for age or other mutation types is

limited in the total population of patients with TP53 mutation

and separate analysis for each feature in TP53 amplification and

deletion could not be performed with statistical power. Another

limitation of our study is that TP53 mutation was tested at gene

level. Whether the MM patients in our cohorts had functional

p53 protein in their tumors or not is unknown, which may have

introduced noise to our data analysis. Addressing the functional

p53 protein levels in those patients in future work could

potentially help to gain more statistical power in our analysis

and a better understanding of the functional role of p53 in

newly diagnosed MM patients.

Conclusion

By extensive analysis of 114 newly diagnosed MM patients

with TP53 abnormalities, we observed a positive correlation

between TP53 amplification and MM patient survival. Further

investigation of TP53 and the common mutations in MM

patients will contribute to the better design of biomarkers to

predict MM patient therapy response and survival.

Ethics

Ethics Committee Approval: The study was approved

by the ethics committee of our hospital.

Informed Consent: All patients gave written informed consent.

Authorship Contributions

Surgical and Medical Practices: T.W.; Concept: F.Y., W.C.;

Design: F.Y.; Data Collection or Processing: F.Y., T.W., A.L., Y.L.,

N.L.; Analysis or Interpretation: T.W., Y.L., N.L., W.C., H.W.;

Writing: F.Y., W.C.

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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253

Yılmaz Başaran İ and Karaöz E: iPSCs and Multiple Myeloma

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2020.0682


Generation of Induced Pluripotent Stem Cells from Patients with

Multiple Myeloma

Multipl Myelom Hastalarından Uyarılmış Pluripotent Kök Hücre Üretilmesi

İrem Yılmaz Başaran 1 , Erdal Karaöz 2,3,4,5

1Eskişehir Osmangazi University, Cellular Therapy and Stem Cell Production Application and Research Center, Eskişehir, Turkey

2STEMBIO Cell Technologies Inc., İstanbul, Turkey

3İstinye University, Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey

4İstinye University, Center for Stem Cell and Tissue Engineering Research and Practice, İstanbul, Turkey

5İstinye University, 3D Bioprinting Design and Prototyping R&D Center, İstanbul, Turkey

6Liv Hospital, Center for Regenerative Medicine and Stem Cell Manufacturing (LivMedCell), İstanbul, Turkey

Abstract

Objective: Patient-specific induced pluripotent stem cells (iPSCs)

have potential in human disease modeling and regenerative medicine.

The in vitro phenotype of disease-specific iPSC-derived cells can

be used to bridge the knowledge gap between clinical phenotype

and molecular or cellular pathophysiology and to understand the

pathology of diseases, along with further applications, such as creating

new strategies for drug screening or developing novel therapeutic

agents. The aim of our study was to generate iPSCs from multiple

myeloma (MM) patients.

Materials and Methods: Mesenchymal stem cells (MSCs) isolated

from MM patients were induced for pluripotency via the Sendai virus.

Fibroblasts were used as a control. Microscopic analysis was performed

daily. For colony selection, live staining was done using alkaline

phosphatase staining. Reprogramming experiments were confirmed by

flow cytometry, immunofluorescence (IF) staining, and gene expression

analyses. To confirm the spontaneous differentiation potential, an in

vitro embryonic body (EB) formation assay was performed.

Results: Fibroblasts and MSCs obtained from MM patients were

reprogrammed using the Sendai virus, which contains reprogramming

vectors with the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc.

Microscopic analysis revealed that the generated iPSCs possessed

classical embryonic stem cell-like morphological characteristics.

Reprogramming experiments further showed that both cell lines can

be reprogrammed up to the pluripotent stage, which was confirmed by

flow cytometry, IF staining, and gene expression analyses. Spontaneous

differentiation potential was confirmed by in vitro EB formation assays.

Conclusion: iPSCs have been successfully obtained from MM patients

for the first time. These cells could clarify the molecular mechanisms

behind this disease.

Keywords: Induced pluripotent stem cells, Multiple myeloma,

Mesenchymal stem cells, Sendai virus

Öz

Amaç: Hastaya özgü uyarılmış pluripotent kök hücreler (uPKH)

insan hastalık modellemesi ve rejeneratif tıpta büyük bir potansiyele

sahiptir. Hastalığa özgü uPKH’den türetilen hücreler, klinik fenotip

ile moleküler veya hücresel patofizyoloji arasındaki bilgi boşluğunu

kapatmak ve ilaç taraması için yeni stratejiler oluşturmak ve yeni

terapötik ajanlar geliştirmek gibi stratejilerle hastalık patolojilerini

anlamada faydalı olacaktır. Çalışmamızın amacı multipl myelom (MM)

hastalarından uPKH üretmektir.

Gereç ve Yöntemler: MM hastalarından izole edilen MKH’ler Sendai

virüs yoluyla uyarılarak pluripotensi aşamasına döndürülmüştür.

Çalışmada fibroblastlar kontrol olarak kullanılmıştır. Her gün

mikroskobik analiz yapılmış, koloni seçimi için alkalin fosfataz canlı

boyaması yapılmıştır. Yeniden programlama deneyleri akış sitometrisi,

immünofloresan (IF) boyama ve gen ekspresyon analizleri ile teyit

edilmıştir. Spontan farklılaşma potansiyelini doğrulamak için in vitro

embriyonik cisimcik (EC) oluşum deneyi yapılmıştır.

Bulgular: Fibroblastlar ve MM hastalarından izole edilmiş MKH’ler;

dört Yamanaka faktörü olan Oct3/4, Sox2, Klf4 ve c-Myc ile yeniden

programlama vektörleri içeren Sendai virüsü kullanılarak yeniden

programlanmıştır. İlk olarak, mikroskobik analiz ile üretilen uPKH’lerin

klasik embriyonik kök hücre (EKH) benzeri morfolojik özelliklere sahip

olduğu ortaya konmuştur. İkinci olarak, her iki hücre hattının da

pluripotent aşamaya kadar yeniden programlanabildiği akış sitometrisi,

IF boyama ve gen ekspresyon analizleri ile teyit edilmiştir. İn vitro

embriyonik cisimcik (EC) oluşum deneyleri ile spontan farklılaşma

potansiyeli gösterilmiştir.

Sonuç: uPKH’ler MM hastalarından ilk kez başarıyla elde edilmiştir ve

bu hücrelerin MM hastalığının arkasındaki moleküler mekanizmaları

netleştirebileceği düşünülmektedir.

Anahtar Sözcükler: Uyarılmış pluripotent kök hücre, Multipl myelom,

Mezenkimal kök hücre, Sendai virüs



Address for Correspondence/Yazışma Adresi: Erdal Karaöz, PhD, İstinye University, Center for Stem Cell and

Tissue Engineering Research and Practice, İstanbul, Turkey

Phone : +90 212 481 36 55

E-mail : ekaraoz@hotmail.com ORCID: orcid.org/0000-0002-9992-833X

Received/Geliş tarihi: November 15, 2020

Accepted/Kabul tarihi: March 16, 2021

254



Introduction

Yamanaka and Takahashi made a discovery in the world of

life science by transforming mouse somatic fibroblasts into

pluripotent cells as a result of transferring 4 gene sets (Sox2,

Oct4, Klf4, and c-Myc) in 2006 [1]. Since that day, induced

pluripotent stem cells (iPSCs) are considered to be one of the

main sources for regenerative medicine, similarly to embryonic

stem cells (ESCs). Because of their pluripotent features, both

cell types are building blocks of regenerative medicine.

However, in contrast to ESCs, there are no ethical limitations

or immunological problems when using iPSCs [2]. Additionally,

iPSCs with disease genotypes have been used for human disease

modeling [3].

To date, iPSCs have been generated from many different sources

[1,4,5,6,7,8,9], including mesenchymal stem cells (MSCs). MSCs

were shown to be more efficient in reprogramming compared to

other somatic cells [10,11].

In the last decade, iPSCs have proven to be a powerful in

vitro system for studying diseases [12,13], especially genetic

disorders [1,14]. Patient-specific iPSCs have powerful potential

in regenerative medicine and notably in human disease

modeling [15]. The in vitro phenotype of disease-specific iPSCderived

cells can enable us to comprehend the differences

and/or similarities between molecular/cellular pathophysiology

and clinical phenotype. This technology can also facilitate

and improve the understanding of disease pathology. To date,

many disease models have been established with iPSCs. There

are efforts for drug screening tests and genetic modifications

of cells for the treatment of diseases [15]. On the other hand,

in many diseases, patient-specific iPSCs have been shown to

exhibit the characteristics of the diseases [13,16].

The use of iPSCs is also very important in research on the

cancer microenvironment [17,18,19]. Multiple myeloma (MM)

progresses with the uncontrolled increase and accumulation of

malignant plasma cells in the bone marrow (BM) [20]. MM bone

disease is observed due to the increase of osteoclastic activity

via the factors synthesized from malignant plasma cells and

the decrease in the differentiation of osteoblasts originating

from MSCs. Imbalance in this process leads to overproduction

of the many responsible chemokines and cytokines and various

signaling cascades are also involved in this complex process

[21,22,23]. Advanced lesions and fractures occur as a result of

this imbalance in bone formation and destruction.

The construction and differentiation of osteoblasts from

MSCs is controlled by many factors and pathways in the BM

microenvironment. Various inhibitory substances released by

plasma cells in the BM microenvironment in MM stop bone

formation as a result of disruption of different stages of

osteoblastogenesis [24,25,26]. Furthermore, there are many

factors in MM disease that disrupt osteoblastogenesis with

different pathways. Many of these factors may be indirectly

secreted or secreted by MM cells [27,28]. In previous studies,

osteogenic differentiation defects were detected in BM-derived

MSCs (BM-MSCs) obtained from MM patients, even in vitro,

where MM cells did not have all the inhibitory factors secreted

[29,30,31,32,33].

In recent years, various approaches have been developed in the

treatment of MM bone disease, especially regarding the use of

MSCs [34,35]. However, the limited proliferation of BM-MSCs

obtained from MM patients and the low capacity of osteoblastic

differentiation under in vivo and in vitro conditions will prevent

possible autologous MSC treatments in the future. In addition to

the purpose of revealing the molecular development stages of

diseases and helping to design disease-specific or personalized

drugs, iPSC technology is expected to show potential for future

use in cell therapy or tissue engineering in many disease models.

With the development of iPSC technology, it will be possible in

the future to obtain genetically repaired autologous stem cells

from patients or reproduce and replace the missing tissue.

Recently, different types of cells collected from patients with

various diseases have been used for generating iPSCs, but

this has not included patients with MM. Based on all this

information, we aimed to obtain the MM disease model for

the first time by reprogramming BM-MSCs obtained from MM

patients in our study. Such iPSCs have serious potential to begin

with because of their MM patient cell origin and the inclusion

of disease genotype in a stem cell. MM-iPSCs would undeniably

contain the genotype that causes the disease. With this study,

patient-specific cells will make patient-specific disease modeling

possible, and defects in MSCs can be studied by programming

them into the pluripotent stage. This research will lead to other

studies being carried out for the first time in the literature.


Selection of Patients and Control Groups

In this study, MSCs were isolated from BM obtained from the

iliac crest of newly diagnosed MM patients (n=3). Biopsies were

performed for diagnosis, staging, and evaluation of ongoing

treatment.

Control samples to generate iPSCs were derived from newborn

babies’ foreskin fibroblasts after obtaining informed consent

approved under standard protocols.

MSC Isolation from MM Patients and Cell Culture

The isolation and culturing of human BM-MSCs were performed

as previously described by Karaöz et al. [36]. Briefly, BM

aspirates were obtained from the iliac crest of MM patients.

Samples were then diluted to 1:3 with phosphate-buffered

255



saline (PBS). Histopaque-1077 (1.0777 g/mL; Sigma-Aldrich,

St. Louis, MO, USA) was used for gradient centrifugation.

Low-density mononuclear cells were collected and plated in

tissue culture flasks.

iPSC Generation

For the generation of iPSCs, the CytoTune-iPS Reprogramming

Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. The

manufacturer’s instructions were followed for setting up the

generation procedure (Figure 1). Two days before transduction,

the cells were plated into 2 wells of a 6-well plate (day -2).

On the day of transduction (day 0), cell medium was aspired

and Yamanaka factors were added to cells, which were then

incubated overnight. The cells were then cultured with their

specific culture media for 6 days. When the colonies had grown

to an appropriate size for transferring, live staining was done

using alkaline phosphatase (ALP Live Stain, Thermo Fisher) for

selecting reprogrammed colonies. The selected colonies were

then harvested. Manually picked colonies were transferred onto

fresh MEF plates. The next day, the medium was changed to iPSC

medium (DMEM-F12 + 20% KnockOut Serum Replacement,

100 µM MEM non-essential amino acids, 1x GlutaMAX,

100 µM β-mercaptoethanol, 0.2% Primocin, and 4 ng/mL FGF)

and was replaced everyday thereafter. Colony formation was

monitored and photographed every day.

iPSC Culture

iPSCs were passaged to avoid overgrowth and to maintain

them in an undifferentiated state. Before splitting the colonies,

differentiated colonies were removed under a microscope

in sterile conditions. Differentiated areas were excised and

discarded before bulk passaging. Colonies were mechanically

cut into pieces using a needle for passaging. Colonies were

usually ready to be passaged in 2-3 days.

For feeder-free culture, picked colonies were added to freshly

prepared plates coated with Geltrex (Invitrogen, Life Tech.,

Carlsbad, CA, USA). The medium was gradually changed to

StemPro ® hESC serum-free medium (Invitrogen, Life Tech.) as

explained in Table 1. StemPro® was used every day thereafter.

The colonies were passaged at a 1:3 ratio. Continued passaging

was done with the StemPro® EZPassage Disposable Stem Cell

Passaging Tool (Invitrogen, Life Tech.).

Characterization of iPSCs

Cell Staining

The same method used for immunofluorescence (IF) staining

of MSCs (Supplemental Materials and Methods) was applied.

The following primary antibodies were used for staining: Oct4,

NANOG, TRA1-60, TRA1-81, and Sox2 (Table 2). DAPI was used

for nuclear staining.

Flow Cytometry

The expressions of pluripotency-associated markers were

analyzed by flow cytometry. Feeder-free cultured iPSCs were

Table 1. Media percentages of MEF conditioned medium

and StemPro® hESC serum-free medium in the first days of

feeder-free culture.

Days in

Geltrex

MEF conditioned

medium

1 st day 75% 25%

2 nd day 50% 50%

3 rd day 25% 75%

4 th day - 100%

MEF: Mouse embryonic fibroblast.

StemPro® hESC serumfree

medium

Table 2. Primary antibodies used for characterization of iPSCs.

Antibody/

marker

Dilution Source Cat. no.

Oct4 1:50 Abcam ab18976

NANOG 1:50 Santa Cruz Biotechnology SC-293121

TRA1-81 1:50 Santa Cruz Biotechnology SC-21706

Sox2 1:100 Santa Cruz Biotechnology SC-17320

iPSC: Induced pluripotent stem cell.

Figure 1. Experimental timeline for the reprogramming experiment for MM-MSCs.

256



passaged by TrypLE (Life Technologies, Waltham, MA, USA) to be

prepared as a suspension. The cells were stained with antibodies

for SSEA4, Tra1-81, and Oct3/4 (BD Biosciences Pharmingen,

San Diego, CA, USA).

Gene Expression Analysis

Cell-specific gene expressions (Lin28, Nr6A, Klf4, FoxD3, Myc,

Utf1, Msx1, Gata6, endogenous Oct4, endogenous Sox2, Nanog,

and Rex1) in the undifferentiated cells were determined by PCR

as previously described [37]. Gene expression level detection was

done with LightCycler 480 DNA SYBR Green I Master (Roche,

Mannheim, Germany) with specific primers on a LightCycler

480 real-time PCR instrument (Roche). The PCR reactions

performed for GAPDH (reference gene) were as follows: 45

cycles of denaturation, 10 s at 95 °C; annealing and extension,

30 s at 60 °C. Analysis of the results was performed using Roche

LightCycler 480 software.

In Vitro Embryonic Body Formation

Spontaneous embryonic body (EB) generation was used for

testing the in vitro differentiation capacity of iPSCs. Cells were

cultured with medium without bFGF2 in bacteriological culture

dishes for 21 days. Formation was monitored daily.

Results

iPSC Generation and Culture

The first colonies were obtained on the 6 th day of culture after

transduction. The structure of these colonies had a scattered

appearance compared to ESC colonies, but the boundaries

became more apparent in the following days. After the colonies

reached a certain size, they were transferred to new feeder

cell layers by mechanical passaging. These new colonies were

observed to form tight cell assemblies with clearly defined

boundaries observed as ESCs. Colony-like structures were

photographed under the microscope as they grew over the days

(Figure 2). When differentiated parts were identified, those parts

were cleaned and the culture was continued. During culturing,

the colonies kept their borders.

ESC markers such as SSEA-4, TRA-1-81, and Oct3/4 were positive

for cells in flow cytometry analysis (Figure 5). iPSCs cultured

on feeder layers were further characterized by IF methods. The

colonies were positive for pluripotent cell markers Oct4, TRA1-

60, Nanog, TRA1-81, and Sox2 (Figure 6).

According to expression analysis, a significant increase was

observed between the 1 st and 3 rd weeks for iPSC cultures. The

significant increase in c-Myc and Klf4 gene expressions in

the 2 nd week decreased in the 3 rd week. Since these genes are

transmitted by viruses, the initial expression was ectopic and

turned into the internal expression of the cells at the 3 rd week

(Figure 7).

Expressions of pluripotent genes were shown in all obtained

iPSCs. Significant increases were observed in the Oct4,

Nanog, Sox2, Rex1, Utf1, and Lin28 genes. Using the Sendai

virus, the Oct4, Sox2, c-Myc, and Klf4 genes were transferred

and their expressions were provided with the help of ectopic

vectors. According to these transferred vectors, the Oct4, Sox2,

c-Myc, and Klf4 expressions may not be the endogenous gene

expressions of the cells. However, the increased expression of

highly specific pluripotent genes such as Nanog, Lin28, and

Utf1 constitutes the most serious evidence that cells acquire a

pluripotent cell character (Figure 8). EB formation was obtained

after the 4 th day of suspended culturing (Figure 9).

Discussion

iPSCs carry immense potential for future cellular therapies.

However, they were also shown to carry the characteristics of

the cells they originated from and their niche [38] through their

Following the mechanical passaging of colonies cultured on

MEF, colonies were successfully grown in Geltrex-coated culture

dishes. It was observed that the colonies retained their classical

morphology (Figure 3).

Characterization

The resulting colonies were stained against ALP while on the

feeder layer in culture dishes. Colonies were marked with

ALP-FITC dye without loss of viability. With this labeling, cells in

colonies with ESC characteristics were stained (Figure 4). Green

colonies were selected under fluorescence microscopy and the

first cell lines were formed by physical passaging.

Figure 2. Development of the first iPSC colonies produced after

Sendai virus transfection was monitored. A) On the 6 th day

after transfection, B) 7 th day after transfection, C) 8 th day after

transfection, and D) 9th day after transfection. Scale bars: 50 µm

(A, B, C, and D).


257



epigenetic memory [39,40]. The first type of reprogrammed cells

was found to be fibroblasts. Other types of human cells have

also been tried for reprogramming, which might be potentially

easier [40].

In this study, we attempted the reprogramming of MSCs

obtained from MM patients’ BM. Our study uses a standardized

reprogramming approach to evaluate the reprogramming of

two cell lines in various stages of differentiation: terminally

differentiated fibroblasts as a control and multipotent

MSCs obtained from MM patients. Both types of cells were

reprogrammed with the CytoTune-iPS 2.0 Sendai Reprogramming

Kit that contains Yamanaka factors. Yamanaka factors have been

reported many times in the literature as adequate for effective

reprogramming [4,41,42,43]. The efficiency of iPSC generation

using the Sendai virus is much higher than that of conventional

vectors [43]. The elimination of the Sendai virus is also easier

than that of conventional vectors, which allows the obtaining

of transgene-free iPSCs.

First of all, microscopic analysis revealed that the generated

iPSCs possessed classical ESC-like morphological characteristics.

Secondly, reprogramming experiments demonstrated that both

cell lines can be reprogrammed up to the pluripotent stage,

which was confirmed by flow cytometry, IF staining, and gene

expression analyses. To confirm the spontaneous differentiation

potential, an in vitro EB formation assay was performed.

iPSCs have been successfully obtained from MM patients for

the first time here. One of the major findings of our study is

the rapid reprogramming of MSCs, which started as early as

the 6 th day with the appearance of the first colony-forming cell

accumulations. Considering the results of previous studies, this

rapid reprogramming can be attributed to the multipotent nature

of MSCs, which implies that the effectiveness of reprogramming

is related to the differentiation stage of the cell line. Adegani

et al. [44] demonstrated that human MSCs of various sources

such as adipose tissue and BM-MSCs intrinsically expressed core

pluripotency factors such as Lin28, Klf4, and Sox2 at higher

levels with Nanog at moderate levels and Oct4 at low levels,

which allows them to reprogram easily.

Figure 3. Development of new iPSC colonies obtained by

mechanical passaging method was observed in culture plates.

A, B, C, D, E, F, G, and H) iPSC colonies cultured in the feeder

layer were picked up and cultured under feeder-free conditions. I

and J) Microscopic views of iPSC colonies in the culture plate, not

feeder free, are monitored. A) 2 nd day of P0, B) 14 th day of P0, C)

3 rd day of P1, D)4 th of P1, E) 2 nd day of P2, F) 5 th day of P2, G) 5 th

day of P3, H) 7 th day of P3, I) 2 nd day of P0 on Geltrex, J) 5 th day of

P0 on Geltrex. Scale bars: 20 µm (A), 100 µm (B, D), 200 µm (C, F,

G, H, I, and J), 50 µm (E).


Our data show that human iPSCs can be derived from MSCs

more rapidly than fibroblasts. Obtaining MSCs from patients

does not require great effort because BM aspirates are taken

almost daily for diagnostic purposes in hematology clinics. MSCs

can be isolated from these samples. As a result, we generated

iPSCs from MM-MSCs for the first time. As we know from

previous studies, the osteogenic differentiation of MM-MSCs

is weak. Our next goal is to explore the differences between

the osteogenic differentiation potential of healthy donors’

MSCs-iPSCs and MM-MSCs-iPSCs. We are planning further

studies to understand the pathogenesis of this disease, because

MM-iPSCs could clarify the molecular mechanisms behind

the disease. Therefore, further studies should be developed

to understand the molecular mechanisms of this disease.

Understanding the pathogenetic mechanisms underlying the

disease is crucial for effective management and improving the

258



Figure 4. Combined images of light and fluorescent microscopes in which produced iPSC colonies reacting positively with ALP are

observed. A and D) Brightfield; B and E) FITC; C and F) overlay. Scale bars: 200 µm.

iPSC: Induced pluripotent stem cell; ALP: alkaline phosphatase.

Figure 5. Flow cytometric analysis of pluripotency marker antigens (SSEA-4, Tra-1-81, and Oct 3/4) in normal fibroblast iPSCs and

MM-MSCs-iPSCs.


259



quality of MM patients’ lives [22]. Based on current knowledge,

the investigation of novel targeted drugs and an understanding

of the role of novel targeted therapies in this disease are of

great interest [23]. For more successful results, researchers are

developing complex 3D environments using MM patients’ cells

[45]. iPSCs offer unprecedented opportunities for drug discovery

and screening with their ability to differentiate into all kinds of

cells found in the body.

Conclusion

The data obtained from this study confirm that iPSCs can be

derived from MSCs more rapidly than fibroblasts and iPSCs

have been successfully obtained here from MM patients for the

first time. iPSCs generated from MM-MSCs could clarify the

molecular mechanisms behind this disease. Thus, further studies

should be developed to understand the molecular mechanisms

of this disease. Our next goal is to discuss the differences

between the osteogenic differentiation potential of healthy

donors’ MSCs-iPSCs and MM-MSCs-iPSCs.

Supplemental Materials and Methods

Fibroblast Isolation

Foreskin samples were obtained from circumcision procedures

under sterile conditions and fibroblasts were derived using a

previously described culture method [46].

Characterization of MSCs

To confirm the phenotypic characteristics in vitro, MSCs at

passage 3 (P3) were analyzed. For the characterization, flow

cytometry analysis, IF staining, and differentiation studies were

performed.

Flow Cytometry

To confirm that MM-MSCs maintain their phenotypic

characteristics in vitro, undifferentiated MSCs were analyzed

by flow cytometry. Analyses were performed on a FACSCalibur

(Becton Dickinson, San Jose, CA, USA) with Cell Quest

software (BD Biosciences, Bedford, MA, USA). MM-MSCs were

immunophenotyped with antibodies against human antigens

(CD45, CD59, CD14, CD117, CD11b, CD34, CD44, CD90, CD15,

CD33, CD105, CD73, CD29, CD38, CD138, and CD166), as well

as their isotype controls immunglobulin G [(IgG1), (IgG1/G2a)]

(BD Biosciences).

Figure 6. Immunofluorescence staining of pluripotency marker

antigens Oct4 (A, B; green), TRA1-60 (C, D; red), Nanog (E, F;

green), TRA1-81 (G, H; red) and Sox2 (I, J; red) in fibroblasts and

MM-MSCs-iPSCs. All markers were positive for the colonies.

Scale bars: 20 µm (A), 100 µm (B, D), 200 µm (C, F, G, H, I, and J),

50 µm (E).

Figure 7. Pluripotent gene expression analysis of colonies formed

after viral infection. Gene expressions were monitored for

3 weeks (w1, w2, w3). As a result, it was seen that iPSCs express

pluripotent markers.

260



Immunofluorescence Staining

For cellular marker identification, cells at P3 were seeded

onto poly-L-lysine-coated 8-well chamber slides (BD

Biosciences). Cells were cultured for another 1-2 days

and then stained. For the determination of the expressed

protein profiles, IF staining was performed with fluorescence

dye-attached antibodies. IF analyses were performed as

previously described [47]. Briefly, samples were rinsed in PBS

and then fixed. Triton X-100 (0.025%; Merck, Darmstadt,

Germany) was used for permeabilization and cells were

incubated for 30 min with blocking serum (Santa Cruz

Biotechnology, Heidelberg, Germany) in PBS at 37 °C to

suppress nonspecific binding of IgGs. Following washing, the

primary antibodies (α-smooth muscle actin, CD29, vimentin,

nestin, CD34, CD44, fibronectin, vinculin, tenascin) were

used for incubating the cells overnight at 4 °C. The next day,

samples were incubated with secondary antibodies for 25 min

at room temperature. After the washing steps, the cells were

mounted with DAPI (Santa Cruz Biotechnology). Samples were

examined under a fluorescence microscope (Leica DMI 4000,

Leica Microsystems, Wetzlar, Germany).

Acknowledgments

Figure 8. Measurement of the expression of pluripotent genes

by real-time polymerase chain reaction. The HPRT gene was

used as the reference gene. Gene expression values are expressed

according to fold values relative to the HPRT gene. iPSCs obtained

from fibroblasts were used as a control in gene expression analysis.

This study was supported by the Scientific and Technological

Research Council of Turkey (TÜBİTAK, Grant 112S296). The

authors would like to thank Dr. Özgür Mehtap for providing BM

aspirates from patients and Cansu Demir, Ayça Dikmen MD, and

Gökhan Duruksu MD for their technical assistance.

Ethics

Ethics Committee Approval: The study received ethical

approval from the Kocaeli University Faculty of Medicine’s

Ethics Committee (KAEK 2012/38) for the collection of human

samples.


Concept: İ.Y.B., E.K.; Design: İ.Y.B., E.K.; Data Collection or

Processing: İ.Y.B., E.K.; Analysis or Interpretation: İ.Y.B., E.K.;

Literature Search: İ.Y.B., E.K.; Writing: İ.Y.B., E.K.


authors.

Financial Disclosure: TUBİTAK (112S296).

Figure 9. Embryoid body (EB) formation of iPSCs. A, B) EBs

generated from fibroblasts. C, D) EBs generated from MM-MSCs.

Scale bars: 200 µm (A and C), 100 µm (B and D).

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Zhai H. et al: Induced Apoptosis in Acute Myeloid Leukemia

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2020.0769


LncRNA-DUXAP8 Regulation of the Wnt/β-Catenin Signaling

Pathway to Inhibit Glycolysis and Induced Apoptosis in Acute

Myeloid Leukemia

Akut Myeloid Lösemide Glikolizi Önlemek ve Apoptozu İndüklemek için Wnt/β-Catenin

Sinyal Yolunun LncRNA-DUXAP8 ile Düzenlenmesi

Hong Zhai, Junting Zhao, Juan Pu, Pan Zhao, Jin Wei

The Affiliated Hospital of North Sichuan Medical College, Department of Hematology, Nanchong, China

Abstract

Objective: Acute myeloid leukemia (AML) is a malignancy of the

hematopoietic system, accounting for approximately 70% of acute

leukemias. Long noncoding RNA-DUXAP8 (lncRNA-DUXAP8) has been

found to be abnormally expressed in a variety of tumors. However, its

function and mechanism in AML have not been studied. We investigate

the effect of lncRNA-DUXAP8 on AML and its mechanism so as to

provide a new theoretical basis for the diagnosis and treatment of AML.

Materials and Methods: The expression of lncRNA-DUXAP8 in

AML bone marrow tissues and the THP-1, HL-60, TF-1, AML193,

and U937 cell lines was detected by qRT-PCR. It was then altered by

transfecting plasmids overexpressing si-DUXAP8 and lncRNA-DUXAP8,

respectively. CCK8 and cell colony assay were performed to evaluate

the proliferation ability of AML cells. In addition, flow cytometry was

used to observe the apoptosis process. Glucose and lactate kits were

utilized to detect glucose consumption and lactate levels. Finally,

western blotting was performed to detect the expression of proteins

related to the Wnt/β-catenin signaling pathway in cells.

Results: LncRNA-DUXAP8 was downregulated in both AML bone marrow

tissues and cell lines. Upon interfering with lncRNA-DUXAP8 in AML cell

line THP-1, AML cell proliferation and glycolysis were promoted while

cell apoptosis was inhibited. The opposite results were obtained after

overexpressing lncRNA-DUXAP8. Meanwhile, western blotting confirmed

that interference with lncRNA-DUXAP8 stimulated the expression of

proteins Wnt5a, β-catenin, c-Myc, and cyclin-D1 in the Wnt/β-catenin

pathway. Moreover, overexpression of lncRNA-DUXAP8 inhibited the

expression of Wnt/β-catenin pathway proteins. Finally, LiCl, an activator

of the Wnt/β-catenin pathway, reversed the regulation of AML cells by

lncRNA-DUXAP8 upregulation compared with the DUXAP group.

Conclusion: This study showed that lncRNA-DUXAP8 regulated the

Wnt/β-catenin signaling pathway to inhibit glycolysis and induce

apoptosis in AML. This experiment has provided new angles and an

experimental basis for treating patients with AML.

Keywords: Acute myeloid leukemia, LncRNA-DUXAP8, Apoptosis,

Glycolysis, Wnt/β-catenin signaling pathway

Öz

Amaç: Akut miyeloid lösemi (AML), hematopoietik sistemin bir

malinitesidir ve akut lösemilerin yaklaşık %70’ini oluşturur. Uzun

kodlamayan RNA-DUXAP8’in (lncRNA-DUXAP8) çeşitli tümörlerde

anormal şekilde ifade edildiği bulunmuştur. Ancak, AML’deki işlevi

ve mekanizması çalışılmamıştır. AML’nin tanı ve tedavisi için yeni

bir teorik temel sağlamak amacıyla lncRNA-DUXAP8’in AML ve

mekanizması üzerindeki etkisini araştırdık.

Gereç ve Yöntemler: AML kemik iliği dokularında ve THP-1, HL-60, TF-1,

AML193 ve U937 hücre dizilerinde lncRNA-DUXAP8 ifadesi qRT-PCR ile

tespit edildi. Daha sonra sırasıyla si-DUXAP8 ve lncRNA-DUXAP8’i aşırı

ifade eden plazmitlerin transfekte edilmesiyle değiştirildi. AML hücrelerinin

çoğalma yeteneğini değerlendirmek için CCK8 ve hücre kolonisi testi

yapıldı. İlaveten, apoptoz sürecini gözlemlemek için akım sitometri

kullanıldı. Glukoz tüketimini ve laktat düzeylerini saptamak için glukoz ve

laktat kitleri kullanıldı. Son olarak, hücrelerde Wnt/β-katenin sinyal yolu

ile ilgili proteinlerin ifadesini saptamak için western blot testi yapıldı.

Bulgular: LncRNA-DUXAP8, hem AML kemik iliği dokularında

hem de hücre dizilerinde baskılanmıştı. AML hücre dizisi THP-1’de

lncRNA-DUXAP8’e müdahale edilmesi üzerine, hücre apoptozu inhibe

edilirken AML hücre proliferasyonu ve glikoliz kolaylaştırıldı. lncRNA-

DUXAP8’in aşırı ifadesinden sonra tersi sonuçlar elde edildi. Bu arada,

western blot yöntemi, lncRNA-DUXAP8 ile etkileşimin Wnt/β-katenin

yolağında Wnt5a, β-katenin, c-Myc ve siklin-D1 proteinlerinin

ifadesini stimüle ettiğini doğruladı. Ayrıca, lncRNA-DUXAP8’in aşırı

ifadesi, Wnt/β-katenin yolu proteinlerinin ifadesini inhibe etti. Son

olarak, Wnt/β-katenin yolunun bir aktivatörü olan LiCl, DUXAP

grubuyla karşılaştırıldığında lncRNA-DUXAP8 upregülasyonu ile AML

hücrelerinin regülasyonunu tersine çevirdi.

Sonuç: Bu çalışma, lncRNA-DUXAP8’in, AML’de glikolizi inhibe

etmek ve apoptozu indüklemek için Wnt/β-katenin sinyal yolunu

düzenlediğini gösterdi. Bu deney, AML’li hastaları tedavi etmek için

yeni açılar ve deneysel bir temel sağlamıştır.

Anahtar Sözcükler: Akut myeloid lösemi, LncRNA-DUXAP8, Apoptoz,

Glikoliz, Wnt/β-katenin sinyal yolu



Address for Correspondence/Yazışma Adresi: Jin Wei, M.D., The Affiliated Hospital of North Sichuan

Medical College, Department of Hematology, Nanchong, China

E-mail : hematology405@163.com ORCID: orcid.org/0000-0003-2205-1901

Received/Geliş tarihi: December 28, 2020

Accepted/Kabul tarihi: August 16, 2021

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Introduction

Acute myeloid leukemia (AML) is an aggressive malignancy

of the hematopoietic system caused by the abnormal

proliferation of myeloid cells in the bone marrow and other

hematopoietic tissues [1,2]. AML might occur at any age and

accounts for 15%-20% of pediatric leukemias and 80% of

adult leukemias [3]. In recent years, breakthroughs have been

made in treatment strategies such as intensive chemotherapy

and even hematopoietic stem cell transplantation. However, the

overall clinical prognosis of AML, especially in elderly patients,

generally remains disappointing [4,5]. Poor clinical prognosis

of AML is closely related to drug resistance, recurrence,

comorbidities, and/or treatment-related mortality in the early

stage of chemotherapy [6]. Based on the highly heterogeneous

characteristics of AML clones and subclones, a large number of

clinical and experimental studies have identified a wide range

of AML molecular biological expression profiles, which in turn

may facilitate prediction and improvement of the prognosis

of patients [7]. Results suggest that analyzing AML molecular

heterogeneity and screening corresponding specific molecular

targets are significant steps in evaluating the clinical prognosis

of AML and developing therapeutic strategies [8].

Long noncoding RNAs (lncRNAs) are a class of endogenous

RNAs more than 200 nucleotides in length that are completely

lacking or have extremely weak polypeptide coding capacity [9].

Numerous studies have confirmed that lncRNAs located in the

nucleus are mainly involved in biomacromolecule-chromatin

interactions and transcriptional regulation, as well as RNA

processing. Furthermore, lncRNAs located in the cytoplasm can

affect not only mRNA stability and translation but also numerous

cell signaling pathways [10]. Recent reports have found that

lncRNA-HOTAIRMI, which is transcribed from the human HOXA

gene cluster, is able to control myeloid cell development by

regulating retinoic acid-induced expression of the HOXA1 and

HOXA4 genes during bone marrow formation [11]. Some lncRNAs

play a key role not only during normal hematopoiesis but also

during leukemogenesis [12,13]. One known as lncRNA-HOTAIR

exerts a carcinogenic effect by mediating cell proliferation or

apoptosis via regulation of c-Kit expression, and it may serve

as a biological marker of AML prognosis [14]. Studies have

shown that in elderly AML patients with normal cytogenetics,

the abnormal expression profile of lncRNAs is closely related

to clinical characteristics and the abnormal expression of some

lncRNAs is closely related to the curative effect and survival

time [15].

Recent studies have reported that lncRNA-DUXAP8 (double

homeobox A pseudogene 8) with a length of 2107 bp is located

on chromosome 22q11. It is abnormally highly expressed in a

variety of tumor tissues and can promote the proliferation of

hepatocellular carcinoma [16], bladder cancer [17], pancreatic

cancer [18], renal cell carcinoma [19], neuroglioma cells [20],

and non-small-cell lung cancer [20]. LncRNA-DUXAP8 is also

closely related to the poor prognosis of these tumors. When

analyzing pathological data in the clinic, it was found that

lncRNA-DUXAP8 was significantly highly expressed in gastric

cancer samples with high TNM grades and lymph node metastasis.

It was shown that lncRNA-DUXAP8 was closely related to the

prognosis of patients, as well, confirming that lncRNA-DUXAP8

may play an important role in the development of gastric cancer

as a molecular target for diagnosis and prognosis [21]. The

above results suggest that lncRNA-DUXAP8, as an oncogene,

can promote the proliferation and invasion of malignant tumor

cells and inhibit apoptosis. Therefore, we hypothesize that

lncRNA-DUXAP8 is also able to regulate the development of

AML, although its specific function and mechanism of action

need to be further explored. In this study, we investigate the

effect of lncRNA-DUXAP8 (henceforth DUXAP8) on AML and

its mechanism so as to provide a new theoretical basis for the

diagnosis and treatment of AML.


Specimen Collection and Disposal

Bone marrow tissues from patients diagnosed with AML

and normal bone marrow tissues from healthy donors in our

hospital from January 2016 to June 2018 were collected. The

clinicopathological characteristics and laboratory features

(age, gender, karyotype, etc.) were recorded. The diagnostic

and staging criteria used for all patients with first diagnosed

leukemia were the 2016 World Health Organization staging

criteria [22]. In addition, all patients had not been treated with

chemotherapy or radiotherapy. All subjects had no history of

major systemic disease and they voluntarily signed informed

consents. The study was approved by the Ethics Committee of

the Affiliated Hospital of North Sichuan Medical College.

Cell Culture

Human normal bone marrow cells (HS-5) and five AML cell

lines (THP-1, HL-60, TF-1, AML193, and U937) were purchased

from the American Type Culture Collection (ATCC, Manassas, VA,

USA). All cells were cultured in DMEM medium (Gibco, Waltham,

MA, USA) with 10% fetal bovine serum (FBS; Gibco) and 1%

penicillin/streptomycin and placed in an incubator with 5% CO 2

at 37 °C.

Cell Transfection

Overexpression vectors carrying DUXAP8 interference or

full-length sequences as well as the corresponding unloaded

si-NC, si-DUXAP8, vector, and DUXAP were synthesized and

cloned by Sangon Biotech Co., Ltd. (Shanghai, China) and were

then identified by enzyme digestion and sequencing. THP-1 cells

in the logarithmic growth phase were seeded in 12-well plates.

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When the cell confluence reached 50%-60%, si-NC, si-DUXAP8,

vector, and DUXAP were transfected into THP-1 cells according

to conventional operational steps using Lipofectamine 2000.

The cells were divided into five groups as follows: (1) sham:

THP-1 cells without transfection; (2) si-NC: THP-1 cells

transfected with negative siRNA; (3) si-DUXAP8: THP-1 cells

transfected with DUXAP8 siRNA; (4) vector: THP-1 cells

transfected with a negative vector; (5) DUXAP8: THP-1 cells

transfected with pcDNA-DUXAP8. After 6 h of transfection,

fresh media were replaced to culture cells for subsequent

experiments.

CCK8 Assay

The CCK8 assay was used to detect the level of cell proliferation.

After the transfected cells had been cultured for 12 h, they were

plated in 96-well plates at a concentration of 1x10 4 cells/well.

After that, 10 µL of CCK8 solution (10% concentration) was

respectively added at 24, 48, and 72 h. The optical density at

450 nm was measured with an enzyme-labeling instrument. Six

duplicated wells were set at each time point in each group. The

experiment was repeated three times.

Colony Formation Assay

Cells from different groups were respectively inoculated in

6-well plates with 300 cells/well. Afterwards, 2 mL of cultivation

liquid was added to each well. Cells were cultured in incubators

for 10 days until cell clones were visible and then fixed in 4%

paraformaldehyde for 15 min and stained with 1% crystal violet

for 4 h. After washing and drying, they were observed and

photographed. The experiment was repeated three times.

Flow Cytometry

The Annexin V-PE/7-AAD double-staining method was utilized

to detect cell apoptosis. After the transfected cells had been

cultured for another 48 h, they were digested with trypsin and

washed with PBS two or three times. Then 500 µL of binding

buffer and 5 µL of Annexin V-PE were added to about 5x10 5

cells. After thorough mixing, 5 µL of 7-AAD staining solution

was added and the solution was mixed again. After a 10-min

reaction at room temperature in the dark, quantitative detection

was performed immediately with a FACScan flow cytometer.

The experiment was repeated three times.

Detection of Glucose Consumption and Lactate Production

Cells in the logarithmic growth phase were seeded in 6-well

plates with 1.0x10 5 cells/well. After the transfected cells were

cultured for 24 h, 100 µL of supernatant from each group

was collected. Supernatants and standards from both the

control group and the experimental group were placed on ice.

Afterwards, the cells were digested with trypsin and counted

for calibration. For determining the glucose content, glucose

standards with 0, 1, 2, 3, 4, and 5 µL were first added, followed

by double-distilled water to increase the volume of solution

in each EP centrifuge tube to 10 µL. After that, the standard

curve was obtained, and then 10 µL of supernatant was taken

from the experimental group and the control group. To each EP

centrifuge tube was added 1 mL of glucose detection reagent

with thorough mixing. Tubes were then placed in a water bath

at 37 °C for 10 min and 200 µL of mixed solution was removed

and added to an ELISA plate. The glucose concentration in each

well was detected at the wavelength of 505 nm with an ELISA

analyzer.

The method for determination of lactic acid content was

similar to the glucose detection method. First, 0, 1, 2, 3, and 4

µL of lactic acid standard was respectively added to the ELISA

plates. Afterwards, the volume of each well was made up to 4

µL with double-distilled water to prepare the standard curve

and then 4 µL of supernatant from the experimental group and

the control group was respectively mixed with 200 µL of lactic

acid detection reagent and added into each EP centrifuge tube.

The lactic acid concentration of each well was detected at the

wavelength of 530 nm with the ELISA analyzer. The experiment

was repeated three times.

qRT-PCR Assay

Based on published references, primer sequences of GAPDH

were designed. Total RNA from tissues and cells was respectively

extracted using an RNA extraction kit in order to determine

its purity and concentration. RNA was reverse-transcribed

into cDNA with the TaKaRa reaction system (Shiga, Japan).

Subsequently, cDNA was used as a template for the quantitative

real-time polymerase chain reaction (qRT-PCR). The reaction

system was programmed as follows: pre-denaturation at 95 °C

for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for

40 s, and 74 °C for 30 s. Measurements were performed on an

ABI 7500 qRT-PCR instrument (Applied Biosystems, Bedford,

MA, USA). The primer sequences used in this study were as

follows: DUXAP8, forward: 5’-AGGATGGAGTCTCGCTGTATTGC-3’

and reverse: 5’-GGAGGTTTGTTTTCTTCTTTT-3’. GAPDH,

forward: 5’-AGGAAGAGCACAAGGAAGGCA-3’ and reverse:

5’-GGTTGCACATAGACGAGGACT-3’. The relative expression of

DUXAP8 was calculated using the 2 -∆∆CT method with GAPDH as

an internal reference. The experiment was repeated three times.

Western Blotting

Transfected THP-1 cells were lysed with cell lysate to isolate total

intracellular protein. Protein concentration was determined by

the BCA quantitative method. First of all, proteins were separated

by 12% polyacrylamide-gel electrophoresis (SDS-PAGE).

Subsequently, proteins were transferred onto polyvinylidene

fluoride membrane and blocked with 5% skimmed milk for

2 h at room temperature. Afterwards, the membranes were

incubated with primary antibodies Wnt5a rabbit polyclonal

266



antibody (ab235966, 1:1000), β-catenin rabbit monoclonal

antibody (ab32572, 1:1000), c-Myc rabbit monoclonal antibody

(CST9402, 1:1000), and cyclin-D1 rabbit monoclonal antibody

(ab16663, 1:1000) overnight at 4 °C. The membranes were

washed with TBST three times, followed by incubation with the

corresponding secondary antibody for 2 h at room temperature.

Finally, the membrane was washed three times with TBST for

electrochemical luminescence and color development. Relative

protein expression was calculated using β-actin as an internal

reference. The experiment was repeated three times.


Statistical analysis was performed using SPSS 20.0 software

(IBM Corp., Armonk, NY, USA). Data were presented as mean

± standard deviation. Comparisons among multiple groups

were performed using one-way analysis of variance (ANOVA),

while comparisons between two groups were performed with

Student’s t-test. Kaplan-Meier analyses were used to analyze

the impact of DUXAP8 expression on overall survival (OS). Values

of p<0.05 were considered statistically significant.

Results

Underexpression of DUXAP8 in AML

A retrieval from the Cancer Genome Atlas (TCGA) database

revealed that DUXAP8 was downregulated in AML patients

(Figure 1A). The results of qRT-PCR confirmed that the expression

level of DUXAP8 was significantly reduced in the bone marrow

tissues of AML patients compared with normal bone marrow

(Figure 1B), which showed that DUXAP8 was involved in the

development of AML. To further confirm this, we selected five

human AML cell lines, THP-1, HL-60, TF-1, AML193, and U937,

as well as normal human bone marrow cell line HS-5 as a control

group. qRT-PCR also revealed that the expression of DUXAP8

in AML cell lines THP-1, HL-60, TF-1, AML193, and U937 was

obviously decreased compared with HS-5 cells (Figure 1C).

Low Expression of DUXAP8 Is Associated with Poor Prognosis

The clinical characteristics of DUXAP8-low and DUXAP8-

high groups are shown in Table 1. DUXAP8 expression was

lower in patients with the AML-M2 subtype (p=0.005) and

poor/intermediate risk (p=0.000). Significant differences

were also detected for both karyotype and karyotype

classification (p=0.000 and p=0.013, respectively). In addition,

Kaplan-Meier plots indicated that the OS of the DUXAP8-low

group was significantly shorter than that of the DUXAP8-high

group (Figure 2).

DUXAP8 Inhibited Biological Functions in AML Cells

In order to further explore the biological functions exerted by

DUXAP8 in AML, DUXAP8 was overexpressed and interfered

with. The biological functions of viability, proliferation, and

apoptosis of THP-1 cells were then detected using the CCK8

assay, cell colony assay, and flow cytometry, respectively. In

addition, glucose consumption and lactate production were

detected using a glucose kit and lactate kit. Afterwards, the

expression levels of Wnt/β-catenin signaling pathway-related

proteins (Wnt5a, β-catenin, c-Myc, cyclin-D1) were verified by

western blotting. Since DUXAP8 had the lowest expression in the

THP-1 cell line, this cell line was used for subsequent experiments.

The results suggested that compared to the si-NC group, the

proliferation ability, cell viability, glucose consumption, and

lactate production in the si-DUXAP8 group were significantly

increased while apoptosis was inhibited. On the contrary,

Figure 1. Underexpression of DUXAP8 in acute myeloid leukemia (AML) bone marrow tissues and AML cell lines. (A) The Cancer Genome

Atlas database reveals downregulation of DUXAP8 in AML patients; (B) decrease in expression level of DUXAP8 in AML patients’ tissues

compared to normal human bone marrow tissues, ***p<0.001: significant difference compared to normal bone marrow; (C) reduction

of expression levels of DUXAP8 in AML cell lines THP-1, HL-60, TF-1, AML193, and U937 compared to HS-5 cells, *p<0.05: significant

difference compared to HS-5 group, **p<0.01: significant compared to HS-5 group, ***p<0.001: significant compared to HS-5 group.

267



Figure 2. The Kaplan-Meier plots of DUXAP8 expression for

overall survival (OS) in acute myeloid leukemia.

compared with the vector group, the proliferation ability,

cell viability, glucose consumption, and lactate production of

THP-1 cells were significantly decreased while apoptosis was

promoted (Figures 3A-3E). Meanwhile, according to western

blotting, the protein expression levels of Wnt5a, β-catenin,

c-Myc, and cyclin-D1 were greatly increased in the cells of the

si-DUXAP8 group compared to the si-NC group. In contrast,

the protein expression levels of Wnt5a, β-catenin, c-Myc,

and cyclin-D1 were obviously decreased in the cells of the

DUXAP8 group compared to the vector group (Figures 3F and

3G). These experimental results confirmed that overexpression

of DUXAP8 inhibited glycolysis and induced apoptosis of AML

cells, ultimately inhibiting the activation of the Wnt/β-catenin

signaling pathway.

Table 1. Comparison of clinical and genetic characteristics of patients with AML.

Total

DUXAP8 expression

Low expression High expression

χ 2 /z p

Age (years) 37 (12-81) 37.5 (15-81) 36.5 (12-79) 0.068 0.946

Gender (%) 0.44 0.507

Male 26 (65.0) 12 (60.0) 14 (70.0)

Female 14 (35.0) 8 (40.0) 6 (30.0)

FAB subtypes (%) 16.978 0.005

M0 2 (5) 0 (0) 2 (10.0)

M1 1 (2.5) 1 (5.0) 0 (0)

M2 13 (32.5) 10 (50.0) 3 (15.0)

M3 3 (7.5) 3 (15.0) 0 (0)

M4 10 (25.0) 2 (10.0) 8 (40.0)

M5 11 (27.5) 4 (20.0) 7 (35.0)

Karyotype (%) 27.185 0.000

Normal karyotype 8 (20) 5 (25.0) 3 (15.0)

t(15;17) 3 (7.5) 3 (15.0) 0 (0)

t(8;21) 6 (15.0) 5 (25.0) 1 (5.0)

inv 5 (12.5) 2 (10.0) 3 (15.0)

t(6;9) 3 (7.5) 3 (15.0) 0 (0)

11q23 4 (10.0) 2 (10.0) 2 (10.0)

Complex karyotype 11 (27.5) 0 (0) 11 (55.0)

Karyotype classification (%) 8.681 0.013

Favorable 11 (27.5) 7 (35.0) 4 (20.0)

Intermediate 19 (47.5) 5 (25.0) 14 (70.0)

Poor 10 (25.0) 8 (40.0) 2 (10.0)

Risk stratification (%) 17.143 0.000

Poor/intermediate 12 (30.0) 12 (60.0) 0 (0)

Favorable 28 (70.0) 8 (40.0) 20 (100)

Median WBC, x10 9 /L (range) 22 (1-195.9) 27.6 (1.0-138) 17.1 (1.7-195.9) 1.123 0.262

FAB: French-American British; WBC: white blood cell count.

268



LiCl as an Activator of the Wnt/β-Catenin Pathway Reversed the

Regulation of DUXAP8 in AML Cells

The above experimental results confirmed that the biological

functions exerted by DUXAP8 in AML are directly related to the

Wnt/β-catenin pathway. In order to further clarify its molecular

mechanism, we not only overexpressed DUXAP8 in THP-1 cells

but also added the Wnt/β-catenin pathway activator LiCl. The

results showed that compared with the DUXAP8 group, the

proliferation level, cell viability, glucose consumption, and

lactate production level of cells in the DUXAP8 + LiCl group

were greatly increased while apoptosis was effectively inhibited

(Figures 4A-4E). Meanwhile, the protein expression levels of

Wnt5a, β-catenin, c-Myc, and cyclin-D1 were significantly

increased in THP-1 cells (Figures 4F and 4G). These experimental

results confirmed that DUXAP8 may inhibit AML cell function

by regulating the activation of the Wnt/β-catenin pathway.

Discussion

Numerous reports have pointed out that lncRNA plays an

oncogene role in the development of tumors by regulating

Figure 3. DUXAP8 regulates biological functions of acute myeloid leukemia cells. (A) qRT-PCR was performed to detect DUXAP8

expression level in THP-1 cells after transfection with si-NC, si-DUXAP8, vector, and DUXAP8; (B) CCK8 assay for THP-1 cell proliferation;

(C) colony assay for THP-1 cell viability; (D) flow cytometry to evaluate THP-1 apoptosis rate; (E) glucose and lactate kits were utilized

to detect glucose consumption and lactate production levels in THP-1 cells, **p<0.01: significant difference compared to si-NC group,

***p<0.001: significant compared to si-NC group, ##p<0.0: significant compared to vector group, ###p<0.001: significant compared

to vector group; (F, G) protein expression levels of Wnt5a, β-catenin, c-Myc, and cyclin-D1 were obviously decreased in the cells of the

DUXAP8 group compared to the vector group, see previous explanation of statistical significance.

269



cancer cell metastasis and cell growth as well as inhibiting

cancer apoptosis, or by interacting with other genes to cause

DNA damage [23,24]. Recent studies have found that lncRNA

as an antisense gene locus in PU.1 can negatively regulate

the expression of hematopoietic transcription factor PU.1 to

maintain normal hematopoietic development, thereby inhibiting

leukemia [25]. In addition, antisense lncRNA-IRAIN, which is

transcribed from the insulin-like growth factor type I receptor

serving as the gene locus, plays a negative regulatory role in

high-risk AML patients [26]. Previous studies have revealed

that DUXAP8 promotes bladder cancer cell proliferation by

regulating PTEN [17]. By downregulating DUXAP8 in lung

cancer cells, it is not only possible to inhibit the proliferation

of pancreatic cancer cells through silencing CDKN1A and KLF2,

but also to induce their apoptosis [18]. However, its biological

functions and regulatory mechanism in AML are still unclear. In

this study, it has been demonstrated by the TCGA database and

qRT-PCR assay that DUXAP8 is expressed at lower levels in AML,

and AML patients with low DUXAP8 expression showed worse

prognosis. On the basis of this result, we then investigated

the effects as well as the specific mechanism of DUXAP8 on

biological characteristics of AML cells.

Rapid proliferation and metastasis of tumor cells require

much energy and, therefore, energy metabolism is of great

importance [27,28]. The mainstream view is that tumor cells

consume large amounts of glucose to supply tumor cells via

glycolysis under oxygen-sufficient conditions, known as the

classical Warburg effect [29]. To date, glycolysis is still widely

considered as an energy source for tumor cells [30]. In the

present study, it was suggested that the glucose consumption

and lactate production levels in AML THP-1 cells increased

significantly after interference with DUXAP8 expression, while

they decreased significantly with overexpression. This result

implied that DUXAP8 was able to effectively inhibit glycolysis in

Figure 4. LiCl as an activator of the Wnt/β-catenin pathway reverses the regulation of DUXAP8 on acute myeloid leukemia cells. (A)

CCK8 assay to detect the cell proliferation of THP-1 after transfection with vector, DUXAP8, and DUXAP8 + LiCl; (B) cell colony assay to

detect the cell viability of THP-1 after transfection with vector, DUXAP8, and DUXAP8 + LiCl; (C) flow cytometry to detect the apoptosis

rate of THP-1 after transfection with vector, DUXAP8, and DUXAP8 + LiCl; (D, E) glucose and lactate kits were utilized to detect glucose

consumption and lactate production levels of THP-1 after transfection with vector, DUXAP8, and DUXAP8 + LiCl, *p<0.05: significant

difference compared to the vector group, **p<0.01: significant compared to the vector group, & p<0.05: significant compared to the

DUXAP8 group, && P<0.01: significant compared to the DUXAP8 group; (F, G) protein expression levels of Wnt5a, β-catenin, c-Myc, and

cyclin-D1 were significantly increased in THP-1 cells, see previous explanation of statistical significance.

270



AML. It is well known that abnormal proliferation and apoptosis

are among the markers of tumor cells. Therefore, the core of

antitumor methods is to inhibit the proliferation of tumor cells

and promote their apoptosis [31,32]. In subsequent experiments,

we applied the CCK8 assay, cell colony assay, and flow cytometry

to respectively detect AML cell proliferation, cell viability, and

apoptosis. The results showed that cell proliferation and viability

were significantly promoted and apoptosis was inhibited in

AML cells after interfering with DUXAP8. Opposite results were

displayed after overexpressing DUXAP8. Thus, DUXAP8 might

inhibit the proliferation of AML cells as well as promoting their

apoptosis.

After identifying the effect of DUXAP8 on the biological

characteristics of AML, it is necessary to focus on its specific

mechanism in subsequent experiments. Various reports have

confirmed that many signaling pathways (e.g., the mTOR

signaling pathway) play key roles in all kinds of cellular processes,

including the regulation of gene expression, cell growth, and

proliferation. Studies have found that cell proliferation and

metastasis are important factors for cancer development. It

should be noted that the Wnt/β-catenin signaling pathway is

a crucial signaling pathway for regulation of cell proliferation

and metastasis, often abnormally activated in the development

of many cancers including prostate cancer, oral squamous cell

carcinoma, and cutaneous squamous cell carcinoma [33,34,35].

Additional studies also confirmed that the Wnt/β-catenin

signaling pathway is able to accelerate the glycolytic process

so as to further provide energy for tumor cells [36,37]. Previous

studies have confirmed that DUXAP8 inhibits biological function

by inhibiting proliferation and glycolysis while promoting the

apoptosis of AML cells. Therefore, this signaling pathway may be

a target pathway. Since Wnt5a, β-catenin, c-Myc, and cyclin-D1

are key factors in the Wnt/β-catenin signaling pathway [38],

it is essential to increase their expressions for Wnt/β-catenin

activation [39]. All in all, activation of this signaling pathway

contributes to promoted survival of tumor cells, which leads to

unlimited proliferation during the tumor phase.

In this study, western blotting was performed to detect the

correlation between DUXAP8 and the Wnt/β-catenin signaling

pathway. The results of the western blot assay showed that upon

interfering with the expression of DUXAP8, expression levels

of Wnt5a, β-catenin, c-Myc, and cyclin-D1 were upregulated;

however, after overexpressing DUXAP8, these expression levels

were significantly downregulated. In addition, the upregulation

of Wnt5a, β-catenin, c-Myc, and cyclin-D1 indicated that the

Wnt/β-catenin signaling pathway was inhibited. Therefore, it

was speculated that DUXAP8 might exert biological functions

in terms of inhibiting glycolysis and inducing apoptosis in AML

by inhibiting the activation of the Wnt/β-catenin signaling

pathway. Subsequent experiments further validated this

speculation. By adding LiCl to THP-1 cells while overexpressing

DUXAP8, the biological function of the AML cell lines was

detected. These results confirmed that LiCl as an activator of

the Wnt/β-catenin pathway reversed the regulation of DUXAP8

in AML cells. These results suggest that DUXAP8 might inhibit

glycolysis and induce apoptosis in AML by regulating the

Wnt/β-catenin signaling pathway.

Conclusion

The current study found that DUXAP8 was expressed at lower

levels in AML. DUXAP8 was able to inhibit the activation

of the Wnt/β-catenin signaling pathway and weaken the

proliferation, viability, and glycolytic process of AML cells, as

well as exacerbating apoptosis. These results suggest that

DUXAP8 plays an important role in AML development and

may become a therapeutic target for AML. However, there are

some shortcomings of this study; in particular, the functional

mechanism of DUXAP8 was not verified in animals. This will

need to be further explored in subsequent studies.

Ethics

Ethics Committee Approval: The study was approved by the

Ethics Committee of the Affiliated Hospital of North Sichuan

Medical College.

Informed Consent: All subjects had no history of major systemic

disease and they voluntarily signed informed consent forms.


Surgical and Medical Practices: H.Z., J.Z., J.W.; Concept: H.Z.,

J.W.; Design: H.Z., J.W.; Data Collection or Processing: H.Z., J.Z.,

J.P., J.W.; Analysis or Interpretation: H.Z., J.Z., J.P., P.Z., J.W.;

Literature Search: H.Z., J.Z., J.P., P.Z., J.W.; Writing: H.Z., J.W.


authors.



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Tombak A. et al: Ibrutinib Experience in Patients with CLL

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2021.0007


Efficacy and Safety of Ibrutinib Therapy in Patients with Chronic

Lymphocytic Leukemia: Retrospective Analysis of Real-Life Data

Kronik Lenfositik Lösemili Hastalarda İbrutinib Tedavisinin Etkililiği ve Güvenilirliği: Gerçek

Hayat Verilerinin Retrospektif Analizi

Anıl Tombak 1 , Funda Pepedil Tanrıkulu 2 , Salih Sertaç Durusoy 3 , Hüseyin Derya Dinçyürek 4 , Emin Kaya 5 ,

Elif Gülsüm Ümit 6 , İrfan Yavaşoğlu 7 , Özgür Mehtap 8 , Burak Deveci 9 , Mehmet Ali Özcan 10 , Hatice Terzi 11 ,

Müfide Okay 12 , Nilgün Sayınalp 12 , Mehmet Yılmaz 3 , Vahap Okan 3 , Alperen Kızıklı 3 , Ömer Özcan 13 , Güven Çetin 13 ,

Sinan Demircioğlu 14 , İsmet Aydoğdu 15 , Güray Saydam 16 , Eren Arslan Davulcu 16 , Gül İlhan 17 , Mehmet Ali Uçar 18 ,

Gülsüm Özet 18 , Seval Akpınar 19 , Burhan Turgut 19 , İlhami Berber 5 , Erdal Kurtoğlu 20 , Mehmet Sönmez 21 ,

Derya Selim Batur 21 , Rahşan Yıldırım 22 , Vildan Özkocamaz 23 , Ahmet Kürşad Güneş 24 , Birsen Sahip 25 , Şehmus Ertop 25 ,

Olga Meltem Akay 26 , Abdülkadir Baştürk 27 , Mehmet Hilmi Doğu 28 , Aydan Akdeniz 1 , Ali Ünal 29 , Ahmet Seyhanlı 30 ,

Emel Gürkan 4 , Demet Çekdemir 31 , Burhan Ferhanoğlu 32

1Mersin University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Mersin, Turkey

2Başkent University Adana Application and Research Center, Adana, Turkey

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

4Çukurova University Faculty of Medicine, Department of Hematology, Adana, Turkey

5İnönü University Turgut Özal Medical Center, Department of Hematology, Malatya, Turkey

6Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey

7Adnan Menderes Univercity Faculty of Medicine, Department of Hematology, Aydın, Turkey

8Kocaeli University Faculty of Medicine,Department of Hematology, Kocaeli, Turkey

9Medstar Antalya Hospital, Clinic of Hematology, Antalya, Turkey

10Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey

11Cumhuriyet University Faculty of Medicine, Department of Hematology, Sivas, Turkey

12Hacettepe University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Ankara, Turkey

13Bezmialem Vakıf University Faculty of Medicine, Department of Hematology, İstanbul, Turkey

14Necmettin Erbakan University Meram Faculty of Medicine, Department of Hematology, Konya, Turkey

15Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa, Turkey

16Ege University Hospital, Clinic of Internal Medicine, Division of Hematology İzmir, Turkey

17Mustafa Kemal University Faculty of Medicine, Department of Internal Medicine, Hatay, Turkey

18Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey

19Namık Kemal University Faculty of Medicine, Department of Hematology, Tekirdağ, Turkey

20Antalya Training and Research Hospital, Clinic of Hematology, Antalya, Turkey

21Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey

22Ataturk University Faculty of Medicine, Department of Hematology, Erzurum, Turkey

23Uludağ University Faculty of Medicine, Division of Hematology, Bursa, Turkey

24Şanlıurfa Mehmet Akif İnan Training and Research Hospital, Clinic of Hematology, Şanlıurfa, Turkey

25Zonguldak Bülent Ecevit University Faculty of Medicine, Department of Hematology, Zonguldak, Turkey

26Koç University Faculty of Medicine, Department of Hematology, İstanbul, Turkey

27Konya Training and Research Hospital, Clinic of Internal Medicine, Konya, Turkey

28İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey

29Erciyes University Faculty of Medicine, Department of Internal Medicine, Kayseri, Turkey

30Ege University Faculty of Medicine, Department of Hematology, İzmir, Turkey

31Anadolu Medical Center, Bone Marrow Transplantation Center, Department of Hematology, Kocaeli, Turkey

32İstanbul University-Cerrahpaşa Cerrahpaşa Faculty of Medicine, Department of Internal Medicine Section of Haematology, İstanbul, Turkey



Address for Correspondence/Yazışma Adresi: Anıl Tombak, M.D., Mersin University Faculty of Medicine,

Department of Internal Medicine, Division of Hematology, Mersin, Turkey

Phone : +90 532 346 07 67

E-mail : aniltombak@mersin.edu.tr ORCID: orcid.org/0000-0002-7195-1845



273



Abstract

Objective: This study aimed to retrospectively evaluate the efficacy,

safety, and survival outcome of single-agent ibrutinib therapy in

chronic lymphocytic leukemia patients.

Materials and Methods: A total of 136 patients (mean age ± standard

deviation: 64.6±10.3 years, 66.9% males) who had received at least

one dose of ibrutinib were included in this retrospective multicenter,

noninterventional hospital-registry study conducted at 33 centers

across Turkey. Data on patient demographics, baseline characteristics,

laboratory findings, and leukemia-cell cytogenetics were retrieved.

Treatment response, survival outcome including overall survival (OS)

and progression-free survival (PFS), and safety data were analyzed.

Results: Overall, 36.7% of patients were categorized as Eastern

Cooperative Oncology Group (ECOG) class 2-3, while 44.9% were in

Rai stage 4. Fluorescence in situ hybridization revealed the presence

of del(17p) in 39.8% of the patients. Patients received a median of 2.0

(range: 0-7) lines of pre-ibrutinib therapy. Median duration of therapy

was 8.8 months (range: 0.4-58.0 months). The 1-year PFS and OS rates

were 82.2% and 84.6%, respectively, while median PFS time was 30.0

(standard error, 95% confidence interval: 5.1, 20.0-40.0) months and

median OS time was 37.9 (3.2, 31.5-44.2) months. Treatment response

(complete or partial response), PFS time, and OS time were better

with 0-2 lines versus 3-7 lines of prior therapy (p<0.001, p=0.001, and

p<0.001, respectively), with ECOG class 0-1 versus class 2-3 (p=0.006,

p=0.011, and p=0.001, respectively), and with Rai stage 0-2 versus 3-4

(p=0.002, p=0.001, and p=0.002, respectively). No significant difference

was noted in treatment response rates or survival outcome with respect

to the presence of comorbidity, bulky disease, or del(17p). While 176

adverse events (AEs) were reported in 74 (54.4%) patients, 46 of those

176 AEs were grade 3-4, including pneumonia (n=12), neutropenia

(n=11), anemia (n=5), thrombocytopenia (n=5), and fever (n=5).

Conclusion: This real-life analysis confirms the favorable efficacy and

safety profile of long-term ibrutinib treatment while emphasizing

the potential adverse impacts of poorer ECOG performance status,

heavy treatment prior to ibrutinib, and advanced Rai stage on patient

compliance, treatment response, and survival outcomes.

Keywords: Chronic lymphocytic leukemia, Ibrutinib, Bruton’s tyrosine

kinase inhibitor

Öz

Amaç: Kronik lenfositik lösemi hastalarında tek ajan ibrutinib

tedavisinin etkinliğini, güvenliğini ve sağkalım sonuçlarını geriye

dönük olarak değerlendirmek.

Gereç ve Yöntemler: Otuz üç merkezde yapılan bu retrospektif,

çok merkezli, girişimsel olmayan hastane kayıt çalışmasına en az

bir doz ibrutinib uygulanan 136 hasta (ortalama ± standart sapma

yaş 64,6 10,3, % 66,9’u erkek) dahil edildi. Hastaların demografik

verileri, bazal karakteristikleri, laboratuvar bulguları, lösemi hücre

sitogenetiği ile ilgili veriler kaydedildi. Tedavi yanıtı, genel sağkalım

(OS), progresyonsuz sağkalım (PFS) ve güvenlik verileri analiz edildi.

Bulgular: Hastaların %36,7’sinde ECOG 2-3, % 44,9’u Rai evre 4 idi.

FISH ile hastaların %39,8’inde del(17p) varlığını gösterdi. Hastalar

medyan 2 (0 ila 7 arasında) sıra pre-ibrutinib tedavisi aldı. Medyan

tedavi süresi 8,8 aydı (0,4-58 ay). Bir yıllık PFS ve OS oranları

sırasıyla %82,2 ve %84,6, medyan (SE, %95 güven aralığı) PFS süresi

30 (5,1, 20-40) ay ve OS süresi 37,9 (3,2, 31,5-44,2) aydı. Tedavi

yanıtı (CR veya PR), PFS ve OS süreleri; ibrutinib öncesi 3-7 basamak

tedaviye karşı 0-2 basamak tedavi alanlarda (p<0,001, p=0,001 ve

p<0,001, sırayla), ECOG 2-3’e göre ECOG 0-2 olanlarda (p=0,006,

p=0,011 ve p=0,001, sırasıyla), Rai evre 0-2 olanlarda Rai evre 3-4

olanlara göre (p=0,002, p=0,001 and p=0,002, sırasıyla) daha iyiydi.

Komorbidite, hacimli hastalık veya del(17p) varlığına göre tedaviye

yanıt oranlarında veya sağkalım sonuçlarında önemli bir fark

kaydedilmedi. 74 hastada (%54,4) 176 advers olay (AE) saptandı; 176

AE’nin 46’sı derece 3-4 idi. Bunlar; pnömoni (n=12), nötropeni (n=11),

anemi (n=5), trombositopeni (n=5) ve ateş (n=5) idi.

Sonuç: Bu gerçek hayat analizi, uzun vadeli ibrutinib tedavisinin

olumlu etkililiğini ve güvenlik profilini doğrularken, kötü ECOG

performans durumunun, ibrutinib’den önce ağır şekilde tedavi verilmiş

olmasının ve ileri evre hastalığın, hasta uyumu, tedavi yanıtı ve

sağkalım üzerindeki potansiyel olumsuz etkilerini ortaya koymuştur.

Anahtar Sözcükler: Kronik lenfosittik lösemi, İbrutinib, Bruton tirozin

kinaz inhibitörü

Introduction

Owing to novel therapeutics such as combination

chemotherapy with fludarabine and cyclophosphamide (FC)

and chemoimmunotherapy with rituximab (FCR), the survival

outcome and long-term remission rates of chronic lymphocytic

leukemia (CLL) patients have improved significantly over the last

decade, particularly in younger, low-risk CLL patients [1,2,3,4,5].

However, older patients with higher-risk genetic abnormalities

or del(17p) still have inferior survival outcomes, while significant

toxicities of chemotherapeutic regimens and poor survival

rates with the use of conventional salvage regimens following

relapse after FCR are also considered challenging factors in the

management of CLL [3,4,6,7,8].

Given the importance of B-cell-receptor signaling in CLL and

the central role of Bruton’s tyrosine kinase (BTK) in this pathway,

targeted therapy with kinase inhibitors has become an alternative

to conventional therapy for CLL [9,10,11]. The introduction of

ibrutinib, an irreversible inhibitor of BTK, enabled significant

improvement in the survival outcomes of CLL patients [10,11].

The results from three phase III trials demonstrated improved

progression-free survival (PFS) and overall survival (OS) with

ibrutinib compared to FCR or chlorambucil [12,13,14], while data

from the RESONATE trial indicated the association of ibrutinib

with significantly improved PFS, OS, and overall response rate

(ORR) when compared to ofatumumab in previously treated

CLL patients with several high-risk prognostic factors [15].

Accordingly, ibrutinib has become the standard of care in

274



relapsed/refractory patients and is now being recommended

for use in front-line treatment of patients regardless of age or

del(17p) status [16,17,18,19,20,21].

Given the potential differences in baseline characteristics and

treatment responses of patients recruited in clinical trials and

those treated outside of clinical trials, there is considerable

interest in real-world experience with the use of novel targeted

drugs in the management of CLL patients, particularly for

drugs such as ibrutinib that are recommended to be used

continuously until progression [10,22,23,24,25]. This real-life

multicenter study was therefore designed to retrospectively

evaluate efficacy and safety along with survival outcomes of

single-agent ibrutinib therapy in CLL patients who were treated

outside the setting of clinical trials.


Study Population

A total of 136 adult patients diagnosed with CLL (≥18

years old; mean age ± standard deviation: 64.6±10.3 years;

66.9% male patients) who had received at least one dose of

single-agent ibrutinib therapy after January 2013 were

included in this retrospective multicenter, noninterventional

hospital-registry study conducted between December 2018

and March 2019 at 33 centers across Turkey. Patients who

had sensitivity to an active ingredient or component of the

medication or who had ibrutinib treatment before December

2012 were excluded.

The study was conducted in full accordance with local good

clinical practice guidelines and current legislations, while

permission was obtained from the relevant institutional ethics

committee for the use of patient data for publication purposes.

Data Collection

Data on patient demographics (age, gender), baseline

characteristics (comorbidity, bulky disease, organomegaly,

infection, Eastern Cooperative Oncology Group [ECOG]

performance status, Rai stage, previous treatments), and

laboratory findings including hemoglobin, platelet count,

leukocyte count, lymphocyte count, erythrocyte sedimentation

rate, lactate dehydrogenase level, beta-2 microglobulin

and IgG levels, Coombs test, and leukemia-cell cytogenetics

(metaphase karyotyping, interphase fluorescence in situ

hybridization [FISH] analysis) were retrieved from hospital

records. Treatment responses including partial response (PR),

complete response (CR), stable disease (SD), and progressive

disease as well as final treatment response (PR and CR) were

evaluated according to the relevant International Workshop

Group on CLL response criteria [25]. Assessment of response

was performed at least 2 months after achieving “maximum

response”. The OS (duration, rate), PFS (duration, rate), and

adverse events (AEs) were also analyzed for patients who

received single-agent ibrutinib treatment within the study

period. PFS was defined as the period from the date of ibrutinib

initiation to the first recurrence/death or the last follow-up.

OS was defined as the period from the date of diagnosis to

death or last follow-up.


Statistical analysis was conducted using IBM SPSS Statistics 22.0

for Windows (IBM Corp., Armonk, NY, USA). Descriptive statistics

were used to summarize baseline characteristics. Pearson’s

chi-square (χ 2 ) test was used for the comparison of categorical

data. Survival analysis was performed via Kaplan-Meier analysis

and comparisons were made via log-rank test. Data were

expressed as mean ± standard deviation, median (minimummaximum),

95% confidence interval (CI), and/or percentage (%)

as appropriate.

Results

Baseline Characteristics

The mean patient age was 64.6±10.3 (range: 39-94) years and

61.9% of patients were male. Diabetes mellitus (25.7%) and

hypertension (22.9%) were the most common comorbidities,

while hepatosplenomegaly was noted in 33.8% of patients.

Overall, 36.7% of patients were categorized as ECOG

performance status class 2-3 and 44.9% were in Rai stage 4

(44.9%), while FISH testing revealed the presence of del(17p) in

39.8% of the patients (Table 1).

Prior Lines of Therapy and Related Treatment Responses

Patients received a median of 2.0 (range: 0-7) lines of

pre-ibrutinib therapy. CR rates were 27.8%, 32.8%, 10.7%, and

15.4% for patients having received 1, 2, 3, and ≥4 lines of prior

therapy (Table 2).

Characteristics of Ibrutinib Therapy

For the majority of patients, ibrutinib was administered orally at

a daily dose of 420 mg. The treatment indications were B signs

and stage 4 disease in 52.2% and 41.2% of patients, respectively

(Table 3).

Median duration of ibrutinib therapy was 8.8 months (range:

0.4-58.0 months), while dose reduction, dose delay, treatment

discontinuation, and AEs occurred in 16.9%, 26.5%, 24.3%, and

54.4% of patients, respectively (Table 3).

Lymphocyte counts increased within the first month of

treatment, followed by a gradual decrease starting from the

second month and resolving at the sixth month (Table 3).

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Table 1. Baseline characteristics of patients.

Patient demographics

Age (years) Mean ± SD 64.6±10.3

Gender, n (%)

Male 91 (66.9)

Female 45 (33.1)

Clinical findings n (%)

Comorbidities 1 70 (51.5)

Diabetes mellitus 18 (25.7)

Hypertension 16 (22.9)

Coronary artery disease 8 (11.4)

Hepatitis B infection 6 (8.6)

Other (each <3%) 22 (30.9)

Bulky disease 2 29 (21.3)

Organomegaly 2 90 (66.2)

Hepatosplenomegaly 46 (33.8)

Splenomegaly 37 (27.2)

Hepatomegaly 2 (1.5)

Infection 3 14 (10.3)

Pneumonia 4 (28.6)

Urinary tract infection 3 (21.4)

ECOG status 4 1 51 (37.5)

2 35 (25.7)

0 23 (16.9)

3 15 (11.0)

Rai stage 5 2 24 (17.6)

4 61 (44.9)

3 32 (23.5)

Laboratory findings

1 4 (2.9)

0 1 (0.7)

Hemoglobin (n=128), median (min-max) 10.2 (4.7-15.3)

Platelets (n=128), median (min-max) 108000 (5000-494000)

Leukocytes (n=128), median (min-max) 29380 (400-433849)

Lymphocytes (n=127), median (min-max) 20040 (294-355077)

LDH (n=107), median (min-max) 244 (89-3132)

Beta-2 microglobulin (n=54),

median (min-max)

5.2 (0.3-16.2)

ESR (n=93), median (min-max) 23 (1.0-247.0)

IgG (n=89), n (%)

Coombs test (n=108), n (%)

Cytogenetic (n=48), n (%)

FISH (n=103), n (%)

>500 59 (43.4)

<500 30 (22.1)

Negative 101 (74.3)

Positive 7 (5.1)

Normal 41 (85.4)

Trisomy 12 7 (14.5)

17p del 41 (39.8)

11q del 8 (7.7)

13q del 8 (7.7)

SD: Standard deviation; ECOG: Eastern Cooperative Oncology Group; LDH: lactate

dehydrogenase; ESR: erythrocyte sedimentation rate; IgG: immunoglobulin G; FISH:

fluorescence in situ hybridization; min: minimum; max: maximum.

Missing data for 1 2, 2 1, 3 82, 4 12, and 5 14 patients.

Treatment Response and Survival Outcome with Respect To

Prognostic Factors

Final treatment response (CR or PR) was better in patients with

0-2 lines versus 3-7 lines of prior therapy (79.3% vs. 41.5%,

p<0.001), in patients with ECOG performance status class 0-1

versus class 2-3 (75.0% vs. 50.0%, p=0.006), and in patients

with Rai stage 0-2 versus 3-4 (88.9% vs. 57.0%, p=0.002). No

significant difference was noted in final treatment response

rates with respect to presence of comorbidity, bulky disease, or

del(17p) status (Table 4).

After a median of 69.0 (range: 9.0-296.0) months of follow-up,

mortality had occurred for 29 of 136 patients (21.3%), while 107

(81.3%) patients survived. Sepsis (31.0%) was the most common

cause of death, followed by cardiac arrest (13.8%), pneumonia

(10.3%), and Richter’s syndrome (10.3%) (Table 5).

Overall, 1-year PFS and OS rates were 82.2% and 84.6%,

respectively (Table 5), while median (standard error [SE], 95% CI)

PFS time was 30.0 (5.1, 20.0-40.0) months and median (SE, 95%

CI) OS time was 37.9 (3.2, 31.5-44.2) months (Table 6, Figure 1).

Mean PFS time was longer in patients with 0-2 lines versus

3-7 lines of prior therapy (39.2±4.4 vs. 20.5±2.9 months, logrank

p=0.001, Figure 2), in patients with ECOG performance

Table 2. Prior lines of therapy and related treatment responses.

Median (min-max)

Number of prior lines

of therapy 1 2.0 (0.0-7.0)

Time to last treatment

response before

ibrutinib 2 6.0 (0.0-120.0)

Last treatment

response before

ibrutinib 3 19

(14.0)

Prior lines of therapy

None

Treatment response

CR PD PR SD Total

2

(66.7)

1 4 5

(27.8)

2 5 20

(32.8)

3 6 3

(10.7)

>4

Total

2

(15.4)

32

(26.0)

30

(22.1)

0

(0.0)

3

(16.7)

7

(11.5)

13

(46.4)

6

(46.2)

29

(23.6)

65

(47.8)

1

(33.3)

9

(50.0)

28

(45.9)

7

(25.0)

5

(38.5)

50

(40.7)

17

(12.5)

0

(0.0)

1

(5.6)

6

(9.8)

5

(17.9)

0

(0.0)

12

(9.8)

131

(100.0)

3

(2.4)

18

(14.6)

61

(49.6)

28

(22.8)

13

(10.6)

123

(100.0)

PR: Partial response; CR: complete response; SD: stable disease; PD: progressive

disease; min: minimum; max: maximum.

Missing data for 1 1, 2 46, 3 2 (also excluding 3 patients with first-line ibrutinib therapy),

4

10, 5 27, and 6 44 patients.

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Table 3. Characteristics of ibrutinib therapy.

Dose, n (%)

420 mg 131 (96.3)

280 mg 3 (2.2)

140 mg 2 (1.5)

Treatment indication, n (%)

Stage 4 disease 56 (41.2)

Stage 3 disease 29 (21.3)

Rapid doubling time 22 (16.2)

B signs 71 (52.2)

Bulky disease 12 (8.8)

Richter’s syndrome 4 (2.9)

Rapidly progressive disease 1 (0.7)

Treatment duration (months)

Mean ± SD 12.2±11.1

Median (min-max) 8.8 (0.4-58.0)

Number of treatment cycles (n=133)

Mean ± SD 11.2±10.5

Median (min-max) 8 (1-58)

Dose reduction, n (%) (n=135) 23 (16.9)

Dose delay, n (%) (n=136) 36 (26.5)

Discontinuation, n (%) (n=111) 33 (24.3)

Adverse events, n (%) 74 (54.4)

Lymphocyte levels n Median (min-max)

Week 1 97 30000 (350-528000)

Month 1 109 29984 (340-441000)

Month 2 98 12200 (105-337000)

Month 3 75 8400 (400-313000)

Month 6 57 4740 (250-129370)

Month 12 30 3530 (1100-82000)

Month 18 17 2810 (980-73000)

Month 24 4 12275 (4000-171000)

SD: Standard deviation; min: minimum; max: maximum.

status class 0-1 versus class 2-3 (37.0±4.0 vs. 21.7±3.3 months,

log-rank p=0.011, Figure 3), and in patients with Rai grade 0-2

versus 3-4 (47.5±5.4 vs. 24.7±3.0 months, log-rank p=0.001,

Figure 4) (Table 6).

Mean OS time was also longer in patients with 0-2 lines versus

3-7 lines of prior therapy (45.9±4.19 vs. 22.1±3.1 months, logrank

p<0.001, Figure 2), in patients with ECOG performance

status class 0-1 versus class 2-3 (43.7±3.9 vs. 22.1±3.49 months,

log-rank p=0.001, Figure 3), and in patients with Rai stage 0-2

versus 3-4 (52.0±4.1 vs. 28.6±3.4 months, log-rank p=0.002,

Figure 4) (Table 6).

No significant difference was noted in PFS time and OS time

with respect to presence of comorbidity, bulky disease, del(17p)

status, or overall FISH findings (Table 6).

Safety Profile

Overall, 176 AEs were reported in 74 (54.4%) patients, and 46 of

those 176 AEs were grade 3-4 AEs, including pneumonia (n=12),

neutropenia (n=11), anemia (n=5), thrombocytopenia (n=5), and

fever (n=5) in most cases. The atrial fibrillation rate was low

(n=2) (Table 7).

Discussion

Our findings revealed the favorable efficacy and safety profile

of ibrutinib in CLL patients (mean age of 64.6 years, del(17p)

mutation in 28.7%, Rai stage 3/4 in 68.4%) with 1-year PFS

and OS rates of 82.2% and 84.6% at a median follow-up of

69.0 months, respectively. The final treatment response (CR or

PR) was better and survival times (PFS and OS) were longer

for patients with fewer than <2 lines of prior therapy, ECOG

performance class 0-1, and Rai stage 0-2 while there was no

significant impact of comorbidity, bulky disease, or del(17p)

status on treatment response or survival outcomes.

Figure 1. Overall 1-year progression-free survival (PFS) and overall survival (OS) rates.

277



Figure 2. One-year progression-free survival (PFS) and overall survival (OS) rates in patients with 0-2 lines versus 3-7 lines of prior therapy.

Figure 3. One-year progression-free survival (PFS) and overall survival (OS) rates in patients with Eastern Cooperative Oncology Group

(ECOG) performance status class 0-1 versus class 2-3.

Figure 4. One-year progression-free survival (PFS) and overall survival (OS) rates in patients with Rai grade 0-2 versus 3-4.

278



Data from a real-life retrospective study including 32

ibrutinib-treated patients (11 had CLL) in Turkey revealed that in

patients with CLL, ibrutinib treatment (median: 4 months) was

associated with an ORR of 85.6% (28.5% CR and 57.1% PR) and

occurrence of diarrhea in 3 (27.3%), pneumonia in 3 (27.3%),

and thrombocytopenia and/or neutropenia in 2 (18.2%) patients

[26]. The authors considered ibrutinib a good treatment option

for CLL and other B-cell lymphomas, with an acceptable sideeffect

profile and a high and promising CR/PR response rate [26].

Similarly, according to real-life data from the UK CLL

Forum obtained from 315 CLL patients with a median

of 16 months of follow-up, the authors noted 1-year

discontinuation-free survival (DFS) of 73.7% and 1-year OS of

83.8% with no significant difference in DFS and OS rates with

respect to del(17p) status, whereas there was an association

of better pre-treatment performance status (0/1 vs. 2+) with

superior DFS (77.5% vs. 61.3%) and OS (86.3% vs. 76.0%) and

an association of 1 prior line of therapy versus 2+ prior lines of

therapy with a significant 1-year PFS advantage (94% vs. 82%)

[22]. The same authors also noted no significant difference

between more or less heavily pre-treated patients in terms of

prognostic factors such as performance status and del(17p),

while emphasizing the likelihood of older patients and those

with del(17p) to have inferior DFS and OS when treated with

ibrutinib beyond the second line [22].

In a multicenter Swedish study providing real-life data from

95 CLL patients (median age: 69 years, del(17p)/TP53 mutation

in 63%, Rai stage 3/4 in 65%), the authors reported that

once-a-day ibrutinib treatment was well tolerated and

associated with an ORR of 84%, PFS of 77%, and OS rate of

83% at a median follow-up of 10.2 months [23]. However,

in contrast to our findings, the authors indicated that

del(17p)/TP53 mutation remained a therapeutic challenge

given the significantly shorter PFS and OS in patients with

del(17p)/TP53 mutation [23].

In addition, data from a mutation analysis study of 63 patients

who were still on ibrutinib after 3 years in an early-access

program at 29 French centers revealed detection of BTK and

PLCG2 mutations in 57% and 13% of the next-generation

sequencing samples (n=30) and the authors reported that after

a median follow-up of 8.5 months from sample collection, the

presence versus the lack of a BTK mutation was significantly

associated with subsequent CLL progression [27]. The same

authors emphasized a need for clinical trials to evaluate whether

patients with BTK mutation may benefit from an early switch to

another treatment [27].

In a real-life study on the efficacy of ibrutinib as a single agent in

180 patients with CLL recruited from three independent cohorts

from Italy, 73 patients were reported to have discontinued

Table 4. Treatment response with respect to prognostic factors.

Ibrutinib-treated patients (n=136) a

Pre-ibrutinib lines of therapy

17p deletion

ECOG

Rai

Comorbidity

Bulky disease

Final treatment response

(CR or PR) Total p

No

0-2 17 (20.7) 65 (79.3) 82

3-7 24 (58.5) 17 (41.5) 41

Total 41 82 123

Present 13 (35.1) 24 (64.9) 37

Absent 27 (43.5) 35 (56.5) 62

Total 40 59 99

0 or 1 18 (25.0) 54 (75.0) 72

2 or 3 22 (50.0) 22 (50.0) 44

Total 40 76 116

0-2 3 (11.1) 24 (88.9) 27

3-4 37 (43.0) 49 (57.0) 86

Total 40 73 113

Present 24 (38.1) 39 (61.9) 63

Absent 16 (27.1) 43 (72.9) 59

Total 40 82 122

Present 9 (36.0) 16 (64.0) 25

Absent 32 (32.7) 66 (67.3) 98

Total 41 82 123

ECOG: Eastern Cooperative Oncology Group; FISH: fluorescence in situ hybridization; PR: partial response; CR: complete response.

a

Missing data for 13 patients.

Pearson chi-square.

Yes

<0.001

0.409

0.006

0.002

0.197

0.751

279



ibrutinib for progression or for AEs, while NOTCH1-mutated

patients were reported to have less redistribution lymphocytosis

at 3 months on ibrutinib, to show inferior nodal response

at 6 months, and to have significantly shorter PFS and OS

[28]. The same authors noted that NOTCH1 M plus lower

BAX/BCL-2 ratio identified a CLL subset showing the worst PFS

and OS, emphasizing the likelihood of either new small-molecule

combination approaches or antibodies targeting NOTCH1 being

more appropriate therapeutic options for NOTCH1-mutated

patients [28].

Notably, based on data from a study conducted in Poland

on the potential significance of the mutational status of 30

selected genes for disease outcome in a real-life cohort of 45

heavily pretreated patients with CLL, the authors reported that

despite the accumulation of several poor prognostic factors

such as TP53 (40.0%), NOTCH1 (28.8%), SF3B1 (24.4%), ATM

(15.6%), MED12 (13.3%), CHD2 (11.1%), XPO1 (11.1%), NFKBIE

(11.1%), BIRC3 (8.9%), SPEN (8.9%), POT1 (8.9%), EGR2 (6.7%),

and RPS15 (6.7%) in their cohort, ibrutinib treatment showed

long-term clinical benefits in terms of 36-month PFS (64.0%)

and OS (68.2%) rates and the ORR (51.1%) [29].

Higher treatment response and better PFS and OS outcomes

in patients previously treated with 0-2 lines of therapy versus

more heavily treated patients in the current study seem to be

consistent with data from other real-life studies [22]. Fewer

lines of prior therapy were also reported to be associated with

significantly improved PFS and OS outcomes and higher CR rates

Table 5. Survival outcome with respect to prognostic factors.

Duration of follow-up, median (min-max) 69.0 (9.0-296.0)

Survivor, n (%) 107 (78.7)

Non-survivor, n (%) 29 (21.3)

Cause of death, n (%)

Sepsis 9 (31.0)

Cardiac arrest 4 (13.8)

Pneumonia 3 (10.3)

Richter’s syndrome 3 (10.3)

Sudden death 1 (3.4)

Cerebral hemorrhage 1 (3.4)

Fungal sinusitis and pneumonia 1 (3.4)

Mucor infection 1 (3.4)

Cerebral aspergillosis 1 (3.4)

Respiratory arrest 1 (3.4)

Stroke 1 (3.4)

Total 26

Missing 3

One-year survival rate (%)

PFS 82.2

OS 84.6

PFS: Progression-free survival; OS: overall survival; min: minimum; max: maximum.

and 5-year PFS and OS rates in treatment-naive (TN) patients

compared to relapsed/refractory (R/R) patients, emphasizing

the deepening of responses with continued ibrutinib therapy

and the likelihood of superior efficacy of initiating ibrutinib in

earlier lines of therapy [16].

Dose reduction (16.9%), dose delay (26.5%), and treatment

discontinuation (24.3%) rates in the current study also seem

to be consistent with previous real-life data on ibrutinib

discontinuation rates (10.5% to 17.5%), dose reductions

(26.0%), and temporary treatment breaks (>14 days, 13.0%)

or permanent treatment discontinuation (17.5% to 41%)

[22,23,30,31]. Notably, neither the dose reductions nor the

temporary treatment breaks were reported to be associated

with survival outcome, whereas permanent cessation of

ibrutinib was associated with reduced 1-year OS survival

[22]. Similar to our findings, poorer 1-year DFS (16.2%) and

OS (9.3%) in patients with poorer pre-treatment performance

status (PS 2+) were reported while also noting a higher

likelihood of treatment breaks within the first year of therapy

in the PS 2+ group [22].

In a recent FILO Group study on the OS benefits of symptom

monitoring in real-world CLL patients treated with ibrutinib, the

authors reported that drug intolerance and toxicities (26.3%)

rather than progressive disease accounted for most drug

withdrawals [27] and they indicated the higher likelihood of

stopping ibrutinib due to toxicities in the real-life setting when

compared to ibrutinib discontinuation rates due to toxicity

(10%) and CLL progression (13.5%) as reported in RESONATE

and RESONATE-2 pooled analysis [32]. The potential role of

certain factors in this discrepancy has been suggested, such

as the clinical experience of physicians in managing toxicity,

the availability of alternative therapy, and the characteristics

of real-life populations in terms of performance status and

comorbidities [31].

In a recent French study on patterns of use and safety of

ibrutinib in real-life practice in 102 patients, half of whom

were CLL patients, the authors reported that 42.1% of patients

permanently discontinued ibrutinib in the first year, mostly

for progression (51.2%) or adverse drug reactions (ADRs)

(32.6%), while 47.1% of patients experienced at least one

ibrutinib-associated serious ADR (SADR; hematological,

infectious, and vascular disorders in particular) [33]. These

authors also reported the probability of developing an

ibrutinib-associated SADR to be 35.1% (95% CI: 26.3-45.7) at

3 months, 44.8% (95% CI: 35.2-55.8) at 6 months, and 54.3%

(95% CI: 44.0-65.2) at 12 months, further indicating a significant

association of age of ≥80 years (hazard ratio [HR]: 2.03; 95%

CI: 1.02-4.05) and being treated for CLL (HR: 1.81; 95%

CI: 1.01-3.25) with a higher risk of SADR occurrence [33].

280



Based on data from a Greek single-center retrospective realworld

study including 58 CLL patients (11 first-line, 47 R/R)

treated with ibrutinib monotherapy (for a median of 6.6 and 16.3

months, respectively), treatment discontinuation was reported

to be associated with AEs (due to atrial fibrillation in 3.5% of

patients) in 9% of the first-line and 10.6% of the R/R patients,

while it was due to disease progression in 13 (24.5%) patients

[34]. These authors concluded that CLL patients had outcomes

similar to those of clinical trials if treated homogeneously

according to standard guidelines, resulting in fewer unneeded

discontinuations and shrinkage of the treatment armamentarium

[34]. The superior efficacy of ibrutinib with significantly

improved ORR, PFS, and OS compared to ofatumumab in R/R

patients or compared to chlorambucil as frontline therapy in TN

patients was established in the RESONATE trials, which included

extended follow-up analyses [9,13,15,24,35,36,37,38].

Accordingly, our findings support favorable treatment responses

and survival outcomes with the use of off-trial ibrutinib,

similar to data from multicenter prospective pivotal trials

on ibrutinib, despite the fact that patients included in the

pivotal clinical trials were often younger, had better ECOG

classifications, and presented with milder lymphadenopathy

[22,23]. Nonetheless, our findings support the potential roles

of poorer ECOG performance status and having been heavily

treated before ibrutinib in the likelihood of observing higher

treatment discontinuation rates and inferior survival outcome

in real-world settings, given the more stringent rules for dose

modifications or interruptions and thus higher levels of drug

compliance in clinical trials [22].

While del(17p) status had no significant impact on survival

outcome in the current study, poorer survival outcome was

reported for patients with del(17p) in the 3-year follow-up of

a phase 1b-2 multicenter study [37] and in the RESONATE-17

study [39], as well as in a real-life study [23]. However, subgroup

analysis of the RESONATE study also showed that the presence

of del(17p) was not associated with inferior PFS outcomes with

similar ORRs (89% and 91%, respectively) and 18-month PFS

rates (71% and 79%, respectively) in patients with del(17p)

Table 6. Further analysis of survival outcome with respect to prognostic factors.

Progression-free survival time (months)

Overall survival time (months)

Mean SD

95% CI

95% CI

95% CI

95% CI

Median SE

Mean SD

Median SE

LB UB LB UB LB UB LB UB

Overall 33.3 3.1 27.2 39.5 30.0 5.1 20.0 40.0 37.9 3.2 31.5 44.2 - - - -

Lines of pre-ibrutinib therapy

0-1-2 39.2 4.4 30.5 47.9 - - - - 45.9 4.1 37.9 53.9 - - - -

3-4-5-6-7 20.5 2.9 14.9 26.2 17.1 2.6 12.1 22.1 22.1 3.1 16.0 28.1 17.1 3.4 10.5 23.7

p 1 0.001 <0.001

17p deletion

Present 20.05 2.9 14.3 25.7 14.1 4.4 5.4 22.7 22.1 3.2 15.7 28.4 19.4 4.3 10.9 27.8

Absent 33.8 4.2 25.7 42.0 30.7 7.6 15.8 45.5 40.0 4.0 32.1 478 . . . .

p 1 0.224 0.123

ECOG status

0 or 1 37.0 4.0 29.1 44.8 30.0 - - - 43.7 3.9 36.1 51.3 -. - - -.

2 or 3 21.7 3.3 15.3 28.1 23.9 7.4 9.3 38.5 22.1 3.49 15.4 28.8 17.1 5.0 7.3 27.0

p 1 0.011 0.001

Rai

0, 1, 2 47.5 5.4 36.9 58.19 . . . . 52.0 4.1 43.9 59.9 . . . .

3, 4 24.7 3.0 18.9 30.6 22.4 4.7 13.3 31.5-6 28.6 3.4 22.1 35.2 30.0 11.1 8.5 51.7

p 1 0.001 0.002

Comorbidities

Present 26.6 3.3 20.1 33.0 22.4 4.0 14.6 30.3 29.4 3.6 22.4 36.4 23.9 8.0 8.2 39.6

Absent 39.5 4.9 29.8 49.1 . . . . 45.5 4.5 36.8 54.2 . . . .

p 1 0.203 0.074

Bulky disease

Present 26.6 3.0 20.7 32.5 30.0 4.7 20.8 39.2 26.2 3.0 20.3 32.2 30.0 4.7 20.7 39.3

Absent 32.3 3.5 25.5 39.1 22.4 7.5 7.8 37.1 38.3 3.6 31.3 45.38 . . . .

p 1 0.543 0.918

SD: Standard deviation; CI: confidence interval; LB: lower bound; UB: upper bound; ECOG: Eastern Cooperative Oncology Group.

281



and those without del(17p) [35]. Likewise, 3-year PFS in

ibrutinib-treated CLL patients was reported to be 53% for

patients with del(17p), 66% for those with del(11q), and 58%

for patients without these abnormalities [40]. In a phase 1b-2

multicenter study of 85 CLL patients, the authors reported

ibrutinib to promote durable responses irrespective of the dose,

with similar ORRs (71%) in the 420-mg and 840-mg cohorts along

with 26-month PFS and OS rates of 75% and 83%, respectively

[11]. The authors also noted no significant impact of traditional

high-risk prognostic features, including del(17p), on the

treatment response rates [11].

Notably, del(17p) has been suggested to be a poor prognostic

factor in patients who receive frontline ibrutinib with no

negative impact of del(17p) on OS in the R/R setting, while

R/R disease, age, performance status, and comorbidities were

reported as determinants of poor OS in ibrutinib-treated

patients with CLL [41]. Moreover, the frequency of high-risk

genomic abnormalities including del(17p) has been suggested

to dramatically increase with increasing lines of chemotherapy,

and treatment with single-agent ibrutinib earlier in the disease

course before the development of these abnormalities has

therefore been considered to improve patient outcomes [16].

Table 7. Safety profile.

# of AEs Grade 1 Grade 2 Grade 3 Grade 4 Grade 5

Tiredness 29 24 5 - - -

Anemia 19 6 8 4 1 -

Pneumonia 19 1 6 11 1 -

Neutropenia 18 3 4 6 5 -

Diarrhea 17 11 6 - - -

Thrombocytopenia 10 1 4 4 1 -

Rash 7 5 2 - - -

Decreased appetite 6 5 1 - - -

Fever 6 1 - 5 - -

Arthralgia 6 1 5 - - -

Nausea 6 5 1 - - -

ALT/AST elevation 4 4 - - - -

Gastrointestinal complaints 3 1 2 - - -

Stomatitis 2 1 1 - - -

Itching 2 1 - 1 - -

Lymphopenia 2 1 1 - - -

Neutropenic fever 2 - - 2 - -

Arrhythmia 2 - 2 - - -

Eye complaints 2 2 - - - -

Atrial fibrillation 2 - 2 - - -

Hypothyroidism 1 1 - - - -

Elevated creatinine 1 1 - - - -

Intracranial hemorrhage 1 - - - - 1

Deep vein thrombosis 1 - - 3 - -

Muscle cramps 1 - 2 - - -

Ataxia 1 1 - - - -

Confusion 1 1 - - - -

Dyspnea 1 1 - - - -

Cough 1 - - 1 - -

Fungal infection 1 - - 1 - -

Cellulitis 1 - 2 - - -

Hyperpigmentation 1 1 - - - -

Zona 1 1 - - - -

AEs: Adverse events; ALT: aspartate transaminase; AST: alanine transaminase.

282



Indeed, targeted therapies such as ibrutinib are considered to

challenge the value of classic prognostic factors defined in the

original CLL International Prognostic Index, emphasizing the

need for new risk models applicable to CLL patients treated with

all currently approved targeted therapies [41,42,43,44].

In the current study, lymphocyte counts increased within

the first month of treatment, followed by a gradual decrease

starting from the second month. This is consistent with the

transient increase in absolute lymphocyte count expected

within the first few weeks of ibrutinib therapy, which may

persist for several weeks of treatment and does not signify

disease progression [24,45]. Nonetheless, some authors reported

the association of prolonged treatment-related lymphocytosis

with higher likelihood of ibrutinib responders to carry favorable

prognostic markers (i.e., del13q and mutated IGHV) and a

trend toward improved PFS [35,45], while more rapid and more

frequent normalization of lymphocyte counts was also reported

in patients with unmutated immunoglobulin genes [11].

The safety profile of ibrutinib-treated patients in the current

study seems consistent with previous reports, with most

AEs being mild to moderate in severity and neutropenia,

hypertension, pneumonia, and anemia being the most

commonly reported grade 3-4 events [11,15,37,39]. Overall,

176 AEs were reported for 74 (54.4%) of the patients in

the current study, with 46 of those 176 AEs being grade

3-4 AEs including pneumonia (n=12), neutropenia (n=11),

anemia (n=5), thrombocytopenia (n=5), and fever (n=5) in

most cases. The results from the RESONATE trial with up to

5 years of follow-up also showed that the safety profile of

ibrutinib over time remains acceptable and manageable and

that extended treatment with ibrutinib is tolerable with no

long-term safety signals and a reduction in the majority

of grade >3 AEs over time, while effective management of

AEs during the first year of treatment is considered critical

given the highest discontinuation rates within this period

[16,24,40].

Consistent with previous real-life data obtained from

ibrutinib-treated CLL patients that identified infection as the

main cause of death and the common reason for permanent

discontinuation of ibrutinib [22,23], our findings revealed sepsis

as the leading cause of death among ibrutinib-treated CLL

patients. Nonetheless, it should be noted that in a systematic

review and meta-analysis of phase III trials with 1227 patients

(617 in the ibrutinib arm and 610 in the control arm), the authors

concluded that there was no significant increase in the risk of

infection associated with ibrutinib in patients with CLL [46].

Study Limitations

Although the occurrence of atrial fibrillation is generally

between 7% and 15% in this age group in real-world analyses,

our finding of atrial fibrillation occurrence of only 2% may

be explained by the retrospective design of the current study.

While the cardiac arrest (14%) and sudden death (3%) rates

in our study population indicate a high rate of cardiac death

(17%), none of these deaths were related to ibrutinib treatment

and they were associated with the high proportion of elderly

patients with comorbidities in the study cohort.

Conclusion

This real-life analysis of CLL patients confirms the favorable

efficacy and safety profile of long-term ibrutinib treatment as

reported by prospective clinical trials, while emphasizing the

potential adverse impact of poorer ECOG performance status,

having been heavily treated prior to ibrutinib initiation, and

advanced Rai stages but not comorbidity, bulky disease, or

del(17p) status on patient compliance, treatment responses, and

survival outcomes.

Acknowledgments

This study was supported by Janssen Pharmaceutica Turkey.

The authors would like to thank Prof. Şule Oktay, MD, PhD, and

Çağla Ayhan, MD, from KAPPA Consultancy Training Research

Ltd. (İstanbul, Turkey), who provided editorial support.

Ethics

Ethics Committee Approval: The study was conducted in full

accordance with local good clinical practice guidelines and

current legislations, while permission was obtained from the

relevant institutional ethics committee for the use of patient

data for publication purposes.


Surgical and Medical Practices: A.T.; Concept: A.T.; Design: A.T.;

Data Collection or Processing: A.T., F.P.T., S.S.D., H.D.D., E.K.,

E.G.Ü., İ.Y., Ö.M., B.D., M.A.Ö., H.T., M.O., N.S., M.Y., V.O., A.K.,

Ö.Ö., G.Ç., S.D., İ.A., G.S., E.A.D., G.İ., M.A.U., G.Ö., S.A., B.T., İ.B.,

E.K., M.S., D.S.B., R.Y., V.Ö., A.K.G., B.S., Ş.E., O.M.A., A.B., M.H.D.,

A.A., A.Ü., A.S., E.G., D.Ç., B.F.; Analysis or Interpretation: A.T.;

Literature Search: A.T.; Writing: A.T.


authors.



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285

Visal Okur F. et al: Uric Acid and Sinusoidal Obstruction Syndrome

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2021.0174


Pre-Conditioning Serum Uric Acid as a Risk Factor for Sinusoidal

Obstruction Syndrome of the Liver in Children Undergoing

Hematopoietic Stem Cell Transplantation

Hematopoetik Kök Hücre Transplantasyonu Yapılan Çocuklarda Karaciğer Sinüzoidal

Obstrüksiyon Sendromu için Bir Risk Faktörü Olarak Rejim Öncesi Serum Ürik Asit Düzeyi

Fatma Visal Okur 1 , Murat Karapapak 1 , Khaled Warasnhe 1 , Umut Ece Arslan 2 , Barış Kuşkonmaz 1 , Duygu Çetinkaya 1 *

1Hacettepe University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology and Pediatric BMT Unit, Ankara, Turkey

2Hacettepe University, Institute of Public Health, Department of Health Research, Ankara, Turkey

Abstract

Objective: Uric acid (UA), a known danger signal released from

injured cells, is a valuable sign of inflammation. We aimed to evaluate

the association of serum UA levels before the start of conditioning

regimens with the risk of hepatic sinusoidal obstruction syndrome

(SOS) development after hematopoietic stem cell transplantation

(HSCT).

Materials and Methods: Two hundred and twenty-two children who

underwent allogeneic HSCT at the Pediatric BMT Unit of Hacettepe

University between 2000 and 2014 were included in this retrospective

study. Serum UA levels were measured before conditioning as an

indicator of the pre-transplant inflammatory status of the patients.

Patients with and without a diagnosis of SOS were compared regarding

primary diagnosis, previously described risk factors for SOS, and preconditioning

serum UA.

Results: SOS was diagnosed in 42 patients who had higher

pre-conditioning serum UA levels compared to those who did not.

Pre-transplant serum creatinine, gamma-glutamyl transferase,

bilirubin, ferritin, and C-reactive protein levels did not differ

significantly among patients with and without SOS; however, serum

albumin was lower in the patients who developed SOS. Receiver

operating characteristic analysis revealed that a pre-conditioning UA

level higher than 3.32 mg/dL was predictive of SOS. When applied

to a multivariate model, only pre-conditioning UA and albumin

levels remained significant risk factors for SOS (UA: odds ratio

[OR], 2.54; 95% confidence interval [CI], 1.26-5.12, p=0.009; albumin:

OR, 0.45, 95% CI, 0.22-0.95, p=0.037).

Conclusion: Our results suggest that pre-conditioning serum UA is

an independent risk factor for SOS, and it might be used as an early

predictor of hepatic SOS together with previously described clinical

and laboratory parameters.

Keywords: Sinusoidal obstruction syndrome, Hematopoietic stem cell

transplantation, Uric acid, Inflammation

Öz

Amaç: Hasarlı hücrelerden salınan bilinen bir tehlike sinyali olan

ürik asit (ÜA), önemli bir enflamasyon belirtecidir. Bu çalışmayı

hazırlama rejimine başlamadan önce bakılan serum ürik asit seviyesi

ile hematopoetik kök hücre transplantasyonu (HKHT) sonrası hepatik

sinüzoidal obstrüksiyon sendromu (SOS) gelişimi riski arasındaki ilişkiyi

değerlendirmeyi amaçladık.

Gereç ve Yöntemler: Bu retrospektif çalışmaya Hacettepe Üniversitesi

Pediatrik KİT Ünitesi’nde 2000-2014 yılları arasında allojenik HKHT

uygulanan 222 çocuk hasta dahil edildi. Serum ÜA seviyeleri,

hastaların transplantasyon öncesi enflamatuvar durumunun bir

göstergesi olarak hazırlama rejimi öncesi ölçülmüştür. SOS tanısı olan

ve olmayan hastalar, birincil tanı, SOS için daha önce tanımlanan risk

faktörleri ve rejim öncesi serum ÜA açısından karşılaştırıldı.

Bulgular: SOS tanısı alan 42 hastada rejim öncesi ÜA düzeyleri

olmayan hastalara göre daha yüksek saptandı. Nakil öncesi bakılan

serum kreatinin, gama-glutamil transferaz, bilirubin, ferritin ve

C-reaktif protein parametreler açısından daha düşük olan serum

albümini dışında, SOS gelişen hastalar ile gelişmeyen hastalar arasında

anlamlı farklılık gözlenmedi. ROC analizi, 3,32 mg/dL’den yüksek rejim

öncesi ÜA düzeyinin SOS gelişimini öngördüğünü ortaya koydu. Çok

değişkenli bir modele uygulandığında, yalnızca rejim öncesi ÜA ve

albümin seviyeleri SOS için önemli risk faktörleri belirlendi (ÜA; odds

ratio (OR), 2,54; %95 güven aralığı (GA), 1,26 ila 5,12; p=0,009 ve

albumin; OR, 0,45; %95 GA, 0,22 ila 0,95; p=0,037).

Sonuç: Sonuçlarımız, rejim öncesi serum ÜA düzeyinin SOS için

bağımsız bir risk faktörü olduğunu ve önceden tanımlanmış klinik/

laboratuvar parametreleriyle birlikte hepatik SOS’nin gelişimi

açısından erken bir belirteç olarak kullanılabileceğini göstermektedir.

Anahtar Sözcükler: Sinüzoidal obstrüksiyon sendromu, Hematopoetik

kök hücre transplantasyonu, Ürik asit, Enflamasyon



Address for Correspondence/Yazışma Adresi: Fatma Visal Okur, M.D., Hacettepe University Faculty of Medicine,

Department of Pediatrics, Division of Pediatric Hematology and Pediatric BMT Unit, Ankara, Turkey

E-mail : fvokur@hacettepe.edu.tr ORCID: orcid.org/0000-0002-1679-6205

Received/Geliş tarihi: March 9, 2021

Accepted/Kabul tarihi: April 19, 2021

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Introduction

Hepatic sinusoidal obstruction syndrome (SOS), also known as

hepatic veno-occlusive disease, is a serious complication of

hematopoietic stem cell transplantation (HSCT) and is clinically

characterized by fluid retention, painful hepatomegaly, and

hyperbilirubinemia. It is the most frequent and well-studied of

the early-onset vascular endothelial syndromes that develop

after HSCT. It occurs in 5%-15% of patients after allogeneic

HSCT. Variations in its incidence are attributed to multiple factors

such as the diagnostic criteria used, center experience, year of

HSCT, and patient type [1]. Although the reported incidence

of SOS has decreased with new transplantation strategies, the

severe form of SOS is still associated with significant mortality

and early identification of SOS remains challenging [2].

Patient-, disease-, and transplant-related clinical risk factors

have been well established, including pre-existing liver

dysfunction, disease type and/or disease status, young or old

age, allogeneic HSCT, and myeloablative conditioning regimen,

but precise prediction of SOS in individuals remains elusive.

Early identification and monitoring of high-risk patients using

predictive markers will lead to timely treatment implications,

which might have a significant impact on survival [3].

SOS is initiated with the sinusoidal endothelial cell damage

caused mainly by cytotoxic effects of intensive conditioning

regimens including chemotherapy and/or radiotherapy given

before transplantation. Prophylactic immunosuppressive

therapies, growth factors used to support engraftment,

developing infections, and transplantation itself can also

cause endothelial damage [4,5]. Clinical evidence suggests

that endogenous ‘danger signals’ from injured cells have

a role in the pathogenesis of SOS through induction of a

non-infectious inflammatory reaction in the allogeneic setting

[6]. Uric acid (UA) is an endogenous danger signal released

from injured cells that induces the maturation of dendritic

cells and expansion of alloreactive T-cells via activation of the

NOD-like receptor protein (NLRP)3 inflammasome [7].

Recently, a preclinical study showed the role of NLRP3

inflammasome-mediated IL-1 production in acute

graft-versus-host disease (aGVHD) [8]. There is a limited

number of clinical studies about the association between

serum UA level and aGVHD and their results are controversial

[9,10]. The role of serum UA in the pre-transplant period as

a risk factor for SOS remains unclear [4,6,11,12]. Although

laboratory/clinical markers of endothelial injury preceding

SOS development have been described, they have not been

adopted to clinical use due to practical limitations and there is

still a need for dynamic laboratory parameters that can predict

SOS development. Therefore, we evaluated the association

between pre-transplant serum UA levels as sensitive markers

of inflammation with SOS development after allogeneic HSCT

in pediatric patients.


Two hundred and twenty-two children (median age: 7

years, range: 0.3-19; male/female: 150/72) who underwent

allogeneic HSCT in the Pediatric BMT Unit of Hacettepe

University between 2000 and 2014 were included in the

retrospective data analysis. Serum UA levels measured

before the initiation of the conditioning regimen (day -9)

and after the conditioning regimen was completed (day 0

before transplantation) were analyzed to assess any changes

in serum UA that would be indicative of the pre-transplant

inflammatory status of individual patients. Pre-conditioning

serum creatinine, transaminases, gamma-glutamyl transferase

(GGT), and total bilirubin levels were recorded for all patients

together with iron overload and cytomegalovirus (CMV)

serology for the exclusion of other potential causes of

hyperuricemia and hepatic dysfunction. Inflammatory markers

(C-reactive protein [CRP], albumin) were also noted at the

same time point. The association of serum UA levels of the pretransplant

period with the development of SOS was assessed.

HSCT was performed according to standard institutional

transplantation procedures. The patients received either

myeloablative (intravenous busulfan-based with no area under

the curve [AUC] targeting) or reduced-intensity conditioning

(fludarabine-based) regimens depending on the primary disease

or disease status. GVHD prophylaxis consisted of cyclosporine

and methotrexate with or without rabbit anti-thymocyte

globulin. Enoxaparin, vitamin E, and ursodeoxycholic acid were

administered for SOS prophylaxis to all patients beginning with

conditioning. None of the patients received allopurinol before

or after transplant. Defibrotide was started when the clinical

manifestations of SOS developed with the exclusion of other

potential diagnoses. Hepatic SOS was diagnosed when two or

more European Society for Blood and Marrow Transplantation

(EBMT) diagnostic criteria (refractory thrombocytopenia,

weight gain >5% above baseline, hepatomegaly, ascites, and

bilirubin value ≥2 mg/dL) were present and the EBMT severity

grading system was used [12,13]. The study was approved by

the institutional ethics committee.

The associations between clinical variables including

patient- and transplant-related risk factors (primary disease,

conditioning regimen, HLA compatibility, CMV status, ferritin,

GGT, pre/post-conditioning serum UA levels, albumin, and CRP)

and hepatic SOS were analyzed using a logistic regression model.

Only the variables with p<0.2 in the univariate analysis were

subjected to multivariate analysis. The variables with p<0.05

were considered significant. Comparisons within and between

the groups were performed using the Wilcoxon signed-rank

and Mann-Whitney U test, respectively. Median follow-up time

and overall survival were estimated using Kaplan-Meier limit

estimation. The Cox proportional hazards model for multivariate

analyses of survival was used. Receiver operating characteristic

287



(ROC) curve analysis was performed to calculate sensitivity,

specificity, and AUC for peak pre-conditioning serum UA of

≥3.32 mg/dL.

Results

The patient and transplant characteristics are summarized in

Table 1. There were 222 children enrolled in the study with a

median age of 7 years (range: 0.3-19). Sixty-eight percent of

the children were male and most patients were transplanted for

non-malignant diseases (69%), mainly non-malignant

hematological diseases and primary immunodeficiencies, while

the rest of the patients were transplanted for hematological

Table 1. Patient and transplant characteristics.

Variable

Total (n=222)

Age

Median, years (range) 7 (0.3-19)

Sex

Male

Female

Diagnosis

Hematologic malignancies

Non-malignant hematologic

Primary immunodeficiencies

Conditioning regimen

Myeloablative

Reduced intensity

Donor

Matched related

Mismatched related

Stem cell source

Bone marrow

Peripheral stem cell

Umbilical cord

Bone marrow and umbilical cord

Acute GVHD

Grade I-II

Grade III-IV

Veno-occlusive disease

Yes

No

150 (68%)

72 (32%)

69 (31%)

111 (50%)

42 (19%)

156 (70%)

66 (30%)

198 (89%)

24 (11%)

148 (67%)

60 (27%)

5 (2%)

9 (4%)

24 (59%)

17 (41%)

42 (19%)

180 (81%)

Serum creatinine* (mg/dL), median (range) 0.26 (0.01-1.43)

Transaminases* (IU/mL), median (range)

ALT

AST

24 (3-235)

31 (4-199)

Total bilirubin* (mg/dL), median (range) 0.45 (0.06-3.6)

Recipient CMV status: positive 183 (82%)

Ferritin* (µg/L), median (range) 879.4 (7-11365)

GVHD: Graft-versus-host disease; ALT: alanine aminotransferase; AST: aspartate

aminotransferase; CMV: cytomegalovirus.

*: Values for all these parameters represent pre-conditioning levels.

malignancies (31%). Most patients received a bone marrow

graft (71%) from an HLA-matched family donor (89%)

following myeloablative conditioning (70%). Median values of

pre-transplant serum creatinine, transaminases, and total

bilirubin levels were all within the normal range for age (Table 1).

When the median serum UA levels of all patients were evaluated,

pre-conditioning UA levels were found to be significantly higher

than the post-conditioning levels (3.1 mg/dL, range: 0.68-6.9 at

day -9 vs. 2.8 mg/dL, range: 0.61-6.94 at day 0 post-conditioning;

p=0.02). After grouping patients by the development of

hepatic SOS, the 42 patients (19%) who developed mostly

mild/moderate hepatic SOS (91%) were found to have higher

median pre-conditioning UA levels compared to those who did

not develop SOS (3.6 mg/dL vs. 3.0 mg/dL; p=0.01). Pre-transplant

serum creatinine, GGT, total bilirubin, ferritin, CRP, and albumin

were checked, and while most of these variables did not differ

significantly regarding SOS groups (p>0.05), albumin was found

to be lower in patients with SOS compared to those without

SOS (p=0.02) (Table 2). Among the 42 patients diagnosed with

SOS, 25 (60%) had mild, 13 (31%) had moderate, and 4 (9%) had

severe disease. Thirty patients out of 42 recovered from SOS,

while the 4 patients with severe SOS and 8 patients with other

early transplant-related complications died.

While a significant decrease in the serum UA levels of the

patients who did not develop SOS was observed on the day of

HSCT following conditioning (3.05±0.09 mg/dL vs. 2.89±0.09

mg/dL; p=003), there was no significant change in the serum

UA levels of patients who developed SOS (3.75±0.25 mg/dL

vs. 3.2±0.18 mg/dL; p>0.05). The development of SOS was

associated with higher pre-conditioning UA levels (p=0.002).

The ROC curve was drawn to determine a cutoff value and

evaluate the predictive value of pre-conditioning UA for

hepatic SOS development. As shown in Table 3, the cutoff

value of UA in the pre-transplant period before the start of

conditioning for hepatic SOS was 3.32 mg/dL with an AUC of

62.4%, sensitivity of 62%, and specificity of 61%. Hence, the

pre-conditioning UA level seems to be predictive of hepatic

SOS. We observed a difference in the frequency of SOS

among the patients when compared regarding the UA cutoff

value (above the cutoff: 26.8% vs. below the cutoff: 12.8%;

p=0.008) Next, we conducted univariate analysis to investigate

the associations among pre-conditioning serum UA, previously

described risk factors, and hepatic SOS, as illustrated in Table

4. When applied to a multivariate model, pre-conditioning UA

remained significant as a risk factor for SOS (UA: OR, 2.54;

95% CI, 1.26-5.12; p=0.009). The odds of SOS incidence in

patients with UA higher than 3.32 mg/dL were 2.5 times higher

than among patients with UA below the cutoff. Pre-transplant

serum albumin was also associated with SOS (OR, 0.45; 95%

CI, 0.22-0.95; p=0.037) among all other risk factors included

in the model.

288



Table 2. Comparison of patients with and without hepatic sinusoidal obstruction syndrome.

Variable

Primary disease

Hematologic malignancy


Primary immunodeficiency

Conditioning regimen

MAC

RIC

HLA compatibility

MSD

MMRD

Recipient CMV status

Positive

Negative

Hepatic SOS

(n=42)

13 (31%)

22 (52%)

7 (17%)

31 (74%)

11 (26%)

39 (93%)

3 (7%)

29 (69%)

13 (31%)

No Hepatic SOS

(n= 180)

56 (31%)

89 (49%)

35 (20%)

123 (68%)

57 (32%)

159 (88%)

21 (12%)

154 (86%)

26 (14%)

Ferritin (µg/L) 842.7 (28.2-11365) 891.7 (7-9505) 0.77

Serum creatinine (mg/dL) 0.3 (0.03-0.6) 0.26 (0.02-1.4) 0.29

Total bilirubin (mg/dL) 0.43 (0.1-2.7) 0.46 (0.06-3.6) 0.74

GGT (U/L) 16.15 (8-487.3) 17.4 (2.9-398.7) 0.99

Albumin (g/dL) 3.9 (2.8-5.2) 4.1 (2.6-5.6) 0.02

CRP (mg/dL) 0.49 (0.1-8.8) 0.64 (0.1-24.6) 0.86

Pre-conditioning uric acid (mg/dL) 3.6 (0.7-6.9) 3.0 (0.7-6.4) 0.01

Post-conditioning uric acid (mg/dL) 3.0 (1.2-5.4) 2.7 (0.6-6.9) 0.10

SOS: Sinusoidal obstruction syndrome; MAC: myeloablative conditioning; RIC: reduced-intensity conditioning; HLA: human leukocyte antigen; MSD: matched sibling donor; MMRD:

mismatched related donor; CMV: cytomegalovirus; GGT: gamma-glutamyl transferase; CRP: C-reactive protein.

Table 3. ROC analysis for pre-conditioning uric acid.

Parameter AUC Cutoff point Sensitivity (%) Specificity (%)

Pre-conditioning uric acid (mg/dL) 0.624 3.32 62 61

ROC: Receiver operating characteristic; AUC: area under the curve.

p

0.90

0.42

0.58

0.01

Table 4. Risk factors for hepatic sinusoidal obstruction syndrome by logistic regression analysis.

Factors

Primary disease

Hematologic malignancy


Primary immunodeficiency

Univariate

HR (95% CI)

0.94 (0.44-2.01)

0.81 (0.32-2.06)

p

0.87

0.91

0.66

Multivariate

HR (95% CI)

- -

Conditioning regimen 1.38 (0.63-3.01) 0.42 - -

HLA compatibility 0.58 (0.16-2.05) 0.40 - -

Recipient CMV status 0.38 (0.17-0.82) 0.014* 0.59 (0.26-1.36) 0.21

Ferritin (µg/L) 1.00 (1.00-1.00) 0.72 - -

CRP (mg/dL) 1.01 (0.88-1.16) 0.88 - -

Albumin (g/dL) 0.46 (0.22-0.66) 0.039* 0.45 (0.22-0.95) 0.037*

GGT (U/L) 1.00 (0.99-1.01) 0.093* 1.00 (0.99-1.01) 0.26

Pre-conditioning UA ³3.32 mg/dL 2.49 (1.25-4.98) 0.009* 2.54 (1.26-5.12) 0.009*

HR: Hazard ratio; CI: confidence interval; HLA: human leukocyte antigen; CMV: cytomegalovirus; CRP: C-reactive protein; GGT: gamma-glutamyl transferase; UA: uric acid.

*: Serum ferritin, GGT, albumin, and CRP values represent pre-conditioning levels. Variables with p<0.2 in the univariate analysis were subjected to multivariate analysis, in which

p<0.05 was accepted as significant.

p

289



The probability of 10-year overall survival after HSCT was

75% for all patients enrolled with a median survival of 114

months. There was a significant difference between survival

rates of patients who developed SOS (64%; mean survival:

77.5±8.5 months) and those who did not (77%; mean survival:

82±3.2 months) (p=0.047). However, the cutoff value of

pre-conditioning UA of 3.32 mg/dL had no significant effect

on the survival of the patients (OR, 0.86, 95% CI, 0.63-1.18;

p=0.35).

Discussion

SOS of the liver is thought to result from conditioning

regimen-related cytotoxic injury to the hepatic sinusoidal

endothelium and hepatocytes intensified by cytokine-mediated

alloimmunity. Although different combinations of biomarkers

for endothelial injury and hemostasis have been described to

predict the occurrence of SOS by various studies, early and

precise prediction of SOS is still challenging, probably due

to the lack of well-defined specific marker panels [14,15]. In

the present study, we aimed to retrospectively investigate the

association of serum UA levels measured before initiation of

the conditioning regimen with hepatic SOS in 222 pediatric

patients who underwent allogeneic HSCT. Our results indicate

that high pre-conditioning serum UA level is an independent

pre-transplant risk factor for SOS development after allogeneic

HSCT.

Even though danger-associated molecular patterns released

from injured cells including extracellular adenosine triphosphate,

high mobility group box chromosomal protein 1, and UA are

recognized to play a role in aGVHD pathogenesis, the role of UA,

as a pro-inflammatory mediator in allogeneic immune responses,

is still ambiguous [11] (Figure 1). A phase I study reported that

patients with aGVHD had higher serum UA levels during the

pre-transplant period compared to patients without aGVHD

Figure 1. Potential explanation for the role of uric acid in the development of SOS in the inflammatory setting. High levels of uric

acid activate the RAGE/HMGB1 signaling pathway in sinusoidal endothelial cells and increase NF-κB expression. NF-κB overexpression

increases the secretion of pro-inflammatory cytokines, i.e. TNF-α and IL-6, from damaged sinusoidal endothelial cells, resulting in

cytokine derangement within the hepatic sinusoids leading to immune activation. HMGB1 also increases the expression of adhesion

molecules (VCAM and ICAM) and PAI-1, resulting in the activation of the pro-coagulant cascade and sinusoidal obstruction [24,27].

SOS: Sinusoidal obstruction syndrome; RAGE: receptor for advanced glycation end products; HMGB1: high mobility group box chromosomal protein

1; IL-6: interleukin-6; TNF-α: tumor necrosis alpha; NF-κB: nuclear factor κB; ICAM: intercellular adhesion molecule; VCAM: vascular cell adhesion

protein; PAI-1: plasminogen activator inhibitor-1.

290



[10]. The incidence of grade II to IV aGVHD was significantly

decreased in the group treated with urate oxidase during

conditioning. On the contrary, a retrospective study indicated

a significant association between aGVHD and low serum UA

levels [9]. The discrepancies among previous reports on the

role of UA in the development of aGVHD probably arise from

both the paradoxical actions of UA as a pro- and anti-oxidant

[16] and its complex role in inflammation. Preclinical studies

suggest that decreasing serum UA levels during conditioning

before HSCT may suppress recipient antigen-presenting

cell activation and T-cell response [7,17]. Thus, considering

the current understanding of SOS pathogenesis, UA, as an

endogenous danger signal released from injured cells, seems

to be an attractive target for SOS prediction and preventive

measures. There is no previous preclinical or clinical study that

investigated the impact of UA as a pro-inflammatory mediator

on SOS and its predictive role. Thus, it would be reasonable

to consider that a common mechanism of initiation and/or

maintenance of inflammation through endogenous ‘danger

signals’ from injured cells underlies the development of SOS

after HSCT.

The results of this study support our initial hypothesis

about the association of high pre-transplant serum UA

levels with SOS and are in parallel with previous reports

about the significant role of high UA levels in transplant

outcomes such as aGVHD and survival [18,19,20]. Unlike

most other studies assessing the prognostic value of several

biomarkers at different time points after transplantation,

either before or during the early stage of hepatic SOS, we

preferred to evaluate serum UA levels before initiation of

conditioning, which could be a critical period for the early

detection of the inflammatory background of individual

patients. Interestingly, serum UA levels were higher in the

patients with SOS even before the start of the conditioning

regimen compared to patients without SOS. In addition,

the UA levels decreased after conditioning in the patients

without SOS, while they remained high in those with SOS.

This seems to conflict with previous studies reporting

elevated serum UA levels following conditioning because

of its cytotoxicity [10,21]. High pre-conditioning UA levels

that remained unchanged following conditioning together

with low serum albumin levels in our patients with SOS

might be attributed to ongoing subtle inflammation, occult

tissue injury, and accelerated cell turnover related to primary

disease/disease status, previous therapies, and infections

[22]. Post-conditioning decrease in UA levels in patients

without SOS might be explained by the suppressive effect

of myeloablative/lymphodepleting conditioning regimens

on the recipient’s immune system, indicating the presence

of a fine balance between pro-inflammatory and antiinflammatory

mechanisms during the peri-transplant period.

Newly developing bone marrow aplasia could also contribute

to this change in UA levels. Haen et al. [23] reported that

serum UA levels remained low until incipient hematopoietic

recovery in HSCT patients and leukemia patients undergoing

induction chemotherapy. They emphasized the role of UA as

a potential marker for bone marrow activity during aplasia,

besides its role in immune activation and inflammation [23].

Endothelial damage related to conditioning, acting as a second

hit, potentiates immune activation leading to alloimmunization

and the development of SOS. Dysregulation of cytokine

homeostasis is common after conditioning. Pro-inflammatory

cytokines including tumor necrosis alpha, interleukin

(IL)-1, and IL-6 have been reported to activate xanthine oxidase,

thus stimulating UA production, which is involved in the

pathogenesis of early non-infectious transplant complications,

such as SOS. Even in the absence of clinically defined

hyperuricemia (serum UA of >7 mg/dL) based on the solubility

limit of urate in body fluids, this positive feedback loop may

cause a further release of cytokines and endogenous adjuvants

that contribute to the development of endothelial cell injury in

an inflammatory setting [24,25], as in the case of our patients

with high pre-conditioning UA who developed hepatic SOS over

the transplant course. There are several reports investigating

the association of serum UA and transplant outcomes, which

have defined their own cutoff values for their own patient

cohorts [18,26]. Our determined cutoff value of 3.32 mg/dL for

pre-conditioning UA was derived from ROC analysis and it was

predictive of hepatic SOS development after allogeneic HSCT.

Multivariate analysis confirmed the association of serum UA

higher than 3.32 mg/dL with the risk of hepatic SOS. Low serum

albumin in the pre-transplant period was also identified as a

risk factor for SOS. Serum albumin serves as a laboratory marker

of inflammatory status, and the prognostic value of low serum

albumin for transplant outcomes has been revealed by previous

studies.

Study Limitations

One of the limitations of our study is its retrospective nature.

Therefore, serial measurements of serum UA and other

pro-inflammatory and/or endothelial injury markers over the

transplant course could not be performed. Also, the design of

our study did not allow us to investigate whether UA plays an

active role in the pathogenesis of hepatic SOS through induction

of a non-infectious inflammatory reaction in the allogeneic

setting or is simply a biomarker of inflammatory status. Since

our patient population mostly included pediatric patients who

received bone marrow grafts from matched related donors

after myeloablative conditioning for non-malignant disorders,

we could not draw any conclusion about the impact of serum

UA on SOS development in different transplant settings such

as unrelated and haploidentical transplants, which carry a

291



higher risk for SOS. However, we think that the association

of serum UA higher than the cutoff value with SOS in such

a restricted population could be accepted as proof of its

predictive strength.

Conclusion

This is the first report about the association of pre-conditioning

serum UA level and hepatic SOS in HSCT recipients and it

supports the use of pre-transplant serum UA level as a risk

factor for SOS. There is a need for mechanistical studies to

understand the precise role of UA in the pathogenesis of SOS as

an inflammatory mediator in the allogeneic transplant setting.

Further studies with independent cohorts and in different

transplant settings may also help to clarify the role of UA

in predicting high-risk patients for SOS together with other

defined clinical and laboratory markers of endothelial injury.

Ethics

Ethics Committee Approval: The study was approved by the

institutional ethics committee.


Concept: F.V.O., D.C.; Design: F.V.O., D.C.; Data Collection or

Processing: B.K., M.K., K.W.; Analysis or Interpretation: U.E.A.


authors.



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293

Zengin E. et al: Systemic Thrombolysis with rtPA in Children

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2021.0038


Thrombolysis with Systemic Recombinant Tissue Plasminogen

Activator in Children: A Multicenter Retrospective Study

Çocuklarda Sistemik Rekombinant Doku Plazminojen Aktivatörü ile Tromboliz: Çok Merkezli

Bir Retrospektif Çalışma

Emine Zengin 1 , Nazan Sarper 1 , Arzu Yazal Erdem 2 , Işık Odaman Al 3 , Melike Sezgin Evim 4 , Neşe Yaralı 2 , Burcu Belen 5 ,

Arzu Akçay 6 , Ayşen Türedi Yıldırım 7 , Tuba Hilkay Karapınar 3 , Adalet Meral Güneş 4 , Sema Aylan Gelen 1 , Hale Ören 8 ,

Lale Olcay 5 , Birol Baytan 4 , Hüseyin Gülen 7 , Gülyüz Öztürk 6 , Mehmet Fatih Orhan 9 , Yeşim Oymak 3 , Sibel Akpınar 10 ,

Özlem Tüfekçi 8 , Meryem Albayrak 11 , Burçak Tatlı Güneş 12 , Aylin Canpolat 13 , Namık Özbek 2

1Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey

2University of Health Sciences Turkey, Ankara City Hospital, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey

3University of Health Sciences Turkey, Dr. Behçet Uz Pediatrics and Pediatric Surgery Training and Research Hospital, İzmir, Turkey

4Uludağ University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Bursa, Turkey

5Başkent University Ankara Hospital, Clinic of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey

6Acıbadem Mehmet Ali Aydınlar University Acıbadem Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey

7Celal Bayar University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Manisa, Turkey

8Dokuz Eylül University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, İzmir, Turkey

9Sakarya University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Sakarya, Turkey

10University of Health Sciences Turkey, Kanuni Sultan Süleyman Training and Research Hospital, Clinic of Pediatrics, Division of Pediatric

Hematology, İstanbul, Turkey

11Kırıkkale University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Kırıkkale, Turkey

12University of Health Sciences Turkey, Tepecik Training and Research Hospital, Department of Pediatrics, Division of Pediatric Hematology, İzmir,

Turkey

13İstanbul Medeniyet University Göztepe Training and Research Hospital, Clinic of Pediatrics, Division of Pediatric Hematology, İstanbul, Turkey

Abstract

Objective: This study aimed to evaluate systemic thrombolysis

experiences with recombinant tissue plasminogen activator (rtPA).

Materials and Methods: Retrospective data were collected from

13 Turkish pediatric hematology centers. The dose and duration of

rtPA treatment, concomitant anticoagulant treatment, complete

clot resolution (CCR), partial clot resolution (PCR), and bleeding

complications were evaluated. Low-dose (LD) rtPA treatment was

defined as 0.01-0.06 mg/kg/h and high-dose (HD) rtPA as 0.1-0.5

mg/kg/h.

Results: Between 2005 and 2019, 55 thrombotic episodes of 54

pediatric patients with a median age of 5 years (range: 1 day to 17.75

years) were evaluated. These patients had intracardiac thrombosis

(n=16), deep vein thrombosis (DVT) (n=15), non-stroke arterial

thrombosis (n=14), pulmonary thromboembolism (PE) (n=6), and

stroke (n=4). The duration from thrombus detection to rtPA initiation

was a median of 12 h (range: 2-504 h) and it was significantly longer

Öz

Amaç: Geriye dönük çok merkezli bir çalışma düzenleyerek, çocuk

hematoloji uzmanlarının sistemik rekombinant doku plazminojen

aktivatörü (rtPA) kullanımı ile ilgili deneyimlerinin ortaya konması

amaçlandı

Gereç ve Yöntemler: Türkiye’deki 13 pediatrik hematoloji

merkezinden geriye dönük veriler toplandı. rtPA tedavisinin dozu ve

süresi, eşzamanlı antikoagülan tedavi, tam pıhtı erimesi (TPE), kısmi

pıhtı erimesi (KPE) ve kanama komplikasyonları değerlendirildi. Düşük

doz (DD) rtPA tedavisi 0,01-0,06 mg/kg/saat ve yüksek doz (YD)

0,1-0,5 mg/kg/saat olarak tanımlandı.

Bulgular: 2005-2019 yılları arasında ortanca yaşı 5 yıl (1 gün-17,75

yıl) olan 54 hastanın 55 trombotik epizodu değerlendirildi. Hastaların

tanıları; intrakardiyak tromboz (n=16), derin ven trombozu (DVT) (n=15),

inme dışı arteriyel tromboz (n=14), pulmoner tromboemboli (PE) (n=6)

ve inme (n=4) idi. Trombüs saptanmasından rtPA başlangıcına kadar

geçen süre medyan 12 saat (2 sa-504 sa) idi ve inme, inme olmayan



Address for Correspondence/Yazışma Adresi: Emine Zengin, Prof., M.D., Kocaeli University Faculty of Medicine,

Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey

Phone : +90 532 574 05 02

E-mail : eminezengin@hotmail.com ORCID: orcid.org/0000-0002-7362-6130



294



Abstract

in cases of DVT and PE compared to stroke, non-stroke arterial

thrombosis, and intracardiac thrombosis (p=0.024). In 63.6% of the

episodes, heparin was initiated before rtPA treatment. LD and HD

rtPA were administered in 22 and 33 of the episodes, respectively.

Concomitant anticoagulation was used in 90% and 36% of the

episodes with LD and HD rtPA, respectively (p=0.0001). Median total

duration of LD and HD rtPA infusions was 30 h (range: 2-120 h) and

18 h (2-120 h), respectively (p=0.044). Non-fatal major and minor

bleeding rates were 12.5% and 16.7% for LD and 3.2% and 25.8% for

HD rtPA, respectively. At the end of the rtPA infusions, CCR and PCR

were achieved in 32.7% and 49.0% of the episodes, respectively. The

most successful site for thrombolysis was intracardiac thrombosis. HD

versus LD rtPA administration was not correlated with CCR/PCR or

bleeding (p>0.05).

Conclusion: Systemic thrombolytic therapy may save lives and organs

effectively if it is used at the right indications and the right times in

children with high-risk thrombosis by experienced hematologists with

close monitoring of recanalization and bleeding.

Keywords: Recombinant tissue plasminogen activator, Thrombolysis,

Childhood thrombosis

Öz

arteriyel tromboz ve intrakardiyak tromboza kıyasla DVT ve PE için

anlamlı olarak daha uzundu (p=0,024). Atakların %63,6’sında rtPA

tedavisinden önce heparin başlandı. Atakların 22’sine DD ve 33’üne

YD rtPA uygulandı. DD ve YD rtPA ataklarının sırasıyla %90’ında ve

%36’sında eş zamanlı antikoagülasyon kullanıldı (p=0,0001). DD ve

YD rtPA infüzyonlarının medyan toplam süresi sırasıyla 30 sa (2 sa-

120 sa) ve 18 sa (2 sa-120 sa) idi (p=0,044). Ölümcül olmayan majör

ve minör kanama oranları DD için sırasıyla %12,5, %16,7 ve YD rtPA

için %3,2, %25,8 idi. rtPA infüzyonlarının sonunda, atakların sırasıyla

%32,7’sinde TPE ve %49’unda KPE elde edildi. Tromboliz için en başarılı

bölge intrakardiyak trombozdu. YD’ye karşı DD rtPA uygulaması, TPE/

KPE veya kanama ile korele değildi (p>0,05).

Sonuç: Yüksek riskli trombozlu çocuklarda sistemik trombolitik tedavi,

deneyimli hematologlar tarafından rekanalizasyon ve kanamanın yakın

takibi altında doğru endikasyon ve doğru zamanda kullanıldığında

etkin bir şekilde hayat ve organ kurtarabilir.

Anahtar Sözcükler: Rekombinant doku plazminojen aktivatörü,

Tromboliz, Çocukluk trombozu

Introduction

The incidence of pediatric thrombosis is increasing due to

advancements in neonatal and pediatric intensive care. Central

venous catheters (CVCs), infections, surgery, immobility, trauma,

congenital heart disease (CHD), vasculitis, underlying cancer,

and thrombophilia are important risk factors for thrombosis

[1,2,3]. Endovascular and surgical interventions for CHD save

many lives, but the development of thrombosis is a frequent

complication of these procedures [4]. Anticoagulation with

unfractionated heparin (UFH) or low-molecular-weight heparin

(LMWH) is the routine management of pediatric acute thrombosis.

Heparins are used to prevent propagation, embolization, and

recurrence of acute thrombosis [5]. Anticoagulation does not

provide rapid recanalization of the occluded vessels and 49% of

surviving infants and children suffering from arterial thrombosis

have long-term sequelae including extremity or organ loss

or epilepsy [1,3]. Postthrombotic syndrome (PTS) is the most

common long-term complication of deep vein thrombosis

(DVT) of the extremities. It is a chronic condition characterized

by the development of venous insufficiency that manifests

clinically with swelling, pain, cramping, stasis, dermatitis,

and ulceration of the affected extremity [6]. The incidence

of pediatric PTS ranges from 3% to 70% in median 2-year

follow-up [6,7]. In cases of pediatric arterial thrombosis and

DVT, mortality is nearly 4-9% and 2%, respectively, with cranial

and intracardiac thrombosis causing the most mortality [1,8].

Since 1990, off-label use of recombinant tissue plasminogen

activator (rtPA) in newborns and children has shown that

systemic thrombolytic therapy is very effective if it is used at

the right indications in selected patients under close monitoring

[9,10,11,12,13,14]. Endovascular thrombolysis requires expert

interventional radiologists and general anesthesia, and the

small vessel diameters of children frequently limit such

procedures; in addition, this intervention may damage the

vascular endothelium and induce new thrombosis. However,

the intravenous administration of thrombolytics is practical

[5]. In this retrospective study, the experiences of pediatric

hematology centers with systemic thrombolysis are presented.

This study may help in the timely referral of pediatric patients

to hematologists, intensive care physicians, or cardiologists for

thrombolysis in cases of organ-, limb-, and life-threatening

thrombi.


This retrospective study was approved by the institutional ethics

committee and approval was obtained from the patients’ legal

guardians at admission to the hospital for the release of medical

data for scientific purposes without including patient identity.

A questionnaire form was designed and submitted to all Turkish

pediatric hematology centers via electronic mail to collect the

medical data of patients 0-18 years old who received systemic

rtPA for thrombolysis. rtPA administrations for CVC clearance

were not included in the study. Major surgery or central nervous

system bleeding within 10 days and inability to maintain a

platelet count of at least 50,000/µL with transfusion support

or fibrinogen of at least 100 mg/dL were exclusion criteria.

Concomitant heparin administration was at the decision of

the attending physician. UFH dose of 5-10 U/kg/h and LMWH

295



(enoxaparin) dose of 0.5 mg/kg twice daily were the prophylactic

doses. Patients’ age, sex, underlying disease, location of

the thrombus, period from thrombus detection to rtPA

administration, previous anticoagulant treatments, initial and

maximum rtPA doses, concomitant anticoagulant treatments,

total duration of rtPA infusions, bleeding complications,

thrombus resolution, amputations, organ loss, and outcomes

after 3 months of anticoagulation were evaluated. Low-dose

(LD) rtPA treatment was defined as 0.01-0.06 mg/kg/h and

high-dose (HD) as 0.1-0.5 mg/kg/h [5,15]. The centers reported

that for older children and adolescents receiving LD continuous

infusion, the maximum dose per hour was not defined and rtPA

doses exceeding 2 mg/h were administered. Major bleeding

was defined as any intracranial or retroperitoneal bleeding, or

bleeding resulting in a drop in hemoglobin of 2 g/dL, or requiring

transfusion or leading to death as defined by the International

Society on Thrombosis and Haemostasis bleeding scale [16].

Minor bleeding was defined as oozing at the site of intravenous

or other indwelling catheters or minor epistaxis, hematoma,

hematuria, or melena not resulting in a drop in hemoglobin.

Diagnosis of thrombosis and monitoring for clot resolution

performed by color Doppler ultrasound examination,

echocardiography, and/or computed tomographic angiography

was acceptable. Complete clot resolution (CCR) was defined

as objective radiologic imaging confirming recanalization

of the occluded vessel and/or arterial thrombosis clinical

examination showing equal pulse pressure, capillary return, and

warm extremities. Partial clot resolution (PCR) was defined as

objective radiologic imaging confirming partial recanalization

of the occluded vessels. No resolution (NR) was defined as

objective radiologic imaging confirming no change or thrombus

extension in the occluded vessel.

Centers reported performing laboratory monitoring by

measuring prothrombin time, activated partial thromboplastin

time, fibrinogen, anti-factor Xa, fibrin degradation products,

D-dimer, platelet counts, and hemoglobin levels every 6-12 h.

Patients were strictly monitored clinically for bleeding (oozing

from puncture sites, gastrointestinal system bleeding, and signs

of cerebral bleeding), any new emboli, allergic and anaphylactoid

reactions, laryngeal edema, orolingual angioedema, rash, and

urticaria. Centers also monitored blood pressure, heart rate,

and respiration rate. If minor bleeding developed, the rtPA

dose was either deescalated or rtPA was withdrawn transiently.

They stopped the treatment and infused fresh frozen plasma

(FFP) and/or red blood cells if major bleeding developed. If

recanalization was seen in imaging studies, rtPA was stopped.


For calculation of median and percentage, descriptive analysis

was used; for comparison of two independent variables, the

Mann-Whitney U test was used; and for comparison of more

than two independent variables, Kruskal-Wallis H tests were

used. Values of p<0.05 were defined as significant. All analyses

were performed using SPSS version 13 (SPSS Inc., Chicago, IL,

USA).

Results

Thirteen pediatric hematology centers participated in the study.

Questionnaire forms for patients aged 0-18 years who received

systemic rtPA (alteplase) between May 2005 and December 2019

were evaluated. The study included 55 thrombotic episodes of

54 patients (24 male, 30 female) with a median age of 5 years

(range: 1 day to 17.75 years). Two episodes (intracardiac and

diffuse pulmonary thromboembolism) of a patient with CHAPLE

syndrome were included. Eight of these cases were already

published [11,17]. Table 1 shows the characteristics of patients

and treatment outcomes. Patients with intracardiac and nonstroke

arterial thrombosis were younger than patients with

pulmonary thromboembolism, DVT, and stroke (p=0.001).

Out of 16 patients with intracardiac thrombus, five were

preterm babies in the neonatal intensive care unit. They had

umbilical catheters and ventilator support, and two of them

also had septicemia. Five patients had CHD (one of them also

had leukemia) and another two patients had cardiomyopathy.

There were also patients with chronic pulmonary disease

in the intensive care unit, acute lymphoblastic leukemia,

lipin-1 deficiency (rhabdomyolysis + acute renal failure with

hemodialysis), and CHAPLE syndrome (CD55 deficiency).

Out of 15 patients with DVT, five had hematological diseases (all

with CVCs) and three had metabolic diseases (propionic academia

with CVC and Candida septicemia, hyperhomocysteinemia,

and hyperlipidemia). There were also cases of Behçet’s disease,

pyelonephritis, exposure to trauma during football activity,

Down syndrome, epileptic seizures, idiopathic Budd-Chiari

syndrome, and a patient taking oral contraceptive pills.

In 14 patients with non-stroke arterial thrombosis, the

underlying diseases were prematurity-associated in five

patients (septicemia, respiratory distress syndrome, necrotizing

enterocolitis, bronchopulmonary dysplasia) and one baby

also had CHD. They also had umbilical catheters as a

thrombus-provoking factor. CHD was also present in another

four patients: Fallot tetralogy, ventricular septal defect (VSD)

+ patent foramen ovale + pulmonary hypertension, a newborn

with aortic stenosis who underwent cardiac catheterization,

and an infant with VSD who underwent transcatheter repair.

Other patients had Kawasaki syndrome with coronary artery

thrombus and pulmonary stenosis (n=2), while there was also

an infant with bronchiolitis in the pediatric intensive care unit

and an adolescent in the postpartum period with inherited

homozygous antithrombin (AT) deficiency.

296



Table 1. Characteristics of the patients and treatment outcomes with systemic recombinant tissue plasminogen activator.

Sex,

male/female

Age, median

(range)

Site of

thrombosis

Underlying

disease or

provoking

factors

Infectionrelated

thrombosis, n

Patients with

central venous

catheters, n

Patients with

malignancy, n

Period from

thrombus

detection

to rtPA

administration

(h), median

(range)

rtPA dose (mg/

kg/h), median

(range)

Intracardiac thrombosis

n=16

Deep venous

thrombosis

n=15

Non-stroke

arterial

thrombosis

n=14

Pulmonary

thromboembolism

n=6

Stroke

n=4

7/9 7/8 5/9 4/2 2/2 0.799

3 years

(15 days-17.75 years)

7 right atrium

3 right ventricle

3 left ventricle

2 left atrium

1 aortic and mitral valves

5 prematurity†

4 CHD

2 dilated CMP

1 CHD + leukemia

1 leukemia

1 lipin-1 deficiency

1 chronic pulmonary

disease†

1 CHAPLE syndrome

15 years

(1 day-17 years)

Jugular, subclavian,

axillary, vena cava

inferior, vena porta,

iliac, renal

3 leukemia

1 AML-HSCT-pRTA-CS

1 Fanconi anemia-HSCT

1 hyperlipidemia

1 OC

1 Behçet’s disease

1 pyelonephritis†

1

hyperhomocysteinemia

1 propionic acidemia†

1 epilepsy

1 trauma

1 Down syndrome +

obesity

1 idiopathic Budd-

Chiari syndrome

0.6 years

(1 day-17 years)

5 lower extremity

4 upper extremity

2 coronary artery

2 renal artery

1 subclavian artery

5 prematurity†

4 CHD

2 Kawasaki

syndrome

1 inherited AT

deficiency

1 bronchiolitis in

PICU†

1 infantbacteriemia†

15 years

(5-17 years)

Pulmonary

1 Evans-CS

1 obesitypostpartum

1 nephrotic

syndrome

1 Behçet’s disease

1 MS-CS

1 CHAPLE

syndrome

2 2 5 0 1

9 5 4 0 2

2 4 0 0 1

11.5 years

(2.5-13.5 years)

2 venous sinus

1 MCA

1 circle of Willis

+ left ophthalmic

artery

1 leukemia

1 aplastic anemia-

HSCT-progesterone

1 AOMmastoiditis†

1 idiopathic

2 (2-504) 48 (2-360) 7 (2-48) 60 (12-360) 2 (2-12) 0.024

0.25 (0.01-0.5) 0.06 (0.03-0.5) 0.2 (0.01-0.5) 0.175 (0.03-0.3) 0.055 (0.03-0.1) 0.160

Patients on concomitant anticoagulation

UFH, n (%) 1 (6) 3 (20) 3 (21) 1 (16.6) 2 (50)

LMWH, n (%) 4 (25) 7 (46.6) 7 (50) 2 (33.3) 2 (50)

Duration of

rtPA infusion

(h), median

(range)

15 (2-36) 18 (6-120) 30 (6-120) 41 (24-48) 27 (6-96) 0.015

p

0.001

0.046

297



Table 1. Continued

Minor bleeding,

n (%)

Major bleeding,

n (%)

Bleedingrelated

death,

n (%)

Early outcome with rtPA

Intracardiac thrombosis

n=16

Deep venous

thrombosis

n=15

Non-stroke

arterial

thrombosis

n=14

Pulmonary

thromboembolism

n=6

3 (18.7) 6 (40) 3 (21.4) 0 0

0 0 1 (7.1) 3 (50) 0

0 0 0 0 0

CCR, n (%) 10 (62.5) 2 (13.3) 3 (21) 3 (50) 0

Stroke

n=4

PCR, n (%) 6 (37.5) 9 (60) 6 (42) 3 (50) 3 (75)

CCR after 3

months of

anticoagulation

Total CCR after

3 months, n

(%)

Sequelae/death

5 (31.2) 8 (53.3) 5 (35) 2 (33.3) 3 (75)

15 (93) 10* (66.6) 8** (57) 5 (83.3) 3 (75)

0 sequelae

1 death (prematurity

septicemia)

1 cirrhosis***

2 venous failure

1 death with VOD

1 lost to follow-up

3 amputations

-1 leg

-1 arm

-1 finger

1 kidney atrophy

2 deaths related

to prematurity

complications

1 pulmonary

hypertension

1 unilateral

blindness

AML: Acute myeloblastic leukemia, AOM: acute otitis media, CCR: complete clot resolution, CHD: congenital heart disease, CMP:

cardiomyopathy, CS: corticosteroid, HSCT: hematopoietic stem cell transplantation, LMWH: low-molecular-weight heparin, MCA: middle

cerebral artery, MS: multiple sclerosis, OC: oral contraceptives, rtPA: recombinant tissue plasminogen activator, PCR: partial clot resolution,

PICU: pediatric intensive care unit, pRTA: proximal renal tubular acidosis, UFH: unfractionated heparin, VOD: veno-occlusive disease.

† Patients with infection, * underwent left iliac vein endovascular thrombectomy after systemic rtPA, ** patient with Kawasaki syndrome

underwent coronary artery endovascular thrombectomy after systemic rtPA, *** underwent liver transplantation.

p

0.209

0.006

0.355

In cases of pulmonary thromboembolism, underlying diseases

were nephrotic syndrome, immune dysregulation (Evans

syndrome; the patient received corticosteroid), Behçet’s disease,

multiple sclerosis with corticosteroid treatment, CHAPLE

syndrome (CD55 deficiency), and obesity and pregnancy in a

17-year-old patient.

Underlying diseases of the patients with stroke were

hematological disease in two (leukemia; a female adolescent

with aplastic anemia who underwent hematopoietic stem cell

transplantation, having a CVC and receiving depot progesterone),

infection in one (acute mastoiditis), and idiopathic thrombus in

a toddler.

As provoking factors for thrombus, there were CVCs, infections,

and malignancy in 20 (36.4%), 10 (18.2%), and 7 (12.7%) of

the thrombotic episodes, respectively. There was more than one

thrombotic risk factor for some patients (Table 1).

In 63.6% (35/55) of the episodes, heparin was initiated before

rtPA treatment. LMWH was preferred in 33 episodes and UFH

in only two episodes. Six patients received FFP to replace

plasminogen before administration of rtPA; two of them

were infants. The period from thrombus detection to rtPA

initiation was a median of 12 h (2-504 h); this period was

significantly longer for DVT and pulmonary thromboembolism

compared to stroke, non-stroke arterial, and intracardiac

thrombi (p=0.024). When all 55 episodes were evaluated, no

correlation was found between this period and thrombus

resolution (p=0.75).

There was a wide range of administered rtPA doses and

durations. In 14 episodes, dose escalation, in four episodes dose

de-escalation, and in two episodes temporary withdrawal of

rtPA due to bleeding was applied. The median initial and median

maximum rtPA doses were both 0.1 mg/kg/h (0.01-0.5). The total

duration of rtPA infusions was a median of 18 h (2-120 h) and

298



it was significantly shorter for cardiac thrombosis compared to

pulmonary and arterial thrombosis (p=0.015).

In this study, 33 patients received HD rtPA. Only one center

used a standard rtPA treatment protocol. They used HD rtPA

for 6 h and repeated infusion after 24 h if thrombus resolution

was not achieved. They also transfused FFP at 10-20 mL/kg

concomitantly. They did not use concomitant heparin. This

center had five patients in this study. For four patients, they

had to repeat rtPA infusions 3-6 times (totally for 18-36 h)

and reported no bleeding complications. One of their patients

with femoral and popliteal artery thrombus had CCR after only

6 h of rtPA infusion. Out of their five patients (two cardiac,

one peripheral artery, one pulmonary thromboembolism, one

DVT), two achieved CCR. Eight more patients from other centers

received HD rtPA for only 2-6 h. When all patients receiving

HD rtPA for ≤6 h (total duration) were evaluated, 2/9 achieved

CCR with two minor bleeding complications. Patients receiving

HD rtPA for 6 h at a time with or without repeated daily doses

had two minor bleeding complications (2/13) and only two had

CCR (2/13).

In Table 2, patients receiving HD rtPA infusion for longer than

6 h/day are presented. Patients 5, 6, and 16 received rtPA

for 7-12 h daily whereas the others received rtPA without

interruption. These 20 patients received HD rtPA for 12-120

h totally and ten achieved CCR; they had one major and six

minor bleeding complications. In eight episodes, concomitant

Table 2. Outcomes and bleeding complications of patients using high-dose rtPA longer than 6 h/day.

Patient number

Age/sex

1

14 years/M

2

3.5 months/M

3

24 months/F

4

13.5 years/F

5

45 months/M

6

35 months/M

7

14.5 years/F

8

17 years/F

9

2.5 months/F

10

5 years/M

11

8 years/M

Underlying disease/site of thrombosis

Trauma/right iliac and femoral veins

Preterm, NEC, BPD, hydrocephaly after

intracranial bleeding, acute gastroenteritis,

microthrombus of left-hand fingers

Tetralogy of Fallot/right popliteal artery

HSCT for aplastic anemia, CVC, depot

progesterone use/left internal carotid

artery, posterior communicant artery, left

ophthalmic artery

Propionic acidemia, Candida septicemia,

catheter/vena cava superior

JMML, catheter/left jugular vein

Down syndrome/portal vein, Budd-Chari

syndrome

Obesity, postpartum/pulmonary arteries,

main femoral and popliteal veins

Preterm SGA, sepsis, RDS, CVC/left iliac and

femoral arteries

Nephrotic syndrome/diffuse pulmonary

embolism

Kawasaki syndrome/left coronary artery

aneurism

rtPA dose,

mg/kg/h

duration

0.06/6 h

0.3/6 h

0.5/6 h

Total 18 h

Concomitant

medicine

Bleeding

complications

during rtPA

treatment

UFH first 6 h No No

0.1/120 h LMWH for 3 days No No

0.3/4 h

0.4/12 h

Total 16 h

0.03/6 h

0.1/12 h

Total 18 h

0.5/6 h/day

0.2/12 h/day*

Total 18 h

0.1/6 h/day

0.1/7 h/day*

Total 13 h

- Minor CR

UFH

No

LMWH Minor CR

- Minor PR

0.2/18 h FFP No PR

0.2/24 h - No PR

0.3/24 h FFP No No

0.03/12 h

0.15/6 h

0.03/28 h

Total 46 h

0.1/5 h

0.2/4 h

0.3/9 h

Total 18 h

- No CR

- Minor PR

Clot

resolution

PR/left

blindness

299



Table 2. Continued

Patient number

Age/sex

12

1 day/F

Underlying disease/site of thrombosis

Late prematurity/left subclavian artery (at

birth)

rtPA dose,

mg/kg/h

duration

Concomitant

medicine

Bleeding

complications

during rtPA

treatment

0.4/48 h UFH Minor No

Clot

resolution

13

6 months/F

14

17.5 years/M

15

11.7 years/M

16

10.5 years/F

17

12 years/M

18

15 days/F

19

3.5 years/F

20

12 years/M

32 weeks preterm, pneumonia, convulsion,

hemiparesis, CVC/left atrium

Dilated cardiomyopathy, progressive

muscular dystrophy/left ventricle

VSD/right ventricle

Chronic pulmonary disease, septicemia,

tracheostomy, CVC, mechanic ventilation/

right atrium

VSD + pulmonary stenosis

Preterm, septicemia, umbilical catheter/

right atrium

Hypertension, right kidney atrophy/left

renal artery

Behçet’s disease/right atrium, pulmonary

embolism vena cava inferior, bilateral iliac

veins, vena porta, vena hepatica

0.1/12 h - No CR

0.3/6 h

0.4/16 h

0.5/6 h

Total 28 h

- No CR

0.4/12 h - Minor

0.1/6 h/day

0.2/12 h/day*

Total 18 h

0.3/12 h

0.4/12 h

0.5/12 h

Total 36 h

0.3/8 h

0.4/12 h

Total 20 h

CR

LMWH No PR

- No CR

- No CR

0.1/72 h UFH No

0.3/4 h

0.25/3 h

0.3/39 h

Total 46 h

CR

UFH Major CR

BPD: Bronchopulmonary dysplasia, CVC: central venous catheter, F: female, JMML: juvenile myelomonocytic leukemia, LMWH: low-molecular-weight heparin, M: male, NEC: necrotizing

enterocolitis, UFH: unfractionated heparin, VSD: ventricular septal defect.

* These three patients received rtPA for 6-12 h daily whereas others received rtPA without interruption.

heparin was used. FFP was used in only two episodes.

The numbers were small for statistical comparison of the

infusion period concerning thrombus resolution and bleeding

complications. In HD rtPA treatment, a longer infusion period

compared to ≤6 h seemed more effective (CCR 10/20 versus

2/9), but with more bleeding complications (six minor and

one major bleeding episode in 20 patients versus two minor

bleeding episodes in 9 patients).

Concomitant heparin was administered in 58.2% (32/55) of the

thrombotic episodes (10 UFH and 22 LMWH), in 24 episodes at the

therapeutic dose and in eight episodes at the prophylactic dose.

Therapeutic-dose heparin was preferred in patients receiving

LD rtPA (p<0.05). In intracardiac thrombosis, concomitant

anticoagulant administration was less frequent compared to

stroke or DVT (p=0.046). In 18 episodes of this study, CCR was

achieved, and in 11 of those, concomitant heparin was not used.

In 10 of these episodes, the thrombi were intracardiac.

Four major non-fatal bleedings (7.2%) developed; three were

in patients with pulmonary thromboembolism (pulmonary

hemorrhage, melena + hematemesis, hematuria) and one

in a patient with coronary artery thrombosis (hematuria).

Underlying diseases in the patients developing major bleeding

were Kawasaki syndrome, Behçet’s disease, Evans syndrome, and

multiple sclerosis; they all received concomitant heparin and

three of them received LD rtPA for 18-48 h. FFP and packed red

cells or only FFP or fibrinogen concentrate were administered as

supportive treatment in major bleeding episodes. Antifibrinolytics

were not used for any patient. There were minor bleedings in

12 episodes (21.8%) that could be easily controlled; these were

epistaxis, gingival bleeding, venipuncture site bleeding, CVC

300



exit site or incision site bleeding, subcutaneous hematoma,

and bone marrow puncture site bleeding. When patients

with and without rtPA-related bleedings were compared, no

relationship was found with age, gender, initial rtPA dose,

maximum rtPA dose, site of thrombosis, total rtPA duration,

or concomitant use of anticoagulants. In 22 patients receiving

LD treatment, there were three major and four minor bleeding

complications. In patients receiving LD continuous treatment,

rtPA doses exceeding 2 mg/h were used in older children and

adolescents. When major bleedings in patients receiving LD

were evaluated, a dose above 2 mg/h was administered for only

one adolescent with pulmonary thromboembolism. This patient

received concomitant LMWH and had hematuria and injection

site bleeding. Two other patients on LD treatment and with

major bleeding also received concomitant LMWH.

As shown in Table 1, after rtPA administration, CCR and PCR

were achieved in 18 (32.7%) and 27 (49.0%) of the episodes,

respectively. In intracardiac thrombosis, CCR was achieved

in 62.5% and PCR in 37.5%; thrombolysis was significantly

more successful compared to patients with non-stroke arterial

thrombosis (CCR 21% and PCR 42%) and patients with DVT

(CCR 13.3% and PCR 60%) (p=0.006). Out of 16 patients with

intracardiac thrombosis, CCR was achieved in two patients

even with late rtPA administrations (7 and 21 days after

diagnosis). The total duration of rtPA infusion was significantly

shorter in intracardiac thrombosis compared to pulmonary

thromboembolism and non-stroke arterial thrombosis.

Physicians discontinued treatment when CCR was achieved.

Two patients achieving only PCR after systemic thrombolysis

with rtPA underwent endovascular thrombectomy: a patient

with Kawasaki syndrome having coronary artery thrombus and

another patient with left iliac and femoral vein thrombosis. The

latter patient also developed a thrombus in the contralateral

extremity in the following week under LMWH treatment.

rtPA was administered again but due to NR with the second

thrombus, endovascular thrombectomy was performed.

Off-label rivaroxaban was started with special approval from

the health authority.

There were two patients with unilateral renal artery thrombosis.

The patient with inherited AT deficiency presented with left

renal artery thrombosis in the postpartum period and left kidney

atrophy developed. A 3.5-year-old patient with preexistent

right kidney atrophy presented with left renal artery thrombosis

and achieved CCR. There was an 11-year-old patient with

pyelonephritis associated with left renal vein thrombosis (RVT);

rtPA treatment was started 7 days after the diagnosis and PCR

was achieved. This patient also already had right kidney atrophy

at presentation. Unilateral blindness developed in a patient

after stroke; the etiology of the thrombus was otitis media and

mastoiditis. Amputations were performed for three patients with

arterial thrombosis of extremities or fingers after unsuccessful

thrombolytic treatments. Cirrhosis developed in a patient with

idiopathic Budd-Chiari syndrome; this patient underwent

liver transplantation. Three patients died due to prematurity

complications of septicemia + necrotizing enterocolitis +

bronchopulmonary dysplasia and one transplanted patient with

juvenile myelomonocytic leukemia died due to veno-occlusive

disease. Anticoagulant treatments were continued with LMWH

for a median of 3 months (1-6 months) in all surviving patients

except three patients who received coumadin and rivaroxaban

and one non-compliant patient. Acetylsalicylic acid was also

used for seven patients with arterial, cardiac, and venous

thrombosis. The patient with CHAPLE syndrome also received

eculizumab. When the patients’ outcomes were evaluated after

3 months of anticoagulation, CCR was increased to 74.5% from

32.7%.

In Table 3, LD and HD rtPA treatments are compared in terms of

characteristics of thrombotic episodes, bleeding complications,

and treatment outcomes. Patients’ median age and median

duration from thrombus detection to rtPA administration

were similar between patients receiving LD and HD treatment.

Although the numbers were small for statistical power, there were

more patients with intracardiac thrombosis in the HD group. In

the LD group, the median duration of rtPA treatment was longer

(p=0.044) and concomitant anticoagulant administration was

more frequent (p=0.0001). In LD treatment 90% (20/22) and in

HD treatment 36% (12/33) of the patients received concomitant

anticoagulants. The dose of rtPA was not correlated with clot

resolution or bleeding (p>0.05).

Discussion

The lack of prospective trials for the treatment of pediatric

thrombosis with rtPA is a serious dilemma for pediatricians.

The Thrombolysis in Pediatric Stroke Study started in 2010

but was ended by the National Institutes of Health in 2013

due to lack of accrual [18]. In the pediatric antithrombotic

therapy guidelines published in 2012, thrombolysis was

recommended for the following indications [19]: a) limbthreatening

or organ-threatening arterial thrombosis (via

proximal extension) or femoral artery thrombosis that fails to

respond to initial UFH therapy; b) bilateral RVT with evidence of

renal impairment; c) symptomatic peripheral arterial catheterrelated

thromboembolism; d) right atrial thrombosis related

to CVC, especially >2 cm and mobile; and e) thrombotic giant

coronary artery aneurism in cases of Kawasaki syndrome. The

authors were more cautious in thrombolysis of childhood

acute ischemic stroke outside of research protocols [19]. Some

authors also included the following conditions among strong

indications for thrombolysis: superior vena cava syndrome;

pulmonary embolism with hypotension or shock or resulting

in right heart strain or myocardial necrosis; extensive venous

thrombosis with total occlusion of venous flow, increased

301



Table 3. Comparison of systemic thrombolysis with low- and high-dose recombinant tissue plasminogen activator.

Median age

Age range

Site of thrombosis, n

Period from thrombus detection to rtPA

administration (h), median (range)

Median (range) duration of rtPA

treatment (h)

Concomitant heparin, n (%)

rtPA-associated bleeding, n (%)

Clot resolution, n (%)

Low-dose rtPA

0.01-0.06 mg/kg/h

n=22

9.60 years

15 days-17 years

DVT 8

Non-stroke arterial 6

Intracardiac 3

Stroke 3

Pulmonary 2

High-dose rtPA

0.1-0.5 mg/kg/h

n=33

3.95 years

1 day-17.75 years

DVT 7

Non-stroke arterial 8

Intracardiac 13

Stroke 1

Pulmonary 4

p

0.275

0.264

12 (2-240) 18 (2-504) 0.575

30 (2-120) 18 (2-120)* 0.044

20 (90)

(UFH, n: 5 + LMWH, n: 15)

Minor: 4 (18.1)

Major: 3 (13.6)

Partial: 12 (54.5)

Complete: 6 (27.2)

12 (36)

(UFH, n: 5 + LMWH, n: 7)

Minor: 8 (24.2)

Major: 1 (3.0)

Partial: 16 (48.4)

Complete: 12 (36.3)

DVT: Deep venous thrombosis, LMWH: low-molecular-weight heparin, rtPA: recombinant tissue plasminogen activator, UFH: unfractionated heparin.

*In five patients, rtPA was used for 6 h at a time and repeated every 24 h.

0.0001

0.567

0.509

compartment pressures, and compromise of arterial blood

flow; and CHD with shunt thrombosis and cerebral sinovenous

thrombosis with neurological impairment and no improvement

with anticoagulation or progressive thrombosis [5].

Tarango and Manco-Johnson [5] recommended systemic

thrombolysis with LD treatment for 6-72 h and HD treatment

for 2-6 h at a time, repeating the same doses if indicated

over a period of 72 h. In the current retrospective study, only

one center applied HD treatment following this protocol,

administering rtPA for 6 h at a time and repeating the infusion

every 24 h if there was no clot resolution. However, they

repeated the infusions for 6 days for some patients, longer than

recommended by Tarango and Manco-Johnson [5]. Other centers

used continuous infusions of LD and HD rtPA for a maximum

of 120 h or stopped infusions earlier when CCR was achieved

or bleeding complications occurred. When HD rtPA infusions

for longer than 6 h/day were compared to HD for 2-6 h/day,

bleeding complications were more frequent in infusions for >6

h (six minor bleedings and one major bleeding in 20 thrombotic

episodes versus two minor bleedings in 13 thrombotic episodes).

However, with LD continuous infusion (2-120 h), there were also

four minor and three major bleeding episodes in 22 thrombotic

episodes. In this series, major bleeding was more frequent with

LD rtPA compared to HD rtPA. Centers administered continuous

LD without any dose limitation per hour, exceeding 2 mg/h in

older children and adolescents. Although bleeding episodes

were non-fatal in this retrospective study, to be on the safe

side, administration of continuous LD rtPA for longer than

72 h and/or exceeding 2 mg/h cannot be recommended, similar

to administration of HD rtPA for longer than 6 h at a time.

Similar to our study, LD rtPA was shown to be effective and

safe in a group of patients including preterm neonates and

children [15]. In the present study, when LD and HD rtPA

treatments were compared, there was no difference in clot

resolution, but with LD rtPA, the median duration of treatment

was longer and physicians preferred concomitant heparin

administration more frequently. This may be explained by

physicians’ fear of bleeding complications in HD treatment.

CCR was achieved in 32.7% and PCR in 49.0% of the

thrombotic episodes at the end of rtPA infusions. Similar to

our results, in a study of 46 children receiving systemic rtPA,

Ansah et al. [12] found that CCR and PCR/NR were not related

to initial and maximum rtPA doses, duration of rtPA, or mean

time from diagnosis to treatment. They reported that CCR

and PCR were achieved in 46% and 22%, respectively, with a

slightly higher CCR rate than that seen in our study. They also

reported that bleeding complications occurred in 33% of the

patients and bleeding-related death in 4.3%, and bleeding

complications were related to median initial rtPA dose

(0.10 mg/kg/h vs. 0.03 mg/kg/h) [12]. In the present study,

in 63.6% of the episodes, heparin was initiated before rtPA

treatment and concomitant heparin was administered to 90%

of the patients receiving LD rtPA. Concomitant heparin was

used to prevent proximal clot extension during rtPA infusion.

302



The median duration of rtPA treatment was 30 h (range:

2-120 h) with LD treatment and 18 h (2-504 h) with HD. In

Wang et al.’s [15] study, the duration of treatment was 4-48

h with LD and 1-24 h with HD; complete lysis was achieved

in 97% of 29 patients with arterial thrombus, intracardiac

thrombus, and DVT presented within 2 weeks. It is not easy to

compare these studies and draw any conclusions due to the

heterogeneity of the patients.

In the present series, early CCR and PCR rates were between

0% and 62.5% and between 37.5% and 75.0%, respectively, for

different thrombus locations, but thrombolysis was significantly

more successful for intracardiac thrombi and pulmonary

thromboembolism (62.5% and 50% CCR, respectively).

Fortunately, after two endovascular thrombectomies following

unsuccessful systemic rtPA administrations, and with 3 months

of anticoagulation, CCR ranged between 57% and 93% for

different thrombus locations.

In renal artery thrombus, rtPA treatment resulted in CCR in

one of two patients. rtPA was also recommended for bilateral

RVT with evidence of renal impairment [19]. In a study of five

newborns with unilateral RVT, clot resolution was not successful

with rtPA plus heparin [20]. However, in another newborn, CCR

was achieved when rtPA was used even 6 days after the onset

of thrombus [21].

In three of four patients with stroke, PCR was achieved with

thrombolysis. In the patient with left median cerebral artery

thrombosis, PCR was achieved with rtPA administration even

12 h after diagnosis. Low rates of bleeding complications

were reported with rtPA in cases of pediatric ischemic stroke

[2,14], and reports about successful thrombolysis in pediatric

stroke with systemic rtPA administered within the first 4 h are

increasing [2,13,22,23].

For a 12-year-old patient with Behçet’s disease, rtPA was

life-saving. Symptoms of thrombosis in multiple sites had

developed within 15 days and systemic vasculitis was diagnosed.

This patient was classified among the patients with pulmonary

thromboembolism in this study, but he also had thrombi in the

right atrium, vena cava inferior, bilateral iliac veins, vena porta,

and vena hepatica. Administration of rtPA at 0.3 mg/kg/h for

46 h and concomitant UFH led to CCR. In this patient, there

was major bleeding, which was controlled with temporary drug

withdrawal and transfusion. There were four major non-fatal

bleeding episodes (7.2%) in 55 systemic rtPA administrations.

In addition to blood components, tranexamic acid may be used

for bleeding complications. The use of antifibrinolytic agents

for treating symptomatic intracranial hemorrhage that develops

as a complication of thrombolytic therapy was also suggested

[24]. Antifibrinolytics are available for urgent use compared to

blood components.

In this series, CCR was achieved in only 13.3% of DVT episodes.

In a pediatric study of 26 cases, there was no CCR in DVT,

whereas CCR was achieved for 81% of arterial thrombi [10].

Successful thrombolysis in DVT with rtPA was reported in some

pediatric studies [12,25]. In massive iliofemoral DVT with total

occlusion of venous flow and/or compromise of arterial flow,

there is a strong indication for systemic/catheter-directed

(endovascular) thrombolysis or percutaneous mechanical

thrombectomy to achieve venous patency [5]. Invasive

interventions for thrombolysis of iliofemoral thrombosis are

also technically possible in children aged 1-18 years and these

approaches may reduce the risk or severity of PTS compared

to standard anticoagulation alone [26]. Among the patients of

the present study, endovascular thrombectomy was performed

for one patient with iliofemoral thrombosis after unsuccessful

systemic thrombolysis and extension of the thrombus to the

contralateral extremity. However, thrombolysis with systemic

rtPA was evaluated in this study.

In vitro studies show that rtPA has more rapid clot lysis

activity and high affinity for fibrin, and it induces the binding

of plasminogen to fibrin; due to these characteristics, it is

recommended in newborns and children over other thrombolytics

[19,27]. In the present study, alteplase was used with a

half-life of 3-5 min; a short half-life provides bleeding control

by transient drug withdrawal or decreasing the dose. UFH

should be preferred for concomitant anticoagulation due to its

short half-life. As a general practice, thrombolysis is attempted

for vessel occlusions of up to 14 days due to the decreased

response in cases of older thrombi [5]. Similar to our study,

Ansah et al. [12] found no correlation between clot resolution

and period from diagnosis to rtPA administration. In their study,

the mean time from diagnosis to treatment was 36.0±16.8 h

versus 18.1±5.3 h for CCR and PCR/NR, respectively.

Study Limitations

There are limitations of this study due to its retrospective

multicenter nature and the heterogeneity of the patient

population. For some patients, thrombolysis was initiated 2 weeks

after thrombus presentation. Participating centers generally

had no standard protocols for thrombolysis. Only some centers

used concomitant heparin, either in therapeutic or prophylactic

doses. Imaging studies for diagnosis and monitoring were not

standardized. Data on serum fibrinogen, fibrin degradation

products, anti-factor Xa, prothrombin time, and activated

partial thromboplastin time were not presented in this study.

Thrombosis requiring thrombolysis is rare in children compared

to adults and each center was already trying to develop its own

experiences with thrombolysis during the 14-year study period.

Prospective multicenter pediatric studies must be planned using

standard protocols with the administration of continuous LD

rtPA for 6-72 h and HD for not more than 6 h at a time.

303



Conclusion

Systemic thrombolytic therapy with LD or HD rtPA may save

lives and organs effectively if it is used for the right indications

and at the right times in children with high-risk thrombosis. This

therapy should be applied by experienced hematologists under

close monitoring of recanalization and bleeding.

Acknowledgments

Thanks to all contributing centers and patients’ guardians for

sharing patient data. Thanks also to the steering committee

of the Turkish Pediatric Hematology Association for their help

in achieving communication and collaboration among the

participating pediatric hematology centers.

Ethics

Ethics Committee Approval: This retrospective study was

approved by the institutional ethics committee.

Informed Consent: Approval was obtained from the patients’

legal guardians at admission to the hospital for the release of

medical data for scientific purposes without including patient

identity.


Surgical and Medical Practices: E.Z., N.S., A.Y.E., I.O.A., M.S.E.,

N.Y., B.B., A.A., A.T.Y., T.H.K., A.M.G., S.A.G., H.Ö., L.O., B.B., H.G.,

G.Ö., M.F.O., Y.O., S.A., Ö.T., M.A., B.T.G., A.C., N.Ö.; Concept: E.Z.;

Data Collection or Processing: E.Z., A.Y.E., I.O.A., M.S.E., N.Y., B.B.,

A.A., A.T.Y., T.H.K., A.M.G., H.Ö., L.O., B.B., H.G., G.Ö., M.F.O., Y.O.,

S.A., Ö.T., M.A., B.T.G., A.C., N.Ö.; Analysis or Interpretation: E.Z.;

Literature Search: E.Z., N.S.; Writing: E.Z., N.S.


authors.



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305

Sönmez B. et al: Hematologic Effects of Radioactive Iodine Therapy

RESEARCH ARTICLE

DOI: 10.4274/tjh.galenos.2021.2021.0092


Assessment of Long-Term Hematologic Effects in Differentiated

Thyroid Cancer Patients Treated with Radioactive Iodine

Radyoaktif İyotla Tedavi Edilen Diferansiye Tiroid Kanserli Hastalarda Uzun Dönem

Hematolojik Etkilerin Değerlendirilmesi

Bircan Sönmez 1 , Özlen Bektaş 2 , Nergiz Erkut 2 , Mehmet Sönmez 2

1Karadeniz Technical University Faculty of Medicine, Department of Nuclear Medicine, Trabzon, Turkey

2Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey

Abstract

Objective: Radioactive iodine (RAI) therapy may cause hematologic

abnormalities. The aim of this study is to evaluate long-term

hematologic effects in differentiated thyroid cancer (DTC) patients

after RAI therapy.

Materials and Methods: A total of 1389 patients with DTC who were

treated with RAI were retrospectively evaluated. Complete blood cell

counts before RAI therapy and at last follow-up and hematologic

malignancy development were obtained from the electronic records.

Results: In the long-term analysis, thrombocytopenia and lymphopenia

were observed significantly in patients over 60 years of age.

Thrombocytopenia was observed more frequently in men. Leukopenia,

thrombocytopenia, and lymphopenia were observed significantly

with doses of >175 mCi. Thrombocytopenia and lymphopenia were

observed significantly with multiple dose administration. Higher

frequencies of anemia, thrombocytopenia, leukopenia, neutropenia,

and lymphopenia were found in patients with advanced-stage

disease. However, patients with advanced-stage disease had higher

doses and more multiple doses than patients with early-stage disease.

The rate of hematologic malignancy was found to be higher than in

the general population.

Conclusion: We suggest that cytopenia be surveyed more carefully in

patients older than 60 years of age. The most important risk factor for

lower platelets after RAI therapy is male gender. Clinically, the most

important predictor for cytopenia is advanced disease stage, which is

related to the combined effects of applied high dose activity, multiple

dose applications, and high tumor burden.

Keywords: Radioactive iodine, Thyroid cancer, Cytopenia, Hematologic

malignancy, Long-term hematologic effects, Thrombocytopenia,

Neutropenia

Öz

Amaç: Radyoaktif iyot (RAİ) tedavisi hematolojik anormalliklere yol

açabilir. Bu çalışmada, diferansiye tiroid kanserli (DTC) hastalarda RAİ

tedavisi sonrası uzun dönemli hematolojik etkilerin değerlendirilmesi

amaçlanmıştır.

Gereç ve Yöntemler: Bin üç yüz seksen dokuz RAİ ile tedavi edilmiş

DTC’li hasta retrospektif olarak değerlendirildi. Tedavi öncesi, son takip

ve hematolojik malignite geliştikten sonra ölçülen tam kan sayımları

elektronik kayıtlardan elde edildi.

Bulgular: Uzun dönemli analizde trombositopeni ve lenfopeni özellikle

60 yaş üstü bireylerde gözlendi. Trombositopeni erkek hastalarda

daha sıktı. Lökopeni, trombositopeni ve lenfopeni >175 mCi dozlarda

anlamlı oranda artmıştı. Trombositopeni ve lenfopeni çoklu doz

uygulaması olanlarda anlamlı oranda izlendi. Anemi, trombositopeni,

lökopeni, nötropeni ve lenfopeni, ileri evre hastalıklı hastalarda daha

sıktı. Ancak ileri evre hastalığa sahip hastalar, erken evrelilere göre

hem daha yüksek doz hem de çoklu doz tedavi almışlardı. Hematolojik

malignite oranı genel popülasyondan daha yüksek oranda idi.

Sonuç: Özellikle 60 yaş üstü hastalar sitopeniler açısından daha yakın

izlenmelidir. RAİ tedavi sonrası en önemli risk faktörü erkek cinsiyettir.

Klinik olarak sitopeniler için en önemli belirleyici ileri evre hastalıktır,

ki bu da, daha yüksek doz ve çoklu doz tedavi uygulanmış olmasının ve

daha fazla tümör yükünün kombine etkisi ile ilişkilidir.

Anahtar Sözcükler: Radyoaktif iyot, Tiroid kanseri, Sitopeni,

Hematolojik malignite, Uzun süreli hematolojik etkiler, Trombositopeni,

Nötropeni



Address for Correspondence/Yazışma Adresi: Özlen Bektaş, M.D., Karadeniz Technical University Faculty of Medicine,

Department of Hematology, Trabzon, Turkey

Phone : +90 532 543 05 75

E-mail : ozlenbektas@hotmail.com ORCID: orcid.org/0000-0002-2670-022X

Received/Geliş tarihi: February 2, 2021

Accepted/Kabul tarihi: March 22, 2021

306



Introduction

Radioactive iodine (RAI) therapy is a commonly used therapeutic

option for patients with differentiated thyroid cancer (DTC) for

ablation of residual thyroid tissue after thyroidectomy or for

the treatment of recurrent and metastatic disease [1]. RAI doses

of 30-100 mCi are used for ablation of residual thyroid tissue.

Higher and repeated doses (up to 200 mCi for single doses and

600 mCi for cumulative doses) can be applied for locoregional

recurrence or metastatic disease [1,2,3].

After RAI therapy, side effects such as sialadenitis, nasolacrimal

duct obstruction, keratoconjunctivitis, amenorrhea, and

hematologic abnormalities can be observed in the first few

months [4]. Most of these early side effects are usually temporary

and have little long-term clinical significance. However, several

meta-analyses have reported an increased incidence of second

primary malignancies (SPMs) in patients with DTC treated with

RAI [5,6,7].

Temporary anemia, leukopenia, and thrombocytopenia may

occur within the first month after a single RAI therapy [8].

Several studies reported improvement in complete blood cell

(CBC) counts at 6 months to 1 year after treatment [9,10],

while in other studies, the decrease in leukocytes [11], platelets

[8,11,12], and lymphocytes [12] persevered.

In our study, we aimed to evaluate the potential long-term

effect of RAI on the hematologic system in patients with DTC

who received RAI therapy.


A total of 1389 patients who were treated with RAI therapy after

total thyroidectomy during 2005-2018 were retrospectively

evaluated. Exclusion criteria were i) bone marrow infiltration,

ii) receiving external beam radiotherapy and/or chemotherapy

at any time, iii) concurrent or pretreatment hematologic

malignancies, and iv) patients developing solid cancer. The

inclusion criterion was the development of hematologic

malignancy after RAI therapy. At the time of the initial

treatment, all patients were staged according to the 8 th version

of the AJCC TNM classification. Each patient was categorized

according to receiving single or repeated doses of RAI therapy.

Radioiodine therapy were performed with fixed doses as follows:

<100 mCi (dose I) for remnant ablation, 125-150 mCi (dose II)

for those with lymph node metastasis, and >175 mCi (dose III)

for metastatic disease. Levothyroxine (L-T4) was discontinued

4-6 weeks and triiodothyronine (L-T3) was discontinued 2

weeks before RAI therapy. A low-iodine diet for 2 weeks was

recommended to all patients before RAI therapy. L-T4 treatment

was restarted 48 h after RAI therapy. Two or more doses were

applied to the patients with locoregional recurrence and/or

distant metastasis. CBC results before RAI therapy and at the

last follow-up and the development of hematologic malignancy

were obtained from the electronic records. The criteria for

anemia were hemoglobin of <12 g/dL in women and <13.0 g/dL

in men, while neutropenia was defined at a level of <1500/µL,

leukopenia at <4000/µL, thrombocytopenia at <150x10 9 /µL, and

lymphopenia at <1000/µL.

This study was approved by the Local Ethics Committee of

Karadeniz Technical University.


Data analysis was performed using SPSS 22.0 for Windows.

Descriptive statistics were shown as mean ± standard deviation

or median (minimum-maximum) for continuous variables and

as number of cases (%) for categorical variables. Significance

between baseline and last follow-up laboratory values was

analyzed with the paired samples t-test. Significance between

groups was analyzed with the Mann-Whitney U test when

the number of independent groups was two and with the

Kruskal-Wallis test when the group number was greater than

two. If there was a significant difference in the outcome of the

Mann-Whitney U or Kruskal-Wallis tests, post hoc tests were

used to determine the variables responsible for this significance.

Correlation between continuous variables was examined with

Spearman’s correlation test for non-parametric variables and

with Pearson’s correlation analysis for parametric variables.

Odds ratios were analyzed with binary and multinominal logistic

regression. Values of p<0.05 were considered statistically

significant.

Results

While 263 patients (18.9%) were male, 1126 (81.1%) were female.

Mean age was 47.44±12.52 (range: 8-82) years. Mean follow-up

period and time of last follow-up CBC results was 60.47±36.60

(range: 6.67-386.33) months. Baseline characteristics of patients

are summarized in Tables 1 and 2.

Upon comparing the differences between baseline and last

follow-up hematologic values, a significant decrease was

observed in hemoglobin and platelets at the last follow-up

(p=0.000, p=0.012). The change between baseline and last

follow-up lymphocyte values was significant when patients

with hematologic malignancies were excluded (p=0.002)

(Table 3).

A negative correlation was found between age and white blood

cell (WBC) count, neutrophils, and platelets (r=-0.094, p=0.000;

r=-0.093, p=0.001; r=-0.126, p=0.000, respectively) and a

positive correlation was found between age and hemoglobin

according to the last follow-up laboratory values of the patients

(r=0.076, p=0.004).

307



It was observed that platelets were significantly lower in

patients over 60 years of age (p<0.001). All series in women

except platelets and lymphocytes were found to be significantly

lower than in men. It was observed that lymphocytes decreased

significantly when the dose was increased. Dose 1 with dose

2 and dose 1 with dose 3 made the differences (p=0.001 and

p=0.008, respectively). Hemoglobin, platelets, and lymphocytes

also decreased as the stage increased. Stages I and IV for

hemoglobin, platelets, and lymphocytes (p=0.041, p=0.001,

p=0.006, respectively) and stages II and IV for hemoglobin

(p=0.046) made the differences (Table 4).

Table 1. Baseline characteristics of patients.

Patients’ characteristics Mean ± SD or n (%)

Age 47.44±12.5

<60 years 1150 (82.8%)

≥60 years 239 (17.2%)

Histology

Micropapillary 355 (25.6%)

Papillary 954 (68.7%)

Follicular 59 (4.2%)

Hurthle cell 8 (0.6%)

Poorly differentiated 6 (0.4%)

Follicular and papillary 5 (0.4%)

Hurthle cell and papillary 1 (0.1%)

Hyalinizing trabecular cancer 1 (0.1%)

Dose I (<100) 1150 (82.8%)

Dose II (125-150) 173 (12.5%)

Dose III (>175) 66 (4.8%)

Repeated RAI doses

1 1329 (95.7%)

2 49 (3.5%)

3 8 (0.6%)

4 3 (0.2%)

Staging

I 1224 (88.1%)

II 149 (10.7%)

IV 16 (1.2%)

Lymph node involvement 109 (7.8%)

Metastasis 20 (1.4%)

RAI: Radioactive iodine.

Before treatment, anemia was present in 223 patients

(16.1%), leukopenia in 33 patients (2.4%), thrombocytopenia

in 21 patients (1.5%), neutropenia in 10 patients (0.7%),

and lymphopenia in 20 patients (1.4%). When the last

follow-up cytopenia status of the patients was examined, it

was observed that rates of thrombocytopenia and lymphopenia

were significantly higher in patients over 60 years of age

(p=0.013, p=0.018, respectively). Males had thrombocytopenia

more often than females (p=0.006). Higher doses were a risk

factor for leukopenia, thrombocytopenia, and lymphopenia

and the risk increased with RAI of >175 mCi. The frequencies

of thrombocytopenia and lymphopenia increased with repeated

doses (p=0.003, p=0.003, respectively). Cytopenia was more

frequent in all 5 series in stage >II (stage IV for this study, since

there were no stage III patients in the study group) compared to

the earlier stages (Table 5).

Acute myeloid leukemia (AML) developed in 0.2% cases

(n=3), chronic lymphocytic leukemia (CLL) in 0.3% (n=4), and

myelodysplastic syndrome (MDS) in 0.1% (n=2) (Table 6). The

mean time until the development of hematologic malignancy

was 38.13±33.82 (range: 5.46-104.45) months. When

hematologic malignancies were evaluated according to their

prevalence in the world, the prevalence in our sample was found

to be higher than that of the general population.

Discussion

RAI therapy is one of the standard treatments for DTC. Orally

administered RAI diffuses into the circulation through the

gastrointestinal tract. The whole body is exposed to highly

energetic β- and γ-radiation during its transport to, accumulation

in, and destruction of thyroid tissue and the urinary excretion of

the RAI [13]. Cell renewal, apoptosis, and redistributions of the

hematopoietic cells are affected by this radiation [14].

In this study, we have evaluated the long-term hematologic

complications of RAI therapy in DTC patients. When the

differences between pretreatment and last follow-up laboratory

values were examined, it was observed that final hemoglobin

and platelets were significantly lower than the baseline levels

regardless of gender, applied activity, and number of applications,

which suggested that RAI therapy can decrease blood cell

levels regardless of cytopenia. There was no significant change

between the baseline and last follow-up lymphocyte values

Table 2. Applied doses according to stage.

Dose I Dose II Dose III 1 dose ≥1 dose

Stage I 1041 (85.5%) 145 (11.8%) 38 (3.1%) 1190 (97.2%) 34 (2.8%)

Stage II 109 (73.2%) 27 (18.1%) 13 (8.7%) 135 (90.6%) 14 (9.4%)

Stage III NA NA NA NA NA

Stage IV 0 (0%) 1 (6.3%) 15 (93.8%) 4 (25%) 12 (75%)

NA: Not applicable.

308



when patients with hematologic malignancies were included.

We considered that patients with CLL could have increased last

follow-up mean lymphocyte counts and repeated the statistical

analysis without patients with hematologic malignancies. It was

observed that lymphocyte counts were significantly lower when

hematologic malignancies were excluded (p=0.002). In the

literature, one of the most common complications in long-term

follow-up after RAI therapy is a decrease of platelets [11,15].

Lymphocytes are the most radiosensitive of all hematologic

cells [14]. Granulocytes/monocytes are also more sensitive than

erythroid series [13]. However, a decrease in the erythroid series

is an expected finding. Molinaro et al. [11] did not observe a

change in hemoglobin during 1 year of follow-up, while Schober

et al. [15] showed that thrombocytopenia and erythrocytopenia

were the most common types of cytopenia in a period of 65

months. Sönmez et al. [8] showed a significant decrease in

hemoglobin during 1 year of follow-up.

Table 3. Changes in hematologic parameters with RAI therapy.

Baseline

Last follow-up

Changes between baseline

and last follow-up values

Hemoglobin (g/dL) 13.50±1.5 (8.4-19.0) 13.3±1.4 (7.9-18.0) 0.18±1.22 <0.001

Platelets (10 3 /µL) 260.3±64.9 (16-733) 256.9±63.0 (31-710) 3.42±5.66 0.012

WBC (10 3 /µL) 6.89±1.79 (0.43-17.75) 6.98±3.62 (1.97-83.02) -0.10±3.57 0.284

Neutrophils (10 3 /µL) 4.0±1.40 (0.2-11.97) 3.942±1.43 (0-17.64) 0.06±1.51 0.141

Lymphocytes (10 3 /µL) 2.26±0.74 (0.17-14.38) 2.24±1.52(0-45.30) 0.02±1.33

RAI: Radioactive iodine; WBC: white blood cell count.

*When hematologic malignancies were excluded, mean baseline lymphocyte value was 2.25/µL, mean last follow-up lymphocyte value was 2.18/µL, and mean

difference between baseline and last follow-up was 0.63±0.76 (p=0.002).

p

0.570

0.002*

Table 4. Analysis of hematologic parameters at last follow-up.

Age

Hemoglobin (10 3 /µL) WBC (10 3 /µL) Platelets (10 3 /µL) Neutrophils (10 3 /µL) Lymphocytes (10 3 /µL)

<60 years 13.34±1.45 6.98±3.56 261.46±63.34 3.95±1.39 2.2±0.76

≥60 years 13.28±1.36 7.02±3.9 235.02±57.09 3.9±1.63 2.42±3.36

p 0.537 0.875 <0.001 0.636 0.305

Gender

Female 12.97±1.20 69.04±32.09 262.63±62.34 39.03±14.48 22.46±16.50

Male 14.85±1.35 73.48±50.00 232.38±60.38 41.06±13.52 22.05±0.74

p <0.001 0.008 <0.001 0.007 0.948

Doses

I 13.36±1.41 69.76±31.45 257.49±60.76 39.23±13.70 2.88±1.63

II 13.19±1.52 71.13±57.74 256.16±74.07 40.44±16.91 2.02±0.62

III 12.89±1.66 65.23±23.48 231.10±76.57 40.57±21.10 18.54±0.57

p 0.239 0.188 0.332 0.734 0.001 a , 0.008 b

Repeated doses

1 13.33±1.42 70.03±36.72 257.18±62.41 39.35±14.11 15.45±42.43

>1 13.35±1.81 66.67±21.09 250.83±76.80 40.90±18.42 19.07±6.06

p 0.798 0.181 0.468 0.778 0.003

Staging

I 13.32±1.44 70.36±37.93 258.46±61.71 39.52±14.12 22.62±16.01

II 13.46±1.26 66.77±16.90 249.72±67.86 38.60±14.55 21.02±6.06

III NA NA NA NA NA

IV 13.33±1.43 62.67±30.36 205.31±93.25 39.39±25.09 16.67±0.71

p 0.041 c , 0.046 d 0.179 0.001 c 0.389 0.006 c

WBC: White blood cell count; NA: not applicable.

a

: Significance occurred between doses 1 and 2; b : significance occurred between doses 1 and 3; c : significance occurred between stages I and IV; d : significance occurred between

stages II and IV.

309



Table 5. Analysis of cytopenia.

Age

Anemia Leukopenia Thrombocytopenia Neutropenia Lymphopenia

<60 years 188 (16.3%) 21 (1.8%) 20 (1.7%) 4 (0.3%) 13 (1.1%)

≥60 years 35 (14.6%) 5 (2.1%) 11 (4.6%) 3 (1.3%) 8 (3.3%)

OR (95% CI) NS NS 0.43 (0.19-0.98) NS 0.41 (0.15-1.12)

p NS NS 0.013 NS 0.018

Gender

Female 198 (17.6%) 21 (1.9%) 19 (1.7%) 6 (0.5%) 21 (1.9%)

Male 25 (9.5%) 5 (1.9%) 12 (4.6%) 1 (0.4%) 0 (0%)

OR (95% CI) 0.46 (0.32-1.72) NS 2.79 (1.34-5.82) NS NS

p 0.002 NS 0.006 NS NS

Doses

I 178 (15.5%) 18 (1.6%) 22 (1.9%) 5 (0.4%) 14 (1.2%)

II 40 (23.1%) 4 (2.3%) 4 (2.3%) 1 (0.6%) 3 (1.7%)

III 18 (27.3%) 4 (6.1%) 5 (7.6%) 1 (1.5%) 4 (6.1%)

OR (95% CI)

I vs. II

NS NS NS NS NS

p NS NS NS NS NS

OR (95% CI)

I vs. III

NS 4.06 (1.33-12.35) 4.20 (1.54-11.48) NS 5.23 (1.67-16.37)

p NS 0.014 a 0.005 a NS 0.004 a

OR (95% CI)

II vs. III

NS NS NS NS NS

p NS NS NS NS NS

Repeated doses

1 220 (16.6%) 23 (1.7%) 26 (2%) 7 (0.5%) 17 (1.3%)

>1 16 (26.7%) 3 (5%) 5 (8.3%) 0 (0%) 4 (6.7%)

OR (95% CI) NS NS 0.22 (0.80-0.59) NS 0.18 (0.06-0.56)

p NS NS 0.003 NS 0.003

Staging

I 206 (16.8%) 20 (1.6%) 23 (1.9%) 5 (0.4%) 15 (1.2%)

II 23 (15.4%) 2 (1.3%) 4 (2.7%) 0 (0%) 2 (1.3%)

III NA NA NA NA NA

IV 7 (43.8%) 4 (25%) 4 (25%) 2 (12.5%) 4 (25%)

OR (95% CI)

I vs. II

NS NS NS NS NS

p NS NS NS NS NS

OR (95% CI)

I vs. IV

4.10 (1.51-11.15) 20.07 (5.96-67.62) 17.41 (5.22-58.05)

34.83 (6.22-

194.96)

P 0.006 b <0.001 b <0.001 b <0.001 b <0.001 b

OR (95% CI)

II vs. IV

26.87 (7.77-92.92)

4.74 (1.59-14.10) 0.41 (0.01-0.25) 0.08 (0.02-0.37) NS 0.41 (0.01-0.25)

P 0.005 c <0.001 c 0.001 c NS <0.001 c

OR: Odds ratio; CI: confidence interval; NA: not applicable; NS: not significant.

a

: Significance occurred for dose III; b : significance occurred for stage IV; c : significance occurred for stage IV.

310



Table 6. Distribution of hematologic malignancies.

Age

AML CLL MDS

<60 years 2 1 2

≥60 years 1 3 0

Gender

Female 2 3 1

Male 1 1 1

Doses

I 2 4 1

II 1 0 0

III 0 0 1

Repeated doses

1 3 4 2

>1 0 0 0

Staging

I 3 4 1

II 0 0 0

III NA NA NA

IV 0 0 1

AML: Acute myeloid leukemia; CLL: chronic lymphocytic leukemia; MDS:

myelodysplastic syndrome.

We observed that hemoglobin levels were higher among

patients of older age. As age increases, the number of women

entering menopause also increases. Considering that women

constituted a significant majority of our cases (81%), this may

explain the hemoglobin increase. We found that platelet counts

were significantly lower and rates of clinical thrombocytopenia

and lymphopenia were significantly higher in patients over 60

years of age. A decrease in platelets with age after RAI has been

previously reported [16]. The decrease in bone marrow reserve

with age may explain the thrombocytopenia and lymphopenia.

We found gender-specific differences for certain types of cells.

Mean hemoglobin, WBC count, and neutrophils were lower in

women than in men. The reason for the difference in hemoglobin

is that the normal range of hemoglobin of women is lower than

that of men. When the presence of anemia was evaluated, no

difference was observed between men and women. However,

thrombocytopenia was observed more frequently in men than in

women, which indicates that platelets in men are more sensitive

to RAI therapy than in women. Prinsen et al. [16] reported more

frequent thrombocytopenia in men after RAI. Hu et al. [12]

showed in a study with 385 patients that there was a decrease

in WBC and lymphocyte counts without gender difference and

a greater decrease in platelets in women than in men during

the 6-month follow-up. Fewer cases and the shorter follow-up

period of that study may be the main reasons for the difference

from our results.

We observed that cytopenia occurred more frequently with

higher applied activity of RAI. Leukopenia, thrombocytopenia,

and lymphopenia occurred more frequently at RAI doses of

>175 mCi. A similar result was previously reported about

thrombocytopenia and excessive cumulative dose [16]. Padovani

et al. [17] showed low hemoglobin and platelets, especially at

> 250 mCi. When we evaluated the effects of multiple doses, we

observed that thrombocytopenia and lymphopenia were more

common.

In our study, platelet and lymphocyte counts were found to

be lower in patients with stage IV disease. Since there were no

patients with stage III in our study, an evaluation could not be

performed with that stage. Additionally, as the stage increased,

an increase in the frequency of cytopenia was detected in all

series. However, it must be noted that advanced-stage patients

received higher dose activities and more multiple-dose therapy.

It is known that after RAI treatment, the blood RAI concentration

shows a diphasic course. In the first 24-48 h, a rapid decrease

in inorganic 131 I in the blood is observed due to rapid clearance

by the kidneys, functional tumor tissue, and remaining thyroid

tissue. In the next 2-10 days, a protein-bound 131 I peak is

observed due to release by residual thyroid tissue and/or

functional tumor tissue. This can cause RAI to be carried in the

body for days [18]. Therefore, higher tumor burden may cause

large amounts of RAI to be released into the circulation with a

long duration of stay and, as a result, increased bone marrow

toxicity may occur. It has been reported that thrombocytopenia

is observed more frequently in individuals with large tumor

masses [16]. The strongest results in our study were obtained

for disease stage and cytopenia. We suggest that the clinical

effect of advanced disease stage on cytopenia was due to

the combined effect of three factors: higher applied activity,

multiple RAI administration, and higher tumor burden.

The incidence of AML is 1.6-2.8 cases per 100,000 in men and

1.0-2.2 cases per 100,000 in women [19], and the incidence

of CLL is approximately 4.2 cases per 100,000 people in the

world population [20]. Acute and chronic leukemias have been

reported after RAI therapy [18,21]. The incidence of leukemia

increases with >600 mCi activity, >45 Gy, and treatment with

short intervals [11,22]. Leukemia has been reported very rarely

at <300 mCi activity [11]. Cumulative dose was reported to

be the strongest risk factor for leukemia [22,23], aplastic

anemia, and MDS [23]. When hematologic malignancies

were evaluated according to their incidence in the world, the

incidence in our sample was found to be higher than that of

the general population. Regarding hematologic malignancies,

no relationship was observed with age; furthermore,

diagnoses of MDS, AML, and CLL were more frequent in the

low-dose RAI group including single dose administration and

early-stage disease. We did not observe the reported risk factors

for hematologic malignancies in our patient group. Although

311



RAI is known to be a risk factor for leukemia, SPMs cannot be

ruled out. Karaköse et al. [24] detected 70 SPMs in 1196 patients

with thyroid cancer. Thirty-two of them were diagnosed with a

second malignancy after thyroid cancer and 38 of them were

diagnosed with another malignancy before thyroid cancer.

RAI treatment was given to 25 patients in each group. SPM

independent of RAI was detected in 45 patients (3.8%) [24]. The

incidence of the malignancy in Turkey is 0.2%. As to be expected,

it was reported that the prevalence of SPM was increased by

20 times in patients with thyroid cancer [24]. Silva-Vieira et al.

[25] detected SPMs in 4.8% of patients with thyroid cancer who

did not receive RAI treatment. The risk of leukemia is higher

in patients with thyroid cancer, and especially in those treated

with RAI. The 5- to 10-year absolute leukemia development risk

is 0.23%-0.26% in patients with thyroid cancer treated with

RAI [26]. The results of our study are similar as the AML rate was

0.22% and CLL was 0.28% in patients receiving RAI.

Study Limitations

The strengths of this study are the long follow-up periods and

the large number of cases. The main limitation of our study is its

retrospective nature. As a result, we could not evaluate whether

the patients were using drugs that would affect hematologic

parameters or if they had a disease other than malignancy

affecting hematologic parameters during laboratory testing, or

menopause status in women. Additionally, patients’ cytopenia

status could not be evaluated by peripheral blood smear.

Conclusion

Thrombocytopenia and lymphopenia were observed more

frequently in patients older than 60 years of age and we

suggest that patients of this age group who receive RAI should

be surveyed particularly more carefully for these types of

cytopenia. We also observed that higher doses of RAI therapy,

multiple doses of RAI administration, and higher tumor burden

may cause CBC abnormalities and cytopenia. The most important

risk factor for lower platelet counts after RAI therapy was male

gender. Clinically, the most important predictor for cytopenia

is advanced disease stage, which is related to the combined

effects of applied high-dose activity, multiple dose applications,

and high tumor burden.

Ethics

Ethics Committee Approval: This study was approved by the

Local Ethics Committee of Karadeniz Technical University.


Data Collection or Processing: B.S.; Analysis or Interpretation: Ö.B.,

N.E., M.S.; Writing: Ö.B.


authors.



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313

Gündüz E. et al: Castleman Disease

PERSPECTIVE

DOI: 10.4274/tjh.galenos.2021.2021.0440


A Rare Lymphoproliferative Disease: Castleman Disease

Nadir Bir Lenfoproliferatif Hastalık: Castleman Hastalığı

Eren Gündüz 1 , Nihal Özdemir 2 , Şule Mine Bakanay 3 , Sema Karakuş 4

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

2İstinye University Medical School, Department of Hematology, İstanbul, Turkey

3Ankara Yıldırım Beyazıt University Medical School, Department of Hematology, Ankara, Turkey

4Başkent University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Castleman disease is a rare lymphoproliferative disease also known

as angiofollicular lymph node hyperplasia. It is classified as hyaline

vascular and plasmacytic variants histologically but characteristics

of both types can coexist. Most unicentric cases of the disease are

hyaline vascular while most multicentric cases are of the plasmacytic

type. Although the pathogenesis is not completely understood, the

role of interleukin (IL)-6 in unicentric disease and the roles of IL-6

and human herpes virus-8 in multicentric disease are well defined.

Unicentric disease is typically localized and symptoms are minimal

and treated locally. Multicentric disease is systemic and clinically

characterized by generalized lymphadenopathy, splenomegaly,

anemia, and systemic inflammatory symptoms. Systemic therapies

are primarily given. Several malignant diseases including lymphomas,

POEMS syndrome, follicular dendritic cell sarcomas, paraneoplastic

pemphigus, Kaposi sarcoma, and amyloidosis can be associated with

Castleman disease. In this paper, recent information about Castleman

disease, which is a rare disease, is summarized.

Keywords: Castleman disease, Diagnosis, Treatment

Öz

Castleman hastalığı, anjiyofolliküler lenf nodu hiperplazisi olarak da

bilinen nadir bir lenfoproliferatif hastalıktır. Histolojik olarak hiyalin

vasküler ve plazmasitik varyant olarak sınıflandırılır ancak nadiren

iki tipe ait özellikler bir arada bulunabilir. Unisentrik hastalığı olan

olguların çoğu hiyalin vasküler, multisentrik hastalığı olan olguların

çoğu ise plazma hücreli histolojik tipindedir. Patogenezi tam olarak

anlaşılmamıştır fakat unisentrik hastalıkta interlökin (IL)-6’nın,

multisentrik hastalıkta IL-6 ve human herpes virüs-8’in rolü iyi

tanımlanmıştır. Unisentrik hastalık tipik olarak lokalizedir, semptomlar

minimaldir ve tek başına lokal tedavi uygulanır. Multisentrik hastalık

sistemik bir hastalıktır ve klinik olarak yaygın lenfadenopati,

splenomegali, anemi ve sistemik inflamatuar semptomlarla

karakterizedir. Başlıca sistemik tedaviler uygulanır. Lenfomalar, POEMS

sendromu, folliküler dendritik hücreli sarkomlar, paraneoplastik

pemphigus, Kaposi sarkomu, amiloidoz Castleman hastalığı ile ilişkili

olabilir. Bu yazıda nadir bir hastalık olan Castleman hastalığı ile ilgili

güncel bilgiler özetlenmiştir.

Anahtar Sözcükler: Castleman hastalığı, Tanı, Tedavi

Introduction

Castleman disease (CD), also known as angiofollicular lymph

node hyperplasia and giant lymph node hyperplasia, was first

reported by Benjamin Castleman in 1954. It is a rare disease

diagnosed in 6600-7700 individuals each year in the United

States [1,2].

CD is classified as unicentric CD (UCD), involving a single lymph

node or a single region of nodes, and multicentric CD (MCD),

involving multiple lymphatic regions [3]. UCD is more common

[1] and has been reported to occur in younger individuals than

MCD [4,5,6,7,8]. MCD can occur in any region of the body and

has poorer prognosis.

MCD is further divided into three subgroups: human

herpes virus-8 (HHV-8)-associated MCD; polyneuropathy,

organomegaly, endocrinopathy, monoclonal gammopathy, and

skin changes (POEMS)-associated MCD; and idiopathic MCD

(iMCD) (Figure 1) [9].

Diagnosis

Standard investigations for CD usually begin with lymph

node biopsy followed by radiological investigation, preferably

with positron emission tomography/computed tomography

(PET/CT), complete blood count, serum chemistry, markers

of inflammation, serum cytokine levels, viral serology

for HHV-8 and human immunodeficiency virus (HIV),



Address for Correspondence/Yazışma Adresi: Eren Gündüz, M.D., Eskişehir Osmangazi University

Faculty of Medicine, Department of Hematology, Eskişehir, Turkey

E-mail : erengunduz26@gmail.com ORCID: orcid.org/0000-0002-7660-4092

Received/Geliş tarihi: July 31, 2021

Accepted/Kabul tarihi: October 25, 2021

314



protein electrophoresis, immunofixation, and quantitative

immunoglobulins [10,11].

The diagnostic criteria for iMCD and TAFRO syndrome,

explained below, are summarized in Table 1 [12]. Diagnosis of

HHV-8-associated MCD requires HHV-8 detection and

plasmablastic histopathologic findings on lymph node biopsy

[13]. POEMS-associated MCD is diagnosed if only one of the two

mandatory major criteria of polyneuropathy and monoclonal

plasma proliferative disorder is present with lymph node biopsy

diagnostic of CD [14].

Table 1. Diagnostic criteria for idiopathic multicentric Castleman

disease.

Major criteria

1. Lymph nodes with histopathologic features consistent with iMCD

spectrum

2. Enlarged lymph nodes (≥1 cm in short-axis diameter) in ≥2 lymph

node regions

Minor criteria

Laboratory

1. Elevated CRP or ESR

2. Anemia

3. Thrombocytopenia or thrombocytosis

4. Hypoalbuminemia

5. Renal dysfunction or proteinuria

6. Polyclonal hypergammaglobulinemia

Clinical

1. B symptoms

2. Hepatomegaly or splenomegaly

3. Fluid accumulation

4. Eruptive cherry hemangiomatosis

5. Violaceous papules

6. Lymphocytic interstitial pneumonitis

Supporting features

1. Elevated IL-6, VEGF, IgA, IgE, LDH, and/or B2M

2. Reticulin fibrosis of bone marrow

3. Disorders associated with iMCD: paraneoplastic pemphigus,

bronchiolitis obliterans organizing pneumonia, autoimmune

cytopenia, polyneuropathy, inflammatory myofibroblastic tumor

Exclusion criteria

1. Infection: HHV-8, EBV, CMV, toxoplasmosis, HIV, active

tuberculosis

2. Autoimmune/autoinflammatory: systemic lupus erythematosus,

rheumatoid arthritis, adult-onset Still disease, juvenile idiopathic

arthritis, autoimmune lymphoproliferative syndrome

3. Malignancy: lymphoma, multiple myeloma, POEMS syndrome,

primary lymph node plasmacytoma, follicular dendritic cell sarcoma

Diagnosis requires both major criteria and at least 2 of 11

minor criteria with 1 laboratory criterion

iMCD: Idiopathic multicentric Castleman disease; CRP: C-reactive protein; ESR:

erythrocyte sedimentation rate; IL-6: interleukin-6; VEGF: vascular endothelial growth

factor; IgA: immunoglobulin A; IgE: immunoglobulin E; LDH: lactate dehydrogenase;

B2M: beta-2 microglobulin; HHV-8: human herpesvirus-8; EBV: Epstein-Barr virus;

CMV: cytomegalovirus; HIV: human immunodeficiency virus; POEMS: polyneuropathy,

organomegaly, endocrinopathy, monoclonal protein, and skin changes.

Differential Diagnosis

Autoimmune diseases (immunoglobulin G4-related disease,

rheumatoid arthritis, systemic lupus erythematosus,

adult-onset Still disease), neoplastic disorders (lymphoma,

desmoid tumors, retroperitoneal sarcoma, paragangliomas,

sarcomas, hemangiopericytoma, bronchial adenoma,

neurofibroma, chest wall tumors, schwannoma), and infectious

disorders (HIV, Epstein-Barr virus [EBV], cytomegalovirus,

tuberculosis, toxoplasmosis) must be considered in the

differential diagnosis [3,14,15,16,17,18,19].

Pathogenesis

Excessive cytokine production underlies the pathogenesis of

CD. UCD and POEMS-associated MCD are caused by somatic

mutations in monoclonal stromal and plasma cells [20]. In

HHV-8-associated MCD, HHV-8 leads to a viral cytokine

storm driven by interleukin-6 (IL-6) [11,12,21,22]. The exact

mechanism of iMCD is unknown, but elevated IL-6 associated

with autoimmune mechanisms, ectopic cytokine secretion by

tumor cells, and/or viral signaling by a non-HHV-8 virus have

been proposed [23].

Pathology

The types of CD (hyaline vascular or hypervascular, plasmacytic,

and mixed) are characterized by distinctive lymphoid

architectural changes in all nodal compartments. The hyaline

vascular variant is the most common type of UCD. MCD is

predominantly of the plasmacytic variant with a few cases

showing plasmablastic characteristics (Table 2) [24].

Figure 1. Classification of Castleman disease.

POEMS: Polyneuropathy, organomegaly, endocrinopathy, monoclonal

protein, and skin changes; HHV-8: human herpesvirus-8; TAFRO:

thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly;

HIV: human immunodeficiency virus.

315



Clinical and Laboratory Features

UCD may be clinically silent and laboratory findings are

typically unremarkable. On the other hand, MCD presents

with diffuse lymphadenopathy, systemic inflammation, and

organ dysfunction [25]. Comorbid malignancies, lymphoma

in iMCD, and Kaposi sarcoma in HHV-8-associated MCD have

been described [26,27,28]. Patients with MCD may demonstrate

anemia, leukocytosis, thrombocytopenia, thrombocytosis,

elevated C-reactive protein, elevated IL-6, elevated erythrocyte

sedimentation rate, elevated IgG, hypoalbuminemia, renal

dysfunction, and elevated liver enzymes [1,26]. Clinical and

laboratory features of CD are summarized in Table 3.

Table 2. Histopathology of Castleman disease.

Histological subtypes

Frequency (%)

Unicentric

Hyaline vascular 75 <10

Mixed <10 45

Plasmacytic 20 45

Multicentric

Specific Presentations of Castleman Disease

Paraneoplastic Pemphigus

The presence of mouth ulceration is highly suggestive of

pemphigus and the severity of the disease correlates with lung

involvement. It is more frequent in the context of UCD [11].

POEMS Syndrome

POEMS syndrome refers to the presence of peripheral

neuropathy, organomegaly, endocrinopathy, monoclonal

gammopathy, and skin changes. Other frequent clinical findings

are papilledema, pleural effusions, ascites, sclerotic bone lesions,

and thrombocytosis [21].

TAFRO Syndrome

TAFRO syndrome corresponds to a subtype of iMCD characterized

by thrombocytopenia (T), anasarca (A), fever (F), reticulin

fibrosis (R), and organomegaly (O) [11]. The outcome may be

worse than in other cases of iMCD and no specific treatment has

Table 3. Clinical and laboratory features of Castleman disease.

Systemic symptoms (fever,

sweating, weight loss,

effusion, autoimmune,

respiratory)

Lymphadenopathy

UCD iMCD NOS iMCD TAFRO

+/-

None or

compression

Mostly central,

frequently bulky

++

And rarely peripheral

neuropathy

Peripheral and central,

usually small

+++

And anasarca

Peripheral and

central, usually small

POEMSassociated

MCD

++

Peripheral and

central

Organomegaly +/- ++ +++ +++ +++

Abnormal inflammation

markers (ESR, CRP,

cholinesterase, ferritin,

albumin)

Anemia, thrombocytopenia,

abnormal liver function tests

+/- +++

+/-

++

Sometimes

thrombocytosis

+++

Procalcitonin is also

elevated

+++ +/-

++ +++

Hypergammaglobulinemia +/- +++ +/- +, small M spike +++

Renal dysfunction - +

Autoimmune phenomena

Rare, but

paraneoplastic

pemphigus can

be seen

++

Autoimmune hemolytic

anemia, paraneoplastic

pemphigus, immune

thrombocytopenia,

interstitial lung disease

+++

Intravascular

coagulation and

fibrinolysis

+/- +/-

+ ++

HHV-8-associated

MCD

+++

And Kaposi sarcoma

Peripheral and

central, usually small

+++

HHV-8 DNA is

detected in plasma

DAT positivity 46%,

monoclonal

gammopathy 28%

Clinical course Benign Variable Very aggressive Aggressive Aggressive

Lymphoma risk + + +/- +/- ++

UCD: Unicentric Castleman disease; iMCD NOS: idiopathic multicentric Castleman disease-not otherwise specified; iMCD TAFRO: idiopathic multicentric Castleman diseasethrombocytopenia,

anasarca, fever, reticulin fibrosis, and organomegaly; POEMS MCD: multicentric Castleman disease-polyneuropathy, organomegaly, endocrinopathy, monoclonal

gammopathy, and skin changes; HHV-8: human herpes virus-8; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; DAT: direct antiglobulin test.

316



been identified [22]. Diagnostic criteria for TAFRO syndrome are

summarized in Table 4.

Hemophagocytic Lymphohistiocytosis

MCD and especially HHV-8-related MCD may be characterized by

hemophagocytic lymphohistiocytosis at the initial presentation

or upon relapse [25,26].

Autoimmune Cytopenia

Autoimmune hemolytic anemia is a relatively frequent

complication of MCD. Immune thrombocytopenia has been

reported in 5% to 20% of MCD cases [25,27,28].

Table 4. Diagnostic criteria for TAFRO syndrome.

Criteria

Histopathological criteria (all must be met)

Typical lymph node pathology

LANA-1 negative for HHV-8

Major criteria (3 of 5 must be met)

Thrombocytopenia (<100000/µL)

Anasarca (pleural effusion and ascites in computed tomography)

Fever (>38 °C)

Reticulin fibrosis

Organomegaly

Minor criteria (at least 1)

Hyper/normoplasia in megakaryocytes

Elevated alkaline phosphatase without significant transaminase

elevation

TAFRO: Thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly;

LANA-1: latency-associated nuclear antigen-1; HHV-8: human herpes virus-8.

Peripheral Neuropathy

Demyelinating peripheral neuropathy is frequently observed

with CD. There is no clear association between the severity of

the peripheral neuropathy and the subtype of CD [29].

Renal Involvement

Renal involvement is frequently observed in MCD, mainly in the

plasmacytic and mixed subtypes, being reported in up to 25% of

MCD cases. Glomerular lesions, AA amyloidosis, and interstitial

nephritis are the most common renal pathology findings [30].

Treatment

Surgical resection provides radical treat ment for the majority

of patients with UCD. Radiotherapy is an important alternative

when surgical resection is contraindi cated or technically

difficult. Other treatment options are embolization, rituximab,

or siltuximab/tocilizumab in the event of inflammation [23].

Treatment of MCD still remains complex because MCD is a rare

clinical entity and there is a lack of randomized controlled

trials. Multiple therapeutic approaches have been used,

including conventional cytotoxic chemotherapy (single-agent

or combined), antiviral treatment, glucocorticoids, thalidomide,

interferon-alpha, and molecular targeted therapies. Determination

of HHV-8 status is also important [10]. Therapeutic

approaches for MCD are listed in Table 5.

The use of prednisone or other glucocorticosteroids will frequently

ameliorate symptoms, partially improve lymphadenopathy,

and correct laboratory abnormalities. However, the impact is

generally temporary. Lasting remissions are rare and the disease

Table 5. Therapeutic approaches for multicentric Castleman disease.

Disease

First-line treatment

Second-line

treatment and

beyond

Idiopathic

MCD

Siltuximab

Tocilizumab

Glucocorticoids

Rituximab

Cyclosporine

Sirolimus

IVIG

Thalidomide

Lenalidomide

Bortezomib

R-CVP, R-CHOP

Autologous stem cell transplantation

POEMS-associated MCD

iMCD-like treatment if bone lesion

absent

Myeloma like treatment if bone lesion

present

As mentioned above

HHV-8 associated

MCD

Combined antiretroviral treatment if

HIV-positive

Rituximab

Etoposide

Liposomal doxorubicin

Interferon

Antiviral treatment

MCD: Multicentric Castleman disease; IVIG: intravenous immunoglobulin; R: rituximab; CVP: cyclophosphamide, vincristine, prednisolone; CHOP: cyclophosphamide, doxorubicin,

vincristine, prednisolone; iMCD: idiopathic multicentric Castleman disease; HHV-8: human herpesvirus-8; POEMS: polyneuropathy, organomegaly, endocrinopathy, monoclonal

protein, and skin changes; HIV: human immunodeficiency virus.

317



may require the long-term use of corticosteroids, increasing the

risk of bacterial infections [31,32]. The use of corticosteroids

alone may be better reserved as a temporary intervention in

acute situations where more definitive therapy has not yet been

decided or will be delayed [24].

Currently, chemotherapy is the first option for most symptomatic

patients. However, data are insufficient to favor one treatment

for all patients. Oral chlorambucil and cyclophosphamide have

been effective and are generally well tolerated [23,31,33].

Vinblastine [10] and oral etoposide [13] may also have activity.

Therapy with a single alkylating agent may be most appropriate

for fragile patients or cases in which a prompt response is not

required.

Combination chemotherapy regimens such as cyclophosphamide,

vincristine, and prednisone or cyclophosphamide, doxorubicin,

vincristine, and prednisone have significant activity [10,14,31].

When combination chemotherapy is used, patients need to be

closely monitored because of the increased risk of infection.

Patients with HIV-associated CD may be at especially high risk

for complications with standard combination chemotherapy

[34]. A treatment algorithm is shown in Figure 2 [34,35].

Figure 2. Treatment algorithm for idiopathic multicentric Castleman disease.

ECOG: Eastern Cooperative Oncology Group.

318



Conclusion

CD is a rare lymphoproliferative disease that can mimic many

malignant and nonmalignant conditions. Lymph node biopsy

is essential to establish a definitive diagnosis and greater

awareness of the disease among clinicians would facilitate early

diagnosis.


Concept: E.G., N.Ö., Ş.M.B., S.K.; Design: E.G., N.Ö., Ş.M.B., S.K.;

Literature Search: E.G.; Writing: E.G.


authors.



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320

Omma A. et al: Convalescent Plasma Reduces Antibodies in COVID-19

BRIEF REPORT

DOI: 10.4274/tjh.galenos.2021.2021.0277


Convalescent Plasma Reduces Endogenous Antibody Response in

COVID-19: A Retrospective Cross-Sectional Study

Konvalesan Plazma COVID-19’da Endojen Antikor Yanıtını Azaltır: Retrospektif Kesitsel Bir

Çalışma

Ahmet Omma 1 , Abdulsamet Erden 1 , Serdar Can Güven 1 , İhsan Ateş 2 , Orhan Küçükşahin 3

1Ministry of Health Ankara City Hospital, Department of Internal Medicine, Division of Rheumatology, Ankara, Turkey

2Ministry of Health Ankara City Hospital, Department of Internal Medicine, Ankara, Turkey

3Yıldırım Bayezıt University Faculty of Medicine, Department of Internal Medicine, Division of Rheumatology, Ankara, Turkey

Abstract

Objective: The aim of this study is to investigate post-COVID-19

antibody titers in patients who received convalescent plasma (CP) in

addition to standard-of-care treatment.

Materials and Methods: Hospitalized COVID-19 patients who received

CP in addition to standard care were retrospectively investigated.

Patients who received CP with a recorded total COVID-19 antibody

test result after treatment were included. From among hospitalized

COVID-19 patients who received only standard care with a recorded

total COVID-19 antibody test result, a control group matched for age,

gender, and comorbidities was formed. Total COVID-19 antibody index

levels were compared.

Results: Thirty-three CP recipients were enrolled in the study. The

control group consisted of 34 age-, gender-, and comorbiditymatched

standard-care patients. Median total COVID-19 antibody

index levels were significantly reduced in the CP group.

Conclusion: Although CP therapy may have benefits for disease

outcome, its potential ability to hamper long-term immunity may be

a problem.

Keywords: COVID-19, Convalescent plasma, Antibody, Immunity

Öz

Amaç: Çalışmanın amacı tedavi sürecinde standart tedavilere ek olarak

konvalesan plazma (KP) tedavisi uygulanan COVID-19 hastalarında

takipte oluşan COVID-19 antikor düzeylerini incelemektir.

Gereç ve Yöntemler: Yatarak tedavi alan COVID-19 hastaları içinde

standart tedavilere ek olarak KP tedavisi alanlar retrospektif olarak

incelenmiştir ve takipte COVID-19 antikor düzeyleri bakılmış olanlar

çalışmaya dahil edilmiştir. Aynı zaman zarfında yatarak takip edilen,

standart tedavi alan ve takipte COVID-19 antikor düzeyi bakılmış

olan COVID-19 hastaları arasından yaş, cinsiyet ve komorbidite sıklığı

eşleştirilmiş bir kontrol grubu oluşturulmuştur. İki grup arasında

COVID-19 antikor düzeyleri karşılaştırılmıştır.

Bulgular: KP tedavisi alan ve takipte antikor düzeyleri bakılmış olan

33 COVID-19 hastası çalışmaya dahil edildi. Kontrol grubunda 34 hasta

mevcuttu. Median total COVID-19 antikor indeks düzeyleri standart

yaklaşıma ek olarak KP alan grupta anlamlı olarak düşük saptandı.

Sonuç: KP tedavisinin COVID-19 hastalarında sonuçlar üzerine olumlu

etkileri görülmekle beraber hastalık sonrasında azalmış antikor yanıtı,

uzun dönem bağışıklık üzerine olumsuz etkilerin bir göstergesi olabilir.

Anahtar Sözcükler: COVID-19, Konvelesan plazma, Antikor, Bağışıklık

Introduction

Convalescent plasma (CP) therapy is the transfusion of plasma

containing polyclonal antiviral antibodies, obtained from

recently ill donors who have fully recovered with sufficient

antibody responses. Potential mechanisms of action for CP are

virus neutralization, antibody-dependent virolysis, antibodydependent

antigen presentation, antibody-dependent cellular

toxicity, and complement activation [1]. Enhancement of viral

clearance is the foremost effect expected from CP therapy;

therefore, administration in the early stages of the infection with

high viral load and insufficient endogenous immunoglobulin

(Ig) response may be more beneficial [2,3]. CP has previously

been used for prophylaxis after contact in viral hepatitis,

mumps, measles, and polio and is used as a therapeutic agent for

influenza, severe acute respiratory syndrome (SARS), and Middle

East respiratory syndrome (MERS) [4,5,6,7,8,9,10]. Likewise, the

effectiveness of CP therapy with early administration has been



Address for Correspondence/Yazışma Adresi: Serdar Can Güven, M.D., Ministry of Health

Ankara City Hospital, Department of Internal Medicine, Division of Rheumatology, Ankara, Turkey

Phone : +90 533 711 29 51

E-mail : drserdarguven@gmail.com ORCID: orcid.org/0000-0003-1965-9756

Received/Geliş tarihi: April 30, 2021

Accepted/Kabul tarihi: July 25, 2021

321



demonstrated for coronavirus disease 2019 (COVID-19). In a

retrospective cohort study based on the US national registry,

the unadjusted mortality within 30 days after CP therapy was

lower among patients who received a transfusion within 3 days

after receiving a diagnosis of COVID-19 than among those who

received a transfusion 4 or more days after the diagnosis [11].

In addition, Libster et al. [12] demonstrated reduced progression

to severe respiratory disease with early CP administration.

Contradictory results regarding the efficacy of CP in COVID-19

also exist [13].

Endogenous antibodies produced by the host in COVID-19

have protective effects against reinfection. Although early

administration of CP seems to have beneficial effects on

outcomes in COVID-19, it is unclear whether CP therapy alters

the endogenous antibody production and hampers long-term

humoral immunity against the virus. In this study, we aim to

investigate post-COVID antibody titers in patients who received

CP in addition to standard-of-care (SOC) treatment and compare

them to those of patients who received only SOC treatment.


Study Design

This study was conducted as a single-center, retrospective, casecontrol

study. Ethical approval of the study was obtained from

the Ethics Committee of Ankara City Hospital.

Patients

Hospitalized COVID-19 patients who received CP therapy in

addition to the SOC approach from Ankara City Hospital’s

Internal Medicine Inpatient Clinic between August 15 and

December 31, 2020, were retrospectively investigated. The

COVID-19 diagnosis was confirmed with the presence of a

recorded positive SARS-CoV-2 real-time reverse transcription

polymerase chain reaction (RT-PCR) test from a nasopharyngeal

swab for every patient. Among patients with positive PCR

results, subjects who received a total of at least 400 mL of

CP obtained from donors (200-250 mL administered on two

consecutive days or two alternate days) with recorded test

results for total COVID-19 antibody against the S1 antigen

(Siemens Atellica-IM Total [COV2T]) after PCR positivity were

included in the study. Index values of COVID-19 total Ig over

1 are accepted as positive for this kit. Our center reported

all values over 10 as >10; therefore, patients with Ig levels

of >10 were recorded as having levels of 10. Data regarding

demographics and comorbidities were recorded for all CP

recipients. From among hospitalized COVID-19 patients who

received only SOC treatment during the same period of

time with recorded total COVID-19 antibody test results, an

age-, gender-, and comorbidity-matched control group was

formed.

Interventions

The SOC approach comprised oxygen support, hydroxychloroquine,

favipiravir, low-molecular-weight heparin, anticoagulants,

and additional anti-inflammatory treatment when indicated

in accordance with the COVID-19 guidelines of the Turkish

Ministry of Health [14]. Likewise, indications for hospitalization,

CP therapy administration, intubation, and discharge were also

set in accordance with the Turkish Ministry of Health guidelines

[14].

Outcomes

Total COVID-19 antibody index levels and days from symptom

onset at the time of COVID-19 antibody work-up were recorded

for both groups.


Statistical analyses were performed using SPSS 22 (IBM Corp.,

Armonk, NY, USA). The normality of variables was investigated

by Shapiro-Wilk test. Continuous variables were presented as

median and interquartile range (IQR). Categorical variables were

presented as number and percentage. The Mann-Whitney U

test was used for comparison of continuous variables according

to normality. For comparisons of categorical variables, the

chi-square test was used. Values of p<0.05 were considered

statistically significant.

Results

Of 67 recipients of CP in addition to SOC treatment, 33 patients

with total COVID-19 antibody test results were enrolled in

this study. The control group consisted of 34 age-, gender-,

and comorbidity-matched SOC patients with total COVID-19

antibody test results. Demographics, comorbid diseases,

duration of symptoms at the time of COVID-19 antibody workup,

and total COVID-19 antibody titers are presented in Table 1.

No significant differences were observed in the demographics

or frequency of comorbid diseases between groups. Days from

symptom onset at the time of antibody work-up was 28.5

(29.75) in the SOC group and 25 (12.5) in the SOC + CP group

[median (IQR), p=0.292]. In the SOC + CP group, the interval

between CP administration and antibody work-up was 21 (12.5)

days [median (IQR)]. Median (IQR) total COVID-19 Ig levels were

significantly reduced in the CP group (Table 1).

Discussion

Our results demonstrate a significantly reduced total COVID-19

antibody response in CP recipients.

CP is obtained from recovered COVID-19 patients who

have developed humoral immunity, containing neutralizing

antibodies (NAbs) for severe acute respiratory syndrome

coronavirus 2 (SARS-CoV-2) capable of pathogen clearance

322



Table 1. Demographics, frequency of comorbid diseases, days from symptom onset at the time of COVID-19 antibody work-up,

and total COVID-19 antibody index levels in patient groups.

Standard-ofcare

group

(n=34)

Convalescent plasma plus

standard-of-care group

(n=33)

Gender, male, n (%) 25 (73.5) 23 (69.7) 0.728

Age, years, median (IQR) 54.50 (25) 52 (17) 0.730

Presence of any comorbid disease, n (%) 22 (64.7) 14 (42.4) 0.067

Hypertension, n (%) 12 (35.3) 10 (30.3) 0.664

Diabetes, n (%) 8 (23.5) 6 (18.2) 0.590

Asthma or COPD, n (%) 1 (2.9) 1 (3) 0.983

CHD, n (%) 6 (17.6) 3 (9.1) 0.305

Total COVID-19 antibody index levels, median (IQR) 10 (0.06) 7.71 (7.71) 0.023

Days from symptom onset at the time of COVID-19 antibody work-up, median (IQR) 28.5 (29.75) 25 (12.5) 0.292

n: Number, IQR: interquartile range, COPD: chronic obstructive pulmonary disease, CHD: chronic heart disease, COVID-19: coronavirus disease 2019.

p

from peripheral circulation and pulmonary tissues [15].

NAbs particularly bind to the S1-receptor binding domain

(S1-RBD) of the S protein, which binds to angiotensin-converting

enzyme receptors, preventing the entrance of the virus into

the cells. Furthermore, CP contains various IgG and IgM type

non-neutralizing antibodies (non-NAbs), similar to fresh frozen

plasma. In addition to enhancing viral clearance, both NAbs

and non-NAbs have immunomodulatory effects via limiting

immune complex formation and complement cascade activation

[16,17]. Furthermore, both NAbs and non-NAbs in CP reduce

innate immune activation and regulate the activation of these

cells downstream of the Fcγ receptors in B lymphocytes and

antigen-presenting cells. Again, by regulating T lymphocyte

interactions of these cells, they cause humoral tolerance against

SARS-CoV-2 and reduce antibody formation [18]. Theoretically,

CP administration during the active infection period may suppress

the endogenous antibody response due to these aforementioned

effects on both B lymphocytes and innate immunity. Our results

have demonstrated reduced levels of COVID-19 antibodies in CP

recipients after a median of 28.5 days from symptom onset in

the SOC group and 25 days in the CP group. Since CP is generally

administered within the first week of symptoms and the IgG

half-life in circulation is 10 to 21 days, we may assume that

even with the presence of considerable exogenous COVID-19

antibodies in circulation, the index COVID-19 antibody levels

were still lower in the CP group, possibly indicating deterioration

in endogenous antibody production [19].

CP therapy may enhance viral clearance and provide better

disease outcomes, particularly when administered in early

stages of infection [12,20]. However, an altered long-term

humoral immunity due to suppression of endogenous antibody

production may be speculated as a risk of CP therapy. Therefore,

it should not be overlooked that after immunoglobulins in CP

are metabolized by the host, an absence of immunological

memory for SARS-CoV-2 may occur [21].

Study Limitations

The small sample size and retrospective nature of this study

were the major limitations. Disease severity at admission or

onset of CP treatment was not evaluated. Furthermore, there

may have been undetected variations in Ig levels of CP solutions

since Ig levels were not measured. Finally, our center reported

COVID-19 total Ig values over 10 as >10; therefore, patients

with higher Ig levels were not evaluated precisely, which would

have further reflected the altering effects of CP on endogenous

Ig production. Nevertheless, to our best knowledge, this is the

first study to evaluate effects of CP therapy on endogenous

antibody production in COVID-19.

Conclusion

In conclusion, although CP therapy may have benefits for

disease outcomes, its potential to hamper long-term immunity

and increase the risk of reinfection may be a problem, since the

pandemic is still far from being under control globally.


Concept: A.O., A.E., S.C.G., İ.A., O.K.; Design: A.O., A.E., S.C.G., İ.A., O.K.;

Data Collection or Processing: A.O., A.E., S.C.G., İ.A., O.K.; Analysis

or Interpretation: A.O., A.E., S.C.G., İ.A., O.K.; Literature Search: A.O.,

A.E., S.C.G., İ.A., O.K.; Writing: A.O., A.E., S.C.G., İ.A., O.K.


authors.



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Kang ES, Cho D, Müller MA, Drosten C, Kang CI, Chung DR, Song JH, Peck

KR. Challenges of convalescence plasma infusion therapy in Middle East

respiratory coronavirus infection: a single centre experience. Antivir Ther

2018;23:617-622.

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Lai KY, Koo CK, Buckley T, Chow FL, Wong KK, Chan HS, Ching CK, Tang

BS, Lau CC, Li IW, Liu SH, Chan KH, Lin CK, Yuen KY. Convalescent plasma

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324

Li M. et al: A Rare Case of Lymphoblastic Leukemia

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.galenos.2021.2020.0503


Simultaneous Presentation of Hairy Cell Leukemia and Acute

Lymphoblastic Leukemia

Tüylü Hücreli Lösemi ve Akut Lenfoblastik Löseminin Eş Zamanlı Tanısı

Mingyong Li 1 , Yuan He 1 , Kang Jiang 2 , Juan Zhang 2

1Sichuan Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu, China

2Shimian People’s Hospital, Ya’an, China

Figure 1. Peripheral and bone marrow smear revealed cells with cytoplasmic projections (top left corner, 1000 x ) and primitive cells (left

upper diagonal line, 1000 x ). These cells were negative for peroxidase staining (right bottom of the top left picture, 1000 x ). Flow cytometry

of the marrow confirmed two clonal B-cell populations (top right and bottom row; see text for details).



Address for Correspondence/Yazışma Adresi: Juan Zhang, M.D., Shimian People’s Hospital, Ya’an, China

Phone : +86 18080928906

E-mail : zhangjuan82@126.com ORCID: orcid.org/0000-0002-7396-2097

Received/Geliş tarihi: August 21, 2020

Accepted/Kabul tarihi: September 29, 2020

325

Li M. et al: A Rare Case of Lymphoblastic Leukemia


A 59-year-old man was admitted to the hospital because of

repeated systemic bone pain for more than 1 month. His complete

blood count revealed hemoglobin of 54 g/L, white blood cells of

12.35x10 9 /L, neutrophils of 0.247x10 9 /L, and a platelet count of

34x10 9 /L. Peripheral and bone marrow smears revealed cells with

cytoplasmic projections (Figure 1, top left corner) and primitive

cells (left upper diagonal line). These cells were negative for

peroxidase staining (right bottom of the top left picture). Flow

cytometry of marrow confirmed two clonal B-cell populations

(top right): the first (red) was CD34 + CD10 + CD19 + cCD79a +

(bottom left) and HLA-DR + D33 + CD38 + (dim)CD2 - CD7 - CD13 -

CD14 - CD15 - CD20 - CD56 - CD117 - cIgM - cMPO - cCD3 - (not shown),

diagnostic of B-lineage acute lymphoblastic leukemia (B-ALL).

A second population (purplish-red) was CD103 + CD11c + (bottom

right), CD19 + CD25 + CD123 + CD22 + CD20 + sIgM + CD23 + CD5 - CD10 -

CD38 - FMC7 - slambda - (not shown), and skappa light chain +

(bottom right), representing hairy cell leukemia (HCL). The BRAF

V600E mutation was detected in the bone marrow aspirate

sample. Therefore, the patient was diagnosed with simultaneous

B-ALL and classic HCL. The association of ALL and HCL, either

synchronous or metachronous, has rarely been reported [1].

In such cases, immunophenotyping with multiparameter flow

cytometry is useful. This case highlights the indolent course of

HCL, which can coexist with the acute process of ALL.

Keywords: Hairy cell leukemia, Acute lymphoblastic leukemia,

Anemia

Anahtar Sözcükler: Tüylü hücreli lösemi, Akut lenfoblastik

lösemi, Anemi


Data Collection or Processing: Y.H.; Analysis or

Interpretation: M.L.; Literature Search: K.J.; Writing: J.Z.


authors.

Financial Disclosure: This work was supported by grants from

the National Natural Science Foundation of China (81802075).

Reference

1. Francischetti IMB, Calvo KR. Hairy cell leukemia coexistent with chronic

lymphocytic leukemia. Blood 2019;133:1264.

326

Mallik N. and Sachdeva M.U.S: Bone Marrow Oxalosis

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.galenos.2021.2020.0557


Bone Marrow Oxalosis: Crystal Flowers in the Bone Marrow Garden

Kemik İliği Oksalozisi: Kemik İliği Bahçesinde Kristal Çiçekler

Nabhajit Mallik,

Man Updesh Singh Sachdeva

Postgraduate Institute of Medical Education and Research, Department of Hematology, Chandigarh, India

Figure 1. Bilateral bone biopsy core showed replacement of bone marrow with extensive interstitial and paratrabecular deposition of

crystals accompanied by fibrosis (a, 100 x ; b and c, 400 x ). Crystals were translucent and rod-shaped, arranged in a rosette-like pattern, and

birefringent under polarized light, consistent with calcium oxalate crystals (d and e, 100 x ; f, 400 x ).

A 20-year-old male with a known case of chronic kidney disease

had been diagnosed with nephrolithiasis at the age of 8 months.

Since then, he had recurrent renal stones leading to end-stage

renal disease and was scheduled for a renal transplant. A

computed tomography scan showed bilateral small kidneys with

right-sided nephrolithiasis. Complete blood counts revealed

hemoglobin level of 70 g/L, white blood cell count of 8.1x10 9 /L,

and platelet count of 395x10 9 /L. Bilateral bone marrow biopsies

were performed to investigate the cause of persistent anemia,

and one of the cores showed replacement of bone marrow with

extensive interstitial and paratrabecular deposition of crystals

accompanied by fibrosis (Figures 1a, 100 x , and 1b-1c, 400 x ). The

crystals appeared translucent and rod-shaped, and they were

arranged in a rosette-like pattern. They were birefringent under



Address for Correspondence/Yazışma Adresi: Man Updesh Singh Sachdeva, M.D., Postgraduate Institute of

Medical Education and Research, Department of Hematology, Chandigarh, India

Phone : 0091-9815802080

E-mail : drmanupdeshpgi@yahoo.co.in ORCID: orcid.org/0000-0002-4406-8299

Received/Geliş tarihi: September 11, 2020


327

Mallik N. and Sachdeva M.U.S: Bone Marrow Oxalosis


polarized light, consistent with calcium oxalate crystals (Figures

1d-1e, 100 x , and 1f, 400 x ).

Systemic oxalosis results in the deposition of calcium oxalate

crystals mainly in the myocardium, cardiac conduction system,

kidneys, bones, or bone marrow [1]. This case demonstrates

that although bone marrow examination may not be a routine

modality for the diagnosis of hyperoxaluria, it should definitely

be considered in young patients with renal failure and childhood

recurrent nephrolithiasis who present with cytopenia/refractory

anemia.

Keywords: Oxalosis, Crystals, Bone marrow

Anahtar Sözcükler: Oksalozis, Kristaller, Kemik iliği


Concept: N.M., M.U.S.S.; Design: N.M., M.U.S.S.; Data Collection

or Processing: N.M., M.U.S.S.; Analysis or Interpretation: N.M.,

M.U.S.S.; Literature Search: N.M., M.U.S.S.; Writing: N.M.,

M.U.S.S.


authors.



Reference

1. Sriram K, Kekre NS, Gopalakrishnan G. Primary hyperoxaluria and systemic

oxalosis. Indian J Urol 2007;23:79-80.

328

LETTERS TO THE EDITOR


Hematological Findings and Clinical Severity in Pediatric Patients

with COVID-19

Pediatrik Hastalarda COVID-19’da Hematolojik Bulgular ve Klinik Ciddiyet

Pathum Sookaromdee 1 , Viroj Wiwanitkit 2

1Private Academic Consultant, Bangkok, Thailand

2Patil University, Pune, India

To the Editor,

We would like to share some ideas on “Can Hematological

Findings of COVID-19 in Pediatric Patients Guide Physicians

Regarding Clinical Severity?” [1]. Arıkan et al. [1] concluded

that the “neutrophil-to-lymphocyte ratio (NLR) was higher in

severe/critical cases… Red cell distribution width (RDW)

statistically significantly increased in severe cases”. We agree that

basic hematological parameters might be good indicators for

monitoring the severity of many medical problems. Regarding

coronavirus disease-19 (COVID-19), NLR and RDW might or

might not be useful as predictive parameters for severity in

children with COVID-19. RDW might not be useful in many

settings. In our setting in Southeast Asia, there is a very high

incidence of thalassemia. There are also many pediatric cases

of COVID-19. Since these children usually have high RDW as a

background hematological finding [2], RDW is not useful for

the prediction of COVID-19 severity in these cases. Therefore,

RDW is not a useful predictive parameter in any setting with

a high incidence of thalassemia. The NLR ratio might be a

better predictive parameter. However, it is necessary to set a

population-specific reference value for clinical application [3].

Keywords: Hematology, Severity, COVID-19, Pediatric

Anahtar Sözcükler: Hematoloji, Ciddiyet, COVID-19, Pediatrik

Informed Consent: Not applicable.


Concept: P.S., V.W.; Design: P.S., V.W.; Data Collection or

Processing: P.S., V.W.; Analysis or Interpretation: P.S., V.W.;

Literature Search: P.S., V.W.; Writing: P.S., V.W.


authors.



References

1. Arıkan KO, Şahinkaya S, Böncüoğlu E, Kıymet E, Cem E, Kara AA, Bayram

N, Devrim I. Can hematological findings of COVID-19 in pediatric patients

guide physicians regarding clinical severity? Turk J Hematol 2021;38:243-

245.

2. Flynn MM, Reppun TS, Bhagavan NV. Limitations of red blood cell

distribution width (RDW) in evaluation of microcytosis. Am J Clin Pathol

1986;85:445-449.

3. Wang J, Zhang F, Jiang F, Hu L, Chen J, Wang Y. Distribution and reference

interval establishment of neutral-to-lymphocyte ratio (NLR), lymphocyteto-monocyte

ratio (LMR), and platelet-to-lymphocyte ratio (PLR) in Chinese

healthy adults. J Clin Lab Anal 2021;35:e23935.



Address for Correspondence/Yazışma Adresi: Pathum Sookaromdee, M.D., Private Academic Consultant,

Bangkok, Thailand

E-mail : pathumsook@mail.com ORCID: orcid.org/0000-0002-8859-5322

Received/Geliş tarihi: August 27, 2021


DOI: 10.4274/tjh.galenos.2021.2021.0488

329



REPLY FROM THE AUTHORS

We thank the author for their interest in our manuscript,

and we acknowledge their concerns regarding use of red cell

distribution width (RDW) in clinical diagnosis of severity of

COVID-19 infected children in places with very high incidence

of thalassemia like Indochina. While we agree that pediatric

children usually have a high RDW as a background hematological

disease, in our study none of the patients had thalassemia

as underlying disease. And published studies support usage

hematological parameters including RDW in COVID-19 infected

cases.

In a published study , a progressive increase of RDW was observed

with advancing COVID-19 severity. Also in multivariate analysis,

elevated RDW was associated with 9-fold increased odds of

severe COVID-19 [1].

In an other published study, RDW was found to be a prognostic

predictor for patients with severe COVID-19, and authors

concluded that the increase in reticulocyte may contribute to

elevated RDW [2].In a study conducted in 1641 patients, authors

concluded that elevated RDW at the time of hospital admission

and an increase in RDW during hospitalization were associated

with increased mortality risk for patients with COVID-19 [3].

In our findings, the NLR ratio was higher in severe/critical

cases compared to cases of asymptomatic, mild, and moderate

severity. In a study conducted in 245 COVID-19 patients

multivariate analysis demonstrated that there was 8% higher

risk of in-hospital mortality for each unit increase in NLR (odds

ratio [OR] =1.08; 95% confidence interval [95% CI], 1.01 to

1.14; P=0.0147) [4].

Finally we conclude that hematological parameters of COVID-19

infected pediatric cases may alert physicians about clinical

severity. But hematological markers especially RDW should be

used correctly as patient-based approach in COVID-19 infected

pediatric patients .

References

1. Henry BM, Benoit JL, Benoit S, Pulvino C, Berger BA, Olivera MHS, Crutchfield

CA, Lippi G. Red Blood Cell Distribution Width (RDW) Predicts COVID-19

Severity: A Prospective, Observational Study from the Cincinnati SARS-

CoV-2 Emergency Department Cohort. Diagnostics (Basel) 2020;10:618.

2. Wang C, Zhang H, Cao X, Deng R, Ye Y, Fu Z, Gou L, Shao F, Li J, Fu W,

Zhang X, Ding X, Xiao J, Wu C, Li T, Qi H, Li C, Lu Z. Red cell distribution

width (RDW): a prognostic indicator of severe COVID-19. Ann Transl Med

2020;8:1230.

3. Foy BH, Carlson JCT, Reinertsen E, Padros I Valls R, Pallares Lopez R,

Palanques-Tost E, Mow C, Westover MB, Aguirre AD, Higgins JM. Association

of Red Blood Cell Distribution Width With Mortality Risk in Hospitalized

Adults With SARS-CoV-2 Infection. JAMA Netw Open 2020;3:e2022058.

4. Liu Y, Du X, Chen J, Jin Y, Peng L, Wang HHX, Luo M, Chen L, Zhao Y.

Neutrophil-to-lymphocyte ratio as an independent risk factor for mortality

in hospitalized patients with COVID-19. J Infect 2020;81:e6-e12.

Kamile Ötiken Arıkan, Şahika Şahinkaya, Elif Böncüoğlu,

Elif Kıymet, Ela Cem, Aybüke Akaslan Kara, Nuri Bayram,

İlker Devrim

330



Hematological Malignancy Patients, COVID-19, and Favipiravir

Hematolojik Malignite Hastaları, COVID-19 ve Favipiravir

Rujittika Mungmunpuntipantip 1 , Viroj Wiwanitkit 2

1Private Academic Consultant, Bangkok, Thailand

2D.Y. Patil University, Pune, India

To the Editor,

We would like to share our ideas on the “Clinical Characteristics

and Outcomes of COVID-19 in Turkish Hematological

Malignancy Patients.” As Civriz Bozdağ et al. [1] noted in

that study: “Treatments with hydroxychloroquine alone or

in combination with azithromycin were associated with a

higher rate of mortality in comparison to favipiravir use”.

First, favipiravir is an antiviral drug proposed for management

of COVID-19. Compared to other alternative drugs, including

hydroxychloroquine and azithromycin, favipiravir should be

more specifically applied in the management of viral infections.

To determine the exact usefulness of favipiravir, clinical trials

and observational studies are needed for assessment of drug

efficacy and safety. Comparative studies between favipiravir and

other therapies would also be useful and would yield more data

on favipiravir. The report cited here is a good example of this.

Another example is a previous comparative study of favipiravir

therapy and other antiviral drugs [2].

Regarding the effects of favipiravir, some additional

considerations should be discussed. Other concurrent therapies,

such as steroid medications, can also affect the clinical outcome

[3]. Additional analysis of the possible effects of concurrent use

of steroids with favipiravir, hydroxychloroquine, azithromycin,

or their combination would be interesting. Additionally, whether

the efficacy of the drug is associated with specific types and

severities of hematological malignancies is a point for further

research. The day of initiation of favipiravir treatment is also

an important factor for therapeutic outcome [4]. According to

a recent report by Doi et al. [5], early and late administrations

of favipiravir result in different clinical outcomes. More data on

the exact period between onset of disease and start of favipiravir

are required. Regarding the present report [1], Civriz Bozdağ et

al. [1] might consider additional factor analysis regarding the

time effect for assessment of the effect of favipiravir. If future

study designs are to be based on the current report of Civriz

Bozdağ et al. [1], it may be necessary to conduct an additional

prospective study from the onset of COVID-19 to the start of

favipiravir administration.

Keywords: Malignancy, COVID-19, Favipiravir

Anahtar Sözcükler: Malignite, COVID-19, Favipiravir

Ethics

Informed Consent: Informed consent is not applicable for this

letter to the editor.


Concept: R.M., V.W.; Design: R.M., V.W.; Data Collection or

Processing: R.M., V.W.; Analysis or Interpretation: R.M., V.W.;

Literature Search: R.M., V.W.; Writing: R.M., V.W.


authors.



References

1. Civriz Bozdağ S, Cengiz Seval G, Yönal Hindilerden İ, Hindilerden F, Andıç N,

Baydar M, Aydın Kaynar L, Koçak Toprak S, Göksoy HS, Balık Aydın B, Demirci

U, Can F, Özkocaman V, Gündüz E, Güven ZT, Özkurt ZN, Demircioğlu S,

Beksaç M, İnce İ, Yılmaz U, Eroğlu Küçükdiler H, Abishov E, Yavuz B, Ataş Ü,

Mutlu YG, Baş V, Özkalemkaş F, Üsküdar Teke H, Gürsoy V, Çelik S, Çiftçiler R,

Yağcı M, Topçuoğlu P, Çeneli Ö, Abbasov H, Selim C, Ar MC, Yücel OK, Sadri

S, Albayrak C, Demir AM, Güler N, Keklik M, Terzi H, Doğan A, Yegin ZA, Kurt

Yüksel M, Sadri S, Yavaşoğlu İ, Beköz HS, Aksu T, Maral S, Erol V, Kaynar L,

İlhan O, Bolaman AZ, Sevindik ÖG, Akyay A, Özcan M, Gürman G, Ünal Ş,

Yavuz Y, Diz Küçükkaya R, Özsan GH. Clinical characteristics and outcomes

of COVID-19 in Turkish hematological malignancy patients. Turk J Hematol

2021. doi: 10.4274/tjh.galenos.2021.2021.0287. Epub ahead of print. PMID:

34521187.

2. Çınarka H, Günlüoğlu G, Çörtük M, Yurt S, Kıyık M, Koşar F, Tanrıverdi E,

Arslan MA, Baydili KN, Koç AS, Altın S, Çetinkaya E. The comparison of

favipiravir and lopinavir/ritonavir combination in COVID-19 treatment. Turk

J Med Sci 2021;51:1624-1630.

3. Murohashi K, Hagiwara E, Kitayama T, Yamaya T, Higa K, Sato Y, Otoshi

R, Shintani R, Okabayashi H, Ikeda S, Niwa T, Nakazawa A, Oda T, Okuda

R, Sekine A, Kitamura H, Baba T, Komatsu S, Iwasawa T, Kaneko T, Ogura

T. Outcome of early-stage combination treatment with favipiravir and

methylprednisolone for severe COVID-19 pneumonia: a report of 11 cases.

Respir Investig 2020;58:430-434.

4. Fujii S, Ibe Y, Ishigo T, Inamura H, Kunimoto Y, Fujiya Y, Kuronuma K, Nakata

H, Fukudo M, Takahashi S. Early favipiravir treatment was associated with

early defervescence in non-severe COVID-19 patients. J Infect Chemother

2021;27:1051-1057.

331



5. Doi Y, Hibino M, Hase R, Yamamoto M, Kasamatsu Y, Hirose M, Mutoh Y,

Homma Y, Terada M, Ogawa T, Kashizaki F, Yokoyama T, Koba H, Kasahara

H, Yokota K, Kato H, Yoshida J, Kita T, Kato Y, Kamio T, Kodama N, Uchida

Y, Ikeda N, Shinoda M, Nakagawa A, Nakatsumi H, Horiguchi T, Iwata M,

Matsuyama A, Banno S, Koseki T, Teramachi M, Miyata M, Tajima S, Maeki

T, Nakayama E, Taniguchi S, Lim CK, Saijo M, Imai T, Yoshida H, Kabata

D, Shintani A, Yuzawa Y, Kondo M. A prospective, randomized, open-label

trial of early versus late favipiravir therapy in hospitalized patients with

COVID-19. Antimicrob Agents Chemother 2020;64:e01897-20.



Address for Correspondence/Yazışma Adresi: Rujittika Mungmunpuntipantip, M.D., Private Academic

Consultant, Bangkok, Thailand

E-mail : rujittika@gmail.com ORCID: orcid.org/0000-0003-0078-7897

Received/Geliş tarihi: October 10, 2021

Accepted/Kabul tarihi: November 5, 2021

DOI: 10.4274/tjh.galenos.2021.2021.0582

REPLY FROM THE AUTHORS

In our study, we reported the clinical characteristics and

outcomes of 340 adult and pediatric COVID-19 patients with

hematological malignancies from March to November 2020.

The aim of the study was to evaluate the factors related to

severity and mortality of COVID-19 retrospectively; the study

was not statistically designed to show the impact of treatment

on outcomes. There was no approved treatment for COVID-19

so treatment plans in our country entailed hydroxychloroquine

(HCQ) alone or in combination with azithromycin as initial

treatment and favipiravir for further progress of pneumonia in

follow-up. In the later months, favipiravir was introduced to

the initial treatment schedule for COVID-19, which resulted

in the formation of different treatment groups within our

retrospective data.

In multivariate analysis related to mortality, besides

hematological disease status, decreased life expectancy related

to primary hematological disease, neutropenia, and admission

to the intensive care unit, the type of COVID-19 treatment was

a significant factor. Patients treated with HCQ alone had 4.9-

fold higher mortality risk in comparison to patients treated

with favipiravir alone, while those treated with HCQ plus

favipiravir had 2.0-fold risk and those treated with HCQ plus

azithromycin had 2.1-fold risk. Favipiravir is an antiviral drug

so a greater impact may be expected in comparison to HCQ.

However, it should be kept in mind that the treatment policy in

our country was changed after the negative results published

on HCQ treatment. Furthermore, we did not have any other

patients treated with different antiviral agents in our study.

For patients treated with favipiravir as the initial treatment, all

were treated with the drug on the day of COVID-19 diagnosis,

so we do not have a second group for a comparison of the

impact of the timeline, unfortunately. Anticytokine treatments

were administered based on individual centers’ policies so the

data were heterogenous for further analyses. We agree that

prospective studies would better show the outcome differences

between favipiravir and other antiviral therapies.

Sinem Civriz Bozdağ, Güldane Cengiz Seval,

İpek Yönal Hindilerden, Fehmi Hindilerden, Neslihan Andıç,

Mustafa Baydar, Lale Aydın Kaynar, Selami Koçak Toprak,

Hasan Sami Göksoy, Berrin Balık Aydın, Ufuk Demirci,

Ferda Can, Vildan Özkocaman, Eren Gündüz,

Zeynep Tuğba Güven, Zübeyde Nur Özkurt, Sinan Demircioğlu,

Meral Beksaç, İdris İnce, Umut Yılmaz, Hilal Eroğlu Küçükdiler,

Elgün Abishov, Boran Yavuz, Ünal Ataş, Yaşa Gül Mutlu,

Volkan Baş, Fahir Özkalemkaş, Hava Üsküdar Teke,

Vildan Gürsoy, Serhat Çelik, Rafiye Çiftçiler, Münci Yağcı,

Pervin Topçuoğlu, Özcan Çeneli, Hamza Abbasov, Cem Selim,

Muhlis Cem Ar, Orhan Kemal Yücel, Sevil Sadri, Canan Albayrak,

Ahmet Muzaffer Demir, Nil Güler, Muzaffer Keklik,

Hatice Terzi, Ali Doğan, Zeynep Arzu Yegin,

Meltem Kurt Yüksel, Soğol Sadri, İrfan Yavaşoğlu,

Hüseyin Saffet Beköz, Tekin Aksu, Senem Maral,

Veysel Erol, Leylagül Kaynar, Osman İlhan, Ali Zahit Bolaman,

Ömür Gökmen Sevindik, Arzu Akyay, Muhit Özcan,

Günhan Gürman, Şule Ünal Cangül, Yasemin Yavuz,

Reyhan Diz Küçükkaya, Güner Hayri Özsan

332



A Peculiar Disease in a Young Woman Wanting to Get Pregnant

Gebe Kalmak İsteyen Genç Kadında Garip Bir Hastalık

Tülin Tiraje Celkan 1 , Şeyma Fenercioğlu 2 , Ayşe Gonca Kaçar 3

1İstinye University Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey

2İstanbul IVF Center, İstanbul, Turkey

3İstanbul University-Cerrahpaşa, Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey

To the Editor,

Plasminogen has an important role in intravascular and

extravascular fibrinolysis [1,2,3]. Severe hypoplasminogenemia is

associated with ligneous conjunctivitis, but ligneous lesions can

also occur in many different mucosal membranes like the cervix

and endometrium [4,5]. Despite numerous clinical evaluations,

biopsies, and laboratory tests, these kinds of diagnoses remained

elusive for many years [6]. By presenting this case, we want to

increase the awareness of this peculiar disease for its proper

diagnosis.

A 32-year-old nulliparous woman was referred to our clinic.

Her history revealed persistent conjunctivitis that started when

she was 10 years old. She had married 5 years ago and the

couple wanted to have a baby. Despite extensive and detailed

investigations, no definitive reason for lack of conception

was found other than right fallopian tube occlusion. After

serological and genetic tests were done in our clinic, she was

diagnosed with plasminogen deficiency (Plg gene mutation

Lys38Glu and plasminogen level of <0.01 mg/dL, while normal

levels are 0.06-0.25 mg/dL). The patient’s family health history

revealed that she was the only symptomatic member of the

family. She was referred to a gynecologist due to infertility and

cervicitis. Colposcopic and ultrasound examination (Figures 1

and 2) of the uterus revealed a woody membranous lesion that

covered the inner part of the uterine cavity. The pathological

evaluation of membranes from endocervical curetting

showed woody fibrin accumulation, patchy ulceration, and

polymorphonuclear leukocyte exudate [5]. Her menstrual cycles

were normal and she had no problems during intercourse [6].

As plasma-derived plasminogen concentrate (NCT02312180)

was not available in Turkey, fresh frozen plasma (FFP) was given

both intravenously and directly into the uterine cavity in an

off-label way. Our clinical practice with FFP and alteplase, a

recombinant human tissue-type plasminogen activator (rTPA)

administered by bronchoscopic method, was published recently

[7]. Therefore, a similar regimen was utilized for this case. FFP

was given intravenously on alternate days and 50 mL of FFP

with 5 mg of rTPA was also administered into the uterine cavity

and held there for 4 hours. When a regular cavity view was

achieved after FFP and rTPA treatment (Figures 1 and 2), frozen

embryo transfer was performed three times. Unfortunately,

pregnancy could not be achieved despite the pre-preparation

of the genital tract for embryonal transfer with FFP and rTPA.

Pantanowitz et al. [8] also mentioned infertility in women with

Figures 1 and 2. Colposcopic and ultrasound examination of the uterus revealed a woody membranous lesion that covered the inner

part of the uterine cavity; a regular cavity view was achieved after treatment.

333



plasminogen deficiency in their work. Our local treatment

response was successful in the bronchial tree, but not in the

genital tract. In our opinion, the failure of the treatment can be

attributed to plasmin due to its role in the degradation of the

follicular wall during ovulation [8]. Impaired ovarian function

may be the cause of infertility besides the woody membranous

lesions in the uterine cavity.

Awareness about plasminogen deficiency and its symptoms are

essential for early diagnosis and treatment of this challenging

disease [9]. Currently, plasma-derived plasminogen concentrate

is reported to resolve all complications, except infertility

[6]. However, 25% of female patients have only genital tract

ligneous infiltration of the cervix and uterus accompanying

infertility. Therefore, new therapy modalities, like our therapy,

should be developed and tried in this group of patients to

reduce morbidity and mortality.

Keywords: Ligneous membranes, Ligneous cervicitis, Plasminogen,

Rare disease

Anahtar Sözcükler: Ligneous membranlar, Ligneous servisit,

Plasminojen, Nadir hastalık

Informed Consent: Obtained.


Concept: T.T.C., Ş.F., A.G.K.; Design: T.T.C., Ş.F., A.G.K.; Data

Collection or Processing: T.T.C., Ş.F., A.G.K.; Analysis or

Interpretation: T.T.C., Ş.F., A.G.K.; Literature Search: T.T.C., Ş.F.,

A.G.K.; Writing: T.T.C., Ş.F., A.G.K.


authors.



References

1. Schuster V, Hügle B, Tefs K. Plasminogen deficiency. J Thromb Haemost

2007;5:2315-2322.

2. Mehta R, Shapiro AD. Plasminogen deficiency. Haemophilia 2008;14:1261-

1268.

3. Celkan T. Plasminogen deficiency. J Thromb Thrombolysis 2017;43:132-138.

4. Miles LA, Lighvani S, Baik N, Khaldoyanidi S, Mueller BM, Parmer RJ. New

insights into the role of Plg-RKT in macrophage recruitment. Int Rev Cell

Mol Biol 2014;309:259-302.

5. Pantanowitz L. Ligneous cervicitis. BJOG 2004;111:635.

6. Baithun M, Freeman-Wang T, Chowdary P, Kadir RA. Ligneous cervicitis and

endometritis: a gynaecological presentation of congenital plasminogen

deficiency. Haemophilia 2018;24:359-365.

7. Kilinc AA, Tarcin G, Kurugoglu S, Schuster V, Cokugras H, Celkan T. A novel

combined treatment for plasminogen deficiency with lung involvement.

Pediatr Pulmonol 2020;55:E1-E3.

8. Pantanowitz L, Bauer K, Tefs K, Schuster, V, Balogh K, Pilch BZ, Adcock

D,Cirovic C, Kocher O. Ligneous (pseudomembranous) inflammation

involving the female genital tract associated with type-1 plasminogen

deficiency. Int J Gynecol Pathol 2004;23:292-295.

9. Magdaleno-Tapial J, Hernández-Bel P, Valenzuela-Oñate C, Gimeno-Ferrer F,

Rodriguez-Lopez R, Hernandez-Bel L, Sabater-Marco V, Alegre-de Miquel V.

Congenital plasminogen deficiency with long standing pseudomembranous

conjunctival and genital lesions. JAAD Case Rep 2018;5:44-46.



Address for Correspondence/Yazışma Adresi: Tülin Tiraje Celkan, M.D., İstinye University Faculty of

Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey

E-mail : tirajecelkan@yahoo.com ORCID: orcid.org/0000-0001-7287-1276

Received/Geliş tarihi: March 17, 2021


DOI: 10.4274/tjh.galenos.2021.2021.0191

334



Pulmonary Embolism Secondary to Intravenous Immunoglobulin

in a Child with Leukemia

Lösemili Bir Çocukta İntravenöz İmmünoglobuline İkincil Pulmoner Emboli

Işıl Seren Oğuz 1 , Zühre Kaya 2 , Serap Kirkiz 2 , Ülker Koçak 2

1Gazi University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey

2Gazi University Faculty of Medicine, Unit of Pediatric Hematology-Oncology, Ankara, Turkey

To the Editor,

Pulmonary embolism (PE) secondary to intravenous

immunoglobulin (IVIG) is a rare life-threatening complication

that occurs in 1% of patients with hematologic malignancies

[1]. This complication has mainly been described in adults with

chronic lymphocytic leukemia and multiple myeloma [1]. To our

knowledge, there have been no previous reports of PE resulting

from IVIG administration in a child with acute lymphoblastic

leukemia (ALL).

Our patient was an 11-year-old boy with high-risk ALL who

was treated with the ALL-BFM-95 protocol, as described

previously [2]. Since the patient had an HLA-matched

sibling donor, we planned a third high-dose chemotherapy

regimen followed by allogeneic stem cell transplantation

(allo-SCT). Hypogammaglobinemia was detected prior to

allo-SCT. The IVIG preparation was administered at 400

mg/kg (total dose: 20 g). The product information for this IVIG

advises a slow infusion at 0.3 mL/kg/h for the first 30 min, to

gradually increase to 4.8 mL/kg/h if no reaction occurs and to

be completed within 4 h.

Approximately 1.5 h after our patient’s infusion ended, he

developed shortness of breath and oxygen desaturation

(SpO 2

92%). He had no fever or hypotension, and chest

radiography was normal. Complete blood count results were

within normal limits, but the patient’s D-dimer level was

slightly elevated at 0.67 mg/L (normal: <0.5 mg/L). Oxygen

support was initiated and, although there was no fever, we

ordered a COVID-19 PCR test. The PCR test was negative, but

when the patient’s blood oxygen level did not improve during

follow-up, chest computed tomography angiography was

performed. Partial filling defects consistent with thrombus

were observed in the segmental and subsegmental branches of

the pulmonary artery, in the lower lobes of both lungs (Figure

1a). The patient was diagnosed with PE. Low-molecular-weight

heparin was initiated, with 100 IU/kg divided into two doses

and given subcutaneously. On the first day of treatment, the

dyspnea improved. The patient’s oxygen requirements began to

decrease on day 2, and on day 3 his SpO 2

was 98%.

The PE was suspected to be IVIG-induced because the patient

was in complete remission at the time of the event and did

not have febrile neutropenia, inciting drug therapy, or a central

venous catheter. Risk factors for inherited thrombophilia were

excluded. Doppler ultrasound for lower extremity thrombosis,

echocardiography findings, and levels of antithrombin 3,

antiphospholipid antibodies, C3, and C4 were all normal. The PE

gradually resolved with heparin during the second week after

diagnosis, similar to a recent report (Figure 1b) [3].

Figure 1. a) Partial filling defects in the segmental and subsegmental branches of the pulmonary artery in the lower lobe of both lungs.

b) Resolution of thrombus in the segmental and subsegmental branches of the pulmonary artery in the lower lobe of both lungs.

335



The pathophysiologic mechanisms of PE due to IVIG are poorly

understood. Some reports have suggested possible contributors

to the development of PE in these patients including increased

hyperviscosity (i.e., infusion rate not exceeding 200 mL/h or

0.08 mL/kg/min) secondary to rapid infusion, resulting in a

hypercoagulable state, and serum complement or platelet

activation due to exogenous immunoglobulin G [4,5,6,7].

A comprehensive review noted that most thromboembolic

complications occurred within 24 h of IVIG administration

[8]. Our experience suggests that leukemia specialists should

be aware of the potential for PE complications after IVIG

administration in children with leukemia.

Keywords: Immunoglobulin, Pulmonary embolism, Leukemia,

Children

Anahtar Sözcükler: İmmünoglobulin, Pulmoner emboli, Lösemi,

Çocuk

Ethics

Informed Consent: Informed consent was obtained from the

patient’s family.


Surgical and Medical Practices: I.S.O., Z.K., S.K., Ü.K.;

Concept: I.S.O., Z.K.; Design: I.S.O., Z.K.; Data Collection or

Processing: I.S.O., Z.K.; Analysis or Interpretation: I.S.O., Z.K.;

Literature Search: I.S.O., Z.K.; Writing: I.S.O., Z.K.


authors.



References

1. Ammann EM, Jones MP, Link BK, Carnahan RM, Winiecki SK, Torner JC,

McDowell BD, Fireman BH, Chrischilles EA. Intravenous immune globulin

and thromboembolic adverse events in patients with hematologic

malignancy. Blood 2016;127:200-207.

2. Kocak U, Gursel T, Kaya Z, Aral YZ, Albayrak M, Keskin EY, Belen B,

Isık M, Oner N. ALL-BFM 95 treatment in Turkish children with acute

lymphoblastic leukemia--experience of a single center. Pediatr Hematol

Oncol 2012;29:130-140.

3. Degliuomini M, Cooley V, Mauer E, Gerber LM, Acharya S, Kucine N.

Assessment of provider practices regarding venous thromboembolism

management and prevention in pediatric acute leukemia patients. J Thromb

Thrombolysis 2021;52:209-213.

4. Lee YJ, Shin JU, Lee J, Kim K, Kim WS, Ahn JS, Jung CW, Kang WK. A case of

deep vein thrombosis and pulmonary thromboembolism after intravenous

immunoglobulin therapy. J Korean Med Sci 2007;22:758-761.

5. Bilal J, Riaz IB, Hill JL, Zangeneh TT. Intravenous immunoglobulin-induced

pulmonary embolism: it is time to act! Am J Ther 2016;23:1074-1077.

6. Basta M. Intravenous immunoglobulin-related thromboembolic events - an

accusation that proves the opposite. Clin Exp Immunol 2014;178:153-155.

7. Flannery MT, Humphrey D. Deep venous thrombosis with pulmonary

embolism related to IVIG treatment: a case report and literature review.

Case Rep Med 2015;2015:971321.

8. Paran D, Herishanu Y, Elkayam O, Shopin L, Ben-Ami R. Venous and arterial

thrombosis following administration of intravenous immunoglobulins.

Blood Coagul Fibrinolysis 2005;16:313-318.



Address for Correspondence/Yazışma Adresi: Işıl Seren Oğuz, M.D., Gazi University Faculty of Medicine,

Department of Pediatrics, Ankara, Turkey

E-mail : isilserenoguz@hotmail.com ORCID: orcid.org/0000-0001-6296-5017



DOI: 10.4274/tjh.galenos.2021.2021.0400

336



Severe Lymphocytosis in a Case of Diffuse Large B-Cell

Lymphoma Treated by Ibrutinib

İbrutinib ile Tedavi Edilen Bir Diffüz Büyük B Hücreli Lenfoma Olgusunda Ciddi Lenfositoz

Semra Paydaş, Ertuğrul Bayram, Mehmet Türker, Turan Özer

Çukurova University Faculty of Medicine, Department of Medical Oncology, Adana, Turkey

To the Editor,

We would like to report extreme leukocytosis in a case of diffuse

large B-cell lymphoma (DLBCL) treated by ibrutinib [1]. To the

best of our knowledge, this is the first case of a very high white

blood cell (WBC) count in a patient with DLBCL.

A 63-year-old man was diagnosed with stage IV-B DLBCL with

bone marrow (BM) infiltration. He had double-hit lymphoma

and Ki67 was 90%. Two cycles of a dose-adjusted rituximab,

etoposide, prednisone, vincristine, cyclophosphamide, and

doxorubicin (R-EPOCH) regimen were given with clinical and

radiologic improvement. However, lymphomatous skin lesions

confirmed by cytologic examination developed. Computerized

tomography (CT) scans showed progressive disease. In the

original biopsy sample, PD-L1 expression was detected in 30%

of lymphoma cells. A rituximab-vinorelbine-gemcitabineprednisolone

regimen was given for two cycles as secondline

treatment. However, rapid progression developed and an

ibrutinib-nivolumab combination was planned and prescribed.

Four days after ibrutinib treatment, WBC and lymphocyte/

monocyte counts increased rapidly and peaked at 100x10 9 /L

on the 20 th day of treatment. Mononuclear cells were larger

than mature lymphocytes with nucleolus-like bodies. Flow

cytometric surface analysis showed CD10: 96%, HLA-DR: 55.8%,

cCD79a: 53.6%, CD45: 100%, CD19: 13%, CD20: 0% expression.

Nivolumab was given on the 14 th day of ibrutinib treatment. CT

scans showed regression of abdominal lymph nodes. However,

febrile complication developed and he died due to infection.

response rate was found to be 57.9% and 49.7% in cases of

newly diagnosed DLBCL and relapsed/refractory cases of DLBCL,

respectively [7]. We did not find any reports about lymphocytosis

in cases of DLBCL treated by ibrutinib. Our patient had 100% BM

infiltration and mononuclear cells increased rapidly with a peak

at the 20 th day of ibrutinib treatment (Figure 1). The very high

volume of mononuclear cells was due to the BM infiltration

and also the presence of very high tumor burden. The CD20

negativity in peripheral blood may be due to the previous

rituximab treatment. To the best of our knowledge, this is the

first case of a very high lymphocyte count in a patient with

DLBCL treated by ibrutinib. The mechanism of this lymphocytosis

is associated with mobilization of malignant cells related to the

action of lymphocyte trafficking, and egress of lymphocytes

from the protective stromal microenvironment occurred in our

case as also seen in cases of CLL treated by Bruton’s tyrosine

kinase [8].

We suggest that peripheral blood cell counts may increase in

patients with DLBCL with BM infiltration and/or high tumor

burden as seen in CLL.

It is well known that lymphocytes with CD19 and CD5 positivity

and CD3 negativity increase in such cases [2]. Lymphocytosis

in cases of chronic lymphocytic leukemia (CLL) is due to

tumor cells moving from infiltrated tissues to the blood. This

phenomenon is associated with a class effect and driven by

the efflux of cells from tissue compartments, paralleled by a

substantial decrease in total tumor burden [2,3,4].

Lymphocytosis has been reported in 34% of cases of mantle cell

lymphoma (MCL) [5]. Furtado et al. [6] reported lymphocytosis

in cases of MCL treated by ibrutinib among patients with higher

BM infiltration. The efficacy and safety of ibrutinib in cases

of DLBCL have been analyzed in a meta-analysis; the overall

Figure 1. White blood cell (WBC), lymphocyte, and monocyte

counts during ibrutinib treatment (/mm 3 ).

337



Keywords: Ibrutinib, Lymphocytosis, Diffuse large B cell

lymphoma

Anahtar Sözcükler: İbrutinib, Lenfositoz, Diffüz büyük B hücreli

lenfoma

Ethics

Informed Consent: Obtained.


Concept: S.P., E.B., M.T., T.Ö.; Design: S.P., E.B., M.T., T.Ö.; Data

Collection or Processing: S.P., E.B., M.T., T.Ö.; Analysis or

Interpretation: S.P., E.B., M.T., T.Ö.; Literature Search: S.P., E.B.,

M.T., T.Ö.; Writing: S.P., E.B., M.T., T.Ö.


authors.



References

1. Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA, Grant B,

Sharman JP, Coleman M, Wierda WG, Jones JA, Zhao W, Heerema NA,

Johnson AJ, Sukbuntherng J, Chang BY, Clow F, Hedrick E, Buggy JJ, James

DF, O’Brien S. Targeting BTK with ibrutinib in relapsed chronic lymphocytic


2. Chang BY, Francesco M, De Rooij MF, Magadala P, Steggerda SM, Huang

MM, Kuil A, Herman SE, Chang S, Pals ST, Wilson W, Wiestner A, Spaargaren

M, Buggy JJ, Elias L. Egress of CD19+CD5+ cells into peripheral blood

following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in

mantle cell lymphoma patients. Blood 2013;122:2412-2424.

3. Herman SE, Niemann CU, Farooqui M, Jones J, Mustafa RZ, Lipsky A, Saba N,

Martyr S, Soto S, Valdez J, Gyamfi JA, Maric I, Calvo KR, Pedersen LB, Geisler

CH, Liu D, Marti GE, Aue G, Wiestner A. Ibrutinib induced lymphocytosis in

patients with chronic lymphocytic leukemia: correlative analyses from a

phase II study. Leukemia 2014;28:2188-2196.

4. Deeks ED. Ibrutinib: A review in chronic lymphocytic leukaemia. Drugs

2017;77:225-236.

5. Wang ML, Rule S, Martin P, Goy A, Auer R, Kahl BS, Jurczak W, Advani

RH, Romaguera JE, Williams ME, Barrientos JC, Chmielowska E, Radford J,

Stilgenbauer S, Dreyling M, Jedrzejczak WW, Johnson P, Spurgeon SE, Li L,

Zhang L, Newberry K, Ou Z, Cheng N, Fang B, McGreivy J, Clow F, Buggy JJ,

Chang BY, Beaupre DM, Kunkel LA, Blum KA. Targeting BTK with ibrutinib in

relapsed or refractory mantle cell lymphoma. N Engl J Med 2013;369:507-

516.

6. Furtado M, Wang ML, Munneke B, McGreivy J, Beaupre DM, Rule S. Ibrutinibassociated

lymphocytosis corresponds to bone marrow involvement in

mantle cell lymphoma. Br J Haematol 2015;170:131-133.

7. Hou K, Yu Z, Jia Y, Fang H, Shao S, Huang L, Feng Y. Efficacy and safety of

ibrutinib in diffuse large B-cell lymphoma: a single-arm meta-analysis. Crit

Rev Oncol Hematol 2020;152:103010.

8. Davids MS, Brown JR. Ibrutinib: a first in class covalent inhibitor of Bruton’s

tyrosine kinase. Future Oncol 2014;10:957-967.



Address for Correspondence/Yazışma Adresi: Semra Paydaş, M.D., Çukurova University Faculty of Medicine,

Department of Medical Oncology, Adana, Turkey

E-mail : sepay@cu.edu.tr ORCID: orcid.org/0000-0003-4642-3693

Received/Geliş tarihi: June 9, 2021


DOI: 10.4274/tjh.galenos.2021.2021.0362

Leg Ulcers Associated with Anagrelide

Anagrelid ile İlişkili Bacak Ülserleri

Tuba Oskay 1 ,

Mehmet Özen²

1Bayındır Health Group, Department of Dermatology, Ankara, Turkey

2Bayındır Health Group, Department of Hematology, Ankara, Turkey

To the Editor,

A 77-year-old woman was referred to the dermatology

department with a complaint of leg ulcers that had existed

for a year. She had been treated with oral anagrelide at 0.5

mg twice a day for two years with the diagnosis of essential

thrombocytosis (ET). Besides itching in her hands and legs,

she had advanced non-healing leg ulcers that made walking

difficult. A dermatologic examination revealed ulcers of 2x2 cm

with demarcated, regular margins on the back of the left foot

and ulcers of 2x1.5 cm covered by sloughing necrotic tissue over

the lateral malleolus of the left foot, as well as punched-out

tiny ulcers on the lateral sides of both feet (Figures 1a and 1b).

She also had comorbidities of chronic obstructive lung disease,

hypertension, and deep vein thrombosis.

Her physical examination was unremarkable except for mild

splenomegaly. Laboratory investigations including vasculitis

and metabolic work-up showed no abnormalities. The complete

blood count test revealed hemoglobin of 12 g/dL, a white blood

338



cell (WBC) count of 7710 µL, and a platelet count of 285000/µL

(pretreatment platelet level: 685000/µL). The WBC differential

counts were 77.2% neutrophils, 12.1% lymphocytes, 7.1%

monocytes, and 2% eosinophils. Serum lactate dehydrogenase,

C-reactive protein, and erythrocyte sedimentation rate were

265 (normal range: 60-200 U/L), 12 (normal: 6 mg /L), and 18

mm/h (normal: 20 mm/h), respectively.

Bacterial and fungal cultures showed no growth. Doppler

ultrasound examination of the arterial and venous systems

was normal. The ulcers were resistant to surgical debridement,

various topical wound dressings, and antibiotic treatment. As

the leg ulcers were thought to be caused by anagrelide use,

the causative drug was discontinued and aspirin was given to

control the platelet level. After three months, her lesions had

significantly improved (Figures 1c and 1d). During two years of

follow-up, no recurrence was noted.

ET is a myeloproliferative disorder characterized by prolonged

peripheral thrombocytosis as well as thrombosis and hemorrhage

susceptibility. Despite the fact that ET is characterized by

skin involvement, therapies for ET also result in cutaneous

manifestations. Although they are generally well tolerated,

long-term treatments frequently cause cutaneous and mucosal

side effects [1,2].

Anagrelide has been preferred as a platelet-lowering drug in

patients with myeloproliferative neoplasms in recent years due

to its rapid onset of action and selective thrombocytopenic

effect. It works by preventing the maturation of platelets

from megakaryocytes, reducing platelet production, having

a selective effect on megakaryocytes, and preserving myeloid

and erythroid lineages. Although anagrelide is known to be

a phosphodiesterase (PDE) inhibitor that prevents platelet

aggregation, its exact mechanism of action is unclear [3,4].

Clinical trials have shown that anagrelide is just as effective

as other medications in reducing platelet counts without

additional complications [5]. Anagrelide appears to have a

higher response rate and may be better tolerated than other

therapies. Furthermore, anagrelide is not mutagenic and there

has been no evidence of leukemogenicity when compared to

other drugs [3,4,5].

The most frequent side effects of anagrelide are headaches and

tachycardia, which are caused by its inhibitory actions on PDE

3. The majority of frequent side effects are dose-dependent,

low to moderate, and easily tolerable. Anagrelide-related

cutaneous manifestations such as a transient rash, hair loss, skin

discoloration, itching, and xerosis have rarely been reported.

To our knowledge, only two previous cases of leg ulcers in

anagrelide-treated patients have been published [6,7]. However,

because one of those patients had previously taken hydroxyurea

(HU), it is unclear whether anagrelide caused the leg ulcer.

HU-related painful leg ulcers have been documented frequently

[2,8,9].

Although the underlying cause of these leg ulcers remains

unknown, they may be due to microcirculation difficulties and

the effects of PDE inhibitors on skin vascularity. The presence of

a temporary relationship and the elimination of other possible

causes support the diagnosis of drug-induced leg ulcers with

treatment focused on discontinuing the drug, as in our case.

We believe that early diagnosis of anagrelide-induced leg

Figure 1. a) Ulcer on the back of the left foot, b) Healed lesions on the left foot, c) Ulcers on the lateral malleolus and lateral side of the

left foot, d) Healed ulcers on the left foot.

339



ulcers is essential for these individuals to avoid functional and

psychological problems.

Keywords: Anagrelide, Leg ulcers, Essential thrombocytemia

Anahtar Sözcükler: Anagrelid, Bacak ülserleri, Esansiyel trombositemi

Ethics

Informed Consent: The patient presented in this manuscript

gave her written informed consent for the publication of her

case details.


Surgical and Medical Practices: T.O., M.Ö.; Concept: T.O., M.Ö.;

Design: T.O., M.Ö.; Data Collection or Processing: T.O., M.Ö.;

Analysis or Interpretation: T.O., M.Ö.; Literature Search: T.O.,

M.Ö.; Writing: T.O., M.Ö.


authors.



References

1. Cozzani E, Iurlo A, Merlo G, Cattaneo D, Burlando M, Pierri I, Gugliotta L,

Parodi A. Essential thrombocythemia: the dermatologic point of view. Clin

Lymhoma Myeloma Leuk 2015;15:739-747.

2. Birgegård G. Long-term management of thrombocytosis in essential

thrombocythaemia. Ann Hematol 2009;88:1-10.

3. Wagstaff AJ, Keating GM. Anagrelide: a review of its use in the management

of essential thrombocythaemia. Drugs 2006;66:111-131.

4. Dingli D, Tefferi A. A critical review of anagrelide therapy in essential

thrombocythemia and related disorders. Leuk Lymphoma 2005;46:641-650.

5. Gisslinger H, Gotic M, Holowiecki J, Penka M, Thiele J, Kvasnicka HM,

Kralovics R, Petrides PE; ANAHYDRET Study Group. Anagrelide compared

with hydroxyurea in WHO-classified essential thrombocythemia: the

ANAHYDRET Study, a randomized controlled trial. Blood 2013;121:1720-

1728.

6. Ruiz-Argüelles GJ, Ruiz-Delgado GJ, Ruiz- Reyes G, Chernoff SG. Anagrelide

induced relapse of a hydroxyurea-induced leg ulcer in a patient with

primary thrombocythemia. Mayo Clin Proc 1998;73:1125.

7. Rappoport L, Körber A, Grabbe S, Dissemond J. Appearance of leg ulcers

associated with intake of anagrelide. Dtsch Med Wochenschr 2007;132:319-

321.

8. Simeonovski V, Breshkovska H, Duma S, Dohcheva-Karajovanov I, Damevska

K, Nikolovska S. hydroxyurea associated cutaneous lesions: a case report.

Open Access Maced J Med Sci 2018;6:1458-1461.

9. Randi ML, Ruzzon E, Tezza F, Luzzatto G, Fabris F. Toxicity and side effects

of hydroxyurea used for primary thrombocythemia. Platelets 2005;16:181-

184.



Address for Correspondence/Yazışma Adresi: Tuba Oskay, M.D., Bayındır Health Group, Department of

Dermatology, Ankara, Turkey

E-mail : tboskay@hotmail.com ORCID: orcid.org/0000-0001-8578-9511



DOI: 10.4274/tjh.galenos.2021.2021.0399

340



Acute Basophilic Leukemia Arising from Chronic Myeloid

Leukemia with Isolated Thrombocytosis

İzole Trombositozlu Kronik Miyeloid Lösemiden Kaynaklanan Akut Bazofilik Lösemi

Yun Zhang 1 , Xiaosu Kang 2 , Xiliang Chen 1 , Ting Li 3

1The District People’s Hospital of Zhangqiu, Department of Clinical Laboratory, Jinan, China

2Shandong College of Traditional Chinese Medicine, Yantai, China

3Beijing Ludaopei Hospital, Department of Laboratory and Pathology, Beijing, China

To the Editor,

Acute basophilic leukemia (ABL) is a very uncommon form of

acute myeloid leukemia (AML), accounting for <1% of all cases

of AML [1]. Most cases have been described as evolving from

other hematological diseases, such as chronic myeloid leukemia

(CML) and myelodysplastic syndromes [2,3]. CML is one of the

classical types of myeloproliferative neoplasms, characterized

by the existence of a reciprocal translocation between

chromosomes 9 and 22, t(9;22)(q34:q11), resulting in the BCR-

ABL1 fusion gene. Here, we report a very rare case of CML in

basophilic blast crisis in a 54-year-old female patient with an

8-year history of CML. It is worth noting that the current World

Health Organization (WHO) guidelines would regard this case as

CML in the basophilic blast phase rather than ABL.

This 54-year-old female patient had an 8-year history of

chronic-phase chronic myeloid leukemia (CML-CP). At the

initial diagnosis of CML, a complete blood count showed

hemoglobin of 121 g/L and white blood cells of 11.2x10 9 /L, with

basophilia and very few immature myeloid cells, accompanied

by marked thrombocytosis of 1186x10 9 /L. BCR-ABL1 (p210

fusion protein) was detected by RT-PCR. Hence, a diagnosis of

CML-CP was made. She then started regular oral imatinib at 600

mg/day for 5 years. During that period, she achieved complete

remission (CR) several times, and 3 years ago, she had a final

bone reexamination that revealed CR with hematological,

cytogenetic, and molecular response to imatinib. However,

compliance was poor and she discontinued the imatinib

treatment. She was subsequently admitted to the hematology

department with a low-grade fever for 2 weeks and leukocytosis

for 1 day. Physical examination showed no hepatosplenomegaly.

Blood tests revealed a total leukocyte count of 32.68x10 9 /L

with 3% basophils, absolute basophil count of 0.98x10 9 /L,

hemoglobin concentration of 109 g/L, and platelet count of

382x10 9 /L. A peripheral blood (PB) smear revealed 32% blasts

(one-quarter of the blasts containing a variable number of

coarse basophilic granules), 2% basophils, and 10% basophilic

precursors (Figure 1A). Bone marrow (BM) aspiration showed

hypercellularity with 51.5% blasts (40% of the blasts were

metachromatic blasts), 7.5% basophils, and 8% basophilic

precursors such as basophilic metamyelocytes and myelocytes.

Simultaneously, a small amount of dwarf megakaryocytes and

numerous agranular blast cells were also observed (Figure

1B). Basophilic granules in the blasts and basophils exhibited

metachromasia with toluidine blue (Figure 1C). Flow cytometric

analysis demonstrated two distinct populations of blastoid cells

with one population of CD34+, CD33+, HLA-DR+, CD117+, and

CD123+ cells accounting for 31.32%, suggesting immature

blast cells, and the second population with 15.99% of indicated

blasts showing differentiation to basophils, which were CD33+,

CD34+, CD123+, partially CD9+, and partially HLA-DR+.

Cytogenetic analysis showed 46,XX,t(9;22)(q34; q11),i(17)(q10)

[18]/46,XX,t(9;22)(q34;q11)[2]. Molecular study showed blasts

positive for BCR-ABL (p210 fusion protein) rearrangement.

According to the consensus reports for classification [4],

given the history of CML along with the characteristics of

dwarf megakaryocytes and t(9;22)(q34;q11) with i(17)(q10),

the findings supported the diagnosis of CML in the basophilic

blast phase. The patient started two cycles of initial treatment

[regimen of idarubicin (60 mg, days 1-3) and cytarabine (0.15

g, days 1-7)] with imatinib (600 mg, by mouth once a day). She

then received two cycles of early consolidation chemotherapy of

cytarabine (2 g, intravenously, every 12 h, days 1, 3, and 5), two

cycles of cytarabine (0.15 g, days 1-7) and homoharringtonine

(3 mg, days 1-7), and another two cycles of idarubicin (20 mg,

days 1-3) and cytarabine (0.1 g, days 1-7), and she achieved CR.

She then began regular oral imatinib at 400 mg/day. To date,

she remains in continuous CR.

The term “basophilic leukemia” was first used in 1906 by

Joachim. In general, basophilic leukemias should be divided

into de novo and secondary forms and acute and chronic

variants [4]. ABL was recognized as a distinct entity in the

most recent WHO classification of myeloid malignancies [1]. In

certain circumstances, the boundary between secondary ABL

and BP-CML with increase of basophils is thin; the recently

proposed diagnostic criteria for ABL are blasts of ≥20% and

immature basophils of ≥40% of nucleated BM or PB cells [4].

Given the clinical history of CML-CP and the percentages and

341



characteristics of blasts and basophils in PB and BM, our case

may be considered as CML in basophilic blast crisis. Basophilic

blasts are typically characterized by a high N:C ratio, round

or irregular nuclei with dispersed chromatin, and moderately

basophilic cytoplasm containing a variable number of coarse

basophilic granules, which characteristically stain positive in

metachromatic staining with toluidine blue. In addition, the

blasts are frequently negative for naphthol AS-D chloroacetate

esterase (CAE). The lack of CAE reactivity can be helpful in

distinguishing blasts of ABL from mast cells [1].

In general, the diagnosis of secondary ABL and CML in

basophilic blast crisis is dependent on history, morphology,

and molecular profile in combination with immunophenotypic

results. However, morphologically, basophilic blasts can be

a heterogeneous group varying from agranular features to

significantly coarse basophilic presentation. In our case, the

majority of the blasts showed no obvious basophilic granules

in BM smears. To our knowledge, these two disease entities may

have different genetic characteristics. There are many reports of

cytogenetic abnormalities in ABL, including the following: t(X;6)

(p11.2;q23.3) resulting in MYBGATA1 [5]; a normal karyotype

with U2AF1 mutation [6]; t(16;21)(p11;q22) generating the

FUS-ERG fusion gene [7]; t(6;12)(q13;p13.3) with loss of

ETV6 [8]; and loss of TP53 in the setting of conversion from

acute myeloblastic leukemia [9]. Interestingly, Pidala et al.

[10] reported secondary ABL from CML with the development

of t(7;8)(q32;q13), while our case showed typical t(9;22)

translocation and i(17)(q10). The exact significance of the t(7;8)

translocation event is not known. Meanwhile, flow cytometric

immunophenotyping plays a key role in the definitive diagnosis.

Basophilic blasts are usually CD9+, CD25+, CD13+, CD33+,

CD123+, CD203c+, and CD11b+, while they are occasionally

positive for membrane CD22 and negative for other monocytic

markers and CD117 [1]. Additionally, the proposed diagnostic

criteria of ABL with blasts of ≥20% and immature basophils of

≥40% of nucleated BM or PB cells are helpful for distinguishing

it from CML in basophilic blast crisis [4]. CML in the basophilic

blast phase must also be distinguished from de novo ABL and

other AML subtypes with basophilia, such as AML with t(6;9)

(p23;q34.1), AML with BCR-ABL1, and, more rarely, a subtype of

lymphoblastic leukemia with prominent coarse granules.

Figure 1. Peripheral blood smear revealed 32% blasts (one-quarter of the blasts containing a variable number of coarse basophilic

granules), 2% basophils, and 10% basophilic precursors (A, Wright-Giemsa stain, 1000 x ). Bone marrow aspiration showed hypercellularity

with 51.5% blasts (40% of the blasts were metachromatic blasts), 7.5% basophils, and 8% basophilic precursors such as basophilic

metamyelocytes and myelocytes; simultaneously, a small amount of dwarf megakaryocytes and numerous agranular blast cells were also

observed (B, Wright-Giemsa, 1000 x ). Basophilic granules in the blasts and basophils exhibited metachromasia with toluidine blue (C).

342



In conclusion, here we have presented an extremely uncommon

case of CML in basophilic blast crisis. Due to the rarity and

nonspecific morphology of this disease, the combination of

clinical history, flow cytometry, and cytogenetic analysis is useful

in making a confirmed diagnosis. Since CML in the basophilic

blast phase is especially rare, with very few case reports and small

collections of cases documented in the literature, more data

are needed to ensure the accurate diagnosis and appropriate

therapeutic schedule for this unique entity.

Keywords: Acute basophilic leukemia, Chronic myeloid leukemia,

Isolated thrombocytosis

Anahtar Sözcükler: Akut bazofilik lösemi, Kronik myeloid

lösemi, İzole trombositoz

Ethics

Ethics Committee Approval: All procedures performed in this

study involving human participants were in accordance with

the ethical standards of the institutional and national research

committee and with the 1964 Declaration of Helsinki and its

later amendments or comparable ethical standards.

Informed Consent: Informed consent was obtained from this

patient.


Concept: Y.Z., X.K., X.C., T.L.; Design: Y.Z., X.K., X.C., T.L.; Data

Collection or Processing: Y.Z.; Writing: T.L.

Conflict of Interest: The authors confirm that they have no

conflicts of interest.

References

1. Arber DA, Brunning RD, Orazi A, Porwit A, Peterson LC, Thiele J. Acute myeloid

leukaemia, NOS. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA,

Stein H, Thiele J (eds). WHO Classification of Tumors of Haematopoietic and

Lymphoid Tissues. Lyon, IARC Press, 2016.

2. Parkin JL, McKenna RW, Brunning RD. Ultrastructural features of basophil

and mast cell granulopoiesis in blastic phase Philadelphia chromosome

positive leukemia. J Natl Cancer Inst 1980;65:535-539.

3. Yamagata T, Miwa A, Eguchi M, Kitagawa S, Muroi K, Hatake K, Suda T,

Sakamoto S, Miura Y. Transformation into acute basophilic leukemia in a

patient with myelodysplastic syndrome. Br J Haematol 1995;89:650-652.

4. Valent P, Sotlar K, Blatt K, Hartmann K, Reiter A, Sadovnik I, Sperr WR,

Bettelheim P, Akin C, Bauer K, George TI, Hadzijusufovic E, Wolf D, Gotlib

J, Mahon FX, Metcalfe DD, Horny HP, Arock M. Proposed diagnostic criteria

and classification of basophilic leukemias and related disorders. Leukemia

2017;31:788-797.

5. Quelen C, Lippert E, Struski S, Demur C, Soler G, Prade N, Delabesse

E, Broccardo C, Dastugue N, Mahon FX, Brousset P. Identification of a

transforming MYB-GATA1 fusion gene in acute basophilic leukemia: a new

entity in male infants. Blood 2011;117:5719-5722.

6. Carruale A, Muntone G, Rojas R, Bonfigli S, Virdis P, Longu F, Valdes G, Piras

G, Uras A, Palmas A, Caocci G, La Nasa G, Fozza C. Acute basophilic leukemia

with U2AF1 mutation. Blood Cells Mol Dis 2019;76:63-65.

7. Toda Y, Nagai Y, Shimomura D, Kishimori C, Tsuda K, Fukutsuka K, Hayashida

M, Ohno H. Acute basophilic leukemia associated with the t(16;21)

(p11;q22)/FUS-ERG fusion gene. Clin Case Rep 2017;5:1938-1944.

8. Kritharis A, Brody J, Koduru P, Teichberg S, Allen SL. Acute basophilic

leukemia associated with loss of gene ETV6 and protean complications. J

Clin Oncol 2011;29:e623-626.

9. Eveillard M, Desjonqueres A. Acute basophilic leukemia. Blood

2014;123:3071.

10. Pidala J, Pinilla-Ibarz J, Cualing HD. A case of acute basophilic leukemia

arising from chronic myelogenous leukemia with development of t(7;8)

(q32;q13). Cancer Genet Cytogenet 2008;182:46-49.





Address for Correspondence/Yazışma Adresi: Ting Li, M.D., Beijing Ludaopei Hospital, Department of

Laboratory and Pathology, Beijing, China

E-mail : litingsysu@163.com ORCID: orcid.org/0000-0002-2027-9462

Received/Geliş tarihi: September 22, 2021


DOI: 10.4274/tjh.galenos.2021.2021.0546

343



Treatment with Venetoclax for Chronic Lymphocytic Leukemia

with the Highest Known White Blood Cell Count: Safe and Effective

Bilinen En Yüksek Beyaz Küre Sayılı Kronik Lenfositik Lösemi için Venetoclax ile Tedavi:

Güvenli ve Etkili

Mehmet Sönmez, Merve Kestane, Osman Akıdan, Nergiz Erkut, Özlen Bektaş

Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey

To the Editor,

Venetoclax is a potent, selective inhibitor of B-cell lymphoma

2 (BCL-2) that is a key regulator of apoptosis and is used for

the management of chronic lymphocytic leukemia (CLL) either

alone or in combination. Inhibition of BCL-2 induces apoptosis,

leading to rapid tumor debulking and high response rates in cases

of CLL. The toxicity profile of venetoclax includes manageable

hematologic toxicities such as neutropenia, gastrointestinal

adverse events, and tumor lysis syndrome (TLS). The risk of TLS

can be reduced by a slow dose ramp-up, strict monitoring, and

adequate prophylaxis [1,2,3].

An 86-year-old female patient presented to the hospital

with fatigue, weight loss, night sweats, shortness of breath,

and weakness. She had been diagnosed with CLL 10 years

previously and she subsequently received four lines of

chemoimmunotherapy. During the COVID-19 pandemic she did

not visit the hospital for CLL follow-up. Her white blood cell

count was 925,190/µL with 90% lymphocytes. Hemoglobin level

was 5.2 g/dL and platelet count was 128,000/µL. Lymphocytes

expressed CD5, CD19, CD23, CD200, and CD20. Other laboratory

investigations including urea, electrolytes, and liver function

tests were all within normal limits. In computed tomography

scans of the neck, thorax, abdomen, and pelvis, she was observed

to have splenomegaly (150 mm) and paraaortic, supraclavicular,

axillar, and mediastinal multiple, differently sized (32x21

to 22x13 mm) lymphadenopathies. Venetoclax treatment

was planned with a 5-week dosing ramp-up (20 mg, 50 mg,

100 mg, 200 mg, 400 mg) with adequate tumor lysis prophylaxis

(allopurinol and 1.5-2 L of fluid daily) and close monitoring. In

the third week, COVID-19 infection was detected. The venetoclax

treatment was continued without any change and the patient

was discharged with no complications. Data obtained from

laboratory monitoring are presented in Figure 1.

We observed rapid tumor debulking without complications and

the disappearance of symptoms probably caused by venetoclaxinduced

apoptosis. We conclude that venetoclax is an effective

and safe treatment option for CLL [4,5,6].

Keywords: Chronic lymphocytic leukemia, Venetoclax, Highest

white blood cell count

Anahtar Sözcükler: Kronik lenfositik lösemi, Venetoclax, En

yüksek beyaz küre sayısı

344



Figure 1. Trends of tumor lysis syndrome parameters during the first 3 months of treatment. Changes of (a) white blood cell (WBC), (b)

lactate dehydrogenase (LDH), (c) creatinine, (d) calcium, (e) phosphate, and (f) uric acid levels.

345




Design: M.S., O.A., N.E., Ö.B.; Data Collection or Processing: M.K.;

Analysis or Interpretation: M.S., M.K., O.A., N.E., Ö.B.; Literature

Search: M.S., M.K., O.A., N.E., Ö.B.; Writing: M.S., M.K.


authors.



References

1. Eradat H. Venetoclax for the treatment of chronic lymphocytic leukemia.

Curr Hematol Malig Rep 2019;14:469-476.

2. Molica S. Venetoclax: a real game changer in treatment of chronic

lymphocytic leukemia. Int J Hematol Oncol 2020;9:IJH31.

3. Roberts AW. Therapeutic development and current uses of BCL-2 inhibition.

Hematology Am Soc Hematol Educ Program 2020;2020:1-9.

4. Fürstenau M, Langerbeins P, De Silva N, Fink AM, Robrecht S, von Tresckow

J, Simon F, Hohloch K, Droogendijk J, van der Klift M, van der Spek E, Illmer

T, Schöttker B, Fischer K, Wendtner CM, Tausch E, Stilgenbauer S, Niemann

CU, Gregor M, Kater AP, Hallek M, Eichhorst B. COVID-19 among fit

patients with CLL treated with venetoclax-based combinations. Leukemia

2020;34:2225-2229.

5. Mato AR, Thompson M, Allan JN, Brander DM, Pagel JM, Ujjani CS, Hill BT,

Lamanna N, Lansigan F, Jacobs R, Shadman M, Skarbnik AP, Pu JJ, Barr PM,

Sehgal AR, Cheson BD, Zent CS, Tuncer HH, Schuster SJ, Pickens PV, Shah

NN, Goy A, Winter AM, Garcia C, Kennard K, Isaac K, Dorsey C, Gashonia

LM, Singavi AK, Roeker LE, Zelenetz A, Williams A, Howlett C, Weissbrot H,

Ali N, Khajavian S, Sitlinger A, Tranchito E, Rhodes J, Felsenfeld J, Bailey N,

Patel B, Burns TF, Yacur M, Malhotra M, Svoboda J, Furman RR, Nabhan C.

Real-world outcomes and management strategies for venetoclax-treated

chronic lymphocytic leukemia patients in the United States. Haematologica

2018;103:1511-1517.

6. Koenig KL, Huang Y, Dotson EK, Sheredy S, Bhat SA, Byrd JC, Desmond E,

Ford J, Iarocci S, Jones JA, Lucas MS, Moran ME, Wiczer TE, Woyach JA, Awan

FT, Rogers KA. Safety of venetoclax rapid dose escalation in CLL patients

previously treated with B-cell receptor signaling antagonists. Blood Adv

2020;4:4860-4863.



Address for Correspondence/Yazışma Adresi: Mehmet Sönmez, Prof. M.D., Karadeniz Technical University

Faculty of Medicine, Department of Hematology, Trabzon, Turkey

Phone : +90 532 344 63 68

E-mail : mesonmez@yahoo.com ORCID: orcid.org/0000-0002-2176-4371


Accepted/Kabul tarihi: November 5, 2021

DOI: 10.4274/tjh.galenos.2021.2021.0435

346

AUTHOR INDEX 2021

38 th Volume Index / 38. Cilt Dizini

AUTHOR INDEX 2021 - YAZAR DİZİNİ 2021

Abdullah Hacıhanefioğlu.................................211

Abdulsamet Erden............................................. 321

Abdülkadir Baştürk........................................... 273

Abdülkadir Erçalışkan....................................... 226

Abibatou Sall...................................................... 153

Abraham Pouliakis................................................22

Achmet Ali..............................................................15

Adalet Meral Güneş.......................................... 294

Afrodite Chairopoulou........................................22

Aggeliki Tsakania..................................................22

Ahmet Emre Eşkazan.................................79, 226

Ahmet Kürşad Güneş........................................ 273

Ahmet Muzaffer Demir............................ 64, 241

Ahmet Omma...................................................... 321

Ahmet Seyhanlı.................................................. 273

Ahmet Yasir Yıldırım..........................................171

Ahu Senem Demiröz......................................... 226

Aijun Liu............................................................... 246

Ajay Gogia..............................................................81

Akshay Ramesh Gore........................................ 239

Ali Bülent Antmen.............................................101

Ali Ünal........................................................195, 273

Ali Zahit Bolaman.................................. 41, 77, 96

Alp Atasoy.............................................................171

Alperen Kızıklı..................................................... 273

Alphan Küpesiz....................................................101

Anastasia Livada...................................................22

Andreas Papachronis...........................................22

Angela Guarina.................................................. 175

Angela Maggio................................................... 175

Angela Michelutti...............................................119

Angela Petrone................................................... 175

Angelica Barone................................................. 175

Anıl Tombak......................................................... 273

Anthippi Gafou.....................................................22

Antonis Aggelidis..................................................22

Areti Skordilaki......................................................22

Arzu Akçay..................................................222, 294

Arzu Yazal Erdem............................................... 294

Aslı Çiftçibaşı Örmeci........................................171

Aslıhan Pekmezci..................................................79

Aspasia Argyrou....................................................22

Assunta Tornesello............................................ 175

Atakan Turgutkaya...............................................41

Athina Mougiou....................................................22

Atilla Uslu............................................................. 111

Awa Oumar Touré.............................................. 153

Aybüke Akaslan Kara........................................ 243

Aydan Akdeniz.................................................... 273

Ayfer Gedük.........................................................211

Aylin Canpolat.................................................... 294

Ayşe Gonca Kaçar.............................................. 333

Ayşe Hilal Eroğlu Küçükdiler.............................41

Ayşe Korkmaz..................................................... 222

Ayşen Türedi Yıldırım........................................ 294

Barış Kuşkonmaz................................................ 286

Başak Koç..............................................................101

Berna Oğuz...........................................................101

Berrin Balık Aydın..............................................171

Bin Song..................................................................74

Bircan Sönmez.................................................... 306

Birol Baytan......................................................... 294

Birsen Sahip.................................................. 41, 273

Blaise Félix Faye................................................. 153

Bo Gao.....................................................................74

Boju Pan............................................................... 169

Burak Deveci..............................................195, 273

Burcu Belen......................................................... 294

Burcu Kolukısa.........................................................1

Burcu Özkan.........................................................101

Burçak Tatlı Güneş............................................ 294

Burhan Ferhanoğlu....................................89, 273

Burhan Turgut.................................................... 273

Bülent Zülfikar....................................................101

Can Balkan............................................................101

Can Baykal..............................................................49

Can Boğa.............................................................. 159

Can Çevikol...........................................................101

Can Zafer Karaman..............................................77

Cem Selim........................................................41, 77

Cemaleddin Öztürk............................................ 111

Ceren Uzunoğlu................................................. 233

Cheng Cheng...................................................... 230

Chiara Gorio........................................................ 175

Christina Pappa.....................................................22

Chrysoula Alepi.....................................................22

Chu-Cheng Wan...................................................74

Daniela Damiani..................................................119

Demet Aydoğdu..................................................101

Demet Çekdemir................................................ 273

Demet Nak..............................................................69

Deniz Aslan.......................................................... 161

Derya Selim Batur............................................. 273

Diama Samb........................................................ 153

Dilek Gürlek Gökçebay........................................72

Dimitra Moschandreou.......................................22

Dongbei Li............................................................ 230

Duygu Çetinkaya............................................... 286

Eda Albayrak....................................................... 222

Eftihia Kontekaki..................................................22

Ela Cem................................................................. 243

Eleftheria Zervou..................................................22

Elena Chiocca..................................................... 175

Elena Facchini..................................................... 175

Elgin Özkan.............................................................69

Elias Kyriakou.........................................................22

Elif Böncüoğlu.................................................... 243

Elif Gülsüm Ümit............................................... 273

Elif Kıymet........................................................... 243

Elif Ümit..................................................................64

Elifcan Aladağ..................................................... 138

Elisavet Grouzi.......................................................22

Emel Gürkan........................................................ 273

Emel Merve Yenihayat......................................211

Emin Kaya...................................................195, 273

Emin Ümit Bağrıaçık......................................... 145

Emine Zengin...................................................... 294

Emre Çeliksoy.........................................................15

Emre Osmanbaşoğlu............................................89

Enes Cömert........................................................ 165

Erdal Karaöz........................................................ 254

Erdal Kurtoğlu.................................................... 273

Eren Arslan Davulcu.......................................... 273

Eren Gündüz........................................................ 314

Ertuğrul Bayram................................................. 337

Esma Evrim Doğan...................................165, 181

Esra Turan Erkek................................................. 181

Ethan Burns......................................................... 218

Fahri Şahin..................................................101, 195

Fang Ye................................................................. 246

Faruk Güçlü Pınarlı........................................... 145

Farzad Kompani....................................................94

Fatma Arıkan....................................................... 233

Fatma Visal Okur................................................ 286

Fergün Yılmaz............................................195, 233

Figen Esen...............................................................15

Filiz Vural.............................................................. 195

Fotini Sakellaridi...................................................22

Fragoula Roussinou..............................................22

Francesca Compagno....................................... 175

Francesca Romano............................................ 175

Funda Pepedil Tanrıkulu.................................. 273

Gabriela Boscarol............................................... 175

Georges Martinis...................................................22

AUTHOR INDEX 2021

Georgia Kakava.....................................................22

Gianluca DellOrso.............................................. 175

Giovanna Russo.................................................. 175

Giuseppe Bertoni............................................... 175

Giuseppe Lassandro.......................................... 175

Giuseppe Puccio................................................. 175

Gizem Kumru Şahin.......................................... 167

Gökhan Özgür..................................................... 195

Gökhan Sargın.......................................................41

Gül İlhan............................................................... 273

Gülay Kadıoğlu................................................... 165

Gülçin Hilal Alay...................................................15

Güldane Cengiz Seval....................... 69, 111, 195

Gülen Tüysüz........................................................101

Gülsan Türköz Sucak........................................ 195

Gülsüm Özet........................................................ 273

Gülyüz Öztürk..................................................... 294

Günhan Gürman................................................. 111

Günseli Orhun........................................................15

Güray Saydam...........................................195, 273

Güven Çetin......................................................... 273

Habib Moshref Razavi...................................... 151

Hakan Göker.............................33, 138, 195, 204

Hakan Gürkan..................................................... 241

Hakan Özdoğu.................................................... 195

Hakkı Onur Kırkızlar.............................................64

Hale Ören............................................................. 294

Haluk Demiroğlu............................... 33, 138, 204

Haruna Sano..........................................................83

Hasan Emre Kocabay........................................ 167

Hatice Terzi.......................................................... 273

Hayrunnisa Albayrak.........................................211

Helin Masyan.........................................................89

Hikmet Akar...........................................................85

Hirohisa Fujikawa.................................................92

Hong Zhai............................................................ 264

Hong-bin Zhang................................................ 126

Honglei Wang..................................................... 236

Huan Wang.......................................................... 246

Hui Liu................................................................... 236

Humaira Sarfraz................................................. 218

Hüseyin Derya Dinçyürek................................ 273

Hüseyin Gülen.................................................... 294

Hüseyin Tokgöz...................................................101

Ibrahim Muhsen................................................. 218

Ilaria Fotzi............................................................ 175

Ilkın Muradov...................................................... 226

Ioanna Apostolidou.............................................22

Ioanna Dendrinou................................................22

Irene D’Alba......................................................... 175

Işık Odaman Al................................................... 294

Işıl İnanır.................................................................85

Işıl Seren Oğuz.................................................... 335

İbrahim Celalettin Haznedaroğlu....................33

İhsan Ateş............................................................ 321

İhsan Karadoğan................................................ 195

İlgen Şaşmaz........................................................101

İlhami Berber...................................................... 273

İlkay Anaklı.............................................................15

İlker Devrim......................................................... 243

İlknur Mansuroğlu............................................. 165

İpek Tamsel...........................................................101

İrem Yılmaz Başaran......................................... 254

İrfan Yavaşoğlu.......................... 41, 77, 234, 273

İsmet Aydoğdu............................................85, 273

Jiaqiong Xu.......................................................... 218

JiaWei Zhao............................................................87

Jin Wei.................................................................. 264

Jing Wang............................................................ 126

Jingyao Wang..................................................... 188

Juan Chang.............................................................74

Juan Pu................................................................. 264

Juan Zhang.......................................................... 325

Junqing Xu........................................................... 188

Junting Zhao....................................................... 264

Kaan Kavaklı.........................................................101

Kadir Uluç Anıl....................................................171

Kamile Ötiken Arıkan....................................... 243

Kang Jiang........................................................... 325

Keisuke Kidoguchi................................................83

Kemal Aygün........................................................211

Kenan Keven....................................................... 167

Kenichiro Ebisuda.................................................92

Kerim Sarıyılmaz................................................ 222

Khaled Warasnhe............................................... 286

Klara Dalva............................................................ 111

Konstantinos Lebesopoulos...............................22

Konstantinos Stamoulis......................................22

Kostas Malekas......................................................22

Kurtuluş Didem Yazganoğlu..............................49

Lale Olcay............................................................. 294

Lampothea Labrianou.........................................22

Lei Wang............................................................... 188

Lempousi Dimitra.................................................22

Leylagül Kaynar.................................................. 195

Li Li......................................................................... 188

Li-xia Zhang........................................................ 126

Licai An................................................................. 188

Lin Chen................................................................ 230

Lu Zhang........................................................87, 169

Lu-Lu Zhang...........................................................74

Lucia Dora Notarangelo................................... 175

Luis M. Allende................................................... 145

Macoura Gadji.................................................... 153

Maddalena Casale............................................. 175

Maddalena Marinoni........................................ 175

Mahmut Yeral..................................................... 159

Man Updesh Singh Sachdeva........................ 327

Manorama Bhargava........................................ 239

Marco Spinelli..................................................... 175

Marco Zecca........................................................ 175

Margherita Cavallin...........................................119

Maria Baka..............................................................22

Maria Gavalaki......................................................22

María J. Díaz-Madroñero................................ 145

Marianna Politou..................................................22

Mario Tiribelli.......................................................119

Maryame Ahnach.................................................98

Maurizio Miano.................................................. 175

Mehmet Akif Baltacı.......................................... 111

Mehmet Ali Özcan............................................. 273

Mehmet Ali Uçar................................................ 273

Mehmet Baysal................................................... 241

Mehmet Baysal......................................................64

Mehmet Can Uğur............................................. 181

Mehmet Fatih Orhan........................................ 294

Mehmet Hilmi Doğu......................................... 273

Mehmet Kılıç..........................................................15

Mehmet Özen..................................................... 338

Mehmet Sönmez....................101, 273, 306, 344

Mehmet Türker................................................... 337

Mehmet Yılmaz.........................................195, 273

Melek Büyük........................................................171

Melek Işık............................................................. 145

Melike Sezgin Evim........................................... 294

Meltem Kurt Yüksel........................................... 111

Meral Beksaç.................................................69, 111

Mert Öztaş........................................................... 226

Merve Gökçen Polat..........................................211

Merve Kestane.................................................... 344

Meryem Albayrak.............................................. 294

Mesut Bulakçı......................................................101

Milena Motta...................................................... 175

Mine Araz................................................................69

Mine Miskioğlu......................................................85

Mingyong Li........................................................ 325

Minoru Saito..........................................................92

Moeinadin Safavi..................................................94

Moussa Seck........................................................ 153

Muhit Özcan........................................................ 111

Murat Karapapak............................................... 286

Mustafa Şahin.......................................................85

Mustafa Velet..................................................... 195

Mutlu Mercan........................................................15

Müfide Okay........................................................ 273

Nabhajit Mallik................................................... 327

Nalan Neşe..............................................................85

Namık Özbek....................................................... 294

Nazan Sarper...................................................... 294

Nergiz Erkut...............................................306, 344

Nesimi Büyükbabani............................................49

Neşe Yaralı....................................................72, 294

AUTHOR INDEX 2021

Nihal Özdemir..................................................... 314

Nihan Alkış........................................................... 241

Nihan Nizam....................................................... 181

Nihan Oruklu....................................................... 145

Niki Vgontza...........................................................22

Nilgün Sayınalp...........................................33, 273

Ningning Li.......................................................... 246

Nitin Sood............................................................ 239

Nuh Filizoğlu....................................................... 224

Nur Soyer............................................................. 195

Nuri Bayram........................................................ 243

Oktay Bilgir.......................................................... 181

Olga Meltem Akay......................................89, 273

Orhan Küçükşahin............................................. 321

Orhun Çığ Taşkın...................................................89

Osman Akıdan..................................................... 344

Osman İlhan................................................111, 167

Osman Özcebe.......................................................33

Ozan Salim........................................................... 195

Ömer Özcan......................................................... 273

Ömür Gokmen Sevindik....................................171

Önder Arslan........................................................ 111

Özgür Mehtap............................................ 211, 273

Özlem Polat............................................................15

Özlem Tüfekçi..................................................... 294

Özlen Bektaş...............................................306, 344

Pan Zhao............................................................... 264

Panagiotis Siourounis..........................................22

Paola Giordano................................................... 175

Paola Saracco...................................................... 175

Pathum Sookaromdee..................... 99, 229, 329

Peng Yin................................................................ 126

Perihan Ergin Özcan............................................15

Pervin Topçuoğlu.......................................111, 195

Peyker Temiz...........................................................85

Pınar Göçün Uyar.............................................. 145

Pınar Tarkun.........................................................211

Piero Farruggia................................................... 175

Pin Hu......................................................................74

Polat Koşucu........................................................101

Qing Xiao.............................................................. 126

Rafet Eren...................................................165, 181

Rafiye Çiftçiler.............................................33, 204

Rahşan Yıldırım.................................................. 273

Raquel Ruiz-García........................................... 145

Renato Fanin........................................................119

Ritu Gupta..............................................................81

Rong Fu................................................................ 236

Ruihua Mi............................................................ 230

Rujittika Mungmunpuntipantip...... 94, 157, 331

Saba Kiremitçi.................................................... 167

Safa Barış..................................................................1

Sai Ravi Pingali................................................... 218

Salih Aksu...............................................................33

Salih Özgüven..................................................... 224

Salih Sertaç Durusoy........................................ 273

Saliou Diop.......................................................... 153

Samuel Wang.........................................................57

Sara Di Giusto......................................................119

Saumyaranjan Mallick.........................................81

Saverio Ladogana.............................................. 175

Selami Koçak Toprak.......................................... 111

Sema Aylan Gelen............................................. 294

Sema Karakuş...................................................... 314

Semra Cemre Atalar.............................................89

Semra Paydaş.............................................163, 337

Serap Kirkiz.................................................145, 333

Serdal Uğurlu...................................................... 226

Serdar Beken....................................................... 222

Serdar Can Güven.............................................. 321

Serena Valsami......................................................22

Serkan Ünal..........................................................211

Seval Akpınar...................................................... 273

Sevgi Beşışık...........................................................15

Sevgi Kalayoğlu Beşışık.....................................171

Shahrukh K. Hashmi......................................... 218

Shinya Kimura.......................................................83

Shu-liang Guo.................................................... 126

Sıla Kılıç Sayar.......................................................49

Sibel Akpınar....................................................... 294

Silverio Perrotta................................................. 175

Silvia Salvatore................................................... 175

Sinan Demircioğlu............................................. 273

Sinan Mersin........................................................211

Sinem Civriz Bozdağ.................................111, 195

Sinem Namdaroğlu........................................... 181

Smeeta Gajendra......................................228, 239

Sofia Tsagia............................................................22

Spiridon Koliofotis...............................................22

Sudhir Kirar............................................................81

Süha Süreyya Özbek..........................................101

Syeda A. Mina..................................................... 218

Şahika Şahinkaya............................................... 243

Şebnem Batur..................................................... 226

Şehmus Ertop............................................... 41, 273

Şevkiye Selin Aytaç............................................101

Şeyma Fenercioğlu............................................ 333

Şule Mine Bakanay........................................... 314

Tajamul H. Mir.................................................... 155

Taner Arpacı.........................................................101

Tarık Onur Tiryaki...............................................171

Tayfur Toptaş....................................................... 233

Theofanis Adraktas...............................................22

Ting Li.................................................................... 341

Tongtong Wang.................................................. 246

Tuba Hilkay Karapınar...................................... 294

Tuba Oskay........................................................... 338

Tuğana Akbaş.......................................................101

Turan Özer............................................................ 337

Turgut Seber.........................................................101

Tülin Tiraje Celkan............................................. 333

Tülin Tuğlular...................................................... 233

Ugo Ramenghi.................................................... 175

Uğur Şahin............................................................ 111

Umut Ece Arslan................................................ 286

Umut Yılmaz..........................................................79

Ülker Koçak................................................145, 335

Ümran Çalışkan...................................................101

Vahap Okan......................................................... 273

Vasiliki Giannopoulou.........................................22

Vasiliki Koika..........................................................22

Vasiliki Pliatsika.....................................................22

Vasiliki Sochali.......................................................22

Vasiliki Voulgaridou.............................................22

Verda Tuna..............................................................15

Veysel Haksöyler................................................ 163

Vildan Özkocamaz............................................. 273

Viroj Wiwanitkit......97, 99, 157, 229, 329, 331

Wanlu Ma......................................................87, 169

Wei Geng.................................................................74

Wenming Chen................................................... 246

Xia Zhang................................................................74

Xiaosu Kang......................................................... 341

Xiaoxia Chu......................................................... 188

Xiliang Chen........................................................ 341

Xudong Wei......................................................... 230

Yahya Büyükaşık............................... 33, 138, 204

Yalçın Gül................................................................79

Yanchen Li............................................................ 246

Yasushi Kubota......................................................83

Yaşa Gül Mutlu....................................................171

Yeşim Oymak....................................................... 294

Yıldız İpek............................................................. 233

Yi-Gang Guo..........................................................74

Yi-ying Xiong...................................................... 126

Yinghui Liu........................................................... 188

Yingying Chen.................................................... 236

Yu Liu........................................................................74

Yuan He................................................................ 325

Yuki Nishimura......................................................83

Yun Zhang............................................................ 341

Yunus Murat Akçabelen.....................................72

Yusuke Miyazato...................................................92

Zafeiria Alexandropoulou..................................22

Zahit Bolaman.................................................... 234

Zehra Narlı Özdemir.......................................... 111

Zhang-zhi Li...........................................................74

Zhaoyun Liu......................................................... 236

Zohreh Nozarian...................................................94

Zuhal Demirci......................................................101

Zühre Kaya..................................................145, 335

SUBJECT INDEX 2021

38 th Volume Index / 38. Cilt Dizini

SUBJECT INDEX 2021 - KONU DİZİNİ 2021

1. Acute Leukemia

Acute basophilic leukemia/Akut bazofilik lösemi 343

Acute kidney injury/Akut böbrek yetmezliği 167

Acute leukemia/Akut lösemi 138

Acute lymphoblastic leukemia/Akut lenfoblastik lösemi 94, 326

Acute myeloid leukemia/Akut myeloid lösemi 111, 119, 231, 264

Acute myeloid leukemia with myelodysplasia-related changes/

Myelodisplazi ilişkili değişiklikler gösteren akut myeloid lösemi 188

Allogeneic hematopoietic stem cell transplantation/Allojeneik

hematopoetik kök hücre nakli 126

Anemia/Anemi 326

Apoptosis/Apoptoz 264

Autoimmune lymphoproliferative syndrome/Otoimmün

lenfoproliferatif sendrom 145

B-cell neoplasms/B-hücreli neoplaziler 173

BCL2/BCL2 119

Blast crisis/Blast krizi 76

Bortezomib/Bortezomib 96

CD200/CD200 119

Chemotherapy/Kemoterapi 94, 126

Children/Çocuk 336

Chronic lymphocytic leukemia/Kronik lenfositik lösemi 167

Chronic myeloid leukemia/Kronik myeloid lösemi 76, 343

Clinical characteristics/Klinik özellikler 188

Convalescent plasma therapy/Nekahat plazma tedavisi 76

COVID-19/COVID-19 76, 331

Cystoisospora belli/Cystoisospora belli 173

Dicentric (7, 12)/Disentrik (7, 12) 241

ETV6/RUNX1/ETV6/RUNX1 241

Extranodal NK/T-cell lymphoma/Ekstranodal NK/T-hücreli lenfoma

126

Favipiravir/Favipiravir 331

Flowcytometric immunophenotyping/Akım sitometrik immün

fenotipleme 228

Flower cells/Çiçek hücreler 228

Fluorescence in situ hybridization/Floresan in situ hibridizasyon 241

Foamy macrophage/Köpüksü makrofaj 94

Gastric hemorrhage/Mide kanaması 169

Gastrointestinal plasmacytoma/Gastrointestinal plazmasitom 169

Glycolysis/Glikoliz 264

Hairy cell leukemia/Tüylü hücreli lösemi 326

Hematopoietic stem cell transplantation/Hematopoetik kök hücre

nakli 138

Immune cytopenia/İmmün sitopeni 145

Immunoglobulin/İmmünoglobulin 336

Immunohistochemistry/İmmünohistokimya 151, 228

Interferon α, -1b/İnterferon α, -1b 231

Interleukin-2/İnterlökin-2 231

Isolated thrombocytosis /İzole trombositoz 343

Lenalidomide/Lenalidomid 231

Leukemia/Lösemi 224, 336

Leukemic infiltration/Lösemik infiltrasyon 167

LncRNA-DUXAP8/LncRNA-DUXAP8 264

Lymphocytes/Lenfositler 173

Lymphoid cell neoplasms/Lenfoid hücre neoplazileri 173

Lymphoma/Lenfoma 145

Malignancy/Malignite 331

Measurable residual disease/Ölçülebilir kalıntı hastalık 111

Minimal residual disease/Minimal kalıntı hastalık 231

Mixed-phenotype acute leukemia/Karışık-fenotip akut lösemi 241

Monoclonal gammopathy of undetermined significance/Önemi

belirlenemeyen monoklonal gammopati 151

Morphologic abnormalities/Morfolojik anormallikleri 153

Multiparameter flow cytometry/Çok renkli akım sitometri 111

Multiple myeloma/Multipl myelom 153, 169, 173

Myeloid sarcoma/Myeloid sarkom 224

Other lymphoproliferative disorders/Diğer lenfoproliferatif hastalıklar

173

Plasma cell/Plazma hücresi 153

Plasma cell inclusions/Plazma hücre inklüzyonları 151

Plasma cell leukemia/Plazma hücreli lösemi 96

Plasma cell myeloma/Plazma hücre myelom 228

Prognosis/Prognoz 119

Prolonged watery diarrhea/Uzamış sulu ishal 173

Pulmonary embolism/Pulmoner emboli 336

Radiotherapy/Radyoterapi 126

Refractory/relapsed/Dirençli/nüks 231

Risk scoring/Risk skoru 138

Russell bodies/Russell cisimsikleri 151

Survival/Sağkalım 119

Testicular vein/Testiküler ven 224

Treatment/Tedavi 96, 188

Wnt/β-catenin signaling pathway/ Wnt/β-katenin sinyal yolu 264

2. Anemia

ADAMTS13/ADAMTS-13 155

Allogeneic stem cell transplantation/Allojeneik kök hücre nakli 195

Aplastic anemia/Aplastik anemi 195



Bentley score/Bentley skoru 64

Complication/Komplikasyon 159

SUBJECT INDEX 2021

COVID-19/COVID-19 99

COVID-19-induced iTTP/COVID-19 ilişkili iTTP 155

Cytopenia/Sitopeni 306

French score/Klinik skorlama sistemleri 64

Hematologic malignancy/Hematolojik malignite 306

Hydroxychloroquine/Hidroksiklorokin 99

Hydroxychloroquine-induced TTP/Hidroksiklorokin ilişkili TTP 155


Long-term hematologic effects/Uzun süreli hematolojik etkiler 306


Neutropenia/Nötropeni 306

Paroxysmal nocturnal hemoglobinuria/Paroksismal noktürnal

hemoglobinuri 195

PLASMIC score/PLASMIC skor 64

Radioactive iodine/Radyoaktif iyot 306

Sickle cell trait/Orak hücre taşıyıcılığı 159

SLE/SLE 99

Splenic infarct/Dalak infarktı 159

Thrombocytopenia/Trombositopeni 306

Thrombotic microangiopathy/Trombotik mikroanjiyopati 64, 155

Thrombotic thrombocytopenic purpura/Trombotik trombositopenik

purpura 64

Thyroid cancer/Tiroid kanseri 306

Transplantation/Transplantasyon 195

TTP/TTP 99

Von Willebrand factor/Von Willebrand faktör 155

3. Bleeding Disorders

ACVRL1 mutation/ACVRL1 mutation 242

Anemia/Anemia 242

Celiac/Çölyak 175

Children/Çocuklar 175

Cirrhosis/Cirrhosis 242

Eltrombopag/Eltrombopag 181

Epistaxis/Epistaxis 242

Hemophilic arthropathy/Hemofilik artropati 101

Hereditary hemorrhagic telangiectasia/Hereditary hemorrhagic

telangiectasia 242

HJHS/HJHS 101

Immune/İmmün 175

Joint scores/Eklem skorları 101

Pediatric/Pediatrik 175

Splenectomy/Splenektomi 181

Thrombocytopenia/Trombositopeni 175, 181

Ultrasonography/Ultrasonografi 101

4. Chronic Leukemia


Acute lymphoblastic leukemia/Akut lenfoblastik lösemi 326

Anemia/Anemi 326

Atrial fibrillation/Atriyal fibrilasyon 83

Bleeding/Kanama 83

Bruton’s tyrosine/Bruton tirozin 274

Cardiac tamponade/Kardiyak tamponad 83

Chronic lymphocytic leukemia/Kronik lenfositik lösemi 167, 274, 344

Chronic myelomonocytic leukemia/Kronik myelomonositik lösemi 227

CLL/CLL 82

Giant cell arteritis/Dev hücreli arterit 227


Highest white blood cell count/En yüksek beyaz küre sayısı 344

Ibrutinib/İbrutinib 83, 274

Kinase inhibitor/Kinaz inhibitörü 274

Leukemia/Lösemi 227


Vasculitis/Vaskülit 227

Venetoclax/Venetoclax 344

5. Coagulation

Antithrombin/Antitrombin 15, 157

Arterial/Arteriyel 222

Childhood thrombosis/Çocukluk trombozu 295


Fresh frozen plasma/Taze dondurulmuş plazma 157

Fresh frozen plasma/Taze donmuş plazma 15

Hemophilic arthropathy/Hemofilik artropati 101

HJHS/HJHS 101

Hypercoagulopathy/Hiperkoagülopati 15

Inherited antithrombin deficiency/Kalıtsal antitrombin eksikliği 162

Ischemia/İskemi 222

Joint scores/Eklem skorları 101

Ligneous cervicitis/Ligneous servisit 334

Ligneous membranes/Ligneous membranlar 334

Mortality/Mortalite 15

Newborn/Yenidoğan 222

p.Asp374Val mutation/p.Asp374Val mutasyonu 162

Plasminogen/Plasminojen 334

Rare disease/Nadir hastalık 334

Recombinant tissue plasminogen activator/Rekombinant doku

plazminojen aktivatörü 295

SERPINC1/SERPINC1 162

Thrombolysis/Tromboliz 295

Ultrasonography/Ultrasonografi 101

6. Hematological Malignancies

23-Valent polysaccharide pneumococcal vaccine/23-valan polisakkarit

pnömokok aşısı 92


Acute lymphoblastic leukemia/Akut lenfoblastik lösemi 94

Acute myeloid leukemia/Akut myeloid lösemi 111, 119, 231, 264

Adverse effect/Yan etki 41

SUBJECT INDEX 2021

AL amyloidosis/AL amiloidoz 234


Anagrelide/Anagrelid 340



Appendix/Apendiks 89



B2-microglobulin/B2-mikroglobulin 33

BCL2/BCL2 119

Behçet’s disease/Behçet hastalığı 92


Blood cultures/Kan kültürleri 57

Blood/Kan 229

Bortezomib/Bortezomib 96, 211

Breast cancer/Meme kanseri 85

Brentuximab/Brentuximab 85


Cancer/Kanser 229

Castleman disease/Castleman hastalığı 314

CD200/CD200 119

CD30+ T-cell lymphoproliferative disorders/CD30+ T-hücreli

lenfoproliferatif bozukluklar 49

Chemotherapy/Kemoterapi 94

Children/Çocuk 336

Chronic lymphocytic leukemia/Kronik lenfositik lösemi 274, 344

Chronic myeloid leukemia/Kronik myeloid lösemi 76, 80, 343


CLL/CLL 82

Clone proliferation/Klonal proliferasyon 236



COVID-19, Drug-drug interaction/COVID-19 İlaç-ilaç etkileşimleri 80

COVID-19 infection/COVID-19 enfeksiyonu 78

Cutaneous anaplastic large cell lymphoma/Kutanöz anaplastik büyük

hücreli lenfoma 85

Cystoisospora/Cystoisospora 229


Diagnosis/Tanı 314


Diffuse large B cell/Diffüz büyük B hücreli 338

Equivalent/Eşdeğer 211

Essential thrombocytemia/Esansiyel trombositemi 340



FDG/FDG 69

Febrile neutropenia/Febril nötropeni 57



Generic/Jenerik 41

Genomic abnormality/Genomik anormallik 246



Haematology/Hematoloji 57


Hematopoietic stem cell Transplantation/Hematopoetik kök hücre

transplantasyonu 286


Hodgkin lymphoma/Hodgkin lymphoma 204

Ibrutinib/Ibrutinib 81, 274, 338



Induced pluripotent stem cells/Uyarılmış pluripotent kök hücre 254

Inflammation/Enflamasyon 286



International Staging System/Uluslararası Evreleme Sistemi 33

Intravascular large B-cell lymphoma/İntravasküler büyük B-hücreli

lenfoma 89


Ixazomib/İksazomib 87, 235

Kappa light chain/Kappa hafif zincir 234


Laboratory medicine/Laboratuvar tıbbı 57

Leg ulcers/Bacak ülserleri 340

Lenalidomide/Lenalidomid 41, 231

Leukemia/Lösemi 224, 227, 336


LncRNA/LncRNA 236


Lymphocytosis/Lenfositoz 338

Lymphoma/Lenfoma 145, 338

Lymphomatoid papulosis/Lenfomatoid papüloz 49

MALAT1/MALAT1 236



Mesenchymal stem cells/Mezenkimal kök hücre 254

Microbiology/Mikrobiyoloji 57



Mobilization failure/Mobilizasyon başarısızlığı 204

Monotherapy/Monoterapi 85


Multiple myeloma/Multipl myelom 33, 211, 218, 234, 246, 254

Mycosis fungoides/Mikozis fungoides 49

Myelodysplastic syndrome/Miyelodisplastik sendrom 92


Myeloma/Myelom 229

Myeloma and other plasma cell dyscrasias/Myeloma ve diğer plazma

hücre diskrazileri 69

SUBJECT INDEX 2021

Neutropaenic sepsis/Nötropenik sepsis 57


Non-Hodgkin lymphoma/Non-Hodgkin lymphoma 204

Original/Orijinal 41


hemoglobinuri 195, 236

Peripheral neuropathy/Periferik nöropati 218


Positron emission tomography/Pozitron emisyon tomografi 69

Primary cutaneous anaplastic large cell lymphoma/Primer kutanöz

anaplastik büyük hücreli lenfoma 49

Primary mediastinal large B-cell lymphoma/Primer mediastinal büyük

B-hücreli lenfoma 78


Proteasome inhibitors/Proteozom inhibitörleri 218


QTc prolongation/QTc uzaması 80



Rituximab-induced thrombocytopenia/Rituksimab ile indüklenen

trombositopeni 87

SARS-CoV-2/SARS-CoV-2 80

Sendai virus/Sendai virüs 254

Severe inflammatory reaction/Şiddetli enflamatuvar reaksiyon 92

Severe inflammatory syndrome/Şiddetli enflamatuvar yanıt sendromu

92

Shoulder pad/Omuz yastığı 234

Sinusoidal obstruction syndrome/Sinüzoidal obstrüksiyon sendromu

286

Skin rash/Deri döküntüsü 82

Stem cell mobilization/Kök hücre mobilizasyonu 204


Sweet syndrome/Sweet sendromu 235

Systemic anaplastic large cell lymphoma/Sistemik anaplastik büyük

hücreli lenfoma 49




TP53/TP53 246


Treatment/Tedavi 41, 96, 314

Trisomy 8/Trizomi 8 92

Tyrosine kinase inhibitör/Tirozin kinaz inhibitörü 80

Uric acid/Ürik asit 286



Waldenstrom macroglobulinemia/Waldenström makroglobülinemisi

87


7. Immunohematology




Acute myeloid leukemia/Akut myeloid lösemi 111, 119, 231

Antibody/Antikor 321

Autoimmunity/Otoimmünite 1

BCL2/BCL2 119



CD200/CD200 119

Celiac/Çölyak 175

Children/Çocuk 336


Clinical severity/Klinik şiddeti 245

Convalescent plasma/Konvelesan plazma 321

COVID-19/COVID-19 245, 321

Diagnosis/Tanı 314

Eosinophilia/Eozinofili 166

Hematological parameters/Hematolojik parametreler 245


nakli 138


Immune dysregulation/İmmün disregülasyon 1

Immune/İmmün 175

Immunity/Bağışıklık 321





Kimura disease/Kimura hastalığı 166



Lymphadenopathy/Lenfadenopati 166

Lymphoproliferation/Lenfoproliferasyon 1


Minimal change disease/Minimal değişiklik hastalığı 166







Precision medicine/Hassas tıp 1

Primary immune deficiencies/Primer immün yetmezlikler 1



Red cell distribution width/Kırmızı küre dağılım aralığı 245


SUBJECT INDEX 2021



trombositopeni 87



92




Targeted therapy/Hedeflenmiş tedavi 1


Treatment/Tedavi 314



87

8. Infection Disorders









Blood cells/Kan hücreleri 98


Blood smear/Kan yayması 97

Blood smear/Periferik yayma 72

Blood/Kan 229

Cancer/Kanser 229

Chronic myeloid leukemia/Kronik myeloid lösemi 76, 80



Coronavirus/Koronovirüs 72

COVID-19/COVID-19 15, 76, 97, 98, 99, 157, 321, 329, 331

COVID-19, Drug-drug interaction/COVID-19 ilaç-ilaç etkileşimleri 80





Dysplasia/Displazi 72





Haematology/Hematoloji 57, 329

Hematology/Hematoloji 329





Infectious diseases/Bulaşıcı hastalıklar 98


lenfoma 89

Laboratory/Laboratuvar 97





Multiple myeloma/Multipl myelom 173


Myeloma/Myelom 229










92

Severity/Ciddiyet 329

SLE/SLE 99

Thrombotic microangiopathy/Trombotik mikroanjiyopati 155


TTP/TTP 99



9. Lymphoma















hücreli lenfoma 85

Diagnosis/Tanı 314



126

SUBJECT INDEX 2021


Ibrutinib/İbrutinib 338



lenfoma 89




Lymphoma/Lenfoma 145, 338







173








hücreli lenfoma 49


10. Molecular Hematology



Acute myeloid leukemia/Akut myeloid lösemi 111, 119, 264




Anemia/Anemia 242



BCL2/BCL2 119


CD200/CD200 119











telangiectasia 242





LncRNA/LncRNA 236


MALAT1/MALAT1 236





Multiple myeloma/Multipl myelom 246, 254




hemoglobinüri 236








92



Therapy/Tedavi 188

TP53/TP53 246



11. Multiple Myeloma

Adverse effect/Yan etki 41



Blood/Kan 229

Bortezomib/Bortezomib 96, 211

Cancer/Kanser 229




FDG/FDG 69


fenotipleme 228




Generic/Jenerik 41



SUBJECT INDEX 2021



Ixazomib/Iksazomib 87







Multiple myeloma/Multipl myelom 33, 153, 169, 173, 211, 218, 234,

246, 254

Myeloma/Myelom 229



Original/Orijinal 41










trombositopeni 87




TP53/TP53 246



87

12. Myelodysplastic Syndromes










92

Therapy/Tedavi 188


13. Myeloproliferative Disorders



Chronic myeloid leukemia/Kronik myeloid lösemi 76, 80, 343



COVID-19, Drug-drug interaction/COVID-19 ilaç-ilaç etkileşimleri 80





14. Neutropenia





















15. Stem Cell Transplantation









126


nakli 138










SUBJECT INDEX 2021



hemoglobinuri 195





286




16. Thrombosis


Antithrombin/Antitrombin 15




Children/Çocuk 336













Leukemia/Lösemi 227, 336









SLE/SLE 99




purpura 64

TTP/TTP 99



17. Thrombocytopenia





Celiac/Çölyak 175











Immune/İmmün 175







SLE/SLE 99


Thrombocytopenia/Trombositopeni 175, 181, 306



purpura 64


TTP/TTP 99


18. Other




Acute myeloid leukemia/Akut myeloid lösemi 231





Anemia/Anemia 242




Atrial fibrillation/Atriyal fibrilasyon 83



Bleeding/Kanama 83

SUBJECT INDEX 2021





Bone marrow/Kemik iliği 328





Cardiac tamponade/Kardiyak tamponad 83





Chronic lymphocytic leukemia/Kronik lenfositik lösemi 167, 274, 344


CLL/CLL 82


Complication/Komplikasyon 159


COVID-19/COVID-19 97, 98, 99, 329

Crystals/Kristaller 328


hücreli lenfoma 85


Diagnosis/Tanı 314






FDG/FDG 69




Haematology/Hematoloji 57



Hematopoietic stem cell Transplantation/Hematopoetik kök hücre



telangiectasia 242



Ibrutinib/Ibrutinib 82, 274







lenfoma 89












Ligneous cervicitis/Ligneous servisit 334

Ligneous membranes/Ligneous membranlar 334

LncRNA/LncRNA 236





MALAT1/MALAT1 236





Multiple myeloma/Multipl myelom 169, 211, 218, 234









Oxalosis/Oksalozis 328


hemoglobinuri 195, 236



Plasminogen/Plasminojen 334








Rare disease/Nadir hastalık 334



SUBJECT INDEX 2021



trombositopeni 87



92



Sickle cell trait/Orak hücre taşıyıcılığı 159


286

Skin rash/Deri döküntüsü 82

SLE/SLE 99

Splenic infarct/Dalak infarktı 159



hücreli lenfoma 49









TTP/TTP 99



87


19. Pathology


Acute lymphoblastic leukemia/Akut lenfoblastik lösemi 94, 326

Acute myeloid leukemia/Akut myeloid lösemi 119



Anemia/Anemi 326


BCL2/BCL2 119




Bone marrow/Kemik iliği 328



CD200/CD200 119


Chronic lymphocytic leukemia/Kronik lenfositik lösemi 167


Clinical severity/Klinik şiddeti 245



COVID-19/COVID-19 97, 98, 245, 329

Crystals/Kristaller 328

Diagnosis/Tanı 314





fenotipleme 228






Hematological parameters/Hematolojik parametreler 245


Ibrutinib/İbrutinib 338



Ixazomib/Iksazomib 87




LncRNA/LncRNA 236






MALAT1/MALAT1 236







173

Oxalosis/Oksalozis 328


hemoglobinüri 236







Red cell distribution width/Kırmızı küre dağılım aralığı 245


trombositopeni 87

SUBJECT INDEX 2021




Therapy/Tedavi 188



87

20. Autoimmune Disorders





Celiac/Çölyak 175











Immune/İmmün 175





SLE/SLE 99


Thrombocytopenia/Trombositopeni 175, 181



purpura 64

TTP/TTP 99



21. Transfusion

Acquired coagulopathies/Edinilmiş koagülapatiler 22




Blood coagulation/Kan pıhtılaşması 22

Chronic myeloid leukemia/Kronik myeloid lösemi 76



COVID-19/COVID-19 15, 76, 157, 321






Replacement therapies/Yerine koyma tedavileri 22

Transfusion medicine/Transfüzyon tıbbı 22

Advisory Board of This Issue (December 2021)

Ahmet Emre Eşkazan, Turkey

Ahmet Muzaffer Demir, Turkey

Ahu Demiröz, Turkey

Ali Zahit Bolaman, Turkey

Atakan Tekinalp, Turkey

Ayşegül Ünüvar, Turkey

Barna Gabor, Hungary

Buket Altınok Güneş, Turkey

Burhan Ferhanoğlu, Turkey

Cristina Tarango, USA

Elif Ümit, Turkey

Emre Tekgündüz, Turkey

Evangelos Terpos, Greece

Fortunato Morabito, Israel

Girish Vankataraman, USA

Gül Nihal Özdemir, Turkey

Hakan Özdoğu, Turkey

Hasan Çakmaklı, Turkey

Işınsu Kuzu, Turkey

İlknur Kozanoğlu, Turkey

John M. Bennett, USA

Maria K. Angelopoulou, Greece

Mario Tiribelli, Italy

Massimiliano Bonifacio, Italy

Mehmet Dal, Turkey

Meliha Nalçacı, Turkey

Meryem Albayrak, Turkey

Musa Karakükçü, Turkey

Naci Tiftik, Turkey

Nazan Sarper, Turkey

Neslihan Andıç, Turkey

Norbert Grzasko, Poland

Nükhet Tüzüner, Turkey

Onur Kırkızlar, Turkey

Ömür Kayıkçı, Turkey

Özgür Mehtap, Turkey

Sema Karakuş, Turkey

Seval Akpınar, Turkey

Sigbjørn Berentsen, Norway

Ümit Yavuz Malkan, Turkey

Volkan Hazar, Turkey

Volkan Karakuş, Turkey

Ya Zhang, China

Yahya Büyükaşık, Turkey

Zoltan Matrai, Hungary

Zühre Kaya, Turkey

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