tjh-2011-2

Issue 2

June 2011

40 TL

ISSN 1300-7777

Volume 28

Review Article Treatment of chronic lymphocytic leukemia Fabienne Mc Clanahan et al.; Heidelberg, Germany

Research Articles Detection of ABL mutations by dHPLC Yücel Erbilgin et al.; İstanbul, Turkey

hTERC analysis by FISH in AML Özge Özer et al.; Ankara, Turkey

T regulatory cells in multiple myeloma Ayşe Pınar Erçetin et al.; İzmir, Turkey

NO metabolism in patients with SCD Esra Güzeldemir et al.; Kocaeli, Ankara, Turkey

Perforin A91V and acquired HLH

Hamza Okur et al.; Ankara, Kırıkkale, Turkey

Acquired methemoglobinemia in infants Mehmet Mutlu et al.; Trabzon, Turkey

Cover Picture: Kevser Turay Yardımcı

2

Editor-in-Chief

International Review Board

Aytemiz Gürgey Associate Editors

Mutlu Arat Muzaffer Demir Reyhan Diz Küçükkaya Mehmet Ertem Hale Ören Mehmet Ali Özcan Ayşegül Ünüvar Celalettin Üstün Neşe Yaralı Akif Selim Yavuz Past Editors

Erich Frank Orhan Ulutin Hamdi Akan Senior Advisory Board

Orhan Ulutin Yücel Tangün Osman İlhan Language Editors

Corinne Can Scott Evans Statistic Editor

Mutlu Hayran Editorial Secretary

İpek Durusu Bengü Timoçin Cover Picture: Kevser Turay Yardımcı was born in 1942, Turkey. She was retired from Marmara University, Istanbul, Turkey.

The Cover Picture was exhibited at 36.

Turkish National Hematology Meeting.

Nejat Akar (Turkey) Görgün Akpek (USA) Serhan Alkan (USA) Çiğdem Altay (Turkey) Koen van Besien (USA) Ayhan Çavdar (Turkey) M.Sıraç Dilber (Sweden) Ahmet Doğan (USA) Peter Dreger (Germany) Thierry Facon (France) Jawed Fareed (USA) Gösta Gahrton (Sweden) Dieter Hoelzer (Germany) Marilyn Manco-Johnson (USA) Andreas Josting (Germany) Emin Kansu (Turkey) Winfried Kern (Germany) Nigel Key (USA) Korgün Koral (USA)

Abdullah Kutlar (USA) Luca Malcovati (Italy) Robert Marcus (United Kingdom) Jean Pierre Marie (France) Ghulam Mufti (UK) Gerassimos A. Pangalis (Greece) Santiago Pavlovsky (Argentina) Antonio Piga (Italy) Ananda Prasad (USA) Jacob M. Rowe (Israel) Jens-Ulrich Rüffer (Germany) Norbert Schmit (Germany) Orhan Sezer (Germany) Anna Sureda (Spain) Ayalew Tefferi (USA) Nüket Tüzüner (Turkey) Catherine Verfaillie (USA) Srdan Verstovsek (USA) Claudio Viscoli (Italy)

Contact Information

All other inquiries should be adressed to

Editorial Correspondence should be addressed to Dr. Aytemiz Gürgey Editor-in-Chief Address: Hacettepe University Faculty of Medicine Pediatric Hematology Department 06100 Sıhhiye, Ankara/Turkey Phone: +90 312 305 41 17 Fax: +90 312 305 41 17 E-mail: agurgey@hacettepe.edu.tr

TURKISH JOURNAL OF HEMATOLOGY Address: İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara/Turkey Phone: +90 312 490 98 97 Fax: +90 312 490 98 68 E-mail: info@tjh.com.tr ISSN: 1300-7777

Turkish Society of Hematology

Sahibi

Muhit Özcan, President Mutlu Arat, General Secretary Hale Ören, Vice President Muzaffer Demir, Research Secretary Teoman Soysal, Treasurer Fahir Özkalemkaş, Member Mehmet Sönmez, Member Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish Journal of Hematology’nin Türk Hematoloji Derneği İkdisadi İşletmesi tarafından yayınlanmasına karar vermiştir.

Web page www.tjh.com.tr Türk Hematoloji Derneği adına Muhit Özcan

Sorumlu Yazı İşleri Müdürü Aytemiz Gürgey

Yayın ve Yönetim Yeri

Türk Hematoloji Derneği Türk Ocağı Cad. 17/6 Cağaloğlu-Eminönü-İstanbul Üç ayda bir yayınlanan uluslararası bilimsel dergidir.

Baskı: ADA Ofset Matbaacılık Tic. Ltd. Şti.

Basım Tarihi: Mayıs 2011

Publisher: AVES Yayıncılık Address: Kızılelma cad. 5/3 34096 Fındıkzade-İstanbul-Turkey Phone: +90 212 589 00 53 Fax: +90 212 589 00 94 E-mail: info@avesyayincilik.com

A-I

AIMS AND SCOPE The Turkish Journal of Hematology is the regular publishing organ of the Turkish Society of Hematology. This periodical journal covers subjects on hematology. The journal is an independent, peer-reviewed international periodical, published quarterly (March, June, September and December) in English language. The Turkish Journal of Hematology is a nonprofit scientific peer reviewed journal.

Editorial Board of Turkish Journal of Hematology works under the principles of The World Association of Medical Editors (WAME), the International Council of Medical Journal Editors (ICMJE), and Committee on Publication Ethics (COPE).

The aim of the Turkish Journal Hematology is to publish original research papers of highest scientific and clinical value on hematology. Additionally, educational material, reviews on basic developments, editorial short notes, case reports, original views and letters from specialists on hematology, and hematology medicine covering their experience and comments as well as social subjects are published.

General Practitioners interested in hematology, and internal medicine specialists, are also our target audience, and we will arrange the Turkish Journal of Hematology according to their needs. The Turkish Journal of Hematology is indexed in - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Tübitak/Ulakbim Turkish Medical Index - Index Copernicus Subscription Information The Turkish Journal of Hematology is sent free of charge to hematologists and academicians in our country as well as to other specialists interested in hematology. All published volumes in full text can be reached free of charge through the web site www.tjh.com.tr Adress: Ilkbahar mah. Turan Güneş Bulvarı 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Web page: www.tjh.com.tr E-mail: info@tjh.com.tr Permissions Requests for permission to reproduce published material should be sent to the editorial office.

Editor: Prof.Dr. Aytemiz Gürgey Adress: Ilkbahar mah. Turan Güneş Bulvarı 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Web page: www.tjh.com.tr E-mail: info@tjh.com.tr Instructions for Authors Instructions for authors are published in the journal and on the web page www.tjh.com.tr Material Disclaimer The author(s) is (are) responsible from the articles published in the Turkish Journal of Hematology. The editor, editorial board and publisher do not accept any responsibility for the articles.

The journal is printed on acid-free paper.

A-II

INSTRUCTION TO AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, case reports, letters to the editor, and images in Hematology on subjects within the scope of hematology, on the condition that they have not been previously published elsewhere. All papers are subject to editorial revision for purpose of conformity to the style adopted by the Journal. Evaluation is a double blind kind of evaluation.

Original research articles Regular Articles Maximum length for a Regular Article is 4,000 words of text. Abstracts must not exceed 300 words with subheadings; objective, material and methods, results, conclusion. Submissions are limited to a total of 7 figures/tables. References should be limited to 50. The sections of a Regular Article should include Abstract, Introduction, Material and Methods, Results, Discussion, References, Figure Legends. Editorial Board of Turkish Journal of Hematology works under the principles of The World Association of Medical Editors (WAME), the International Council of Medical Journal Editors (ICMJE), and Committee on Publication Ethics (COPE).

Brief Reports Short manuscripts definitively documenting either experimental results or informative clinical observations will be considered as brief report. Brief Reports should not exceed 1,000 words of text not counting the abstract, figure legends, and references; abstracts must not exceed 300 words.

Review Articles Review articles should not exceed 4,000 words in length, must include an abstract of 300 words or fewer, and may not have more than 100 references.

Letters to the Editor Letters can include no more than 400 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title.

A-III

Images in Hematology Authors can submit for consideration an illustration (or, where appropriate, two or more related images) which is interesting, instructive and visually attractive, with a few lines of explanatory text and references. The images (e.g. a clinical photograph, radiology, cytology, histology, a laboratory test) should be submitted in a digital format.

Preparation of Manuscript Each of the following sections of the manuscript should be typed on separate pages. Title Page should include (in Turkish when possible): (a) title of the article in a concise but informative style, (b) first name, middle initial, last name of each author, (c) name of department(s) and institution(s) to which the work should be attributed, (d) name and address of author responsible for correspondence for the manuscript, (e) name and address of author to whom requests for reprints should be addressed, (f) source(s) of support in the form of grants, equipments, drugs, etc., and (h) short running title of no more than 40 characters.

Authorship Each author should have participated sufficiently in the work to take public responsibility for the content. Any part of an article critical to its main conclusions must be the responsibility of at least one author. All authors’ signatures should be included in the title page.

The signed statement on absence of conflict of interests between authors is required.

Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, or any other sources. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note must be included stating: This study was supported (in part) by research funding from (company name) to (authors’ initials). Grant support, if received, needs to be stated and the specific granting

institution(s) name(s) and grant numbers provided when applicable.

Authors are expected to disclose, on the title page of their manuscripts, any commercial or other associations that might pose a conflict of interest in connection with the submitted article. All funding sources supporting the work, and institutional or corporate affiliations of the authors, should be acknowledged on the title page.

Ethics When reporting experiments on human subjects indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation. An approval of research protocols by ethic committee in accordance with international agreements (Helsinki Declaration of 1975, revised 2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for the care and use of laboratory animals www.nap.edu/catalog/5140.html/) is required for experimental, clinical and drug studies. Do not use patient names, initials, or hospital numbers, especially in any illustrative material. Manuscripts reporting the results of experimental investigations on human subjects must include a statement to the effect that procedures had received official institutional approval. The statement on the informed consent of patients is required.

We frown upon unethical practices such as plagiarism, duplicate publication, ‘salami’ publication, and efforts to influence the review process with practices such as gifting authorship, inappropriate acknowledgements and references. Also, authors must respect patients’ right to privacy.

Abstract and key words: The second page should include an Abstract which does not exceed 300 words. For manuscripts sent from Turkey, a title and abstract in Turkish are required. The abstract should state the purpose of the study or investigation, basic procedures, methods, main findings, specific data, statistical significance and the principal conclusions.

Provide 3 to 10 key words below the abstract

to assist indexers. Use terms from the Medical Subject Headings List of Index Medicus.

The text should be divided into sections with headings as follows: Objective, Materials and Methods, Results and Conclusion. Other types of articles such as case reports, reviews, perspectives and editorials will be published according to uniform requirements.

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

Materials and Methods: Describe your selection of the observational or experimental subjects clearly. Identify the methods and procedures in sufficient detail to allow other workers to reproduce the results. Give references to established methods (including statistical methods), provide references and brief modified methods, give reasons for using them and evaluate their limitations. Identify all drugs and chemicals used, including generic name(s), dose(s) and route(s) of administration.

Statistics: Describe statistical methods in enough detail to enable a knowledgeable reader with access to the original data to verify the reported results. Give details about randomization, describe treatment complications, give number of observations, and specify any computer program used.

Results: Present your results in logical sequence in the text, tables and illustrations.

Do not repeat in the text all the data in the tables or illustrations; emphasize or summarize only important observations.

Discussion: Emphasize the new and important aspects of the study and the conclusions that follow them. Link the conclusions with the goals of the study but avoid unqualified statements and conclusions not completely supported by your data.

References: Identify references in text, tables and legends by Arabic numerals in parentheses. Number references consecutively in the order in which they are first mentioned in the text. The titles of the journals should be abbreviated according to the style used

A-IV

in Index Medicus; consult List of Journals Indexed in Index Medicus. Include among the references any papers accepted but not yet published, designating the journal and followed by “in press”.

Articles in Journals 1. List all authors Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukemia inhibitory factor (LIF) maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-7.

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;2:742-4.

3. Complete book Adams DO, Edelson PJ, Koren HS. Methods for studying mononuclear phagocytes. San Diego: Academic Press, 1981.

4. Chapter of book Smolen JE, Boxer LA. Functions of Neutrophils. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed.

New York: McGraw-Hill, 1991: 780-94.

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;84:2821-3.

7. CD-ROM Anderson SC, Poulsen KB, Andersüon’s electronic atlaso hematology [CD-ROM].

Philadelphia: Lippincott Williams&Wilkins: 2002.

8. Journal article on the internet Abood S. Quality improvement intititative in nursing homes: The ANA acts in an advisory role. Am J Nurs [serial on the Internet.] 2002 n [cited 2002 Aug 12]:102(6)[about 3 p.]

Available from: http:/www.nursing world.org/ AJN/2002/June/Wawatch.htm.

9. Homepage/Web site Cancer-Pain.org [homepage on the Internet].

New York: Association of Cancer Online Resources, Inc.: c2000-01 [updated 2002 May 16: cited 2002 Jul 9]. Available from: hhtp:// www.cancer-pain.org/.

Tables Type each table on a separate sheet. Number tables consecutively in the order of appearance in the text and supply a brief title for each. Give each column a short or abbreviated heading.

Place explanatory statistical measures of variations such as standard deviation or standard error of mean. Be sure that each table is cited in the text.

Illustrations Figures should be professionally drawn and photographed. Please send sharp, glossy, black and white photographic prints, usually 9 x 13 cm. Affix a label to the back of each figure indicating the number of the figure, first author’s name and top of the figure. Type legends for illustrations double-spaced, starting on a separate page with Arabic numerals corresponding to the illustrations. Explain the internal scale and identify method of staining.

Units of Measurement Measurements should be reported in the metric system in terms of the International System of Units (SI). Consult SI Unit Conversion Guide, New England Journal of Medicine Books 1992, when necessary.

Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for which an abbreviation stands should precede its first use in the text unless it is a standard abbreviation.

ONLINE MANUSCRIPT SUBMISSION PROCESS Manuscripts can be submitted online at www.journalagent.com/tjh/ The online system consists of four main parts: manuscript submission module (MSM), editorial module, admin module and referee module. The editorial module, admin module and referee module work on the background and will not be open to the end user. The term module used in this document refers only to the MSM. As part of the peer-review system, authors will also receive the referee reports and can observe he current status of their manuscript(s) online. An online help is also available during the submission process. The module accepts the body of the manuscript as a whole document; thus, documents should be completed as a .doc or .rtf file before submission. The supported file extensions, fonts and other formats are given in Table 1.

Table 1.

Supported formats .doc (MS Office for Windows or Macintosh) .rtf (rich text format)

Supported fonts Arial Times Helvetica Times New Roman Courier

Tables, figures and pictures Tables should be created in your original wordprocessing software or inserted in the original file from Excel or another compatible software.

Please ensure the table or figure created complies with the limitations mentioned in Table 1. Tables created as a picture file are problematic and are not advised.

Figures should be embedded in the original file, but the system also requires that they be sent separately. The supported image files are given in Table 1.

Symbols Special characters not available on the keyboard can be accessed either from the insert menu (select symbol) or by selecting symbol as a font from the font window of the Formatting toolbar. Please check these characters in your

A-V

original file and proofs as the softwares tend to replace these characters with others if they are unreadable.

Submission online To submit a manuscript, you must first establish a login name and password, which can then be used indefinitely. After you login to the module, the first page accessed allows you to track the status of your current or previous manuscripts. To submit a new manuscript, login with your user name and password, then click on “Author” on your main menu. From this page, select “new article” pull-down menus. The next page is for the details of the institutions of the authors. On the next page where the authors are listed, the instructions and menus allow you to select the appropriate institution for each author from the list. Please remember to check the box next to the corresponding Supported images .bmp .jpg .jpeg .gif .tif

author. The title page requires only the title; special characters listed on the bottom of the title input window can be used when needed.

On the summary page which follows, please write the abstract of your manuscript in the appropriate window. This is followed by a keyword input page, which allows up to 10 keywords. Any special notes to the editor can be recorded on the next page. The body and figures of the manuscript are submitted on the next page. Locate the manuscript in your PC, then write the type of the file you are sending and give a description. Use this page to also send your images. Send your manuscript using the designated button on the next page. All pages are supported with help menus; if you require additional help or experience a problem, please send an e-mail to info@tjh.com.tr

Manuscripts that have passed an initial screening by the. Editors are reviewed by members of the Editorial Board and/or other experts in the field. The Editors select the reviewers and make the final decision on the manuscript.

Referees who review a manuscript remain unknown to the authors. Every manuscript is treated by the Editors and reviewers as privileged information, and they are instructed to exclude themselves from review of any manuscript that may involve a conflict of interest or the appearance of such.

Following initial peer-review, articles judged worthy of further consideration often require revisions. Revised manuscript generally must be received within 3 months of the date on the initial decision. Extensions must be requested from the Associate Editor at least 2 weeks before the 3-month revision deadline expires.

Otherwise Turkish Journal of Hematology will reject manuscripts which do not received within 3 months of the date on the initial revesion decision.

A Copyright transfer and conflict of interest form signed by all authors, must also be submitted by fax to +90 3124909868. Both forms can be found at the web site www.tjh. com.tr Authors of accepted manuscripts will receive electronic page proofs directly from the printer and are responsible for proofreading and checking the entire article, including tables, figures, and references. Page proofs must be returned within 48 hours to avoid delays in publication.

English-language editing All manuscripts are professionally edited by English language editor before publication.

Online Early Turkish Journal of Hematology published abstracts of accepted articles online in advance of their publication in a printed issue.

CONTENTS Review Article 86

Current strategies for the diagnosis and management of chronic lymphocytic leukemia (CLL), with a focus on poor-risk CLL: A review Fabienne Mc Clanahan, Peter Dreger, Heidelberg, Germany

Research Articles 97

ABL gene kinase domain mutation scanning by denaturing high performance liquid chromatography sequencing method

103

Fluorescence in situ hybridization analysis of the hTERC region in acute myeloid leukemia patients

107

The correlation between T regulatory cells and autologous peripheral blood stem cell transplantation in multiple myeloma

115

Nitric oxide in gingival crevicular fluid and nitric oxide synthase expression in the gingiva of patients with sickle cell disease

125

The frequency of A91V in the perforin gene and the effect of tumor necrosis factor-α promoter polymorphism on acquired hemophagocytic lymphohistiocytosis

Yücel Erbilgin, Suzin Çatal, Ahmet Emre Eşkazan, Özden Hatırnaz, Teoman Soysal, Uğur Özbek, İstanbul, Turkey Özge Özer, Tuğçe Bulakbaşı Balcı, Zerrin Yılmaz, Feride İffet Şahin, Ankara, Turkey

Ayşe Pınar Erçetin, Safiye Aktaş, Özden Pişkin, Halil Ateş, Zeynep Filiz Zadeoğluları, Nur Hilal Turgut, Mehmet Ali Özcan, İzmir, Turkey Esra Güzeldemir, Hilal Uslu Toygar, Nebil Bal, Ruksan Anarat, Can Boğa, Kocaeli, Ankara, Turkey

Hamza Okur, Şule Ünal, Günay Balta, Didem Efendioğlu, Eren Çimen, Mualla Çetin, Aytemiz Gürgey, Çiğdem Altay, Fatma Gümrük, Ankara, Kırıkkale, Turkey

131

Acquired methemoglobinemia in infants

Mehmet Mutlu, Erol Erduran, Yakup Aslan, Trabzon, Turkey

Case Reports 135

Splenic artery embolization: An alternative approach in a critically ill patient with autoimmune hemolytic anemia

139

Wiskott-Aldrich syndrome mutation in two Turkish siblings with X-linked thrombocytopenia

142

Are cup-like blasts specific to AML patients with FLT3 ITD and a normal karyotype? An ALL case report and review of the literature

Mine Durusu Tanrıöver, Bora Peynircioğlu, Begüm Ergan Arsava, Arzu Topeli İskit, Ankara, Turkey

Göksel Leblebisatan, Ali Bay, Noriko Mitsuiki, Osamu Ohara, Kenichi Honma, Kohsuke İmai, Shigeaki Nonoyama, Gaziantep, Turkey, Japan Özgür Mehtap, Elif Birtaş Ateşoğlu, Emel Gönüllü, Hakan Keski, Abdullah Hacıhanefioğlu, Kocaeli, Turkey

Letters to the Editor 146

Central retinal artery occlusion as the presenting sign of essential thrombocythemia

149

“Arbitrary” criterion for the diagnosis of acute leukemia

151

Paclitaxel therapy and immune thrombocytopenic purpura: Coincidence or association?

153

Interaction between hereditary spherocytosis and the beta-thalassemia trait: A case report

Gül Arıkan, Ali Osman Saatci, Selda Kahraman, Özden Pişkin, Süleyman Men, Bülent Ündar, İzmir, Turkey Abbas Hashim Abdulsalam, Baghdad, Iraq

Ümmügül Üyetürk, Şerife Hülya Arslan, Meltem Kurt Yüksel, Fevzi Altuntaş, Ankara, Turkey Sunita Sharma, Sonal Jain Malhotra, Richa Chauhan, New Delhi, India

Images in Hematology 155

Hematogones in the bone marrow of a child with rubella virus-associated immune thrombocytopenic purpura concomitant with iron deficiency anemia Faruk Barlık, Emel Özyürek, Feride Duru, Samsun, Turkey

158

Multiple myeloma with multilobated plasma cell nuclei

Nergiz Erkut, Ümit Çobanoğlu, Mehmet Sönmez, Trabzon, Turkey

A-VI

86

Review

Current strategies for the diagnosis and management of chronic lymphocytic leukemia (CLL), with a focus on poor-risk CLL: A review Kronik lenfositik lösemi (KLL) tanı ve tedavisine ilişkin, özellikle yüksek riskli KLL’ye odaklanan güncel stratejiler: Derleyici bir inceleme Fabienne Mc Clanahan, Peter Dreger Department of Internal Medicine V, University Hospital of Heidelberg, Heidelberg, Germany

Abstract

Despite substantial advancement in the understanding and treatment of chronic lymphocytic leukemia (CLL), a standard curative approach does not exist. The choice of treatment is generally based on the existence of biological and genetic factors associated with the prediction of prognosis, individual response to therapy, and duration of remission. About 20% of patients that require treatment have an aggressive disease course and die within a few years, despite early initiation of intensive therapy (poor-risk CLL).

Poor-risk CLL can be predicted by the presence of genomic markers, and the quality and duration of response to purine-analogue-based treatment. Within this patient subgroup alternative treatment approaches such as alemtuzumab or new substances such as flavopiridol or IMiDs® should be considered.

To date, the only treatment bearing curative potential is allogeneic stem cell transplantation; in contrast to conventional immunochemotherapy, it can provide long-term disease control, even in patients with del 17p or other unfavorable biological and clinical risk factors. The aim of this review was to outline the current strategies for the diagnosis and management of CLL, with a focus on high-risk CLL. (Turk J Hematol 2011; 28: 86-96)

Key words: CLL, genetics, poor-risk, treatment, allogeneic stem cell transplantation Received: March 17, 2011

Accepted: April 26, 2011

Özet Kronik lenfositik löseminin (KLL) tanı ve tedavisine yönelik önemli gelişmelere karşın, şifa sağlayıcı (küratif) standart bir yaklaşım henüz bulunmamaktadır. Tedavi seçimi genellikle prognozu öngören biyolojik ve genetik faktörlerin varlığına, tedaviye alınan bireysel yanıt ve iyileşme (remisyon) süresine dayanır. Tedavi edilmesi gereken hastaların yaklaşık %20’si hızla ilerleyen bir klinik gidiş gösterirler ve yoğun tedaviye erkenden başlanmasına karşın birkaç yıl içerisinde yaşamlarını yitirirler (yüksek riskli KLL). Yüksek riskli KLL olasılığı, genomik belirteçler yanında purin-analogu temelli tedaviye alınan yanıtın niteliği ve süresiyle önceden kestirilebilir. Bu hasta altgrubunda, alemtuzumab gibi alternatif tedavi yaklaşımları ya da flavopiridol ve IMiD® grubu gibi yeni ilaçlar göz önüne alınmalıdır.

Günümüzde bu hastalarda şifa sağlayıcı potansiyele sahip tek tedavi seçeneği allogeneik kök hücre naklidir. Bu yöntemle, geleneksel immünokemoterapinin aksine, del 17p veya diğer olumsuz biyolojik ve klinik risk faktörlerine sahip hastalarda bile hastalığın uzun süreli denetimi sağlanabilir. Bu derleyici incelemede, yüksek riskli KLL’ye odaklanarak KLL tanı ve tedavisine ilişkin güncel stratejilerin özetlenmesi amaçlanmıştır. (Turk J Hematol 2011; 28: 86-96) Anahtar kelimeler: KLL, genetik, yüksek risk, tedavi, allogeneik kök hücre transplantasyonu Geliş tarihi: 17 Mart 2011

Kabul tarihi: 26 Nisan 2011

Address for Correspondence: Dr. Peter Dreger, Department Medicine V, University of Heidelberg, Im Neuenheimer Feld 410 69120 Heidelberg, Germany Phone: 004962215639240 E-mail: Peter.Dreger@med.uni-heidelberg.de doi:10.5152/tjh.2011.23

Turk J Hematol 2011; 28: 86-96

Epidemiology and Clinical Features Chronic lymphocytic leukemia (CLL) is one of the most common lymphoid malignancies, accounting for more than 10% of all lymphoid neoplasms, and is the most common adult leukemia in Western countries, with an age-adjusted annual incidence rate of about 4 cases per 100.000 men and women [1,2]. While the median age at diagnosis is over 70 years, approximately 30% of patients are diagnosed at an age of ≤65 years. The disease has a slight male predominance (male:female ratio: 1.5-2:1).

Clinical features are highly variable, and most patients are asymptomatic. As the disease proceeds, fatigue, splenomegaly, hepatomegaly, lymphadenopathy, and extranodal infiltrates are observed.

Another hallmark is immune suppression and deficiency, including hypogammaglobulinemia, autoimmune phenomena, and impaired response to vaccination, which is further exacerbated by therapy-related immunosuppression [3].

Diagnosis To unify the criteria for diagnosis and response assessment a working group sponsored by the National Cancer Institute first published guidelines for the design and conduct of clinical trials on CLL in 1988 [4]. Following an update in 1996, the International Workshop on CLL (iwCLL) revised the guidelines, taking into account the substantial progress that had been made in the understanding and treatment of the disease [5]. The recommendations for diagnosis and treatment discussed in this review are based on the iwCLL criteria.

The diagnosis of CLL requires the presence of cells with a CLL-specific immunophenotype, along with ≥5.000 B-lymphocytes µL–1 in peripheral blood [5].

The presence of CLL cells with a lower B-lymphocyte count in the absence of lymphadenopathy, organomegaly, or cytopenia is now defined as monoclonal B-lymphocytosis (MBL) [6]. The presence of lymphadenopathy or splenomegaly, and a B-lymphocyte count ≤5.000 µL–1 is defined as small lymphocytic lymphoma (SLL). More than 55% prolymphocytes in the blood suggests a diagnosis of B-cell prolymphocytic leukemia (B-PLL) [7].

Flow cytometry is essential for determining the clonality of B-lymphocytes and the following characteristic CLL-cell-surface phenotypes: the presence of CD19, CD5, CD23 and CD43, weak expression of CD20 and CD79b, and kappa or lambda immunoglobulin light chains [8,9]. The expression of CD38 is variable, but has prognostic significance and should therefore be evaluated [10,11]. Flow cytometry is

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

87

also indispensable for differentiating CLL from other lymphoproliferative diseases, such as hairy cell leukemia, leukemic manifestations of mantle cell lymphoma, marginal zone lymphoma, splenic marginal zone lymphoma, and follicular lymphoma.

Bone marrow investigation is generally not required for the diagnosis of CLL, especially in patients without cytopenia and those not requiring treatment. Bone marrow aspiration and biopsy may be indicated, however, when treatment is required, as the extent and pattern (diffuse vs. non-diffuse) of bone marrow infiltration can provide valuable information on tumor burden and factors that may contribute to cytopenia. Post-treatment bone marrow investigation is recommended in patients with persisting cytopenia of unknown origin. In SLL the diagnosis should be verified via histopathological evaluation of a lymph node biopsy specimen.

Clinical Staging and Prognosis As the clinical course of CLL varies widely, staging systems have been developed to stratify patients into different risks groups for predicting survival.

The 2 most commonly used are the Rai and Binet staging systems [12,13]. Both systems consist of parameters that are obtained via clinical examination and standard laboratory tests, and are therefore easy to obtain. Ultrasound and computed tomography are not required, although they increase the accuracy of the assessment of intra-abdominal lymph nodes and organomegaly. Table 1 outlines the parameters that define the clinical stages. To account for extreme heterogeneities observed within each risk group, Wierda et al. proposed a new prognostic index for previously untreated CLL patients for predicting overall survival (OS) [14]: in the Rai staging system age, absolute lymphocyte count, gender, b2-microglobulin concentration, and the number of involved lymph nodes were independently associated with OS. This index was validated in an independent patient cohort by a Mayo Clinic study [15]. A recent observational database study by GIMEMA (Gruppo Italiano Malattie EMatologiche dell´Adulto) also confirmed the utility of the index for predicting time to first treatment [16].

Indications for Treatment As there is no standard curative approach for CLL, the decision to begin treatment is based on the development of symptoms and disease activity.

Newly diagnosed asymptomatic patients should be monitored without therapy, as there is no documented benefit from early anti-leukemic treatment [5].

Patients with symptomatic, advanced, or rapidly pro-

88

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

Turk J Hematol 2011; 28: 86-96

Table 1. Rai and Binet clinical staging Risk group

Rai

Binet

Low

0: lymphocytosis with CLL cells in PB and/or BM, lymphoid cells >30%

A: Hb ≥100 g L-1 (10 g dL-1), platelet count ≥100×109 L-1, and up to 2 lymph node areas involved*

Intermediate 1/2: Lymphocytosis, enlarged lymph nodes at any

site, splenomegaly and/ or hepatomegaly

High

3/4: Disease-related anemia (Hb <110 g L-1 [11 g dL-1]) or thrombocytopenia (plt <100x109 L-1)

B: Hb ≥100 g L-1 (10 g dL-1), platelet count ≥100x109 L-1, and organomegaly greater than that defined for stage A (i.e. ≥3 areas of nodal or organ enlargement) C: Hb <100 g L–1 (10 g dL–1) and/or a platelet count <100x109 L–1, irrespective of organomegaly

*Areas of involvement considered for staging 1. Head and neck, including the Waldeyer ring (this counts as 1 area, even if >1 group of nodes is enlarged) 2. Axillae (involvement of both axillae counts as 1 area) 3. Groins, including superficial femorals (involvement of both groins counts as 1 area) 4. Palpable spleen 5. Palpable liver

gressive disease usually benefit from treatment.

Definitions of disease activity are listed in Table 2.

The decision to begin second-line treatment generally follows the same guidelines.

Factors Guiding the Choice of Treatment Physical fitness and comorbidity Once treatment is indicated, each patient’s individual physical condition and degree of co-morbidity should be evaluated. In completely independent patients with no comorbidity and otherwise normal life expectancy (go-go patients) aggressive chemotherapy aimed at the prolongation of survival is recommended. The goal in patients with some co-morbidity, impaired organ function, and reduced performance status (slow-go patients) is to achieve disease remission via a less aggressive approach. Patients that are severely handicapped and have high comorbidity (no-go patients) might often fare best with supportive palliative care only. Physical fitness can be determined empirically or by using the cumulative illness rating scale (CIRS) [17].

Biological prognostic factors Substantial progress has been made in the identification of biological and genetic factors that are strongly associated with the prediction of prognosis, individual response to therapy, and duration of remission.

Several studies reported that elevated serum thymidine kinase (sTK), b2-microglobulin, and soluble CD23 may predict OS or progression-free survival (PFS) [18-21]. Using fluorescence in-situ hybridization (FISH) cytogenetic aberrations can be identified in more than 80% of CLL patients [22]. The most common chromosomal aberrations are del 13q, del 11q, trisomy 12, del 17p, and del 6q, with 13q deletion indi-

Table 2. Definitions of disease activity At least 1 of the following: 1. Evidence of progressive marrow failure: anemia and/or thrombocytopenia 2. Massive (i.e. ≥6 cm below the left costal margin) or progressive or symptomatic splenomegaly 3. Massive nodes (i.e. ≥10 cm in diameter), or progressive or symptomatic lymphadenopathy 4. Progressive lymphocytosis with an increase >50% over a 2-month period, or lymphocyte doubling time (LDT) <6 months.

5. Autoimmune anemia and/or thrombocytopenia 6. Constitutional symptoms: unintentional weight loss, significant fatigue, fevers, night sweats

cating the best prognosis with a median survival of >10 years and 17p deletion indicating a particularly poor prognosis with a median survival of <3 years, even with modern fludarabine/rituximab-based frontline treatments.

Somatic mutations in the gene encoding the variable region of the heavy chain of immunoglobulins (IGHV) occur in approximately 50% of CLL patients [23]. Patients lacking a mutation in this region (unmutated IGHV) tend to have a more progressive and advanced form of CLL than patients with mutated IGHV. This was first shown retrospectively in patients treated in the pre-purine-analogue era, but has been confirmed in prospective studies using fludarabine combination regimens [11,24]. As testing for IGHV mutation status is complex and expensive, ZAP-70 expression was reported to correlate with the expression of unmutated IGHV [11,24,25]; however, the association between ZAP-70 and IGHV is not definitive. According to the iwCLL guidelines, with the exception of del 17p FISH, these tests should not

Turk J Hematol 2011; 28: 86-96

be used in general practice to guide clinical decision-making; however, it is essential to consider the existence and importance of these tests at the time of first diagnosis.

First-line Treatment Go-go patients without del 17p Following traditional treatment approaches consisting of alkylating agents (mostly cyclophosphamide or chlorambucil) with or without nucleoside analogue-based substances, the combination of monoclonal antibodies with chemotherapy increased the complete response (CR) rate from 4% [26] to 70% [27-30]. A large phase III trial that randomly assigned patients to 6 courses of fludarabine and cyclophosphamide (FC), or rituximab-FC (R-FC) was conducted by the German CLL Study Group (GCLLSG) (CLL8 trial). At 3 years post treatment, 65% of the patients in the chemoimmunotherapy group were progression free, compared to 45% in the chemotherapy group (HR:.56; 95% CI: .46-.69; p<0.0001) [31]. Additionally, 3-year OS was significantly longer in the R-FC group (87% vs.

83%, p=0.01), and R-FC treatment was associated with a significantly higher complete remission (CR) rate and longer duration of response. Although R-FC was more frequently associated with grade 3 and 4 neutropenia and leucopenia, it is now considered the gold standard treatment for physically fit patients without 17p deletion. In another GCLLSG phase II trial previously untreated patients received 6 cycles of bendamustine (90 mg m-2 on d 1 and 2) with rituximab (375 mg m-2 for the first cycle and 500 mg m-2 for subsequent cycles, repeated every 28 d) (R-B regimen) [32]. The overall response (OR) rate was 91%, including 33% of patients with CR. Hematopoietic and overall toxicity of R-B was modest. After 18 months 76% of the patients were still in remission, while median progression-free survival (PFS) had not been reached. R-B can therefore be considered safe and effective. The GCLLSG is currently conducting a randomized phase III trial to make a direct comparison of R-B and R-FC. Variations of the R-FC regimen have been tested extensively. For example, Bosch et al. conducted a phase II trial with 29 previously untreated CLL patients receiving rituximab plus FCM (mitoxantrone), followed by rituximab maintenance every 3 months for up to 2 years [33]. The OR, MRD- (minimal residual disease) negative CR, MRD-positive CR, and PR rates were 93%, 46%, 36%, and 11%, respectively, proving the efficacy of the regimen.

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

89

Another approach was substitution of fludarabine by pentostatine (R-PC). A phase III randomized trial of R-FC versus R-PC in previously untreated and minimally treated CLL patients showed that there werenâ&#x20AC;&#x2122;t any statistical differences between OS, response, or infection rates [34].

The role of autologous stem cell transplantation Autologous hematopoietic stem cell transplantation (autoHSCT) is considered an attractive treatment alternative for a select group of patients.

Pioneers in the field of autoHSCT for CLL are Gribben et al. from the Dana Faber Cancr Center [35]. An update published in 2005 showed that relapses continued to occur after 10 years of followup, translating into a 6-year PFS of 30% and a 6-year OS of 58% [36]. In the MRC pilot study, a large multicenter phase II trial on autoHSCT as a coponent of first-line CLL treatment, the 5-year OS and PFS rates were 78% and 52%, respectively [37]. An update of the GCLLSG CLL3 study, which had a similar design, reported a median OS of 10.5 years and a median PFS of 6.8 years after early autoHSCT [38]. The first, and to date, only phase III randomized trial was conducted by the EBMT; 39 patients with CR after first- or second-line treatment were randomized to consolidating autoHSCT or observation. Median event-free survival (EFS) was 24.4 months (range: 16.7-32 months) in the observation group and 51.2 months (range: 39.8-62.5 months) in the autoHSCT group, indicating a 5-year EFS of 24% and 42%, respectively. While autoHSCT almost doubled event-free survival (EFS) and time to retreatment, there wasnâ&#x20AC;&#x2122;t a significant difference in OS (5-year OS was 84% and 86%, respectively). In addition, several studies indicate that autoHSCT fails to achieve durable MRD negativity [40,41], which means that autoHSCT cannot be considered as a curative treatment in CLL. Moreover, long-term follow-up observations have raised concerns about the increased incidence of therapy-related myeloid neoplasms (MDS and AML) following autoHSCT. In the Dana Faber and MRC series the 5- and 8-year incidence of therapy-related myeloid neoplasms was 12% [36,42], versus a 10-year incidence rate of 8% in the CLL3 trial [38]. Due to these limitations, autoHSCT cannot be recommended as a standard approach in CLL and should only be used in clinical trials.

Go-go patients with del 17p As patients carrying del 17p have a high risk of very poor outcome with fludarabine-based regimens that include bendamustine, alternative treatment

90

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

approaches should be considered. It is well known that alemtuzumab has a similar affect in del 17p and non-del 17p CLL patients [43]. In the ongoing prospective GCLLSG CLL2o study patients with del 17p or refractoriness to fludarabine received subcutaneous alemtuzumab combined with oral dexamethasone, followed by alemtuzumab maintenance or allogeneic hematopoietic stem cell transplantation (alloHSCT). As of September 2010, 80 patients were enrolled in the protocol, of which 31 without prior therapy had del 17p and 17 that relapsed had del 17p [44]. OR and CR were 100% and 23%, respectively, in the del 17p first-line group, which are considerably higher than those in the CLL8 study F-CR group (71% and 5%, respectively). Adverse events were hematotoxicity and grade 3/4 cytomegaly virus (CMV) infection (16%); however, the response duration following alemtuzumab is usually limited, making the search for effective first-line consolidation mandatory. To date, the only treatment with the potential for longterm disease control in the del 17p patient subgroup is alloHSCT, which will be discussed in detail below.

Slow-go patients with and without del 17p Patients with relevant comorbidity may be offered chlorambucil, bendamustine, or a dose-reduced fludarabine-containing regimen, with or without rituximab, depending on the fragility of the patient [45,46]. Alemtuzumab can also be considered in appropriate patients (i.e. those with del 17p). The GCLLSG is currently conducting an open-label 3-arm randomized phase III trial to compare the efficacy and safety of the new monoclonal CD20 antibody RO5072759 (GA101) plus chlorambucil (GClb) to those of rituximab plus chlorambucil (RClb) or chlorambucil only (Clb) in previously untreated patients with comorbidity.

Second-line Treatment For selecting the appropriate indication and regimen for salvage treatment, the same criteria for first-line therapy primarily apply; start only if symptoms or rapid lymphoproliferation are present, and consider comorbidity and the presence of del 17p.

In addition, an important factor guiding the choice of salvage treatment is the quality and duration of response to the previous treatment line; patients that relapse >12 months after purine analogue monotherapy or 24 months after completion of a modern combination treatment might benefit from repetition or modest escalation of the previous regimen, e.g. R-FC after F or FC, and R-B after R-FC.

Turk J Hematol 2011; 28: 86-96

Before starting second-line treatment reassessment of 17p status is highly recommended to avoid unnecessary exposure to ineffective drugs in cases of clonal evolution. In contrast, all patients that relapse sooner must be regarded as having poorrisk CLL and treated accordingly.

Definition of Poor-Risk CLL About 20% of patients with CLL that require treatment have an aggressive course and die within a few years of diagnosis, despite early initiation of intensive therapy. The hallmark of this so called poor-risk CLL is pre-existent or rapid development of resistance to the current standard combination regimens. Poor-risk CLL can be partially predicted by the presence of defined genomic markers.

Molecular markers As outlined above, the existence of del 17p is associated with poor prognosis [22]. Patients with 17p deletion are often resistant to standard chemotherapy regimens. Following initial results of retrospective analyses [47], the adverse impact of del 17p was confirmed by several prospective phase III clinical trials using purine-analogue-based therapy. The LRF CLL4 trial that included 777 CLL patients that required treatment reported that patients with del 17p had significantly poorer response to fludarabinebased treatment and shorter PFS [28]. In a Spanish trial del 17p was associated with a significantly lower CR rate [33], and in the GCLLSG CLL4 (F vs. FC) and CLL8 trials (FC vs. FCR) del 17p was associated with dramatically lower CR, OR, OS, and PFS [31].

Recent data suggest that the vast majority of patients with del 17p had mutations of the remaining allele of the TP53 gene located in the deleted region of 17p [48]. Whereas TP53 mutations in general led to significantly shorter survival (p=0.002), survival was equally poor in patients with TP53 mutation only (5.5 months), TP53 mutation plus del 17p (7.6 months), and del 17p only (5.4 months). In a recent analysis of the GCLLSG CLL4 trial (F vs. FC) TP53 mutation was observed in 8.5% of the patients, of which 4.5% did not have del 17p [49]. None of the patients with TP53 mutation achieved CR, and median PFS and OS were significantly shorter in the group with TP53 mutation (p<0.001). As the outcome of patients with del 17p and/or TP53 mutation-both individually and combined-was very poor, it was recently proposed that these patients be considered as ultra-high risk [50].

In 2010 Oscier et al. published a comprehensive report on the prognostic significance of age, gender, and biomarkers in the prediction of treatment

Turk J Hematol 2011; 28: 86-96

response, PFS, and OS reported by a prospective randomized British CLL4 trial [51]. Using the factors identified as independent predictors for PFS, they subdivided CLL4 patients into 3 risk groups: 6% with known TP53 loss >10% were considered poor risk and 72% without TP53 loss and at least 1 of the following factors-unmutated IGHV, IGHV3-21 usage, 11q deletion, and/ or b-2 microglobulin >4 m Lâ&#x20AC;&#x201C;1-were considered intermediate risk. In all, 22% of patients were considered good risk, defined as none of the above factors and mutated IGHV. The 5-year PFS rates in these 3 groups were 0%, 12%, and 34%, respectively, and the corresponding 5-year OS rates were 9%, 53% and 79%, respectively, which confirmed the poor outcome of patients with del 17p and the prognostic impact of unmutated IGHV, b2-microglobulin, and 11q deletion. As this is a novel approach to risk stratification in CLL, it needs to be validated in patient cohorts treated with newer combination therapies, including monoclonal antibodies.

Response to treatment and duration of remission Independent of the presence of genomic poor-risk markers, the overriding predictor of poor-risk disease is the response to purine-analogue-based treatment. According to the iwCLL guidelines, every clinical response that is not CR or PR (e.g. stable disease, non-response, or progressive disease) should be rated as a treatment failure. Refractory disease is defined as treatment failure or disease progression within 6 months of the completion of the last antileukemic treatment. Early data from the M.D. Anderson Cancer Center on 174 patients with progressive or advanced CLL that received first-line therapy with fludarabine or fludarabine combined with prednisone showed that patients that did not achieve a clinical response had significantly shorter OS [52]. Patients with residual disease or nonresponse after fludarabine combined with cyclophosphamide and rituximab (R-FC) also had significantly reduced OS [30].

Median survival was also significantly reduced in patients that initially responded to R-FC, but then relapsed within 36 months, as compared to those that relapsed â&#x2030;Ľ36 months after R-FC treatment (p<0.0001) [53]. In an analysis of the CLL8 trial R-FC patients with PFS of 12-24 months (n=43) had a median post-relapse OS of <40 months. Outcomes for relapsing patients treated with FC within the same time interval were even worse [54].

The poor-risk associated with resistant disease is reflected in both the iwCLL guidelines and the EBMT (European Group for Blood and Marrow Transplantation) transplant consensus. According to

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

91

iwCLL, patients with resistant disease (defined as a short time to progression after the first treatment) and/or leukemia cells with del 17p should be offered alternative treatment approaches such as alloHSCT. According to the EBMT consensus, patients with non-response or early relapse (within 12 months) after purine analogue treatment relapse within 24 months of having achieved a response with purine-analogue-based combination therapy, or autologous transplantation and TP53 abnormalities requiring treatment are potential candidates for alloHSCT [55].

Treatment of Poor-Risk CLL As outlined in detail above, stable disease, nonresponse, progressive disease, and refractory disease are predictors of poor survival, and such patients should be considered high-risk, regardless of pre-existing biomarkers. This means that patients that have not been considered for alemtuzumab or alloHSCT pre-treatment based on their cytogenetic risk profile are candidates for a more intensive approach based on their inadequate response to first-line treatment. Most importantly, it is crucial to change the treatment components used in the initial failed approach. There are several treatment options, including alemtuzumab, ofatumumab, experimental drugs, and alloHSCT. The phase II GCLLSG CLL2H trial evaluated the safety and efficacy of subcutaneous alemtuzumab in patients with fludarabine-refractory CLL, and reported that OR was 34% (4% CR and 30% PR), median PFS was 7.7 months, and median OS was 19.1 months [56]. Efficacy did not vary significantly between genetic subgroups, indicating that alemtuzumab treatment could overcome the adverse prognostic impact of IGHV mutation status, TP53 mutation, and genomic aberrations. The potential benefit of alemtuzumab in combination with chemotherapy was first observed in 6 patients by Kennedy et al.

[57]; their findings were confirmed by a phase II trial on relapsed or refractory CLL, which reported an OR of 83%, including 11 patients with CR, and resolution of disease in all affected sites [58].

In the ongoing GCLLSG CLL2o trial on the combination of alemtuzumab and high-dose dexamethasone, 31 of the 80 patient enrolled to date were fludarabine-resistant; their OR rate was 47%, none achieved CR, and 12-month OS was 54% [44]. A recently published interim analysis of an international phase II study on the efficacy of the human monoclonal CD20 antibody ofatumumab in 138 patients that were fludarabine- and alemtuzumab-

92

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

Turk J Hematol 2011; 28: 86-96

refractory reported an OR rate of 58%; median PFS and OS were 5.7 and 13.7 months, respectively, indicating that ofatumumab might be a promising treatment option for fludarabine-refractory patients with poor-prognosis CLL [59]. Alternative treatment approaches using new substances, such as flavopiridol or IMiDs®, are currently being tested in clinical trials, and some patients might be eligible for inclusion in phase I or II clinical studies; however, none of the current or novel approaches has the potential for long-term disease control, highlighting the need for effective consolidation once remission is achieved.

The role of Allogeneic Stem Cell Transplantation On the basis of its capacity to induce graft-versus-leukemia (GVL) effects [60], alloHSCT has been shown to provide long-term disease control in selected CLL patients [36,61-68]. Key outcome data from selected prospective clinical trials on reducedintensity conditioning (RIC) are summarized in Table 3. To elucidate the effect of alloHSCT in patients with del 17p, the EBMT performed a retrospective database analysis in which 44 patients with del 17p that received alloHSCT were identified [69].

After a median post-alloHSCT observation time of 39 months, 19 patients were still alive. Three-year OS and PFS rates were 44% and 37%, respectively, and the cumulative incidence of disease progression at 4 years was 34%. During 4 years of follow-up no late relapses occurred in 9 patients, indicating that alloHSCT might have curative potential in patients with del 17p.

The final results of the prospective GCLLSG CLL3X trial on the feasibility and efficacy of RIC

alloHSCT in patients with poor-risk CLL were recently published [70]. After a median follow-up of 46 months (7-102 months), 4-year non-relapse mortality (NRM), EFS, and OS were 23%, 42%, and 65%, respectively. Among the 52 patients for whom MRD monitoring results were available, 27 (52%) were alive and MRD negative 12 months after transplantation. EFS was similar in all genetic subgroups, including patients with del 17p. Multivariate analysis showed that uncontrolled disease at the time of alloHSCT and in vivo T-cell depletion with alemtuzumab, but no del 17p, previous purine analogue refractoriness, and donor source (human leukocyte antigen-identical siblings or unrelated donors) had an adverse impact on EFS and OS, indicating that alloHSCT can result in long-term MRD-negative survival in up to 50% of patients, independent of the underlying genomic risk profile. A recent update of the CLL3X data with work-up of TP53 mutation status showed that the adverse impact of TP53 mutation, similarly as del 17p, can be overcome by alloHSCT [71].

Although controlled trials are lacking, currently available data strongly suggest that alloHSCT is the only therapy with curative potential in high-risk CLL. In contrast to conventional immunochemotherapy, it can provide long-term disease control, even in patients with del 17p or other unfavorable biological and clinical risk factors.

Conclusions Substantial progress has been made in the understanding and treatment of CLL, and advances in

Table 3. Results of prospective clinical trials on reduced-intensity conditioning (RIC) alloHSCT in CLL Schetelig et al. (61)

Sorror et al. (67)

Khouri et al. (65)

Brown et al. (66)

Delgado et al. (68)

Dreger et al. (70)

30

82

39

46

41

90

FB/ATGa

F/TBI2b

FCR +/– ATGc

FBd

FM/CD52e

F/C +/–ATGf

57%

37%

18%

67%

41%

60%

4-year PFS

58%

39% (5y)

44%

34% (2y)

45% (2y)

42%

4-year OS

69%

50% (5y)

48%

54% (2y)

51% (2y)

65%

4-year NRM

15%

23% (5y)

n.r.

17% (2y)

26% (2y)

23%

Extensive chronic GVHD

21%

49-53%

58%

38%

5%

14%

3.7 (2.1-5.6)

5

2.3 (.3-6.7)

1.7

1.3 (0-5.2)

3.8 (0.6-8.5)

Number of patients Conditioning regimen Proportion of alternative

donorsg

Median follow-up [years] (range)

aFludarabine, busulfan, anti-thymocyte globulin (ATG) bFludarabine, total body irradiation 2Gy cFludarabine, cyclophosphamide, rituximab plus ATG in alternative donor transplants dFludarabine, busulfan eFludarabine, melphalan, alemtuzumab fFludarabine, cyclophosphamide plus ATG in alternative donor transplants gDonor other than HLA-identical siblings NRM: non-relapse mortality, GVHD: graft-versus-host disease

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

Turk J Hematol 2011; 28: 86-96

molecular profiling of the disease have enabled physicians to better predict patient risk profiles and response to therapy. Several studies have validated the components and impact of poor-risk CLL, and international guidelines have implemented these criteria in their treatment recommendations. As a result, Poor-risk patients can now be identified with greater accuracy and offered intensified treatment options, such as allo HSCT or alemtuzumab.

Depending on patient performance status, personal preference, and the availability of a stem cell donor, these treatment options offer a tailored treatment approach, providing an opportunity to cure CLL in this poor-risk population.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

5.

Sant M, Allemani C, Tereanu C, De Angelis R, Capocaccia R, Visser O, Marcos-Gragera R, Maynadié M, Simonetti A, Lutz JM, Berrino F. Incidence of hematologic malignancies in Europe by morphologic subtype: results of the HAEMACARE project. Blood 2010;116:3724-34.

Altekruse SF, Kosary CL, Krapcho M, Neyman N, Aminou R, Waldron W, Ruhl J, Howlader N, Tatalovich Z, Cho H, Mariotto A, Eisner MP, Lewis DR, Cronin K, Chen HS, Feuer EJ, Stinchcomb DG, Edwards BK (eds).

SEER Cancer Statistics Review, 1975-2007, National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/ csr/1975_2007/, based on November 2009 SEER data submission, posted to the SEER web site, 2010.

Morrison VA. Infectious complications of chronic lymphocytic leukaemia: pathogenesis, spectrum of infection, preventive approaches. Best Pract Res Clin Haematol 2010; 23: 145-53.

Cheson BD, Bennett JM, Rai KR, Grever MR, Kay NE, Schiffer CA, Oken MM, Keating MJ, Boldt DH, Kempin SJ, et al. Guidelines for clinical protocols for chronic lymphocytic leukemia: Recommendations of the national cancer institute-sponsored working group.

American Journal of Hematology 1988;29:152-63.

Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, Hillmen P, Keating MJ, Montserrat E, Rai KR, Kipps TJ; International Workshop on Chronic Lymphocytic Leukemia. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008;111:5446-56.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

93

Rawstron AC, Bennett FL, O'Connor SJ, Kwok M, Fenton JA, Plummer M, de Tute R, Owen RG, Richards SJ, Jack AS, Hillmen P. Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia. New England Journal of Medicine 2008;359:575-83.

Müller-Hermelink HK, Montserrat E, Catovsky D, Campo E, Harris NL, Stein H. Chronic lymphocytic leukaemia/ small lymphocytic leukaemia. In: Swerdlow SH, Campo E, Harris NL, et al. eds. WHO Classification of tumours of haematopoietic and lymphoid tissues.

4th Edition ed. Lyon: International Agency for Research on Cancer (IARC), 2008.

Moreau EJ, Matutes E, A'Hern RP, et al. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 1997;108:378-82.

Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Catovsky D. Levels of expression of CD19 and CD20 in chronic B cell leukaemias. Journal of Clinical Pathology 1998;51:364-9.

Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N. Ig V Gene Mutation Status and CD38 Expression As Novel Prognostic Indicators in Chronic Lymphocytic Leukemia. Blood 1999;94:1840-7.

Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK, Oscier DG. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002;99:1023-9.

Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, Vaugier G, Potron G, Colona P, Oberling F, Thomas M, Tchernia G, Jacquillat C, Boivin P, Lesty C, Duault MT, Monconduit M, Belabbes S, Gremy F. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis.

Cancer 1981;48:198-206.

Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood 1975;46:219-34.

Wierda WG, O'Brien S, Wang X, et al. Prognostic nomogram and index for overall survival in previously untreated patients with chronic lymphocytic leukemia.

Blood 2007;109:4679-85.

Shanafelt TD, Jenkins G, Call TG, Zent CS, Slager S, Bowen DA, Schwager S, Hanson CA, Jelinek DF, Kay NE. Validation of a new prognostic index for patients with chronic lymphocytic leukemia. Cancer 2009;115:363-72.

Molica S, Mauro FR, Callea V, Giannarelli D, Lauria F, Rotoli B, Cortelezzi A, Liso V, Foà R. The utility of a prognostic index for predicting time to first treatment in early chronic lymphocytic leukemia: the GIMEMA experience. Haematologica 2010;95:464-9.

Extermann M, Overcash J, Lyman GH, Parr J, Balducci L. Comorbidity and functional status are independent in older cancer patients. Journal of Clinical Oncology 1998;16:1582-7.

Hallek M, Langenmayer I, Nerl C, Knauf W, Dietzfelbinger H, Adorf D, Ostwald M, Busch R, Kuhn-Hallek I, Thiel E,

94

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

Emmerich B. Elevated Serum Thymidine Kinase Levels Identify a Subgroup at High Risk of Disease Progression in Early, Nonsmoldering Chronic Lymphocytic Leukemia. Blood 1999;93:1732-7.

Hallek M, Wanders L, Ostwald M, Busch R, Senekowitsch R, Stern S, Schick HD, Kuhn-Hallek I, Emmerich B. Serum ss2-Microglobulin and Serum Thymidine Kinase are Independent Predictors of Progression-Free Survival in Chronic Lymphocytic Leukemia and Immunocytoma. Leukemia & Lymphoma 1996;22:439-47.

Reinisch W, Willheim M, Hilgarth M, Gasché C, Mader R, Szepfalusi S, Steger G, Berger R, Lechner K, BoltzNitulescu G, et al. Soluble CD23 reliably reflects disease activity in B-cell chronic lymphocytic leukemia.

Journal of Clinical Oncology 1994;12:2146-52.

Sarfati M, Chevret S, Chastang C, Biron G, Stryckmans P, Delespesse G, Binet JL, Merle-Beral H, Bron D. Prognostic importance of serum soluble CD23 level in chronic lymphocytic leukemia. Blood 1996;88:4259-64.

Döhner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, Bullinger L, Döhner K, Bentz M, Lichter P. Genomic Aberrations and Survival in Chronic Lymphocytic Leukemia. New England Journal of Medicine 2000;343:1910-6.

Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig VH Genes Are Associated With a More Aggressive Form of Chronic Lymphocytic Leukemia. Blood 1999;94:1848-54.

Wiestner A, Rosenwald A, Barry TS, Wright G, Davis RE, Henrickson SE, Zhao H, Ibbotson RE, Orchard JA, Davis Z, Stetler-Stevenson M, Raffeld M, Arthur DC, Marti GE, Wilson WH, Hamblin TJ, Oscier DG, Staudt LM. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 2003;101:4944-1.

Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, Marcé S, López-Guillermo A, Campo E, Montserrat E. ZAP-70 Expression as a Surrogate for Immunoglobulin-Variable-Region Mutations in Chronic Lymphocytic Leukemia. New England Journal of Medicine 2003;348:1764-75.

Rai KR, Peterson BL, Appelbaum FR, et al. Fludarabine Compared with Chlorambucil as Primary Therapy for Chronic Lymphocytic Leukemia. New England Journal of Medicine 2000;343:1750-7.

Eichhorst BF, Busch R, Hopfinger G, Pasold R, Hensel M, Steinbrecher C, Siehl S, Jäger U, Bergmann M, Stilgenbauer S, Schweighofer C, Wendtner CM, Döhner H, Brittinger G, Emmerich B, Hallek M; German CLL Study Group. ludarabine plus cyclophosphamide versus fludarabine alone in first-line therapy of younger patients with chronic lymphocytic leukemia. Blood 2006;107:885-91.

Catovsky D, Richards S, Matutes E, Oscier D, Dyer MJ, Bezares RF, Pettitt AR, Hamblin T, Milligan DW, Child JA, Hamilton MS, Dearden CE, Smith AG, Bosanquet AG, Davis Z, Brito-Babapulle V, Else M, Wade R, Hillmen P; UK National Cancer Research Institute (NCRI) Haematological Oncology Clinical Studies Group; NCRI Chronic Lymphocytic Leukaemia Working Group.

Turk J Hematol 2011; 28: 86-96

29.

30.

31.

32.

33.

34.

35.

36.

37.

Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. The Lancet 2007;370:230-9.

Flinn IW, Neuberg DS, Grever MR, Dewald GW, Bennett JM, Paietta EM, Hussein MA, Appelbaum FR, Larson RA, Moore DF Jr, Tallman MSFlinn IW, Neuberg DS, Grever MR, Dewald GW, Bennett JM, Paietta EM, Hussein MA, Appelbaum FR, Larson RA, Moore DF Jr, Tallman MS. Phase III Trial of Fludarabine Plus Cyclophosphamide Compared With Fludarabine for Patients With Previously Untreated Chronic Lymphocytic Leukemia: US Intergroup Trial E2997.

Journal of Clinical Oncology 2007;25:793-8.

Tam CS, O'Brien S, Wierda W, Kantarjian H, Wen S, Do KA, Thomas DA, Cortes J, Lerner S, Keating MJ. Longterm results of the fludarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia. Blood 2008;112:975-80.

Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J, Hensel M, Hopfinger G, Hess G, von Grünhagen U, Bergmann M, Catalano J, Zinzani PL, Caligaris-Cappio F, Seymour JF, Berrebi A, Jäger U, Cazin B, Trneny M, Westermann A, Wendtner CM, Eichhorst BF, Staib P, Bühler A, Winkler D, Zenz T, Böttcher S, Ritgen M, Mendila M, Kneba M, Döhner H, Stilgenbauer S; International Group of Investigators; German Chronic Lymphocytic Leukaemia Study Group. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial.

The Lancet 2010;376:1164-74.

Fischer K, Cramer P, Stilgenbauer S, et al. Bendamustine Combined with Rituximab (BR) in First-Line Therapy of Advanced CLL: A Multicenter Phase II Trial of the German CLL Study Group (GCLLSG). ASH Annual Meeting Abstracts 2009;114:3382-91.

Bosch F, Abrisqueta P, Villamor N, Terol MJ, GonzálezBarca E, Ferra C, González Diaz M, Abella E, Delgado J, Carbonell F, García Marco JA, Escoda L, Ferrer S, Monzó E, González Y, Estany C, Jarque I, Salamero O, Muntañola A, Montserrat E. A New, Highly Active Chemoimmunotherapy Regimen for Chronic Lymphocytic Leukemia. Journal of Clinical Oncology 2009;27:4578-84.

Reynolds C, Di Bella N, Lyons RM, et al. Phase III Trial of Fludarabine, Cyclophosphamide, and Rituximab Vs. Pentostatin, Cyclophosphamide, and Rituximab in B-Cell Chronic Lymphocytic Leukemia. ASH Annual Meeting Abstracts 2008;112:327.

Rabinowe SN, Soiffer RJ, Gribben JG, Daley H, Freedman AS, Daley J, Pesek K, Neuberg D, Pinkus G, Leavitt PR, et al. Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia. Blood 1993;82:1366-76.

Gribben JG, Zahrieh D, Stephans K, et al. Autologous and allogeneic stem cell transplantations for poor-risk chronic lymphocytic leukemia. Blood 2005;106:4389-96.

Milligan DW, Fernandes S, Dasgupta R, Davies FE, Matutes E, Fegan CD, McConkey C, Child JA, Cunningham D, Morgan GJ, Catovsky D; National Cancer Research Institute Haematological Studies Group Results of the MRC pilot study show autografting

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

Turk J Hematol 2011; 28: 86-96

38.

39.

40.

41.

42.

43.

44.

45.

46.

for younger patients with chronic lymphocytic leukemia is safe and achieves a high percentage of molecular responses. Blood 2005;105:397-404.

McClanahan F, Stilgenbauer S, Schmitz N, et al. Secondary malignancies after early autologous stem cell transplantation in genetically poor-risk chronic lymphatic leukemia: results from the German CLL Study Group (GCLLSG) CLL3 trial. Haematologica 2009;94(Suppl. 3):iwCLL meeting abstracts, abstract 8.9, 76.

Michallet M, Dreger P, Sutton L, Brand R, Richards S, van Os M, Sobh M, Choquet S, Corront B, Dearden C, Gratwohl A, Herr W, Catovsky D, Hallek M, de Witte T, Niederwieser D, Leporrier M, Milligan D; EBMT Chronic Leukemia Working Party. Autologous hematopoietic stem cell transplantation in chronic lymphocytic leukemia: results of European intergroup randomized trial comparing autografting versus observation. Blood 2010;117:1516-21.

Moreno C, Villamor N, Colomer D, Esteve J, Giné E, Muntañola A, Campo E, Bosch F, Montserrat E. Clinical significance of minimal residual disease, as assessed by different techniques, after stem cell transplantation for chronic lymphocytic leukemia. Blood 2006;107:4563-9.

Ritgen M, Stilgenbauer S, von Neuhoff N, Humpe A, Brüggemann M, Pott C, Raff T, Kröber A, Bunjes D, Schlenk R, Schmitz N, Döhner H, Kneba M, Dreger P. Graft-versus-leukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene status: implications of minimal residual disease measurement with quantitative PCR. Blood 2004;104:2600-2.

Milligan DW, Kochethu G, Dearden C, Matutes E, MacConkey C, Catovsky D; Haematological Studies Group, UK National Cancer Research Institute High incidence of myelodysplasia and secondary leukaemia in the UK Medical Research Council Pilot of autografting in chronic lymphocytic leukaemia. British Journal of Haematology 2006;133:173-5.

Hillmen P, Skotnicki AB, Robak T, Jaksic B, Dmoszynska A, Wu J, Sirard C, Mayer J. Alemtuzumab Compared With Chlorambucil As First-Line Therapy for Chronic Lymphocytic Leukemia. Journal of Clinical Oncology 2007;25:5616-23.

Stilgenbauer S, Cymbalista F, Leblond V, et al.

Subcutaneous Alemtuzumab Combined with Oral Dexamethasone, Followed by Alemtuzumab Maintenance or Allo-SCT In CLL with 17p- or Refractory to Fludarabine - Interim Analysis of the CLL2O Trial of the GCLLSG and FCGCLL/MW. Blood/ ASH Annual Meeting Abstracts 2010;116:920.

Eichhorst BF, Busch R, Stilgenbauer S, et al. First-line therapy with fludarabine compared with chlorambucil does not result in a major benefit for elderly patients with advanced chronic lymphocytic leukemia. Blood 2009;114:3382-91.

Knauf WU, Lissichkov T, Aldaoud A, Liberati A, Loscertales J, Herbrecht R, Juliusson G, Postner G, Gercheva L, Goranov S, Becker M, Fricke HJ, Huguet F, Del Giudice I, Klein P, Tremmel L, Merkle K, Montillo M. Phase III Randomized Study of Bendamustine Compared With Chlorambucil in Previously Untreated

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

95

Patients With Chronic Lymphocytic Leukemia. J Clin Oncol 2009;27:4378-84.

Döhner H, Fischer K, Bentz M, Hansen K, Benner A, Cabot G, Diehl D, Schlenk R, Coy J, Stilgenbauer S, et al. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. Blood 1995;85:1580-9.

Zenz T, Kröber A, Scherer K, Häbe S, Bühler A, Benner A, Denzel T, Winkler D, Edelmann J, Schwänen C, Döhner H, Stilgenbauer S. Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up. Blood 2008;112:3322-9.

Zenz T, Eichhorst B, Busch R, et al. TP53 Mutation and Survival in Chronic Lymphocytic Leukemia. Journal of Clinical Oncology 2010;28:4473-9.

Stilgenbauer S, Zenz T. Understanding and Managing Ultra High-Risk Chronic Lymphocytic Leukemia.

Hematology 2010;2010:481-8.

Oscier D, Wade R, Davis Z, Morilla A, Best G, Richards S, Else M, Matutes E, Catovsky D; Chronic Lymphocytic Leukaemia Working Group, UK National Cancer Research Institute. Prognostic factors identified three risk groups in the LRF CLL4 trial, independent of treatment allocation. Haematologica 2010;95:1705-12.

Keating MJ, O'Brien S, Lerner S, Koller C, Beran M, Robertson LE, Freireich EJ, Estey E, Kantarjian H. LongTerm Follow-Up of Patients With Chronic Lymphocytic Leukemia (CLL) Receiving Fludarabine Regimens as Initial Therapy. Blood 1998;92:1165-71.

Keating MJ, Wierda WG, Tam CS, et al. Long Term Outcome Following Treatment Failure of FCR Chemoimmunotherapy as Initial Therapy for Chronic Lymphocytic Leukemia. ASH Annual Meeting Abstracts 2009;114:2381.

Zenz T, Busch R, Fink A, et al. Genetics of Patients with F-Refractory CLL or Early Relapse After FC or FCR: Results From the CLL8 Trial of the GCLLSG. ASH Annual Meeting Abstracts 2010;116:2427.

Dreger P, Corradini P, Kimby E, Michallet M, Milligan D, Schetelig J, Wiktor-Jedrzejczak W, Niederwieser D, Hallek M, Montserrat E; Chronic Leukemia Working Party of the EBMT. Indications for allogeneic stem cell transplantation in chronic lymphocytic leukemia: the EBMT transplant consensus. Leukemia 2007;21:12-7.

Stilgenbauer S, Zenz T, Winkler D, Bühler A, Schlenk RF, Groner S, Busch R, Hensel M, Dührsen U, Finke J, Dreger P, Jäger U, Lengfelder E, Hohloch K, Söling U, Schlag R, Kneba M, Hallek M, Döhner H; German Chronic Lymphocytic Leukemia Study Group.

Subcutaneous Alemtuzumab in Fludarabine-Refractory Chronic Lymphocytic Leukemia: Clinical Results and Prognostic Marker Analyses From the CLL2H Study of the German Chronic Lymphocytic Leukemia Study Group. Journal of Clinical Oncology 2009;27:3994-4001.

Kennedy B, Rawstron A, Carter C, Ryan M, Speed K, Lucas G, Hillmen P. Campath-1H and fludarabine in combination are highly active in refractory chronic lymphocytic leukemia. Blood 2002;99:2245-7.

Elter T, Borchmann P, Schulz H, Reiser M, Trelle S, Schnell R, Jensen M, Staib P, Schinköthe T, Stützer H,

96

59.

60.

61.

62.

63.

64.

65.

Mc Clanahan et al.

Treatment of chronic lymphocytic leukemia

Rech J, Gramatzki M, Aulitzky W, Hasan I, Josting A, Hallek M, Engert A. Fludarabine in Combination With Alemtuzumab Is Effective and Feasible in Patients With Relapsed or Refractory B-Cell Chronic Lymphocytic Leukemia: Results of a Phase II Trial. Journal of Clinical Oncology 2005;23:7024-31.

Wierda WG, Kipps TJ, Mayer J, Stilgenbauer S, Williams CD, Hellmann A, Robak T, Furman RR, Hillmen P, Trneny M, Dyer MJ, Padmanabhan S, Piotrowska M, Kozak T, Chan G, Davis R, Losic N, Wilms J, Russell CA, Osterborg A; Hx-CD20-406 Study Investigators. Ofatumumab As Single-Agent CD20 Immunotherapy in FludarabineRefractory Chronic Lymphocytic Leukemia. Journal of Clinical Oncology 2010;28:1749-55.

Ben-Bassat I, Raanani P, Gale RP. Graft-versus-leukemia in chronic lymphocytic leukemia. Bone Marrow Transplant 2007;39:441-6.

Schetelig J, Thiede C, Bornhauser M, Schwerdtfeger R, Kiehl M, Beyer J, Sayer HG, Kroger N, Hensel M, Scheffold C, Held TK, Hoffken K, Ho AD, Kienast J, Neubauer A, Zander AR, Fauser AA, Ehninger G, Siegert W; Cooperative German Transplant Study Group.

Evidence of a Graft-Versus-Leukemia Effect in Chronic Lymphocytic Leukemia After Reduced-Intensity Conditioning and Allogeneic Stem-Cell Transplantation: The Cooperative German Transplant Study Group.

Journal of Clinical Oncology 2003;21:2747-53.

Dreger P, Brand R, Hansz J, Milligan D, Corradini P, Finke J, Deliliers GL, Martino R, Russell N, Van Biezen A, Michallet M, Niederwieser D; Chronic Leukemia Working Party of the EBMT. Treatment-related mortality and graft-versus-leukemia activity after allogeneic stem cell transplantation for chronic lymphocytic leukemia using intensity-reduced conditioning. Leukemia 2003;17:841-8.

Pavletic SZ, Khouri IF, Haagenson M, King RJ, Bierman PJ, Bishop MR. Unrelated donor marrow transplantation for B-cell chronic lymphocytic leukemia after using myeloablative conditioning: results from the Center for International Blood and Marrow Transplant research. J Clin Oncol 2005;23:5788-94.

Sorror ML, Maris MB, Sandmaier BM, Storer BE, Stuart MJ, Hegenbart U, Agura E, Chauncey TR, Leis J, Pulsipher M, McSweeney P, Radich JP, Bredeson C, Bruno B, Langston A, Loken MR, Al-Ali H, Blume KG, Storb R, Maloney DG. Hematopoietic Cell Transplantation After Nonmyeloablative Conditioning for Advanced Chronic Lymphocytic Leukemia. Journal of Clinical Oncology 2005;23:3819-29.

Khouri IF, Saliba RM, Admirand J, O'Brien S, Lee MS, Korbling M, Samuels BI, Giralt S, Lima de M, Keating MJ, Champlin RE, Bueso-Ramos C. Graft-versusleukaemia effect after non-myeloablative haematopoi-

Turk J Hematol 2011; 28: 86-96

66.

67.

68.

69.

70.

71.

etic transplantation can overcome the unfavourable expression of ZAP-70 in refractory chronic lymphocytic leukaemia. Br J Haematol 2007;137:355-63.

Brown JR, Kim HT, Li S, Stephans K, Fisher DC, Cutler C, Ho V, Lee SJ, Milford EL, Ritz J, Antin JH, Soiffer RJ, Gribben JG, Alyea EP. Predictors of Improved Progression-Free Survival After Nonmyeloablative Allogeneic Stem Cell Transplantation for Advanced Chronic Lymphocytic Leukemia. Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 2006;12:1056-64.

Sorror ML, Storer BE, Sandmaier BM, Maris M, Shizuru J, Maziarz R, Agura E, Chauncey TR, Pulsipher MA, McSweeney PA, Wade JC, Bruno B, Langston A, Radich J, Niederwieser D, Blume KG, Storb R, Maloney DG. Five-Year Follow-Up of Patients With Advanced Chronic Lymphocytic Leukemia Treated With Allogeneic Hematopoietic Cell Transplantation After Nonmyeloablative Conditioning. Journal of Clinical Oncology 2008;26:4912-20.

Delgado J, Thomson K, Russell N, Ewing J, Stewart W, Cook G, Devereux S, Lovell R, Chopra R, Marks DI, Mackinnon S, Milligan DW; British Society of Blood and Marrow Transplantation. Results of alemtuzumabbased reduced-intensity allogeneic transplantation for chronic lymphocytic leukemia: a British Society of Blood and Marrow Transplantation Study. Blood 2006;107:1724-30.

Schetelig J, van Biezen A, Brand R, Caballero D, Martino R, Itala M, García-Marco JA, Volin L, Schmitz N, Schwerdtfeger R, Ganser A, Onida F, Mohr B, Stilgenbauer S, Bornhäuser M, de Witte T, Dreger P. Allogeneic Hematopoietic Stem-Cell Transplantation for Chronic Lymphocytic Leukemia With 17p Deletion: A Retrospective European Group for Blood and Marrow Transplantation Analysis. Journal of Clinical Oncology 2008;26:5094-100.

Dreger P, Döhner H, Ritgen M, Böttcher S, Busch R, Dietrich S, Bunjes D, Cohen S, Schubert J, Hegenbart U, Beelen D, Zeis M, Stadler M, Hasenkamp J, Uharek L, Scheid C, Humpe A, Zenz T, Winkler D, Hallek M, Kneba M, Schmitz N, Stilgenbauer S; German CLL Study Group. Allogeneic stem cell transplantation provides durable disease control in poor-risk chronic lymphocytic leukemia: long-term clinical and MRD results of the German CLL Study Group CLL3X trial.

Blood 2010;116:2438-47.

Zenz T, Dreger P, Dietrich S, et al. Allogeneic Stem Cell Transplantation Can Overcome the Adverse Prognostic Impact of TP53 Mutation In Chronic Lymphocytic Leukemia (CLL): Results From the GCLLSG CLL3x Trial. ASH Annual Meeting Abstracts 2010;116:2357.

Research Article

97

ABL gene kinase domain mutation scanning by denaturing high performance liquid chromatography sequencing method Denatüre edici yüksek performanslı likit kromotografi yöntemi ile ABL geni kinaz bölgesi mutasyonlarının taranması Yücel Erbilgin1, Suzin Çatal1, Ahmet Emre Eşkazan2, Özden Hatırnaz1, Teoman Soysal2, Uğur Özbek1 1Department 2Adult

of Genetics, Institute of Experimental Medicine, İstanbul University, İstanbul, Turkey Hematology Division, Cerrahpaşa Faculty of Medicine, İstanbul University, İstanbul, Turkey

Abstract Objective: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor imatinib, the development of resistance against imatinib has been observed. The most important mechanisms known to cause resistance are point mutations in the ABL tyrosine kinase and the ATP domain. This study describes the use of denaturing high performance liquid chromatography (dHPLC) as a method to screen for mutations of the ABL gene.

Material and Methods: We used the dHPLC based assay for the screening of ABL point mutations.

Forty chronic myeloid leukemia (CML) patients who showed resistance to imatinib were screened in parallel by dHPLC and direct sequencing.

Results: Nine of the 40 patients (23%) had mutations.

Conclusion: dHPLC can be a useful method for pre-screening. Analyzing the mutations and monitoring (high-risk) patients can improve their prognosis and survival rate. dHPLC can potentially become a valuable tool for regular testing of patients in the future. (Turk J Hematol 2011; 28: 97-102) Key words: Chronic myeloid leukemia, imatinib resistance, mutation, dHPLC Received: February 12, 2010

Accepted: August 19, 2010

Özet Amaç: Bir BCR-ABL tirozin kinaz inhibitörü olan imatinib’in etkinliğine karşın bazı hastalarda ilaca karşı direnç gelişimi gözlenmektedir.. Direnç gelişimine neden olan en önemli mekanizma, ABL geninin tirozin kinaz ve ATP bölgelerindeki nokta mutasyonlarıdır. Bu çalışma ABL gen mutasyonlarının taranmasında denatüre edici HPLC (dHPLC) yönteminin yerini ve önemini açıklama amacıyla yapılmıştır.

Yöntemler ve Gereçler: ABL nokta mutasyonları, dHPLC kullanılarak taranmıştır. Imatinib direnci gösteren 40 KML hastası DHPLC ve paralelinde doğrudan sekanslama ile incelenmiştir.

Bulgular: Kırk hastanın dokuzunda (%23) mutasyon saptandı.

Address for Correspondence: Prof. Uğur Özbek, Department of Genetics, Institute of Experimental Medicine, İstanbul University, İstanbul, Turkey Phone: +90 212 414 20 00-33312 E-mail: uozbek@istanbul.edu.tr doi:10.5152/tjh.2011.24

98

Erbilgin et al.

Detection of ABL mutations by dHPLC

Turk J Hematol 2011; 28: 97-102

Sonuç: dHPLC yararlı bir ön tarama yöntemi olabilir. Mutasyon analizleri ile yüksek riskli hastaların monitörizasyonu prognozu iyileştirebilir ve sağ kalım oranını arttırabilir. Gelecekte dHPLC bu hastaların düzenli aralıklarla izleminde değerli bir araç olarak kullanılabilir.

(Turk J Hematol 2011; 28: 97-102)

Anahtar kelimeler: Kronik miyeloid lösemi, Imatinib direnci, mutasyon, dHPLC Geliş tarihi: 12 Şubat 2010

Kabul tarihi: 19 Ağustos 2010

Introduction Chronic myeloid leukemia (CML) is a proliferative stem cell disorder that affects patients in all age groups [1,2]. The clinical course of CML is characteristically triphasic, starting with a chronic phase (CP) of variable duration, followed by progression to an accelerated phase (AP) and finally resulting in blast crises (BC) [3-5]. CML is the first discovered human cancer associated with a consistent chromosomal abnormality-the chimeric BCR/ABL gene, known as Philadelphia (Ph) chromosome [6,7].

This translocation causes an unregulated tyrosine kinase activity and has a pivotal role in CML pathogenesis. Approximately 95% of CML patients have BCR/ABL rearrangement, indicating it is a suitable drug target for anticancer therapy [8,9].

Imatinib mesylate (IM) is a potent and selective inhibitor of BCR-ABL tyrosine kinase, the first rationally designed, molecularly targeted therapy for a human malignancy [10-13]. Despite the high rates of hematologic and cytogenetic responses, some patients show IM resistance in both phases. Resistance to IM can be defined as the lack of complete hematological response in patients with CP disease or as lack of return to CP for patients in an AP or BC [14,15].

Several underlying mechanisms for IM resistance have been studied in CML patients and cell lines.

Drug resistance has mostly arisen as a result of point mutations in the BCR-ABL gene that reduce drug binding within the kinase domain or due to overexpression of BCR-ABL protein [15-18]. In clonal selection, BCR-ABL mutated cells have a higher survival rate due to the selective pressure of imatinib therapy. A number of mutations have been well characterized in terms of their ability and the degree to which they induce resistance [17,19-22].

Today, second-generation tyrosine kinase inhibitors (TKIs) have become available (Dasatinib, Sprycel, Bristol-Myers Squibb; Nilotinib, Tasigna, Novartis Pharma) in the market [23-25].

In clinical practice, cytogenetic monitoring (as fluorescence in situ hybridization-FISH) and reverse transcription quantitative polymerase chain reaction (RQ-PCR) have become gold standard monitoring assays, and they are becoming increasingly standardized between laboratories [26-28].

Furthermore, CML treatment should be monitored correctly and cABL kinase domain mutations should be investigated. However, there are no widely accepted guidelines at present for the screening of Ph+CML mutations [18,26,28]. Therefore, we report here the use of denaturing high performance liquid chromatography (dHPLC) and sequencing methods to screen for mutations in the nucleotide binding loop (P-loop), the catalytic domain and the activation loop of the ABL gene.

Materials and Methods Patients Peripheral blood (PB) samples were obtained from 40 imatinib-resistant or -intolerant CML patients, who were diagnosed between 1999 and 2007 in the Adult Hematology Division of Istanbul Medical Faculty, Istanbul. All patients showed resistance to 400 mg/day imatinib. Imatinib resistance was defined as inadequate initial response or loss of hematologic or cytogenetic response at any time during the treatment. All of the patient’s materials were stored at -80°C after homogenization in RLT buffer (Qiagen, GmbH, Germany). One PB sample from a CML patient known to be wild-type for ABL mutations was used as a negative control. We obtained written informed consents from all patients RNA isolation and cDNA synthesis Total RNA was isolated by Qiagen RNeasy Protect kit (Qiagen, GnbH, Germany). RNA samples were treated with DNAse (1 U/µg) for the possible DNA contaminations during isolation. 1 µg of total RNA was used for cDNA synthesis by using random hex-

Erbilgin et al.

Detection of ABL mutations by dHPLC

Turk J Hematol 2011; 28: 97-102

amers and MMLV reverse transcriptase according to the protocol of the manufacturer (Fermentase).

Nested PCR and experimental design of dHPLC analysis Mutation detection in the nucleotide binding loop (P-loop), the catalytic domain and the activation loop of the ABL gene was performed via PCR-based dHPLC using a WAVE DNA fragment analysis system (Transgenomic). We used a nested-PCR approach for amplification of the ABL kinase domain with primers and PCR conditions described before [18]. First PCR was performed with 2 µl of cDNA and exons 12, 13 on BCR mRNA and exon 8 on ABL mRNA were amplified. The ABL kinase domain was divided into two partially overlapping fragments; codons 206- 335 (fragment B) and codons 262- 421 (fragment C) were amplified separately. This procedure ensures that the wild-type ABL transcript is not analyzed [18].

Using Wavemaker software (Transgenomic), we selected optimal temperatures for the two ABL mRNA fragments. Annealed PCR fragments (8-15 µl per sample) were injected into the DNASep HT cartridge for analysis at the following selected temperatures: Fragment B at 61.3, 62.3°C and fragment C at 60.2, 61.1°C. Products were eluted at a constant flow rate of 1.5 ml/min with a linear acetonitrile gradient determined by Navigator software (Transgenomic) based on the size and GC-content of the amplicon. The gradient was formed by combining buffer A (0.1M TEAA) (Transgenomic) and Buffer B (0.1M TEAA with 25% acetonitrile) (Transgenomic). The elution profiles of DNA fragments, monitored by the system’s UV detector, were used to produce chromatographs. A wild-type sample was used as a negative control. Less stable heteroduplexes denature earlier than homoduplexes and, thus, appear first in elution profiles. Patients’ chromatograms were compared with the wild-type profile, and samples with different characters (eluted before normal homoduplexes) were scored as positive. In addition to positive scored samples, crude PCR products from patients scored as “negative” were also directly sequenced to exclude the presence of undetected genetic variations. Sequences were compared with the wild-type sequence and analyzed using CLC combined Workbench software (V.3.6.1, Denmark).

99

Results In this paper, we set up a straightforward, highthroughput dHPLC-based assay to screen for the presence of mutations in the catalytic domain of ABL tyrosine kinase of 40 Turkish CML patients with imatinib resistance. Nine of 40 patients (23%) had point mutations, and mutation characteristics of the patients are shown in Table 1. Four mutations (M244V, G250E, Y253H, V304I) fell within the nucleotide-binding loop (P-loop) that was involved in ATP binding. We observed that two mutations (F359C, K357R) affected the catalytic domain and two mutations (T315I, F317L) were located in imatinib-binding residues. We identified two novel amino acid substitutions (V304I, K357R) affecting codons known as the hotspot region. Representative dHPLC chromatograms for fragments B and C are illustrated in Figure 1.

Discussion The basis of current therapy for CML in the CP includes the three TKIs - imatinib, dasatinib and nilotinib - and allogeneic hematopoietic stem cell transplantation (allo-HSCT). The treatment algorithm appears simple and easy to follow. Imatinib is the recommended first-line treatment for newly diagnosed CP CML at an initial standard dose of 400 mg daily, dasatinib or nilotinib is second line, and alloHSCT is for instances of failure of drug therapy. A partial guide to the choice of second-generation TKIs may be provided by the detection of some mutations [2,5,15,17-19,21,26]. Therefore, it is recommended that molecular laboratories incorporate this analysis Table 1. BCR-ABL mutation characteristics of patients Patient

Base change

Amino acid substitution Status

ABL11

ACT>ATT

T315I

R

ABL13

TTC>TTG

F317L

R

ABL15

GGG>GAG, TTC>TGC

G250E, F359C

R

ABL16

ATG>GTG

M244V

R

ABL17

TAC>CAC

Y253H

R

ABL20

ACT>ATT

T315I

R

ABL25

TTC>TTG

F317L

R

ABL29

GTC>ATC

V304I

N

ABL34

TTC>TTG, AAA>AGA

F317L and K357R

R, N

R: Previously reported; N: Previously undescribed

100

Erbilgin et al.

Detection of ABL mutations by dHPLC

into their testing procedures [18,26]. In this paper, we used direct sequencing and dHPLC. Direct sequencing is the most widespread method applied in the routine monitoring of patients. However, its sensitivity is low (20%), and it may cause false-negative results. Therefore, we used dHPLC technique, which has a sensitivity of 1-5% in mutation detection, in addition to the sequencing [29-31].

We found BCR-ABL mutations localized in the P-loop, in the catalytic domain. The P-loop normally acts as nucleotide-binding loop for the phosphate groups of ATP, and its mutations are the most serious and common mutations. The catalytic domain is involved in the catalytic process [14-16,32-35]. In our study group, we detected (M244V, G250E, Y253H, V304I) substitutions in P-loop domain, (K357R, F359C) mutations in catalytic domain and (T315I, F317L) mutations in the imatinib-binding site.

M244V, G250E, F317L, and F359C mutations might be overcome by dose-escalation [5,14,18,26,36].

However, Y253H mutation confers a highly resistant phenotype, suggesting withdrawal of imatinib in favor of alternative therapeutic strategies [18,26].

Two patients showed T315I mutation, which interfered with a critical hydrogen bond that forms between the ABL kinase and imatinib. It is known to be resistant to imatinib as well as the second-generation TKIs. Other than mutations, clinical relevance of other differences between dasatinib and nilotinib is unknown [5,26,36,37]. dHPLC is a sensitive and simple way to detect low level of mutations, based on differentiation in elution profiles. The results of the dHPLC showed several heteroduplex peaks on the chromatograms of samples (Figure 1). All abnormal elution profiles were parallel to sequencing results. As a result, dHPLC seems to be a reliable method for use in the pre-screening process. Certainly, the sequencing must confirm a mutation and tell us the exact mutation type.

Mutations can occur among imatinib-resistant and -intolerant patients, and sensitive detection of the mutation status might be important for the patients who are resistant to imatinib. The European LeukemiaNET (ELN) recommends mutation analysis in occurrences of suboptimal response or failure of 400 mg daily imatinib treatment. The suboptimal response refers to no cytogenetic response after three months of therapy, less than partial cytoge-

Turk J Hematol 2011; 28: 97-102

c a t c a c t g a g

A

Wild Type CATCATTGAG Mutant

B a a a a c t t c a t Mutant Wild Type

AAAACTGCAT

Figure 1. dHPLC chromatograms and direct sequencing of mutant DNA for fragments B and C. (A) dHPLC profiles of two representative mutants overlaid with the wild-type profiles (left panel), fragment B at 61.3°C; (right panel); ACT>ATT, T315I. (B) dHPLC profiles of two representative mutants overlaid with the wild-type profiles (left panel), fragment C at 60.2°C; (right panel); TTC>TGC, F359C

netic response after six months of therapy, only achieving a partial cytogenetic response at 12 months of treatment, or less than major molecular response after 18 months of treatment. Furthermore, loss of the major molecular response at any time during the treatment or detection of a mutation is so-called suboptimal response. Treatment failure refers to not achieving a complete hematological response after three months of treatment, no cytogenetic response after six months of therapy, less than a partial cytogenetic response after 12 months of treatment, or incomplete cytogenetic response after 18 months of treatment. Losing the complete hematological response or complete cytogenetic response at any time during the treatment and detection of a mutation that is poorly sensitive to imatinib or the second- generation TKIs or clonal chromosome abnormalities are also considered failure. By determining the mutational status of a patient, the right therapy option (second-generation TKIs, alloSCT, experimental drugs, etc.) can be selected for each patient [38]. dHPLC system shows higher sensitivity (95-99%) for detecting minor clones in the mixed follow-up patient samples.

Conventional direct sequencing has lower detection sensitivity to perform such analysis (about 80-90%) [29,31]. Therefore, we suggest using dHPLC screening as a routine approach to screen ABL kinase domain mutations, which can be confirmed by sequence analyses.

Erbilgin et al.

Detection of ABL mutations by dHPLC

Turk J Hematol 2011; 28: 97-102

Acknowledgements This work was funded by T.R. Prime Ministry State Planning Organization (Project no: 2005K120430).

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

12.

References 1.

Rowley JD. Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining.

Nature 1973;243:290-3. [CrossRef] 2. National Comprehensive Cancer Network (2008).

Chronic myelogenous leukemia V.I.2008. NCCN Clinical Practice Guidelines in Oncology. http://www.nccn.org/ professionalsphysician_gls/PDF/cml.pdf. Accessed 19 September 2008.

3. Griffin JD, Todd RF 3rd, Ritz J, Nadler LM, Canellos GP, Rosenthal D, Gallivan M, Beveridge RP, Weinstein H, Karp D, Schlossman SF. Differentiation patterns in the blastic phase of chronic myeloid leukemia. Blood 1983;61:85-91.

4. Bettelheim P, Lutz D, Majdic O, Paietta E, Haas O, Linkesch W, Neumann E, Lechner K, Knapp W. Cell lineage heterogeneity in blast crisis of chronic myeloid leukaemia. Br J Hematol 1985;59:395-409.

5. European LeukemiaNet (2008) Management of chronic myeloid leukemia (CML): recommendations from the European LeukemiaNet (ELN). http://www.leukemia-net.org/content/leukemias/cml/standards_sops/ recommendations/. Accessed 2 Sept 2008.

6. Bartram CR, de Klein A, Hagemeijer A, van Agthoven T, Geurts van Kessel A, Bootsma D, Grosveld G, FergusonSmith MA, Davies T, Stone M, et al. Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia. Nature 1983;306277-80. [CrossRef] 7. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 1984;36:93-9.

8. Melo JV. The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype. Blood 1996;88:2375-84.

9. Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000;96:3343-56.

10. Kantarjian H, Sawyers C, Hochhaus A, et al.

Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002;346:645-52. [CrossRef] 11. Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C, Niederwieser D, Resta D, Capdeville R, Zoellner U, Talpaz M, Druker B,

13.

14.

15.

16.

17.

18.

19.

101

Goldman J, O'Brien SG, Russell N, Fischer T, Ottmann O, Cony-Makhoul P, Facon T, Stone R, Miller C, Tallman M, Brown R, Schuster M, Loughran T, Gratwohl A, Mandelli F, Saglio G, Lazzarino M, Russo D, Baccarani M, Morra E; International STI571 CML Study Group.

Efficacy and safety of a specific inhibitor of the BCRABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031-7.

Ottmann OG, Druker BJ, Sawyers CL, Goldman JM, Reiffers J, Silver RT, Tura S, Fischer T, Deininger MW, Schiffer CA, Baccarani M, Gratwohl A, Hochhaus A, Hoelzer D, Fernandes-Reese S, Gathmann I, Capdeville R, O'Brien SG. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002;100:1965-71.

Rosti G, Martinelli G, Bassi S, Amabile M, Trabacchi E, Giannini B, Cilloni D, Izzo B, De Vivo A, Testoni N, Cambrin GR, Bonifazi F, Soverini S, Luatti S, Gottardi E, Alberti D, Pane F, Salvatore F, Saglio G, Baccarani M; Study Committee, Italian Cooperative Study Group for Chronic Myeloid Leukemia; Writing Committee, Italian Cooperative Study Group for Chronic Myeloid Leukemia. Molecular response to imatinib in late chronic-phase chronic myeloid leukemia. Blood 2004;103:2284-90.

Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J, Sawyers CL. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia.

Cancer Cell 2002;2:117-25. [CrossRef] Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876-80. [CrossRef] Hochhaus A, Kreil S, Corbin AS, La RosĂŠe P, MĂźller MC, Lahaye T, Hanfstein B, Schoch C, Cross NC, Berger U, Gschaidmeier H, Druker BJ, Hehlmann R. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 2002;16:2190-6. [CrossRef] Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J, Taylor K, Herrmann R, Seymour JF, Arthur C, Joske D, Lynch K, Hughes T. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003;102:276-83.

Soverini S, Martinelli G, Amabile M, Poerio A, Bianchini M, Rosti G, Pane F, Saglio G, Baccarani M; Italian Cooerative Study Group on Chronic Myeloid Leukemia; European LeukemiaNet-6th Framework Program of the European Community. Denaturing-HPLC-based assay for detection of ABL mutations in chronic myeloid leukemia patients resistant to Imatinib. Clin Chem 2004;50:1205-13.

Soverini S, Martinelli G, Rosti G, Bassi S, Amabile M, Poerio A, Giannini B, Trabacchi E, Castagnetti F, Testoni N, Luatti S, de Vivo A, Cilloni D, Izzo B, Fava M, Abruzzese E, Alberti D, Pane F, Saglio G, Baccarani M.

102

20.

21.

22.

23.

24.

25.

26.

27.

28.

Erbilgin et al.

Detection of ABL mutations by dHPLC

ABL mutations in late chronic phase chronic myeloid leukemia patients with up-front cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival: a study by the GIMEMA Working Party on Chronic Myeloid Leukemia. J Clin Oncol 2005;23:4100-9.

Willis SG, Lange T, Demehri S, Otto S, Crossman L, Niederwieser D, Stoffregen EP, McWeeney S, Kovacs I, Park B, Druker BJ, Deininger MW. High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy. Blood 2005;106:2128-37.

Kang HY, Hwang JY, Kim SH, Goh HG, Kim M, Kim DW. Comparison of allele specific oligonucleotide-polymerase chain reaction and direct sequencing for high throughput screening of ABL kinase domain mutations in chronic myeloid leukemia resistant to imatinib.

Haematologica 2006;91:659-62.

Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer 2007;7:441-53.

Weisberg E, Manley PW, Breitenstein W, Brüggen J, Cowan-Jacob SW, Ray A, Huntly B, Fabbro D, Fendrich G, Hall-Meyers E, Kung AL, Mestan J, Daley GQ, Callahan L, Catley L, Cavazza C, Azam M, Neuberg D, Wright RD, Gilliland DG, Griffin JD. Characterization of AMN107, a selective inhibitor of native and mutant BcrAbl. Cancer Cell 2005;7:129-41. [CrossRef] Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K, Castaneda S, Cornelius LA, Das J, Doweyko AM, Fairchild C, Hunt JT, Inigo I, Johnston K, Kamath A, Kan D, Klei H, Marathe P, Pang S, Peterson R, Pitt S, Schieven GL, Schmidt RJ, Tokarski J, Wen ML, Wityak J, Borzilleri RM. Discovery of N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem 2004;47:6658-61.

Garg RJ, Kantarjian H, O'Brien S, Quintás-Cardama A, Faderl S, Estrov Z, Cortes J. The use of nilotinib or dasatinib after failure to two prior tyrosine kinase inhibitors (TKI): long-term follow-up. Blood 2009;114:4361-8. [CrossRef] Baccarani M, Pane F, Saglio G. Monitoring treatment of chronic myeloid leukemia. Haematologica 2008;93:161-9.

Baccarani M, Rosti G, Castagnetti F, Haznedaroglu I, Porkka K, Abruzzese E, Alimena G, Ehrencrona H, Hjorth-Hansen H, Kairisto V, Levato L, Martinelli G, Nagler A, Lanng Nielsen J, Ozbek U, Palandri F, Palmieri F, Pane F, Rege-Cambrin G, Russo D, Specchia G, Testoni N, Weiss-Bjerrum O, Saglio G, Simonsson B. Comparison of imatinib 400 mg and 800 mg daily in the front-line treatment of high-risk, Philadelphia-positive chronic myeloid leukemia: a European LeukemiaNet Study. Blood 2009;113:4497-504. [CrossRef] Martinelli G, Iacobucci I, Soverini S, Cilloni D, Saglio G, Pane F, Baccarani M. Monitoring minimal residual disease and controlling drug resistance in chronic myeloid

Turk J Hematol 2011; 28: 97-102

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

leukaemia patients in treatment with imatinib as a guide to clinical management. Hematol Oncol 2006;24:196-204. [CrossRef] Emmerson P, Maynard J, Jones S, Butler R, Sampson JR, Cheadle JP. Characterizing mutations in samples with low-level mosaicism by collection and analysis of DHPLC fractionated heteroduplexes. Hum Mutat 2003;21:112-5. [CrossRef] Antonarakis ES, Sampson JR, Cheadle JP. Temperature modulation of DHPLC analysis for detection of coexisting constitutional and mosaic sequence variants in TSC2. J Biochem Biophys Methods 2002;51:161-4. [CrossRef] Wolford JK, Blunt D, Ballecer C, Prochazka M. Highthroughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC). Hum Genet 2000;107:483-7. [CrossRef] Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002;99:3472-5. [CrossRef] Roumiantsev S, Shah NP, Gorre ME, Nicoll J, Brasher BB, Sawyers CL, Van Etten RA. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc Natl Acad Sci U S A 2002;99:10700-5. [CrossRef] Hofmann WK, Jones LC, Lemp NA, de Vos S, Gschaidmeier H, Hoelzer D, Ottmann OG, Koeffler HP. Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood 2002;99:1860-2. [CrossRef] Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Laï JL, Philippe N, Facon T, Fenaux P, Preudhomme C. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 2002;100:1014-8. [CrossRef] Jabbour E, Kantarjian H, Jones D, Breeden M, GarciaManero G, O'Brien S, Ravandi F, Borthakur G, Cortes J. Characteristics and outcomes of patients with chronic myeloid leukemia and T315I mutation following failure of imatinib mesylate therapy. Blood 2008;112:53-5. [CrossRef] Soverini S, Colarossi S, Gnani A, Castagnetti F, Rosti G, Bosi C, Paolini S, Rondoni M, Piccaluga PP, Palandri F, Giannoulia P, Marzocchi G, Luatti S, Testoni N, Iacobucci I, Cilloni D, Saglio G, Baccarani M, Martinelli G. Resistance to dasatinib in Philadelphia-positive leukemia patients and the presence or the selection of mutations at residues 315 and 317 in the BCR-ABL kinase domain.

Haematologica 2007;92:401-4. [CrossRef] Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, Cervantes F, Deininger M, Gratwohl A, Guilhot F, Hochhaus A, Horowitz M, Hughes T, Kantarjian H, Larson R, Radich J, Simonsson B, Silver RT, Goldman J, Hehlmann R; European LeukemiaNet. Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNET. J Clin Oncol 2009;35:6041-51. [CrossRef]

Research Article

103

Fluorescence in situ hybridization analysis of the hTERC region in acute myeloid leukemia patients Akut myeloid lösemi olgularında floresan in situ hibridizasyon yöntemi ile hTERC bölgesinin analizi Özge Özer, Tuğçe Bulakbaşı Balcı, Zerrin Yılmaz, Feride İffet Şahin Department of Medical Genetics, Faculty of Medicine, Başkent University, Ankara, Turkey

Abstract Objective: The telomerase RNA component (hTERC) gene is located at 3q26. Increased hTERC gene expression has been frequently observed and amplification was shown using fluorescence in situ hybridization (FISH) in different cancers. The aim of this study was to determine whether hTERC gene amplification is detectable by FISH in acute myeloid leukemia (AML) cells.

Material and Methods: FISH and karyotype results at the time of diagnosis of 23 adult AML patients were retrospectively evaluated. Additionally, fixed cells were hybridized with an hTERC region-specific FISH probe to determine gene amplification.

Results: Ten of the 23 patients had a normal karyotype and 6 had an abnormal karyotype. hTERC region amplification was not observed in any of the patients.

Conclusion: Although it was reported that hTERC gene amplification may partially contribute to increased telomerase expression and activity in leukemic cells, it is not possible to make such a conclusion based on the results of the this study, as hTERC amplification was not observed in the study group. This suggests that increased telomerase activity via gene amplification in the development of AML may not be as important a factor as it is in solid tumors. (Turk J Hematol 2011; 28: 103-6) Key words: AML, hTERC, FISH Received: February 11, 2010

Accepted: September 6, 2010

Özet Amaç: Telomeraz RNA komponenti (hTERC) geni 3q26 bölgesinde yer alır. Çeşitli kanserlerde artmış hTERC gen anlatımı sıklıkla saptanmış ve FISH yöntemi ile gen amplifikasyonu gösterilmiştir. Bu çalışmanın amacı akut miyeloid lösemi (AML) hücrelerinde hTERC gen amplifikasyonunun FISH yöntemi ile saptanabilirliğinin araştırılmasıdır.

Yöntem ve Gereçler: Bu çalışmada 23 yeni tanı erişkin AML olgusunun FISH ve karyotip sonuçları geriye dönük olarak değerlendirildi. Ayrıca gen amplifikasyonunu değerlendirebilmek için tespit edilmiş hücreler hTERC bölgesine özgül FISH probu ile melezlendi.

Address for Correspondence: Prof. Feride İffet Şahin, Department of Medical Genetics, Faculty of Medicine, Başkent University, Kubilay Sok. No: 36 06570 Maltepe, Ankara, Turkey Phone: +90 312 232 44 00-302 E-mail: feridesahin@hotmail.com doi:10.5152/tjh.2011.25

104

Özer et al. hTERC analysis by FISH in AML

Turk J Hematol 2011; 28: 103-6

Bulgular: Çalışma kapsamına alınan 23 hastanın 10’unda normal karyotip; 6 hastada anormal karyotip bulundu. Hastaların hiçbirinde hTERC bölgesinde gen amplifikasyonu saptanamadı.

Tartışma: Hasta grubumuzda hTERC amplifikasyonu bulmadık. Bu nedenle, hTERC gen amplifikasyonunun lösemik hücrelerde telomeraz anlatımı ve aktivitesini arttırmada kısmen etkili olduğunun bildirilmesine karşın, çalışmamızın sonuçları ile benzer bir sonuca ulaşmak mümkün olmamıştır. Bu sonuç, AML gelişiminde gen amplifikasyonu yoluyla telomeraz aktivite artışının, solid tümörlerde olduğu kadar önemli bir faktör olmadığı görüşünü destekler niteliktedir. (Turk J Hematol 2011; 28: 103-6) Anahtar kelimeler: AML, hTERC, FISH Geliş tarihi: 11 Şubat 2010

Kabul tarihi: 6 Eylül 2010

Introduction Acute myeloid leukemia (AML) originates from myeloid cells. The disease is characterized by rapid proliferation of abnormal cells, which accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common form of acute leukemia in adults and its incidence increases with age [1]. Morphological, cytochemical, immunophenotypic, cytogenetic, and molecular features of the blasts are important factors in the classification of AML patients [2].

Telomeres, located at the ends of chromosomes, determine the replication capacity of cells. They shorten during each cell division and it is generally thought that telomere length serves as a clock for cells. Telomerase is a ribonucleoprotein complex, consisting of human transcriptase reverse transcriptase (hTERT), proteins (hTP1), and RNA template for telomeric DNA synthesis (hTERC) [3]; this complex synthesizes telomeres and stabilizes their length. Mutations in any of these components may result in a rare bone marrow failure syndrome known as dyskeratosis congenita [4].

Telomerase activity was reported to be low in normal human somatic cells [3]. Proliferating cells become senescent when telomeres are short and when there is no telomerase activity. Cancer cells are known to overcome this senescence pathway and become immortalized. Immortalization is almost always accompanied by expression of telomerase, which is most likely necessary for the continued growth of cancer cells. Telomerase is activated in most malignant tumours, but is usually inactive in normal somatic cells [3]. Despite the questionable role of telomerase reactivation in cell immortalization and carcinogenesis, telomerase may serve as a diagnostic marker for tumor development [5]. Several studies have shown that telomerase activity can be used to differentiate malignant

from normal tissue in various organs; however, the majority of reports are devoted either to telomerase activity or expression [6]. The present study aimed to determine the presence of amplification of the hTERC component of the telomerase complex in cultured bone marrow cells obtained from AML patients.

Materials and Methods The study included bone marrow samples taken at the time of diagnosis from 23 AML adult patients between 2006 and 2008 that analyzed in our cytogenetics laboratory. Conventional cytogenetic analysis and FISH results for each patient were evaluated retrospectively, and additional hybridization with the hTERC (3q26)/ 3q11 probe (Kreatech, The Netherlands) was performed to determine hTERC gene amplification. Slides were dehydrated in 70%, 85%, and 90% alcohol series, consecutively, and dried at room temperature. Denaturation and hybridization were carried out in a HyBrite denaturation/ hybridization system for FISH (Vysis, UK). The probe was denatured at 75°C for 5 min. Hybridization was carried out at 37°C for 14-16 h. After hybridization, the slides were washed in first post hybridization wash buffer at 73°C for 2 min, and then in second post hybridization wash buffer at room temperature for 1 min and counterstained with DAPI. Signals were quantified in ≥200 cells for both hTERC gene and 3q11 signals under oil immersion at 1000× magnification using the recommended filters. Results are expressed as the ratio of hTERC signal (orange) to 3q11 signal (green). The expected ratio is 1:1; which indicates there is no gene amplification and when the ratio of signals is ≥2:1, it indicates hTERC gene amplification (Figure 1).

Our samples were chosen from archive material consisting of patient samples who approved material useage after diagnostic procedures.

Turk J Hematol 2011; 28: 103-6

Results Among the 23 patients, 14 (60.9%) were male and 9 (39.1%) female; median age was 49 years (range: 15-76 years). Conventional cytogenetic and routine FISH results are given in the Table 1.

Cytogenetic analysis was not possible in 7 patients due to culture failure or because we did not find suitable metaphases for chromosome analysis (shown in the Table as N/A). In all, 10 of the 23 patients had a normal karyotype and 6 had an abnormal karyotype. The observed abnormalities included trisomy 8, t(1;3), der(7), t(15;17), del(5), and inv(16). We did not observed any complex karyotypes and the above-mentioned cytogenetic defects were the only abnormality in each patient.

Routine FISH analysis showed that 5 patients had t(15;17), 2 patients had t(8;21), 1 patient had triple signal for the 8q22 region, 1 patient had a deletion in the 5q31 region, and 1 patient had inv(16).

According to FISH analysis, none of the patients had hTERC gene amplification.

Discussion Normal human cells have low levels of telomerase expression; however, when telomere length reaches a critical point abnormal activation of telomerase can lead to immortalization and uncontrolled proliferation. This process is associated with the development of many leukemias and lymphomas [8]. More than 80% of various cancers have increased telomerase activity, and it has been reported that amplification of the telomerase gene might play a role in telomerase reactivation, which

Figure 1. Normal AML cells for hTERC gene amplification analysis using FISH. The specific molecular probe for hTERC was labeled with rhodamine (Spectrum Orange) and the 3q11 reference probe was labeled with fluorescein (Spectrum Green)

Ă&#x2013;zer et al. hTERC analysis by FISH in AML

105

leads to cell immortalization and seems to be a common event in carcinogenesis [9].

It has been reported that increased hTERT and/or hTERC gene expression may be an important mechanism involved in upregulated telomerase activity.

Accordingly, investigation of hTERT and/or hTERC amplification might be useful in the diagnosis and follow-up of cancer patients [10]. In the present study we only examined amplification of the hTERC component, as it is known that hTERC gene amplification alone can result in increased telomerase activity [10]. Further investigation of hTERT amplification may be useful for elucidating the interactions of these genes in the progression of AML. hTERC gene expression can be affected by and regulated by various processes inside cells; namely, gene amplification, transcriptional regulation, and epigenetic modification [11]. It was suggested that the high-level of expression and activity of telomerase observed in acute leukemia cells might be due to hTERC gene amplification, which is localized at 3q26.3. FISH is a useful method for observing gene amplification and was used in the present study to determine hTERC gene amplification. Previous studies have reported that amplification of hTERT and hTERC genes in acute leukemia cells exhibit a variable number of copies-as many as 12-based on FISH analysis [12].

One study that included patients with different types of leukemia subgroups reported that amplification of hTERC and hTERT genes was associated with increased telomerase activity and expression in leukemia cells [12]. Hematologic malignancies differ from other cancers, as they do not originate from cells with low-level telomerase activity.

Serakinci et al. reported that there was no amplification of the hTERT region in patients with monosomy 7 and no deletion at 7q31 [13]. In the present study 1 patient had a derivative chromosome 7 (patient No. 10), which we think originated from a translocation with an undefined chromosome. The fact that we did not observe amplification of the hTERC region in this patient supports the notion that this amplification may not be a characteristic of leukemia cells with an abnormality in chromosome 7.

Nonetheless, the unknown chromosomal component of the translocation and the putative effector genes on the derivative chromosome 7 should not be overlooked.

106

Ă&#x2013;zer et al. hTERC analysis by FISH in AML

Turk J Hematol 2011; 28: 103-6

Table 1. The patients, and their conventional cytogenetic and FISH results Patient Sex Number 1

E

Age Karyotype 76

N/A

2 E 15 47,XY,+8

FISH panel Normal 8q22 triple signal

3

E

24

46,XY,t(1;3) (p36.3;q21) Normal

4

K

64

N/A

Normal

5

E

27

Normal

Normal

6

K

28

N/A

t(15;17) +

7

K

34

N/A

t(15;17) +

8

K

50

Normal

t(15;17) +

9

E

49

Normal

Normal

10 E 47

46,XY,der(7)t(7;?) t(8;21) + (q32;?),t(8;21)(q22;q22)

11

E

33

46,XY,t(15;17)(q22;q21) t(15;17) +

12

E

55

N/A

Normal

13

E

52

N/A

t(8;21) +

14 E 37

46,XY,del(5)(q31) [16]/46,XY[2]

Deletion 5q31

15

Normal

Normal

K

50

16 E 75

46XY,inv(16) (p13q22 ) inv(16) + [4]/46,XY[1]

17

E

27

Normal

Normal

18

E

46

N/A

Normal

19

E

53

Normal

Normal

20

K

39

Normal

Normal

21

K

57

Normal

Normal

22

K

58

Normal

t(15;17) +

23

K

65

Normal

Normal

Given the genetic heterogeneity of AML, investigation of hTERC gene amplification and frequent genetic changes could further delineate the tumor biology and might be useful for determining the prognostic factors in AML. In the present study hTERC gene amplification in the bone marrow samples was not observed, based on FISH analysis.

Given the limited number of cases included, it is not possible to definitively conclude that hTERC gene amplification has any impact on the clinical course of AML or that its amplification based on FISH could be used as a diagnostic marker; however, the present results are in agreement with those of previous studies that have suggested that increased telomerase activity due to telomerase gene amplification may not be as influential in leukemias as in solid

tumors [13]. Additional research (with larger patients groups) on the expression of the hTERC gene and other components of the telomerase complex is warranted.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ. Cancer statistics. CA Cancer J Clin 2006;56:106-30.

2. Flandrin G . Classification of acute myeloid leukemias.

Atlas Genet Cytogenet Oncol Haematol. May 2002.

URL: http://AtlasGeneticsOncology.org/Anomalies/ ClassifAMLID1238.html 3. Norrback KF and Roos G. Telomeres and telomerase in normal and malignant haematopoietic cells. Eur J Cancer 1997;33:774-80.

4. Kirwan M, Dokal I. Dyskeratosis congenita: a genetic disorder of many faces. Clin Genet 2008;73:103-12.

5. Ohyashiki JH, Sashida G, Tauchi T, Ohyashiki K. Telomeres and telomerase in hematologic neoplasia.

Oncogene 2002;21:680-7.

6. Ohyashiki JH, Ohyashiki K, Iwama H, Hayashi S, Toyama K, Shay JW. Clinical implications of telomerase activity levels in acute leukemia. Clin Cancer Res 1997;3:619-25.

7. Davison GM. Telomeres and telomerase in leukaemia and lymphoma. Transfusion and Apheresis Science 2007;37:43-7.

8. Januszkiewicz D, Wysoki J, Lewandowski K, Pernak M, Nowicka K, Rembowska J, Nowak J. Lack of correlation between telomere length and telomerase activity and expression in leukemic cells. Int J Mol Med 2003;12:935-8.

9. Cao Y, Bryan TM, Reddel RR. Increased copy number of the TERT and TERC telomerase subunit genes in cancer cells. Cancer Sci 2008;99:1092-9.

10. Cairney CJ, Keith WN. Telomerase redefined: integrated regulation of hTR and hTERT for telomere maintenance and telomerase activity. Biochimie 2008;90:3-23.

11. Nowak T, Januszkiewicz D, Zawada M, Pernak M, Lewandowski K, Rembowska J, Nowicka K, Mankowski P, Nowak J. Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase. Oncology Reports 2006;16:301-5.

12. Serakinci N, Koch JE. Telomerase activity in human leukemic cells with or without monosomy 7 or 7q-.

BMC Med Genet 2002;29:3-11.

Research Article

107

The correlation between T regulatory cells and autologous peripheral blood stem cell transplantation in multiple myeloma Multipl miyelomda T düzenleyici hücreleri ile otolog çevre kanı kök hücre nakli arasındaki korelasyonun araştırılması Ayşe Pınar Erçetin, Safiye Aktaş, Özden Pişkin, Halil Ateş, Zeynep Filiz Zadeoğluları, Nur Hilal Turgut, Mehmet Ali Özcan Department of Institute of Oncology, Faculty of Medicine, Dokuz Eylul University, İzmir, Turkey

Abstract Objective: Multiple myeloma (MM) is characterized by malignant proliferation of plasmocytes and their precursors. T regulatory cells (Tregs) have a role in immunosuppression and control of autoimmunity, and are currently an important topic in the study of immune response to tumor cells. The correlation between Tregs and autologous peripheral blood stem cell transplantation (APBSCT) in MM has not been studied. The aim of this study was to compare CD4+CD25+FOXP3+ Treg, CD200, and PD-1 levels in MM patients that did and did not undergo APBSCT. Materials and Methods: Peripheral blood samples were collected from 28 MM patients ranging in age from 41 to 78 years for analysis of CD4CD25+ FOXP3+ Tregs, PD-1 (CD279), and CD200. Peripheral blood mononuclear cells were isolated via density gradient centrifugation. Four-color flow cytometry was performed. Using a sequential gating strategy, Tregs were identified as CD4+CD25+FOXP3+ T-cells. Results were analyzed using the Mann Whitney U non-parametric test and a compare means test. p values <0.05 were considered statistically significant.

Results: The study included 28 MM patients (10 female and 18 male). In all, 11 of the patients underwent APBSCT. The level of Tregs identified as CD4+CD25+FOXP3+ T-cells was higher in the patients that underwent APBSCT (p=0.042). CD200 and PD-1 levels did not significantly differ between the 2 groups (p=0.711 and p=0.404, respectively). There weren’t any statistically significant differences in CD200, PD-1, or CD4+CD25+FOXP3+ T-cell levels between the patients that did and did not undergo APBSCT (p>0.05).

Conclusion: Treg levels were higher in the patients that underwent APBSCT. Tregs are crucial for the induction and maintenance of peripheral tolerance to self-antigens. In addition, Tregs can suppress immune responses to tumor antigens; however, APBSCT and Treg levels were not correlated with CD200 or PD-1 expression. Relationship of Tregs with prognosis needs to be determined by studies that include larger cohorts. (Turk J Hematol 2011; 28: 107-14) Key words: T Regulatory cells, multiple myeloma, autologous bone marrow transplantation, CD200, programmed death-1 Received: May 10, 2010

Accepted: November 26, 2010

Address for Correspondence: Dr. Ayşe Pınar Erçetin, Department of Institute of Oncology, Faculty of Medicine, Dokuz Eylul University, İzmir, Turkey Phone: +90 232 412 58 73 E-mail: pinarercetin@gmail.com doi:10.5152/tjh.2011.26

108

Erçetin et al.

T regulatory cells in multiple myeloma

Turk J Hematol 2011; 28: 107-14

Özet Amaç: Multipl miyelom (MM) plazmositlerin ve bunların öncü hücrelerinin habis proliferasyonu ile kendini gösteren bir hastalıktır. T düzenleyici (regülatör) hücreler (Treg) immünsüpresyonda ve otoimmün sistemin denetiminde rol oynarlar. Ayrıca tümör hücrelerine karşı oluşan immün yanıttaki rolleri de güncel bir araştırma konusudur. Treg hücreleri MM’da otolog çevre kanı kök hücre nakli (APBSCT) ile ilişkili olarak daha önce araştırılmamıştır. Bu çalışmanın amacı, CD4+ CD25+ FoxP3+ Treg hücreleri ile, CD200 ve PD–1 ‘in APBSCT yapılmış ve yapılmamış MM hastalarındaki düzeylerini karşılaştırmaktır.

Yöntem: Yaşları 41 ile 78 arasında 28 MM hastasından CD4+ CD25+ FoxP3+ Treg hücreleri ile, CD200 ve PD–1 (CD279) analizi için çevre kanı örnekleri alındı. Mononükleer hücreler dansite gradient santrifüj yöntemi ile ayrıldı. Dört renkli akış sitometri ile uygulandı. Ardışık kapılamalar (gating) ile Treg hücreleri CD4+ CD25+ FoxP3+ T hücreleri olarak tanımlandı. Sonuçlar Mann Whitney U nonparametrik testi ve Compare Means testleri ile analiz edildi. P değerleri <0.05 olanlar istatistiksel açıdan anlamlı kabul edildi.

Bulgular: Bu çalışma 28 MM hastası (10 kadın, 18 erkek) içermektedir. Otolog çevresel kan kök hücre nakli 11 hastaya uygulanmıştır. CD4+ Treg hücreler CD4+ CD25+ FoxP3+ olarak saptanmış olup APBSCT alan hastalarda daha yüksek bulunmuştur (p=0.042). CD200 ve PD-1 iki grup arasında istatistiksel anlamlı bir sonuç göstermemiştir (sırasıyla; p=0.711 ve p=0.404). APBSCT yapılan ve yapılmayan gruplar arasında CD200, PD-1 ve CD4+CD25+FOXP3+ düzeyleri karşılaştırıldığında istatistiksel anlamlı bir sonuç bulunmamıştır (p>0.05).

Sonuç: Bu çalışmada Treg hücreleri APBSCT yapılmış hastalarda daha yüksek düzeyde bulunmuştur.

Treg hücrelerinin bireyin kendi hücrelerine karşı periferik toleransın indüklenmesinde ve korunmasında çok önemli rolü olduğu bilinmektedir. Buna ek olarak, Treg hücreleri tümör antijenlerine karşı oluşturulacak immün yanıtı baskılayabilmektedir. Ne var ki, çalışmamızda APBSCT veya Treg hücre düzeyleri CD200 ve PD-1 ekspresyonları ile korelasyon göstermemiştir. Prognoz ile ilişkileri daha fazla sayıda olgu grupları içeren çalışmalarla aydınlatılabilir. (Turk J Hematol 2011; 28: 107-14) Anahtar kelimeler: T Regülatör hücreler, multipl miyelom, otolog kemik iliği transplantasyonu, CD200, programlı ölüm-1 Geliş tarihi: 10 Mayıs 2010

Kabul tarihi: 26 Kasım 2010

Introduction Multiple Myeloma (MM) is characterized by malignant proliferation of plasmocytes and their precursors. It is an incurable clonal B-cell malignancy with terminally differentiated plasma cells [1,2]. The DurieSalmon and International Staging System (ISS) are used for MM staging [3,4]. Major advances in the treatment of MM include immunomodulatory drugs (lenalidomide and thalidomide) and proteosome inhibitors (bortezomib) [5]. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance to MM cells via both direct cell contact and soluble factors. Thalidomide and immunomodulatory derivatives, as well as the proteosome inhibitor bortezomib, which can overcome clinical drug resistance, act not only directly on MM cells, but also on the BM microenvironment to inhibit up-regulation of IL-6 and VEGF secretion triggered by the binding of MM cells to BM stem cells [6,7].

T regulatory cells (Tregs) play a role in immunosuppression and control of autoimmunity, and are

currently an important topic in the study of immune response to tumor cells. Various subsets of Tregs have been identified based on their expression of cell surface markers, production of cytokines, and mechanisms of action. Naturally occurring thymicderived CD4+CD25+ Tregs are a T-cell population with immunosuppressive properties that constitute 5%-10% of peripheral CD4+ T-cells [8,9]. Recent studies have reported that the transcription factor forkhead box P3 (FOXP3) is an exclusive intracellular marker for Tregs (10). In addition, FOXP3 is also a crucial transcription factor in the development and function of CD4+CD25+ Tregs. FOXP3 blocks the ability of Rel-family transcription factors NFAT and NFκB to induce their target genes, and as a consequence it acts as a transcriptional repressor of IL-2 and other cytokine genes (IL-4 and IFN-γ), thereby programming cells not to exert immune stimulatory functions. In brief, CD4+ Tregs are identified by their CD4+CD25+FOXP3+ expression pattern [8-10]. Dysfunctional Tregs were observed in MM; as such, Tregs have become a novel target for research [11].

Turk J Hematol 2011; 28: 107-14

CD200 is a type 1 transmembrane glycoprotein with an immunosuppressive effect on T-cellmediated immunity and delivers immunoregulatory signals following engagement of CD200R. It is expressed by thymocytes, activated T-cells, B-cells, dendritic cells, endothelial cells, and neurons. Its up regulation is thought to be related to tumor cell evasion of the immune system [12,13]. CD200 is currently considered a new prognostic factor in MM [14]. Programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2 deliver inhibitory signals that regulate the balance between T-cell activation, tolerance, and immunopathology. PD-1 is expressed on T-cells, B-cells, natural killer cells, and dendritic cells. PD-1 is not expressed on resting T-cells, but is inducibly expressed after activation [15]. PD-1 typically has a greater effect on cytokine production than on cellular proliferation, with significant effects on IFN-γ, TNF-α, and IL-2 production [16]. PD-1 expression is upregulated on tumor-infiltrating lymphocytes, which may also contribute to tumor immunosuppression, and stimulates T-cell apoptosis. Its inhibition is related to tumor progression.

PD-L1 negatively regulates CD4+CD25+FOXP3+ Tregs by limiting STAT-5 phosphorylation [17].

T-cells non-responsive to tumor cells have been reported in MM (1,2). As such, immunosuppressive CD4+CD25+FOXP3+ Tregs, PD-1, and CD200-a novel prognostic factor in MM-were selected as the subjects of the present study. CD4+CD25+FOXP3+ Tregs have a high level of expression in patients that undergo allogeneic bone marrow transplantation [18]. CD200 is also considered a novel prognostic factor in MM, with high expression [14].

Additionally, PD-1 is related to tumor immunosuppression, but PD-1 levels in MM patients that underwent autologous peripheral blood stem cell transplantation (APBSCT) haven’t been reported. Based on this knowledge, our hypothesis was that CD4+CD25+ FOXP3+ Tregs, CD200, and PD-1 would have higher levels of expression in MM patients that underwent APBSCT. As such, the aim of the present study was to compare the leve CD4CD25+ FOXP3+ Treg, CD200, and PD-1 levels in MM patients that did and did not undergo APBSCT.

Materials and Methods This prospective study’s protocol was approved by the Dokuz Eylul University Medical Faculty Ethics

Erçetin et al.

T regulatory cells in multiple myeloma

109

Committee. Blood samples were not collected from a biobank. Fresh blood samples were collected from volunteer MM patients that were diagnosed, treated, and followed-up at our hematology department. MM patients that did and did not undergo APBSCT were included in the study. For MM patients that did not undergo APBSCT allogeneic stem cell transplantation was an exclusionary criterion.

Patients that did not volunteer to participate were excluded from the study.

Patient characteristics This study included 28 MM patients aged 41-78 years. Documentation about the aim of the study and procedures for collecting blood samples not associated with MM treatment were prepared in advance. Written informed consent was obtained from each volunteer participant. In all, 10 patients were female and 18 were male. All patients received first-line therapies and 11 patients underwent APBSCT. Blood was collected 40-110 d following transplantation. All of the APBSCT patients received the same mobilization protocol, which included high-dose cyclophosphamide. Patient clinical parameters and therapies were recorded at the time of sample collection and analysis. Patients that did not undergo APBSCT were treated with a combination of vincristine, adriamycin, and dexamethasone (VAD) (n=3) or cyclophosphamide and dexamethasone combination (CYP+DEX) (n=8). Only 2 cases were treated with a melphalan prednisolone combination (MP). Sample collection and analysis were performed on the same day so that the parameters and therapies at the time of sample collection and analysis would not differ. Frozen samples were not used in the study.

Sample collection and preparation CD4CD25+Foxp3+ Tregs have a high level of expression in solid tumors, but as MM is a hematological malignancy sufficient quantities for flow cytometric analysis were obtained from peripheral blood samples [19-21]. We did not collect samples from bone marrow because, it would has been a corrosive procedure for the patients to get enough sample.

Peripheral blood samples were collected into sterile EDTA containers for analysis of CD4CD25+ FOXP3+ Tregs, PD-1 (CD279), and CD200. Samples were diluted with phosphate buffer saline solution. Peripheral

110

Erçetin et al.

T regulatory cells in multiple myeloma

blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation in Biocoll separating solution (density: 1.077 g mL-1, Biochrom) and underwent immediate analysis by flow cytometry.

Flow cytometry Four-color flow cytometry was performed on an Epics XL (Beckman Coulter) and analyzed with Expo 32 ADIC XL 4 Color software. Directly conjugated monoclonal antibodies (mAbs) against CD4FITC, CD25-pyhcoerythryrin (PE) (eBioscience), and PD-1- FITC/CD200-PE (BD Biosciences), and appropriate IgG isotype controls were used to stain mononuclear cells. Isolated PBMCs underwent immediate staining procedures. Frozen or ripe samples were not used for flow cytometry staining or analysis. Fixation and permeabilization procedures were used for intracellular indirect staining.

Co-staining of intracellular FOXP3 was performed via application of the PE-Cy5 conjugated anti-FOXP3 clone PCH101 (eBioscience). Staining protocols were performed according to the manufacturers’ instructions. During all staining procedures nonspecific conjugation was blocked via use of normal rat serum. Using a sequential gating strategy Tregs were identified as CD4+CD25+FOXP3+ T-cells.

CD200 and PD-1 levels were also determined using a sequential gating strategy.

Statistical analysis Results were analyzed using the Mann-Whitney non-parametric U test, Pearson’s correlation analysis, and the Kaplan Meier survival analysis test. P values <0.05 were considered statistically significant.

Survival was compared according Treg high and low levels. Mean values were taken as the cut off for high ands low Treg levels. Kaplan Meier survival curves were established regardless of patient transplantation status for comparison of Treg high and low populations. Both groups included transplant or nontransplant patients. The study did not include a sufficient number of patients for forming subgroups.

Results This study included 28 MM patients (10 female and 18 male). In all, 11 of the patients underwent APBSCT. The clinical and histological features of the patients that did [APBSCT (+)] and did not [APBSCT

Turk J Hematol 2011; 28: 107-14

(-)] undergo APBSCT are shown in Tables 1 and 2, respectively.

Flow cytometry results CD4+CD25+FOXP3+ Treg, CD200+, and PD-1+ T-cell levels were determined using sequential and appropriate gating strategies. CD4+CD25+FOXP3+ Treg levels ranged between 0% and 11% in all the cases, between 0.07% and 9% in the APBSCT (+) cases, and between 0% and 11% in the APBSCT (-) cases. CD200+ cell levels ranged between 0.1% and 7.08% in all the cases, between 0.1% and 6.72% in the APBSCT (+) cases, and between 0.1% and 7.08% in the APBSCT (-) cases. The level of cells expressing PD-1 ranged between 0% and 50.66% in Table 1. Clinical and histological features of the APBSCT (+) cases (n=11)

Female

5

45.5%

Male

6

54.5%

Kappa (K)

5

45.5%

Light Chain-Type

Lamda (L)

5

45.5%

NA

1

4.0%

IgG

7

63.6%

Ig Heavy Chain-Type

IgA

2

18.2%

NA

2

18.2%

Stage 1

2

18.2%

ISS

Stage 2

5

45.5%

Stage 3

2

18.2%

NA

1

9.1%

IA

1

9.1%

IIA

3

27.3%

Durie-Salmon Stage

IIIA

3

27.3%

IIIB

3

27.3%

NA

2

9.1%

Diffuse

5

45.5%

Bone Marrow Biopsy

Nodulary

5

45.5%

Diffuse and Nodulary

1

9.1%

VAD

6

54.5%

Therapy

CYP + DEX

4

36.4%

NA

1

9.1%

CR

1

9.1%

PR

6

54.5%

Therapy Response

SD

2

18.2%

Refractory

1

9.1%

NA

1

9.1%

Gender

Erçetin et al.

T regulatory cells in multiple myeloma

Turk J Hematol 2011; 28: 107-14

Table 2. Clinical and histological features of the APBSCT (-) cases (n=17)

population did not significantly differ between the APBSCT (+) and APBSCT (-) patients (p=0.353).

CD4+ T-cell and CD4+CD25+FOXP3+ Treg levels were not correlated with transplantation status (p=0.137). CD4+ T-cells were correlated with CD200and PD-1-expressing T-cells, according to transplantation status (p=0.011 and p=0.021, respectively).

Based on correlation analysis performed separately

5

29.4%

Male

12

70.6%

Kappa (K)

14

82.4%

Light Chain-Type

Lamda (L)

2

11.8%

NA

1

5.8%

IgG

12

70.6%

Ig Heavy Chain-Type

IgA

3

17.6%

NA

2

12.8%

Stage 1

7

41.2%

Stage 2

3

17.6%

Stage 3.

3

17.6%

NA

4

23.6%

IA

7

41.2%

IIA

5

29.4%

Durie-Salmon Stage

IIB

2

11.8%

IIIA

1

5.9%

NA

2

12.8%

Diffuse

9

52.9%

Bone Marrow Biopsy

Nodulary

3

17.6%

Diffuse and Nodulary

3

17.6%

NA

2

12.8%

VAD

3

17.6%

CYP + DEX

8

47.1%

Therapy

MP

2

11.8%

7.00

NA

4

29.5%

6.50

CR

3

17.6%

6.00

PR

7

41.2%

Therapy Response

SD

1

5.9%

Refractory

2

11.8%

NA

4

23.6%

ISS

all the cases, between 0% and 53.02% in the APBSCT (+) cases, and between 0% and 50.66% in the APBSCT (-) cases. Flow cytometry results for CD4+ T-cells, CD4+CD25+FOXP3+ Tregs, PD-1 (+), and CD200 (+) T-cells are shown in Table 3.

Statistical results The level of CD4+ Tregs identified according to CD4+CD25+FOXP3+ was higher in the APBSCT (+) patients (p=0.042) (Figure 1). CD200 and PD-1 levels did not significantly differ between the APBSCT (+) and APBSCT (-) patients (p=0.711 and p=0.404, respectively) (Figures 2 and 3). The CD4+ T-cell

T regilatory cells %

Female

11.00 10.50 10.00 9.50 9.00 8.50 8.00 7.50 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

Outliers are hidden Extreme values are hidden (-)

(+)

Autologous Peripheral Blood Stem Cell Transplantation

Figure 1. Comparison of CD4+CD25+FOXP3+ Treg levels between APBSCT (+) (n=11) and APBSCT (-) (n=17) patients’ peripheral blood samples (n=28). The percentage of Tregs in the peripheral blood of APBSCT (+) patients was significantly higher than that in the peripheral blood of APBSCT (-) patients

(-)

(+)

5.50 5.00 4.50 CD200 %

Gender

111

4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Autologous Bone Marrow Transplantation

Figure 2. Comparison of CD200-expressing lymphocyte cell levels between the peripheral blood samples of APBSCT (+) (n=11) and APBSCT (-) (n=17) patients (total n=28). There wasn’t a statistically significant difference in CD200-expressing lymphocyte cell levels between the peripheral blood samples of the APBSCT (+) and APBSCT (-) patients (p>0.05)

112

Erçetin et al.

T regulatory cells in multiple myeloma

Turk J Hematol 2011; 28: 107-14

for APBSCT (+) and APBSCT (-) patients, CD4+ T-cell levels were positively correlated with CD200and PD-1-expressing T-cells in the APBSCT (-) patients (p=0.013 and p=0.035, respectively). There wasn’t a statistically significant difference in CD200, PD-1, or CD4+CD25+FOXP3+ levels between the APBSCT (+) and APBSCT (-) patients (p>0.05). According to Pearson’s correlation analysis, CD4+CD25+FOXP3+ Tregs were not correlated with CD200- or PD-1expressing T-cells in the APBSCT (-) patients (p>0.05); however, a significant correlation was observed between CD200- and PD-1-expressing T-cells (p=0.005). Kaplan Meier survival analysis showed

that MM patients with high Treg concentrations had longer event-free survival than MM patients with low Treg concentrations (Figure 4).

Discussion

In the present study Treg levels were higher in the APBSCT (+) patients. Tregs are crucial for the induction and maintenance of peripheral tolerance 55.00 50.00 45.00

Table 3. Flow cytometry results for CD4+ T-cells, CD4+ CD25+FOXP3+ Tregs, and PD-1+ and CD200+ T-cells CD200 (+)

PD-1 (+)

35.00

PD-1 %

APBSCT Patient CD4 (+) CD4 (+)

no.

T-cells CD25 (+)

FOXP3 (+)

40.00

30.00 25.00

1

21.1%

0.5%

4.2%

2.2%

2

10.4%

2.8%

1.9%

1.0%

3

6.7%

9.0%

0.9%

0.4%

4

6.3%

6.6%

0.8%

0.6%

+

5

7.4%

1.4%

1.1%

0.2%

5.00

6

12.7%

4.9%

0.1%

0.0%

0.00

7

19.1%

1.4%

6.7%

12.1%

8

17.24%

1.8%

1.54%

3.83%

9

7.34%

1.47%

6.72%

4.84%

10

8.23%

0.07%

0.79%

50.66%

11

8.78%

3.53%

6.62%

4.63%

12

5.6%

11.0%

0.1%

0.2%

13

4.9%

1.1%

1.2%

1.4%

14

3.8%

0.0%

0.5%

3.0%

15

4.7%

1.4%

0.5%

0.9%

16

8.3%

1.9%

4.6%

1.0%

17

11.0%

0.5%

0.9%

0.1%

18

17.8%

3.9%

1.3%

1.6%

-

19

68.03%

0.73%

3.2%

44.42%

20

12.0%

2.33%

0.98%

5.35%

21

11.79%

0.51%

0.52%

4.61%

22

21.47%

0.19%

2.57%

16.77%

23

17.41%

1.69%

2.11%

4.57%

24

27.94%

0.71%

2.88%

26.79%

25

37.98%

0.04%

1.28%

2.5%

26

11.48

0.08%

7.08%

53.02

27

28.2%

0.08%

5.05%

2.64%

28

14.24%

0.05%

1.01%

6.88%

20.00

(-)

15.00

(+)

10.00

Autologous Peripheral Blood Stem Cell Transplantation

Figure 3. Comparison of PD-1-expressing lymphocyte cell levels between the peripheral blood samples of APBSCT (+) (n=11) and APBSCT (-) (n=17) patients (n=28). There wasn’t a statistically significant difference in PD-1-expressing lymphocyte cell levels between the peripheral blood samples of the APBSCT (+) and APBSCT (-) patients (p>0.05) Survival Functions 1.0

+

Treg low high + low-censored + high-censored

+ +

0.8 Cum Survival

+ 0.6 + 0.4 +

+

0.2

0.0 0.00

20.00

40.00 60.00 80.00 Follow up (months)

100.00

Figure 4. Event-free survival analysis of all the MM patients. MM patients with higer Treg concentrations had longer survival than MM patients with low Treg concentrations

Erçetin et al.

T regulatory cells in multiple myeloma

Turk J Hematol 2011; 28: 107-14

to self-antigens. In addition, Tregs can suppress immune responses to tumor antigens; however, in the present study APBSCT and Treg levels were not correlated with CD200 or PD-1 expression.

Relationship of T regs with prognosis needs to be determined by studies that include larger cohorts.

The CD4+CD25+FOXP3+ trio is used in the present study because according to the literature it is the most widely used method. Accordingly, flow cytometry was used in the present study because of its ability to detect levels of this combination.

Atanackovic et al. reported that Treg reconstitution resulted in accumulation of donor-derived CD4+CD25+FOXP3+ cells in the BM of MM patients following APBSCT. They suggested that Tregs might contribute to the prevention of graft versus host disease via their immunosuppressive effect [18].

Similarly, high Treg populations were noted in the APBSCT (+) patients post surgery in the present study, suggesting that Tregs may play a role in stimulation of the immune system after APBSCT. CD200 is thought to induce Tregs that inhibit the immune response of tumor-specific effector T-cells [22]. In contrast, Treg levels in the present study were not correlated with CD200-expressing T-cells in the present study. In future studies this incongruity could be solved by studying BM, which better reflects the tumor microenvironment. Moreaux et al. reported that MM cases without CD200 expression have significantly longer event free-survival than CD200-expressing cases [14]; however, no such relationship was observed in the present study. Moreaux et al.’s study included 112 cases and MM cells in BM were investigated, whereas the present study included 28 cases and PBMCs were investigated, which might account for the different results obtained in each study.

Benson et al. reported that monoclonal antibody anti-PD-1 could be a novel immunotherapy for MM because its inhibition upregulates T-cell activation and immune response [23]. Accordingly, the relationship between PD-1 and novel prognostic factor CD200 should be considered. A significant correlation was observed between CD200- and PD-1expressing T-cells in the present study, but the role of this correlation on immune response in MM should be analyzed in future studies.

Acknowledgement This study was supported by the Dokuz Eylul University Research Foundation (project no. 2009.

KB. SAG.022) and the Izmir Blood Diseases and Cancer Association.

113

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Barillé-Nion S, Barlogie B, Bataille R, Bergsagel PL, Epstein J, Fenton RG, Jacobson J, Kuehl WM, Shaughnessy J, Tricot G. Advances in biology and therapy of multiple myeloma. Hematology Am Soc Hematol Educ Program 2003;248-78.

Schwartz RN, Vozniak M. Current and emerging treatments for multiple myeloma. J Manag Care Pharm 2008;14:12-9.

Durie BG, Salmon SE. A clinical staging system for multiple myeloma. Cancer 1975;36:842-54. [CrossRef] Greipp PR, San Miguel JF, Durie BG. International Staging System for multiple myeloma. J Clin Oncol 2005;23:3412-20. [CrossRef] Mollee P. Current trends in the diagnosis, therapy and monitoring of the monoclonal gammopathies. Clin Biochem Rev 2009;30:93-103.

Bommert K, Bargou RC, Stuhmer T. Signalling and survival pathways in multiple myeloma. Eur J Cancer 2006;42:1574-80. [CrossRef] Ander KC. Targeted therapy of multiple myeloma based upon tumor microenvironmental interactions.

Exp Hematol 2007;35:155-62.

Cools N, Ponsaerts P, Van Tendeloo VF, Berneman ZN. Regulatory T cells and human disease. Clin Dev Immunol 2007;2007:89195. [CrossRef] Mottet C, Golshayan D. CD4+CD25+FOXP3+ Regulatory T cells: from basic research to potential therapeutic use. Swiss Med Wkly 2007;137:625-34.

Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3.

Science 2003;299:1057-61. [CrossRef] Prabhala RH, Neri P, Bae JE, Tassone P, Shammas MA, Allam CK, Daley JF, Chauhan D, Blanchard E, Thatte HS, Anderson KC, Munshi NC. Dysfunctional T regulatory cells in multiple myeloma. Blood 2006;107:301-4.

Minas K, Liversidge J. Is the CD200/ CD200 receptor interaction more than just a myeloid cell inhibitory signal. Crit Rev Immunol 2006; 26:213-30.

Moreaux J, Veyrune JL, Reme T, De Vos J Klein B. CD200: a putative therapeutic target in cancer. Biochem Biophys Res Commun 2008;366:117-22. [CrossRef] Moreaux J, Hose D, Reme T, Jourdan E, Hundemer M, Legouffe E, Moine P, Bourin P, Moos M, Corre J, Möhler T, De Vos J, Rossi JF, Goldschmidt H, Klein B. CD200 is a new prognostic factor in multiple myeloma. Blood 2006;108:4194-7. [CrossRef] Keir ME, Francisco LM, Sharpe AH. PD-1 and its ligands in T cell immunity. Immunology 2007;19:309-14. [CrossRef] Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy

114

Erรงetin et al.

T regulatory cells in multiple myeloma

by PD-L1 blockade. Proc Natl Acad Sci USA 2002;99:12293-7. [CrossRef] 17. Franceschini D, Paroli M, Francavilla V, Videtta M, Morrone S, Labbadia G, Cerino A, Mondelli MU, Barnaba V. PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV. J Clin Invest 2009;119:551-64. [CrossRef] 18. Atanackovic D, Cao Y, Luetkens T, Panse J, Faltz C, Arfsten J, Bartels K, Wolschke C, Eiermann T, Zander AR, Fehse B, Bokemeyer C, Kroger N. CD4+CD35+FOXP3+ T regulatory cells reconstitute and accumulate in the bone marrow of patients with multiple myeloma following allogeneic stem cell transplantation. Haematologica 2008;93:423-30. [CrossRef] 19. Gallimore A, Godkin A. Regulatory T cells and tumour immunity-observations in mice and men. Immunology 2008;123:157-63. [CrossRef]

Turk J Hematol 2011; 28: 107-14

20. Ozer H, Han T, Henderson ES, Nussbaum A, Sheedy D. Immunoregulatory T cell function in multiple myeloma. J Clin Invest 1981;67:779-89. [CrossRef] 21. Lehner T. Special regulatory T cell review: The resurgence of the concept of contrasuppression in immunoregulation. Immunology 2008;123:40-4. [CrossRef] 22. Gorczynski RM, Lee L, Boudakov I. Augmented Induction of CD4+CD25+ Treg using monoclonal antibodies to CD200R. Transplantation 2005;79:1180-3.

[CrossRef] 23. Benson DM Jr, Bakan CE, Mishra A, Hofmeister CC, Efebera Y, Becknell B, Baiocchi RA, Zhang J, Yu J, Smith MK, Greenfield CN, Porcu P, Devine SM, RotemYehudar R, Lozanski G, Byrd JC, Caligiuri MA. The PD-1 / PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel, monoclonal anti-PD-1 antibody. Blood 2010;116:2286-94. [CrossRef]

Research Article

115

Nitric oxide in gingival crevicular fluid and nitric oxide synthase expression in the gingiva of patients with sickle cell disease Orak hücre anemili hastaların dişetlerindeki nitrik oksit sentetaz ekspresyonu ve dişeti oluğu sıvılarındaki nitrik oksit düzeyinin değerlendirilmesi Esra Güzeldemir1, Hilal Uslu Toygar2, Nebil Bal3, Ruksan Anarat4, Can Boğa5 1Department

of Periodontology, Faculty of Dentistry, Kocaeli University, Kocaeli, Turkey of Periodontology, Faculty of Dentistry, Baskent University, Ankara, Turkey 3Department of Pathology, Faculty of Medicine, Baskent University, Ankara, Turkey 4Department of Biochemistry, Faculty of Medicine, Baskent University, Ankara, Turkey 5Department of Hematology, Faculty of Medicine, Baskent University, Ankara, Turkey 2Department

Abstract Objective: This study aimed to biochemically measure the production of nitric oxide in gingival crevicular fluid and immunohistochemically measure the expression of inducible nitric oxide synthase in the gingiva of patients with sickle cell disease. Additionally, we aimed to obtain insight into the immunopathology of sickle cell disease by comparing inducible nitric oxide synthase levels in patients with sickle cell disease and controls using gingiva and gingival crevicular fluid.

Materials and Methods: The study included 20 sickle cell disease patients and 20 healthy controls.

Immunohistochemical analysis was used to measure inducible nitric oxide synthase expression in gingiva and nitric oxide levels in gingival crevicular fluid were spectrophotometrically measured.

Results: Nitric oxide levels in the patients and controls did not differ significantly (21.2±4.5 and 23.1±2.3 μM L-1, respectively, p>0.05). There weren’t any statistically significant differences in infiltrated inflammatory cells, density of inflammatory cells that stained with inducible nitric oxide synthase, or nitric oxide expression in gingiva between the patient and control groups (p>0.05).

Conclusion: To the best of our knowledge this is the first study to examine the expression of inducible nitric oxide synthase in the gingiva and gingival crevicular fluid in patients with sickle cell disease. Using the gingiva and gingival crevicular fluid we were unable to observe sickle cell disease-associated inducible nitric oxide synthase expression and a difference in nitric oxide levels.

(Turk J Hematol 2011; 28: 115-24)

Key words: Sickle cell disease, nitric oxide, nitric oxide synthase, gingival crevicular fluid, gingiva Received: June 22, 2009

Accepted: June 16, 2010

Address for Correspondence: Assoc. Prof. Esra Güzeldemir, Department of Periodontology, Faculty of Dentistry, Kocaeli University, Yuvacık Yerleşkesi, Yuvacık, Başiskele 41190 Kocaeli, Turkey Phone: +90 262 344 22 22 E-mail: esragd@yahoo.com doi:10.5152/tjh.2011.27

116

Güzeldemir et al.

NO metabolism in patients with SCD

Turk J Hematol 2011; 28: 115-24

Özet Amaç: Çalışmamızda, orak hücre anemili hastaların, dişeti dokusunda immünohistokimyasal yöntemle nitrik oksit sentetaz ekspresyonunun ve dişeti oluğu sıvısında biyokimyasal yöntemle nitrik oksit düzeyinin değerlendirilmesi amaçlanmıştır.

Yöntemler ve Gereçler: Çalışmaya, 20 orak hücre anemili hasta ve her yönden sağlıklı 20 kontrol kişi alınmıştır. Dişeti dokusundaki nitrik oksit sentetaz ekspresyonu immünohistokimyasal yöntemlerle, dişeti oluğu sıvısındaki nitrik oksit düzeyi ise spektrofotometrik yöntemlerle ölçülmüştür.

Bulgular: Hasta ve kontrol gruplarında, dişeti oluğu sıvısındaki nitrik oksit düzeyleri arasında istatistiksel olarak anlamlı bir fark bulunmamıştır (21.2±4.5 ve 23.1±2.3 μM L-1, p>0.05). Gruplar arasında, inflamatuvar hücre yoğunluğu, nitrik oksit sentetazla boyanan inflamatuvar hücrelerin yoğunluğu ve nitrik oksitle boyanan bu hücrelerde görülen ekspresyonun yoğunluğu açısından anlamlı bir fark görülmemiştir (p>0.05).

Sonuç: Araştırmalarımıza göre, bu çalışma, orak hücre anemili hastaların dişeti dokusunda ve dişeti oluğu sıvısında nitrik oksit düzeyini ve nitrik oksit sentetaz ekspresyonunu değerlendiren ilk çalışmadır. Çalışmamızda dişeti dokularında ve dişeti oluğu sıvısında, orak hücre anemisi ile ilişkili olarak, farklı nitrik oksit düzeyleri ve ekspresyonu görülmemiştir. (Turk J Hematol 2011; 28: 115-24) Anahtar kelimeler: Orak hücre anemisi, nitrik oksit, nitrik oksit sentetaz, dişeti oluğu sıvısı, dişeti Geliş tarihi: 22 Haziran 2009

Kabul tarihi: 16 Haziran 2010

Introduction Sickle cell disease (SCD) is the result of hemoglobin S homozygosity, which causes rigid sickle erythrocytes. Interactions between abnormal rigid erythrocytes and the vascular wall result in endothelial activation, which may lead to endothelial damage and tissue ischaemia in SCD. Chronic inflammation during both the steady state and vasoocclusive crisis occurs via the action of cytokines and other inflammatory factors [1-4]. The clinical manifestations of the disease, such as painful crises, anemia, bone necrosis, and leg ulceration, arise due to chronic hemolysis, vaso-occlusion, ischemia, and tissue damage.

Nitric oxide (NO) is a short-lived free radical that influences physiological processes in virtually every tissue and is derived from L-arginine via the action of constitutive NO synthase (NOS) or inducible NO synthase (iNOS), whose activity is induced in several cell types, including human gingival fibroblasts. iNOS expression results in increased production of NO and is activated during cellular distress, systemic inflammation, hypoxia, and oxidative stress.

NO is a molecule involved in vascular regulation, homeostasis, bone formation and resorption, neurotransmission, and immune function. It plays a role in host defense response to infection in oral tissues [5]. NO mediates physiologic processes in the immune, inflammatory, nervous, cardiovascular, and pulmonary systems.

In infection and the resultant inflammatory responses, NO plays a dual role: it is a central mediator of host response, but also is an important agent in the pathogenesis of host damage [6]. Decreased levels of L-arginine, and consumption of NO by cellfree plasma hemoglobin and by reactive oxygen species (ROS) appear to impair NO bioavailability, resulting in oxidative damage. Because SCD is associated with oxidative stress, increased expression of endothelial cell adhesion molecules, and blood cell adhesion, which are characteristic features of an inflammatory response, several researchers have studied the role of iNOS in SCD [7].

The intermittent vascular occlusion that occurs in SCD leads to ischemia-reperfusion injury and activation of inflammatory processes, including enhanced production of ROS and increased expression of iNOS. Increased iNOS expression and a consequent increase in tissue nitrite and nitrate production have been observed in the kidneys and liver of SCD patients [8], mice [2,3,8,9], and pigs [4].

Additionally, it was shown that iNOS inhibition significantly limits tissue ischemia-reperfusion injury in the liver and kidneys [10,11]; however, inhibition of iNOS attenuated ischemia-reperfusion injury in the rat heart [12] and did not affect ischemia-reperfusion injury in the rat lung [13]. Increased levels of iNOS expression were shown in the kidneys of transgenic mice [9], but Nath et al. [14] reported the lack of upregulation of iNOS in the intrarenal vasculature and increased expression of iNOS in the

Güzeldemir et al.

NO metabolism in patients with SCD

Turk J Hematol 2011; 28: 115-24

glomeruli and distal tubules of sickle mice. The role of iNOS and its expression seems to vary by organ.

In addition, NO metabolites (NOx; nitrate [NO3-] and nitrite [NO2-]) in SCD were evaluated in blood samples in vivo [15-17] and in vitro [18]. It was reported that NOS activity plays a role in stimulating production of NOx. iNOS was observed in dental tissues, and increased iNOS production was reported in gingival crevicular fluid (GCF) [19] and gingival tissues [20-23] in different periodontal diseases, but iNOS activity was not observed in the gingival tissues in sterile animals [24]. Periodontal diseases are inflammatory disorders that cause tissue damage and loss via the complex interactions between pathogenic bacteria and host immune response.

Oxidative stress also plays a role in the pathogenesis of periodontal diseases; this causes an imbalance between the antioxidant and pro-oxidant systems. ROS and reactive nitrogen species (NO, NO-2, and ONOO-) are also contributors to this process.

NO synthesis and iNOS activity increase in inflamed periodontal tissues [20,25-28] as a result of macrophage infiltration in periodontal tissues [29].

The literature includes only a few studies that evaluated iNOS activity in inflamed periodontal tissues. iNOS activity was reported to increase in ligatureinduced periodontitis [27], experimental periodontitis [30], and chronic periodontitis [20,26,31]. Higher levels of iNOS expression were observed in chronic periodontitis tissues than in clinically healthy gingival tissues [20,23,26,27,31] and a previous study of ours showed that iNOS increased in the gingival tissue of aggressive periodontitis patients (unpublished data).

In various animal models iNOS was susceptible to infections by a variety of pathogens [32-35]. It was shown that NO plays an important role in the pathogenesis of chronic inflammation [36]. iNOS is expressed almost exclusively during inflammatory conditions, which has led to the hypothesis that iNOS has detrimental effects on inflamed tissues and promotes the inflammatory response [37].

While data regarding dental and periodontal conditions is SCD patients are limited, there are no data regarding NO levels and the presence/expression of iNOS in the gingival tissues and GCF of SCD patients. GCF is inflammatory exudates that can be easily collected from gingival crevices of teeth by means of paper strips. Host cell-derived inflammatory tissue response and bacterial products in GCF

117

were assessed in studies related to markers of pathogenesis and the diagnosis of periodontal disease [38].

To the best of our knowledge the present study is the first to examine NO levels and the presence/ expression of iNOS in the gingiva and GCF of patients with SCD. Findings regarding NO levels and the expression of iNOS in different tissues and organs in SCD patients are inconsistent. The role of iNOS and its expression appears to vary in each organ in patients with SCD. It is well documented that iNOS is expressed in inflamed gingiva in different conditions and diseases. Ongoing chronic inflammatory reactions in SCD patients result in increased iNOS expression; therefore, the aim of the present study was to evaluate NO levels and the presence/expression of iNOS in the gingival tissues and GCF in SCD patients, as well as to obtain insight into the immunopathology of SCD in gingiva and and GCF, in terms of iNOS metabolism.

Methods Study population This study was conducted at Başkent University, Adana Medical and Research Center between January and August 2007. The Başkent University Ethics Committee (D-KA07/01) approved the study protocol. Samples were evaluated at Başkent University, Adana Medical and Research Center, Departments of Pathology and Biochemistry. All the participants provided informed consent. Evaluation of each participant consisted of personal, medical, and dental history, and panoramic radiographs and dental examinations. The inclusion criterion was age ≥18 years.

The study included 20 homozygous SCD patients in the steady stae that had not received blood transfusions in the preceding 3 months. The patients’ genotypes were confirmed by electrophoresis. The SCD patients were seen regularly at the Başkent University Hematology Clinic. As part of a standard medical examination they were sent to the Department of Periodontology for dental and periodontal examination and consultation.

The control group included 20 age- and sexmatched volunteers without a history of SCD. Exclusion criteria were as follows: history of hepatitis, HIV infection, or diabetes, requirement for antibiotic

118

Güzeldemir et al.

NO metabolism in patients with SCD

prophylaxis, pregnancy or lactation, and long-term use of anti-inflammatory drugs. All the participants were free of systemic, and dental and periodontal diseases. Both groups consisted of non-smokers.

GCF sampling and analysis Assessment of total NO metabolites (NOx: nitrate [NO3-] and nitrite [NO2-]) is commonly used as a measure of NO production in biologic fluids. GCF samples were obtained using standard paper strips (Periopaper, Ora Flow Inc., NY, USA ) following isolation of the tooth with cotton rolls and removal of any supragingival deposits on tooth surfaces. The collection region was gently air-dried for 2 s to reduce any contamination by plaque and saliva.

The strip was left at the entrance of the gingival sulcus for 30 s. GCF volume was calculated using a Periotron 8000 (Periotron 8000, Pro Flow Inc., Amityville, NY, USA ) device, which was pre-calibrated with a dry, sterile strip. Samples with evidence of bleeding were not included. After volume determination, GCF samples were placed in sterile Eppendorf tubes that were wrapped securely and stored at -20°C until analyzed. The same periodontist (E.G.) performed all clinical GCF sampling.

Quantification of nitrate/nitrite content in GCF There are numerous direct and indirect analytical methods for the quantification of NO in aqueous solutions, but only a few are applicable to biological fluids, as NO is highly reactive, has a short half-life in vivo, and is produced in small quantities in cells.

The method used in the present study is based on the quantification of the stable end products of nitric oxide-nitrate and nitrite. Nitrate determination requires chemical and enzymatic reduction to nitrite. This method is easy, quick, inexpensive, and more sensitive (to micromolar range) than other methods [39].

To each GCF sample in an Eppendorf tube was added 130 µL of distilled water. The samples were mixed to extract nitrate into the water. To measure nitrate levels 100 μL of the extract was obtained from the mixture and evaluated using the nitrite/ nitrate colorimetric test (Nitrite/Nitrate Colorimetric Test, Roche, Germany). The colorimetric reaction was allowed to proceed for 10 min at room temperature, and then the optical density (absorbance, OD) at 540 nm was measured at room temperature

Turk J Hematol 2011; 28: 115-24

using a spectrophotometer (Elisa, Merck, Germany).

The measured OD values were converted to concentrations using a standard curve established by the serial dilution of nitrate. Analyses were performed on coded samples by 1 of the authors (R.A.) that was blinded to the participants’ diagnoses.

Gingival biopsy collection and analysis The patients underwent gingival biopsy under local anesthesia. Local anesthetic agent without adrenalin was administered following application of a topical anesthetic agent. An inverse bevel incision was used to obtain tissue from the underside of the papilla. After washing with sterile 0.15 M saline solution, gingival biopsy specimens were fixed in 10% formalin solution and sent to the Pathology Department. Gingival tissue biopsy samples were collected by the same periodontist (E.G.) and analyses were performed on coded samples by another author (N.B.) that was blinded to the participants’ diagnoses.

After routine paraffin tissue procedures, tissues were embedded in paraffin blocks. Then, 4-5-μm thick sections were obtained from the paraffin blocks, and serial sections were immunostained with an AEC (3-amino-9-ethylcarbazole) substrate system and biotin-streptavidin complex system (3-amino-9-ethyl-carbazole [AEC]+ready-to-use substrate-chromogen K3469, DAKO, Denmark). AEC is a widely used chromogen for immunohistochemical staining. Deparaffinization and rehydration of the sections were followed by blocking endogenous peroxidase activity via incubation of the sections in 3% H2O2 for 30 min at room temperature. After rinsing with distilled water the sections were treated in a microwave oven with 10 mM citrate buffer (pH 6.0) at 500°C for 5 min, 600°C for 4 min, and then 700°C for 3 min for antigen retrieval. The slides were left to cool at room temperature for 50 min. After rinsing with phosphate-buffered saline (PBS) for 2-3 min the slides were kept in 3% H2O2 for 20 min, and then rinsed with PBS for 5 min. Non-specific binding was reduced with protein blocking serum for 20 min. Sections were incubated with iNOS primary polyclonal antibody (ready-to-use rabbit polyclonal antibody RB-1605-R7, Lab-Vision, CA, USA) at room temperature overnight. After rinsing with PBS for 2-3 min the sections were incubated with biotinylated goat antipolyvalent (Thermo Scientific Lab Vision

Güzeldemir et al.

NO metabolism in patients with SCD

Turk J Hematol 2011; 28: 115-24

TP-060-HL, Fremont, CA, USA) for 20 min. The sections were then washed with PBS for 2-3 min, treated with streptavidin peroxidase (ready-to-use TS-060-HR, Thermo Scientific, Lab Vision, Fremont, CA, USA) for 20 min, and washed with PBS for 2-3 min. To visualize antibody binding a biotin-streptavidin complex system was used, followed by washing with distilled water, and then the sections were counterstained with Mayer’s hematoxylin.

Immunohistochemical slides were used for morphological identification of neutrophils, lymphocytes, and macrophages. Immunohistochemical slides were examined for positive staining using light microscopy. Inflammation in each case was rated according to a modified scoring system [40-41] shown in Table 1, as follows: no inflammation (0), minimal inflammation (1), mild inflammation (2), moderate inflammation (3), and diffuse inflammation (4). In all, 5 high-power fields (40× magnification) per patient and control sample were examined, and the percentage of inflammatory cells expressing iNOS was scored as 0 (0%-1%), 1 (2%-20%), 2 (21%-50%), and 3 (≥51%). In addition, the intensity of staining was evaluated, as follows: 0 (absent), 1 (weak), 2 (strong).

Because AEC was used as a color reagent, orangebrown staining in cytoplasm was considered as positive for iNOS activity in inflammatory cells.

Statistical analysis Statistical analyses were performed using SPSS v.11.0 for Windows (Statistical Product and Service Solutions, SSPS Inc, Chicago, IL, USA). Normality was analyzed using the Shapiro-Wilk test. The Mann-Whitney U test and independent samples t test was used to compare differences between the patients and controls. Spearman's rank correlation coefficient was used to analyze the relationships between all parameters. P values <0.05 were considered statistically significant.

Results In all, 20 homozygous SCD patients in the steady state and 20 healthy age- and sex-matched controls without a history of SCD were evaluated.

Determination of NO production Total NO end metabolites (NOx: nitrate [NO3-] and nitrite [NO2-]) were quantified in the GCF of 20

119

patients with SCD and 20 controls. NOx in GCF ranged from 4.45 to 29.37 μM L-1 in SCD patients and 17.81 to 27.88 μM L-1 in the controls. Mean NO concentration in GCF was higher in the controls (23.1±2.3 μM L-1) than in the SCD patients (21.2±4.5 μM L-1); the difference was not statistically significant (p=0.098) (Table 2). Sampling volume was 33.9±21.2 μL in the SCD patients and 25.8±11.1 μL in the controls; a significant difference was not observed (p=0.139).

Assessment of iNOS expression In total, 37 gingival biopsy tissue specimens were evaluated for iNOS levels and expression; 17 from SCD patients and 20 from controls. Inflammation intensity, density of iNOS(+) inflammatory cells, and iNOS expression were measured (Table 1).

Inflammation intensity indicates the intensity of inflammatory cell infiltration, iNOS expression indicates the level of iNOS expression in iNOS-expressed inflammatory cells, and density of iNOS(+) inflammatory cells is the percentage of cells expressing iNOS [31].

All tissues from both groups had few, but evident inflammatory cells present in connective tissue.

There weren’t any differences in inflammation intensity, percentage of cells expressing iNOS, or iNOS expression intensity between the patient and Table 1. Evaluation of gingival biopsy specimen sections Inflammation Intensity

Density of iNOS (+) Inflammatory Cells

iNOS Expression

Absent

0

Absent

0

Absent

0

Minimal

1

<20%

1

Weak

1

Slight

2

20%-50%

2

Strong

2

Moderate

3

≥51%

Diffuse

4

3

Table 2. Volume and concentration of total NO products (nitrite/nitrate) in the SCD patients and controls

SCD Patients Controls p (n=20) (n=20) mean±SD mean±SD median (range) median (range)

Concentration (μM L-1)

21.2±4.5 21.69 (14.45-29.37)

Sample volume (μ L-1)

33.9±21.2

32.50 (8-100)

23.1±2.3 0.098 22.85 (17.81-27.88) 25.8±11.1 0.139 22.50 (11-55)

120

Güzeldemir et al.

NO metabolism in patients with SCD

control samples (Table 3); however, all immunohistochemical parameters were strongly correlated in the SCD group (inflammation intensity and density of iNOS(+) inflammatory cells: r=0.604, p<0.05; inflammation intensity and iNOS expression: r=0.609, p<0.01; iNOS expression and density of iNOS(+) inflammatory cells: r=0.979, p<0.001) and in the control group (inflammation intensity and density of iNOS(+) inflammatory cells: r=0.669, p<0.01; inflammation intensity and iNOS expression: r=0.741, p<0.001; iNOS expression and density of iNOS(+) inflammatory cells: r=0.943, p<0.001) (Table 4).

In the present study immunohistochemical staining of the samples obtained from SCD patients (Figure a) and controls (Figure b) confirmed the lack of inflammatory iNOS-expressing cells in connective tissue (40× magnification). Stained cells were scattered in the connective tissues. Some of the basal layer of epithelium cells exhibited sparse positive staining.

Discussion To the best of our knowledge the present study is the first to examine the NO level in the gingiva and the presence/expression of iNOS in GCF in SCD patients. The present findings indicate that NO levels and iNOS expression in SCD patients did not significantly differ from those in the controls. iNOS expression was observed in chronic inflammatory diseases of the airway, blood vessels, bowel,

A

Turk J Hematol 2011; 28: 115-24

kidneys, heart, skin, and gingiva. It was suggested that the serum NO level is elevated in megaloblastic anemia [42] and that the plasma NO level in betathalassemia minor patients is low at the time of diagnosis [43]. In addition, excessive salivary NO plays a potential role in modifying oral mucosal diseases, as a physiopathological regulator [44]. Many recent studies have reported increased NO production in Table 3. Median inflammation intensity, density of iNOS(+) inflammatory cells, and iNOS expression in the SCD patients and controls SCD Patients Controls p (n=17) (n=20) median (range) median (range)

Inflammation intensity

2.0 (0-4)

1.0 (0-4)

0.964

Density of iNOS (+) inflammatory cells

0.0 (0-2)

0.5 (0-2)

0.537

iNOS expression

0.0 (0-2)

0.5 (0-2)

0.641

Table 4. Correlation between tissue specimen immunohistochemical parameters in the SCD patients and controls

SCD Patients

Inflammation intensity

Density of iNOS iNOS (+) expression inflammatory cells 0.604*

Density of iNOS (+)

inflammatory cells

Controls

Inflammation intensity

0.669**

Density of iNOS (+) inflammatory cells

0.609** 0.979*** 0.741*** 0.943***

Significant correlations: *p<0.05, **p<0.01,Significant correlations: *p<0.05, **p<0.01, ***p<0.001, ***p<0.001

B

Figure 1. Immunohistochemical staining of SCD patient (A) and control tissues (B) confirmed the lack of inflammatory iNOS-expressing cells in connective tissue (40× magnification)

Turk J Hematol 2011; 28: 115-24

certain temporomandibular disorders [5]. NO has been implicated in the pathogenesis of apical infection [45], periapical granulomas [46], and inflamed human dental pulp [47]. Increased iNOS expression in gingiva was shown in periodontitis [20,22,26,28,31], ligature-induced periodontitis [27], experimental periodontitis [30,46], inflamed gingiva [23], localized aggressive periodontitis [21], and recently by our group in aggressive periodontitis (unpublished data).

Increased levels of NO in diabetic-periodontitis patients were observed in GCF [19]. Accordingly, we previously observed that inflammation and iNOS expression were higher in the gingiva of patients with both diabetes and periodontitis [48]. iNOS expression has been reported in a variety of pathogenic states characterized by oxidative stress, and inflammation and/or ischemic reperfusion [7,49]. SCD is also strongly associated with oxidative stress, increased expression of endothelial cell adhesion molecules, and blood adhesion. Increased iNOS expression and NO production have been observed in the kidneys and the liver in SCD patients [8], in mice [2,3,8,9], and in pigs [4]. During the clinically asymptomatic state, NO bioavailability is reduced in SCD patients due to scavenging of NO by cell-free hemoglobin released from hemolyzed sickle erythrocytes [50] and increased arginase activity [51]. During NO synthesis by NOS, if L-arginine levels decrease or consumption of NO increases via cell-free plasma hemoglobin and ROS, then oxidative damage occurs, HbS denatures, and superoxide production increases. It was shown that iNOS inhibition significantly limits tissue ischemiareperfusion injury in the liver and kidneys [10-11]; however, inhibition of iNOS attenuated ischemiareperfusion injury in the rat heart [12], but not in the rat lung [13]. Increased iNOS expression was reported in the kidneys of transgenic mice [9], but Nath et al. [14] reported the lack of iNOS upregulation in intrarenal vasculature and increased iNOS expression in the glomeruli and distal tubules of sickle mice. Previous attempts to demonstrate increased iNOS expression in SCD-affected tissues have yielded inconclusive results [52-55].

NO reacts with oxygen, forming nitrite (NO2-) and nitrate (NO3-). These oxidative metabolites of NO-referred to as NOx-are often used as surrogate markers of NO production because of their instability in oxygenated environments. A deficient plasma

GĂźzeldemir et al.

NO metabolism in patients with SCD

121

concentration in microcirculation promotes prolonged vascular transit time. In a clinical study plasma NOx concentrations in steady-state SCD patients were significantly higher than in controls and there was no difference compared to the onset of painful crises [16]. Lopez et al. [56] studied adult SCD patients with vaso-occlusive crises and reported that both plasma arginine and NOx levels were significantly lower in the SCD patients than in the controls. NOx levels in children that presented during vaso-occlusive crisis were similar to baseline levels [57], in contrast to Lopez et al.â&#x20AC;&#x2122;s [58] finding of low NOx levels in adults during vaso-occlusive crisis. Most studies on NOx evaluated the effects of different treatment modalities in SCD patients.

Although oral problems have been described in SCD patients, they are not as common as other medical problems. Oral problems, including unilateral infarct of the mandible [59,60], pulpal necrosis [59,61], osteonecrosis [62], facial swelling [63], diastema and hypodontia [64], gingival enlargement [63], palatal pallor [65], increased risk for caries [66], osteomyelitis of the mandible [67], unilateral anesthesia [60,62], midfacial overgrowth [65], and orofacial pain [65,67,68], have been reported [69]. As iNOS activity and expression are strongly correlated with immune and inflammatory systems, to eliminate the likelihood of conflicting results in the present study only participants that were free of clinically detectable infection and inflammation were included; the participants had no ongoing dental, periodontal, or orofacial disorders.

There are conflicting findings regarding NO levels and iNOS expression in different tissues and organs in SCD patients. This may be because NO bioavailability is controlled by several mechanisms, such as NO scavenging by plasma hemoglobin and superoxide, arginine concentration and availability by plasma arginase, NO inactivation via ROS derived from xanthine oxidase, NADPH oxidase, hemoglobin S and auto-oxidation, iNOS mRNA stability, post translational modification, presence of endogenous inhibitors of NOS activity, and local concentrations of arginase-inhibiting compounds [49,70]. Thusly, NO bioavailability and iNOS expression, and their roles seem to vary by organ and sometimes vary in different regions of the same organ.

In conclusion, the present findings indicate that NO levels and iNOS expression in the gingiva and GCF in SCD patients did not differ from those in

122

Güzeldemir et al.

NO metabolism in patients with SCD

healthy controls. Further studies are needed to clarify the role of iNOS in different tissues in SCD patients.

Acknowledgments This study was supported by the Başkent University Research Foundation (D-KA07/01). All procedures were performed at Başkent University Laboratories (external quality control-EQAS-BIO_ RAD Lab Code Number3584-USA). The authors thank Bahar Tasdelen, MD from Mersin University, Faculty of Medicine, Department of Biostatistics, Mersin, Turkey for helping with the statistical analyses and Ms. Mine Yıldırım for editing the English language manuscript.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

5.

6.

7.

8.

Canalli AA, Franco-Penteado CF, Saad ST, Conran N, Costa FF. Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation. Haematologica 2008;93:605-9. [CrossRef] Diwan BA, Gladwin MT, Noguchi CT, Ward JM, Fitzhugh AL, Buzard GS. Renal pathology in hemizygous sickle cell mice. Toxicol Pathol 2002;30:254-62. [CrossRef] Yu L, Gengaro PE, Niederberger M, Burke TJ, Schrier RW. Nitric oxide: a mediator in rat tubular hypoxia/ reoxygenation injury. Proc Natl Acad Sci U S A 1994;91:1691-5. [CrossRef] Isobe M, Katsuramaki T, Hirata K, Kimura H, Nagayama M, Matsuno T. Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver. Transplantation 1999;68:803-13. [CrossRef] Ugar-Cankal D, Ozmeric N. A multifaceted molecule, nitric oxide in oral and periodontal diseases. Clin Chim Acta 2006;336:90-100.

Brown GC. Nitric oxide inhibition of cytochrome oxidase and mitochondrial respiration: implications for inflammatory, neurodegenerative and ischaemic pathologies. Mol Cell Biochem 1997;174:189-92.

Wood KC, Granger DN. Sickle cell disease: role of reactive oxygen and nitrogen metabolites. Clin Exp Pharmacol Physiol 2007;34:926-32. [CrossRef] Aslan M, Ryan TM, Townes TM, Coward L, Kirk MC, Barnes S, Alexander CB, Rosenfeld SS, Freeman BA. Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization. J Biol Chem 2003;278:4194-204.

Turk J Hematol 2011; 28: 115-24

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Bank N, Aynedjian HS, Qiu JH, Osei SY, Ahima RS, Fabry ME, Nagel RL. Renal nitric oxide synthases in transgenic sickle cell mice. Kidney Int 1996;50:184-90. [CrossRef] Lee VG, Johnson ML, Baust J, Laubach VE, Watkins SC, Billiar TR. The roles of iNOS in liver ischemia-reperfusion injury. Shock 2001;16:355-60. [CrossRef] Ling H, Gengaro PE, Edelstein CL, Martin PY, Wangsiripaisan A, Nemenoff R, et al. Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice. Kidney Int 1998;53:1642-6. [CrossRef] Liu P, Hock CE, Nagele R, Wong PY. Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rats. Am J Physiol 1997;272:H2327-36.

Moore TM, Khimenko PL, Wilson PS, Taylor AE. Role of nitric oxide in lung ischemia and reperfusion injury.

Am J Physiol 1996;271:H1970-7.

Nath KA, Shah V, Haggard JJ, Croatt AJ, Smith LA, Hebbel RP, et al. Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease. Am J Physiol Regul Integr Comp Physiol 2000;279:R1949-55.

Nahavandi M, Wyche MQ, Perlin E, Tavakkoli F, Castro O. Nitric Oxide Metabolites in Sickle Cell Anemia Patients after Oral Administration of Hydroxyurea; Hemoglobinopathy. Hematology 2000;5:335-9.

Rees DC, Cervi P, Grimwade D, O'Driscoll A, Hamilton M, Parker NE, et al. The metabolites of nitric oxide in sicklecell disease. Br J Haematol 1995;91:834-7. [CrossRef] Nahavandi M, Tavakkoli F, Wyche MQ, Perlin E, Winter WP, Castro O. Nitric oxide and cyclic GMP levels in sickle cell patients receiving hydroxyurea. Br J Haematol 2002;119:855-7. [CrossRef] Nahavandi M, Tavakkoli F, Millis RM, Wyche MQ, Habib MJ, Tavakoli N. Effects of hydroxyurea and L-arginine on the production of nitric oxide metabolites in cultures of normal and sickle erythrocytes. Hematology 2006;11:291-4. [CrossRef] Skaleric U, Gaspirc B, McCartney-Francis N, Masera A, Wahl SM. Proinflammatory and antimicrobial nitric oxide in gingival fluid of diabetic patients with periodontal disease. Infect Immun 2006;74:7010-3. [CrossRef] Lappin DF, Kjeldsen M, Sander L, Kinane DF. Inducible nitric oxide synthase expression in periodontitis. J Periodontal Res 2000;35:369-73. [CrossRef] Gaspirc B, Masera A, Skaleric U. Immunolocalization of inducible nitric oxide synthase in localized juvenile periodontitis patients. Connect Tissue Res 2002;43:4138. [CrossRef] Batista AC, Silva TA, Chun JH, Lara VS. Nitric oxide synthesis and severity of human periodontal disease.

Oral Dis 2002;8:254-60. [CrossRef] Hirose M, Ishihara K, Saito A, Nakagawa T, Yamada S, Okuda K. Expression of cytokines and inducible nitric oxide synthase in inflamed gingival tissue. J Periodontol 2001;72:590-7. [CrossRef] Lohinai Z, Stachlewitz R, Virag L, Szekely AD, Hasko G, Szabo C. Evidence for reactive nitrogen species formation in the gingivomucosal tissue. J Dent Res 2001;80:470-5. [CrossRef]

Güzeldemir et al.

NO metabolism in patients with SCD

Turk J Hematol 2011; 28: 115-24

25. Kendall HK, Haase HR, Li H, Xiao Y, Bartold PM. Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts. J Periodontal Res 2000;35:194-200. [CrossRef] 26. Matejka M, Partyka L, Ulm C, Solar P, Sinzinger H. Nitric oxide synthesis is increased in periodontal disease. J Periodontal Res 1998;33:517-8. [CrossRef] 27. Lohinai Z, Benedek P, Fehér E, Györfi A, Rosivall L, Fazekas A, Salzman AL, Szabó C. Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat. Br J Pharmacol 1998;123:353-60. [CrossRef] 28. Daghigh F, Borghaei RC, Thornton RD, Bee JH. Human gingival fibroblasts produce nitric oxide in response to proinflammatory cytokines. J Periodontol 2002;73:392-400.

[CrossRef] 29. Paquette DW, Williams RC. Modulation of host inflammatory mediators as a treatment strategy for periodontal diseases. Periodontol 2000 2000;24:239-52. [CrossRef] 30. Popkov VL, Fil'chukova IA, Lapina NV, GalenkoYaroshevskii VP, Dukhanin AS. Activity of nitric oxide synthase and concentration of nitric oxide end metabolites in the gingiva under experimental pathological conditions. Bull Exp Biol Med 2005;140:391-3. [CrossRef] 31. Gullu C, Ozmeric N, Tokman B, Elgun S, Balos K. Effectiveness of scaling and root planing versus modified Widman flap on nitric oxide synthase and arginase activity in patients with chronic periodontitis.

J Periodontal Res 2005;40:168-75. [CrossRef] 32. Alayan J, Ivanovski S, Gemmell E, Ford P, Hamlet S, Farah CS. Deficiency of iNOS contributes to Porphyromonas gingivalis-induced tissue damage.

Oral Microbiol Immunol 2006;21:360-5. [CrossRef] 33. Gyurko R, Boustany G, Huang PL, Kantarci A, Van Dyke TE, Genco CA, Gibson FC 3rd. Mice lacking inducible nitric oxide synthase demonstrate impaired killing of orphyromonas gingivalis. Infect Immun 2003;71:4917-24. [CrossRef] 34. Gyurko R, Shoji H, Battaglino RA, Boustany G, Gibson FC 3rd, Genco CA, Stashenko P, Van Dyke TE. Inducible nitric oxide synthase mediates bone development and P. gingivalis-induced alveolar bone loss. Bone 2005;36:472-9. [CrossRef] 35. Garlet GP, Cardoso CR, Campanelli AP, Garlet TP, AvilaCampos MJ, Cunha FQ, Silva JS. The essential role of IFN-gamma in the control of lethal Aggregatibacter actinomycetemcomitans infection in mice. Microbes Infect 2008;10:489-96. [CrossRef] 36. Zamora R, Vodovotz Y, Billiar TR. Inducible nitric oxide synthase and inflammatory diseases. Mol Med 2000;6:347-73.

37. Hickey MJ. Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment. Clin Sci (Lond) 2001;100:1-12. [CrossRef] 38. Ozmeric N. Advances in periodontal disease markers.

Clin Chim Acta 2004;343:1-16. [CrossRef] 39. Ricart-Jane D, Llobera M, Lopez-Tejero MD. Anticoagulants and other preanalytical factors inter-

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

123

fere in plasma nitrate/nitrite quantification by the Griess method. Nitric Oxide 2002;6:178-85.

Mirbod SM, Ahing SI, Pruthi VK. Immunohistochemical study of vestibular gingival blood vessel density and internal circumference in smokers and non-smokers.

J Periodontol 2001;72:1318-23. [CrossRef] Brennan PA, Palacios-Callender M, Sinclair D, Spedding AV, Zaki GA. Does expression of inducible nitric oxide synthase correlate with severity of oral epithelial dysplasia? J Craniomaxillofac Surg 2000;28:44-8. [CrossRef] Erkurt MA, Aydogdu I, Bayraktar N, Kuku I, Kaya E. The levels of nitric oxide in megaloblastic anemia. Turk J Hematol 2009;26:197-200.

Bayraktar N, Erkurt MA, Aydogdu I, Basaran Y. The levels of nitric oxide in beta-thalassemia minor. Turk J Hematol 2008; 25:187-9.

Ohashi M, Iwase M, Nagumo M. Elevated production of salivary nitric oxide in oral mucosal diseases. J Oral Pathol Med 1999;28:355-9. [CrossRef] Shimauchi H, Takayama S, Narikawa-Kiji M, Shimabukuro Y, Okada H. Production of interleukin-8 and nitric oxide in human periapical lesions. J Endod 2001;27:749-52. [CrossRef] Hama S, Takeichi O, Saito I, Ito K. Involvement of inducible nitric oxide synthase and receptor for advanced glycation end products in periapical granulomas. J Endod 2007;33:137-41. [CrossRef] Di Nardo Di Maio F, Lohinai Z, D'Arcangelo C, De Fazio PE, Speranza L, De Lutiis MA, Patruno A, Grilli A, Felaco M. Nitric oxide synthase in healthy and inflamed human dental pulp. J Dent Res 2004;83:312-6. [CrossRef] Pan Z, Guzeldemir E, Toygar HU, Bal N, Bulut S. Nitric Oxide Synthase in Gingival Tissues of Patients With Chronic Periodontitis and With and Without Diabetes.

Journal of Periodontology 2010;81:109-20. [CrossRef] Wood KC, Hsu LL, Gladwin MT. Sickle cell disease vasculopathy: a state of nitric oxide resistance. Free Radic Biol Med 2008;44:1506-28. [CrossRef] Reiter CD, Wang X, Tanus-Santos JE, Hogg N, Cannon RO, 3rd, Schechter AN, et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002;8:1383-9. [CrossRef] Schnog JJ, Jager EH, van der Dijs FP, Duits AJ, Moshage H, Muskiet FD, Muskiet FA. Evidence for a metabolic shift of arginine metabolism in sickle cell disease. Ann Hematol 2004;83:371-5.

Wood KC, Hebbel RP, Lefer DJ, Granger DN. Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction.

Free Radic Biol Med 2006;40:1443-53.

Osei SY, Ahima RS, Fabry ME, Nagel RL, Bank N. Immunohistochemical localization of hepatic nitric oxide synthase in normal and transgenic sickle cell mice: the effect of hypoxia. Blood 1996;88:3583-8.

Bank N, Kiroycheva M, Singhal PC, Anthony GM, Southan GJ, Szabo C. Inhibition of nitric oxide synthase ameliorates cellular injury in sickle cell mouse kidneys. Kidney Int 2000;58:82-9.

124

GĂźzeldemir et al.

NO metabolism in patients with SCD

55. Kaul DK, Liu XD, Fabry ME, Nagel RL. Impaired nitric oxide-mediated vasodilation in transgenic sickle mouse. Am J Physiol Heart Circ Physiol 2000;278: H1799-806.

56. Lopez BL, Kreshak AA, Morris CR, Davis-Moon L, Ballas SK, Ma XL. L-arginine levels are diminished in adult acute vaso-occlusive sickle cell crisis in the emergency department. Br J Haematol 2003;120:532-4.

57. Morris CR, Kuypers FA, Larkin S, Vichinsky EP, Styles LA. Patterns of arginine and nitric oxide in patients with sickle cell disease with vaso-occlusive crisis and acute chest syndrome. J Pediatr Hematol Oncol 2000;22:515-20.

58. Lopez BL, Barnett J, Ballas SK, Christopher TA, DavisMoon L, Ma X. Nitric oxide metabolite levels in acute vaso-occlusive sickle-cell crisis. Acad Emerg Med 1996;3:1098-103.

59. Kavadia-Tsatala S, Kolokytha O, Kaklamanos EG, Antoniades K, Chasapopoulou E. Mandibular lesions of vasoocclusive origin in sickle cell hemoglobinopathy.

Odontology 2004;92:68-72.

60. Bishop K, Briggs P, Kelleher M. Sickle cell disease: a diagnostic dilemma. Int Endod J 1995;28:297-302.

61. Demirbas Kaya A, Aktener BO, Unsal C. Pulpal necrosis with sickle cell anaemia. Int Endod J 2004;37:602-6.

62. Kelleher M, Bishop K, Briggs P. Oral complications associated with sickle cell anemia: a review and case

Turk J Hematol 2011; 28: 115-24

63.

64.

65.

66.

67.

68.

69.

70.

report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;82:225-8.

Scipio JE, Al-Bayaty HF, Murti PR, Matthews R. Facial swelling and gingival enlargement in a patient with sickle cell disease. Oral Dis 2001;7:306-9.

Oredugba FA. Hypodontia in an adolescent with the HbSC genotype: a case report. Int J Paediatr Dent 2005;15:455-8.

Rada RE, Bronny AT, Hasiakos PS. Sickle cell crisis precipitated by periodontal infection: report of two cases.

J Am Dent Assoc 1987;114:799-801.

Laurence B, George D, Woods D, Shosanya A, Katz RV, Lanzkron S, Diener-West M, Powe N. The association between sickle cell disease and dental caries in African Americans. Spec Care Dentist 2006;26:95-100.

Shroyer JV, 3rd, Lew D, Abreo F, Unhold GP. Osteomyelitis of the mandible as a result of sickle cell disease. Report and literature review. Oral Surg Oral Med Oral Pathol 1991;72:25-8.

O'Rourke CA, Hawley GM. Sickle cell disorder and orofacial pain in Jamaican patients. Br Dent J 1998;185:90-2.

Carr MM. Dental management of patients with sickle cell anemia. J Can Dent Assoc 1993;59:180-2, 185.

Suschek CV, Schnorr O, Kolb-Bachofen V. The role of iNOS in chronic inflammatory processes in vivo: is it damage-promoting, protective, or active at all? Curr Mol Med 2004;4:763-75.

Research Article

125

The frequency of A91V in the perforin gene and the effect of tumor necrosis factor-α promoter polymorphism on acquired hemophagocytic lymphohistiocytosis Perforin geninde A91V frekansı ve tümör nekrozis-α faktör promotor polimorfizminin edinsel hemofagositik lenfohistiositoza etkisi Hamza Okur1, Şule Ünal1, Günay Balta1, Didem Efendioğlu2, Eren Çimen1, Mualla Çetin1, Aytemiz Gürgey1, Çiğdem Altay3, Fatma Gümrük1 1Department

of Pediatrics, Division of Pediatric Hematology, Faculty of Medicine, Hacettepe University, Ankara, Turkey of Pediatrics, Division of Neonatology, Faculty of Medicine, Kırıkkale University, Kırıkkale, Turkey 3The Turkish Academy of Sciences Honorary Member, Ankara, Turkey 2Department

Abstract Objective: Numerous acquired etiological factors, such as infections, malignancies, and collagen tissue disorders, are involved in the development of acquired hemophagocytic lymphohistiocytosis (AHLH).

Not everyone with the same etiological factors developments AHLH, which suggests the role of additional genetic or environmental predisposing factors that remain to be identified.

Materials and Methods: Perforin gene A91V missense transition (C>T change at position 272 in exon 2 of the perforin gene) and TNF-α gene promoter-1031 T>C nucleotide substitution are 2 candidate genetic predisposing factors due to their potential to alter inflammatory responses. In the present study these changes were investigated in healthy controls and AHLH patients.

Results: A91V transition was observed in 7 of the 159 (4.4%) controls. Among the 44 AHLH patients, 5 (11.3%) were heterozygous and the difference in the frequency of A91V transition, although striking (odds ratio: 2.8), was not statistically significant (p=0.09). All A91V-positive patients had infection.

TNF-α-1031 T>C polymorphism was examined in 164 healthy controls and 40 AHLH patients, and the CC risk-elevating genotype was noted in 7 (4.3%) of the controls and 1 (2.5%) of the AHLH patients. The frequency of C and T alleles was 22.5% (n=18) and 77.5% (n=62) among the AHLH patients, and 22% (n=72) and 78% (n=259) among the controls, respectively. There wasn’t a statistically significant difference between the groups in terms of allele frequencies (p>0.05).

Conclusion: The present results indicate that compared to controls, A91V mutation was 2.8-fold more prevalent (according to the odds ratio) in the AHLH patients. A91V mutation is not uncommon in the general population and increases the risk of AHLH in patients with an underlying condition, especially those with an underlying infection. (Turk J Hematol 2011; 28: 125-30) Key words: Acquired hemophagocytic lymphohistiocytosis, infection-related HLH, perforin, A91V mutation, TNF-α polymorphism Received: September 23, 2010

Accepted: February 22, 2011

Address for Correspondence: Prof. Fatma Gümrük, Department of Pediatrics, Division of Pediatric Hematology, Faculty of Medicine, Hacettepe University, Ankara, Turkey Phone: +90 312 305 11 70 E-mail: fgumruk@hacettepe.edu.tr doi:10.5152/tjh.2011.28

126

Okur et al.

Perforin A91V and acquired HLH

Turk J Hematol 2011; 28: 125-30

Özet Amaç: Edinsel hemofagositik lenfohistiositozun (EHL) gelişmesinde enfeksiyonlar, habis hastalıklar, kollajen doku hastalıkları gibi çok çeşitli etmen rol oynamaktadır. Aynı tetikleyici faktörü bulunan hastaların tümünde EHL’un gelişmemesi EHL’ye yatkınlık yapan ek genetik ve çevresel faktörlerin varlığına işaret etmektedir.

Yöntem ve Gereçler: Perforin geninde A91V yanlış anlam değişikliği (perforin geninde ekzon 2, pozisyon 272’de C>T değişikliği) ve tumor nekrozis faktör (TNF)-α geninin promoter bölgesinde –1031T>C nükleotid değişikliği inflamatuvar yanıtı değiştirebilen ve bu nedenle EHL’ye yatkınlığa neden olabilen iki potansiyel adaydır. Çalışmamızda EHL’li hastalar ve kontrollerde bu değişiklikler incelenmiştir.

Bulgular: 159 sağlıklı Türk popülasyonunda A91V değişikliği 7 (%4.4) kişide saptanmıştır. 44 EHL olgusunun beşinde (%11.3) bu değişiklik saptanmış olup, fark dikkat çekici olmakla birlikte istatistiksel anlamlılık göstermemiştir (p=0.09); odds oranı 2.8 olarak hesaplanmıştır. A91V pozitif olan hastaların tümünde enfeksiyon altta yatan etiolojik nedendi. TNF-α -1031T>C polimorfizmi 164 sağlıklı birey ve 40 EHL’li hastada çalışıldı. Kontrollerin 7’sinde (%4.3) ve EHL bulunan hastaların 1’inde (%2.5) riski artıran CC genotipi saptandı. C ve T allel frekansları sırasıyla EHL’de 18 (%22.5) ve 62 (%77.5), kontrollerde 72 (%22) ve 259 (%78) olarak bulundu. Allel frekansları açısından gruplar arasında fark saptanmadı (p>0.05).

Sonuçlar: Çalışmamızın sonuçları edinsel HLH’li hastalarda sağlıklı kontrollara göre A91V sıklığının 2.8 kat odds oranına göre daha sık olduğunu, A91V’nin sağlıklı Türk populasyonunda nadir olmadığını ve özellikle enfeksiyonu olanlarda EHL’ye yatkınlık yapabileceğini göstermektedir.

(Turk J Hematol 2011; 28: 125-30)

Anahtar kelimeler: Edinsel hemofagositik lenfohistiositoz, enfeksiyon ilişkili HLH, perforin, A91V mutasyonu, TNF-α, polimorfizmi Geliş tarihi: 23 Eylül 2010

Kabul tarihi: 22 Şubat 2011

Introduction Hemophagocytosis is a mysterious abnormal cellular condition that accompanies several disorders.

One such disorder is genetically transmitted familial (primary) hemophagocytic lymphohistiocytosis (HLH), which is characterized by high fever, hepatosplenomegaly, cytopenia, hyperferritinemia, hypertriglyceridemia and/or hypofibrinogenemia, a high level of the alpha chain of soluble interleukin-2 (sCD25), low natural killer (NK) cell activity, and hemophagocytosis in bone marrow, cerebrospinal fluid (CSF), or lymph nodes [1]. Mutations in 4 different genes, namely perforin on chromosome 10q21, munc 13-4 on 17q25, syntaxin 11 on 6q24, and syntaxin-binding protein 2 (STXBP2) on 19p13.2 are reported to be responsible for familial HLH [2-5].

Another hemophagocytosis-related disorder is acquired (secondary) HLH (AHLH), which is related to all of the above criteria of the Histiocyte Society, except for a genetic defect in 1 of the familial HLHassociated genes. AHLH is commonly associated with various etiologic agents, such as infectious agents-particularly viruses-malignancy, and autoimmune diseases such as juvenile rheumatoid arthritis [6-9]; however, why some patients with an underlying disorder develop AHLH and others don’t in the

presence of the same etiological factor is not known and requires further investigation.

Recently, 272C>T nucleotide changes in exon 2 of the perforin gene, which leads to alanine 91 valine (A91V) amino acid substitution, was described as a polymorphism because of its high incidence rate, which varies from 3% to 17% in healthy populations [10,11]. Subsequently, it was reported that A91V causes conformational changes and impairs the process by which perforin protein becomes the active form [12,13]. A91V is reported to be associated with mutations in the perforin and munc 13-4 genes [14]. In addition, it was suggested that homozygous A91V transition is a cause of infectioninduced FHL, and it was also suggested that the transition is a predisposing factor in such disorders as autoimmune lymphoproliferative syndrome-a childhood acute lymphoblastic leukemia-and type 1 diabetes [15-19]. As such, it is very likely that A91V plays a role in the pathogenesis of AHLH by reducing the cytotoxicity of cytotoxic T lymphocytes and NK cells [11-13].

On the other hand, it was reported that a number of cytokines are elevated in HLH, such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6, IL-10, IL-12, IL-16, and IL-18.

Hypercytokinemia results from uncontrolled activa-

Okur et al.

Perforin A91V and acquired HLH

Turk J Hematol 2011; 28: 125-30

tion of histiocytes and T cells [8]. Clinical and laboratory findings of HLH were reported to be due to organ infiltration by histiocytes and lymphocytes, as well as hypercytokinemia. Among these cytokines, TNF-α is a major cytokine that plays a critical role in the pathogenesis of inflammatory, malignant, and autoimmune disorders. The C allele of -1031 T>C polymorphism in the TNF-α gene promoter was reported to increase susceptibility to AHLH in a Korean population [20].

AHLH is relatively common in Turkey, but not much is known about its predisposing factors [6-9].

The present study aimed to investigate the frequency of the A91V mutation in healthy controls and to determine the roles of A91V and TNF-α-1031 T>C changes in the pathogenesis of AHLH.

Materials and Methods Patients and controls The study included 44 unrelated AHLH patients treated at 2 hospitals (42 from Hacettepe University and 2 from Kırıkkale University) between 2005 and 2009, and 164 healthy controls. The controls were blood bank donor samples that were used following receipt of written informed consent by the donors.

Among the patients, 44 (32M/12) fulfilled the Histiocyte Society diagnostic criteria for AHLH. The patients ranged in age from 1 d to 16 years (median age: 2 years). The study protocol was approved by the Hacettepe University Ethics Committee (approval number: TBK 05/19-25) and written informed consent was obtained from all the participants.

Genotyping Genomic DNA was isolated from EDTA-preserved peripheral venous blood cells using automatic isolation methods (MagnaPure Large Volume Nucleic Asit Isolation Kit, Roche). Individuals were genotyped for A91V transition using custom-designed probes for the LightCyclerTM instrument (Roche Applied Sciences, Mannheim, Germany), and for the TNF-α-1031.

T>C polymorphism using the 5’ nuclease polymerase chain reaction (TIBMOLBIOL, Germany), as previously described [21]. Statistical analyses were performed using Fisher’s exact and chi-square tests.

127

Results The etiologic agents in the AHLH patients are shown in Table 1. In all, 3 patients had skin eruption, fever, and lymphocyte predominance in peripheral blood. These patients were accepted as having unknown viral infections. There was first-degree consanguinity between the parents of 15 patients and the parents of 3 other patients were from the same village, suggesting possible consanguinity; therefore, the families were further analyzed via haplotype analysis for HLH mutations, including perforin, munc 13-4, and syntaxin 11 genes, and none had haplotype homozygosity for these genes.

A91V status of the patients Among the 159 healthy controls, 7 (4.4%) had A91V transition (Table 2). The controls that had the Table 1. Etiologic agents and perforin A91V transition in the AHLH patients Underlying Disease

Number of Patients

A91V mutation positive

EBV infection

6

2

Leukemia

5

Autoimmune disease

4

Combined E. coli and Pseudomonas aeruginosa sepsis

3

1

Pneumonia

3

1

Respiratory tract infection

3

Sepsis (Streptococcus viridians)

1

MAS

2

CMV

2

HHV-6

1

Parvovirus B19

1

Brucella

1

Salmonella

1

Unknown-possible viral infection

3

Metabolic disorder*

3

Hepatitis of unknown origin

1

Inflammatory bowel disease

1

Langerhans cell histiocytosis

1

Myelodysplastic syndrome

1

Neonatal hemochromatosis

1

1

EBV: Epstein-Barr virus; MAS: macrophage activation syndrome; HHV-6: human herpes virus 6; CMV: cytomegalovirus; JMML: juvenile myelomonocytic leukemia *The underlying metabolic disorders were glycogen storage disease, Gaucher disease, and propionic acidemia

128

Okur et al.

Perforin A91V and acquired HLH

Turk J Hematol 2011; 28: 125-30

A91V mutation were confirmed to be free of any disease at the time the results were obtained. In all, 5 (11.3%) of the AHLH patients were heterozygous for the A91V transition (Table 2). The difference in the frequency of the transition between the AHLH and control groups was not statistically significant (Fisher’s exact test, p=0.090), although it was strikingly higher in the AHLH group (odds ratio: 2.8).

Among the 5 patients with A91V, 2 had infectious mononucleosis, 1 had Escherichia coli and P. aeruginosa sepsis, 1 had pneumonia, and 1 had a viral infection of unknown origin (Table 1).

TNFα promoter-1031 T>C polymorphism TNF-α-1031 T>C polymorphism findings are given in Table 2. Among the 164 controls, 7 (4.3%) had homozygous risk of elevated CC and 58 (35.4%) had heterozygous TC genotypes. Among the 40 AHLH patients examined for the polymorphism, 1 (4%) had the CC genotype and 16 (40%) had the TC genotype. Among the 164 controls that were analyzed for TNF polymorphisms and the 40 patients whose DNA was available for analysis, there wasn’t a statistically significant difference between allele status (p=0.78). The frequency of C and T alleles was 22.5% (n=18) and 77.5% (n=62) among the AHLH patients, and 22% (n=72) and 78% (n=259) among the controls, respectively. There wasn’t a statistically significant difference between groups in terms of T and C allele frequencies (p>0.05).

Discussion The present study obtained initial data on the frequency of A91V in Turkey (4.4%), indicating that it was quite common in the controls, as previously reported [10,15]. In the present study 5 (11.3%) AHLH patients with infection had A91V in the heterozygote state, suggesting that the transition may be a predisposing factor for infection-associated AHLH; the difference between the 2 groups was not statistically significant. Nonetheless, the difference

between the AHLH patients and healthy controls in terms of A91V status was striking (odds ratio: 2.8); this problem might be overcome by increasing the number of participants in future studies. Among the 5 patients that had A91V, 2 had severe EBV infection, 1 had neonatal sepsis due to E. coli and P. aeruginosa, 1 had severe pneumonia, and 1 had a probable viral infection of unknown origin, which may indicate the additive effect of carrier state and infections in the development of HLH. Although the number of patients in the present study was small, the statistically non-significant but higher rate of A91V in the AHLH patients suggests that especially in the presence of infection as a triggering factor A91V transition may play a predisposing role in the emergence of full-blown AHLH. Additionally, the present results would have been more useful had perforin expression analysis been performed in all of the patients and controls.

TNF-α promoter-1031 T>C polymorphism, which is reported to have a significant affect on transcription, was not observed to increase the risk of AHLH in the present study. A study on patients with AHLH reported that this cytokine polymorphism and the TNFα-1031 C allele increased the risk of AHLH [19].

Discrepancies between the reported results may be due to differences in the genetic pools of the study populations included in these 2 studies.

Additional research with different populations and larger patient cohorts will help in elucidating the contribution of this polymorphism to the development of AHLH. In conclusion, the prevalence of A91V was relatively high in the present study. The rate of A91V transition was 4.4% among the controls and 11.3% among the AHLH patients; the difference was not statistically significant, but the incidence of A91V transition was approximately 3-fold higher in the patients according to the odds ratio. TNF-α polymorphisms did not contribute to AHLH according to the present findings. The presence of A91V might contribute to the development of AHLH, especially in

Table 2. Distribution of perforin gene A91V transition, and TNF-α promoter-1031 T>C polymorphism genotypes and alleles Group Perforin A91V TNF-α-1031 T>C

Genotype

Total Studied A91V+ Total Studied CC TC TT

(n) n (%) (n) n (%) n (%) n (%)

Allele C n (%)

T n (%)

AHLH

44

5 (11.3)

40

1 (2.5)

16 (40)

23 (57.5)

18 (22.5)

62 (77.5)

Control

159

7 (4.4)

164

7 (4.3)

58 (35.4)

99 (60.3)

72 (22)

256 (78)

Okur et al.

Perforin A91V and acquired HLH

Turk J Hematol 2011; 28: 125-30

the presence of severe concomitant infection; therefore, A91V transition should be screened in patients-particularly those with infection-induced AHLH-to ensure that patients are closely monitored.

Additional research with larger AHLH patient groups is needed to more clearly delineate the role of A91V transition in AHLH. Acknowledgements This study was supported by the Hacettepe University Research Fund [A91V mutation (0801101001)] and TUBA [([A. Gurgey), (C. Altay), (TNF-α polymorphism)].

Electronic Database Information Accession numbers and URLs for data presented in the study are as follows: GenBank: http://www.ncbi.nih.gov/Genbank.

PRF1 human: [NM005041] Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm.nih.gov/Omim.

Familial hemophagocytic lymphohistiocytosis numbers: OMIM #267700 and OMIM #603553.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

Henter JI, Horne A, Aricó M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis.

Pediatr Blood Cancer 2007;48:124-31. [CrossRef] Stepp SE, Dufourcq-Lagelouse R, Le Deist F, Bhawan S, Certain S, Mathew PA, Henter JI, Bennett M, Fischer A, de Saint Basile G, Kumar V. Perforin gene defects in familial haemophagocytic lymphohistiocytosis.

Science 1999;286:1957-9.

Feldmann J, Callebaut I, Raposo G, Certain S, Bacq D, Dumont C, Lambert N, Ouachée-Chardin M, Chedeville G, Tamary H, Minard-Colin V, Vilmer E, Blanche S, Le Deist F, Fischer A, de Saint Basile G. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell. 2003;115:461-73. [CrossRef] zur Stadt U, Schmidt S, Kasper B, Beutel K, Diler AS, Henter JI, Kabisch H, Schneppenheim R, Nürnberg P, Janka G, Hennies HC. Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

129

6q24 and identification of mutations in syntaxin 11.

Hum Mol Genet. 2005;14:827-34. [CrossRef] Côte M, Ménager MM, Burgess A, Mahlaoui N, Picard C, Schaffner C, Al-Manjomi F, Al-Harbi M, Alangari A, Le Deist F, Gennery AR, Prince N, Cariou A, Nitschke P, Blank U, El-Ghazali G, Ménasché G, Latour S, Fischer A, de Saint Basile G. Munc 18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells. J Clin Invest 2009;119:3765-73. [CrossRef] Gurgey A, Secmeer G, Tavil B, Ceyhan M, Kuskonmaz B, Cengiz B, Ozen H, Kara A, Cetin M, Gumruk F. Secondary hemophagocytic lymphohistiocytosis in Turkish children. Pediatr Infect Dis J. 2005;24:1116-7.

[CrossRef] Rouphael NG, Talati NJ, Vaughan C, Cunningham K, Moreira R, Gould C. Infection associated with haemophagocytic lymphohistiocytosis. Lancet 2007;7:814-22.

[CrossRef] Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis. Eur J Pediatr 2007;166:95-109.

Oren H, Gulen H, Ucar C, Duman M, Irken G. Successful treatment of infection associated hemophagocytosis syndrome with intravenous immunoglobulin. Turk J Hematol 2003;20:95-9.

Zur Stadt U, Beutel K, Weber B, Kabisch H, Schneppenheim R, Janka G. A91V is a polymorphism in the Perforin gene not causative of FHLH phenotype.

Blood 2004;104:1909-10. [CrossRef] Molleran Lee S, Villanueva J, Sumegi J, Zhang K, Kogawa K, Davis J, Filipovich AH. Characterisation of diverse PRF1 mutations leading to decreased natural killer cell activity in North American families with haemophagocytic lymphohistiocytosis. J Med Genet 2004;41:137-44. [CrossRef] Trambas C, Gallo F, Pende D, Marcenaro S, Moretta L, De Fusco C, Santoro A, Notarangelo L, Arico M, Griffiths GM. A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of perforin. Blood 2005;106:932-7. [CrossRef] Risma K, Frayer R, Filipovich A, Sumegi J. Aberrant maturation of mutant Perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis. J Clin Invest 2006;116:182-92.

Zhang K, Johnson JA, Biroschak J, Villanueva J, Lee SM, Bleesing JJ, Risma KA, Wenstrup RJ, Filipovich AH. Familial haemophagocytic lymphohistiocytosis in patients who are heterozygous for the A91V perforin variation is often associated with other genetic defects.

Intern J Immunogenet 2007;34:231-3. [CrossRef] Mancebo E, Allende LM, Guzmán M, Paz-Artal E, Gil J, Urrea-Moreno R, Fernández-Cruz E, Gayà A, Calvo J, Arbós A, Durán MA, Canet R, Balanzat J, Udina MA, Vercher FJ. Familial hemophagocytic lymphohistiocytosis in an adult patient homozygous for A91V in the Perforin gene with tuberculosis. Haematologica 2006;91:1257-60.

130

Okur et al.

Perforin A91V and acquired HLH

16. Santoro A, Cannella S, Trizzino A, Lo Nigro L, Corsello G, Arico MA. A single amino acid change A91V in Perforin: a novel, frequent predisposing factor to childhood acute lymphoblastic leukemia? Haematologica 2005;90:697-8.

17. Mehta PA, Davies SM, Kumar A, Devidas M, Lee S, Zamzow T, Elliott J, Villanueva J, Pullen J, Zewge Y, Filipovich A; Children's Oncology Group. Perforin polymorphism A91V and susceptibility to B-precursor childhood acute lymphoblastic leukemia: a report from the Children’s Oncology group. Leukemia 2006;20:1539-41. [CrossRef] 18. Clementi R, Chiocchetti A, Cappellano G, Cerutti E, Ferretti M, Orilieri E, Dianzani I, Ferrarini M, Bregni M, Danesino C, Bozzi V, Putti MC, Cerutti F, Cometa A, Locatelli F, Maccario R, Ramenghi U, Dianzani U. Variation of the Perforin gene patients with autoimmunity/lymphoproliferation and defective Fas function.

Blood 2006;108:3079-84. [CrossRef] 19. Orilieri E, Cappellano G, Clementi R, Cometa A, Ferretti M, Cerutti E, Cadario F, Martinetti M, Larizza D,

Turk J Hematol 2011; 28: 125-30

Calcaterra V, D'Annunzio G, Lorini R, Cerutti F, Bruno G, Chiocchetti A, Dianzani U. Variations of the Perforin gene in patients with type 1 diabetes. Diabetes 2008;57:1078-83. [CrossRef] 20. Chang YH, Lee DS, Jo HS, Cho SI, Yoon HJ, Shin S, Yoon JH, Kim HY, Hong YJ, Hong SI, Cho HI. Tumor necrosis factor alpha promoter polymorphism associated with increased susceptibility to secondary hemophagocytic lymphohistiocytosis in the Korean population. Cytokine 2006;36:45-50. [CrossRef] 21. di Giovine FS, Camp NJ, Cox A, et al. Detection and population analysis of IL-1 and TNF gene polymorphisms. In Cytokine Molecular Biology: A Practical Approach. Blackwill F, editor. Oxford: Oxford University Press. 2000;21-46.

22. Karapınar B, Yılmaz D, Aydınok Y, Türkoğlu E, Hekimgil M, Kavaklı K. Intense myelofibrosis in a child: unusual result of EBV-associated hemophagocytic lymphohistiocytosis. Turk J Hematol 2007; 24:32-5.

Research Article

131

Acquired methemoglobinemia in infants Süt çocuklarında edinsel methemoglobinemi Mehmet Mutlu, Erol Erduran, Yakup Aslan Department of Pediatrics, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey

Abstract Objective: This study aimed to determine the etiologic factors of acquired methemoglobinemia in infants younger than three months in our region.

Material and Methods: This study was carried out retrospectively in infants with methemoglobinemia admitted to Karadeniz Technical University, Pediatric Clinic, during the period 2000-2009. Infants with methemoglobinemia were identified according to the medical records or ICD-10 code.

Results: Nine infants with acquired methemoglobinemia (8 male, 1 female) were included in the study.

Seven cases were associated with the use of prilocaine for circumcision, one case with the use of prilocainelidocaine for local pain therapy, and one case with neonatal sepsis caused by Staphylococcus aureus.

Conclusion: Prilocaine should not be used in infants less than three months of age because of the risk of methemoglobinemia. Ascorbic acid is an effective therapy if methylene blue is not obtained.

It should not be forgotten that sepsis caused by S. aureus may cause methemoglobinemia in infants.

(Turk J Hematol 2011; 28: 131-4)

Key words: Methemoglobinemia, infant, local anesthetics, sepsis Received: April 8, 2010

Accepted: May 11, 2010

Özet Amaç: Bölgemizde üç aydan küçük bebeklerde meydana gelen akkiz methemoglobinemilerin etiyolojisini araştırmak.

Yöntem ve Gereçler: Bu çalışma, 2000-2009 yılları arasında pediatri kliniğine başvuran ve ICD-10 koduna veya hasta kayıt defterine göre akkiz methemoglobinemi tanısı alan hastaların kayıtlarının retrospektif incelenmesi ile gerçekleştirildi.

Bulgular: Sekizi erkek olmak üzere, dokuz akkiz methemoglobinemili infant çalışmaya alındı. Yedi vaka; sünnet için prilokain kullanımı ile ilişkili iken, bir vaka; lokal anestezi için kullanılan prilokain– lidokain ile ve diğer vaka ise; Staphylococcus aureus sepsisi ile ilişkili idi.

Sonuç: Üç aydan küçük infantlarda prilokain methemoglobinemi riski nedeniyle kullanılmamalıdır ve metilen mavisi elde edilemediği durumlarda vitamin C effektif bir tedavi şeklidir. Staphylococcus aureus’a bağlı sepsisin infantlarda methemoglobinemiye neden olabileceği unutulmamalıdır.

(Turk J Hematol 2011; 28: 131-4)

Anahtar kelimeler: Methemoglobinemi, infant, lokal anestezikler, sepsis Geliş tarihi: 8 Nisan 2010

Kabul tarihi: 11 Mayıs 2010

Address for Correspondence: Asst. Prof. Mehmet Mutlu, Department of Pediatrics, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey Phone: +90 462 377 55 68 E-mail: drmehmetmutlu38@hotmail.com doi:10.5152/tjh.2011.29

132

Mutlu et al.

Acquired methemoglobinemia in infants

Introduction Methemoglobinemia is an uncommon cause of cyanosis. It should be suspected in an infant with cyanosis who has no respiratory distress or cyanotic congenital heart disease. Cyanosis is recognized in patients with methemoglobin (MetHb) concentrations higher than 1.5 g/dl [1,2]. It develops due to both acquired and congenital factors. Congenital causes of methemoglobinemia are NADH-cytochrome b5 reductase and NADPH-MetHb reductase deficiencies or presence of abnormal Hb such as Hb M [2].

Acquired methemoglobinemia is caused by some drugs such as local anesthetics, some foods or water containing high levels of nitrate, gastrointestinal infection, and sepsis [2-4]. Most of the acquired methemoglobinemia cases in clinical practice are due to local anesthetics. In this report, nine infants with methemoglobinemia are presented and the etiologic factors are discussed.

Materials and Methods This study was carried out retrospectively in infants with methemoglobinemia admitted to Karadeniz Technical University, Pediatric Clinic, during the period 2000-2009. Infants with methemoglobinemia were identified according to the medical records or ICD-10 code. Medical charts of all infants with methemoglobinemia were reviewed for birth weight (g), gender, postnatal age, Hb levels, presenting symptoms, etiologic causes, type of feeding, MetHb levels, treatment, recovery time of methemoglobinemia, and glucose6-phosphate dehydrogenase (G6PD) deficiency.

Blood MetHb levels were measured by using a Rapidlab® 1200 Blood Gas Analyzer (Siemens Healthcare Diagnostics) with spectrophotometric method. Blood MetHb levels were measured at fourhour intervals. Sepsis was considered in infants having two or more of the following criteria associated with positive blood culture: (a) fever or hypothermia, (b) tachycardia, (c) tachypnea or apnea, and (d) abnormal white blood cells or increase in band/total neutrophils. Written informed consent was obtained from the patients’ family.

Results Nine infants with methemoglobinemia (8 male, 1 female) were included in the study. Mean birth

Turk J Hematol 2011; 28: 131-4

weight and postnatal age were 3655±270 (33004100) g and 33.7±16.6 d (5-60) days, respectively.

Cyanosis was present in all infants, fever in three (33%), and hypotonia and refusal to breast-feed in two (22%). Mean percentage of MetHb levels of the infants was 28.8±9.8 (14.7-44%). Seven cases were associated with the use of a local anesthetic agent (prilocaine; Citanest® 0.5%) for circumcision. The applied prilocaine doses were not determined. One case was associated with the use of prilocaine–lidocaine (EMLA®, 5%, Astra Zeneca) for local pain therapy after scalding to 3% of the body surface, and three cases were associated with neonatal sepsis.

No phenol disinfectant was used for floor cleaning in the hospital during this period.

Staphylococcus hominis was grown in blood cultures of two infants (Case 3 and Case 5) associated with the use of prilocaine. Staphylococcus aureus was grown in the blood culture of Case 6. Acute phase reactants (procalcitonin and C-reactive protein [CRP]) were positive in these neonates, and they met sepsis criteria. They were treated with vancomycin. All of the neonates were receiving only breast milk. Their mothers denied the use of any medications and consumption of any foods containing a high nitrate level.

Two neonates were treated with methylene blue (1-2 mg/kg) intravenously, and the others were treated with vitamin C (300 mg) intravenously because methylene blue was not obtained.

Repeated doses were not necessary. Mean recovery time was 10.2±3.5 [4-16] hours. The plasma G6PD levels were normal in all neonates.

Cytochrome b5 reductase activity could not be measured in the neonates because of the lack of technical facilities. Demographic, clinic and laboratory characteristics of the neonates associated with methemoglobinemia are shown in Table 1.

Discussion Methemoglobinemia is characterized by oxidation of the iron in Hb from a ferrous (Fe++) to a ferric (Fe+++) form. Methemoglobinemia occurs in healthy persons but the intraerythrocytic MetHbreducing system maintains its concentration at less than 1% of the total Hb [5]. There are two MetHbreducing systems (NADH-cytochrome b5 reductase and NADPH-MetHb reductase) in erythrocytes.

Under physiological conditions, NADH-cytochrome

Mutlu et al.

Acquired methemoglobinemia in infants

Turk J Hematol 2011; 28: 131-4

133

Table 1. Demographic, clinic and laboratory characteristics of the patients with methemoglobinemia

Birth Postnatal Gender Complaints Hb MetHb Etiologic Treatment

Weight (g) age (day) (gr/dL) (%) causes

Recovery G6PD time (h) level (MetHb <5%)

Case 1

4000

45

Male

Cyanosis

10.9

24.1

Prilocaine

300 mg C vit

8

Normal

Case 2

3700

60

Male

Cyanosis

11.2

39.9

Prilocaine

300 mg C vit

12

Normal

Prilocaine Sepsis caused by S. hominis??

300 mg C vit +Antibiotics

4

Normal

l

Prilocaine

300 mg C vit

12

Normal

Prilocaine- lidocaine Sepsis caused by S. hominis??

300 mg C vit +Antibiotics

8

Normal

8

Normal

Methylene blue (1 mg/kg)

16

Normal

300 mg C vit

12

Normal

Methylene blue (1 mg/kg)

12

Normal

Case 3 4100 5 Male Cyanosis, hypotonia, 14.7 14.7

fever, refusal to

breast-feed

Case 4

3300

22

Male

Cyanosis

8.8

33.7

Case 5 3600 35 Male Cyanosis, fever, 13.5 19.0

refusal to

breast-feed

Case 6 3300 17 Female Fever, hypotonia, 13.5 20.9

cyanosis

Case 7 3600 45 Male Cyanosis 10.5 44 Prilocaine

Case 8

3700

37

Male

Cyanosis

11.5

30.4

Sepsis 300 mg C vit caused by +Antibiotics S. aureus

Prilocaine

Case 9 3600 48 Male Cyanosis 11.8 26.8 Prilocaine

G6PD: Glucose-6-phosphate dehydrogenase, Hb: Hemoglobin, MetHb: Methemoglobin

b5 reductase is responsible for the removal of 95-99% of the MetHb [5]. NADPH-MetHb reductase usually plays only a minor role in the removal of MetHb. This enzyme system utilizes NADPH, which is produced by G6PD. NADPH-MetHb reductase has a more important role in MetHb regulation in infants with cytochrome b5 reductase deficiency. MetHb has an increased affinity for oxygen, and causes a leftward shift in the oxygen dissociation curve, and hypoxemia and lactic acidosis develop [1,2,6]. If MetHb level is higher than 10%, cyanosis occurs, and a MetHb level of 70% is lethal. Cyanosis was observed in all of our infants and all of them improved after the appropriate treatments.

Prilocaine is widely used in children and adults as a local anesthetic agent, and its metabolite o-toluidine can cause methemoglobinemia. The dose of prilocaine and the age of the infant are the most important predictive factors for higher MetHb formation [7]. Infants are more vulnerable to methemoglobinemia because they have higher gastric pH than that in older children, and this causes greater bacterial proliferation of the intestinal flora that can convert the ingested nitrate to nitrite [6].

Fetal Hb is the predominant form of Hb in infants,

and is oxidized more readily to MetHb by nitrite [6].

The cytochrome b5 MetHb reductase activity is reduced 50% in infants compared with older children and adults [6,8]. Guay [9] suggested that prilocaine should not be used in children younger than six months and the doses should not exceed 2.5 mg/kg in children older than six months. In this study, MetHb in seven neonates was associated with the use of prilocaine for circumcision, and all of them were younger than two months.

Symptomatic methemoglobinemia may develop due to the application of EMLAÂŽ on the injured skin [10]. Methemoglobinemia was observed in Case 5 due to the application of EMLAÂŽ for local pain therapy.

Blood MetHb levels may be increased in patients with sepsis [3,4]. Large amounts of nitric oxide (NO) are released in sepsis and septic shock [11,12]. NO interacts with Hb, and leads to methemoglobinemia [13]. S. aureus has a respiratory type of nitrate reductase enzyme system. This enzyme system may convert nitrate to nitrite, and methemoglobinemia may develop [14]. Vancomycin-related methemoglobinemia has not been reported in the literature. We believe that methemoglobinemia was related with sepsis rather than vancomycin [15];

134

Mutlu et al.

Acquired methemoglobinemia in infants

nevertheless, methemoglobinemia related with vancomycin could not be completely ruled out. As far as we know, S. hominis does not have this enzyme system. Therefore, we believe that prilocaine was responsible for the methemoglobinemia in the neonates (Case 3-Case 5).

Methylene blue accelerates the NADPHdependent MetHb reduction pathway [16].

Methylene blue was given (1-2 mg/kg) in only two infants because it was not obtained for the others.

Ascorbic acid directly reduces MetHb and it is effective when used alone [17]. Intravenous 300 mg ascorbic acid was used in seven cases, and all cases recovered between 8-16 hours.

Prilocaine should not be used in infants less than three months of age because of the risk of methemoglobinemia. Ascorbic acid is an effective therapy if methylene blue is not obtained. It should not be forgotten that S. aureus may cause methemoglobinemia in infants.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Turk J Hematol 2011; 28: 131-4

5.

6.

7.

8.

9.

10.

11.

12.

13.

References 1.

2.

3.

4.

Ash-Bernal R, Wise R, Wright SM. Acquired methemoglobinemia: a retrospective series of 138 cases at 2 teaching hospitals. Medicine (Baltimore) 2004;83:265-73.

[CrossRef] moglobinemia: a rare cause of cyanosis in the newborna case report. Pediatrics 2003;112:e158-61.

Ohashi K, Yukioka H, Hayashi M, Asada A. Elevated methemoglobin in patients with sepsis. Acta Anaesthesiol Scand 1998;42:713-6. [CrossRef] May RB. An infant with sepsis and methemoglobinemia.

J Emerg Med 1985;3:261-4.

14.

15.

16.

17.

Mansouri A, Lurie AA. Concise review: methemoglobinemia. Am J Hematol 1993;42:7-12. [CrossRef] Greer FR, Shannon M. American Academy of Pediatrics Committee on Nutrition; American Academy of Pediatrics Committee on Environmental Health. Infant methemoglobinemia: the role of dietary nitrate in food and water.

Pediatrics 2005;116:784-6. [CrossRef] Vasters FG, Eberhart LH, Koch T, Kranke P, Wulf H, Morin AM. Risk factors for prilocaine-induced methaemoglobinaemia following peripheral regional anaesthesia. Eur J Anaesthesiol 2006;23:760-5. [CrossRef] Panin G, Pernechele M, Giurioli R, Secchieri S, Milanesi O, Pellegrino PA, Chiandetti L. Cytochrome b5 reductase activity in erythrocytes and leukocytes as related to sex and age. Clin Chem 1984;30:701-3.

Guay J. Methemoglobinemia related to local anesthetics: a summary of 242 episodes Anesth Analg 2009;108:837-45. [CrossRef] Book A, Fehlandt C, Krija M, Radke M, Pappert D. Methemoglobin intoxication by prilocaine in EMLA. Accidental intoxication of an infant with scald injuries.

Anaesthesist 2009;58:370-4. [CrossRef] Ochoa JB, Udekwu AO, Billiar TR, Curran RD, Cerra FB, Simmons RL, Peitzman AB. Nitrogen oxide levels in patients after trauma and during sepsis. Ann Surg 1991;214:621-6. [CrossRef] Lorente JA, Delgado MA, LandĂ­n L. Septic shock and nitric oxide. Enferm Infecc Microbiol Clin 1997;3:14-9.

Wennmalm A, Benthin G, Petersson AS. Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin. Br J Pharmacol 1992;106:507-8.

Burke KA, Lascelles J. Nitrate reductase activity in hemedeficient mutants of Staphylococcus aureus. J Bacteriol 1976;126:225-31.

Mutlu M, Erduran E, Aksoy A, Aslan Y. Methemoglobinemia associated with Staphylococcus aureus sepsis in a newborn. JNPM 2010;3:63-5.

Hoffman R, Benz E, Shattil S, Furie B, Cohen H. Hematology Basic Principles and Practice. New York: Churchill Livingstone, 2005;650-7.

Gibson QH. The reduction of methemoglobin by ascorbic acid. Biochem J 1943;37:615-8.

Case Report

135

Splenic artery embolization: An alternative approach in a critically ill patient with autoimmune hemolytic anemia Splenik arter embolizasyonu: Otoimmün hemolitik anemili kritik derecede ağır bir hastada yaklaşım seçeneği Mine Durusu Tanrıöver1, Bora Peynircioğlu2, Begüm Ergan Arsava3, Arzu Topeli İskit4 1Department

of Internal Medicine, General Internal Medicine Section, Faculty of Medicine, Hacettepe University, Ankara, Turkey 2Department of Radiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey 3Department of Pulmonology, Faculty of Medicine, Hacettepe University, Ankara, Turkey 4Department of Internal Medicine, General Internal Medicine Section and Medical Intensive Care Unit, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Abstract Assessment of general health status and hematological parameters usually precedes the use of invasive diagnostic and therapeutic procedures in critically ill patients. Angiography can be effective and safe as a substitute for major surgical procedures, or as a bridging therapy in such cases. We present a critically ill patient with hemolytic anemia that underwent splenic artery embolization as a bridging therapy. We aimed to emphasize that minimally invasive approaches and multidisciplinary care can be utilized in the treatment of critically ill patients with accompanying hematological disease.

(Turk J Hematol 2011; 28: 135-8)

Key words: Critical care medicine, therapeutic embolization, hemolytic anemia Received: December 14, 2009

Accepted: April 22, 2010

Özet Kritik derecede ağır bir hastanın genel durum bozukluğu ve hematolojik parametreleri, invazif tanı ve tedavi girişimlerinin uygulanmasına izin vermeyebilir. Bu tür olgularda, anjiyografik yaklaşımlar büyük cerrahi girişimlerin yerini tutacak veya köprü tedavisi görevi görecek güvenilir ve etkili bir tedavi yöntemi olabilir. Burada, köprü tedavisi olarak dalak arteri embolizasyonu yapılan hemolitik anemili kritik durumda ağır bir hasta sunulmaktadır. Bu olgu, ciddi bir kan hastalığının eşlik ettiği bu tür hastalarda multidisipliner bakımın önemini ve daha az invazif yaklaşımların seçilmesinin uygun olabileceğini vurgulamaktadır. (Turk J Hematol 2011; 28: 135-8) Anahtar kelimeler: Kritik hasta tıbbı, terapötik embolizasyon, hemolitik anemi Geliş tarihi: 14 Aralık 2009

Kabul tarihi: 22 Nisan 2010

Address for Correspondence: M.D. Mine Durusu Tanrıöver, Department of Internal Medicine, General Internal Medicine Section, Faculty of Medicine, Hacettepe University, Ankara, Turkey Phone: +90 312 305 30 29 E-mail: mdurusu@hacettepe.edu.tr doi:10.5152/tjh.2011.30

136

Tanrıöver et al.

Splenic artery embolization in a critically ill patient

Introduction Autoimmune hemolytic anemia is a disorder in which immunoglobulin (Ig) G-coated erythrocytes are sequestered and destroyed in the spleen.

Splenectomy can result in partial remission in patients refractory to medical therapy. Invasive procedures, especially major surgery, usually cannot be performed in critically ill patients because of the tendency for bleeding, labile hemodynamic state, and poor general health status. Angiography can be an effective and safe therapeutic modality in such cases.

Case Report

A 70-year-old male patient presented to the hematology outpatient clinic with jaundice and dark urine, which were present for 1 month. His hemoglobin was 8.5 g dL-1 at presentation (Table 1).

Anisocytosis and poikilocytosis were evident based on peripheral smear. The patient was diagnosed with autoimmune hemolytic anemia, and Deltacortril® (Pfizer, Istanbul, Turkey) 5 mg d-1 p.o. and Folbiol® (IE Ulagay, Istanbul, Turkey) 5 mg d-1 p.o. were started. One month later the patient’s hemoglobin level was 6.8 g dL-1 (Table). He was admitted to the emergency department and after his hemoglobin dropped to 3.8 g dL-1 he was transferred to the medical intensive care unit (ICU).

Upon admission to the ICU the patient’s arterial blood pressure was 125/55 mmHg and pulse was Table 1. The patient’s laboratory findings

Normal At the time At the time

range of diagnosis of admission

1 month after

diagnosis Hemoglobin

14-16 g dL-1

8.5 g dL-1

6.8 g dL-1

MCV

80.7-95.5 fL

97.7 fL

100.8 fL

RDW

11.8%-14.3%

14.5%

16.5%

0.6%-2.6%

7.3%

Reticulocyte count Serum total bilirubin

0.1-1.2 mg dL-1 3.41 mg dL-1

Serum indirect bilirubin

0-1.2 mg dL-1

Serum LDH

240-480 U L-1

Haptoglobin

496 U L-1

13.18 12.31 mg dL-1 521 U L-1

36-195 mg dL-1 <5.83 mg dL-1

MCV: Mean corpuscular volume; RDW: red cell distribution width; LDH: lactate dehydrogenase

Turk J Hematol 2011; 28: 135-8

106 bpm. Jaundice was evident. The liver was 16 cm long and there was dullness to percussion over Traube’s space. Transfusion could not be performed because the patient’s serum reacted with all erythrocyte suspensions tested. He received intravenous Prednol-L® (Mustafa Nevzat, Istanbul, Turkey) 1 g d-1 for 3 d and IG-Vena N® (Biosano Laboratorio, Santiago, Chile) 1g·kg·d-1 for 2 d. The patient underwent 2 cycles of plasmapheresis. Immunosuppressive therapy was continued with Prednol-L® (Mustafa Nevzat) 1 mg·kg·d-1 and Sandimmun Neoral® (Novartis Pharma A.G., Basel, Switzerland) 100 mg p.o. b.i.d. Rituximab was considered as an alternative for the treatment of refractory hemolytic anemia; however, the drug could not be immediately obtained. The patient was referred to the surgery department for splenectomy, but as the surgeons required a hemoglobin level >9 g dL-1 and a compatible erythrocyte suspension could not be found, the patient did not undergo surgery. The patient was then referred to the interventional radiology department for splenic artery embolization.

The patient’s hemodynamic parameters were normal just before catheterization: blood pressure was 133/63 mmHg, pulse rate was 82 bpm, body temperature was 36.1ºC, and pulse oximeter oxygen saturation was 99% while the patient breathed through a nasal oxygen cannula. On the 8th day in the ICU selective splenic artery injection was performed via the left main femoral artery via celiac artery catheterization. After mapping the splenic artery branches, the post hilar branches of the splenic artery were catheterized superselectively using a microcatheter. Approximately 75% of the spleen parenchyma was embolized using polyvinyl alcohol particles (300-500 µm in diameter) until the flow was stagnated. Follow-up splenic artery injections showed that there was slow blood flow only to the part of the parenchyma that was not embolized (Figure 1) and the procedure was finalized.

Computed tomography could not be performed before or after the intervention because the patient’s serum creatinine level was 1.3 mg dL-1. Although only 40-50 mL of intravenous contrast material was used during the procedure, the serum creatinine level continued to increase post procedure.

Immediately following the angiographic procedure the patient’s hemoglobin level was 3.8 g dL-1; however, levels as low as 1.4 g dL-1 were recorded

Turk J Hematol 2011; 28: 135-8

A

Tanrıöver et al.

Splenic artery embolization in a critically ill patient

137

shock ensued. The patient required intubation and mechanical ventilation, as well as hemodialysis because of acute renal failure. Septic shock and disseminated intravascular coagulation developed and the patient died on the 40th day in the ICU-14 days following splenectomy.

Written informed consent was obtained from the patient's family.

Discussion

B

Figure 1. A significant decrease in splenic blood flow following splenic artery injections is observed

during the patient’s hospitalization. One week post embolization the patient’s hemoglobin level reached 6.0 g dL-1 without transfusion. On the 15th day of hospitalization a compatible erythrocyte suspension became available and the patient’s hemoglobin level increased to 8.5 g dL-1 after transfusion of 4 units. The patient underwent surgery on the 26th day of hospitalization. Massive bleeding of 2.5 L occurred during splenectomy and hypovolemic

The presented case shows that splenic artery embolization can be performed successfully in patients that require splenectomy, but cannot undergo the procedure due to problematic hematological parameters and poor general health status. The indications for splenectomy and splenic artery embolization are variable. In traumatic injuries of the spleen minimal bleeding and maximal functional splenic tissue are sought. On the other hand, in patients with hypersplenism and autoimmune hemolysis the aim is to decrease the sequestration and destruction functions of the spleen while preserving its immune functions. Splenic artery embolization may be preferred over surgery when the aim is to preserve the immune functions of the spleen.

Splenic artery embolization was first described in the 1970s and has since been used in different clinical settings, including traumatic spleen rupture, portal hypertension-related ascites, immune thrombocytopenic purpura, hemoglobinopathies, and hereditary spherocytosis [1-5]. Use of splenic embolization in patients with autoimmune hemolytic anemia is rarely reported [6]. The procedure may fail in some patients if an adequate quantity of spleen parenchyma cannot be embolized or if autoimmune hemolysis is IgM mediated.

Significant bleeding is the most serious complication related to splenectomy. Preoperative splenic artery embolization has been suggested as a potential way to reduce this complication [7]. Embolization can decrease the need for transfusion in patients with immune thrombocytopenic purpura when performed prior to splenectomy [8]. Contrast-induced nephropathy is a potential adverse outcome of the embolization procedure, particularly in patients with such risk factors as chronic renal disease, diabetes mellitus, advancing age, congestive heart failure, hypotension, and shock.

138

Tanrıöver et al.

Splenic artery embolization in a critically ill patient

Partial splenic artery embolization was developed as a minimally invasive endovascular procedure in order to avoid complications due to hypersplenism and associated comorbidities [9,10].

Polyvinyl alcohol and gelatin particles are the most commonly used materials, They can reach the most distal points at the smallest arteriole level via blood flow and obliterate the arterioles. Use of small particles may result in unintended embolization of small arteries, including the pancreatic branches [9,10].

Splenic artery embolization was used as an alternative treatment option in the presented case, which not only decreased the requirement for transfusion, but also offered a time period of relative wellbeing until surgery. The patient’s decline began with major bleeding of 2.5 L during splenectomy, which resulted in hypovolemic shock and ultimately death. This raises the question of what would have happened if the patient had not undergone splenectomy, but was followed-up conservatively with splenic embolization only.

In conclusion, splenic artery embolization might be a valuable option for hemolytic anemia as a bridging therapy to surgery or as a stand-alone alternative therapy, especially in critically ill patients with disorders that contraindicate surgery.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Turk J Hematol 2011; 28: 135-8

References 1.

Raikhlin A, Baerlocher MO, Asch MR, Myers A. Imaging and transcatheter arterial embolization for traumatic splenic injuries: review of the literature. Can J Surg 2008;51:464-72.

2. N'Kontchou G, Seror O, Bourcier V, Mohand D, Ajavon Y, Castera L, Grando-Lemaire V, Ganne-Carrie N, Sellier N, Trinchet JC, Beaugrand M. Partial splenic embolization in patients with cirrhosis: efficacy, tolerance and longterm outcome in 32 patients. Eur J Gastroenterol Hepatol 2005;17:179-84. [CrossRef] 3. Pratl B, Benesch M, Lackner H, Portugaller HR, Pusswald B, Sovinz P, Schwinger W, Moser A, Urban C. Partial splenic embolization in children with hereditary spherocytosis. Eur J Haematol 2008;80:76-80.

4. Miyazaki M, Itoh H, Kaiho T, Ohtawa S, Ambiru S, Hayashi S, Nakajima N, Oh H, Asai T, Iseki T. Partial splenic embolization for the treatment of chronic idiopathic thrombocytopenic purpura. AJR Am J Roentgenol 1994;163:123-6.

5. Meral A, Sevinir B, Sadikoğlu Y, Nacarküçük E, Günay U. Partial splenic embolization in beta-thalassemia major.

A case report. Turk J Pediat 2000;42:76-9.

6. Campbell R, Marik PE. Severe autoimmune hemolytic anemia treated by paralysis, induced hypothermia and splenic embolization. Chest 2005;127:678-81. [CrossRef] 7. Totte E, Van Hee R, Kloeck I, Hendrickx L, Zachee P, Bracke P, Hermans P. Laparoscopic splenectomy after arterial embolization.

Hepatogastroenterology 1998;45:773-6.

8. Baú PC, Cavazolla SA, Souza HP, Garicochea B. Preoperative embolization of the splenic artery in patients that underwent splenectomy for immune thrombocytopenic purpura. Acta Cir Brasil 2007;22:470-3. [CrossRef] 9. Wang HY, Shih SC, Lin SC, Chang WS, Wang TE, Lin FJ, Yang FS. Partial splenic embolization: 12-month hematological effects and complications. Hepatogastroenterology 2008;55:1838-42.

10. Yoshida H, Mamada Y, Taniai N, Tajiri T. Partial splenic embolization. Hepatol Res 2008;38:225-33. [CrossRef]

Case Report

139

Wiskott-Aldrich syndrome mutation in two Turkish siblings with X-linked thrombocytopenia X’e bağlı trombositopenili iki Türk kardeşte Wiskott Aldrich sendromu mutasyonu

Göksel Leblebisatan1, Ali Bay2, Noriko Mitsuiki3, Osamu Ohara3, Kenichi Honma4, Kohsuke İmai4, Shigeaki Nonoyama4 1Department

of Pediatric Hematology, Gaziantep Children’s Hospital, Gaziantep, Turkey of Pediatric Hematology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey 3Kazusa NA Research Institute, Japan 4National Defense Medical Collage, Japan 2Department

Abstract Wiskott-Aldrich syndrome (WAS) is a clinical condition characterized by thrombocytopenia, eczema, and life-threatening infections. In some cases autoimmunity-related problems and even malignancy might be seen; however, some patients have milder clinical manifestations due to mutations in the same gene family, such as in X-linked thrombocytopenia (XLT), which is generally not associated with serious symptoms of disease, except for thrombocytopenia. Herein we report 2 siblings with chronic thrombocytopenia that were diagnosed with XLT based on a missense mutation in the WASP gene (223G>A, Val75Met). To the best of our knowledge this mutation has not been previously reported in a Turkish patient with XLT. (Turk J Hematol 2011; 28: 139-41) Key words: X-linked thrombocytopenia, Wiskott-Aldrich syndrome, WASP gene Received: January 25, 2011

Accepted: April 11, 2011

Özet Wiskott Aldrich Sendromu (WAS) trombositopeni, egzema ve hayatı tehdit edici enfeksiyonlar ile karakterize klinik durumdur. Bazı vakalarda otoimmünite ile ilgili problemler hatta malign hastalıklar da görülebilmektedir. Bunların yanında aynı gen ailesinden köken alan bazı mutasyonlarda daha hafif klinik seyir görülmektedir. X’e bağlı trombositopeni bunlardan biri olup genellikle trombositopeni dışındaki ciddi semptomları taşımaz. Burada kronik trombositopenisi olan iki erkek kardeş, WASP geninde bir missens mutasyona bağlı X’e bağlı trombositopeni tanısı almaları nedeniyle sunulmuştur.

Bu mutasyon bildiğimiz kadarıyla X’e bağlı trombositopeni tanılı Türk hastalarda daha önce bildirilmemiştir. (Turk J Hematol 2011; 28: 139-41) Anahtar kelimeler: X’e bağlı trombositopeni, Wiskott Aldrich sendromu, WASP geni Geliş tarihi: 25 Ocak 2011

Kabul tarihi: 11 Nisan 2011

Address for Correspondence: Dr. Göksel Leblebisatan, Department of Pediatric Hematology, Gaziantep Children’s Hospital, Gaziantep, Turkey Phone: +90 342 341 01 52 E-mail: gokselleb@yahoo.com doi:10.5152/tjh.2011.31

140

Leblebisatan et al.

Familial thrombocytopenia in a Turkish family

Introduction X-linked thrombocytopenia (XLT) is a congenital disease characterized by small platelets and a clinical spectrum that generally does not include other features of Wiskott-Aldrich syndrome (WAS), such as eczema and recurrent infections. XLT belongs to a disorder group related with the WAS gene family, which is expressed selectively in the hematopoietic stem cell-derived lineage that encodes WAS protein (WASP) functioning in cellular motility, the cytoskeleton, signaling, and apoptosis. According to the literature, the condition is common in Europe and Japan [1]; however, XLT with the detected mutational analysis is not common in Turkey. Herein we present 2 brothers with XLT mutation (223G>A, Val75Met), which to the best of our knowledge has not been previously described in a Turkish family.

Case Reports Patient 1 Patient 1 was a 7-year-old boy that has had ecchymosis and mucosal bleeding since the age of 2 years. Laboratory investigation showed moderate thrombocytopenia (platelet count: 20.000-50.000 mm-3) and peripheral blood smear showed thrombocytopenia and small platelets (MPV: 5 fL). The patient’s history of eczema, and recurrent or lifethreatening infection, which are observed in the classical WAS clinical picture, was negative. Other findings associated with WAS, including lymphopenia, staphylococcal superinfections, and opportunistic infections, were not observed during followup. The patient was previously followed-up at another clinic as chronic idiopathic thrombocytopenic purpura, and received such treatments as intravenous immunoglobulin therapy until last 2 years.

Upon presentation to our hematology clinic at Gaziantep Children’s Hospital, physical examination findings were normal, except for generalized petechial lesions. As XLT was considered, he was treated with thrombocyte suspensions when prolonged mucosal bleeding occurred and when the hemoglobin level dropped. No treatments were administered to alter the patient’s platelet count.

The patient had multiple head traumas that resulted in scalp hematomas, but no intracranial bleeding.

Turk J Hematol 2011; 28: 139-41

Patient 2 Patient 2 was the 5-year-old bother of patient 1 and had identical symptoms since the age of 1 year, with severe thrombocytopenia (platelet count: ≅10,000 mm-3). Patient did not have other findings associated with WAS, such as eczema, and recurrent or life-threatening infections. He received the same treatment as his brother (patient 1). Their father and mother are first cousins.

After the patient’s were followed-up by the hematology department because of the familial pattern and microthrombocytes, XLT was suspected.

After written informed consent was obtained from the patients’ parents, mutational analysis was performed by Kazusa DNA Research Institute and National Defense Medical Collage in Japan, according to their published method (2). A missense mutation [WAS: Exon 2, G→A, V→M (c.223G>A, Val75Met)] was observed at exon 2 after purified genomic DNA samples were amplified with primer pairs designed to span each exon and exon/intron junction; the specific causative mutation was identified via direct sequencing. At the time this report was written the 2 patients were being monitored closely for bleeding and symptoms of infection.

They did not require antibiotic prophylaxis or stem cell transplantation, but splenectomy was considered as a treatment to improve their bleeding status by increasing platelet count.

Discussion WAS gene-related conditions are divided into 3 clinical groups: WAS, XLT, and X-linked neutropenia [3,4]. Whereas mutations that distort WASP expression and result in the classical WAS clinical symptoms, including eczema and life-threatening infections, in addition to microthrombocytopenia, mutations that result in defective WASP production, such as missense mutations, cause the milder phenotype XLT, which has a better prognosis; however, mutation, WASP production, and the genotype-phenotype relationship show individual differences.

The WASP gene encodes WASP, a 502 amino acid protein that plays a critical role actin cytoskeleton organization, signaling, and immune cell function [5]. This protein has multiple functional domains used in actin polymerization, cellular motility, intracellular signaling, and apoptosis [6]. The actin cyto-

Leblebisatan et al.

Familial thrombocytopenia in a Turkish family

Turk J Hematol 2011; 28: 139-41

skeleton is active in the basic mechanisms of cell adhesion and migration. Major dynamic rearrangement of the actin cytoskeleton facilitates cell protrusion, retraction, adhesion, and detachment for migration through tissues and endothelial barriers via WASP participation in the transduction of signals from the cell surface to the actin cytoskeleton (7).

A recent study on XLT mutations that included 178 patients from 11 European countries and Japan identified 62 unique mutations located primarily on exon 1 and exon 2-the majority of which were missense mutations. One of the 3 most common mutations is valine at position 75 (Val75Met), as was observed in the presented patients. This mutation was observed in 22 patients from 16 families in 7 countries [1]. A study that included 262 patients from 227 families (including Turkish cases) reported nonsense mutations 950 G>T at exon 9 and 1124 C>T at exon 10, missense mutations 138 T>A at exon 1, 290 C>T at exon 2, 1115C>A at exon 10, and 1542G>C at exon 12, as well as 1 splice mutation at exon 10 in the WASP gene of Turkish patients other than the mutation in the presented cases [8]. A novel mutation at exon 4 was also reported in a Turkish patient [9]. The presented cases did not have a novel mutation, but to the best of our knowledge this is the first report of XLT mutation (223G>A, Val75Met) in a Turkish family.

Patients with classical WAS require stem cell transplantation, whereas those with milder variants (XLT) may not. Overall survival among German XLT patients was reported to be excellent, though some exhibited features of WAS, including mild eczema and infections. Because the genotype-phenotype relationship is not unique, in terms of mutations and WASP production, treatment must be evaluated on an individual basis. Proper treatment is based on clinical suspicion, followed by a correct diagnosis.

Treatment options include splenectomy to increase the platelet count; however, it is associated with a high infection rate and cases without an increase in thrombocytes following splenectomy have been reported. Splenectomized patients require life-long antibiotic prophylaxis. Stem cell transplantation must be considered on a patient-by-patient basis, as some XLT patients have excellent survival, where others have significant morbidity and mortality secondary to life threatening bleedings [1].

We presented 2 brothers with XLT and a previously unreported mutation from Turkey in order to

141

emphasize that in patients with chronic thrombocytopenia associated with a low platelet count genetic analysis of WAS is necessary for the differential diagnosis of diseases of chronic thrombocytopenia and for preventing administration of unnecessary treatments.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

Albert MH, Bittner TC, Nonoyama S, Notarangelo LD, Burns S, Imai K, Espanol T, Fasth A, Pellier I, Strauss G, Morio T, Gathmann B, Noordzij JG, Fillat C, Hoenig M, Nathrath M, Meindl A, Pagel P, Wintergerst U, Fischer A, Thrasher AJ, Belohradsky BH, Ochs HD. X-linked thrombocytopenia (XLT) due to WAS mutations: clinical characteristics, long-term outcome, and treatment options.

Blood 2010;115:3231-8. [CrossRef] Imai K, Morio T, Zhu Y, Jin Y, Itoh S, Kajiwara M, Yata J, Mizutani S, Ochs HD, Nonoyama S. Clinical course of patients with WASP gene mutations. Blood 2004;103:45664. [CrossRef] Ochs HD, Filipovich AH, Veys P, Cowan MJ, Kapoor N. Wiskott-Aldrich syndrome: diagnosis, clinical and laboratory manifestations, and treatment. Biol Blood Marrow Transplant 2009;15:84-90. [CrossRef] Ochs HD, Thrasher AJ. The Wiskott-Aldrich syndrome. J Allergy Clin Immunol 2006;117:725-38. [CrossRef] Gulácsy V, Freiberger T, Shcherbina A, Pac M, Chernyshova L, Avcin T, Kondratenko I, Kostyuchenko L, Prokofjeva T, Pasic S, Bernatowska E, Kutukculer N, Rascon J, Iagaru N, Mazza C, Tóth B, Erdös M, van der Burg M, Maródi L. The J Project Study Group. Genetic characteristics of eighty-seven patients with the Wiskott-Aldrich syndrome.

Mol Immunol 2011;48:788-92. [CrossRef] Rengan R, Ochs HD. Molecular biology of the WiskottAldrich syndrome. Rev Immunogenet 2000;2:243-55.

Thrasher AJ. New insights into the biology of WiskottAldrich syndrome (WAS). Hematology Am Soc Hematol Educ Program 2009;132-8. [CrossRef] Jin Y, Mazza C, Christie JR, Giliani S, Fiorini M, Mella P, Gandellini F, Stewart DM, Zhu Q, Nelson DL, Notarangelo LD, Ochs HD. Mutations of the Wiskott-Aldrich Syndrome Protein (WASP): hotspots, effect on transcription, and translation and phenotype/genotype correlation. Blood 2004;104:4010-9. [CrossRef] Doğu F, Ariga T, Ikincioğullari A, Bozdoğan G, Aytekin C, Metin A, Babacan E. A case of Wiskott-Aldrich syndrome with de novo mutation at exon 4. Turk J Pediatr 2006;48:66-8. [CrossRef]

142

Case Report

Are cup-like blasts specific to AML patients with FLT3 ITD and a normal karyotype? An ALL case report and review of the literature

Cup-like blastlar FLT3 ITD ve normal karyotipli AML hastalarına özgü müdür? ALL’li bir hastanın sunumu ve literatür taraması Özgür Mehtap, Elif Birtaş Ateşoğlu, Emel Gönüllü, Hakan Keski, Abdullah Hacıhanefioğlu Department of Adult Hematology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey

Abstract Cup-like morphology is defined as cup-like nuclear invagination spanning ≥25% of the nuclear diameter in >10% of blasts. Studies have shown that FLT3 ITD and normal cytology are strongly associated with cup-like morphology in acute myeloid leukemia (AML) patients. Herein we describe a patient with cup-like blasts that was diagnosed and treated for common acute lymphoblastic leukemia (ALL). In contrast to the literature, the presented case was Philadelphia chromosome positive and FLT3 ITD negative. (Turk J Hematol 2011; 28: 142-5) Key words: ALL, AML, cup-like blast, FLT3 ITD Received: October 14, 2010

Accepted: March 5, 2011

Özet Cup-like morfoloji, blastların %10’unda fazlasında bulunan, blast çekirdeği çapının, en az %25’ini kapsayan, çekirdeğin içe doğru kıvrılması olarak tanımlanır. Çalışmalarda AML hastalarındaki cuplike morfolojinin, FLT3 ITD ve normal sitogenetik ile güçlü bir ilişkisi olduğu gösterilmiştir. Bu çalışmada Common akut lenfoblastik lösemi tanısı alan ve tedavisi buna göre düzenlenen, cup-like blastları olan bir hasta sunulmuştur. Literatürün aksine bizim hastamızda Philadelphia kromozomu pozitif ve FLT3 ITD negative tespit ettik. (Turk J Hematol 2011; 28: 142-5) Anahtar kelimeler: ALL, AML, cup-like blast, FLT3 ITD Geliş tarihi: 14 Ekim 2010

Kabul tarihi: 05 Mart 2011

Address for Correspondence: M.D. Özgür Mehtap, Department of Adult Hematology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey Phone: +90 262 303 89 14 E-mail: ozgurmehtap@gmail.com doi:10.5152/tjh.2011.32

Mehtap et al.

Cup-like blasts in a patient with ALL

Turk J Hematol 2011; 28: 142-5

Introduction Diagnosis and prognosis of acute myeloid leukemia (AML) depends on evaluation of cytomorphology, and immunophenotyping, cytogenetic, and molecular analyses [1]. Although FAB classification provides a morphological classification of AML, the correlation between morphology and clinical features are imperfect, except in some subtypes of AML (e.g. AML with inv (16) and acute promyelocytic leukemia) [1]. A few studies on cup-like indentation in myeloid blasts and its association with FMS-like tyrosine kinase 3 (Flt3) gene mutations have been published [2-5]. Flt3 protein is expressed on immature hematopoietic and lymphoid progenitors, and seems to play an important role in early stem cell survival and myeloid differentiation [6,7]. The protein is highly expressed in most AML patients and in up to 50% of leukemic blasts in acute lymphoblastic leukemia (ALL) patients [8]. Recently, internal tandem duplication (ITD) mutations of the FLT3 gene have been described in approximately 20%-30% of adult patients with AML [8-10].

Kussick et al. defined cup-like morphology as >10% of blasts with cup-like nuclear invagination spanning ≥25% of the nuclear diameter [5]. This morphology was has only been reported in AML patients, and to the best of our knowledge the present study is the first to report this morphology in an ALL patient.

biopsy specimen showed blasts that stained positive for CD20 and CD34, and negative for MPO, confirming the diagnosis of ALL (Figure 2). BCR-ABL was positive based on RT-PCR following the diagnosis of ALL. DNA-based PCR results were negative for mutant Flt3 ITD (internal tandem duplication). The patient was treated according to the CALGB protocol and in addition received imatinib mesylate.

After the induction phase of the CALGB protocol the patient was in complete remission, which was defined as <5% blasts in the bone marrow. Written informed consent was obtained from the patient.

Discussion A number of studies on cup-like morphology in AML blasts reported similar findings-that Flt3-ITD and normal cytology are strongly associated with cup-like morphology in AML patients (Table 1). Flt3/ITD mutation causes constitutive activation of the receptor’s tyrosine kinase activity, which results in self activation that stimulates the RAS and STAT5 signal transduction pathways, leading to cell proliferation and progression to AML [11]. Many studies reported that Flt3/ITD mutation is closely related to the clinical response and poor prognosis of AML [12,13].

Chen et al. reported a higher frequency of normal cytogenesis and Flt3 gene mutations of internal tandem duplication in patients with cuplike blasts than normal morphology, although it was statisti-

Case A 41-year-old male presented with acute onset of generalized weakness, fatigue, and mucosal bleeding. Physical examination showed marked splenomegaly and petechiae. Full blood count showed that hemoglobin was 7.6 g dL-1, a white cell count of 62.700 µL-1, and a platelet count of 28.400 µL-1.

Peripheral blood smear showed agranular blasts with an abundance of large nuclear indentations and prominent invagination cup-like blasts (Figure 1).

Flow cytometric immunophenotyping results were positive for 89.38% CD10, 89.56% CD19, 78.86% CD20, 88.5% CD22, 88.18% anti-TdT, 79.73% CD34, 90.41% HLA-DR, and 0.26% sIgM, suggesting common ALL. MPO and CD117 were negative according to flow cytometry.

Histopathological and immunohistochemical evaluation of the patient’s bone marrow trephine

143

Figure 1. Cup-like blasts

Figure 2. MPO negativity and CD20 positivity in bone marrow

144

Mehtap et al.

Cup-like blasts in a patient with ALL

Turk J Hematol 2011; 28: 142-5

cally insignificant [14]. In addition to the presence of Flt3/ITD and normal karyotype, the other characteristic feature of cup-like morphology is complete or partial loss of HLA-DR and CD34 expression in AML blasts (Table 1). Kronschinsky et al. reported that cup-like nuclei could occur in both non-monocytic and monocytic AML [4]. They reported that there was an association between cup-like nuclear morphology and negative HLA-DR expression only in non-monocytic FAB types [4]. Oelschlaegel et al. studied the relationship between HLA-DR negative AML blasts, and CD34 expression, Flt3/ITD, and cuplike nuclear morphology [3]. They reported that loss of HLA- DR expression in AML blasts was strongly correlated with cup-like morphology and Flt3/ITD (p<0.001) [3].

Recent studies reported that cup-like morphology in AML is highly associated with the presence of nucleophosmin (NPM1) gene mutation [4,14,15].

Exon 12 mutations in the NPM1 gene cause abnormal cytoplasmic accumulation of the NPM1 phosphoprotein in primary AML patients with a normal karyotype [16]. NPM1 mutations occur in approximately 35% of AML patients [16]. Chen et al. observed NPM1 mutation in 19 of 22 (86%) AML patients with cup-like morphology [15]. According to the literature, the frequency of Flt3/ITD and NPM1 mutations (or both) in patients with cup-like morphology is high. Three studies reported that there weren’t any significant differences in overall survival between AML patients with cup-like nuclei and the control group [4,14,15].

In contrast to the above-mentioned studies, the presented case was diagnosed as ALL with cup-like invagination in blast cells; cup-like morphology was observed in 20% of blasts. In contrast to AML patients, the presence of Flt3/ITD is rare in ALL patients [8]. ITD mutations of the FLT3 gene have

been reported in ≤3% of adult patients with ALL [10,17,18]. Considering the relationship between Flt3 gene mutation and cup-like morphology in previous studies, we evaluated the presented case for Flt3/ITD mutation, but found none. As with Flt3/ITD, NPM1 mutation in ALL patients is rare. In fact, a published pilot study from India reported that 1 of 114 (0.08%) ALL patients had NPM1 mutation [19]; however, the presented case was not screened for this mutation due to socioeconomic factors. The presented patient was Philadelphia chromosome positive, whereas according to the literature AML patients with cup-like morphology usually have a normal karyotype.

To the best of our knowledge this is the first report of an ALL patient with cup-like blasts. The question that remains to be answered is as follows: are cup-like blasts in ALL a very rare event or are they overlooked?

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

Table 1. Characteristics of patients in studies

Kussick Kroschisky Weina Chen Weina Chen et al.5 et al.4 et al.14 et al.15

Flt3/ITD

84%*

70.9%*

88%**

86%*

Cytogenetic abnormality

8.3%*

18.5%*

30%**

14%*

Lack of HLA-DR

70%*

33.5%**

50%*

59%*

Lack of CD34

68%*

93%*

71%*

82%*

*Significant compared to non-cup-like morphology **Insignificant compared to non-cup-like morphology

4.

5.

Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues: World Health Organization Classification of Tumours.

Lyon, France: IARC Press; 2008;28-30.

Jalal S, Leach M, McKay P. Possible significance of cuplike blasts in acute myeloid leukaemia. Br J Haematol.

2010;148:182.

Oelschlaegel U, Mohr B, Schaich M, Schäkel U, Kroschinsky F, Illmer T Ehninger G, Thiede C. HLA-DR negative patients without acute promyelocytic leukemia show distinct immunophenotypic, genetic, molecular, and cytomorphologic characteristics compared to acute promyelocytic leukemia. Cytometry B Clin Cytom.

2009;76:321-7.

Kroschinsky FP, Schäkel U, Fischer R, Mohr B, Oelschlaegel U, Repp R Schaich M, Soucek S, Baretton G, Ehninger G, Thiede C DSIL (Deutsche Studieninitiative Leukämie) Study Group. Cup-like acute myeloid leukemia: new disease or artificial phenomenon?

Haematologica 2008;93:283-6.

Kussick SJ, Stirewalt DL, Yi HS, Sheets KM, PogosovaAgadjanyan E, Braswell S Norwood TH, Radich JP, Wood BL. A distinctive nuclear morphology in acute

Mehtap et al.

Cup-like blasts in a patient with ALL

Turk J Hematol 2011; 28: 142-5

6.

7.

8.

9.

10.

11.

12.

13.

14.

myeloid leukemia is strongly associated with loss of HLA-DR expression and FLT3 internal tandem duplication. Leukemia 2004;18:1591-8.

Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, Cıvın IC. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci. 1994;9:459-63.

Shurin MR, Esche C, Lotze MT. FLT3: receptor and ligand: biology and potential clinical application.

Cytokine Growth Factor Rev. 1998;9:37-48.

Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002;100:1532-42.

Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. NatRev Cancer 2003;3:650-65.

Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K Sonoda Y, Fujimoto T, Misawa S. Internal tandem duplication of the Flt3 gene found in acute myeloid leukemia. Leukemia 1996;10:1911-8.

Choudhary C, Müller-Tidow C, Berdel WE, Serve H. Signal transduction of oncogenic Flt3. Int J Hematol, 2005;82:93-9.

Yanada M, Matsuo K, Suzuki T, Kiyoi H, Naoe T. Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis. Leukemia 2005;19:1345-9.

Shih LY, Huang CF, Wu JH, Lin TL, Dunn P, Wang PN, Kuo MC, Lai CL, Hsu HC. Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse. Blood 2002;100:2387-92.

Chen W, Rassidakis GZ, Li J, Routbort M, Jones D, Kantarjian H. High frequency of NPM1 gene mutations

15.

16.

17.

18.

19.

145

in acute myeloid leukemia with prominent nuclear invaginations ("cuplike" nuclei). Blood. 2006;108:1783-4.

Chen W, Konoplev S, Medeiros LJ, Koeppen H, Leventaki V, Vadhan-Raj S, Jones D, Kantarjian HM, Falini B, BuesoRamos CE. Cuplike nuclei (prominent nuclear invaginations) in acute myeloid leukemia are highly associated with FLT3 internal tandem duplication and NPM1 mutation. Cancer 2009;115:5481-9.

Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi P, Meani N, Pettirossi V, Saglio G, Mand elli F, Lo-Coco F, Pelicci PG, Martelli MF; GIMEMA Acute Leukemia Working Party. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med. 2005;352:254-66.

Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T, Sonoda Y, Abe T, Kahsima K, Matsuo Y, Naoe T. Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies: a study on a large series of patients and cell lines.

Leukemia 1997;11:1605-9.

Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, Kobayashi M, Bessho F, Yanagisawa M, Hayashi Y. Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children. Br J Haematol. 1999;105:155-62.

Nageswara Rao Dunna, Sailaja Kagita, Surekha Damineni, Senthil Rajappa, Raghunadharao Digumarti, Vishnupriya Satti. NPM1/FLT3 ITD Gene Mutation Profiling in Acute leukemia: A Pilot study HGM2008.

Genes Chromosomes and Disease 2010;11:1811-6.

146

Letter to the Editor

Central retinal artery occlusion as the presenting sign of essential thrombocythemia Eansiyel trombositeminin başvuru belirtisi olarak santral retinal arter tıkanıklığı Gül Arıkan1, Ali Osman Saatci1, Selda Kahraman2, Özden Pişkin2, Süleyman Men3, Bülent Ündar2 1Dokuz

Eylül University, School of Medicine, Department of Ophthalmology, İzmir, Turkey Eylül University, School of Medicine, Department of Hematology, İzmir, Turkey 3Dokuz Eylül University, School of Medicine, Department of Radiology, İzmir, Turkey 2Dokuz

To the Editor, Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by an elevated platelet count without an obvious cause. In addition to overproduction, platelets are also functionally abnormal; therefore, thromboembolic and hemorrhagic complications can occur in such patients [1].

Reports of retinal arterial occlusion [2,3] and venous occlusion [4-9] in cases with ET are rare. Herein we report a male patient with unilateral central retinal artery occlusion (CRAO) as the presenting sign of previously undiagnosed ET. A 28-year-old male presented due to painless loss of vision in his right eye that began 6 h earlier.

His medical history was unremarkable, except for smoking. On examination, visual acuity was no light perception in the right eye and 20/20 in the left eye.

Ocular motility was full. A right afferent pupillary defect was noted. Intraocular pressure was 14 mmHg OU. Slit lamp examination results were unremarkable. Fundus examination of the right eye

showed massive retinal opacification, markedly attenuated arteries, a box-car appearance in the retinal arterioles, and a cherry-red spot in the macula without emboli (Figure 1). The left fundus was normal. Anterior chamber paracentesis was performed in a surgical suite immediately following the diagnosis of CRAO. Written informed consent was obtained from the patient.

A thorough medical examination was carried out to clarify the etiology of CRAO. Carotid Doppler ultrasonography showed complete occlusion of the right common carotid artery (CCA) and internal carotid artery (ICA). Computed tomographic angiography also showed total occlusion of the right CCA and ICA, as well as retrograde filling of the intracranial portion of the right ICA from the anterior communicating artery (Figure 2). Echocardiography was normal. Abdominal ultrasonography showed splenomegaly. The patient’s platelet count was >1,000,000 mm-3 (normal range: 156,000-373,000 mm-3) in consecutive assays. White blood cell count was 8500 mm-3, hemoglobin was 16.2 g dL-1,

Address for Correspondence: Prof. Ali Osman Saatci, Mustafa Kemal Sahil Bulvarı No: 73, A Blok, Daire: 9 Narlıdere 35320 İzmir, Turkey Phone: +90 232 412 30 51 E-mail: osman.saatci@yahoo.com doi:10.5152/tjh.2011.33

Arıkan et al.

Retinal artery occlusion in essential thrombocythemia

Turk J Hematol 2011; 28: 146-8

Figure 1. The fundus of the right eye at presentation shows the cherry-red spot and attenuated arterioles

H R

A P

L

Figure 2. Coronal oblique computed tomographic angiography of the entire supra-aortic region. The right common carotid artery is occluded 2 cm distal to its origin (arrow). The right internal carotid artery is also occluded; right intracranial carotid circulation is provided by collateral pathways

hematocrit was 48.3%, mean corpuscular volume was 88 fL, and ferritin was 58 ng mL-1.

Coagulation tests showed prothrombin 20210A gene mutation heterozygosity. Protein C, protein S, antithrombin III, serum homocysteine, activated protein C resistance, lupus anticoagulant, ANA, antiDS DNA, anticardiolipin IGM and IGG antibody, and sedimentation and C reactive protein test results were within normal limits. Factor V Leiden mutation was

147

negative. Peripheral blood smear showed abundant platelet clumps, and bone marrow biopsy showed megakaryocytic hyperplasia and reticulin fibrosis (grade 1). The patient was heterozygous for JAK 2 mutation and was Philadelphia chromosome negative. Cytogenetic analysis showed that the patient’s karyotype was normal. The patient was diagnosed as ET. The patient subsequently stopped smoking, and hydroxyurea therapy and low-dose aspirin were initiated. The patient’s platelet count returned to normal after 1 month of the treatment. Unfortunately, his visual acuity did not improve and he was lost to follow-up due to relocation to another city.

CRAO is a very rare clinical presentation in younger ages and a thorough systemic investigation is required to determine the underlying cause [10].

The pathogenesis of CRAO was not clear in the presented case. Embolism-despite no visible emboli-or thrombosis due mainly to ET might have been the cause; however, this patient, who was a smoker, had prothrombin 20210A mutation heterozygosity in addition to previously undiagnosed ET, and it is likely that the combined effect of these factors might have contributed to serious carotid stenosis and subsequent CRAO. A PubMed search showed that only 2 cases of retinal arterial occlusion associated with ET have been published [2,3]. Singer [2] reported a 37-yearold male with recurrent attacks of migrating emboli in the retinal arteries that were associated with amaurosis fugax; he was subsequently diagnosed as ET. Strassman et al. [3] reported an elderly woman with unilateral CRAO that had been diagnosed as ET before. Clear carotid artery stenosis was not observed, but echocardiography showed a thickened calcific aortic valve. The researchers thought that platelet thrombi caused CRAO, but that an embolus coming from the calcified aortic valve could not be ruled out as a possible cause. Retinal venous occlusion was also reported in patients with ET [4-9]; in some cases venous vascular occlusion was unilateral [4-7] and in others it was bilateral [8,9]. Venous occlusion might also present as multiple occlusions in the same eye [8].

ET is among the underlying hematologic disorders in patients with CRAO. Our aim was to share our observations with the ophthalmic and hematologic communities, and to increase the awareness of the possible etiological role of ET in CRAO.

148

Arıkan et al.

Retinal artery occlusion in essential thrombocythemia

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

Spivak JL. Polycythemia vera and other myeloproliferative diseases. In: Fauci AS, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, Kasper DL, Hauser SL, Longo DL, eds. Harrison’s Principles of Internal Medicine.

14th ed. New York: McGraw-Hill , 1991;683-4.

Singer G. Migrating emboli of retinal arteries in thrombocythaemia. Br J Ophthalmol 1969;53:279-81. [CrossRef] Strassman I, Silverstone BZ, Seelenfreund MH, Sheer A, Berson D. Essential thrombocythemia: a rare case of central retinal artery occlusion. Metab Pediatr Syst Ophthalmol 1991;14:18-20.

Yoshizumi MO, Townsend-Pico W. Essential thrombocythemia and central retinal vein occlusion with neo-

Turk J Hematol 2011; 28: 146-8

vascular glaucoma. Am J Ophthalmol 1996;121:728-30.

Nobacht S, Cruysberg JR, Deutman AF. Peripheral retinal nonperfusion associated with essential thrombocytosis. Am J Ophthalmol 1999;127:101-2. [CrossRef] 6. Asensio Sánchez VM, Manteca Jiménez G, Cano Navarro E. Essential thrombocythemia and retinal vein thrombosis. Arch Soc Esp Oftalmol 2004;79:629-32.

[CrossRef] 7. Tache JE, Saffra N, Marshak H, Aithal S, Novetsky A, Huang YW. Retinal vein thrombosis as the presenting symptom of essential thrombocythemia. Am J Med Sci 2005;329:139-40.

[CrossRef] 8. Imasawa M, Iijima H. Multiple retinal vein occlusions in essential hrombocythemia. Am J Ophthalmol 2002;133:152-5. [CrossRef] 9. Liu M, Lee AG, Rice L, Lambert HM. Bilateral retinal vascular occlusive disease in essential thrombocythemia. Retina 1999;19:563-4. [CrossRef] 10. Brown GC, Magargal LE, Shields JA, Goldberg RE, Walsh PN. Retinal arterial obstruction in children and young adults. Ophthalmology 1981;88:18-25.

5.

Letter to the Editor

149

“Arbitrary” criterion for the diagnosis of acute leukemia Akut lösemi tanısına yönelik “rastgele” kriter Abbas Hashim Abdulsalam Department of Hematology Unit, Teaching Laboratories, Al-Yarmouk Teaching Hospital, Baghdad, Iraq

To the Editor, According to the WHO classification of acute leukemia, diagnosis is based on an arbitrary cut-off point of 20% blasts, as the percentage of bone marrow total or non-erythroid cells, or as the percentage of peripheral blood cells. This cut-off point is also currently used in under-resourced laboratories in which the FAB classification is more commonly used.

This cut-off point seems to be universally accepted, and for the time being represents the bestknown criterion for defining acute leukemia; however, “arbitrary” may still precede the criterion due to the follow: 1. This percentage does not represent a specific biological event in the continuum of an increasing blast count, but is merely, to the best to our knowledge, a cut-off point that facilitates relatively clear classification and therapeutic planning.

Nonetheless, some high-risk MDS patients with only 10% bone marrow blasts are actively treated.

2. A significant difference in the blast percentage cut-off point between peripheral blood and bone marrow is well established in MDS; RAEB1 (blast count <5% in peripheral blood and 5%-9% in bone marrow) and RAEB-2 (blast

count of 5%-9% in peripheral blood and 10%19% in bone marrow). In acute leukemia no such differentiation exits.

3. Morphological findings of pathological “clonal” blasts (type II that contain Auer rods, PseudoChédiak-Higashi or other specific inclusions that are not seen in reactive marrow) refer to the diagnosis of RAEB-2 or AML; here again the arbitrary cut-off point of 20% blasts results in a specific diagnosis.

4. The original FAB classification was based for many years on the arbitrary cut-off point of 30% bone marrow blasts and in the past some patients with 20%-29% blasts remained stable for months without chemotherapy.

5. Although there is a general consensus concerning the criterion for identifying myeloblasts as agranular (type I) and granular blasts (types II and III), there is some disagreement concerning their definitions, and in practice it may be a matter of convention (subjective method) to differentiate them from the continuum of cells, such as determining whether the cell is a type III blast or a promyelocyte.

6. Blasts refer to myeloblasts, lymphoblasts, monoblasts, promonocytes, and megakaryoblasts.

Address for Correspondence: Abbas Hashim Abdulsalam, MD, Department of Hematology Unit, Teaching Laboratories, Al-Yarmouk Teaching Hospital, Baghdad, Iraq Phone: 964 7904 188690 E-mail: dr.abbas77@yahoo.com doi:10.5152/tjh.2011.34

150

Abdulsalam AH. Arbitrary diagnosis of acute leukemia

7. Diagnosis of AML-M3 and its variant is not related to the percentage of blasts.

8. Cases with <20% blast cells may still be diagnosed as acute leukemia if they present with certain recurrent cytogenetic abnormalities, as in AML M4 with inv (16) or t (16;16) (p13;q22) and AML M2 with t (8;21).

9. The utilization of a 20% blast threshold is not really an issue in ALL because most patients at the time of diagnosis already have <50% blasts.

In AML-M0 and M1 this cut-off point is also not useful.

In conclusion: Morphology is the first-line diagnostic criterion for acute leukemia; however, labo-

Turk J Hematol 2011; 28: 149-50

ratory diagnosis of acute leukemia in modern hematologic practice increasingly relies on objective techniques to detect a specific ultrastructural or genetic abnormality. As such, the era of 20% blasts as a diagnostic criterion for acute leukemia may not stand the time any longer than that of the old FAB group 30% blasts lower threshold.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Letter to the Editor

151

Paclitaxel therapy and immune thrombocytopenic purpura: Coincidence or association?

Paclitaxel tedavisi ve immun trombositopeni: Rastlantı mı, ilişki mi? Ümmügül Üyetürk1, Şerife Hülya Arslan2, Meltem Kurt Yüksel2, Fevzi Altuntaş2 1Department

of Clinical Oncology, Dr. Abdurrahman Yurtarslan Oncology Education and Research Hospital, Ankara, Turkey 2Department of Hematology, Dr. Abdurrahman Yurtarslan Oncology Education and Research Hospital, Ankara, Turkey

To the Editor, Thrombocytopenia is defined as a platelet count <150,000 µL-1. Primary causes of thrombocytopenia are decreased production of platelets in the bone marrow and increased destruction of platelets in the spleen [1]. Thrombocytopenia is most commonly observed in cancer patients as a result of direct tumor infiltration in the bone marrow, and bone marrow toxicity due to chemotherapy and/or radiotherapy. Clinical diagnosis of druginduced thrombocytopenia can be made when thrombocytopenia is reversed following withdrawal of the suspected drug and doesn’t develop during follow-up.

Drug-induced thrombocytopenia can be caused by various medications. Drug-induced thrombocytopenia is the result of bone marrow suppression and destruction of platelets in peripheral blood due to immune or non-immune mechanisms [1,2].

Paclitaxel-induced thrombocytopenia percentage is reported to be 4-20%, and 1-7 % if the grade of trombocytopenia is grade 3-4. [3]. Our aim was to present a rare case of immune thrombocytopenic purpura (ITP) following paclitaxel treatment.

Modified radical mastectomy was performed in a 47-year-old female patient following a biopsy of a mass in the upper lateral quadrant that was diagnosed as invasive ductal carcinoma. The tumor was grade II, T3N0M0 invasive ductal carcinoma. CerbB2 score was 1, level of estrogen receptor expression was 60% and progesteron receptor expression was 60%. The patient was treated with 4 courses of cyclophosphamide, epirubicin, and 5-fluorouracil, as well as radiotherapy to the surgical area, followed by weekly treatment with paclitaxel. After taking paclitaxel for 7 weeks the patient presented with rashes on her leg. The patient’s hemogram results were as follows: leukocyte count: 5700 µL-1; hemoglobin: 11.9 g dL-1; platelet count: 7000 µL-1.

Paclitaxel treatment was discontinued due to suspicion of drug-induced bone marrow suppression.

Written informed consent was obtained from the patient.

The patient did not come for regular follow-up visits, but presented 2 months after paclitaxel treatment was withdrawn; hemogram results at that time were as follows: leukocyte count: 6270 µL-1; hemoglobin: 12.9 g dL-1; platelet count: 9740 µL-1.

She was hospitalized. Peripheral blood smear did

Address for Correspondence: Dr. Ümmügül Üyetürk, Department of Clinical Oncology, Dr. Abdurrahman Yurtarslan Oncology Education and Research Hospital, Ankara, Turkey Phone: +90 312 336 09 09-3005 E-mail: ummuguluyeturk@yahoo.com.tr doi:10.5152/tjh.2011.35

152

Üyetürk et al.

Paclitaxel therapy and immune thrombocytopenic purpura

not show atypical cells, but several single platelets were observed. Aggregated platelets were not observed. Brucella agglutination, salmonella group agglutination, ANA, TORCH panel, HBsAg, anti-HCV, anti-HIV, and urea breath test results were negative.

Thyroid function test and abdomen ultrasonography results were normal. Bone marrow biopsy was performed on the third day of hospitalization. Analysis of the biopsy specimen showed an increase in the number of hematopoietic cells of erythroid, myeloid, and megakaryocytic lineage. Findings for blastic cell infiltration and carcinoma metastases were negative. Due to the persistence of thrombocytopenia 2 months after the discontinuation of paclitaxel and lack of any other cause for ITP, the patient was diagnosed with immune thrombocytopenic purpura (ITP) and prednisolone treatment (1 mg kg-1) was initiated. The patient’s platelet count began to increase within 24 h. After 1 week of prednisolone treatment the platelet count reached 150,000 µL-1.

ITP is an acquired disorder caused by antibodies against platelets and progresses as platelet destruction increases [1]. The literature contains only a few cases of ITP that developed following paclitaxelcontaining chemotherapy [4]. The occurrence of ITP in the presented case during the period in which the disease was controlled ruled out the diagnosis of malignancy-related paraneoplastic syndrome. The patient’s bone marrow was not hypocellular; therefore, diagnosis of bone marrow suppression secondary to drug use was also eliminated. There was no clear explanation for the patient’s persistent ITP 2 months after paclitaxel was withdrawn. There is an association between paclitaxel 's elimination half-life and the time to platelet recovery in cases of drug-induced thrombocytopenia [5,6]. As the mean elimination half-life of paclitaxel is 5.8 h, one could expected that platelet recovery after discontinuation of paclitaxel would normally occur within 1 week; however, case reports describe

Turk J Hematol 2011; 28: 151-2

thrombocytopenia persisting beyond the traditional 4-5 half-lives of the drug. Quinidine's half-life is approximately 6 h, but in 1 report recovery time was as long as 15-30 d following withdrawal of the drug.

It was theorized that the antibody originally directed against the drug-protein complex might have broadened its spectrum so that an antigen on the platelets became the target [5].

The presented patient’s ITP might have been related to a paclitaxel-induced immunological mechanism or coincidence. There is a need for further clinical studies involving large numbers of patients to more fully understand this subject. In conclusion, great care should be exercised in patients using paclitaxel, with regard to ITP and neutropenia [6].

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

5.

6.

Hillman RS, Ault KA, Rinder HM. Thrombocytopenia. In: Hematology in clinical practice. 4th ed. New. York: McGraw-Hill, 2005:339-56.

Casciato DA. Hematologic complications. Casciato DA, Territo MC(Ed) Manual of clinical oncology. 6th ed.

Lippincott Williams&Wilkins,2009:658-83.

Solimando DA. Drug ınformation handbook for oncology.

6th ed. Lexİ- Comp Inc, 2007;804-9.

Demirer T, Celebi H, Arat M, Ustün C, Demirer S, Dilek I, Ozcan M, Ilhan O, Akan H, Gürman G, Koç H. Autoimmune thrombocytopenia in a patient with small cell lung cancer developing after chemotherapy and resolving following autologous peripheral blood stem cell transplantation.

Bone Marrow Transplant. 1999;24:335-7.

Hackett T, Kelton JG, Powers P. Drug-induced platelet destruction. Semin Thromb Hemost 1982;8:116-37.

Rowinsky EK, Eisenhauer EA, Chaudhry V, Arbuck SG, Donehower RC. Clinical toxicities encountered with paclitaxel (Taxol). Semin Oncol. 1993;20:1-15.

Letter to the Editor

153

Interaction between hereditary spherocytosis and the beta-thalassemia trait: A case report Kalıtsal sferositoz ve beta-talasemi taşıyıcılığı arasındaki etkileşim: Olgu sunumu Sunita Sharma, Sonal Jain Malhotra, Richa Chauhan Department of Pathology, Lady Hardinge Medical College, Delhi University, New Delhi, India

To the Editor, Coinheritance of hereditary spherocytosis (HS) and β thalassemia is very rare. HS is a familial haemolytic disorder resulting from primary abnormality of red cell membrane. It is transmitted as an autosomal dominant trait. β thalassemia is also a common inherited disorder. In Indians, the frequency of β thalassemia is reported between 3.5&14.9% [1]. The haemolytic anemia resulting from their coexistence has been shown to be of variable severity in different studies [2-5].

We hereby present a case of a 50 yr old Nepalese female who came with fever, cough & weakness without any organomegaly. CBC findings revealed microcytic hypochromic red cells with high red cell count (>5x106/µl) and mildly increased RDW suggestive of βTT (Table 1). Peripheral smear showed large number of microspherocytes, microcytic hypochromic cells, target cells and occasional red cells with basophilic stippling (Figure 1). Reticulocyte count was 1.5%. Direct Coomb’s test was negative and serum bilirubin was normal (1.2 g/dl). HPLC of Hb revealed an increased Hb A2 (4.8%) and Hb F (6.0%). Her son revealed very few spherocytes in peripheral smear and CBC findings were suggestive

of βTT. Coomb’s test was negative, Hb HPLC showed high HbA2 (5.1%). The incubated osmotic fragility curves of both the patient and her son were shifted to right with a tail of fragile cells. Thus, a diagnosis of HS with βTT was made in both (Figure 2).

Inheritance of HS has been reported in association with α thalassemia, β thalassemia and certain enzyme deficiencies [2-8].

The results are conflicting regarding the degree of hemolysis, when hereditary spherocytosis and

Figure 1. (400X)-Peripheral smear (Wright’s stain) showing large number of microspherocytes and some target cells

Address for Correspondence: Prof. Sunita Sharma, Department of Pathology, Lady Hardinge Medical College, Delhi University, 110001 New Delhi, India Phone: (91)0120-2554266 E-mail: d_sharma1960@yahoo.co.in doi:10.5152/tjh.2011.36

154

Sharma et al.

Interaction of hereditary spherocytosis with beta-thalassemia trait

Table 1. Hematological parameters Parameter

Patient

Son

Husband

10.3

12.1

14.0

5.37

6.26

5.05

Hct %

34.5

39.9

43.0

MCV (fl)

64.2

63.7

85.1

MCH (pg)

19.2

19.3

28.0

MCHC (g/dl)

29.9

30.3

32.9

RDW (%)

20.7

16.1

14.4

TLC /cumm

8,500

4,800

9,000

149

142

154

HbA%

89.2

94

98.9

HbA2%

4.8

5.1

1.5

HbF%

6.0

0.9

0.4

Hb g/dl RBC count x

1012/l

Plt Count x109/l Hb HPLC

Turk J Hematol 2011; 28: 153-4

intermittent jaundice. However, our patient was asymptomatic till date. This corollary can be explained by assuming that the coinheritance of βTT with HS probably had an influence on clinical outcome. The microcytic hypochromic red cells of βTT and spherocytes of HS had opposite properties with regards to their fragility and this probably leads to reduced severity of hemolysis. Hence, if both HS and βTT coexist, the later silences the HS and ameliorates the degree of hemolysis.

Written informed consent was obtained from the patient.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

3.

4.

5.

6.

Figure 2. Incubated Osmotic fragility curve showing shift to the right compared to the control

heterozygous β thalassemia coexist. In our case the peripheral smear showed a large number of microspherocytes, pointing towards moderate HS (20-30/ hpf). Moderate HS is associated with a chronic haemolytic anemia with modest splenomegaly and

7.

8.

Borgna-Pignatti C, Galanello. Thalassemias and related disorders: Quantitative disorders of haemoglobin synthesis. In: Wintrobe’s Clinical Hematology.Lippincott Williams & Wilkins. 2004;1320.

Miraglia del Giudice E, Perrotta S, Nobili B, Pinto L, Cutillo L, Iolascon A. Coexistence of hereditary spherocytosis (HS) due to band 3 deficiency and beta-thalassemia trait: partial correction of HS phenotype. Br J Haematol. 1993;85:553-7.

White BP, Farver M. coexixstence of hereditary spherocytosis and beta-thalassemia: case report of severe haemolytic anemia in an American black. S D J Med. 1991;44:257-61.

Aksoy M, Erdem S. Combination of hereditary spherocytosis and hereditary beta-thalassemia: a family study.

Acta Haematol. 1968;39:183-91.

Andrien JM, Heusden A, Lambotte C, Hugues J. Spherocytose hereditaire et β thalassemia: Coexistence des deux genes au sein d’une famille sicilienne. Acta Paediatrica Belgica. 1971;25:35-43.

Heaton DC, Fellowes AP, George PM. Concurrence of hereditary spherocytosis and alpha thalassemia. Aust N Z J Med. 1991;21:485-6.

Li CK, Ng MH, Cheung KL, Lam TK, Shing MM. Interaction of hereditary spherocytosis and alpha thalassemia: a family study. Acta Haematol. 1994;91:201-5.

McCann SR, Finkel B, Cadman S, Allan DW. Study of a kindred with hereditary spherocytosis and Glyceraldehyde-3-Phosphate Dehydrogenase Deficiency. Blood. 1976;47:171-81.

Images in Hematology

155

Hematogones in the bone marrow of a child with Rubella virus-associated immune thrombocytopenic purpura concomitant with iron deficiency anemia Demir eksikliği anemisine eşlik eden Rubella virüsüne bağlı immune trombositopenik purpuralı bir çocuğun kemik iliğindeki hematogonlar Faruk Barlık1, Emel Özyürek2, Feride Duru2 1Department

of Pediatrics, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey

2Department of Pediatrics, Hematology Section, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey

Hematogones are normal B-cell precursors that are observed in small numbers in the bone marrow; however, significant expansion of hematogones can occur in the bone marrow in healthy newborns, and in individuals with malignant and non-malignant hematologic disorders, including congenital and autoimmune cytopenias, nutritional anemias, viral infections, and Gaucher disease [1-6]. Morphologically, hematogones are small- to medium-sized cells with condensed chromatin and scant cytoplasm devoid of inclusions and vacuoles; some hematogones resemble leukemic lymphoblasts. As the morphological and immunophenotypical features of hematogones are similar to those of the blasts in precursor B acute lymphoblastic leukemia (ALL), sometimes diagnostic problems arise.

Using flow cytometry hematogones can be identified by their peculiar antigenic profile; 3 forms are defined on the basis of their maturational stage [1-6].

Stage 1 hematogones are the most immature form, and along with CD34 and TdT positivity they exhibit dimmer expression of CD45 and CD19, and brighter expression of CD10, as compared to the more mature forms and normal lymphocytes. Stage 1 hematogones lack CD20. Stage 2 hematogones represent the majority of the hematogone popula-

tion. They exhibit high CD34 and TdT experession, with increased expression of CD19, dim expression of CD20, and gradually decreasing expression of CD10. As they mature to stage 3 they exhibit brighter expression of CD20 and CD45, and dimmer expression of CD10, as compared to the more immature forms of hematogones. Recently, stage 3 and some stage 2 hematogones were reported to express CD5 concomitantly with CD19 and CD10. Dim expression of CD22 and bright expression of CD38 are seen in each hematogone stage [7].

A 1-year-old girl presented with a cough and a rash on her body 1 d after developing fever. Her medical and family histories were non-contributory.

On physical examination she had petechiae all over her body and on both tonsils. She did not have organomegaly. The remaining systemic examinations were normal. Her complete blood count results were as follows: hemoglobin: 9.3 g dL-1; hematocrit: 27%; erythrocyte count: 4.01x109 L-1; mean corpuscular volume: 69 fL; red cell distribution width: 15.5%; white blood cell count: 14,100 mm-3; platelet count: 2000 mm-3. There were 80% lymphocytes, 20% neutrophils, 1 or 2 platelets without aggregation, and hypochromic microcytic erythrocytes on

Address for Correspondence: M.D. Emel Özyürek, Department of Pediatrics, Hematology Section, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey Phone: +90 362 312 19 19-3757 E-mail: heozyurek@yahoo.com doi:10.5152/tjh.2011.37

156

Barlık et al.

Hematogones in immune thrombocytopenic purpura

peripheral blood smear. We observed 12% mononuclear cells resembling lymphoblasts (Figure 1).

Immunophenotypic analysis of her bone marrow was performed using flow cytometry. Total lymphoid cells were selected using SSC versus FSC gating (Figure 2a). B-lymphoid cells (48%-49% of total lymphocytes) expressing CD19 and CD22 were observed.

Among all the lymphocytes (Figure 2a), those constituting 38% that showed bright CD10 expression, and coexpression of CD19 and CD5 (Figure 2b and c) were considered as hematogones.

A small number (approximately 4%) of cells that were probably early-stage or stage 1 hematogones also expressed CD34 and TdT (Figure 2d and e).

These cells lacked aberrant marker expression.

Serology was positive for rubella virus immunoglobulin M. Iron panel of the patient showed iron deficiency anemia (serum iron level: 20.6 µg dL-1; serum iron binding capacity: 460 µg dL-1; transferrin saturation: 5.8%; ferritin: 5.8 ng dL-1) and complete blood count parameters in both of the patient’s parents were normal. As such, rubella virus-associated immune thrombocytopenic purpura was considered as the patient’s diagnosis, in addition to iron deficiency anemia.

The patient was given intravenous immunoglobulin (2 g kg-1) and ferrous sulphate treatments. An significant increase in her platelet count was observed within 2 days. A follow up bone marrow smear performed 2 days after the first one or immediately following the therapy showed a decrease in the number of hematogones (3%) by morphological examination as compared to the previous analysis.

But flow cytometric analysis of the second bone marrow sample was not performed/done at that time. She was discharged with a platelet count of

Turk J Hematol 2011; 28: 155-7

153,000 mm-3. One month later her hemoglobin increased to 12 g dL-1, and the ferrous sulphate dose was decreased accordingly. She received iron therapy for a total of 3 months starting from her hospitalisation. During 6 months of follow-up her thrombocyte count fluctuated between 112,000 and 451,000 mm-3.

The presented patient had rubella virus-associated thrombocytopenia and iron deficiency anemia; both conditions are reported to be associated with the appearance of hematogones [7]. Once the diaga

b

c

Figure 1. Hematogones with blast-like features in the bone marrow sample

Barlık et al.

Hematogones in immune thrombocytopenic purpura

Turk J Hematol 2011; 28: 155-7

d

e

157

whereas less mature forms-predominantly stage 1 and some stage 2 hematogones-resemble blasts of ALL. In the morphological examination of the second bone marrow, we observed a significant decrease in the percentage of blast-like cells which were identified as stage 1 and some stage 2 hematogones (12%) by immunophenotypic analysis of the first marrow specimen. This prompt fall in the number of blast-like cells may be explained by the maturation of those early stage hematogones to stage 3 ones which are difficult to differentiate by histopathological examination of a marrow smear.

In conclusion, excessive hematogones in the bone marrow of children may cause problems in diagnostically differentiating acute lymphoblastic leukemia from a benign hematogone proliferative state, and flow cytometric analysis may help differentiate these 2 cell populations.

Written informed consent was obtained from the patient.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

Figure 2. a. On the SSC-FSC scatter plot we gated the total lymphocyte population. b. In the SSC versus CD10 plot there were 3 different populations of hematogones (in stage 1, stage 2, and stage 3, shown with arrows) with very low side scatter and variable expression of CD10. c. Hematogones with bright expression of CD19 and CD5. d. Cells shown with arrows are early-stage or stage 1 hematogones with CD10 and CD34, as well as stage 2 hematogones expressing CD10, but lacking CD34. e. Early hematogones expressing TdT (arrow)

nosis was made and appropriate therapy was initiated, rapid improvement of thrombocytopenia along with a striking reduction in bone marrow hematogones was observed. The cause of the observed rapid reduction in hematogones is not clear, but may have been due to their differentiation into more mature forms-probably due to the effect of high-dose intravenous immunoglobulin and/or iron therapy.

Hematogones are B-cell precursors that exhibit a continuous and complete maturation spectrum. In the presented case stage 2 hematogones comprised the majority of the hematogone population, as was reported in most other cases [7]. The most mature stage 3 hematogones resemble mature lymphocytes and frequently remain unrecognized,

2.

3.

4.

5.

6.

7.

McKenna RW, Washington LT, Aquino DB, Picker LJ, Kroft SH. Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry. Blood 2001;98:2498-507.

Rimsza LM, Douglas VK, Tighe P, Saxonhouse MA, Calhoun DA, Christensen RD, Sola MC. Benign B-cell precursors (hematogones) are the predominant lymphoid population in the bone marrow of preterm infants. Biol Neonate 2004;86:247-53.

Babusíková O, Zelezníková T, Kirschnerová G, Kankuri E. Hematogones in leukaemia during and after therapy. Leuk Lymphoma 2008;49:1935-44. van Lochem EG, Wiegers YM, van dem Beemd R, Hahlen K, van Dongen JJM, Hooijkaas H. Regeneration pattern of precursor-B cells in bone marrow of acute lymphoblastic leukaemia patients depends on the type of preceding chemotherapy. Leukemia 2000;14:688-95.

Vargas SO, Hasegawa SL, Dorfman DM. Hematogones as an internal control in flow cytometric analysis of suspected acute lymphoblastic leukaemia. Pediatr Dev Pathol 1999;2:371-6.

Fisgin T, Yarali N, Duru F, Kara A. CMV-induced immune thrombocytopenia and excessive hematogones mimicking an acute B-precursor lymphoblastic leukaemia. Leuk Res 2003;27:193-6.

Sevilla DW, Colovai AI, Emmons FN, Bhagat G, Alobeid B. Hematogones: a review and update. Leuk Lymphoma 2010;51:10-9.

158

Images in Hematology

Multiple myeloma with multilobated plasma cell nuclei Çok çekirdekli plazma hücreli Multipl Miyelom Nergiz Erkut1, Ümit Çobanoğlu2, Mehmet Sönmez1 1Department 2Department

of Haematology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey of Pathology, School of Medicine, Karadeniz Technical University, Trabzon, Turkey

A 61-year-old male patient was admitted to our hospital with backache and fatigue. Physical examination was normal, except for pallor. Laboratory results at initial evaluation were as follows: hemoglobin: 105 g L; hematocrit: 0.31%; white blood cell (WBC) count: 6.3x109 L; platelet count: 200x109 L; blood urea nitrogen (BUN): 22.1 mmol L; creatinine: 477.3 µmol L; calcium: 2.8 mmol L; total protein: 74 g L; albumin: 43 g L; erythrocyte sedimentation rate (ESR): 44 mm h-1. A monoclonal spike was present on protein electrophoresis. Protein studies (by nephelometry) showed a kappa light chain of 5.97 g L (reference range: 1.7-3.7 g L) and low levels of IgG, IgM, and IgA (8.84 g L, 0.30 g L, 0.53 g L, respectively).

Beta 2 microglobulin was 1.2 mg dL (reference range: 0.07-0.19 mg dL). Urine immunoelectrophoresis showed that the patient had a kappa monoclonal light chain. Bone X-rays showed multiple osteolytic lesions. The bone marrow aspirate and biopsy specimen morphology showed infiltration by atypical and multilobated plasma cell nuclei (Figures 1 and 2).

The bone marrow biopsy specimen stained positive with CD138 (Figure 3).

Multiple myeloma with multinucleated plasma cells is a rare morphological variant, which usually presents with light chain expression, and is charac-

Figure 1. Atypical and multinucleated plasma cell infiltration in bone marrow aspirate

Figure 2. Atypical and multinucleated plasma cell infiltration in the bone marrow biopsy specimen

Address for Correspondence: Dr. Mehmet Sönmez, Department of Haematology, School of Medicine, Karadeniz Technical University, 61080 Trabzon, Turkey Phone: +90 462 377 58 48 Fax: +90 462 328 07 04 E-mail: mesonmez@yahoo.com doi:10.5152/tjh.2011.38

Erkut et al.

Multiple myeloma with multinucleated plasma cell

Turk J Hematol 2011; 28: 158-9

159

terized by an aggressive course and resistance to conventional chemotherapy [1,2]. Informed consent was obtained from the patient.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

2.

Figure 3. Kappa light chain-positive bone marrow biopsy specimen

Fonseca R, San Miquel J. Prognostic factors and staging in multiple myeloma. Hematol Oncol Clin North Am. 2007;21:1115-40.

Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia. 2009;23:3-9.

Advisory Board of This Issue (June 2011) Ahmet Türker Çetin, Turkey Ali Turhan, France Ali Ünal, Turkey Bilgehan Yalçın, Turkey Burhan Ferhanoğlu, Turkey Bülent Eser, Turkey Can Boğa, Turkey Cengiz Beyan, Turkey Duran Canatan, Turkey Emel Özyürek, Turkey Ender Akçağlayan Soydan, Turkey Evren Özdemir, Turkey Feride İffet Şahin, Turkey

Ferit Avcu, Turkey Gina Zini, Italy Güray Saydam, Turkey Hakan Özdoğu, Turkey Hilmi Apak, Turkey İsmet Aydoğdu, Turkey Mehmet Ali Erkurt, Turkey Mehmet Ali Özcan, Turkey Mine Hekimgil, Turkey Mualla Çetin, Turkey Musa Karakükçü, Turkey Mutlu Arat, Turkey Nejat Akar, Turkey

Neşe Yaralı, Turkey Nükhet Tüzüner, Turkey Reyhan Diz Küçükkaya, Turkey Tansu Sipahi, Turkey Teoman Soysal, Turkey Tunç Fışgın, Turkey Türkiz Gürsel, Turkey Yahya Büyükaşık, Turkey Yeşim Aydınok, Turkey Yurdanur Kılınç, Turkey Zühre Kaya, Turkey

Announcements 3-7 June 2011 2011 ASCO Annual Meeting Chicago, USA

9-11 September 2011 Turkish School of Hematology 12 - Stem Cell Course Ankara, Turkey

9-12 June 2011 16th Congress of the European Hematology Association London, United Kingdom

19-22 October 2011 37. Turkish National Hematology Congress Ankara, Turkey

24-26 June 2011 Turkish School of Hematology 11 - Practical Hemostasis Thrombosis Laboratory Course 2 Sivas, Turkey

10-13 December 2011 53rd ASH Annual Meeting and Exposition San Diego, USA 23-25 December 2011 Practical Basic Hematology Laboratory Course 1