Genome wide characterization of minimally differentiated acute myeloid leukemia
Gomes e Silva, F.P.
Citation
Gomes e Silva, F. P. (2009, March 3). Genome wide characterization of minimally differentiated acute myeloid leukemia. Retrieved from https://hdl.handle.net/1887/13569
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Genome wide characterization of minimally
differentiated acute myeloid leukemia
Genome wide characterization of minimally differentiated acute myeloid leukemia
PROEFSCHRIFT
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van Rector Magnificus prof. mr. P.F. van der Heijden, volgens besluit van het College voor Promoties
te verdedigen op dinsdag 3 maart 2009 klokke 13:45 uur
door
Fernando Paulo Gomes e Silva geboren te Lisboa, Portugal
in 1978
iv
Promotiecommissie
Promotor: Prof. Dr. L.H.F. Mullenders Co-promotor: Dr. M. Giphart-Gassler Referent: Dr. E.W.A. Marijt
Overige leden: Prof. Dr. H.C. Kluin-Nelemans
(University Medical Center Groningen) Prof. Dr. J. Morreau
The research described in this thesis was performed in the Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands.
This research was financially supported by grants from the Fundação Calouste Gulbenkian and Fundação para a Ciência e a Tecnologia through its correspondent financing program POCI 2010 (SFRH/ BD/5310/2001).
“Aut viam inveniam aut faciam”
Hannibal, 247-183 BC
Aos meus pais À Teresa, Amélia e Sara
vi
© 2009, Fernando Paulo Gomes e Silva, Leiden, The Netherlands
All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means, electronic or mechanical, without prior permission of the author.
ISBN: 978-90-6464-330-9
Printed by Drukkerij Ponsen & Looijen
Cover picture: “Phlebotomy, 1520” (adapted from Seitz, 1520, as illustrated in Hermann Peter, Der Artz und die Heilkunst, Leipzig, 1900; image courtesy of the National Library of Medicine, USA).
vii Contents
Aim and outline of the thesis 1
Chapter 1 3
General introduction
1.1 Leukemia: historical note 5
1.2 Hematopoiesis 5
1.2.1 The hematopoietic system origins 1.2.2 The hematopoietic stem cell
1.2.2.1 Regulation of the hematopoietic stem cell fate
1.3 Leukemia, general mechanisms 9
1.3.1 Molecular mechanisms of leukemogenesis 1.3.2 The primordial leukemic stem cell
1.4 Clonal hematopoietic disorders and classification 12 1.4.1 Acute leukemia classification
1.5 Acute myeloid leukemia 15
1.5.1 Cytogenetic characterization of acute myeloid leukemia 1.5.2 Epidemiology and etiology of acute myeloid leukemia 1.5.3 Frequently mutated genes in acute myeloid leukemia 1.5.3.1 Signal transduction
1.5.3.2 Differentiation and apoptosis 1.5.3.3 Caretaker genes
1.6 Minimally differentiated acute myeloid leukemia: AML-M0 23 Chapter 2 29 Identification of RUNX1 /AML1 as a classical tumor suppressor gene
Chapter 3 45 Genome wide molecular analysis of minimally differentiated acute myeloid
leukemia
Chapter 4 61 Trisomy 13 correlates with RUNX1 mutation and increased FLT3 expression in
AML-M0 patients
Chapter 5 69 ETV6 mutations and loss in minimally differentiated acute myeloid leukemia
Chapter 6 83 Gene expression profiling of minimally differentiated acute myeloid leukemia: M0
is a distinct entity subdivided by RUNX1 mutation status.
Chapter 7 97 Discussion
Chapter 8 103 Samenvatting
Chapter 9 107 Supplements
Curriculum vitae 109
List of publications 111
viii
1 Aim and outline of the thesis
Acute leukemia is a neoplasm characterized by the uncontrolled proliferation of an altered blood cell population. An average of 3.7 in 100,000 people dies from this disease every year worldwide. The knowledge about leukemia has seen a great expansion in recent years, about some subtypes more than others. This knowledge is not only fundamental for diagnosis, prognosis and treatment but also for the development of new, specific, therapies. Treatment outcome and relapse rates vary greatly between different types of leukemia. Some subtypes have remarkably poor prognosis. Minimally differentiated acute myeloid leukemia, the subject of this thesis, is such a case. Minimally differentiated acute myeloid leukemia was known as AML-M0 in the French-American-British classification. Acute myeloid leukemia (AML) is the predominant form of leukemia in adults. AML-M0 accounts for near 5 percent of all AML cases. The knowledge about AML-M0 is lacking in many respects. The aim of this thesis is to characterize AML-M0 at the genomic and gene expression level making use of the most recent knowledge and techniques in the leukemia field.
In the first part of this thesis different aspects of leukemia in general and AML-M0 in particular are introduced to the reader. In chapter 2 we explored microsatellite analysis as a tool to find putative tumor suppressor genes (TSG) in AML-M0. Using this technique we were able to independently pinpoint the chromosomal region harboring RUNX1 as a candidate region. Following this lead, and based on contemporary publications, RUNX1 point mutations and, for the first time, homozygous deletions were detected. It is also shown that the biallelic nature of RUNX1 mutations is in its majority the result of loss of heterozygosity mechanisms leading to uniparental disomy. This approach to find new tumor suppressor genes was extended by using whole genome SNP analysis, and the findings are explored in chapter 3. This chapter aims to characterize AML-M0 at the mutational level using different techniques and a bigger cohort of patients. SNP analysis was complemented with cytogenetic information and led to the discovery of new chromosomal regions of interest. Using current knowledge in molecular alteration in leukemia we screened RUNX1, CEBPA, SPI1, ETV6, FLT3, RAS, JAK2, NPM1 and PTPN11, among other genes, for mutations. Data integration showed correlations between RUNX1 mutation and TdT expression and RUNX1 mutations and trisomy 13. The latter is dealt in chapter 4, where this correlation is associated to over- expression of FLT3 in chromosome 13. In chapter 5 ETV6 alterations are explored in detail.
We report for the first time ETV6 point mutations and insertions in AML-M0, one of which biallelic. Heterozygous loss of ETV6 either by deletion or translocation was also detected in several patients. Our results show that ETV6 is the second most mutated transcription factor in AML-M0, after RUNX1. Gene expression profiling studies are being used for diagnosis, clinical outcome prediction, and discovery of new subtypes of leukemia. In chapter 6, using expression microarrays, we find that AML-M0 is an entity distinct from other AMLs. The results also show that RUNX1 mutation is related to a specific expression profile and we discuss its implications. Furthermore, several deregulated pathways in AML-M0 are reported. In the final chapter the different findings and there implication for future research are discussed.
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