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Studies on Cebpa Mutations in Acute Myeloid Leukaemia

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Studies on Cebpa Mutations in Acute Myeloid Leukaemia STUDIES ON CEBPA MUTATIONS IN ACUTE MYELOID LEUKAEMIA Claire Louise Green UCL A thesis submitted for the degree of Doctor of Philosophy 2012 1 DECLARATION I, Claire Louise Green, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in this thesis. Signed: Date: 2 ABSTRACT Acute myeloid leukaemia (AML) is a highly heterogeneous disease with regard to clinical outcome, and molecular markers with prognostic impact can be used to stratify patients for risk-adapted therapy. CEBPA mutations have been associated with a favourable prognosis, however several questions remained, in particular whether one (CEBPA-single) or two (CEBPA-double) mutations were necessary for this benefit, and their interaction with other molecular markers. A method of detecting CEBPA mutations in patient samples using denaturing HPLC was developed and the CEBPA status of 1427 young adult AML patients (median age 43 years, range 15-68 years) determined. Overall, 107 (7%) were CEBPA- mutant: 48 (45%) CEBPA-single and 59 (55%) CEBPA-double. The majority of CEBPA- double patients (83%) had an out-of-frame insertion/deletion in the N-terminus and a mutation in the C-terminal DNA-binding/leucine zipper domains (DBD/LZD) that were on different alleles as determined by cloning. By contrast, mutations in CEBPA-single cases were distributed across the gene. CEBPA-double patients were less likely to have a FLT3/ITD (P=.04) and highly unlikely to have an NPM1 mutation (P<.0001) compared to CEBPA-WT/CEBPA-single cases. Eight year overall survival (OS) was higher in CEBPA- double patients compared to CEBPA-WT and CEBPA-single cases (54%, 34%, 31%, respectively, P=.004). In multivariate analyses, CEBPA-double, but not CEBPA-single, was an independent favourable factor for OS (P=.004) and relapse (P=.02). However, this benefit was completely lost in the presence of a FLT3/ITD. The mutant level of 101 mutations was determined by fragment analysis and the majority were of a level consistent with a heterozygous mutation present in most cells. The impact of ten atypical CEBPA mutations on C/EBPα transactivation activity was explored by a luciferase reporter assay. Only mutations affecting the DBD or LZD functional domains had an impact on transactivation activity. This work provides insight into the biology of CEBPA mutations and their use as clinical markers. 3 ACKNOWLEDGMENTS I would like to thank my supervisors, Professors Rosemary Gale and David Linch, who have been so supportive and who gave me the opportunity to work towards a doctorate alongside my technician role in the department. I have learnt so much in the six years that I have spent working with them, and am grateful for the time they have so generously spent providing advice and helpful comments throughout the preparation of this thesis. I would also like to thank Kenneth Koo, who worked with me screening patients for CEBPA mutations. Without his hard work it would have taken even longer than the eighteen months it took to complete all the mutation analysis. These studies would not have been possible if not for all the patients consenting to enter the trials and allowing their samples to be used for research, the clinicians who entered and managed the patients, those who have managed the trials over the years and those who have diligently collected and stored the samples. Another large thank you is owed to my parents. They have provided love and support, giving financial aid and proof reading chapters, as well as kindly refraining from asking when I would be finished writing! Finally, I would like to thank my lovely fiancé, Rich. He has helped me through this whole process, put up with me during the more stressful moments and was patient throughout all the evenings and weekends spent writing. 4 TABLE OF CONTENTS TITLE PAGE ............................................................................................................................... 1 DECLARATION.......................................................................................................................... 2 ABSTRACT .................................................................................................................................. 3 ACKNOWLEDGMENTS ........................................................................................................... 4 TABLE OF CONTENTS ............................................................................................................ 5 TABLE OF FIGURES ............................................................................................................... 10 LIST OF TABLES ..................................................................................................................... 12 COMMONLY USED ABBREVIATIONS .............................................................................. 14 CHAPTER 1: INTRODUCTION ........................................................................................ 16 1.1 Haemopoiesis ................................................................................................................... 16 1.2 AML ................................................................................................................................. 18 1.2.1 Leukaemic stem cells and leukaemogenesis ............................................................. 18 1.2.2 Presentation and aetiology ........................................................................................ 19 1.3 Classification of AML ..................................................................................................... 20 1.4 Factors associated with patient outcome in AML ........................................................ 23 1.4.1 Clinical features ........................................................................................................ 23 1.4.2 Cytogenetics ............................................................................................................. 24 1.4.3 Gene mutations ......................................................................................................... 27 1.4.3.1 FLT3 ................................................................................................................. 28 1.4.3.2 NPM1 ................................................................................................................ 29 1.4.3.3 Other recurrently mutated genes in AML ......................................................... 30 1.4.4 Other molecular markers .......................................................................................... 35 1.5 Treatment of AML .......................................................................................................... 36 1.5.1 Chemotherapy ........................................................................................................... 36 1.5.2 Stem cell transplantation ........................................................................................... 36 1.5.3 Targeted therapies ..................................................................................................... 38 1.5.4 Treatment of acute promyelocytic leukaemia ........................................................... 38 1.6 Combining prognostic factors and risk-adapted therapy ........................................... 39 1.7 Aims of this thesis ........................................................................................................... 40 CHAPTER 2: MATERIALS AND METHODS .............................................................. 41 2.1 Molecular Biology ........................................................................................................... 41 2.1.1 Reagents .................................................................................................................... 41 5 2.1.2 Polymerase chain reaction (PCR) ............................................................................. 42 2.1.3 Agarose gel electrophoresis ...................................................................................... 43 2.1.4 dHPLC analysis on the WAVE platform .................................................................. 44 2.1.5 Direct nucleotide sequencing .................................................................................... 46 2.1.6 Restriction enzyme digests ....................................................................................... 47 2.1.7 LB broth and agar plates ........................................................................................... 47 2.1.8 TOPO TA cloning of PCR products ......................................................................... 47 2.1.9 Transformation of One Shot Max Efficiency DH5α-T1 E. coli competent cells ..... 47 2.1.10 Identification and growth of transformed bacterial clones ..................................... 48 2.2 Cell Culture ..................................................................................................................... 49 2.2.1 Cell line ..................................................................................................................... 49 2.2.2 Cell culture general reagents..................................................................................... 49 2.2.3 Culture of 293T cells ................................................................................................ 49 2.2.4 Transient transfection of 293T cells ........................................................................
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