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When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE, HEALTH & LIFE SCIENCES School of Medicine Abnormalities affecting tyrosine kinase signalling in atypical myeloproliferative disorders by Claire Hidalgo-Curtis Thesis for the degree of Doctor of Philosophy May 2009 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE, HEALTH & LIFE SCIENCES SCHOOL OF MEDICINE Doctor of Philosophy ABNORMALITIES AFFECTING TYROSINE KINASE SIGNALLING IN ATYPICAL MYELOPROLIFERATIVE DISORDERS By Claire Hidalgo-Curtis The myeloproliferative disorders (MPDs) are a group of haematopoietic stem cell diseases, characterised by proliferation of one or more cells of the myeloid lineage. Several lines of evidence have highlighted the importance of aberrant tyrosine kinase signalling in the pathogenesis of these disorders. Cloning of rare chromosomal translocations and point mutation analysis in the MPDs has identified diverse deregulated tyrosine kinase genes, notably PDGFRA, PDGFRB, FGFR1 and JAK2. However the majority of atypical MPDs still remain to be characterised and identification of patients harbouring fusions, particularly those involving the PDGF receptors is of increasing importance, as they are likely to be responsive to targeted therapy with imatinib. I am investigating MPD patients for abnormalities affecting tyrosine kinase signalling, and have used three approaches, translocation cloning, expression analysis and SNP array analysis to detect regions of loss of heterozygosity (LOH). Thus far, by translocation cloning I have identified a previously undescribed partner gene fused to PDGFRB and two new PDGFRA fusion genes. I have also designed two reverse transcriptase PCR (RT-PCR) assays and a cDNA MLPA assay to detect over-expression of specific tyrosine kinases screening approximately 200 patients. Each assay identified all patients previously diagnosed with known fusions. Additionally, two patients identified with overexpression of PDGFRB have been found to have cryptic ETV6-PDGFRB fusions and overexpression of PDGFRA in one patient lead to the discovery of a previously undescribed fusion involving a novel partner gene (KIF5B). Recent evidence has indicated that acquired isodisomy is a novel mechanism by which mutations in cancer may be reduced to homozygosity. Typically, acquired isodisomy is associated with oncogenic changes rather than tumour suppressor genes, eg. the activating JAK2 V617F mutation and 9p aUPD. I have undertaken a screen using Affymetrix 50K SNP arrays for regions of acquired isodisomy as a means to identify genomic regions that may harbour novel oncogenes in different subgroups of MPD patients. Large tracts of homozygosity (defined as >20Mb running to a telomere), strongly suggesting acquired isodisomy, were seen in 40% aMPD patients. The homozygous tracts encompassed diverse genomic regions in aMPD, but two common regions (3 cases for each region) were identified at 7q and 11q. Mutations in the CBL ubiquitin ligase gene were discovered in all three aCML patients with 11q aUPD as well as in an additional 23 MPD patients following further screening. CONTENTS Contents………………………………………………….…………………….….......i List of Figures…………………………………………………………………...…viii List of Tables……………………………………………………………….….......xiii Declaration of Authorship……………………………………………….………...xv Acknowledgments……………………………………………………….……......xvii List of Abbreviations…………………………………………………….………xviii 1 INTRODUCTION 1 1.1 Normal Haematopoiesis 1 1.2 Leukaemia 3 1.2.1 Clonality 4 1.2.2 Classification 4 1.3 The chronic myeloproliferative disorders (CMPD and the myelodysplastic/myeloprolfierative disorders (MDS/MPD) 5 1.4 The paradigm for deregulated tyrosine kinases – Chronic myelogenous leukaemia (CML) 7 1.4.1 Molecular pathogenesis of CML 7 1.4.2 Therapy in CML and the development of Imatinib 9 1.5 The tyrosine kinases 11 1.5.1 Non-receptor tyrosine kinases (NTRKs) 12 1.5.2 Receptor tyrosine kinases (RTKs) 13 1.6 Molecular pathogenesis and deregulated tyrosine kinases 15 1.7 PDGFRA and PDGFRB rearrangements in MPDs 17 1.7.1 Fusions involving PDGFRA 17 1.7.2 Chronic eosinophilic leukaemia (CEL) 18 1.7.3 Fusions involving PDGFRB 19 1.7.4 Chronic myelomonocytic leukaemia (CMML) 21 1.7.5 JAK2 and the myeloproliferative disorders 22 i 1.8 Summary 26 1.9 Statement of aims 26 2 METHODS 28 2.1 Sample processing 28 2.1.1 Preparation of blood and bone marrow samples 28 2.1.2 Enriching cultures for T-cells 29 2.1.3 RNA extraction 30 2.1.4 cDNA synthesis 31 2.1.5 DNA extraction 31 2.1.5.1 DNA extraction from fixed cell suspension 31 2.1.5.2 Extraction from cell pellets 32 2.1.5.3 DNA extraction from Guanidinium thiocyanate 33 2.1.5.4 DNA extraction from mouthbrush samples 34 2.1.5.5 DNA extraction from haematological stained slides 34 2.1.5.6 GenomiPhi V2 DNA amplification kit 35 2.2 Amplification and analysis 35 2.2.1 Reverse transcriptase PCR (RT-PCR) 35 2.2.2 Microsatellite PCR 36 2.2.3 Control gene PCR 36 2.2.4 Powerplex® 16 system 37 2.2.5 Long-range PCR 38 2.2.6 Bubble-PCR 38 2.2.7 Gel Electrophoresis 39 2.2.8 Denaturing high-performance liquid chromatography (dHPLC) 40 2.2.9 Multiplex ligation-dependent probe amplification (MLPA) 41 2.2.10 Exo-SAP and sequencing 42 2.2.10.1 Sequencing of PCR products 43 2.2.10.2 Sequencing following cloning 43 2.2.11 Cloning PCR products 44 2.2.11.1 Ligations 44 ii 2.2.11.2 Transformations 44 2.2.11.3 Qiagen™ Miniprep kit 45 2.2.11.4 Digests and PCR 46 2.2.12 5’Rapid amplification of cDNA ends 46 2.2.13 Pyrosequencing 48 2.3 Fluorescence in situ hybridisation (FISH) 49 2.3.1 Maxiprep DNA isolation from BAC clones 49 2.3.2 Labelling 50 2.3.3 Slide making 51 2.3.4 Probe preparation 51 2.3.5 Slide preparation 51 2.3.6 Post hybridisation and visualisation 52 2.4 Tissue Culture 53 2.4.1 Imatinib sensivity assay 53 2.4.1.1 Thawing of primary haematopoietic cells 53 2.4.1.2 Colony assay 53 2.4.2 Midi prep & nucleotide removal kit 54 2.4.2.1 Midi-prep 54 2.4.2.2 QIAquick nucleotide removal kit 56 2.4.2.3 Transfection by electroporation 56 2.4.2.4 Electroporation 57 2.4.2.5 Antibiotic selection 57 2.4.2.6 IL-3 independent growth 58 2.5 Solutions 58 2.6 List of probes used for FISH 61 3 IDENTIFICATION OF NOVEL FUSION GENES ASSOCIATED WITH VISIBLE CHROMOSOME ABNORMALITIES 62 3.1 Introduction 62 3.2 A novel PDGFRB fusion gene 62 3.2.1 Patient details 62 iii 3.2.2 Two-colour fluorescence in situ hybridisation (FISH) 63 3.2.3 5’Rapid amplification of cDNA ends (5’RACE) 64 3.2.4 Confirmation of the GOLGA4-PDGFRB fusion and cloning of the genomic breakpoints 69 3.2.5 Consequence of the t(3;5) 75 3.2.6 Identification of a second MPD patient with a GOLGA4-PDGFRB fusion 76 3.2.7 Two-colour fluorescence in situ hybridisation (FISH) 78 3.2.8 Imatinib sensitivity assay 79 3.2.9 Discussion 81 3.3 Identification of two novel PDGFRA fusion genes 83 3.3.1 Patient details 83 3.3.2 Bubble PCR 84 3.3.3 Confirming the STRN-PDGFRA fusion 86 3.3.4 Consequence of the STRN-PDGFRA fusion 88 3.3.5 Confirming the ETV6-PDGFRA fusion 89 3.3.6 Two-colour fluorescence in situ hybridisation 91 3.3.7 Consequence of the ETV6-PDGFRA fusion 92 3.3.8 Detection of MRD and the response to imatinib in cases 1 &2 93 3.3.9 Discussion: PDGFRA fusions 93 3.4 A novel FGFR1 fusion gene 96 3.4.1 Patient details 97 3.4.2 Fluorescence in situ hybridisation 97 3.4.3 5’Rapid amplification of cDNA ends (5’RACE) PCR 98 3.4.4 Confirming the CPSF6-FGFR1 fusion 99 3.4.5 Consequence of the t(8;12) 99 3.4.6 Discussion 100 3.5 Discussion 102 4 IDENTIFICATION OF DEREGULATED EXPRESSION OF TYROSINE KINASE GENES 107 iv 4.1 Introduction 107 4.1.1 PDGFRB rearrangements 108 4.1.2 PDGFRA rearrangements 109 4.2 Methods 110 4.2.1 Design of the PDGFRB RT-PCR assay 110 4.2.2 Design of the PDGFRA RT-PCR assay 113 4.3 Results 116 4.3.1 Identification of patients with possible PDGFRB rearrangements 116 4.3.2 Identification of patients with possible PDGFRA rearrangements 120 4.3.3 Over-expression assays – discussion 123 4.4 Multiplex ligation-dependent probe amplification (MLPA) 127 4.4.1 Methods – probe design 128 4.4.2 Methods – Optimisation of the assay 130 4.4.3 Validation of the cDNA MPLA assay 137 4.4.4 Results 138 4.4.4.1 A cryptic ETV6-PDGFRB fusion gene 139 4.4.4.2 Patient details 140 4.4.4.3 cDNA MLPA analysis of patient E1026 141 4.4.4.4 5’RACE PCR to identify the potential PDGFRB fusion in patient E1026 142 4.4.4.5 Confirmation of the ETV6-PDGFRB fusion 144 4.4.4.6 Further ETV6-PDGFRB analysis 146 4.4.4.7 ETV6-PDGFRB – discussion 147 4.4.5 MLPA discussion 149 4.5 Discussion 150 5 SCREENING FOR NEW ONCOGENES MARKED BY ACQUIRED UNIPARENTAL DISOMY 156 5.1 Introduction 156 5.1.1 Loss of heterozygosity (LOH) & acquired uniparental disomy (aUPD) 156 v 5.1.2 aUPD