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The Use of Gene Expression Profiling to Identify Novel Minimal Residual Disease Markers (MRD) in Acute Myeloid Leukaemia (AML) Eleanor Woodward PhD 2010 Cardiff University UMI Number: U585569 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U585569 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed .... Date STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of PhD Signed . ,<Eb— Date a .. STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed . Date ....O73^\0.3r\\o.... STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed . Date..... STATEMENT 4 - BAR ON ACCESS APPROVED I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access approved bv the Graduate Development Committee. Signed Date Acknowledgements I would like to thank Dr Chris Pepper and Dr Richard Darley, without their help and supervision this project would not have been possible. I would also like to thank Professor Alan Burnett for allowing me to perform this research within his department. I would also like to thank those within in the Department of Haematology, in particular to Amanda Gilkes, Dr Elisabeth Walsby, Michelle Lazenby and Val Walsh, and to Dr Peter Giles from the Department of Pathology for providing a considerable amount of support and guidance contributing towards my study. Finally I would like to thank my family and friends for all the support they provided throughout my research, in particular to Tim for explaining the difficult bits, Joseph for reminding me all that really matters is physics and to Doug for providing a roof over my head. Summary Acute myeloid leukaemia (AML) is a heterogeneous disorder characterised by the accumulation of immature haematopoietic cells blocked at various stages of differentiation. Despite improved survival rates over the past decade, relapse occurs in approximately 70% patients undergoing chemotherapy. A potential reason for this is that current clinical protocols do not take account of the level of residual disease present at remission. Therefore, one strategy to reduce relapse rates is to monitor minimal residual disease and continue to treat until the patient is minimal residual disease negative. Current minimal residual disease markers are available for patients with characterised fusion genes but approximately 50% of patients have no detectable chromosomal aberration and therefore are without markers. Gene expression profiling is a powerful tool for disease classification, prognosis and therapeutic predictions. This study aimed to investigate the use gene expression profiling to identify novel minimal residual disease markers for specific AML sub-groups. Patient diagnostic samples were profiled to identify genes specific to AML patients with a favourable translocation in order to establish the “proof-of-principle”. Several genes identified were followed in patient diagnostic and follow- up samples and compared to the markers currently used. Continuing with normal karyotype AML, genes were identified as specific to this sub-group. Several homeobox (HOX) genes and the Wilms’ tumour (WT1) gene were identified and their MRD levels followed in diagnostic and follow-up samples. Only WT1 identified as specific to normal karyotype AML met the necessary criteria to be an MRD marker. Although the majority of genes selected from the GEP in this study proved unsuitable as markers, the identification and validation of a marker already used for MRD monitoring, WT1, demonstrates the ability of gene expression profiling to identify potential minimal residual disease markers in normal karyotype AML. Abbreviations ABL Abelson tyrosine kinase ALCL Anaplastic large cell lymphoma ALL Acute lymphoid leukaemia AML Acute myeloid leukaemia AML1 Acute myeloid leukaemia 1 AML1-ETO Acute myeloid leukaemia 1- Eight twenty-one Oncoproteins AML-CFU Proliferative leukaemic blasts ANG-1 Angiopoietin 1 AP3S1 Adaptor-related protein complex 3, sigma 1 subunit APL Acute promyelocytic leukaemia ARC Activity-regulated cytoskeleton-associated protein ASNase L-asparaginase ASNS Asparagine synthetase ASS1 Argininosuccinate ATRA All-frans retinoic acid ANOVA Analysis of variance BAALC Brain and acute leukemia, cytoplasmic BIN Biological interaction networks BM Bone marrow BMEC Bone marrow endothelial cells BMP Bone morphogenic protein BrdU Bromodeoxyuridine BSA Bovine serum albumin CALD1 Caldesmon 1 CBF Core binding factor CBFa Core binding factor alpha CBFp Core binding factor beta CBFP-MYH11 Core binding factor beta- smooth muscle myosin heavy chain CDK Cyclin-dependent kinase CDKI Cyclin-dependent kinase inhibitor cDNA Complementary DNA CDX2 Caudal type homeobox 2 C/EBP CCAAT/enhancer binding protein C/EBPa CCAAT/enhancer binding protein (C/EBP), alpha CFU Colony-forming unit CFU-GEMM Colony-forming units - granulocyte-eyrthroid- macrophage-megakarocyte CG Control gene CLIP3 CAP-GLY domain containing linker protein 3 CLL Chronic lymphoid leukaemia CLP Common lymphoid progenitors CML Chronic myeloid leukaemia CMP Common myeloid progenitors COL4A5 Collagen, type IV, alpha 5 Cp/Ct Crossing point CP Chronic phase CR Complete remission CSF Colony stimulating factor dATP Deoxyadenosine triphosphate DAVID Database for annotation, visualisation and integrated discovery dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate DMSO Di-methyl sulphoxide DNA Deoxyribonucleic acid DNase Deoxyribonuclease dNTP Deoxynucleotide triphosphate dsDNA Double stranded DNA DTT Dithiothreitol dTTP Deoxythymidine triphosphate DX Diagnostic EAC Europe Against Cancer EB Empirical Bayes EDTA Ethylenediaminetetracetic acid ENU A/-ethyl-A/-nitroso-urea EPO Erythropoietin ET Essential thrombocythemia ETO Eight twenty-one oncoproteins EVI1 Ecotropic viral integration site 1 FAB ' French-American-British FBS Foetal bovine serum FCS Foetal calf serum FDR False discovery rate F-MuLV Friend murine leukemia virus FL Fms-like tyrosine kinase 3 ligand FGF-4 Fibroblast growth factor 4 FISH Fluorescence in situ hybridisation FLT3 Fms-like tyrosine kinase 3 FUP Follow-up G6PD Glucose-6-phosphate dehydrogenase GCT Germ cell tumours GEP Gene expression profiling GO Gemtuzumab ozogamicin G-CSF Granulocyte - colony stimulating factor GM-CSF Granulocyte/macrophage- colony stimulating factor GMP Granulocyte/monocyte progenitor GRIN1 Glutamate receptor, ionotropic, N-methyl D- aspartate 1 HAT Histone acetyltransferase HCL Hairy cell leukaemia HD AC Histone deacetylase HOX Homeobox HGF Hepatocyte growth factor HPC Haematopoietic progenitor cell HSC Haematopoietic stem cell HSCT Haematopoietic stem cell transplant HSPG2 Heparan sulphate proteoglycan 2 Ig Immunoglobulin IGF Insulin-like growth factor IGFBP2 Insulin-like growth factor binding protein 2 IL-3 Interleukin 3 IL5RA lnterleukin-5 receptor, alpha IMDM Iscove's Modified Dulbecco's Medium INK4 Inhibitors of CDK4 ITD Internal tandem duplication IVT In vitro transcription JAK2 Janus Kinase 2 LAIP Leukaemia-associated aberrant immunophenotype LIC Leukaemia-initiating cells LSC Leukaemic stem cell LT-HSC Long-term haematopoietic stem cell mAbs Monoclonal antibodies MAD Median absolute deviation MAP Mitogen-activated protein M-CSF Macrophage colony-stimulating factor M-CSFR Macrophage colony-stimulating factor receptor MDCK Madin-Darby canine kidney MDS Myelodysplastic syndrome MEM Minimum essential media MEP Megakaryocyte/erythrocyte progenitor MEIS1 Myeloid ecotropic viral integration site 1 homolog MgCb Magnesium chloride MFC Multiparameter flow cytometry MLL Mixed lineage leukaemia MLL-ENL Mixed-lineage leukaemia - eleven nineteen leukaemia MOZ-TIF2 Monocytic leukaemia zinc finger - TGF-beta induced factor-2 MNC Mononuclear cell MNI Meningioma 1 MPD Myeloproliferative disorder MPN Myeloproliferative neoplasms MRC Medical Research Council MRD Minimal residual disease MRDv Minimal residual disease value MREC Multicentre research ethics committee mRNA Messenger RNA MSV-LTR Moloney sarcoma virus terminal repeat MuLV Murine leukaemia virus MYH11 Smooth muscle myosin heavy chain NAB2 NGFI-A binding protein 2 NCoR Nuclear receptor corepressor NK Natural killer NMTS Nuclear matrix attachment signal NOD Non-obese diabetic NPM-ALK Nucleophosmin-anaplastic lymphoma kinase 1 NPM-MLF1 Nucleophosmin-myelodysplasia/myeloid leukaemia factor 1 NPM-RARa Nucleophosmin-retinoic acid receptor a NPM1 Nucleophosmin 1 N-ras Neuroblastoma RAS viral (v-ras) oncogene homologue NT5E 5'-nucleotidase, ecto (CD73) NuMA Nuclear mitotic apparatus protein NUP98
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