Molecular Analyses of Malignant Pleural Mesothelioma Shir Kiong Lo National Heart and Lung Institute Imperial College Dovehouse Street London SW3 6LY A thesis submitted for MD (Res) Faculty of Medicine, Imperial College London 2016 1 Abstract Malignant pleural mesothelioma (MPM) is an aggressive cancer that is strongly associated with asbestos exposure. Majority of patients with MPM present with advanced disease and the treatment paradigm mainly involves palliative chemotherapy and best supportive care. The current chemotherapy options are limited and ineffective hence there is an urgent need to improve patient outcomes. This requires better understanding of the genetic alterations driving MPM to improve diagnostic, prognostic and therapeutic strategies. This research aims to gain further insights in the pathogenesis of MPM by exploring the tumour transcriptional and mutational profiles. We compared gene expression profiles of 25 MPM tumours and 5 non-malignant pleura. This revealed differentially expressed genes involved in cell migration, invasion, cell cycle and the immune system that contribute to the malignant phenotype of MPM. We then constructed MPM-associated co-expression networks using weighted gene correlation network analysis to identify clusters of highly correlated genes. These identified three distinct molecular subtypes of MPM associated with genes involved in WNT and TGF-ß signalling pathways. Our results also revealed genes involved in cell cycle control especially the mitotic phase correlated significantly with poor prognosis. Through exome analysis of seven paired tumour/blood and 29 tumour samples, we identified frequent mutations in BAP1 and NF2. Additionally, the mutational profile of MPM is enriched with genes encoding FAK, MAPK and WNT signalling pathways. We also found mutations in several novel genes involved in chromatin remodelling other than BAP1 and SETD2 that may contribute to dysregulation of gene expression in MPM. In conclusion, MPM is a complex disease with heterogeneous molecular aberrations. By combining findings from both transcriptome and exome analyses, we identified alterations in genes involved in common signalling pathways such as FAK, WNT and MAPK. These may have important roles in driving MPM carcinogenesis with therapeutic implications and require further explorations. 2 Abbreviations Abbreviations Full name BAM Binary Alignment Map bp base pairs BRC Biomedical Research Centre BRU Biomedical Research Units CALBG Cancer and Leukaemia Group B CDK Cyclin Dependent Kinases cDNA complementary Deoxyribonucleic Acid CEA Carcinoembryonic Antigen CL4 Claudin-4 COSMIC Catalogue of Somatic Mutations in Cancer CRL4 Cullin4A-RING E3 ubiquitin Ligase cRNA complementary Ribonucleic Acid CSC Cancer Stem Cells CT Computed Tomography CTLA-4 Cytotoxic T Lymphocyte Antigen-4 DM Data Mining DNA Deoxyribonucleic Acid DNMTi DNA Methyltransferase inhibitor dNTP deoxyribonucleotide triphosphate ECM Extracellular Matrix EGFR Epidermal Growth Factor Receptor EMA Epithelial Membrane Antigen EMT Epithelial-Mesenchymal Transition EORTC European Organisation for Research and Treatment of Cancer EPP Extra-Pleural Pneumonectomy ERK Extracellular-signal-Regulated Kinases ExAC Exome Aggregation Consortium FAK Focal Adhesion Kinase FC Fold Change FDR False Discovery Rate FFPE Formalin- Fixed, Paraffin-Embedded gm Grams GO Gene Ontology GS Gene Significance GWAS Genome-wide association study GWAS Genome-Wide Association Study H&E Haematoxylin and Eosin HA Hyaluronate HCA Hierarchical Clustering Algorithm 3 HDACi Histone Deacetylase Inhibitors HGF Hepatocyte Growth Factor HMGB1 High Molecular Group Binding Protein 1 IASLC International Association for the Study of Lung Cancer IGF1 Insulin Growth Factor 1 IHC Immunohistochemistry IMIG International Mesothelioma Interest Group indels insertions/deletions IVT in vitro Transcription KEGG Kyoto Encyclopaedia of Genes and Genomes LCM Laser-Capture Micro-dissection LRP Lipoprotein-Related Protein MAPK Mitogen-Activated Protein Kinases MEMO Molecular Analyses of Malignant Pleural Mesothelioma MEs Module Eigengenes MET MET Proto-oncogene MHC Major Histocompatibility Complex miR micro RNA ml Millilitre MM Module Membership MPM Malignant Pleural Mesothelioma MSAC Mitotic Spindle and Assembly Checkpoint mTOR Mammalian Target of Rapamycin NER Nucleotide Excision Repair NF-κB Nuclear Factor Kappa-light-chain-enhancer of activated B cells ng Nanogram NGS Next Generation Sequencing NIHR National Institute for Health Research NK Natural Killer nt Nucleotides OCT Optimal Cutting Temperature OS Overall Survival PCA Principal Component Analysis PCR Polymerase Chain Reaction PD-1 Programmed Death-1 PDP Podoplanin PEN Polyethylene Napthalate PFS Progression-Free Survival PI3K Phosphoinositide 3-Kinase PM Perfect Match PVCA Principal Variance Component Analysis RBH Royal Brompton and Harefield NHS Foundation Trust Hospital RIN RNA Integrity Number RMA Random Multiple Access RMH Royal Marsden NHS Foundation Trust Hospital 4 RNA Ribonucleic Acid RTK Receptor Tyrosine Kinase RTS Review on Transcriptome Studies SGH St George’s Hospital SHH Sonic Hedgehog siRNA small interfering RNA SMRP Soluble Mesothelin-Related Peptides SNP single Nucleotide Polymorphisms SNV Single Nucleotide Variants SV40 Simian Virus 40 TGF-ß1 Transforming Growth Factor Beta 1 TNF Tumour Necrosis Factor TOM Topological Overlap Matrix TSG Tumour Suppressor Gene TUCP Transcripts of Uncertain Coding Potential UK United Kingdom USA United States of America UV Ultraviolet VCF Varian Call Format WES Whole Exome Sequencing WGCNA Weighted Gene Co-expression Network Analysis WHO World Health Organisation WT1 Wilms’ Tumor-1 protein µg Microgram µl Microliter µm Micrometre 5 Acknowledgements Firstly, I would like to express my sincere gratitude to both my supervisors, Professor Cookson and Dr Popat, for giving me this invaluable opportunity to conduct this research. I am extremely grateful for their guidance, motivation and patience. My sincere thanks also go to Professor Moffatt and Professor Bowcock for their insightful advice and encouragement. I am also very grateful to the immense input from Professor Lathrop, Professor Nicholson, Dr Montero and their team. I would also like to thank the Brunei Government and Lung Unit, Royal Marsden Hospital that funded me to study at the National Heart and Lung Institute, Imperial College. I would also like to acknowledge all the members of the Molecular Genetics & Genomics and Cancer Genomics group in particularly Liz, Hima, Saffron, Youming, Kenny and Spyros for their help and advice throughout my research. Lastly, I would like to thank my family and friends who are always there for me. 6 Declaration of Originality I herewith certify that this dissertation is my original work and that all materials included which are not my own work have been acknowledged. 7 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 8 Table of Contents Abstract .................................................................................................................................................................. 2 Abbreviations ........................................................................................................................................................ 3 Acknowledgements................................................................................................................................................ 6 Declaration of Originality..................................................................................................................................... 7 Copyright Declaration .......................................................................................................................................... 8 Chapter 1 - Introduction .................................................................................................................................... 15 1.1 Background.......................................................................................................................................... 15 1.2 Mesothelial cells and the pleura .......................................................................................................... 17 1.3 Epidemiology ...................................................................................................................................... 19 1.4 Risk factors .......................................................................................................................................... 21 1.4.1 Asbestos ...................................................................................................................................... 21 1.4.2 Other risk factors including genetic susceptibility ...................................................................... 24 1.5 Diagnostic biomarkers ......................................................................................................................... 28 1.6 Molecular changes in MPM ...............................................................................................................