Genomic and Epigenomic Analysis of HPV Positive and HPV Negative Head and Neck Squamous Cell Cancer
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Genomic and Epigenomic Analysis of HPV positive and HPV negative Head and Neck Squamous Cell Cancer Matthias Alexander Lechner, MD MRCS DOHNS UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT A thesis submitted to University College London for the degree of Doctor of Philosophy 2012 1 Declaration The dissertation is the result of my own work and contains nothing which is the outcome of work done in collaboration, except where specifically indicated in the text. I also declare that this dissertation is not substantially the same as any that I submitted for a degree or diploma or other qualification at any other University, and that no part has already been, or is concurrently being, submitted for any degree, diploma, or other qualification. This dissertation comprises a total of 136 pages, excluding references and appendices. The dissertation does not exceed the word limit of 100,000 words, excluding tables, figures, references, and appendices. Matthias Alexander Lechner 2 Acknowledgements I would first like to thank my supervisors Prof. Stephan Beck and Prof. Chris Boshoff for their invaluable guidance, great enthusiasm, support, time and continuous feedback during my research work at UCL, writing this thesis and publishing the papers contained within. I would like to thank members of the Medical Genomics Group (in particular Dr. Lee Butcher, Dr. Andrew Feber, Dr. Chrissie Thirlwell, Dr. Helena Caren and Paul Guilhamon), Viral Oncology Group (in particular Dr. Tim Fenton), and Experimental Cancer Medicine Centre (ECMC) for teaching me various lab techniques which I used in my work. I would like to thank UCL Genomics and UCL Advanced Diagnostics for their technical support. My grateful thanks are due to Dr. Andrew Teschendorff and Dr. Gareth Wilson for their guidance, help and advice on the biostatistical planning and analysis of the methylation data. I would also like to thank James West for his advice on R/Bioconductor related issues and Dr. Stephen Henderson for his guidance on the multidimensional data analysis. I would also like to thank my collaborators at Foundation Medicine, in particular Dr. Garrett Frampton, Dr. Gary Palmer, Dr. Maureen Cronin, Dr. Doron Lipson, Dr. Vincent Miller, Dr. Roman Yelensky, and Dr. Philip Stephens. I would like to thank Dr. Juan Martin-Serrano (Department of Infectious Diseases, King’s College London School of Medicine, London), Dr. Susanne Gollin and Dr. Theresa Whiteside (University of Pittsburgh Cancer Institute, US), Dr. Hans Joenje (VU Medical Centre, Netherlands) and Dr. Thomas Carey (University of Michigan, US) for the provision of plasmids and HNSCC cell lines. I appreciate the invaluable help of Dr. Amrita Jay and the team at the Department of Histopathology at UCLH who helped me to select the FFPE tumour samples used for the methylation and genomic analysis. I owe a vote of thanks to Dr. Nischalan Pillay, who interpreted some of the immunohistochemistry results together with Dr. Amrita Jay. I also owe a great vote of thanks to the Head and Neck Cancer MDT coordinator Ciaran Mariner, who greatly facilitated the gathering of the relevant clinical data. Moreover, I would like to thank the team of head and neck surgeons at UCLH who 3 continuously supported the sample collection and for their enthusiasm for this project and lastly all the patients at UCLH who participated in this study. Furthermore, I would like to thank my clinical supervisor Mr. Francis Vaz (University College London Hospital) for his invaluable clinical teaching and guidance during the years of my PhD and for having acted as a role model of a very successful ENT surgeon. He has greatly supported me to stay involved in clinical work at UCLH along with his colleagues Mr. Paul O’Flynn Mr. Nicholas Kalavrezos, Mr. Colin Liew, Mr. Colin Hopper at UCLH together with Prof. Martin Birchall at the Royal National Throat, Nose and Ear Hospital, who I would like to thank for their teaching, their hints when approaching a clinical problem and their support during this busy time. I owe a great vote of thanks to Dr. Martin Forster (UCL/UCLH) and once again to my supervisors, who supported me to get involved in graduate student teaching at UCL, as a lecturer and tutor on the MSc Cancer programme (on the modules ‘Bed-to-Bedside’ and ‘Cancer Epigenetics’). I also owe a great vote of thanks to two of my UCL course tutors, Angela Cooper and Jackie Watson, and to Dr. Julie Olszewski (graduate tutor at the UCL Cancer Institute). I would like to very much thank the funding bodies which made it possible for me to conduct the presented work, namely the Wellcome Trust for the award of a Clinical Research Fellowship (WT093855MA), the UCLH/UCL Comprehensive Biomedical Research Centre and the Austrian Science Fund for their invaluable initial support by awarding me an Erwin-Schroedinger Fellowship (J2856). Finally, I owe a huge debt of gratitude to my family who have supported me throughout my studies and career to date. 4 I dedicate my work to my late grandfather August Steinleitner. The most beautiful thing we can experience is the mysterious. It is the source of all true art and all science. He to whom this emotion is a stranger, who can no longer pause to wonder and stand rapt in awe, is as good as dead: his eyes are closed. Albert Einstein 5 Contents Title page……………………………………………………………….……….…. 1 Declaration…………………………………………………………………………. 2 Acknowledgements… ……………………………………………………………. 3 Contents……………………………………………………………………...…..... 6 Abstract………………………………………………………….…………….….. 10 Publications…………………………………………………….………………… 12 Facilities……………………………………………………….…………….……. 13 Keywords…………………………………………………………………………. 13 Abbreviations…………………………………………………………………….. 14 List of Figures…………………………………………………………………..... 16 List of Tables ….………………………………………………………………..... 19 1. Chapter 1: General Introduction ........................................ 21 1.1 The role of HPV in Head and Neck Cancer. ................................ 21 1.2 HPV-driven carcinogenesis – Current models in head and neck cancer. ........................................................................................... 25 1.3 Genetics and Epigenetics of Head and Neck Cancer. ............... 29 1.3.1 The Cancer Epigenome of Head and Neck Cancer ................. 30 1.3.2 The Cancer Genome of HNSCC – Current State of Affairs ...... 38 1.3.3 Gene expression changes between HPV+ and HPV- head and neck cancer .............................................................................. 39 1.4 The Epigenome of HPV in HNSCC............................................... 40 1.5 Aims of my thesis and hypotheses tested in this work. ............ 41 2. Chapter 2: Materials, Methodology and Experimental Procedures .......................................................................... 44 2.1 Materials. ....................................................................................... 44 2.1.1 Human Tissue Samples and Clinical Data ............................... 44 2.1.2 Cell lines used for the experiments .......................................... 48 2.2 Core Experimental procedures. ................................................... 49 2.2.1 DNA extraction ......................................................................... 49 2.2.2 RNA extraction ......................................................................... 49 6 2.2.3 DNA and RNA quantification .................................................... 49 2.2.4 Assessment of HPV status ....................................................... 50 2.2.5 p16 staining .............................................................................. 50 2.2.6 Laser-capture microdissection ................................................. 50 2.2.7 Reverse transcription of extracted RNA ................................... 51 2.2.8 E6, E7, L1 and L2 qPCR .......................................................... 51 2.2.9 Gel electrophoresis .................................................................. 54 2.3 Experimental procedures used to obtain results in chapter 3. 54 2.3.1 Whole-genome methylation analysis with MeDIP-seq .............. 54 2.3.2 Data analysis of MeDIP-Seq data ............................................ 56 2.3.3 Genome-wide methylation analysis with Illumina 450k BeadChips ................................................................................ 57 2.3.4 Data analysis of Illumina 450k BeadChips data ....................... 58 2.3.5 Copy number variation (CNV) analysis .................................... 61 2.3.6 Generation of SCC003 clones expressing HPV-16 oncogenes 61 2.3.7 Survival Analysis ...................................................................... 62 2.4 Methodology used and data analysis applied to obtain results in chapter 4 .............................................................................................. 63 2.4.1 MeDIP-Seq data analysis of the HPV genome ......................... 63 2.4.2 Validation of obtained results by bisulfite sequencing (BS-Seq)65 2.4.3 5-AZA treatment of cell lines .................................................... 69 2.5 Methodology used and data analysis applied to obtain results in chapter 5. ............................................................................................. 69 2.5.1 Exon-sequencing of 182 genes often mutated in cancer .......... 69 2.5.2 Validation of selected mutations by Sequenom OncoCarta ..... 70 2.5.3 Confirmation of copy number changes by Infinium CNV Profiling. ................................................................................................