Human Papillomavirus Integration and Methylation Events and Cervical Disease Progression Post-Vaccination A thesis submitted for the Degree of Doctor of Philosophy at Cardiff University By Rachel Louise Baldwin Supervisors Dr Sam Hibbitts Dr Amanda Tristram Professor Gavin Wilkinson HPV Research Group Cardiff University 2017 i ii DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed …………………………………………(candidate) Date ………………… STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of, PhD Signed ………………………………………(candidate) Date …………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed …………………………………………(candidate) Date ………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed …………………………………………(candidate) Date ………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed …………………………………………(candidate) Date ………………… iii iv Acknowledgements I would not be submitting this thesis if it wasn’t for the unending support, advice and belief of Dr Sam Hibbitts, who believed in me when I didn’t believe in myself and supported me at my best and my worst. Thank you also to Dr Amanda Tristram and Professor Gavin Wilkinson who have helped shape my research and this thesis. Thank you also to goes to Tenovus who funded this research. In the HPV Group I have met many wonderful and helpful people; thank you to Tiffany, Áine, Rachel, Dean, Evelyne, Stefan, Sadie, Jon and Ned. A huge thank you is due to Jo Jones and Angharad Edwards, two wonderful friends and teachers who only laughed a little when I thought the Plant Room was where plants are grown! Thank you to my family. To my parents; Mum, Dad, Alan and Carol, thank you for always being my biggest supporters. To my sisters Alana and Antonia, who inspire me to be happy. To my Gran, who sends the best care packages. To my new parents, Margaret and Stephen, thank you for making me part of your family. To Adele, Dini, Adam, Suzy and Claire, who have become my family; the others didn’t have a choice but you guys did - thanks! Finally, thank you to my extraordinary wife, Hayley, who keeps me focused on the bigger picture, inspires me to be the best I can be and repeatedly tells me to “get a grip.” This thesis would not have been possible without your support (nagging) and encouragement (more nagging). In all seriousness, thank you for everything you have done for me over the past four years. This thesis is dedicated to you! v vi Collaborators This thesis would not have been possible if it wasn’t for the support, advice and assistance of numerous collaborators including Dr Sally Roberts (Birmingham University) who advised in the production of the raft culture, provided w12 Ser4B cells and also carried out the HPV E4 IHC. Dr C Dawson from Birmingham University who carried out the SHH and GLI-I IHC and provided details of SHH expression in VIN cultures carried out in Birmingham. Cardiff University’s Central Biotech Services carried out the RNAseq presented in chapter 5. Thanks also go to and to Dr Kevin Ashelford and Dr Peter Giles from Wales Gene Park who assisted in the RNAseq library construction and advised in the analysis. Dr Owen Weeks, an independent pathologist from Cardiff University took histology images presented in Chapter 5. Dr Weeks also assisted with the analysis and interpretation of pathology results. Thank you to Professor Robert Hills from Cardiff University for his help and advice on the statistical analysis presented in this thesis. vii viii Abbreviations 9v-HPV 9-valent HPV APOT Amplification of Papillomavirus Transcripts BAK Bcl-2 Homologous Antagonist/Killer BIDD Biomarkers and Imaging Discovery and Development Committee BLAST Basic Local Alignment Search Tool bp base pairs cDNA complementary DNA CFS common fragile sites CIN Cervical Intraepithelial Neoplasia CNRQ calibrated normalized relative quantities CSW Cervical Screening Wales DAPI 4’, 6’-diamidino-2-phenylindole DIPS Detection of Integrated Papillomavirus Sequences DMEM Dulbecco's Modified Eagle's Medium DMSO dimethyl sulfoxide DNA Deoxyribonucleic Acid dNTP deoxiribose nucleotide triphosphate DSB double stranded breaks E6AP E6 Adaptor Protein E. coli Escherichia coli EGF epithelial growth factor EMBL-EBI European Molecular Biology Laboratory – The European Bioinformatics Institute ix ENA European Nucleotide Archive EZH2 Enhancer of Zeste Homolog 2 FISH fluorescent in situ hybridization FBS foetal bovine serum GLI-1 Zinc Finger Protein GLI1 GMEM Glasgow Modified Eagle’s Medium HC2 Hybrid Capture 2 HEKn human epithelial keratinocytes, neonates HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HI-BCD Human Papillomavirus Integration as a Biomarker of Cervical Disease HP/P high positive/positive HPRT Hypoxanthine Phosphoribosyltransferase HPV Human Papillomavirus HR-HPV High-Risk Human Papillomavirus hTERT Human Telomerase Reverse Transcriptase ICC Invasive Cervical Carcinoma IHC immunohistochemistry IL12A Interleukin-12 Alpha IPTG isopropyl β-D-1-thiogalactopyranoside ISH in-situ hybridization kb kilo base LB Lysogeny Broth LBC liquid based cytology x LCR long control region LLETZ large loop excision of the transformation zone LR-HPV low-risk Human Papillomavirus miRNA micro ribonucleic acid mRNA messenger ribonucleic acid NCBI National Institute of Biotechnology Information NCR non-coding region NHS National Health Service ORF open reading frame PCR polymerase chain reaction qRT PCR quantitative real-time polymerase chain reaction RIN ribonucleic acid integrity number RNA ribonucleic acid RS-PCR Restriction Site Polymerase Chain Reaction SCC Squamous Cell Carcinoma SHH Sonic Hedgehog Protein SOC super optimal broth with catabolite repression TAE tris-acetate-EDTA TBP2 TATA-Binding Protein 2 URR upstream regulatory region VaIN Vaginal Intraepithelial Neoplasia VIN Vulva Intraepithelial Neoplasia VLP virus like particle X-gal 5-bromo-4-chloro-indolyl-β-D-galactopyranoside xi List of Figures Figure Title Page 1.1 A schematic of the linearized HPV genome 3 The E2 negative feedback loop controlling HPV genome 1.2 6 expression. DNA damage and immortalization mechanisms employed 1.3 8 by HPV E6 and E7 oncoproteins. Representation of HPV gene expression in a productive 1.4 15 and a transformed infection. The process of bisulphite conversion of DNA with sodium 1.5 23 bisulphite. The progression of HPV infection to Cancer and the key 1.6 features of the cervical epithelium as seen using 26 histological analysis. Cancer incidence and mortality in women in less developed and more developed regions. Incidence is 1.7 27 shown in blue and mortality is shown in red. Figure adapted from Globocan 2012 (IARC) 1.8 Process of cell growth in an organotypic raft culture. 44 Flow diagram of samples included in a study of young 2.1 52 women attending their first cervical smear. Flow diagram of samples included in a study of HPV 2.2 54 Integration as a Biomarker for Cervical Disease (HI-BCD). xii 2.3 Principle of the E2 PCR assay. 72 2.4 Principle of the DIPS assay 74 Example of NCBI nucleotide blast used to identify the 2.5 location of virus-host integration site for the HPV genome 78 and the host. 2.6 IHC quantification using ImageJ 97 Samples extracted by Protocol 1 and Protocol 2: Yield 3.1 109 and purity of DNA and BetaGlobin PCR comparison Average DNA yield and purity for four extraction 3.2 111 procedures. 3.3 Optimisation of the E2 tiling PCR assay PCR condition. 114 Examples of HPV E2 tiling PCR applied to a selection of 3.4 116 clinical samples. HPV 16 samples selection to reflect the proportion of 3.5 118 cervical cytology in the total cohort. E2 gene disruption as detected by type specific E2 tiling 3.6 120 PCR for HPV 16, 31, 33, 35 and 51. Average E2 viral DNA methylation values for samples 3.7 124 with different cervical cytology grades. Average E2 viral DNA methylation at each CpG site for 3.8 125 samples with varying cervical hitology. 4.1 HR-HPV infections as reported by Papillocheck DNA Chip 140 4.2 143 HPV 16 viral disruptions in samples with no reported xiii disease, CIN II, CIN III and Cancer. Age of participants compared to the average viral 4.3 149 methylation across all 8 CpG points analysed. Comparison of viral DNA methylation levels in samples 4.4 151 with (A) CIN III histology and (B) Cancer histology Viral methylation levels were compared between samples 4.5 153 with disrupted genome and intact E2 genome. Trends in viral DNA methylation in samples with a single 4.6 HPV16 infection compared to samples with HPV 16 plus 154 one or more other HR-HPV types. Viral E2 and E6 gene expression in samples with varying 4.7 158 histological outcomes (n= 15). 5.1 H&E staining of raft sections 172 p16 and Ki-67 IHC analysis of W12 Ser4B (positive 5.2 177 control) and HEKn (negative control) raft cultures. p16 and Ki-67 IHC analysis of vulval and vaginal 5.3 178 organotypic raft cultures. Percentage of HP/P pixels in each of the epithelial 5.4 180 cultures as measured by ImageJ and IHC Profiler.