Genetic analyses, protein expression, type and function with clinical and immunological correlates in human papillomavirus associated head and neck neoplasia Liam Masterson St Edmunds College University of Cambridge A dissertation submitted to the University of Cambridge in candidature for the degree of Doctor of Medicine Feb 2018 1 Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text It does not exceed the prescribed word limit for the Doctor of Medicine Degree Committee. Signed: Liam Masterson Date: 11/02/2018 2 Main supervisor: Dr. Jane Sterling PhD FRCP Internal examiner: Dr. Lucy Truman PhD FRCS External examiner: Prof. Chris Nutting BSc (Hons) MD PhD FRCP FRCR MedFIPEm Assessor to Regius Professor of Physic: Dr. Chris Allen MA MD FRCP 3 Summary In head and neck squamous cell carcinoma (HNSCC), the evidence that human papillomavirus (HPV) is associated with a subgroup of tumours has increased over the last thirty years. Prospective randomised controlled clinical trials have now established that detection of HPV in oropharyngeal tumours (~60-70% of cases in North America and Europe) may confer a survival advantage to the patient. Within the oropharynx, HPV16 constitutes ~90-95% of HPV subtypes associated with malignancy. This contrasts with uterine cervix mucosa, where approximately 15 high- risk subtypes cause >99% of disease. Other factors such as differences in genetic background, host immune response, hormonal and environmental influences (e.g. tobacco smoke, alcohol) all play a part in the pathway and susceptibility to oncogenesis. Analogous to uterine cervix disease, HPV-associated cancers involve wild-type TP53 whilst HPV negative tumours often have mutations in this gene. As most patients with HPV associated OPSCC present at an advanced stage, the detection and genetic analysis of a pre-malignant state would be important as it infers the potential for a screening test (similar to the uterine cervix model). To investigate this, whole transcriptome analysis with verification of results by reverse transcription–quantitative polymerase chain reaction (PCR), was performed on OPSCC fresh tissue biopsy samples. Predictable fold changes of RNA expression in HPV-associated disease included multiple transcripts within the p53 oncogenic pathway (e.g. CDKN2A / CCND1). In addition to this, a testis-specific gene not normally expressed in somatic cells, SYCP2, showed a consistently elevated fold change from baseline in pre-malignant and malignant tissue. A subtle immune defect has long been thought to trigger the susceptibility of some individuals to persistent HPV infection with either low or high risk HPV types. Following on from this, we investigated if clinical outcomes in HPV-related head and 4 neck cancer may be affected by host immune response. Peripheral blood from patients with OPSCC treated by chemoradiotherapy underwent IFN-γ enzyme-linked immunosorbent spot assay (ELISPOT) to examine cell-mediated immune responses to HPV16 E2, E6 and E7. T cell responses against E6 or E7 peptides correlated with HPV DNA/RNA status. Within the HPV16+ OPSCC cohort, enhanced immunoreactivity to antigen E7 was linked to improved survival. In addition, an observed increase in regulatory T cell frequencies after treatment would suggest that immunosuppression may contribute to a reduced HPV specific cell mediated response. A further aspect of this study was to determine the role of HPV and Epstein Barr Virus (EBV) in the pathogenesis of squamous cell carcinoma within the temporal bone region. This is an uncommon tumour which is normally preceded by a history of inflammation within the external auditory canal (EAC) or middle ear / mastoid cavity. Although HPV has been implicated in many head and neck malignancies, its role in SCC of the temporal bone has not been established. Treatment strategies could change if a viral aetiology can be found. HPV16 DNA was detected in ~20% of the cases studied, however, no significant difference in disease specific survival was noted for the papillomavirus positive group. Epstein-Barr virus was not detected. The data presented highlight the functional and biological influence of high risk HPV infection on HNSCC. Further studies are likely to focus on developing non-invasive screening tools, therapeutic strategies based on vaccination or immune modulation or indeed de-escalation of current treatment protocols. 5 Abbreviations ATP adenosine triphosphate bp base pair(s) cDNA complementary DNA CIN cervical intraepithelial neoplasia DAPI 4',6-diamidino-2-phenylindole dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate DMSO dimethyl sulphoxide DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate dUTP deoxyuridine triphosphate EDTA ethylenediaminetetraacetic acid disodium salt dehydrate EGF epidermal growth factor ELISPOT enzyme-linked immunospot FCS foetal calf serum FFPE formalin fixed paraffin embedded FISH fluorescence in situ hybridization GAPDH glyceraldehyde-3-phosphate dehydrogenase gDNA genomic DNA H&E haematoxylin & eosin HLA human leukocyte antigen HNSCC head and neck squamous cell carcinoma HMBS hydroxymethylbilane synthase HPV human papillomavirus HR homologous recombination HSIL high-grade squamous intraepithelial lesion HSV-2 herpes simplex virus type 2 hTERT human telomerase reverse transcriptase IARC International Agency for Research on Cancer IL interleukin 6 IFN-γ interferon- γ kbp kilobase pairs LR-HPV low-risk human papillomavirus LSIL low-grade squamous intraepithelial lesion MHC major histocompatibility complex mRNA messenger RNA nm nanometer nt nucleotides OPSCC oropharyngeal squamous cell carcinoma ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PI propidium iodide RTqPCR real time quantitative polymerase chain reaction RIN RNA integrity number RNA ribonucleic acid RT reverse transcriptase SCC squamous cell carcinoma SDS sodium dodecyl sulphate SSC saline sodium citrate TBE tris borate EDTA UICC Union for International Cancer Control URR upstream regulatory region UV ultraviolet VLP virus like particle WTS whole transcriptome sequencing 7 Acknowledgements As a clinician, entering the mysterious world of laboratory medicine was a truly daunting step. Without doubt, this transition was made possible by the patience and good nature of the fantastic people I have met. I am most indebted to my partner, Teofila, for her daily encouragement, support and steadfast belief. In addition, I thank my parents and siblings, for instilling in me a set of values that I hold dear to this day. I am also especially grateful to my main supervisor, Dr. Jane Sterling and other co-supervisors (Dr. David Winder, Dr. Fred Sorgeloos, Dr. Peter Goon, Mr. Piyush Jani and Mr. David Moffat) for their endless support, advice and guidance during this period. Finally, the future of oncology will almost certainly benefit from the rapid advances made in genomic medicine and immunotherapy. This project has allowed me a unique insight into both these fields and I hope to continue this further with the 100K genome head & neck collaboration. 8 Publications Masterson L, Mahony J, Lechner M. (2016) Expanding the benefits of vaccination to boys and men. Lancet. 388, 2992 Masterson L, Lechner M. (2016) HPV vaccination in boys…. will the UK join the fight? Nature Reviews Clinical Oncology. 13, 721-722 Masterson L, Lechner M, Loewenbein S, Hassan M, Davies-Husband C, Fenton T, Sudhoff H, Jani P, Goon PKC, Sterling JC. (2016) CD8+ T cell response to HPV16 E7 can predict survival outcome in oropharyngeal cancer. European Journal of Cancer. 67,141-151 Masterson L, Winder D, Ball SLR, Vaughan K, Lehmann M, Scholtz LU, Sterling JC, Sudhoff H, Goon PKC. (2016) Molecular analyses of unselected head and neck cancer cases demonstrates that human papillomavirus transcriptional activity is positively associated with survival and prognosis. BMC Cancer. 16, 367 Masterson L, Sorgeloos F, Winder D, Lechner M, Marker A, Malhotra S, Sudhoff H, Jani P, Goon PKC, Sterling JC. (2015) Deregulation of SYCP2 predicts early stage HPV+ oropharyngeal carcinoma – a prospective whole transcriptome analysis. Cancer Science.106 (11), 1568-75 Masterson L, Moualed D, Liu ZW, Howard J, Dwivedi R, Benson R, Tysome JR, Sterling JC, Jani P, Sudhoff H, Goon PKC. (2014) De-escalation treatment protocols for HPV associated oropharyngeal squamous cell carcinoma: a systematic review and meta-analysis of current clinical trials. European Journal of Cancer. 50(15), 2636-48 Masterson L, Dwivedi RC, Allam M, Jani P. An adult with a neck lump: Clinical Review. BMJ 2013; 347: f5473 9 Masterson L, Winder D, Marker A, Sterling J, Sudhoff H, Moffat D, Goon P. (2013) Investigating the role of human papillomavirus in squamous cell carcinoma of the temporal bone. Head