Studies on human papillomavirus (HPV) and other markers in the development and prognosis of HPV associated cancer

Sammanfattning: Background and aims: Human papilloma virus (HPV) is a risk factor for anogenital and oropharyngeal cancer (OPSCC) and commonly transmitted sexually although most infections are cleared without adverse effects. Notably, the past decades the incidences of HPV positive (HPV+), but not HPV negative (HPV-) tonsillar and base of tongue cancer (TSCC and BOTSCC), the two major OPSCC subtypes have both increased. For this reason, we wanted to follow HPV-prevalence. Before HPV vaccination a high prevalence of HPV was shown in the cervix and oral cavity of youth aged 15-23 years at a youth clinic in Stockholm 2008-2010, but later, with rising vaccine coverage, a decrease of HPV vaccine types was observed between 2013-2015. In parallel, in the mid-2010s many studies showed a link between vaginal microbiota and obstetric outcomes, inflammatory disease, as well as sexually transmitted disease. A few years later, several meta-studies and one DNA sequencing study abroad, showed an association between HPV prevalence and microbiota. In this context, of note, most HPV associated anogenital cancers go through three stages of development, pre-malignant stages, dysplasia, high-grade dysplasia/cancer in situ, to malignant stages, invasive cancer, and metastatic cancer. However, these stages are not well studied in HPV+ TSCC and BOTSCC and although there have been many biomarker studies in these tumours additional ones would be of use to better individualize treatment of these cancers. The aim of this thesis was therefore to follow up some of these findings. Approaches. In paper I, we followed up the HPV vaccination coverage and HPV prevalence at a youth clinic in Stockholm, to investigate vaccine effects. In paper II, we investigated possible effects of HPV status, age and vaccination status on the vaginal microbiota of women in a cohort from Uppsala and Stockholm. In paper III, we analysed and compared gene expression in high-grade dysplasia and invasive cancer in HPV+ and HPV- TSCCC/BOTSCC with particular emphasis on HPV status. In paper IV, we did whole-exome-sequencing on primary tumours of HPV+ TSCC/BOTSCC patients with and without recurrences, to identify similarities and differences between the groups as well as to identify markers of prognostic significance or candidates for targeted therapy. Results: In paper I, the proportion of HPV vaccinated women increased from 10.7% 2008-2010 to 82.1% 2017-2018. HPV-vaccine types were reduced overall and more in vaccinated than in unvaccinated women, but other high-risk HPV types still remained high. In paper II, microbial alpha-diversity was significantly higher for HPV+ compared to HPVpatients. Twice as many HPV+ than HPV- women had non-lactobacillus dominant vaginal microbiota compared to L. crispatus dominated vaginal microbiota and oncogenic HPVs were associated with non-lactobacillus dominant vaginal microbiota. In paper III, invasive and non-invasive tumours gene-expression were compared using immunohistochemistry (IHC) and RNA-panels. Forty genes showed differential expression, e.g. SPARC, psoriasin I, collagen-1 and galectin-1 and HPV+ and HPV- dysplasia was similarly differentiated from invasive cancer. In paper IV, a high-impact deletion on CDC27 was observed only in primaries of patients with relapse and 3 variants and 26 mutated genes, were present > 30% of all primaries regardless of prognosis. Conclusions. The presented studies in this thesis reaffirm the efficacy of the HPV vaccine programs in Stockholm, but with a remaining continuous prevalence of nonvaccine HR-HPV types. The results also suggest an influence from the HPV status on the vaginal microbiota make up. Furthermore, the data suggest that that HPV+ and HPVTSCC/ BOTSCC although not identical also likely have similar dysplastic cancer stages. Finally, there are differences between the mutational profiles of HPV+ TSCC/BOTSCC that re-occur compared to those that do not re-occur, but also here there are genes that are similarly altered in primary tumours of patients that are cured or that relapse.