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

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

Changes in Cervical Human Papillomavirus (HPV) Prevalence at a Youth Clinic in Stockholm, Sweden, a Decade After the Introduction of the HPV Vaccine

Aim: This study aimed to follow the impact of human papillomavirus (HPV) catch-up and vaccination on the very high cervical HPV-prevalence in women at a youth clinic in central Stockholm during the period 2008–2018.Background: 2008–2010, cervical HPV-prevalence (69.5%) and HPV16 prevalence (34.7%) were high in non-vaccinated women at a youth clinic in Stockholm. 2013–2015, after the introduction of the quadrivalent-Gardasil® HPV-vaccine, HPV16 and HPV6 prevalence had decreased. Here, cervical HPV-prevalence was investigated 10 years after primary sampling.Material and Methods: 2017–2018, 178 cervical swabs, from women aged 15–23 years old, were tested for 27 HPV types by a bead-based multiplex method. HPV-prevalence data were then related to vaccination status and age and compared to HPV-prevalence in 615 samples from 2008 to 2010 and 338 samples from 2013 to 2015 from the same clinic, and to HPV types in 143 cervical cancer cases during 2003–2008 in Stockholm.Results: The proportion of vaccinated women increased from 10.7% (2008–2010) to 82.1% (2017–2018). The prevalence of all 27 HPVs, all high-risk HPVs (HR-HPVs) and the combined presence of the quadrivalent-Gardasil® types HPV16, 18, 6, and 11, was lower in vaccinated compared to unvaccinated women (67.4 vs. 93.3%, p = 0.0031, 60.1 vs. 86.7%, p = 0.0057 and 5.8 vs. 26.7%, p = 0.002, respectively). Furthermore, HPV16 prevalence in non-vaccinated women 2017–2018 was lower than that in 2008–2010 (16.7 and 34.7%, respectively, p = 0.0471) and similar trends were observed for HPV18 and 11. In both vaccinated and non-vaccinated women, the most common non-quadrivalent-Gardasil® vaccine HR-HPV types were HPV39, 51, 52, 56, and 59. Together they accounted for around 9.8% of cervical cancer cases in Stockholm during 2003–2008, and their prevalence tended to have increased during 2017–2018 compared to 2008–2010.Conclusion: Quadrivalent-Gardasil® vaccination has decreased HPV-vaccine type prevalence significantly. However, non-vaccine HR-HPV types remain high in potentially high-risk women at a youth clinic in Stockholm

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Background and Aim: In 2010 HPV vaccination was subsidized in Sweden and in 2012 a national vaccination program against HPV16, 18, 6 and 11 was launched for girls ages 10-12 years. In parallel was a catch-up vaccination program for young women. To investigate base line HPV cervical and oral prevalence in non-vaccinated youth two studies were performed at a youth clinic in Stockholm 2008-2011. This project initiated 2013 aimed to follow HPV prevalence in youth since the previous studies, in the same population.   Materials and Methods: 117 women, of which 73% were HPV catch-up vaccinated donated 93 cervical samples and 117 oral samples, and 54 unvaccinated men donated 54 oral samples and 47 urinary samples. All samples were tested for 27 HPV types with a PCR based system and the data was compared to that obtained in 2008-2011. The categorical Fishers exact test was used for statistical analysis due to HPV-positive samples being n< 5 for certain types.  Results and Conclusion: HPV16 cervical prevalence was significantly lower in the HPV vaccinated women compared to unvaccinated women (7% and 27% respectively, p=0.033) in the 2013 group. For HPV18 and HPV6 there was a significantly lower prevalence in the 2013 vaccinated group compared to the 2008-2010 unvaccinated group (1.5% vs. 10% respectively, p=0.021 and 1.5% vs. 8% respectively, p=0.048). Overall oral HPV prevalence for both genders, was lower in the 2013 group compared to that of 2009-2011, (2.3% and 9.1% respectively, p=0.005). Male urinary prevalence was low (6%) and not efficient to follow changes in specific HPV types. The data indicate that HPV catch-up vaccination was gradually exhibiting an effect, with significant decrease of cervical HPV16 prevalence

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Background and Aim: In 2010 HPV vaccination was subsidized in Sweden and in 2012 a national vaccination program against HPV16, 18, 6 and 11 was launched for girls ages 10-12 years. In parallel was a catch-up vaccination program for young women. To investigate base line HPV cervical and oral prevalence in non-vaccinated youth two studies were performed at a youth clinic in Stockholm 2008-2011. This project initiated 2013 aimed to follow HPV prevalence in youth since the previous studies, in the same population.   Materials and Methods: 117 women, of which 73% were HPV catch-up vaccinated donated 93 cervical samples and 117 oral samples, and 54 unvaccinated men donated 54 oral samples and 47 urinary samples. All samples were tested for 27 HPV types with a PCR based system and the data was compared to that obtained in 2008-2011. The categorical Fishers exact test was used for statistical analysis due to HPV-positive samples being n< 5 for certain types.  Results and Conclusion: HPV16 cervical prevalence was significantly lower in the HPV vaccinated women compared to unvaccinated women (7% and 27% respectively, p=0.033) in the 2013 group. For HPV18 and HPV6 there was a significantly lower prevalence in the 2013 vaccinated group compared to the 2008-2010 unvaccinated group (1.5% vs. 10% respectively, p=0.021 and 1.5% vs. 8% respectively, p=0.048). Overall oral HPV prevalence for both genders, was lower in the 2013 group compared to that of 2009-2011, (2.3% and 9.1% respectively, p=0.005). Male urinary prevalence was low (6%) and not efficient to follow changes in specific HPV types. The data indicate that HPV catch-up vaccination was gradually exhibiting an effect, with significant decrease of cervical HPV16 prevalence

Analysis of somatic mutations in papillomavirus positive tumours from younger and older oropharyngeal cancer patients

ABSTRACT Background: Human Papilloma Virus positive (HPV+) Oropharyngeal Squamous Cell Carcinoma (OSCC), dominated by tonsillar cancer (TSCC) and base of tongue cancer (BOTSCC) has low mutation-frequency and better survival for younger than older patients.Aim: To examine if HPV+ TSCC and BOTSCC have distinct gene-mutation profiles, for 50 often-mutated genes in cancer, in younger compared to older patients and to test and compare different variant callers to get a deeper understanding of the data.Materials and methods: DNA had previously been extracted from 299 formalin-fixed-paraffin-embedded (FFPE) tumor biopsies and 13 normal samples, and sequenced on the Ion Proton sequencer, a NGS (Next-Generation Sequencing) platform. Alignment and variant calling had been performed via the Ion Torrent Suite software v5 (ITS), and Torrent Variant Caller (TVC).UPPMAX, a High-Performance-Computational cluster (HPC) at Uppsala University was used for storing and computing of the sequenced data. Parallel-processing was used to optimize repetitive steps, saving days of computation time. The descriptive analysis, graphical data representations, and more in-depth analysis, were done in R.Initially, variant calling was performed for 13 tumor/normal paired samples using the novel MuTect2 software from the Genome Analysis Toolkit (GATK) toolset. Variant annotation and statistical analysis was performed on all the 13 paired sequenced samples, using SnpEff. Due to a poor overlap between the above MuTect2 and TVC, after adequate filtering TVC, MuTect, Strelka and VarScan2 were also utilized for comparison.Result and Conclusion: Having only 13 normal samples, normal-tumor paired variant calling to distinguish germ line and somatic variants could not be performed. To obtain an approximation of the amount of germline variation in our cohort, additional variant callers were used for the tumor normal pairs. The data obtained with the TVC caller formed the basis for further analysis of the tumor samples.Notably, comparisons of TVC with MuTect2 output revealed major discrepancies and limited overlap. However, when comparing MuTect2 with MuTect, Strelka, or VarScan 2 regarding overlaps with TVC – the overlap were still limited, but higher degrees of overlaps were disclosed between Strelka, MuTect and MuTec2, indicating that MuTect2 was a successful further development and successor of MuTect.Evidence for presence of distinct gene-mutation profiles correlating to age could not be obtained in the analyzed tumor cohort with the software tool kits applied. Statistical analysis using Wilcoxon-Mann Whitney test did not support a hypothesis of distinct age related mutations for any of the 50 genes analyzed in this tumor cohorts.The highest p-value for the Wilcoxon-Mann Whitney test was for the gene APC, at p ~ 0.054 hints at a possible connection. However, more extensive research with more samples sequenced is necessary to confirm or reject this correlation

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Background and Aim: In 2010 HPV vaccination was subsidized in Sweden and in 2012 a national vaccination program against HPV16, 18, 6 and 11 was launched for girls ages 10-12 years. In parallel was a catch-up vaccination program for young women. To investigate base line HPV cervical and oral prevalence in non-vaccinated youth two studies were performed at a youth clinic in Stockholm 2008-2011. This project initiated 2013 aimed to follow HPV prevalence in youth since the previous studies, in the same population.   Materials and Methods: 117 women, of which 73% were HPV catch-up vaccinated donated 93 cervical samples and 117 oral samples, and 54 unvaccinated men donated 54 oral samples and 47 urinary samples. All samples were tested for 27 HPV types with a PCR based system and the data was compared to that obtained in 2008-2011. The categorical Fishers exact test was used for statistical analysis due to HPV-positive samples being n< 5 for certain types.  Results and Conclusion: HPV16 cervical prevalence was significantly lower in the HPV vaccinated women compared to unvaccinated women (7% and 27% respectively, p=0.033) in the 2013 group. For HPV18 and HPV6 there was a significantly lower prevalence in the 2013 vaccinated group compared to the 2008-2010 unvaccinated group (1.5% vs. 10% respectively, p=0.021 and 1.5% vs. 8% respectively, p=0.048). Overall oral HPV prevalence for both genders, was lower in the 2013 group compared to that of 2009-2011, (2.3% and 9.1% respectively, p=0.005). Male urinary prevalence was low (6%) and not efficient to follow changes in specific HPV types. The data indicate that HPV catch-up vaccination was gradually exhibiting an effect, with significant decrease of cervical HPV16 prevalence

Whole-exome sequencing of HPV positive tonsillar and base of tongue squamous cell carcinomas reveals a global mutational pattern along with relapse-specific somatic variants

To identify predictive/targetable markers in human papillomavirus positive (HPV+) ton-sillar and base of tongue cancer (TSCC/BOTSCC), whole-exome sequencing (WES) of tumours of patients with/without recurrence was performed. Forty primary tumours and adjacent normal tissue were separated by micro-dissection from formalin-fixed paraffin-embedded tissue from patients treated with curative intent 2000–2014 at Karolinska University Hospital. Successful sequencing was obtained in primary tumours of 18 patients without and primaries of 17 with local or distant recurrence, as well as in 10 corresponding recurrences (i.e., five local relapses and five distant metas-tases) from these 17 patients. One variant—a high-impact deletion in the CDC27 gene—was observed only in primaries of 5/17 patients that had a recurrence after full treatment but in none of those without recurrence. In addition, 3 variants and 26 mutated genes, including CDC27, BCLAF1 and AQP7, were present in at least 30% of all primary tumours independent of prognosis. To conclude, a CDC27 deletion was specific and found in ~30% of samples from patients with a local relapse/distant metastasis and could, therefore, potentially be a prospective marker to predict prognosis. Commonly mutated genes, such as BCLAF1, should be further studied in the context of targeted therapy

Prognostic Markers and Driver Genes and Options for Targeted Therapy in Human-Papillomavirus-Positive Tonsillar and Base-of-Tongue Squamous Cell Carcinoma

The incidence of Human-papillomavirus-positive (HPV+) tonsillar and base-of-tongue squamous cell carcinoma (TSCC and BOTSCC, respectively) is increasing epidemically, but they have better prognosis than equivalent HPV-negative (HPV−) cancers, with roughly 80% vs. 50% 3-year disease-free survival, respectively. The majority of HPV+ TSCC and BOTSCC patients therefore most likely do not require the intensified chemoradiotherapy given today to head and neck cancer patients and would with de-escalated therapy avoid several severe side effects. Moreover, for those with poor prognosis, survival has not improved, so better-tailored alternatives are urgently needed. In line with refined personalized medicine, recent studies have focused on identifying predictive markers and driver cancer genes useful for better stratifying patient treatment as well as for targeted therapy. This review presents some of these endeavors and briefly describes some recent experimental progress and some clinical trials with targeted therapy

Prevalence of Oral Human Papillomavirus Infection among Youth, Sweden

Human papillomavirus (HPV) causes cervical, head, and neck cancers. We studied 483 patients at a youth clinic in Stockholm, Sweden, and found oral HPV prevalence was 9.3% and significantly higher for female youth with than without cervical HPV infection (p = 0.043). Most oral HPV types matched the co-occurring cervical types