Tese de Doutoramento Biomedicina (associação) 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Head and Neck Carcinoma is considered the sixth most common cancer worldwide and is responsible for 450,000 deaths/year. These tumours appear in various locations of the upper aero-digestive tract. Most of these neoplasms originate in the squamous epithelium and correspond to 90 to 95% of all squamous cell carcinomas of the head and neck region. Despite their common origin in the squamous epithelium, tumours from different locations within the head and neck region show high heterogeneous phenotypic, etiological, and biological characteristics. The registered risk factors are tobacco and alcohol consumption, described as the most common causes for the appearance of these tumours, and Human Papillomavirus (HPV) infection as a risk factor associated with tumours that arise in the oropharynx. The developed project aimed to obtain information about some specific aspects of squamous cell carcinoma of the head and neck region through a detailed molecular analysis of a case series. The study design was developed in distinct and structured tasks to: 1- obtain data related to clinical and pathological variables, 2- identify the different HPV16 variants present and 3- determine the frequency of PIK3CA gene mutations. The cohort included in the study was selected from a universe of patients diagnosed with squamous cell carcinoma of the head and neck region at the Instituto Português de Oncologia Francisco Gentil de Lisboa IPOLFG, being composed mostly of male patients with oropharyngeal cancer, HPV-positive and with active consumption habits. Additionally, a series of samples from a murine model of oropharyngeal carcinogenesis induced by HPV16 was studied to identify the virus variant present in this experimental model. The sequencing of the complete genome of HPV16 was performed using Next Generation Sequencing methodology, developed from previously described primers, which were adapted to optimize a laboratory workflow that allows the identification of HPV16 variants from multiple tissues. Finally, the frequency of the PIK3CA mutation strongly associated with squamous cell carcinoma of the head and neck region was calculated based on the detection of the presence of four different mutations (E545D, E545K, E542K and H1047L) by real-time PCR methodology. The main data obtained express the clinical characterization of the pathology in the series of patients, describe the different HPV16 strains identified in the total cohort of patients, as well as the mutational profile for each HPV16 gene. Phylogenetic analysis shows lineage A as the most prevalent in our cohort. In the exploratory study of the respective strains, it was not possible to establish any reliable association with clinicopathological variables, including survival variables, due to the small number of cases included in the analysis (n=35). Regarding the genomic comparative analysis of the 35 patients, 243 nucleotide variations were reported, 84 of which integrate an amino acid alteration. The mutational profile agrees with that previously described; a total of 41% (100/243) single nucleotide variation (SNV) were published by other authors based on cervix pathology and/or in the pathology of the head-neck region. The remaining SNV that were not described in the systematic review contribute with new data in different genes of the viral genome, including those genes that until now are less explored, such as the E4, E5 and L2 gene. From the exploratory analysis of biological significance, a total of 23 variants were described, the majority studied in cervical carcinoma. Overall, the greatest variability was observed in the E5 and L2 genes that were not previously studied. Oppositely, the most conserved gene was E7, as previously reported by other authors. The frequency of PIK3CA gene mutations found in our cohort support the high occurrence of these mutations among head and neck tumours. In our cohort, we identified the presence of at least one mutation in the PIK3CA gene in 39% of the patients included, and the E545D mutation was the most frequently detected. It should also be noted that the data from the analysis of the murine model genome allowed for the first time to identify the HPV16 lineage present as A1 (European) sublineage, which was the most prevalent in patients, supporting the hypothesis that this is a lineage with high oncogenic potential in the oropharynx and making this model even more interesting. Overall, our data corroborate those previously reported by other groups, highlighting the importance of molecular analysis to better understand the distribution of HPV16 variants and PIK3CA mutations and their potential influence on carcinogenesis, diagnosis and clinical approach in head and neck cancer