Human papillomavirus - associated head and neck squamous cell carcinomas in North-East Italy

Background: Specific oncogenic types of human papillomaviruses (HPV), most frequently HPV16, are causally associated with a subset of head and neck squamous cell carcinomas (HNSCC). HPV DNA associated tumors appear to be heterogeneous in prevalence over time and geographically, in the oncogenic activity (direct and indirect viral markers) and clinical behavior. However, it remains unclear which biomarkers can reliably determine which HNSCC are truly driven by HPV transformation. Aims: In the present thesis, the first aim was to determine the prevalence of the truly HPV-associated HNSCC tumors in North-East Italy. The second aim, was to investigate the association of HPV DNA positivity with other viral (viral load, oncoE6 protein, HPV antibodies) and cellular (p16INK4a, pRb) markers. The third aim, was to evaluate the prognostic significance of HPV-association tumors for clinical outcome (i.e. survival). Materials and Methods: Overall, 247 fresh frozen and 53 (21%) formalin-fixed paraffin-embedded (FFPE) tumor tissue biopsies and 102 (41%) sera were collected from 247 newly detected HNSCC patients from North-East Italy. Clinical parameters for each patient were obtained from the clinical database. HPV DNA was determined by polymerase chain reaction (PCR) with consensus MY09/MY11 primers and Restriction Fragment Length Polymorphism (RFLP) analysis and/or BSGP5+/6+-PCR/Multiplex Papillomavirus Genotyping (MPG) capable of detecting all known 51 mucosal HPV types. The HPV DNA+ tumor tissues were further analyzed for i) viral load by HPV16 qPCR and quantitative BSGP5+/6+-PCR/MS; ii) detection of HPV E6*I transcripts by RT-PCR; iii) expression levels of cellular protein p16INK4a and pRb by IHC; and iv) presence of HPVE6 protein for types 16 and 18 by the commercial OncoE6TM Oral Test; Antibodies to HPV early and late proteins of the eight most frequent high-risk HPV types were determine din all available sera by bead-based multiplex serology. Results: Overall, HPV DNA+ was 8.5% (21/247), type 16 was detected in 95% (20 cases) and type 58 in 5% (1 case). No multiple infections were detected. The HPV RNA+ was 6% (14/244). Oropharynx was the site with the highest HPV prevalence by DNA (27%) and RNA (20%). In the other anatomic sites, HPV prevalence was < 8%. Among the HPVDNA+ RNA+ tumors, i) 93% of the HPV16+ tissues (13/14) showed high viral load; ii) 60% (6/10) showed both up-regulation of p16INK4a and down-regulation of pRb; iii) and in 100% (8/8) HPV16 E6 oncoprotein was detected. All sera of 7 HPV-driven tumors showed strong positive antibody reactions with HPVE6 and E7 proteins, 6 for type 16 and 1 for type 58, type-concordant with the related tumor. Another single serum HPV16 DNA+ in the tumor, showed positivity for all early HPV16 proteins suggestive of an HPV-driven tumor. Kaplan-Meier analyses for the oropharynx showed a trend for better survival in the HPV-associated group than in the HPV negative ones. Conclusions: A low HPV prevalence was found in HNSCC of the population living in the North-East of Italy. Oropharynx was the preferential site for HPV infection while the HPV prevalence in the other anatomic sites appeared negligeable. We observed that the HPVDNA+ RNA+ samples showed a good correlation with the other markers like high viral load, presence of the E6 oncoprotein, and HPVE6 and E7 seromarkers. In contrast to recent reports we did not find a good correlation between HPVDNA+ RNA+ and the up-regulation of p16INK4a and down-regulation of pRb. Survival analyses showed a better prognosis in the HPV-driven patients with tumors occurring in the oropharynx