The prevalence and pathogenesis of Human Papillomavirus (HPV) in normal tonsil tissue

Background: Human papillomavirus (HPV)-positive oropharyngeal cancers (OPCs) of the base of the tongue and tonsils are increasing. The knowledge of the natural history of the virus within the oropharynx is limited, with prospective studies of oral HPV pathogenesis sparse. The prevalence of oral HPV within healthy individuals has been reported to be as high as ~80%. The risk of infection is linked to alcohol consumption, smoking, age, and certain sexual behaviours. It is men that are at greater risk of oral HPV infection and disease.

Objectives: This study aimed to provide an updated estimate of global oral HPV prevalence within healthy adults; to determine the prevalence of HPV within the non-cancerous palatine tonsil tissues of adults and scrutinise the pathogenesis of the virus; and, to examine the tonsil proteome of HPV-positive individuals with comparison to HPV-negative tissues.

Methods: A systematic review and meta-analysis of literature of adult oral HPV prevalence was performed. We established a clinical study on the NIHR-portfolio. Non-cancerous tonsils, a tonsillar swab, and a lifestyle questionnaire were collected from adults undergoing routine tonsillectomy surgery within five UK hospitals. We developed a protocol to isolate DNA from formalin-fixed (10% NBF) tonsil tissue for molecular analysis. A modified HPV-DNA qPCR screen was used to determine tonsillar HPV-status followed by HPV genotyping. HPV-positive tissues have been characterised for p16 and CK7 histologically. Protein biomarkers within tonsil tissues were analysed using a non-biased technique, SWATH mass spectrometry.

Results: We provide an up-to-date estimate of the global burden of overall oral HPV prevalence by meta-analysis of 5.044% (95% CI 4.897-5.193%); interestingly, men were twice as likely to have oral HPV than women. In the clinical study, we describe the prevalence and viral pathogenesis of HPV within the non-cancerous tonsils of our prospective sample. We have determined a 10.2% oropharyngeal HPV prevalence (N=12/118; comprising 4 males, 7 females, and 1 unknown individual). Proportionally, more men than women were HPV-positive, but this was not significant (p=0.49). We found HPV-status to be mainly concordant in both the left and right tonsil tissues; however, positivity varies across the tissue. Three viral types were detected within these tissues (HPV-18, -16, -73); HPV-18 was most prevalent within these individuals (N=10/12, 83.3%). HPV-16 was only detected in a co-infection with HPV-18. Upregulated proteins in HPV-positive tissues included those with immune function (p<0.01), and functionally, the respiratory electron transport chain was the most enriched pathway. Analysis of dysregulated proteins within HPV-positive males versus HPV-positive females revealed enriched pathways to include rRNA processing and associated functions (adj. p<0.001).

Conclusions: Globally, oral HPV prevalence is a greater burden to men than women. Oropharyngeal HPV is prevalent within adult routine tonsillectomy patients in the UK. We describe changes in protein expression based on HPV-positivity in non-cancerous tonsil tissue, that require future validation to provide a ‘signature’ of HPV-positivity. Future multivariate analysis of HPV prevalence, risk factors, and protein expression, would allow identification of populations at risk of HPV-mediated disease who would benefit from intervention, and monitoring for HPV-induced cellular changes.