The Pathogenesis of Human Papillomavirus (HPV) in Cancer of the Oropharynx

BACKGROUND: Human papillomavirus (HPV) has become increasingly associated with head and neck squamous cell carcinomas (HNSCC), particularly oropharyngeal squamous cell carcinomas (OPSCC). Currently, the number of HPV-positive OPSCCs has now surpassed cervical carcinoma (CC), and is expected to increase for the next 30 years, with one of the most prevalent subtypes, HPV-16, accounting for 87-96% of cases. Unfortunately, there are currently no screening programmes for OPSCC, the reticulated crypts of the palatine tonsils. Current NHS guidelines for determining HPV status in OPSCC is p16INK4A (p16) immunohistochemistry (IHC), which has become the widely accepted method of detection. However, despite its high sensitivity, the use of p16 as a standalone marker for OPSCC has drawn criticism due to its lack of specificity.

AIMS: To characterise HPV-positive and HPV-negative OPSCC tissues using potential biomarkers believed to be indicators of HPV-mediated OPSCC development, as well as develop multiplex immunofluorescent (mIF) assays and aptamers against various HPV proteins.

METHODS: The study employed histological techniques including haematoxylin and eosin (H&E) staining, IHC, and mIF techniques for characterisation of five OPSCC tissues obtained from the Human Biomaterials Resource Centre (HBRC) at the University of Birmingham. These OPSCC samples also underwent p16 and HPV DNA in-situ hybridisation (ISH) as per NHS clinical guidelines for determination of HPV status. Commercially-available HPV antibodies were tested in various tissue types, with molecular screening for HPV confirmation performed by real-time polymerase chain reaction (real-time PCR). Aptamers isolated against HPV proteins (HPV-16 E2, HPV-16 E7-E6, HPV-18 E6, and HPV-18 E7) were developed using systematic evolution of ligands by exponential enrichment (SELEX), and post-SELEX experiments, including comparison against commercially-available HPV antibodies.

RESULTS: Within our sample, both HPV-positive and HPV-negative OPSCCs deviated from the typical tumour profiles. Three OPSCCs were determined to be HPV-negative, despite two of these exhibiting non-keratinising morphology which is typically associated with p16/HPV DNA ISH positivity. The remaining two OPSCCs were determined to be HPV-positive (p16-/HPV DNA ISH-positive), despite exhibiting keratinising morphology which is commonly associated with HPV-negative OPSCCs. Furthermore, staining with prognostic biomarkers using IHC and mIF mostly deviated from the typical staining expected, with higher PD-L1 and CD8 expression observed in HPV-negative OPSCCs, in comparison to HPV-positive OPSCCs. Commercially-available HPV antibodies were unsuccessful, with non-specific staining observed in normal tissues that were confirmed molecularly to be HPV-negative. Aptamers isolated against HPV-16 E2, HPV-16 E7-E6, HPV-18 E6, and HPV-18 E7 proteins underwent successful selection by SELEX, and subsequent molecular docking and computational modelling. This demonstrated that the interactions observed between each HPV aptamer, and their corresponding HPV protein, have been observed in nature, and are suggestive of real interactions. Cell immunofluorescence with HPV aptamers demonstrated minimal background staining, and nuclear and endosomal staining consistent with nuclear and endosomal localisation of E6 and E7 proteins.

CONCLUSIONS: The use of p16 as a surrogate biomarker within HPV-mediated OPSCCs is unsuitable. Using HPV proteins directly could pose as better biomarkers for HPV-positive OPSCC; therefore, we propose aptamers as a novel method for HPV subtype detection.