5543 Background: HPV testing is now widely advocated in the work up of oropharyngeal squamous cell carcinoma (OPSCC). The "gold standard" for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in fresh tumour tissue. For clinical utility, testing strategies have necessarily focused on formalin-fixed paraffin-embedded (FFPE) tissue, but this has been at the expense of reduced sensitivity and specificity for oncogenic HPV. This study evaluates a novel RNA-based in situ hybridisation test against the analytical gold standard; detection of high-risk HPV mRNA, derived from fresh frozen tissue samples, by quantitative reverse transcriptase polymerase chain reaction (qPCR). Methods: A tissue-microarray comprising FFPE cores from 79 OPSCC was tested using High Risk HPV RNAscope (Advanced Cell Diagnostics, USA) for HPV16, 18, 31, 33, 35, 52, and 58. Analytical accuracy and capacity for prognostic discrimination was determined by comparison with HPV RNA qPCR for HPV16, 18 and 33. Demographic and tumour parameters were compared by Chi-squared and Kruskal-Wallis tests. Test sensitivity and specificity were calculated against the standard. Kaplan–Meier survival estimates were constructed to assess prognostication. Results: High risk HPV RNAscope had sensitivity and specificity of 97% and 93% respectively (PPV 91%, NPV 98%) by comparison to the gold standard. Kaplan-Meier estimates of disease specific survival (DSS, p=0.002) and overall survival (OS, p<0.001) by RNAscope were similar to the gold standard (DSS, p=0.006, OS, p=0.002) and superior to p16 immunohistochemistry (IHC) or the combination of p16 IHC and DNA qPCR. Conclusions: We demonstrated that High Risk HPV RNAscope can be used to detect oncogenic HPV in FFPE OPSCC samples and has both excellent analytical and prognostic performance against the gold standard test for oncogenic HPV. These results raise the possibility that High Risk HPV RNAscope could be adopted as an ‘international standard’ test for OPSCC in clinical practice. As the oncology community approaches therapeutic de-escalation based on HPV status, such a reliable and efficacious test may have immediate application.
Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva