Purpose: Human papillomavirus-16 (HPV16) is the causative agent in a biologically distinct subset of oropharyngeal squamous cell carcinoma (OPSCC) with highly favorable prognosis. In clinical trials, HPV16 status is an essential inclusion or stratification parameter, highlighting the importance of accurate testing.Experimental Design: Fixed and fresh-frozen tissue from 108 OPSCC cases were subject to eight possible assay/assay combinations: p16 immunohistochemistry (p16 IHC); in situ hybridization for highrisk HPV (HR HPV ISH); quantitative PCR (qPCR) for both viral E6 RNA (RNA qPCR) and DNA (DNA qPCR); and combinations of the above.Results: HPV16-positive OPSCC presented in younger patients (mean 7.5 years younger, P ¼ 0.003) who smoked less than HPV-negative patients (P ¼ 0.007). The proportion of HPV16-positive cases increased from 15% to 57% (P ¼ 0.001) between 1988 and 2009. A combination of p16 IHC/DNA qPCR showed acceptable sensitivity (97%) and specificity (94%) compared with the RNA qPCR "gold standard", as well as being the best discriminator of favorable outcome (overall survival P ¼ 0.002). p16 IHC/HR HPV ISH also had acceptable specificity (90%) but the substantial reduction in its sensitivity (88%) impacted upon its prognostic value (P ¼ 0.02). p16 IHC, HR HPV ISH, or DNA qPCR was not sufficiently specific to recommend in clinical trials when used in isolation.Conclusions: Caution must be exercised in applying HPV16 diagnostic tests because of significant disparities in accuracy and prognostic value in previously published techniques.

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Background:Human papillomavirus (HPV) testing in oropharyngeal squamous cell carcinoma (OPSCC) is now advocated. Demonstration of transcriptionally active high-risk HPV (HR-HPV) in fresh tumour tissue is considered to be the analytical ‘gold standard'. Clinical testing has focused on formalin-fixed paraffin-embedded (FFPE) tissue at the expense of sensitivity and specificity. Recently, a novel RNA in situ hybridisation test (RNAscope) has been developed for the detection of HR-HPV in FFPE tissue; however, validation against the ‘gold standard' has not been reported.Methods:A tissue microarray comprising FFPE cores from 79 OPSCC was tested using HR-HPV RNAscope. Analytical accuracy and prognostic capacity were established by comparison with the reference test; qRT–PCR for HR-HPV on matched fresh-frozen samples.Results:High-risk HPV RNAscope had a sensitivity and specificity of 97 and 93%, respectively, against the reference test. Kaplan–Meier estimates of disease-specific survival (DSS, P=0.001) and overall survival (OS, P<0.001) by RNAscope were similar to the reference test (DSS, P=0.003, OS, P<0.001) and at least, not inferior to p16 immunohistochemistry +/− HR-HPV DNA-based tests.Conclusion:HR-HPV RNAscope demonstrates excellent analytical and prognostic performance against the ‘gold standard'. These data suggest that the test could be developed to provide the ‘clinical standard' for assigning a diagnosis of HPV-related OPSCC.

Background:There is relatively little methylation array data available specifically for oral squamous cell carcinoma (OSCC). This study aims to compare the DNA methylome across a large cohort of tumour/normal pairs.Methods:DNA was extracted from 44 OSCCs and paired normal mucosa. DNA methylation analysis employed the Illumina GoldenGate high-throughput array comprising 1505 CpG loci selected from 807 epigenetically regulated genes. This data was correlated with extracapsular spread (ECS), human papilloma virus (HPV) status, recurrence and 5-year survival.Results:Differential methylation levels of a number of genes distinguished the tumour tissue sample from the matched normal. Putative methylation signatures for ECS and recurrence were identified. The concept of concordant methylation or CpG island methylator phenotype (CIMP) in OSCC is supported by our data, with an association between ‘CIMP-high' and worse prognosis. Epigenetic deregulation of NOTCH4 signalling in OSCC was also observed, as part of a possible methylation signature for recurrence, with parallels to recently discovered NOTCH mutations in HNSCC. Differences in methylation in HPV-driven cases were seen, but are less significant than that has been recently proposed in other series.Conclusion:Although OSCC seems as much an ‘epigenetic' as a genetic disease, the translational potential of cancer epigenetics has yet to be fully exploited. This data points to the application of epigenetic biomarkers and targets available to further the development of therapy in OSCC.