Validation of a novel diagnostic standard in HPV positive Oropharyngeal Squamous Cell Carcinoma


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Andrew Schache1, Triantafilos Liloglou1, Janet Risk1, Terence Jones1, Xiao-Jun Ma3, Hongwei Wang3, Son Bui3, Yuling Luo3, Philip Sloan2, Richard Shaw1, Max Robinson2
1University of Liverpool, Liverpool, UK, 2Newcastle University, Newcastle, UK, 3Advanced Cell Diagnostics, Hayward, CA, USA

Background

HPV testing is widely advocated in oropharyngeal squamous cell carcinoma (OPSCC) workup. The ‘gold standard’ for oncogenic HPV detection remains demonstration of transcriptionally active high-risk HPV in fresh tumour tissue. For clinical utility however, testing strategies have necessarily focused on formalin-fixed paraffin-embedded (FFPE) tissue at the expense of 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).

Method

A tissue-microarray comprising FFPE cores from 79 OPSCC was tested using High-Risk HPV RNAscope (Advanced Cell Diagnostics) 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.

Conclusion

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 considers therapeutic de-escalation based on HPV status, such a reliable and efficacious test may have immediate application.