High-risk Human Papillomavirus E6/E7 MRNA Detection by a Novel in Situ Hybridization Assay Strongly Correlates with P16 Expression and Patient Outcomes in Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV) is established as causative in oropharyngeal squamous cell carcinomas (OSCCs), being detected in 50% to 80% of tumors by DNA in situ hybridization (ISH) and/or polymerase chain reaction. However, these tests do not assess viral transcription. Many consider E6/E7 messenger ribonucleic acid (mRNA) the best indicator of HPV status, but it has not been detected in situ in OSCC. We constructed tissue microarrays (TMAs) from a cohort of OSCC for which p16 immunohistochemistry and HPV DNA ISH were previously performed on whole sections. We utilized a novel, chromogenic RNA ISH assay called RNAscope to detect E6/E7 mRNA of HPV-16 and other high-risk types on these TMAs. RNA ISH results were obtained for 196 of 211 TMA cases, of which 153 (78.1%) were positive. p16 immunohistochemistry and HPV DNA ISH were positive in 79.0% and 62.4% of cases, respectively. Concordance between RNA and p16, DNA and p16, and RNA and DNA were 96.4%, 78.7%, and 83.5%, respectively. Only 7 cases (3.6%) were discrepant between RNA ISH and p16. In univariate analysis, all 3 tests correlated with better overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) (all P<0.001). In multivariate analysis, OS correlated significantly with RNA (hazard ratio=0.39, P=0.001), DNA (0.53, P=0.03), and p16 (0.30, P<0.001), but DSS and DFS correlated significantly only with p16 (DSS: 0.36, P=0.006; DFS: 0.42, P=0.016). RNA ISH is more sensitive than DNA ISH in detecting HPV in OSCC, and it correlates strongly with p16. Although both tests were comparable, p16 more strongly stratified patient outcomes.