Molecular Etiology and Clinical Significance of Discordant Human Papillomavirus Testing in p16+ Oropharyngeal Cancers

PURPOSE: Routine confirmatory testing of p16+ oropharyngeal cancers (OPCs) for human papillomavirus (HPV) mRNA is nonstandard despite evidence that negative confirmatory tests are common and may indicate poor prognosis. This study aimed to evaluate the utility of confirmatory testing by assessing the etiologies and prognostic significance of negative HPV mRNA tests in a surgically treated cohort of p16+ OPCs enriched for recurrence-prone tumors. METHODS: Fifty tumors that later recurred (cases) and 50 cured tumors (controls) were matched for key clinical features. Patients were predominantly White males with a median age of 62 years. Confirmatory testing was performed with a clinically used E6/E7 RNA in situ hybridization assay (RNAScope). RNA sequencing (RNAseq) was used to define true HPV positivity and profile HPV mRNA levels. HPV genotypes and integration were evaluated by sequencing HPV DNA and detecting HPV-host fusion mRNAs, respectively. RESULTS: < .0007). False-negative confirmatory assays, which occurred in 6% (n = 6) of the cohort, were equally distributed between groups and not prognostic of survival. False negatives included three tumors containing HPV types absent from the RNAScope probe set and an episomal tumor with E6/E7 mRNA below the detection threshold by RNAseq. CONCLUSION: HPV RNAScope appears unlikely to detect HPV status misclassification or be prognostic in typical p16+ OPCs treated surgically in the United States. Confirmatory RNAScope can produce misleading false negatives in this population through various mechanisms and should be used selectively rather than routinely.