Comparative efficacy of p16 versus HPV RNA in situ hybridization in oropharyngeal squamous cell carcinomas

The accurate determination of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) has profound prognostic and therapeutic implications. As the incidence of HPV-associated OPSCC continues to rise globally, particularly in developed nations, the need for precise, reliable biomarkers to distinguish HPV-driven malignancies has become increasingly critical. While p16 immunohistochemistry (IHC) has long served as a surrogate marker for HPV involvement, concerns over its specificity given its ability to overexpress in HPV-negative tumors have prompted interest in more direct molecular assays. Among these, HPV RNA in situ hybridization (ISH) offers a promising technique capable of detecting transcriptionally active viral RNA within the tumor microenvironment, directly reflecting oncogenic activity. This study conducts a comparative analysis of p16 IHC and HPV RNA ISH in a cohort of OPSCC patients, examining concordance rates, diagnostic sensitivity and specificity, and their association with clinical outcomes such as overall survival and recurrence-free interval. While p16 IHC maintains high sensitivity and remains cost-effective, RNA ISH demonstrates superior specificity and a closer correlation with disease progression and patient stratification in treatment protocols. Our results reveal discordance in a subset of cases, underscoring the limitations of relying solely on p16 status for clinical decision-making. Incorporating both assays in a dual-testing algorithm may optimize diagnostic accuracy, enhance prognostic stratification, and better inform tailored therapeutic strategies. The findings highlight the importance of nuanced biomarker selection in HPV-driven head and neck cancers and advocate for standardized molecular pathology workflows that integrate both surrogate and direct detection modalities.