Identifying Truly HPV-Driven Head and Neck Squamous Cell Carcinoma by QuantiGene-Molecular-Profiling-Histology Assay Allows for More Precise Prognosis Prediction

Head and neck squamous cell carcinoma (HNSCC) with discordant diagnostic patterns of HPV⁺/p16^- or HPV^-/p16⁺ correlate with worse prognosis. This study aims to identify truly HPV-driven HNSCCs using a QuantiGene-Molecular-Profiling-Histology (QG-MPH) assay for identifying transcriptionally active HPV. Of 97 FFPE samples analyzed, 68 were valid for HPV DNA detection by PCR and quantification of HPV E7 and p16 ^INK4a mRNA by QG-MPH. p16 ^INK4a mRNA expression was compared with p16 protein expression via immunohistochemistry (p16 IHC). Among the 68 cases, 26 (38.2%) showed increased high-risk HPV E7 mRNA expression (hrHPV E7 mRNA⁺), while 37 (54.4%) were hrHPV DNA⁺. Concordance between HPV DNA and mRNA status was 70.1%. Notably, 79.2% of E7 mRNA⁺ cases were p16 IHC⁺, compared to 55.9% of DNA⁺ cases, demonstrating better concordance between HPV E7 mRNA⁺ status and p16 mRNA expression plus p16 IHC positivity. All patients (19/19) in the HPV E7 mRNA⁺/p16 IHC⁺ group survived the 5-year follow-up, compared to 59.5% (22/37) in the HPV E7 mRNA^-/p16 IHC^- group (<i>p</i> = 0.001). Specifically, the OS rate was 57.1% (8/14) in the group with discordant HPV DNA and p16 IHC results, compared to 40% (3/5) in the group with discordant HPV E7 mRNA and p16 IHC results. These findings highlight the better outcomes for the transcriptionally active HPV cases and indicate the prognostic disadvantage for patients with discordant patterns and the advantages for incorporating the molecular mRNA profiling by QG-MPH to p16 IHC. In conclusion, QG-MPH quantification of E7 and p16 ^INK4a mRNA more precisely identifies truly HPV-driven from non-HPV-driven HNSCC, compared to HPV DNA testing alone or with p16 IHC, which reduces misclassification and provides valuable implications for improved prognosis prediction and therapeutic decision-making.