Development and Validation of a Novel and Rapid Molecular Detection Method for High-Risk Human Papillomavirus in Formalin-Fixed Paraffin-Embedded Tumor Tissue

Abstract The most widely applied algorithm for human papillomavirus (HPV) detection in formalin-fixed paraffin-embedded (FFPE) specimen of oropharyngeal squamous cell carcinoma (OPSCC) consists of p16INK4A (p16) immunostaining followed by PCR-based detection of high-risk HPV DNA on the p16-immunopositive samples. However, in non-oropharyngeal head and neck squamous cell carcinoma (HNSCC) this algorithm fails, hampering correct interpretation of prevalence and prognosis of HPV in these cases. In this study, we developed and validated a molecular HPV detection method for FFPE specimens of oropharyngeal and non-oropharyngeal HNSCC. Sectioning of the FFPE blocks was circumvented by using punch biopsies from tumor enriched regions of FFPE tissue blocks, and combined extraction was applied to obtain high-quality DNA and RNA from the punch biopsy. Next, PCR-based detection of HPV-DNA was performed for 15 HR-HPV types with subsequent detection of E6 mRNA for validation. The combined DNA/RNA FFPE test of tissue cores (HPV-rTcore assay), was assessed in well-characterized cohorts with known HPV status based on earlier work, i.e. a cohort of OPSCCs (n=80) and oral cavity squamous cell carcinomas (n=25), and reached an accuracy of 97% and 100%, respectively. In conclusion, our method is rapid, simple and shows an excellent diagnostic performance for detection of HPV16. Ultimately, it can be applied for large cohort studies to determine the etiologic fraction and prognostic implication of HPV in non-oropharyngeal HNSCC.