Rnascope hpv hr18 probe

All cases were screened for high-risk HPV by immunohistochemistry for p16 (clone INK4a; Ventana, Tucson, AZ; prediluted by manufacturer). Nuclear and cytoplasmic staining in at least 70% of tumor cells was considered positive.(23 (link)) For cases that were p16-positive, in situ hybridization for high-risk HPV DNA was performed. Sections of 5 μm from formalin-fixed and paraffin-embedded tissue blocks were evaluated for the presence of HPV DNA with an automated protocol utilizing the Ventana HR HPV III probe set that captures HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66 (Ventana Medical Systems, Tucson, AZ). Finally, in cases where p16 immunochemistry was positive but in-situ hybridization for high-risk HPV DNA was negative, a second methodology for detection of viral nuclei acids was utilized for confirmation of HPV status. Sections of 5 μm from formalin-fixed and paraffin-embedded tissue blocks were evaluated for the presence of HPV RNA using a manual RNAscope HPV-HR18 Probe (Advanced Cell Diagnostics, Hayward, CA) which recognizes 18 high-risk HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82). HPV positive controls included HPV 16-positive head and neck squamous cell carcinoma as well as the HPV 16-positive SiHa and CaSki cell lines. An HPV-negative head and neck squamous cell carcinoma served as a negative control.