Scarce data exist on host gene methylation in oropharyngeal squamous cell carcinomas (OPCs), which are caused by human papillomavirus (HPV) in approximately 40% of cases. We analyzed the methylation status of two host genes involved in cervical carcinogenesis, FAM19A4 and miR124-2, in formalin-fixed paraffin-embedded (FFPE) tissues of primary HPV-driven OPCs (i.e., HPV-positive and p16INK4A-positive), HPV-negative OPCs, head and neck (HN) squamous cell papillomas, and oral rinse-and-gargles (ORGs) from individuals without HN lesions.

A multiplex real-time methylation-specific PCR on bisulfite-converted DNA was employed (PreCursor-M+, Fujirebio). Hypermethylation for each target was expressed as negative/positive based on the ΔΔCt, as well as by the ΔΔCt ratio (2^-ΔΔCt).

A total of 70 HPV-driven OPCs (66 HPV16+, 94.3%), 71 HPV-negative OPCs, 12 HN papillomas, and 20 ORGs were evaluated. Six of the 153 FFPE-purified DNA samples (3.9%) yielded invalid results. Hypermethylation for at least one target gene was observed in 56 of 69 valid HPV-driven OPCs (81.2%) and 33 of 67 valid HPV-negative OPCs (49.2%; p < 0.0001). None of the papillomas or ORGs were hypermethylated. HPV-driven OPCs showed a significantly higher methylation level compared with HPV-negative OPCs for both FAM19A4 (median ΔΔCt ratio 11.95 vs. 5.49; p = 0.0001) and miR124-2 (median ΔΔCt ratio 15.65 vs. 8.27; p < 0.0001).

Our findings indicate that FAM19A4/miR124-2 hypermethylation occurs exclusively in tumor samples and that methylation levels are significantly higher in HPV-driven OPCs than in HPV-negative OPCs.