Methylation of HPV 16 and EPB41L3 in oral gargles: Associations with oropharyngeal cancer detection and tumor characteristics

Abstract

Oropharyngeal cancer (OPC) incidence is increasing significantly among men and often requires intensive therapy causing significant morbidity. Early detection of OPC is needed, when monotherapy can be safely delivered with less treatment‐associated morbidity, while maintaining high cure rates. We conducted a study of 101 pretreatment male OPC cases matched 1:1 to 101 disease‐free controls for age and smoking history. Oral gargles were collected from cases and controls with additional biopsies or aspirates from cases. The HPV SPF10‐LiPA25 PCR assay was utilized for HPV genotyping. Methylation of three CpG sites (438, 427 and 425) in the EPB41L3 gene and methylation status of the L1 (6,367, 6,389), L2 (4,257, 4,262, 4,266, 4,269, 4,275, 4,282) and E2 (3,412, 3,415, 3,417, 3,433, 3,436) CpG sites of HPV 16 positive specimens was assessed by pyrosequencing. Significant correlations were observed between tumor and oral specimens for all methylation biomarkers (p < 0.01). EPB41L3 and HPV 16 L1, L2 and E2 methylation were significantly (p < 0.0001) higher among cases than controls, regardless of early vs. late disease. When HPV 16 genes and EPB41L3 methylation status were combined in a logistic regression analysis, a sensitivity of 70.3% and a specificity of 90.9% were observed for the detection of OPC from an oral gargle. Our data suggest that methylation biomarkers measured in oral gargles may have utility in identifying OPC early. Future studies are needed to replicate these findings and to inform additional biomarkers that can maximize specificity and sensitivity for early OPC detection.

DNA Methylation Changes in Human Papillomavirus-Driven Head and Neck Cancers

“…HPV DNA methylation in these sites has not been comprehensively investigated in HPV-driven HNC. However, available literature suggests similar methylation patterns in HNC [54,155]. However, a functional relationship between CpG methylation of these sites and tumorigenesis has not been identified to date.…”

“…Cervical cancer studies have suggested that methylation changes in HPV L1 and L2 genes can be used to discriminate cervical cancer from low-grade precancer [153,154]. Even though HNC studies have also reported similar findings, these associations have not been comprehensively investigated [54].…”

Pivotal roles of protein 4.1B/DAL‑1, a FERM‑domain containing protein, in tumor progression (Review)

“…Using transgenic mouse models of pancreatic b-cell carcinogenesis (Rip1Tag2), a loss of 4.1B has been reported in the phenotypic transition from adenoma to carcinoma (75). In addition, EPB41L3 and HPV 16 methylation are markedly higher amongst oropharyngeal cancer (OPC) with high sensitivity and specificity for the detection of OPC from an oral gargle, suggesting that measurements of HPV 16 and EPB41L3 methylation have utility in identifying early OPC (77). High methylation of EPB41L3 is also associated with anal intraepithelial neoplasia (78).…”

Human Papillomavirus and Cellular Pathways: Hits and Targets

“…Similarly, gargle and biopsy samples from OPSCC male patients were analyzed for HPV-status and type as well as methylation at various CpG sites in order to understand the potential for early cancer detection through analysis of methylation biomarkers. A strong correlation was observed between OPSCC biopsy samples and gargles, suggesting a potential early, non-invasive screening method [194].…”