Oropharyngeal cancer (OPC) incidence is significantly increasing among men. Most cases are caused by human papillomavirus (HPV) and/or tobacco. As there are no proven methods for prevention or early detection, OPCs are diagnosed late (~85% at stages III/IV), requiring intensive chemo-radiation therapy which causes significant morbidity, life-long disabilities, and mortality. To increase patient survival and quality of life, biomarkers are needed to diagnose all OPCs at earlier stages. Viral and host (EPB41L3) gene methylation have been shown to predict cervical HPV infections that progress to CIN2/3 and recently were shown to be associated with AIN grade. We hypothesized that these methylation markers can also distinguish OPC from controls when measured in oral gargle specimens.
We conducted a case control study of 100 pre-treatment male OPC cases (base of tongue [n=49], tonsil [n=46], other OP [n=5]) receiving care at the Moffitt Cancer Center from 2014- 2016. 100 disease free men were age and smoking history matched 1:1 to cases. Oral gargle specimens were collected from cases and controls and FFPE specimens from cases. Tumor and oral gargle extracted DNA was used in bisulfite conversion reactions using the EZ DNA methylation kit. Bisulfite modified DNA was purified and amplified by PCR primers. PCR was performed using the PyroMark PCR kit and products were captured by streptavidin beads in 96-well plates and pyrosequenced using PyroGold reagents. All runs included standard curves as positive controls of 0%, 50%, and 100% methylated human DNA and a non-template control.
HPV 16 L1 and EBP41L3 methylation was measurable in oral gargle specimens and discriminated cases from controls. Oral gargle HPV 16 L1 mean methylation was comparable to levels measured in tumor suggesting that the oral gargle represents the tumor (HPV 16 L1 mean methylation was 61.8 + 27.4 in oral gargles vs 64.9 + 25.4 in tumor). Among oral HPV 16+ cases 98.8% had detection of L1 methylation. In contrast, HPV 16 L1 was not methylated in the three oral HPV 16+ control participants. EBP41L3 methylation among controls was significantly lower than cases (0.99 + 0.62 vs 2.59 + 3.92, controls and cases, respectively). Significant differences in methylation levels by case status remained after stratifying HPV and p16 tumor status, indicating the biomarker detected both HPV-related and unrelated OPC cases. Importantly, oral gargle EBP41L3 methylation distinguished early OPC stage (I/II) from controls (mean methylation 2.1 + 1.5 vs 0.99 + 0.6).
These data suggest that EBP41L3 and HPV 16 L1 methylation measured in an oral gargle specimen may have utility as OPC early detection biomarkers.