We studied DNA methylation patterns of human papillomavirus (HPV) and tumor suppressor gene EPB41L3 in 148 anal and perianal biopsies to determine whether high levels of methylation would be associated with anal intraepithelial neoplasia (AIN).
DNA was extracted from tissue specimens and HPV genotype was determined by the PapType kit (Genera Biosystems Ltd). DNA was treated with bisulfite, amplified by PCR and levels of methylation were measured by pyrosequencing. Univariate and bivariate logistic models were fitted for statistically significant HPV and human genes with the outcome measures.
The most prevalent HPV type was HPV16, detected in 54% of the 30 benign biopsies, 33% of the 43 low-grade AIN (lgAIN), 82% of the 59 high grade AIN (hgAIN) and 4 of the 5 anal cancers. A methylation score was developed (0.561*HPV16+0.439*EPB41L3) which had increasing values with severity of disease: the mean was 8.1% in benign, 13.2% in lgAIN, 22.3% in hgAIN and 49.3% in cancers (p<0.0001). The methylation score as a triage classifier at a cut-off of 8.8 gave a sensitivity of 90.6% (95% CI: 82.8, 96.9), specificity of 50.7% (95% CI: 39.7, 61.6) and area under the curve of 0.82 (95% CI: 0.75–0.89) for separating hgAIN and cancer from benign and lgAIN biopsies.
We conclude that methylation of HPV16 and EPB41L3 show highly significant association with increasing severity of AIN and cancer and may be useful as biomarkers in anal disease.