Quantitative methylation analysis reveals gender and age differences in p16INK4a hypermethylation in hepatitis B virus-related hepatocellular carcinoma.

Background: Frequent promoter hypermethylation of the inhibitors in either Rb or p53 pathways is associated with the hepatocellular carcinoma (HCC) development. Objective: To quantitatively assess the gradual changes of the promoter methylation of p14ARF, p15INK4b, p16INK4a and CCND2 genes in hepatitis B virus (HBV) infection-related HCC. Methods: A total of 118 pairs of tumour and their corresponding non-tumour tissues were collected from HCC patients with evidence of HBV infection. The promoter methylation status was analysed by combined DNA methylation-sensitive and methylation- dependent restriction endonuclease digestion, followed by subsequential quantitative PCR assay. Results: Promoter hypermethylation frequencies were gradually increased from 6.25% in normal liver tissues to 21.19% in adjacent non-tumour and to 40.68% in tumour tissues for p16INK4a (P = 0.000), and from none to 10.20% and to 29.59% for CCND2 (P = 0.001). The hypermethylation intensities in HCC tissues were also significantly increased (P = 0.0018 for p16INK4a, P = 0.0001 for CCND2). Altogether, 48.93% cases were found with increased hypermethylation intensity of either p16INK4a and/or CCND2 promoter in tumour tissues, compared with their matched non-tumour tissues. In addition, tumour tissue p16INK4a promoter hypermethylation was significantly higher in male than that in female gender patients in frequency (P = 0.041) and was significantly increased in patients older than 50 years of age in intensity (P = 0.0021). No hypermethylation of p14ARF or p15INK4b was found. Conclusion: Our study demonstrated that from normal liver to the adjacent cirrhotic liver and to the HCC tissues, p16INK4a hypermethylation was gradually increased both in frequency and in intensity, such increase might be gender and age related.