Analysis of DNA Methylation in Plasma for Monitoring Hepatocarcinogenesis

Aim: To explore whether the aberrant DNA methylation status in plasma could be used as a biomarker for hepatocellular carcinoma (HCC) screening among high-risk individuals. Methods: The promoter methylation status of ELF, RASSF1A, p16, and GSTP1 was investigated by methylation-specific polymerase chain reaction (PCR) in 34 paired HCC and nontumor liver tissue from HCC patients and 10 tissues from patients with liver cirrhosis (LC). Plasma samples from 31 HCC patients, 10 LC patients as well as 7 patients with benign hepatic conditions were also collected and characterized using the same method. Results: Among liver specimens, HCC tissues displayed a significantly higher methylation frequency of each gene compared with nontumor tissue (p<0.05). Moreover, the frequency was much higher in tumor tissues than in nontumor tissue, when the data from two or three genes were combined (p=0.001 and p<0.001, respectively). Among plasma samples, either the frequency of at least one methylated gene (p<0.001) or the average number of methylated genes (p<0.05) demonstrated a stepwise increase in patients with benign lesions, LC, and HCC. Furthermore, when positive results, that is, plasma methylation status of at least one gene were combined with the elevated AFP(400) level (serum alpha-fetoprotein [AFP] level at a cutoff of 400ng/mL), the diagnostic sensitivity of HCC could increase to 93.55%. Conclusions: These results suggested that the methylation of tumor suppressor genes may participate in the development and progression of HCC. Additionally, it may be useful to combine the plasma DNA methylation status of a panel of gene markers and the serum AFP for HCC screening.