Predicting Hepatocellular Carcinoma by Detection of Aberrant Promoter Methylation in Serum DNA

Abstract

Purpose: Most hepatocellular carcinomas (HCC) are diagnosed at an advanced stage. Hypermethylation of CpG islands in promoter regions is now recognized as an important early event in carcinogenesis and detection of methylated DNA has been suggested as a potential biomarker for early detection of cancer. There are no studies on epigenetic changes in samples from HCC patients before diagnosis. We explored the possible diagnostic value of aberrant promoter hypermethylation of three tumor suppressor genes in serum DNA for early detection of HCC. Experimental Design: Aberrant promoter hypermethylation was investigated in DNA isolated from the serum of 50 HCC patients who provided repeated blood samples before diagnosis and 50 controls enrolled in a cancer screen program inTaiwan. Methylation-specific PCR was used to determine the methylation status of p16, p15, and ras association domain family 1A (RASSF1A). Results: Among cases, aberrant methylation was found in serum DNA 1to 9 years before clinical HCC diagnosis. RASSF1A had the highest frequency of hypermethylation with 35 (70%) cases having at least one positive sample compared with 22 (44%) for p16 and 12 (22%) for p15. Six subjects were hypermethylation negative for all three genes. For the 50 controls, promoter hypermethylation was found in three and two subjects for RASSF1A and p16, respectively; none had methylation of p15. A receiver operating characteristic curve that included clinical risk factors (age, HBsAg status, anti^hepatitis C virus status, smoking, and alcohol status) and hypermethylation biomarkers gave an overall predictive accuracy of 89% with sensitivity and specificity 84% and 94%, respectively. Conclusions: The analysis of epigenetic changes on RASSF1A, p16, and p15 tumor suppressor genes in serum DNA may be a valuable biomarkers for early detection in populations at high risk of HCC.

Disease Progression from Chronic Hepatitis C to Cirrhosis and Hepatocellular Carcinoma is Associated with Increasing DNA Promoter Methylation

“…Moreover, Liu et al (2006) reported the frequency of p15 methylation in (58%) out of 50 HCC cases versus (50%) out of 50 pericancer tissues but they did not include normal controls in their comparison. In contrast to our results, Zhang et al (2007) reported that aberrant methylation was found in serum DNA 1 to 5 years before clinical HCC diagnosis; they reported P15 methylation in 22% out of 50 HCC cases. However, they reported that out of 50 control cases, none had methylation of p15 which is matched with our results (Zhang et al, 2007).…”

“…In contrast to our results, Zhang et al (2007) reported that aberrant methylation was found in serum DNA 1 to 5 years before clinical HCC diagnosis; they reported P15 methylation in 22% out of 50 HCC cases. However, they reported that out of 50 control cases, none had methylation of p15 which is matched with our results (Zhang et al, 2007). On the other hand, Iyer et al (2009) reported P15 methylation frequency in 14.2% HCC Egyptian patients in tissues and 10.7% for plasma.…”

“…Silencing of P15 through promoter methylation may be involved in virus-induced hepatocarcinogenesis (Yang et al, 2003). On the other hand, analysis of epigenetic changes on p15 tumor suppressor gene in serum DNA may be a valuable biomarker for early detection in populations at high risk of HCC (Zhang et al, 2007). In our study, there was an increase in the methylation frequency of P15 gene between the studied groups being 44.2% out of 208 HCC cases with a statistically significant difference between the studied groups.…”

Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma

“…Detection of methylated DNA has then been suggested as a potential biomarker for early detection of cancer. In this study, HCC tumor tissues demonstrated hypermethylation in RASSF1A gene (88.6%, 31/35) and sera (40%, 14/35), which is in concordance with the findings from other investigators (tissue 60-92.5%; serum 42.5-70%) [18][19][20][21][22][23]. Although the exact mechanism of how the tumor DNA enters systemic circulation is unclear, the present result can still suggest that RASSF1A methylation should be a potential marker of incipient malignancy in the human hepatocarcinogenesis since, like any ideal biomarker, it appears early in the course of disease and is detectable in biological samples that can be obtained noninvasively.…”

“…The hypermethylation of RASSF1A suggests new perspectives for the diagnosis of malignant tumor. The methylation of RASSF1A was detected in the serum DNA of HCC patients by Yeo et al [18] and Zhang et al [19], as well as in the tissue DNA of HCC by Zhang et al [20], Di Gioia et al [21], Tischoff et al [22], and Schagdarsurengin et al [23]. But different groups obtained different data for the detection of hypermethylation of RASSF1A in HCC.…”