EVALUATION OF HYPERMETHYLATION OF RAS ASSOCIATION DOMAIN FAMILY-1 A AND GLUTATHIONE S-TRANSFERASE PROTEIN-1 GENES AS DIAGNOSTIC MARKER FOR HEPATOCELLULAR CARCINOMA

Background and study aims: The molecular pathogenesis of HCC involves well-defined genetic and epigenetic alterations. The Ras association domain family 1A (RASSF1A) and the Glutathione S-transferase P 1 (GSTP1) genes are two tumour suppressor genes that are reported to be silenced by CpG island promoter hypermethylation which is a key to the tumourigenic process in HCC. The aim of this study was to analyze the methylation frequency of RASSF1A and GSTP1 genes in early stages of HCC , chronic hepatitis C and healthy subjects to evaluate its value as a diagnostic marker for early HCC. Patients and methods: Methylation-specific polymerase chain reaction (MSP) was used to detect RASSF1A and GSTP1 promotor methylation in DNA extracted from plasma samples of 25 patients with HCC, 25 patients with chronic hepatitis C and 25 healthy controls. Assessment of alpha fetoprotein (AFP) was performed in all groups by ELISA using commercially available kits. Results: Methylated RASSF1A was detected in 76 % of the HCC group (19/25), in 20% of the chronic hepatitis C patients (5/25) and in 16% of the healthy controls(4/25). The methylation frequencies were significantly higher in patients with HCC compared to the controls (P ≤ 0.001) and chronic hepatitis C patients ( P ≤ 0.001). While methylated GSTP1 was detected in 44% of the HCC group(11/25) ,in 12% of the chronic hepatitis C group (3/25) and in 8% of the controls (2/25). Although the sensitivity and specificity, for each gene as an epigenetic biomarker was moderate (76% and 44% for RASSF1A and GSTP1 respectively), the combination analysis of both genes resulted in an increased sensitivity and specificity to 88%,and 76% respectively) in discriminating HCC from normal control and chronic hepatitis C pateints. As regard AFP, Receiver operating characteristic curves were plotted and showed an optimal cutoff value of 9.5 ng/ml with sensitivity of 88 % and specificity of 58% when the area under the receiver operator characteristic (AUROC) curve was 0.87 with 95% Confidence Interval .Conclusion: The epigenetic changes observed in this study indicate that examination of methylation status of RASSF1A and GSTP1 could be of value for early diagnosis of HCC especially when using a combination of more than one epigenetic marker .