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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 . INTRODUCTION epatocellular carcinoma (HCC) is the fifth most common and the third most fatal malignancy worldwide (1). While the incidence of many cancers is declining, HCC incidence continues on the upward trajectory over the last three decades in many countries. Over 80% of hepatocellular carcinoma cases are attributable to four major causes: infection with hepatitis C (HCV) or B (HBV) viruses, chronic alcohol consumption, and/or exposure to aflatoxin B1 (2). It is widely believed that the continuous epidemic of HCV largely accounts for the observed increase in HCC incidence(3). It has been stated that Egypt has the highest prevalence of hepatitis C worldwide and the epidemic will soon peak (4). Although the prognosis of patients with HCC has marginally improved over the last few decades, the 5-year survival rate remains poor as a result of late diagnosis. The majority of patients with advanced HCC do not survive for longer than 6 months from the time of diagnosis (5 ). Late presentation remains an important obstacle to successful treatment (6). Thus, early diagnosis of HCC represents the best opportunity for reduction of the worldwide burden of this disease on the human population (7). In this regard, biomarkers have been developed for early HCC detection (8 ). Alpha-Fetoprotein (AFP) has been a classic blood tumor marker in HCCs for a long period, but both false-positive and false-negative results occur; markers with high sensitivity and specificity should be developed (9). It is well known that many types of tumours shed cells and cellular material (including DNA) into the blood. Therefore, several strategies have been used for the detection of occult cancers using DNA from blood (10). The molecular pathogenesis of HCC involves well-defined genetic and epigenetic alterations (2).A few number of genetic lesions have been identified in the molecular mechanisms of hepatocarcinogenesis such as p53 and Rb alterations. Hypermethylation of Glutathione Stransferase p1 gene (GSTP1),which codes for as an enzyme that plays roles in detoxication and carcinogenesis; and Ras association domain family 1 A (RASSF1A) gene which blocks cell cycle , have been proved in HCC (11). The inactivation of RASSF1A and GSTP1 promoters by methylation was detected in the plasma DNA as well as in the tissue DNA of HCC patients (12). H Z.U.M.J.Vol. 21; No.3 May; 2015 Evaluation Of Hypermethylation Of Ras 280 The aim of this study was to analyze the methylation frequency of RASSF1A and GSTP1 genes in plasma DNA in early stages of HCC , chronic hepatitis C liver disease and healthy subjects to evaluate its value as a diagnostic marker for early HCC. MATERIALS AND METHODS Patients and Control Subjects. This study included a total of 75 plasma samples. Twenty five of them were obtained from patients with early stages of hepatocellular carcinoma (nineteen males and six females with mean age 54.7 ± 6.8 years). Twenty five from chronic hepatitis C virus pateints (thirteen males and twelve females with mean age (51.3 ± 8.2). Twenty five age and sex matched healthy volunteers were included as control group (Seventeen males and eight females with mean age 44.2±1.1 years). The patients were selected from outpatient clinics of Mansoura University Hospitals between September 2013 and October 2014. Diagnosis of cases of HCC was based on the revised version of the Barcelona-Clinic Liver Cancer (BCLC) system, released by the American Association for the Society of Liver Diseases (AASLD) (13). A total of 5ml blood samples were collected in both EDTA tubes (for plasma) and EDTA-free tubes (for serum).The plasma samples were centrifuged at 2000 rpm (5 minutes), and plasma was carefully transferred into 2 ml microtubes. Samples were centrifuged in a microcentrifuge at 5070 rpm (10 minutes). All samples were processed within two hours after collection. Centrifugation steps were performed at 4°C and supernatants were stored at −80°C until analysis was performed. The sera tubes were coagulated during approximately 1h, after which the serum were separated and stored for Estimation of the level of the hepatic tumour marker AFP : by ELISA . DNA Extraction from Plasma Samples. Circulating cell free DNA was isolated from 1 ml plasma using QIAamp MinElute Virus Spin Kit (QIAGEN Catalog no. 57704) according to Sambrook and Russell , (14) and eluted in 50μL sterile water following the manufacturers' protocol. The concentration of isolated DNA was determined spectrophotometrically using NanoPhotometer® P-Class (Implen, Germany) according to Sambrook et al., (15). Bisulfite Conversion of DNA. Bisulfite modification and MSP were conducted based on the principle that bisulfite treatment of DNA converts unmethylated cytosine residues into uracil, whereas methylated cytosine residues would remain unmodified (16). Thus, after bisulfite conversion, methylated and unmethylated DNA sequences would be distinguishable by sequence-specific primers. Bisulfite treatment was conducted using the EpiTect Bisulfite Kit (QIAGEN Catalog no. 59104) (17). Methylation-specific PCR (MSP): MSP reaction was performed according to method by (16) using The EpiTect MSP Kit (2X) provided by QIAGEN, (cat No. 59305). Bisulfite-modified DNA was amplified using primers specific for the methylated RASSF1A or GSTP1 sequence. Gene-specific primers were purchased from Oligo, Macrogen. Primer sets for the PCR amplification genes were selected based on published sequences (table 1). Z.U.M.J.Vol. 21; No.3 May; 2015 Evaluation Of Hypermethylation Of Ras 281 Table 1: Sequence of all primers used in the experiment . Gene Primer sequence (5′–3′) Product size

zagazig university medical journal

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