Hypermethylation of suppressor of cytokine signaling 1 in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC), the most common primary hepatic tumor, is highly prevalent in the Asia-Pacific region, including Thailand. Many genetic and epigenetic alterations in HCC have been elucidated. The aim of this study was to determine whether aberrant methylation of the suppressor of cytokine signaling 1 gene (SOCS1) occurs in HCCs. Methylation specific-PCR assays were performed to identify the methylation status of SOCS1 in 29 tumors and their corresponding normal liver tissues. An abnormal methylation status was detected in 17 (59%), with a higher prevalence of aberrant SOCS1 methylation significantly correlating with HCC treated without chemotherapy (OR=0.04, 95%CI=0.01-0.31; P=0.001). This study suggests that epigenetic aberrant SOCS1 methylation may be a predictive marker for HCC patients.

Histological type of intrahepatic cholangiocarcinoma differentiated by genetic alteration from AP-PCR fingerprint.

Cholangiocarcinoma (CCA), the malignant neoplasm of the biliary epithelium, is usually fatal due to difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of CCA are not well understood and only a few cytogenetic studies have been published. In this study, genomic instability in 30 Thai cases of intrahepatic cholangiocarcinoma (ICC) was assessed using an arbitrarily primed- polymerase chain reaction (AP-PCR) method. Genetic alterations were analyzed as banding pattern changes between tumors and corresponding normal DNA. The abnormal band present at the highest frequency (23/30 cases, 77%) appeared with the AO16 primer. Statistical analysis also showed that DNA alteration from this primer was significantly associated with the moderately to poorly differentiated histological type (P=0.038). Kaplan-Meier survival curves showed borderline significance for this DNA aberration (P=0.06 by the log-rank test). This DNA fragment may thus be of use to predict degree of malignancy of the disease.

Ubiquitin-specific protease 14 expression associated with intrahepatic cholangiocarcinoma cell differentiation.

The purpose of this study was to identify the gene alterations amplified from AO16 primer and examine whether the expression patterns of USP14 in clinical specimens from patients with intrahepatic cholangiocarcinoma (ICC) is associated with cancer cells. DNA from tumor and corresponding normal tissues of 52 patients was amplified with 33 arbitrary primers. The DNA fragment that altered most frequently in ICC was cloned, sequenced, and identified by comparison with known nucleotide sequences in the genome database. The DNA copy numbers of the allelic alterations in cholangiocarcinoma were determined by quantitative real-time PCR and interpreted as allelic loss or DNA amplification by comparison with the reference gene. Associations between allelic imbalance and clinicopathological parameters of ICC patients were evaluated by X²-tests. The Kaplan-Meier method was used to analyze survival rates. Immunohistochemically, USP14 showed weak cytoplasmic staining in normal bile duct epithelial cells. It was strongly detected in 21 cancer patients (43.8%). There were correlations between USP14 expression level and the clinicopathological features of ICC, histological grade (P < 0.05). However, there were no significant differences in age, gender, tumor size, metastasis, lymph node metastasis, and staging. USP14 expression was related to cholangiocarcinoma cell differentiation. Due to their emerging role in control of multiple signaling pathways and oncoproteins, USP14 inhibitors may be useful for anticancer agents.

RASSF1A promoter hypermethylation as a prognostic marker for hepatocellular carcinoma.

This study was performed to determine whether epigenetic aberrant methylation of RASSF1A might be associated with hepatocarcinogenesis. Methylation specific-PCR was performed to identify RASSF1A promoter hypermethylation in 29 tumors and corresponding normal liver tissues. In addition, RASSF1A mRNA levels were analyzed by quantitative real-time reverse transcription-PCR. Aberrant methylation of RASSF1A was detected in 25 of 29 cases (86%), with loss of RASSF1A expression evident in 8 of 22 cases (36%). No correlation between loss of RASSF1A mRNA and promoter hypermethylation of the RASSF1A gene was observed. There was a significant correlation between the methylation status of RASSF1A and hepatocellular carcinoma (HCC) patients who did not undergo chemotherapy (P = 0.03). Multivariate analysis, adjusted for tumor size, treatment, RASSF1A hypermethylation, and RASSF1A under-expression, showed RASSF1A hypermethylation to be assocaited with a better prognosis for HCC patients (HR= 0.089, 95%CI = 0.013-0.578; P = 0.012). Our findings showed that RASSF1A promoter hypermethylation occurs frequently, and may serve as a good prognostic factor.

Prognostic value of DNA alterations on chromosome 17p13.2 for intrahepatic cholangiocarcinoma.

AIM: To characterize and evaluate DNA alterations among intrahepatic cholangiocarcinoma (ICC) patients. METHODS: DNA from tumor and corresponding normal tissues of 52 patients was amplified with 33 arbitrary primers. The DNA fragment that alters most frequently in ICC was cloned, sequenced, and identified by comparison with known nucleotide sequences in the genome database (www.ncbi.nlm.nih.gov). The DNA copy numbers of the allelic alterations in cholangiocarcinoma were determined by quantitative real-time PCR and interpreted as allelic loss or DNA amplification by comparison with the reference gene. Associations between allelic imbalance and clinicopathological parameters of ICC patients were evaluated by chi2-test. The Kaplan-Meier method was used to analyze survival rates. RESULTS: From 33 primers, an altered DNA fragment (518 bp) amplified from BC17 random primer was found frequently in the tumors analyzed and mapped to chromosome 17p13.2. Sixteen of 52 (31%) cases showed DNA amplification, while 7 (13%) showed allelic loss. Interestingly, DNA amplification on chromosome 17p13.2 was associated with a good prognosis, median survival time (wk) of amp vs no amp was 44.14 vs 24.14, P = 0.002; whereas allelic loss of this DNA sequence corresponded with a poor prognosis, median survival time (wk) of loss vs no loss was 18.00 vs 28.71, P = 0.019). Moreover, Kaplan-Meier curves comparing the DNA alterations with survival depicted highly significant separation that the median survival time equal to DNA amplification, allelic loss, and normal was 44.14 wk, 18.00 wk, and 24.29 wk, respectively (P = 0.005). CONCLUSION: Alterations in the DNA sequence on chromosome 17p13.2 may be involved in cholangio-carcinogenesis, and could be used as a prognostic marker in the treatment of ICC patients.