Pisit Tangkijvanich

Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis

Genome-wide losses of DNA methylation have been regarded as a common epigenetic event in malignancies and may play crucial roles in carcinogenesis. Limited information is available on the global methylation status in normal tissues and other cancer types beyond colonic carcinoma. Here we applied the combined bisulfite restriction analysis PCR to evaluate the methylation status of LINE-1 repetitive sequences in genomic DNA derived from microdissected samples from several human normal and neoplastic tissues. We found that methylation of LINE-1 in leukocytes was independent of age and gender. In contrast, normal tissues from different organs showed tissue-specific levels of methylated LINE-1. Globally, most carcinomas including breast, colon, lung, head and neck, bladder, esophagus, liver, prostate, and stomach, revealed a greater percentage of hypomethylation than their normal tissue counterparts. Furthermore, DNA derived from sera of patients with carcinoma displayed more LINE-1 hypomethylation than those of noncarcinoma individuals. Finally, in a colonic carcinogenesis model, we detected significantly greater hypomethylation in carcinoma than those of dysplastic polyp and histological normal colonic epithelium. Thus, the methylation status is a unique feature of a specific tissue type and the global hypomethylation is a common epigenetic process in cancer, which may progressively evolve during multistage carcinogenesis.

Clinical Characteristics and Prognosis of Hepatocellular Carcinoma Analysis Based on Serum Alpha-fetoprotein Levels

The purpose of this study was to determine whether a relation does exist between clinicopathologic features and the prognosis of hepatocellular carcinoma (HCC) with respect to serum alpha-fetoprotein (AFP) levels at diagnosis. We reviewed the clinical data of 309 pathologically proven HCC cases divided into three groups: group 1 with normal AFP (<20 IU/mL), group 2 with moderately elevated AFP (20–399 IU/mL) and group 3 with markedly elevated AFP (≥400 IU/mL). Of these, there were 76 (24.6%), 78 (25.2%), and 155 patients (50.2%) in groups 1, 2, and 3, respectively. We found that HCC patients with high AFP tended to have greater tumor size, bilobar involvement, massive or diffuse types, and portal vein thrombosis. Nonetheless, we could not establish a correlation between increased AFP and Okudas stages, degree of tumor differentiation, or extrahepatic metastasis. The median survival rates in groups 1 (6 months) and 2 (7 months) were significantly longer than that of group 3 (3 months). On multivariate logistic regression analysis, positive hepatitis B surface antigen (HBsAg) status and bilobar tumor involvement represented the independent factors for predicting high AFP values. We concluded that AFP is useful not only for diagnosis, but also as a prognostic indicator in patients with HCC . However, it cannot be considered a sensitive tumor marker, particularly during the early stages in HBsAg-negative patients.

Association of HLA-DRB1*13 and TNF-α gene polymorphisms with clearance of chronic hepatitis B infection and risk of hepatocellular carcinoma in Thai population

Summary.  Considerable evidence suggests that host genetic factor play an important role in the pathogenesis and clinical outcome of chronic hepatitis B virus (HBV) infection in several ethic groups. Association study was performed included 150 chronic HBV patients, 100 resolved hepatitis B and 150 healthy individuals with similar ethic background. Interestingly, human leucocyte antigen (HLA)‐DR13 show a strong association with the clearance of HBV [odds ratio (OR) = 0.04, 95% confidence interval (CI) = 0.00–0.26, corrected P‐value (Pc) = 0.0008] similar to reports from several ethic groups. TNF‐α promoter polymorphisms (−863, −308 and −238) were also analysed. Only −863 C allele was found to be significantly decreased in chronic HBV patients compared with healthy control (Pc = 0.03, OR = 0.54, 95% CI = 0.35–0.84 respectively). This −863C allele was not in linkage disequilibrium with HLA‐DR13 suggesting that other genetic markers linked with −863C might influence clearance of chronic HBV infection in Thai. When stratified chronic HBV patients into patients without hepatocellular carcinoma (HCC) and with HCC, the −863 A allele was significantly increased in the HCC group compared to healthy control (Pc = 0.003, OR = 2.61, 95% CI = 1.49–4.60). Haplotype analysis (−863/−308/−238) revealed that the homozygosity of the haplotype (CGG/CGG) was a protective marker for HCC (OR = 0.37, 95% CI = 0.17–0.79, Pc = 0.02). One can propose that carriers of −863A genotype are associated with increased levels of TNF‐α in the liver in response to HBV infection and induce hapatocyte damage that may finally lead to HCC development. Additional study is needed to validate these finding and to further explore the genetic pathogenesis of HBV infection.

Diagnostic role of serum glypican-3 in differentiating hepatocellular carcinoma from non-malignant chronic liver disease and other liver cancers

Background and Aims:  The role of glypican‐3 (GPC3), a novel serum marker, in differentiating hepatocellular carcinoma (HCC) from non‐malignant chronic liver disease and other malignant space‐occupying lesions in the liver is largely unknown. The aims of this study were to evaluate its diagnostic role and clinical correlations in patients with HCC.

Association of hepatitis viruses with hepatocellular carcinoma in Thailand.

Abstract: Hepatocellular carcinoma (HCC) represents the most common form of malignant tumor among males in Thailand, an area endemic for hepatitis B virus (HBV) infection. Various risk factors have been associated with the development of HCC, among them exposure to certain toxins, and infection with hepatitis viruses, in particular HBV, as well as HCV in areas non-endemic for HBV infection. To examine the association of hepatitis viruses with HCC, our group investigated 101 patients who had been clinically, mainly via alpha fetoprotein level, and/or histologically diagnosed with hepatocellular carcinoma. We also examined 200 voluntary blood donors as controls. All subjects underwent serological tests for the presence of hepatitis B surface antigen (HBsAg) and anti-HCV with polymerase chain reaction (PCR) used for the detection of HBV and TT virus (TTV) DNA, and reverse transcription (RT)-PCR for the detection of HCV RNA and HGV RNA. Besides showing a clear preponderance of HCC among males, with a peak incidence the age group 51–70 years, the results obtained in the HCC patients demonstrated that the prevalence of HBV was 65%, four times that of HCV (17%), ten times that of HGV (6%), and seven times that of TTV (9%). In the controls, the prevalence of HBV was 0.5%; that of HCV, 0.5%; that of HGV, 5%; and that of TTV, 7%. These findings confirmed that hepatitis B virus was associated with the development of hepatocellular carcinoma among the Thai population, among whom case histories of chronic hepatitis and cirrhosis have also been encountered quite frequently.

Geographic distribution of hepatitis C virus genotype 6 subtypes in Thailand

The nucleotide sequence of hepatitis C virus (HCV) genotype 6 found mostly in south China and south‐east Asia, displays profound genetic diversity. The aim of this study to determine the genetic variability of HCV genotype 6 (HCV‐6) in Thailand and locate the subtype distribution of genotype 6 in various geographic areas. Four hundred nineteen anti‐HCV positive serum samples were collected from patients residing in ‐ the central part of the country. HCV RNA positive samples based on reverse transcriptase‐ polymerase chain reaction (RT‐PCR) of the 5′UTR were amplified with primers specific for the core and NS5B regions. Nucleotide sequences of both regions were analyzed for the genotype by phylogenetic analysis. To determine geographic distribution of HCV‐6 subtypes, a search of the international database on subtype distribution in the respective countries was conducted. Among 375 HCV RNA positive samples, 71 had HCV‐6 based on phylogenetic analysis of partial core and NS5B regions. The subtype distribution in order of predominance was 6f (56%), 6n (22%), 6i (11%), 6j (10%), and 6e (1%). Among the 13 countries with different subtypes of HCV‐6, most sequences have been reported from Vietnam. Subtype 6f was found exclusively in Thailand where five distinct HCV‐6 subtypes are circulating. HCV‐6, which is endemic in south China and south‐east Asia, displays profound genetic diversity and may have evolved over a considerable period of time. J. Med. Virol. 82:257–262, 2010.

Low pretreatment serum HBsAg level and viral mutations as predictors of response to PEG-interferon alpha-2b therapy in chronic hepatitis B.

BACKGROUND Viral genomic mutations have become increasingly recognized as being associated with the outcome of chronic HBV infection. However, the role of viral mutations as a predictor of response to pegylated-interferon (PEG-IFN) therapy has so far remained unclear. STUDY DESIGN Viral mutations in the enhancer II/basal core promoter (BCP)/precore and the pre-S regions were characterized by direct sequencing in pretreatment serum samples of 50 patients with chronic hepatitis B (33 HBeAg-positive and 17 HBeAg-negative), who were treated for 48 weeks with PEG-IFN alpha-2b. RESULTS Sustained virological response at 48 weeks post treatment, defined as HBeAg seroconversion and HBV DNA<2000IU/mL for HBeAg-positive patients, and HBV DNA<200IU/mL for HBeAg-negative patients, was achieved in 12 (36.4%) and 6 (35.3%) of HBeAg-positive and HBeAg-negative patients, respectively. Response to PEG-IFN therapy correlated to low pretreatment HBsAg level but did not correlate with HBV genotype, pretreatment alanine transaminase and HBV DNA levels. In HBeAg-positive hepatitis, PEG-IFN response correlated with the appearance of double BCP mutations (A1762T/G1764A) at baseline (P=0.041). In the HBeAg-negative group, response to PEG-IFN therapy was associated with the presence of pre-S mutation/deletions (P=0.028). Multivariate analysis identified low pretreatment HBsAg level as an independent factor associated with SVR in both groups. CONCLUSIONS Pretreatment quantitative HBsAg determination is useful for predicting response to PEG-IFN therapy. The presence of double BCP and pre-S mutation/deletions at entry may be associated with a high rate of antiviral response in HBeAg-positive and HBeAg-negative hepatitis, respectively.

Association between serum hepatocyte growth factor and survival in untreated hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is a common hepatic malignancy worldwide. Its nature of rapid growth results in a grave prognosis. Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, responsible for their proliferation. The aim of the present study was to investigate the prognostic roles of serum HGF in untreated HCC patients.MethodsFifty-five patients with inoperable HCC were studied. The diagnosis of HCC was based on either liver histopathology or imaging evidence of a liver mass, together with elevated serum alpha-fetoprotein. Serum HGF levels of the patients, at the time of diagnosis, were compared to those of 28 healthy controls. All patients received only palliative treatments and were followed up until they died. Comparison of survival curves between patients with a serum HGF level of 1.0 ng/ml or more and those with lower serum HGF was performed, using the log-rank test. Data values are expressed as means and SD.ResultsFifty-one men and four women with inoperable HCC were recruited. The mean age was 54.15 ± 15.34 years. The serum HGF levels in the inoperable HCC patients were significantly higher than those in the controls (0.58 ± 0.43 vs 0.14 ± 0.04 ng/ml; P < 0.001). The patients’ mean survival time was 5.28 ± 6.73 months (range, 0.1–33 months). Serum HGF levels exhibited a negative correlation with the survival time (P = 0.032). In addition, HCC patients with serum HGF levels of 1.0 ng/ml or more had a shorter survival time than the other HCC patients (P = 0.0025).ConclusionsPatients with inoperable HCC had higher levels of serum HGF than the healthy controls, and serum HGF was negatively correlated with the survival time. Serum HGF levels of 1.0 ng/ml or more in HCC patients are suggestive of a grave prognosis, indicating that HGF plays important and active roles in the disease progression. The detailed mechanisms need to be further investigated.

The predominant genotypes of hepatitis B virus in Thailand

In Thailand, chronic liver disease (CLD) as a consequence of infection with hepatitis B virus (HBV) constitutes a public-health burden. Control and treatment are complicated by the virus exhibiting an unusually high mutation rate, with some genotypes apparently causing more severe disease than others. Restriction-fragment-length-polymorphism (RFLP) analysis of the pre-S region of the viral genome, amplified by PCR, was used to determine which genotypes were most prevalent among Thai patients chronically infected with the virus. The patients were chronic HBV carriers (40) or cases of chronic hepatitis (34), cirrhosis (14) or hepatocellular carcinoma (30). As indicated by the results of earlier studies on CLD patients in South-east Asia, genotype C (68.6%) was clearly predominant. RFLP patterns permitted the C1 (12.7%), C7 (45.7%), C8 (10.2%) and B1 (29.7%) subtypes to be identified. Two samples that could not be typed by RFLP were analysed by direct sequencing, categorized as type C, and tentatively designated as subtype C9. As comparison of the present data with those previously obtained by direct sequencing of PCR products indicates that RFLP analysis is as specific and reliable as sequencing and less expensive and time-consuming, RFLP analysis may be particularly useful for epidemiological studies.

Comparison between quantitative hepatitis B surface antigen, hepatitis B e-antigen and hepatitis B virus DNA levels for predicting virological response to pegylated interferon-α-2b therapy in hepatitis B e-antigen-positive chronic hepatitis B

Aim:  The aim of this study was to compare the clinical applicability of quantitative serum hepatitis B surface antigen (HBsAg), hepatitis B e‐antigen (HBeAg) and hepatitis B virus (HBV) DNA for predicting virological response (VR) to pegylated interferon (PEG‐IFN) therapy.