Jaw-Ching Wu

Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma

The epithelial‐mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E‐cadherin and nonmembranous β‐catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down‐regulation of E‐cadherin, nonmembranous expression of β‐catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C‐associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh‐7). Overexpression of Snail or/and Twist in Huh‐7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppress E‐cadherin transcription. Conclusion: Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays. (HEPATOLOGY 2009.)

Risk factors for early and late recurrence in hepatitis B-related hepatocellular carcinoma ☆

BACKGROUND/AIMS Hepatitis B virus (HBV) levels correlate with the development of hepatocellular carcinoma (HCC), but the role of viral load in HCC recurrence after tumor resection remains unclear. Herein we aimed to investigate the role of viral load in HCC recurrence following tumor resection. METHODS From 1990 to 2002, 193 HBV-related HCC patients who underwent tumor resection in Taipei Veterans General Hospital were enrolled. Serum HBV DNA level and mutations were analyzed for association with early and late recurrence, together with other clinical variables. RESULTS During a follow-up of 58.2+/-44 months, 134 patients had HCC recurrence. Multivariate analysis showed that multinodularity (Hazard ratio [HR], 95% confidence interval [CI]; 2.232, 1.021-4.878), macroscopic venous invasion (4.693, 1.645-13.391), AFP >20 ng/ml (3.891, 1.795-8.475), and cut margin <or= 1cm (3.333, 1.487-7.470) were correlated with early recurrence (within two years of operation) of HCC. In addition, multivariate analysis determined that Ishak hepatic inflammatory activity >6 (4.658, 1.970-11.017), multinodularity (3.266, 1.417-7.526), ICG-15 >10% (2.487, 1.095-5.650) and HBV DNA level >10(6) copies/ml (2.548, 1.040-6.240) were significantly associated with late recurrence (>two years after resection). Patients with high viral loads tended to have higher Ishak inflammatory (7.00+/-3.07 vs. 5.33+/-2.96, p=0.001) and fibrosis scores (4.17+/-2.01 vs. 3.20+/-2.41, p=0.007) than those with lower loads. CONCLUSIONS Tumor factors were associated with early HCC recurrence while high viral loads and hepatic inflammatory activity were associated with late recurrence. Pre- and post-operative antiviral and anti-inflammatory therapies may be crucial in reducing late recurrence.

HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis.

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. HCV NS5A, one of non-structural proteins of HCV, was suggested to play a role in oncogenic transformation. Since the tumor suppressor p53 is important for preventing neoplastic transformation, we investigated the functional effects of HCV NS5A on p53. In vitro and in vivo coimmunoprecipitation and confocal microscopy were used to determine the interaction of NS5A and p53. HCV NS5A binds directly to p53 and colocalizes p53 in the perinuclear region. NS5A inhibits transcriptional transactivation by p53 in a dose-dependent manner by use of a reporter assay. Down regulation of endogenous p21/waf1 expression, which is activated by p53 in Hep3B cells, by NS5A was demonstrated by using FLAG- and FLAG-NS5A Hep3B stable cell lines. The effect of NS5A on p53-mediated apoptosis was determined by flow cytometry in both NS5A permanently and transiently transfected Hep3B cells with exogenous p53. The p53-induced apoptosis was abrogated by NS5A and the inhibition effect correlates well with the binding ability of NS5A to p53. In addition, HCV NS5A protein interacts with and colocalizes hTAFII32, a component of TFIID and an essential coactivator of p53, in vivo. These results suggest that HCV NS5A interacts with and partially sequestrates p53 and hTAFII32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.

Hepatic steatosis in chronic hepatitis C virus infection: Prevalence and clinical correlation

Background and Aims: Hepatic steatosis is a histological characteristic in patients with chronic hepatitis C virus (HCV) infection. The aim of this study was to evaluate the prevalence of hepatic steatosis in Chinese patients with chronic hepatitis C, and to look for possible correlation with various histopathological changes and to look for possible correlation with various clinical and pathologic variables.

Serum levels of interleukin-8 in alcoholic liver disease : relationship with disease stage, biochemical parameters and survival

BACKGROUND/AIMS Interleukin-8 (IL-8), a cytokine produced by a host of cells, including monocytes, macrophages, Kupffer cells and hepatocytes, can activate neutrophils. Peripheral neutrophilia and liver neutrophil infiltration are frequently noted in patients with alcoholic liver disease. However, the relationship between IL-8 and different stages of alcoholic liver disease is uncertain. The aim of this study is to determine if a correlation exists between circulating IL-8 levels and biochemical and histological parameters and survival in alcoholic liver disease. METHODS Serum levels of IL-8 were determined with an enzyme-linked immunosorbent assay in 166 subjects, consisting of 30 healthy controls, 26 patients with non-alcoholic fatty liver, 15 with alcoholic fatty liver, 32 with alcoholic hepatitis, 30 with alcoholic cirrhosis, 28 with chronic hepatitis B and 5 with chronic hepatitis C. RESULTS Serum IL-8 levels were markedly elevated in patients with alcoholic hepatitis (437 +/- 51 pg/ml) when compared with all other groups (p < 0.05). Levels of IL-8 in patients with alcoholic fatty liver, alcoholic cirrhosis and viral hepatitis were higher than those in controls and in patients with non-alcoholic fatty liver. In addition, IL-8 levels were higher in patients who died (p = 0.007), and correlated with biochemical and histological parameters, and severity of liver injury: serum aspartate aminotransferase, alanine aminotransferase, total bilirubin, prothrombin time, indocyanine green retention ratio, tumor necrosis factor-alpha and peripheral neutrophil count in patients with alcoholic hepatitis. After a 2-year follow up, patients with IL-8 above 479 pg/ml had a higher mortality rate in the alcoholic hepatitis group (p = 0.033). CONCLUSIONS These findings suggest that IL-8 is activated in alcoholic liver disease, especially in alcoholic hepatitis, and is closely correlated with liver injury. IL-8 levels can reflect the stage and severity of alcoholic liver disease, and may serve as a predictor of survival in patients with alcoholic hepatitis.

Natural history of hepatitis D viral superinfection: Significance of viremia detected by polymerase chain reaction

BACKGROUND/AIMS Polymerase chain reaction (PCR) is very sensitive. The aim of the study was to reevaluate viral replication in hepatitis D virus (HDV) superinfection by PCR. METHODS HDV and hepatitis B virus (HBV) were detected by PCR in 185 patients. RESULTS The acute hepatitis group had the highest detection rate of HDV RNA compared with chronic hepatitis, cirrhosis, hepatocellular carcinoma, and remission groups (63 of 64 vs. 35 of 47, 17 of 23, 19 of 30, and 7 of 21) and the highest alanine aminotransferase (ALT) levels (mean, 1741 U/L vs. 266 to 27 U/L; P < 0.05). The detection rate of HBV DNA was the lowest in the acute group (41%) compared with 66%, 70%, 80%, and 57% in the remaining groups (P < 0.02). At the chronic stage, 13%-25% of cases had HDV RNA, and 30%-48% of cases had HBV DNA detected by PCR but not by traditional method. HDV RNA was associated with ALT levels in horizontal and longitudinal analyses. CONCLUSIONS HDV superinfection may be divided into the following three phases: acute phase, active HDV replication and suppression of HBV with high ALT levels; chronic phase, decreasing HDV and reactivating HBV with moderate ALT levels; and late phase, development of cirrhosis and hepatocellular carcinoma caused by replication of either virus or remission resulting from marked reduction of both viruses.

Clinical and epidemiological implications of swine hepatitis E virus infection

In nonendemic areas, most patients with acute hepatitis E were infected through traveling to endemic areas. However, some patients did not have a history of foreign travel before infection. Furthermore, high seroprevalence rates of antibody to hepatitis E virus (anti‐HEV) were found in the general adult population in some countries without any recorded outbreak of hepatitis E. The significance of anti‐HEV assay in these subjects remains obscure. To study if swine might be a source of HEV infection, HEV was tested in sera of 235 pigs in Taiwan, and from 5 patients with acute HEV infection who either denied or did not provide any foreign travel history. Three (1.3%) pigs had detectable swine HEV RNA. The swine and human HEV strains from Taiwan formed a monophyletic group, distinct from three previously reported groups: the United States human and swine HEV strains, the Mexico strain, and the largest group composed of the Asian and the African strains. The identity of nucleotide sequences was 84–95% between swine and human HEV strains in Taiwan, and 72–79% between Taiwan strains and those from different areas. The predicted amino acid sequence of a Taiwan swine HEV strain within the peptide 3‐2 used in commercial anti‐HEV assay showed a high identity (91–94%) with those of other human and swine HEV strains. Swine may be a reservoir of HEV and subclinical swine HEV infection may occur. Cross‐reactivity of current anti‐HEV assay may account for the high prevalence rate of anti‐HEV in the general population in nonendemic areas. J. Med. Virol. 60:166–171, 2000.

Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis.

The hepatitis D virus (HDV) genotypes in 46 HDV-infected patients and 12 prostitutes were screened with Xhol restriction fragment length polymorphism (RFLP) analysis of reverse transcription PCR products of viral genomes and verified by phylogenetic analysis. The amplificates of three (6.5%) patients and two (17%) prostitutes showed a novel RFLP pattern different from those of the three known genotypes. Complete HDV genomic sequence identities between isolates with a novel RFLP and the HDV genotypes I, II and III were 72.3, 77.2 and 63.0%, respectively. Importantly, divergence was mostly seen in various regions related to replication or packaging. The novel isolates formed a monophyletic group (P < 0.05) and were most closely related to genotype II.

Survival Rates Are Comparable After Radiofrequency Ablation or Surgery in Patients With Small Hepatocellular Carcinomas

BACKGROUND & AIMS Differences in efficacy of radiofrequency ablation (RFA) and surgical resection (SR) are not clear for patients with hepatocellular carcinoma (HCC). METHODS From 2002 to 2007, 419 patients with HCCs ≤5 cm were enrolled consecutively in the study. Among these patients, 190 and 229 patients received RFA and SR, respectively, as their first treatment. Factors were analyzed in terms of overall survival and recurrence by multivariate analysis and propensity score matching analysis. RESULTS The SR group had younger age, a higher male-to-female ratio, higher prevalence of hepatitis B virus, lower prevalence of hepatitis C virus, better liver function reserve, and larger tumor size than the RFA group. The cumulative 5-year overall survival rates were 79.3% in the SR group and 67.4% in the RFA group. During the follow-up period, tumors recurred in 244 patients in a median time of 14.5 ± 15.7 months. Before propensity-score matching, the RFA group had shorter overall survival time (P = .009) and higher tumor recurrence rate (P < .001) than the SR group. After matching, RFA was comparable to SR in overall survival time (P = .519), but the RFA group still had a greater incidence of tumor recurrence (P < .001). In patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 HCC, RFA was as effective as SR for overall survival time and recurrence. CONCLUSIONS Patients with small HCCs have a higher rate of tumor recurrence following RFA than surgery, but overall survival rates are comparable between therapies. RFA is as effective as surgery in patients with BCLC stage 0 HCC.

Comparison of percutaneous acetic acid injection and percutaneous ethanol injection for hepatocellular carcinoma in cirrhotic patients: a prospective study.

Background: Ultrasound-guided percutaneous ethanol injection (PEI) and percutaneous acetic acid injection (PAI) are effective in the treatment of hepatocellular carcinoma (HCC). We conducted a prospective study to compare the therapeutic efficacy of both these methods. Methods: Sixty-three patients were treated by PAI using 50% acetic acid and 62 by PEI using pure ethanol. There were no significant baseline differences in age, sex, Child-Pugh class, tumour size and number, or other clinico-biochemical parameters between the two groups. Results: During a follow-up period of 24 ± 9 (range 6-38) months, 19 (30%) of the PAI group and 21 (34%) of the PEI group died ( P r = r 0.704). The 1- and 3-year survival rates were 84% and 51% for the PAI group and 81% and 46% for the PEI group ( P r = r 0.651). The corresponding tumour recurrence rates were 51% and 74% for the PAI group, and 54% and 64% for the PEI group ( P r = r 0.787). The treatment sessions were 3.9 ± 1.6 and 6.2 ± 2.3 for the PAI and PEI groups, respectively, in each treatment cycle ( P r = r 0.008). A multivariate analysis using the Cox regression model revealed that ascites (relative risk (RR) 3.1, 95% confidence interval (CI) 1.5-6.3, P r = r 0.002), large (>3 r cm) or multinodular HCCs (RR 2.4, 95% CI 1.1-5.4, P r = r 0.04), and development of tumour recurrence (RR 7.0, 95% CI 3.1-16.0, P r < r 0.001) were independent, poor prognostic factors in both groups. Conclusions: PAI and PEI are equally effective in the treatment of HCC. PAI has the advantage of fewer treatment sessions in each treatment course. Careful pretreatment patient selection may improve survival.BACKGROUND Ultrasound-guided percutaneous ethanol injection (PEI) and percutaneous acetic acid injection (PAI) are effective in the treatment of hepatocellular carcinoma (HCC). We conducted a prospective study to compare the therapeutic efficacy of both these methods. METHODS Sixty-three patients were treated by PAI using 50% acetic acid and 62 by PEI using pure ethanol. There were no significant baseline differences in age, sex, Child-Pugh class, tumour size and number, or other clinico-biochemical parameters between the two groups. RESULTS During a follow-up period of 24 +/- 9 (range 6-38) months, 19 (30%) of the PAI group and 21 (34%) of the PEI group died (P = 0.704). The 1- and 3-year survival rates were 84% and 51% for the PAI group and 81% and 46% for the PEI group (P = 0.651). The corresponding tumour recurrence rates were 51% and 74% for the PAI group, and 54% and 64% for the PEI group (P = 0.787). The treatment sessions were 3.9 +/- 1.6 and 6.2 +/- 2.3 for the PAI and PEI groups, respectively, in each treatment cycle (P = 0.008). A multivariate analysis using the Cox regression model revealed that ascites (relative risk (RR) 3.1, 95% confidence interval (CI) 1.5-6.3, P = 0.002), large (>3 cm) or multinodular HCCs (RR 2.4, 95% CI 1.1-5.4, P = 0.04), and development of tumour recurrence (RR 7.0, 95% CI 3.1-16.0, P < 0.001) were independent, poor prognostic factors in both groups. CONCLUSIONS PAI and PEI are equally effective in the treatment of HCC. PAI has the advantage of fewer treatment sessions in each treatment course. Careful pretreatment patient selection may improve survival.