Jun Imamura

Prospective risk assessment for hepatocellular carcinoma development in patients with chronic hepatitis C by transient elastography

Liver stiffness, noninvasively measured by transient elastography, correlates well with liver fibrosis stage. The aim of this prospective study was to evaluate the liver stiffness measurement (LSM) as a predictor of hepatocellular carcinoma (HCC) development among patients with chronic hepatitis C. Between December 2004 and June 2005, a total of 984 HCV‐RNA positive patients, without HCC or a past history of it, visited the University of Tokyo Hospital. LSM was performed successfully in 866 patients, who gave informed consent. During the follow‐up period (mean, 3.0 years), HCC developed in 77 patients (2.9% per 1 person‐year). The cumulative incidence rates of HCC at 1, 2, and 3 years were 2.4%, 6.0%, and 8.9%, respectively. Adjusting for other significant factors for HCC development, patients with higher LSM were revealed to be at a significantly higher risk, with a hazard ratio, as compared to LSM ≤10 kPa, of 16.7 (95% confidence interval [CI], 3.71‐75.2; P < 0.001) when LSM 10.1‐15 kPa, 20.9 (95% CI, 4.43‐98.8; P < 0.001) when LSM 15.1‐20 kPa, 25.6 (95%CI, 5.21‐126.1; P < 0.001) when LSM 20.1‐25 kPa, and 45.5 (95% CI, 9.75‐212.3; P < 0.001) when LSM >25 kPa. Conclusions: This prospective study has shown the association between LSM and the risk of HCC development in patients with hepatitis C. The utility of LSM is not limited to a surrogate for liver biopsy but can be applied as an indicator of the wide range of the risk of HCC development. (HEPATOLOGY 2009.)

Obesity Is an Independent Risk Factor for Hepatocellular Carcinoma Development in Chronic Hepatitis C Patients

BACKGROUND & AIMS It is not fully elucidated whether obesity enhances hepatocarcinogenesis in patients with chronic hepatitis C. The aim of this study was to investigate the relationship between body weight and risk of hepatocarcinogenesis in chronic hepatitis C patients. METHODS We enrolled 1431 patients with chronic hepatitis C who visited our liver clinic between 1994 and 2004, excluding those with hepatocellular carcinoma (HCC) at their visit or with a previous history of HCC. They were divided into 4 groups according to body mass index (BMI): underweight (< or =18.5 kg/m(2), N = 112); normal (18.5 to less than 25 kg/m(2), N = 1023); overweight (25 to less than 30 kg/m(2), N = 265); and obese (>30 kg/m(2), N = 31). We assessed the impact of obesity on the hepatocarcinogenesis adjusted by multivariate Cox proportional hazard regression with other risk factors found significant in univariate analysis. RESULTS During the follow-up period (mean, 6.1 y), HCC developed in 340 patients, showing cumulative incidence rates of 10.5%, 19.7%, and 36.8% at 3, 5, and 10 years, respectively. The incidence differed significantly among the BMI groups (P = .007). Adjusting for other significant factors, overweight and obesity were shown to be an independent risk factor of HCC, with a hazard ratio of 1.86 (95% confidence interval, 1.09-3.16; P = .022) and 3.10 (95% confidence interval, 1.41-6.81; P = .005) as compared with the underweight patients. CONCLUSIONS The risk of HCC in patients with chronic hepatitis C increases in proportion to BMI in a wide range of its values, from underweight to obese.

Neoplastic seeding after radiofrequency ablation for hepatocellular carcinoma.

BACKGROUND:Neoplastic seeding reportedly occurs in up to 12.5% of patients treated with radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC). The aim of this study is to assess the incidence, risk factors, and prognosis of neoplastic seeding after RFA among a large number of patients with a long-term follow-up.METHOD:From February 1999 to December 2004, 1,031 patients underwent a total of 1,845 treatments with RFA for a total of 3,837 HCC nodules. The following variables were assessed to elucidate the risk factors of neoplastic seeding: age, sex, positivity for viral markers, tumor size, number of tumor nodules, number of RFA sessions, tumor location, percutaneous biopsy prior to RFA, alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin (DCP) and lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) levels, and the degree of tumor differentiation.RESULTS:Neoplastic seeding was detected in 33 patients (3.2% per patient) at intervals of 4.8–63.8 (median, 15.2) months after RFA. On multivariate logistic regression analysis, only the poor differentiation degree was associated with the risk of neoplastic seeding (P = 0.012). Of tumor factors, tumor size, and AFP, DCP, and AFP-L3 levels were significantly associated with the poor differentiation degree. The cumulative survival rates 1 and 2 yr after the detection of neoplastic seeding were 86% and 47%, respectively.CONCLUSION:Poor differentiation degree was the risk factor of neoplastic seeding after RFA for HCC. The surrogate markers for poor differentiation degree were larger tumor size and elevated tumor marker levels. Indication for RFA should be carefully considered for HCC patients under these conditions.

The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30–125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

Treatment strategy for hepatocellular carcinoma: expanding the indications for radiofrequency ablation

BackgroundRadiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) is ordinarily indicated for those with three or fewer nodules, none of which exceeds 3 cm in diameter. This study investigated whether an apparent threshold exists in the diameter and number of nodules in terms of the prognosis of patients with HCC.MethodsWe enrolled 663 naïve patients with HCC who were treated with RFA at our hospital between 1999 and 2005. We analyzed the patients’ prognosis using multivariate Cox proportional regression with the diameter and number of nodules as covariates and Child-Pugh class as a stratification factor. The diameter and number were categorized as ≤2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and >5 cm and 1, 2–3, 4–5, and >5, respectively.ResultsThe adjusted hazard ratio of patients whose largest nodule was ≤2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and >5 cm was 1, 1.51, 2.56, 2.25, and 2.71, respectively. The adjusted hazard ratio of patients with one, two or three, four or five, and more than five nodules was 1, 1.35, 1.70, and 2.12, respectively. Therefore, patients with three or fewer nodules, none of which exceeds 5 cm in diameter, have a 5-year survival of 40%.ConclusionsThe prognosis of the patients worsened gradually as the diameter and number of nodules increased. No apparent threshold in the diameter or number of HCC nodules was detected. RFA can be applied beyond the conventional indications.

Functional consequences of mutations in a putative Akt phosphorylation motif of B-raf in human cancers

Mutations in the B‐raf gene have been reported in a number of human cancers, including melanoma and lung cancer. More than 80% of the reported B‐raf mutations were V599E; however, non‐V599E mutations have been frequently found in non‐small cell lung cancers as compared with melanoma. Some non‐V599E mutations have been found surrounding Thr439, which is thought likely to be one of the three Akt phosphorylation sites in the B‐raf protein. However, as a previous report indicated that Thr439 was not phosphorylated by Akt, the functional consequences of these mutations have been unclear. Here, we examined the effects of cancer‐related B‐raf mutations surrounding Thr439 on the activation of the mitogen‐activated protein/ extracellular signal‐regulated kinase kinase (MEK)/extracellular signal‐regulated kinase (Erk) pathway and the transformation of NIH 3T3 fibroblasts. Among the three reported mutations (K438Q, K438T, and T439P) found in non‐small cell lung carcinoma and melanoma, none elevated the activity of the MEK/Erk cascade as determined by in vitro kinase assays, immunoblots using antibody specific for phosphorylated Erk, or Elk1‐dependent reporter assays. The inhibition of phosphatidylinositol 3‐kinase (PI3K)/Akt signaling by LY294002 increased the Erk activation induced by the mutant B‐raf proteins, as well as by wild‐type B‐raf. Furthermore, the B‐raf mutants did not have increased NIH 3T3‐transforming activities, as determined by colony‐formation assays. These results suggest that the B‐raf mutations surrounding Thr439 found in human cancers are unlikely to contribute to increased oncogenic properties of B‐raf.

TRAIL-induced cell death cooperates with IFN-γ activation in the graft-versus-tumor effect against colon tumors

The graft‐versus‐tumor (GVT) effect that occurs following allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) is currently being subjected to intensive investigation because of clinical evidence for GVT efficacy against leukemia. In this report, we investigate the efficacy and molecular mechanisms of GVT against solid tumors, using a modification of the mouse parent‐to‐F1 BMT model. Mouse Colon26 cells in which tumor necrosis factor related apoptosis‐inducing ligand (TRAIL) receptor expression was stably knocked down were transplanted to investigate the role of the TRAIL‐TRAIL receptor system in the GVT effect. In addition, Fas ligand‐(FasL) deficient mice on a C57BL6 (B6) background were used as donors, to determine the significance of the Fas‐FasL system for the antitumor effect. The group that received B6 DLI followed by preconditioning with 950 rad irradiation underwent tumor reduction associated with the induction of IFN‐γ, TRAIL and tumor‐cell apoptosis. In vitro cultured Colon26 cells were resistant to TRAIL but susceptible to the combination of IFN‐γ and TRAIL in a TRAIL‐dose‐dependent manner. The infusion of lymphocytes from FasL‐defective donors reduced the tumor progression, although efficacy was decreased in the TRAIL receptor knockdown tumors but not in wild‐type ones, compared with infusion of B6‐derived lymphocytes. The findings indicate that GVT activity against subcutaneous colon tumors is efficiently induced by preconditioning with irradiation and allogeneic DLI, and that TRAIL and IFN‐γ act cooperatively in the antitumor effect.

Influence of anti‐HBc seropositivity on the risk of hepatocellular carcinoma in HCV‐infected patients after adjusting for confounding factors

Summary.  It is controversial whether past hepatitis B virus infection constitutes an additional risk of hepatocellular carcinoma (HCC) among patients with hepatitis C virus (HCV). The incidence of HCC between 1994 and 2004 was analysed among 1262 patients who were only positive for HCV. The cumulative incidence of HCC was assessed by Kaplan–Meier analysis and the difference between two groups was assessed by the log‐rank test. The effect of anti‐HBc positivity on the risk of HCC was assessed with multivariate Cox proportional analysis. Anti‐HBc was positive in 522 (41.4%) patients. The proportion of male patients (56.7 vs 46.8%, P < 0.001) and mean age (60.8 vs 56.9 years, P < 0.001) were significantly higher in the anti‐HBc positive group. HCC developed in 339 patients (mean follow‐up 7.0 years), with cumulative incidence rates at 3, 5 and 10 years of 12.7, 24.5 and 41.9% in the anti‐HBc positive group and 10.6, 17.7 and 33.4% in the negative group, respectively (P = 0.005). However, anti‐HBc seropositivity did not reach statistical significance in multivariate analysis including age and gender (hazard ratio, 1.06; 95% CI, 0.85–1.31; P = 0.63). Anti‐HBc positivity and HCC incidence were confounded by male gender and older age.