Development of pre and post-operative models to predict early recurrence of hepatocellular carcinoma after surgical resection.

BACKGROUND & AIMS: Resection is the most widely used potentially curative treatment for patients with early hepatocellular carcinoma (HCC). However, recurrence within 2 years occurs in 30-50% of patients, being the major cause of mortality. Herein, we describe 2 models, both based on widely available clinical data, which permit risk of early recurrence to be assessed before and after resection. METHODS: A total of 3,903 patients undergoing surgical resection with curative intent were recruited from 6 different centres. We built 2 models for early recurrence, 1 using preoperative and 1 using pre and post-operative data, which were internally validated in the Hong Kong cohort. The models were then externally validated in European, Chinese and US cohorts. We developed 2 online calculators to permit easy clinical application. RESULTS: Multivariable analysis identified male gender, large tumour size, multinodular tumour, high albumin-bilirubin (ALBI) grade and high serum alpha-fetoprotein as the key parameters related to early recurrence. Using these variables, a preoperative model (ERASL-pre) gave 3 risk strata for recurrence-free survival (RFS) in the entire cohort - low risk: 2-year RFS 64.8%, intermediate risk: 2-year RFS 42.5% and high risk: 2-year RFS 20.7%. Median survival in each stratum was similar between centres and the discrimination between the 3 strata was enhanced in the post-operative model (ERASL-post) which included 'microvascular invasion'. CONCLUSIONS: Statistical models that can predict the risk of early HCC recurrence after resection have been developed, extensively validated and shown to be applicable in the international setting. Such models will be valuable in guiding surveillance follow-up and in the design of post-resection adjuvant therapy trials. LAY SUMMARY: The most effective treatment of hepatocellular carcinoma is surgical removal of the tumour but there is often recurrence. In this large international study, we develop a statistical method that allows clinicians to estimate the risk of recurrence in an individual patient. This facility enhances communication with the patient about the likely success of the treatment and will help in designing clinical trials that aim to find drugs that decrease the risk of recurrence.

New insights into sorafenib resistance in hepatocellular carcinoma: Responsible mechanisms and promising strategies.

It is disappointing that only a few patients with hepatocellular carcinoma (HCC) obtain a significant survival benefit from the sorafenib treatment, which is currently regarded as a first-line chemotherapeutic therapy in patients with advanced HCC. Most patients are highly refractory to this therapy. Therefore, it is necessary to identify resistant factors and explore potential protocols that can be used to overcome the resistance or substitute sorafenib once the resistance is formed. In fact, a growing body of studies has been focusing on the resistance mechanisms or the method to overcome it. The limitation of sorafenib efficacy has been partially but not fully elucidated. Moreover, some protocols have shown encouraging outcomes but still need to be further verified in clinical trials. In this review, we summarize the recent findings on the potential mechanisms that contribute to sorafenib resistance and discuss strategies that can be used to improve the treatment outcome.

Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

Enzyme kinetic and molecular docking studies for the inhibitions of miltirone on major human cytochrome P450 isozymes.

Previous studies have shown that major tanshinones isolated from Danshen (Salvia miltiorrhiza) inhibited human and rat CYP450 enzymes-mediated metabolism of model probe substrates, with potential in causing herb-drug interactions. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC(50)=1.73 µM) and CYP2C9 (IC(50)=8.61 µM), and weak inhibition on CYP2D6 (IC(50)=30.20 µM) and CYP3A4 (IC(50)=33.88 µM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (K(i)=1.48 µM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with K(i) values of 3.17 µM, 24.25 µM and 35.09 µM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of these human CYP450 isoforms, and provided some information on structure-activity relationships for the CYPs inhibition by tanshinones. Taken together, CYPs inhibitions of miltirone were weaker than dihydrotanshinone, but stronger than cryptotanshinone, tanshinone I and tanshinone IIA.

Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.

Cytotoxic effects of tanshinones from Salvia miltiorrhiza on doxorubicin-resistant human liver cancer cells.

P-Glycoprotein (Pgp) overexpression and alterations in p53 oncogene expression are known to affect chemotherapeutic efficacy in the treatment of human hepatocellular carcinoma (HCC). The present study has demonstrated the anti-HCC potential of cryptotanshinone (1), dihydrotanshinone (2), tanshinone I (3), and tanshinone IIA (4), the active lipophilic constituents of Salvia miltiorrhiza, using MTT and caspase-3 activity assays and poly(ADP-ribose) polymerase cleavage in HepG2, Hep3B, and PLC/PRF/5 cells. THLE-3, a normal human immortalized liver cell line, was used to demonstrate the selective growth inhibitory effect of 3 for a HCC cell line. Compound 1 suppressed doxorubicin efflux, a process mediated by P-glycoprotein, in a Pgp-overexpressed HepG2 subclone (R-HepG2 cells). Despite its moderate cytostatic and pro-apoptotic effects and minimal influence on doxorubicin efflux, 4 provided the best synergism with doxorubicin as determined by the Combination Index, the Loewe additivity model, and the Bliss independence criterion.

Embolization efficacy and treatment effectiveness of transarterial therapy for unresectable hepatocellular carcinoma: a case-controlled comparison of transarterial ethanol ablation with lipiodol-ethanol mixture versus transcatheter arterial chemoembolization.

PURPOSE: To compare the embolization efficacy and treatment effectiveness of transarterial ethanol ablation (TEA) versus those of chemoembolization and evaluate the correlation between embolization efficacy and treatment effectiveness of these treatments for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: A case-controlled study was undertaken with 30 patients in each group matched based on Child-Pugh grade, tumor characteristics, and performance status. Primary endpoints were embolization efficacy (ie, Lipiodol retention within tumor at 2 months) and treatment effectiveness as evaluated by tumor response, disease progression, progression-free survival, and overall survival. The secondary endpoint was correlation between embolization efficacy and treatment effectiveness. RESULTS: Lipiodol retention was greater in the TEA group (89.5% +/- 10.7% vs 47.5% +/- 21.2%; P < .0001). The tumor progression rate at 1 year was higher in the chemoembolization group (five of 30 vs zero of 30; P = .0261). One- and 2-year overall survival rates were higher in the TEA group (93.3% and 80.0%, respectively, vs 73.3% and 43.3%, respectively; P = .0053). One- and 2-year extrahepatic disease progression rates were lower in the TEA group (P = .0002). There were no differences in progression-free survival and intrahepatic disease progression rates at 1 and 2 years. Patients with greater Lipiodol retention (ie, >60%) had better treatment outcomes at 1 year than those with lesser retention, with higher overall survival rates (88.9% vs 66.7%; P = .0192), lower intrahepatic disease progression rates (25.6% vs 59.4%; P = .0169), lower extrahepatic disease progression rates (0.31% vs 35.5%; P = .0047), and higher progression-free survival rates (72.1% vs 36.3%; P = .005). CONCLUSIONS: The embolization efficacy and treatment effectiveness of TEA are probably superior to those of chemoembolization for HCC.

Transarterial ethanol ablation of hepatocellular carcinoma with lipiodol ethanol mixture: phase II study.

PURPOSE: This prospective trial aimed to evaluate the safety and effectiveness of transarterial ethanol ablation (TEA) of intrahepatic lesions of hepatocellular carcinoma (HCC) with a Lipiodol-ethanol mixture. MATERIALS AND METHODS: Seventy-seven patients were recruited and 164 lesions (mean size, 5.2 cm +/- 3.0) were treated. Inclusion criteria included histologic proof of HCC, refusal of (n = 9) or contraindication to (n = 68) surgical resection, Eastern Cooperative Oncology Group performance status no greater than 2, and intrahepatic disease without vascular invasion. The mixture consisted of 33% ethanol by volume, with the total dose of Lipiodol-ethanol mixture limited to 60 mL for each treatment session. The primary endpoint was patient survival. Secondary endpoints were tumor response, adverse effects of treatment, and progression-free survival. Median follow-up time for the whole cohort was 2.3 years. RESULTS: Median overall survival was 2.2 years. Overall survival and progression-free survival rates at 1 year and 2 years were 77.9% and 50.1% and 63.6% and 46.3%, respectively. Complete ablation according to radiologic criteria was achieved in 61 patients (79.2%) and 86% of the 164 treated lesions. Mean tumor volume reduction was 65.22%. Patient survival was significantly better in patients with tumors no larger than 5 cm (Cox proportional-hazards regression, P = .0074). Treatment response was significantly better for patients with tumors no greater than 7 cm (chi2 test, P = .0462; Fisher exact test, P = .0326). Adverse effects included irreversible hepatic decompensation (0.6% of procedures), pain (4.8%), and fever (13.8%). CONCLUSIONS: TEA is a safe and effective means to establish local control of unresectable and resectable intrahepatic lesions of HCC.