Integrating genetic variants into clinical models for hepatocellular carcinoma risk stratification in cirrhosis.

BACKGROUND & AIMS: Identifying individuals at higher risk of developing hepatocellular carcinoma (HCC) is pivotal to improve the performance of surveillance strategies. Herein, we aimed to evaluate the ability of single nucleotide polymorphisms (SNPs) to refine HCC risk stratification. METHODS: Six SNPs in PNPLA3, TM6SF2, HSD17B13, APOE, and MBOAT7 affecting lipid turnover and one variant involved in the Wnt-ß-catenin pathway (WNT3A-WNT9A rs708113) were assessed in patients with alcohol-related and/or HCV-cured cirrhosis included in HCC surveillance programmes (prospective CirVir and CIRRAL cohorts). Their prognostic value for HCC occurrence was assessed using Fine-Gray models combined into a 7-SNP genetic risk score (GRS). The predictive ability of two clinical scores (a routine non-genetic model determined by multivariate analysis and the external aMAP score) with/without the GRS was evaluated by C-indices. The standardised net benefit was derived from decision curves. RESULTS: Among 1,145 patients, 86 (7.5%) developed HCC after 43.7 months. PNPLA3 and WNT3A-WNT9A variants were independently associated with HCC occurrence. The GRS stratified the population into three groups with progressively increased 5-year HCC incidence (Group 1 [n = 627, 5.4%], Group 2 [n = 276, 10.7%], and Group 3 [n = 242, 15.3%]; p <0.001). The multivariate model identified age, male sex, diabetes, platelet count, gamma-glutamyltransferase levels, albuminemia and the GRS as independent risk factors. The clinical model performance for 5-year HCC prediction was similar to that of the aMAP score (C-Index 0.769). The addition of the GRS to both scores modestly improved their performance (C-Indices of 0.786 and 0.783, respectively). This finding was confirmed by decision curve analyses showing only fair clinical net benefit. CONCLUSIONS: Patients with cirrhosis can be stratified into HCC risk classes by variants affecting lipid turnover and the Wnt-ß-catenin pathway. The incorporation of this genetic information modestly improves the performance of clinical scores. IMPACT AND IMPLICATIONS: The identification of patients at higher risk of developing liver cancer is pivotal to improve the performance of surveillance. Risk assessment can be achieved by combining several clinical and biological parameters used in routine practice. The addition of patients' genetic characteristics can modestly improve this prediction and will ultimately pave the way for precision medicine in patients eligible for HCC surveillance, allowing physicians to trigger personalised screening strategies.

Sulfatase 2 Along with Syndecan 1 and Glypican 3 Serum Levels are Associated with a Prognostic Value in Patients with Alcoholic Cirrhosis-Related Advanced Hepatocellular Carcinoma.

Purpose: Sulfatase 2 (SULF2) is an enzyme related to heparan sulfate modifications. Its expression, as for some heparan sulfate proteoglycans expression, has been linked to hepatocellular carcinoma (HCC) at mRNA level and immunohistochemistry staining on biopsy samples. This study aims to evaluate the prognostic value of serum levels of SULF2 in patients with alcoholic cirrhosis with or without HCC. Patients and Methods: Two hundred and eighty-seven patients with alcoholic cirrhosis were enrolled in this study: 164 without HCC, 57 with early HCC, and 66 with advanced HCC at inclusion. We analyzed the association between SULF2 serum levels and prognosis using Kaplan-Meier method and univariate and multivariate analysis using a Cox model. Results: Child-Pugh C Patients have higher serum levels of SULF2 than Child-Pugh A patients. Serum levels of SULF2 were also higher in patients with advanced HCC compared with the other groups. In patients with advanced HCC, high serum levels of SULF2 were associated with less favorable overall survival. Combination of SULF2 with Glypican 3 (GPC3) and Syndecan 1 (SDC1) serum levels enhanced the ability to discriminate worst prognostic in advanced HCC. Conclusion: SULF2 along with GPC3 and SDC1 serum levels have been shown to be associated with a prognostic value in advanced HCC.

miR-126-3p is essential for CXCL12-induced angiogenesis.

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro-angiogenic agents. We analysed the miR-126/CXCL12 axis and compared impacts of both miR-126-3p and miR-126-5p strands effects in CXCL12-induced angiogenesis. Indeed, the two strands of miR-126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D-angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR-126-3p/-5p or antimiR-126-3p/-5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro-angiogenic effects in vitro and ex vivo associated with overexpression of miR-126-3p in HUVEC and rat's aortas. Second, we showed that 2D-angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR-126-3p inhibition. Finally, we observed that SPRED-1 (one of miR-126-3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro-angiogenic potential in vitro. Our results proved for the first time: 1-the role of CXCL12 in modulation of miR-126 expression; 2-the involvement of miR-126 in CXCL12 pro-angiogenic effects; 3-the involvement of SPRED-1 in angiogenesis induced by miR-126/CXCL12 axis.

Transient elastography predicts survival after radiofrequency ablation of hepatocellular carcinoma developing on cirrhosis.

BACKGROUND AND AIM: The prognostic value of transient elastography (TE) in cirrhotic patients with hepatocellular carcinoma (HCC) treated by percutaneous radiofrequency ablation (RFA) is currently unknown. METHOD(S): We included patients with histologically proven cirrhosis and with a first diagnosis of HCC inside Milan criteria treated by percutaneous RFA, and with TE available the year before treatment with 10 shots and interquartile range/median < 30%. Association between variables and clinical events was assessed by the Kaplan-Meier method with the log-rank test and using Cox univariate and multivariate analyses. RESULTS: One hundred fifty-nine patients were included, with a median age of 65 years; 77.4% were men. Causes of cirrhosis were alcohol consumption (48.1%), hepatitis C (43.7%), hepatitis B (12.7%), and non-alcoholic steatohepatitis (32.3%). Median value of TE was 26 kPa (4-75 kPa). Overall survival at 1, 2, and 5 years was, respectively, 93%, 81%, and 44%; overall recurrence was 28%, 49%, and 80%. The TE value was not associated with tumor recurrence (0.13). In contrast, in univariate analysis, TE value, age, Child-Pugh B, and alkaline phosphatase were predictive factors in overall survival. In multivariate analysis, TE value (hazards ratio [HR] = 1.02, 95% confidence interval (IC): 1.01-1.04, 0.001), age (HR = 1.05, 95% IC: 1.03-1.08, P = 0.00006), and Child-Pugh B score (HR = 2.78, 95% IC: 1.27-6.08, P = 0.01) were independently associated with higher risk of death. A TE value ≥ 40 kPa was associated with shorter median overall survival (34 months) compared to a TE value < 40 kPa (59 months, P = 0.0008). CONCLUSION(S): Transient elastography (TE) predicts overall survival but not tumor recurrence in cirrhotic patients with HCC treated by RFA.

A 17-Beta-Hydroxysteroid Dehydrogenase 13 Variant Protects From Hepatocellular Carcinoma Development in Alcoholic Liver Disease.

Recently, a loss of function variant (rs72613567) in 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) has been identified as protective of nonalcoholic (NAFLD) and alcoholic liver disease (ALD). However, the role of this single-nucleotide polymorphism (SNP) in the development of hepatocellular carcinoma (HCC) is currently unknown. A total of 3,315 European patients with HCC (n = 1,109) or without HCC, but with chronic liver disease (CLD; n = 2,206), from four centers were analyzed either by whole-exome sequencing (WES; exploratory cohort, 285 HCC) or genotyped for HSD17B13 rs72613567 (validation cohort, 824 HCC and all CLD). We included a control group of 33,337 healthy European individuals from the Exome Aggregation Consortium. We compared distribution of genotype using the chi-square test and logistic regression. In the exploratory cohort analyzed by WES, frequency of the TA allele of HSD17B13 rs72613567 was significantly decreased in HCC patients compared to healthy controls (P = 1.52 × 10-06 ). In the validation cohort, frequency of TA allele carriers was also decreased in patients with CLD and without HCC (39%) compared to healthy individuals (47%; P < 0.0001). The protective effect of the TA allele of HSD17B13 rs72613567 was identified in patients with ALD (odds ratio [OR] = 0.73; 95% confidence interval [CI], 0.65-0.82; P < 0.0001), NAFLD (OR = 0.64; 95% CI, 0.49-0.83; P = 0.0007), and hepatitis C (OR = 0.71; 95% CI, 0.60-0.85; P = 0.0002). In patients with ALD, the proportion of TA allele carriers with HCC was significantly lower (32%) than in CLD patients without HCC (40%), even after adjustment for age, sex, and fibrosis (OR = 0.64; 95% CI, 0.46-0.87; P = 0.005). Conclusion: The HSD17B13 rs72613567 loss of function variant is protective of HCC development in patients with ALD.

Virologic control and severity of liver disease determine survival after radiofrequency ablation of hepatocellular carcinoma on cirrhosis.

BACKGROUND: We aimed to identify the main determinants of long-term overall survival (OS), including virologic control, and recurrence after radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) on cirrhosis. METHODS: Cirrhotic patients treated by RFA for HCC within Milan criteria were included. Associations between patient features and events were estimated by the Kaplan-Meier method with the log rank test and using uni/multivariate Cox models. RESULTS: 389 cirrhotic patients (Child-Pugh A 86.6%, 473 tumors) were included. OS was 79.8%, 42.4% and 16%, and overall tumor recurrence 45%, 78% and 88% at 2, 5 and 10 years, respectively. In multivariate analysis, age, Child-Pugh, GGT, HCC near major vessels, esophageal varices, alkaline phosphatase and HBV predicted OS. Gender, ALT, AFP and alcohol intake were associated with tumor recurrence. Multinodular HCC (19.5%) was associated with risk of tumor recurrence outside Milan criteria. HBV patients had longer OS than other patients (P = 0.0059); negative HBV PCR at RFA was associated with decreased tumor recurrence (P = 0.0157). Using time-dependent analysis in HCV patients, a sustained virologic response was associated with increased OS (124.5 months) compared to other patients (49.2 months, P < 0.001). CONCLUSION: Virologic response and severity of underlying liver disease were the main determinants of long-term OS after RFA for HCC developing on cirrhosis.

PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.

Few single nucleotide polymorphisms (SNPs) have been reproducibly associated with hepatocellular carcinoma (HCC). Our aim was to test the association between nine SNPs and HCC occurrence. SNPs in genes linked to HCC (DEPDC5, GRIK1, KIF1B, STAT4, MICA, DLC1, DDX18) or to liver damage (PNPLA3-rs738409, TM6SF2-rs58542926) in GWAS were genotyped in discovery cohorts including 1,020 HCC, 2,021 controls with chronic liver disease and 2,484 healthy individuals and replication was performed in prospective cohorts of cirrhotic patients with alcoholic liver disease (ALD, n = 249) and hepatitis C (n = 268). In the discovery cohort, PNPLA3 and TM6SF2 SNPs were associated with HCC (OR = 1.67 [CI95%:1.16-2.40], p = 0.005; OR = 1.45 [CI95%:1.08-1.94], p = 0.01) after adjustment for fibrosis, age, gender and etiology. In contrast, STAT4-rs7574865 was associated with HCC only in HBV infected patients (p = 0.03) and the other tested SNP were not linked with HCC risk. PNPLA3 and TM6SF2 variants were independently associated with HCC in patients with ALD (OR = 3.91 [CI95%:2.52-6.06], p = 1.14E-09; OR = 1.79 [CI95%:1.25-2.56], p = 0.001) but not with other etiologies. PNPLA3 SNP was also significantly associated with HCC developed on a nonfibrotic liver (OR = 2.19 [CI95%:1.22-3.92], p = 0.007). The association of PNPLA3 and TM6SF2 with HCC risk was confirmed in the prospective cohort with ALD. A genetic score including PNPLA3 and TM6SF2 minor alleles showed a progressive significant increased risk of HCC in ALD patients. In conclusion, PNPLA3-rs738409 and TM6SF2-rs58542926 are inherited risk variants of HCC development in patients with ALD in a dose dependent manner. The link between PNPLA3 and HCC on nonfibrotic liver suggests a direct role in liver carcinogenesis.

BRIP1 coding variants are associated with a high risk of hepatocellular carcinoma occurrence in patients with HCV- or HBV-related liver disease.

The molecular mechanisms of hepatocellular carcinoma (HCC) carcinogenesis are still not fully understood. DNA repair defects may influence HCC risk. The aim of the study was to look for potential genetic variants of DNA repair genes associated with HCC risk among patients with alcohol- or viral-induced liver disease. We performed four case-control studies on 2,006 European- (Derivation#1 and #2 studies) and African-ancestry (Validation#1 and #2 studies) patients originating from several cohorts in order to assess the association between genetic variants on DNA repair genes and HCC risk using a custom array encompassing 94 genes. In the Derivation#1 study, the BRIP1 locus reached array-wide significance (Chi-squared SV-Perm, P=5.00×10-4) among the 253 haplotype blocks tested for their association with HCC risk, in patients with viral cirrhosis but not among those with alcoholic cirrhosis. The BRIP1 haplotype block included three exonic variants (rs4986763, rs4986764, rs4986765). The BRIP1 'AAA' haplotype was significantly associated with an increased HCC risk [odds ratio (OR), 2.01 (1.19-3.39); false discovery rate (FDR)-P=1.31×10-2]. In the Derivation#2 study, results were confirmed for the BRIP1 'GGG' haplotype [OR, 0.53 (0.36-0.79); FDR-P=3.90×10-3]. In both Validation#1 and #2 studies, BRIP1 'AAA' haplotype was significantly associated with an increased risk of HCC [OR, 1.71 (1.09-2.68); FDR-P=7.30×10-2; and OR, 6.45 (4.17-9.99); FDR-P=2.33×10-19, respectively]. Association between the BRIP1 locus and HCC risk suggests that impaired DNA mismatch repair might play a role in liver carcinogenesis, among patients with HCV- or HBV-related liver disease.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

The CRP level and STATE score predict survival in cirrhotic patients with hepatocellular carcinoma treated by transarterial embolization.

BACKGROUND: Prognostic biomarkers are needed in a heterogeneous population of patients with intermediate hepatocellular carcinoma (HCC) treated by transarterial (chemo)embolization. We aimed to validate the prognostic value of serum CRP levels and the STATE score, combining CRP, albumin and tumor burden. METHODS: All cirrhotic patients with HCC treated by a first transarterial (chemo)embolization (2007-2013) in our institution were included. Overall survival was assessed using the Kaplan-Meier method, log rank, univariate and multivariate Cox analyses. RESULTS: Among 157 patients included, 87% were men, 86% had Child Pugh A. Etiologies of liver disease included alcohol (57%), hepatitis C (32%), hepatitis B (11%) and/or metabolic syndrome (32%); 89% of patients were classified BCLC B. 33% of the patients had a CRP >1mg/dl and 33% a STATE score conferring poor prognosis (<18). Patients with CRP <1mg/dl had better overall survival than patients with CRP >1mg/dl (20 vs. 8 months, P=0.00186). Median overall survival was 6.73 months for patients with a STATE score <18 vs. 22.23 months for patients with STATE-score ≥18 (P=0.0002). In multivariate analysis, a STATE score <18 was independently associated with increased mortality (HR: 2.06 (CI95%: 1.28-3.34), P=0.0031). CONCLUSION: In cirrhotic patients with HCC who underwent transarterial treatment, serum CRP level and STATE score at baseline can predict overall survival.

Determination of Heavy Metal Concentrations in Normal and Pathological Human Endometrial Biopsies and In Vitro Regulation of Gene Expression by Metals in the Ishikawa and Hec-1b Endometrial Cell Line.

It is well known that several metals, such as lead, mercury, cadmium, and vanadium, can mimic the effects of estrogens (metallo-estrogens). Nevertheless, there are only a few studies that have assessed the effects of toxic metals on the female genital tract and, in particular, endometrial tissue. In this context, we measured the concentrations of several trace elements in human endometrial tissue samples from individuals with hyperplasia or adenocarcinoma and in normal tissues. Hyperplasic endometrial tissue has a 4-fold higher concentration of mercury than normal tissue. Mercury can affect both the AhR and ROS signaling pathways. Thus, we investigated the possible toxic effects of mercury by in vitro studies. We found that mercury increases oxidative stress (increased HO1 and NQO1 mRNA levels) and alters the cytoskeleton in the human endometrial Ishikawa cell line and to a lesser extent, in the "less-differentiated" human endometrial Hec-1b cells. The results might help to explain a potential link between this metal and the occurrence of endometrial hyperplasia.

Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis.

Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.

Activation of the aryl hydrocarbon receptor by carcinogenic aromatic amines and modulatory effects of their N-acetylated metabolites.

Aromatic amines (AAs) are an important class of chemicals which account for 12 % of known carcinogens. The biological effects of AAs depend mainly on their biotransformation into reactive metabolites or into N-acetylated metabolites which are generally considered as less toxic. Although the activation of the aryl hydrocarbon receptor (AhR) pathway by certain carcinogenic AAs has been reported, the effects of their N-acetylated metabolites on the AhR have not been addressed. Here, we investigated whether carcinogenic AAs and their N-acetylated metabolites may activate/modulate the AhR pathway in the absence and/or the presence of a bona fide AhR ligand (benzo[a]pyrene/B(a)P]. In agreement with previous studies, we found that certain AAs activated the AhR in human liver and lung cells as assessed by an increase in cytochrome P450 1A1 (CYP1A1) expression and activity. Altogether, we report for the first time that these properties can be modulated by the N-acetylation status of the AA. Whereas 2-naphthylamine significantly activated the AhR and induced CYP1A1 expression, its N-acetylated metabolite was less efficient. In contrast, the N-acetylated metabolite of 2-aminofluorene was able to significantly activate AhR, whereas the parent AA, 2-aminofluorene, did not. In the presence of B(a)P, activation of AhR or antagonist effects were observed depending on the AA or its N-acetylated metabolite. Activation and/or modulation of the AhR pathway by AAs and their N-acetylated metabolites may represent a novel mechanism contributing to the toxicological effects of AAs. More broadly, our data suggest biological interactions between AAs and other classes of xenobiotics through the AhR pathway.

RANTES/CCL5 mediated-biological effects depend on the syndecan-4/PKCα signaling pathway.

The perpetuation of angiogenesis is involved in certain chronic inflammatory diseases. The accelerated neovascularisation may result from an inflammatory status with a response of both endothelial cells and monocytes to inflammatory mediators such as chemokines. We have previously described in vitro and in vivo the pro-angiogenic effects of the chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES)/CCL5. The effects of RANTES/CCL5 may be related to its binding to G protein-coupled receptors and to proteoglycans such as syndecan-1 and -4. The aim of this study was to evaluate the functionality of syndecan-4 as a co-receptor of RANTES/CCL5 by the use of mutated syndecan-4 constructs. Our data demonstrate that site-directed mutations in syndecan-4 modify RANTES/CCL5 biological activities in endothelial cells. The SDC4S179A mutant, associated with an induced protein kinase C (PKC)α activation, leads to higher RANTES/CCL5 pro-angiogenic effects, whereas the SDC4L188QQ and the SDC4A198del mutants, leading to lower phosphatidylinositol 4,5-bisphosphate (PIP2) binding or to lower PDZ protein binding respectively, are associated with reduced RANTES/CCL5 cellular effects. Moreover, our data highlight that the intracellular domain of SDC-4 is involved in RANTES/CCL5-induced activation of the PKCα signaling pathway and biological effect. As RANTES/CCL5 is involved in various physiopathological processes, the development of a new therapeutic strategy may be reliant on the mechanism by which RANTES/CCL5 exerts its biological activities, for example by targeting the binding of the chemokine to its proteoglycan receptor.

Impact of cytokine gene variants on the prediction and prognosis of hepatocellular carcinoma in patients with cirrhosis.

BACKGROUND & AIMS: Genetic polymorphisms modulate the expression of proinflammatory cytokines. We prospectively assessed the influence of 6 single nucleotide polymorphisms (SNPs) in TNFα, IL6, and IL1ß genes on the risk of hepatocellular carcinoma (HCC) in patients with cirrhosis. METHODS: TNFα (G-238A, C-863A, G-308A), IL6 (C-174G), and IL1ß (C-31T, C-511T) SNPs were assessed in 232 alcoholics and 253 HCV-infected patients with biopsy-proven cirrhosis, prospectively followed-up and screened for HCC. Their influence on HCC development was assessed using the Kaplan-Meier method. RESULTS: These variants did not influence the risk of HCC in alcoholic patients. Conversely, two variants influenced the risk of HCC occurrence in patients with HCV-related cirrhosis, namely the TNFα-308 (A) allele (HR = 2.4 [1.6-3.7], Log-rank <0.0001) and the IL1ß-31 (T) allele (HR = 1.5 [1.1-2.1], Log-rank = 0.004). When stratifying HCV-infected patients into four genotypic associations expected to progressively increase TNFα and IL1ß production, we observed increasing risk of HCC occurrence (Log-rank <0.0001) from group 1 to 4. The TNFα-308 (A) allele was the only genetic trait independently associated with risk of HCC in these patients, along with older age, male gender, BMI, and platelet count. These variables led to construction of a predictive score able to separate patients with HCV-related cirrhosis into three subgroups with progressively increasing 5-year cumulative incidences of 4.7%, 14.1%, and 36.3%, respectively (Log-rank <0.0001). CONCLUSIONS: Genetic heterogeneity in the TNFα and IL1ß gene promoters influences the risk of HCC in patients with HCV-induced cirrhosis. These genetic data, when incorporated into clinical scores, are able to refine selection of risk classes of HCC.

Association between the PNPLA3 (rs738409 C>G) variant and hepatocellular carcinoma: Evidence from a meta-analysis of individual participant data.

UNLABELLED: The incidence of hepatocellular carcinoma (HCC) is increasing in Western countries. Although several clinical factors have been identified, many individuals never develop HCC, suggesting a genetic susceptibility. However, to date, only a few single-nucleotide polymorphisms have been reproducibly shown to be linked to HCC onset. A variant (rs738409 C>G, encoding for p.I148M) in the PNPLA3 gene is associated with liver damage in chronic liver diseases. Interestingly, several studies have reported that the minor rs738409[G] allele is more represented in HCC cases in chronic hepatitis C (CHC) and alcoholic liver disease (ALD). However, a significant association with HCC related to CHC has not been consistently observed, and the strength of the association between rs738409 and HCC remains unclear. We performed a meta-analysis of individual participant data including 2,503 European patients with cirrhosis to assess the association between rs738409 and HCC, particularly in ALD and CHC. We found that rs738409 was strongly associated with overall HCC (odds ratio [OR] per G allele, additive model=1.77; 95% confidence interval [CI]: 1.42-2.19; P=2.78 × 10(-7) ). This association was more pronounced in ALD (OR=2.20; 95% CI: 1.80-2.67; P=4.71 × 10(-15) ) than in CHC patients (OR=1.55; 95% CI: 1.03-2.34; P=3.52 × 10(-2) ). After adjustment for age, sex, and body mass index, the variant remained strongly associated with HCC. CONCLUSION: Overall, these results suggest that rs738409 exerts a marked influence on hepatocarcinogenesis in patients with cirrhosis of European descent and provide a strong argument for performing further mechanistic studies to better understand the role of PNPLA3 in HCC development.

Serum proteoglycans as prognostic biomarkers of hepatocellular carcinoma in patients with alcoholic cirrhosis.

BACKGROUND: Proteoglycans are involved in neoangiogenesis and transduction of oncogenic signals, two hallmarks of carcinogenesis. METHODS: This study sought to assess the prognostic value of serum levels of three proteoglycans (endocan, syndecan-1, and glypican-3) and VEGF in 295 patients with alcoholic cirrhosis: 170 without hepatocellular carcinoma, 58 with early hepatocellular carcinoma, and 67 with advanced hepatocellular carcinoma at inclusion. We analyzed the association between proteoglycan levels and prognosis using Kaplan-Meier and Cox methods. RESULTS: Serum levels of the three proteoglycans and VEGF were increased in patients with advanced hepatocellular carcinoma compared with those without hepatocellular carcinoma or with early hepatocellular carcinoma. In multivariate analysis, high levels of serum endocan (>5 ng/mL) were independently associated with death [HR, 2.84; 95% confidence interval (CI,) 1.18-6.84; P = 0.02], but not with hepatocellular carcinoma occurrence, in patients without hepatocellular carcinoma at baseline. High serum endocan (>5 ng/mL) and syndecan-1 (>50 ng/mL) levels were significantly associated with greater risk of tumor recurrence (P = 0.025) in patients with early hepatocellular carcinoma treated by radiofrequency ablation. In patients with advanced hepatocellular carcinoma, high serum levels of endocan (P = 0.004) and syndecan-1 (P = 0.006) were significantly associated with less favorable overall survival. However, only a high level of serum syndecan-1 (>50 ng/mL) was independently associated with greater risk of death (HR, 6.21 95% CI, 1.90-20.30; P = 0.0025). CONCLUSION: Serum endocan and syndecan-1 are easily assessable prognostic serum biomarkers of overall survival in alcoholic cirrhosis with and without hepatocellular carcinoma. IMPACT: These new biomarkers will be useful to manage patients with hepatocellular carcinoma developed on alcoholic cirrhosis.

PNPLA3 rs738409, hepatocellular carcinoma occurrence and risk model prediction in patients with cirrhosis.

BACKGROUND & AIMS: Several studies have reported an association between the genetic variant rs738409 (G) in the PNPLA3 gene and the risk of cirrhosis in various liver diseases. Our purpose was to assess the influence of this polymorphism on the risk of hepatocellular carcinoma (HCC) occurrence in two distinct longitudinal cohorts of patients with cirrhosis as well as its possible usefulness in HCC-risk model prediction. METHODS: PNPLA3 rs738409 genotypes were assessed in 279 patients with alcoholic- and 253 patients with HCV-related cirrhosis. These patients were followed-up and screened for the risk of HCC, and the influence of rs738409 on the occurrence of liver cancer was assessed using the Kaplan-Meier method, then according to the multivariate Cox model. RESULTS: In patients with HCV-related cirrhosis, rs738409 genotypes did not influence the risk of HCC development (log-rank = 0.7) or death (log-rank = 0.2). Conversely, in patients with alcoholic cirrhosis, the rs738409 (GG) genotype was an independent risk factor for HCC occurrence (HR = 1.72 [1.21-2.45], log-rank = 0.002) as well as older age, male gender, and higher BMI. Combining these features enabled HCC-risk stratification of this population into three groups with the 6-year cumulative incidence ranging from 3.4% (low risk, n = 58), 12.2% (intermediate risk, n = 163), and 51.7% (high risk, n = 58), respectively (HR = 4.3 [2.7-6.4]; log-rank <0.0001). CONCLUSIONS: This study provides key data that affirm the influence of the rs738409 (GG) genotype on the occurrence of HCC in patients with alcoholic cirrhosis. Its combination with clinical features refines the selection of patients at higher risk of liver cancer development.

The AhR twist: ligand-dependent AhR signaling and pharmaco-toxicological implications.

The aryl hydrocarbon receptor (AhR) is a transcription factor which is activated by diverse compounds and regulates the expression of xenobiotic metabolism genes. Recent studies have unraveled unsuspected physiological roles and novel alternative ligand-specific pathways for this receptor. In this review, we discuss these novel aspects and focus on the different responses elicited by the diverse endogenous and/or exogenous AhR ligands. In addition to challenging the relevance of the 'agonist/antagonist' classification of ligands, we introduce the concept of AhR plasticity as a primordial factor in the generation of these pathways. Finally, we suggest several promising perspectives for the pharmacological modulation of these responses.

Inflammatory pathway genes belong to major targets of persistent organic pollutants in adipose cells.

BACKGROUND: Epidemiological studies emphasize the possible role of persistent organic pollutants (POPs) in obesity and the metabolic syndrome. These pollutants are stored in adipose tissue (AT). OBJECTIVES: Our aim was to study the effects of POPs on human adipose cells and rodent AT. METHODS: Using human multipotent adipose-derived stem cells, we carried out large-scale gene expression analysis to identify the major pathways modified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl (PCB) congener 126 (PCB-126), and PCB-153 and to evaluate their toxic effects. The effects of TCDD on gene expression and AT histology were also assessed in mice. RESULTS: The most significantly regulated genes in both precursor cells and adipocytes were those involved in the inflammatory/immune response, cancer, and metabolism pathways. Interestingly, the fold induction and the number of modulated genes were higher in precursors than in adipocytes, suggesting that the former could be more sensitive to the effect of pollutants. When cells were treated with combinations of pollutants, the effects of the AhR ligands TCDD and PCB-126 were dominant compared with those of the non-dioxin-like PCB-153. The effects of AhR ligands were reduced by the AhR antagonist α-naphthoflavone. The regulation of inflammatory pathway was observed in wild-type AT but not in AhR-knockout mice. CONCLUSIONS: Both in vitro and in vivo studies showed that adipose cells were targets of AhR ligands and suggest that inflammation is one of the main regulated pathways. These observations suggest a possible contribution of pollutants to low-grade AT inflammation that accompanies the pathogenesis of metabolic diseases.