Person:

Fuchs, Bryan

Context: Overexpression of epidermal growth factor (EGF) in the liver induces transformation to hepatocellular carcinoma in animal models. Polymorphisms in the EGF gene modulate EGF levels. Objective: To assess the relationship among human EGF gene single-nucleotide polymorphism, EGF expression, and risk of hepatocellular carcinoma. Design, Setting, and Participants: Molecular mechanisms linking the 61G allele polymorphism to EGF expression were examined in human hepatocellular carcinoma cell lines and human liver tissue. A case-control study involving 207 patients with cirrhosis was conducted at the Massachusetts General Hospital (1999-2006) and a validation case-control study involving 121 patients with cirrhosis was conducted at Hôpital Paul Brousse (1993-2006). Restriction fragment-length polymorphism was used to determine the EGF gene polymorphism genotype. Logistic regression analysis was used to assess the association between the EGF polymorphism and hepatocellular carcinoma risk. Main Outcome Measures: Mechanisms by which the EGF gene polymorphism modulates EGF levels and associations among EGF gene polymorphism, EGF levels, and hepatocellular carcinoma. Results: Transcripts from the EGF 61G allele exhibited more than a 2-fold longer half-life than those from the 61*A allele, and EGF secretion was 2.3-fold higher in G/G hepatocellular carcinoma cell lines than A/A cell lines. Serum EGF levels were 1.8-fold higher in G/G patients than A/A patients, and liver EGF levels were 2.4-fold higher in G/G patients than A/A patients. Among the 207 patients with cirrhosis in the Massachusetts study population, 59 also had hepatocellular carcinoma. Analysis of the distribution of allelic frequencies revealed that there was a 4-fold odds of hepatocellular carcinoma in G/G patients compared with A/A patients in the Massachusetts study population (odds ratio, 4.0; 95% confidence interval [CI], 1.6-9.6; P = .002). Logistic regression analysis demonstrated that the number of copies of G was significantly associated with hepatocellular carcinoma after adjusting for age, sex, race, etiology, and severity of cirrhosis (G/G or A/G vs A/A; hazard ratio, 3.49; 95% CI, 1.29-9.44; P = .01). The significant association was validated in the French patients with alcoholic cirrhosis and hepatocellular carcinoma. Conclusion: The EGF gene polymorphism genotype is associated with risk for development of hepatocellular carcinoma in liver cirrhosis through modulation of EGF levels.

Single nucleotide polymorphisms (SNPs) in the epidermal growth factor (EGF, rs4444903), patatin-like phospholipase domain-containing protein 3 (PNPLA3, rs738409) genes, and near the interleukin-28B (IL28B, rs12979860) gene are linked to treatment response, fibrosis, and hepatocellular carcinoma (HCC) in chronic hepatitis C. Whether these SNPs independently or in combination predict clinical deterioration in hepatitis C virus (HCV)-related cirrhosis is unknown. We genotyped SNPs in EGF, PNPLA3, and IL28B from liver tissue from 169 patients with biopsy-proven HCV cirrhosis. We estimated risk of clinical deterioration, defined as development of ascites, encephalopathy, variceal hemorrhage, HCC, or liver-related death using Cox proportional hazards modeling. During a median follow-up of 6.6 years, 66 of 169 patients experienced clinical deterioration. EGF non-AA, PNPLA3 non-CC, and IL28B non-CC genotypes were each associated with increased risk of clinical deterioration in age, sex, and race-adjusted analysis. Only EGF non-AA genotype was independently associated with increased risk of clinical deterioration (hazard ratio [HR] 2.87; 95% confidence interval [CI] 1.31–6.25) after additionally adjusting for bilirubin, albumin, and platelets. Compared to subjects who had 0–1 unfavorable genotypes, the HR for clinical deterioration was 1.79 (95%CI 0.96–3.35) for 2 unfavorable genotypes and 4.03 (95%CI 2.13–7.62) for unfavorable genotypes for all three loci (Ptrend<0.0001). In conclusion, among HCV cirrhotics, EGF non-AA genotype is independently associated with increased risk for clinical deterioration. Specific PNPLA3 and IL28B genotypes also appear to be associated with clinical deterioration. These SNPs have potential to identify patients with HCV-related cirrhosis who require more intensive monitoring for decompensation or future therapies preventing disease progression.

Background: A single nucleotide polymorphism (SNP) in the epidermal growth factor (EGF) gene (rs4444903) has been associated with increased risk of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the relationship between the EGF SNP genotype and the development and prognosis of HCC, in a Japanese population. Methods: Restriction fragment-length polymorphism was used to determine the presence of the EGF SNP genotype in 498 patients, including 208 patients with HCC. The level of EGF messenger ribonucleic acid (mRNA) expression in cancerous tissues was measured by quantitative reverse transcription polymerase chain reaction. The correlation between the EGF SNP genotype and prognosis was statistically analyzed in the patients with HCC. Results: The proportion of the A/A, A/G, and G/G genotypes were 5.3%, 42.8%, and 51.9%, respectively, in the patients with HCC, whereas in those without HCC, they were 8.6%, 35.9%, and 55.5%, respectively, revealing that the odds ratio (OR) of developing HCC was higher in patients with a G allele (OR =1.94, P=0.080 for A/G patients and OR =1.52, P=0.261 for G/G patients, as compared with A/A patients). In particular, when the analysis was limited to the 363 patients with hepatitis C, the OR for developing HCC was 3.54 (P=0.014) for A/G patients and was 2.85 (P=0.042) for G/G patients, as compared with A/A patients. Tumoral EGF mRNA expression in G/G patients was significantly higher than that in A/A patients (P=0.033). No statistically significant differences were observed between the EGF SNP genotype and diseasefree or overall survival. Conclusion: The EGF SNP genotype might be associated with a risk for the development of HCC in Japanese patients but not with prognosis. Of note, the association is significantly stronger in patients with hepatitis C, which is the main risk factor for HCC in Japan.

Background & Aims: Liver biopsy, the current clinical gold standard for fibrosis assessment, is invasive and has sampling errors, and is not optimal for screening, monitoring, or clinical decision-making. Fibrosis is characterized by excessive accumulation of extracellular matrix proteins including type I collagen. We hypothesize that molecular magnetic resonance imaging (MRI) with a probe targeted to type I collagen could provide a direct and non-invasive method of fibrosis assessment. Methods: Liver fibrosis was induced in rats with diethylnitrosamine and in mice with carbon tetrachloride. Animals were imaged prior to and immediately following i.v. administration of either collagen-targeted probe EP-3533 or non-targeted control Gd-DTPA. Magnetic resonance (MR) signal washout characteristics were evaluated from T1 maps and T1-weighted images. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline. Results: EP-3533-enhanced MR showed greater signal intensity on delayed imaging (normalized signal enhancement mice: control = 0.39 ± 0.04, fibrotic = 0.55 ± 0.03, p <0.01) and slower signal washout in the fibrotic liver compared to controls (liver t1/2 = 51.3 ± 3.6 vs. 42.0 ± 2.5 min, p <0.05 and 54.5 ± 1.9 vs. 44.1 ± 2.9 min, p <0.01 for fibrotic vs. controls in rat and mouse models, respectively). Gd-DTPA-enhanced MR could not distinguish fibrotic from control animals. EP-3533 gadolinium concentration in the liver showed strong positive correlations with hydroxyproline levels (r = 0.74 (rats), r = 0.77 (mice)) and with Ishak scoring (r = 0.84 (rats), r = 0.79 (mice)). Conclusions: Molecular MRI of liver fibrosis with a collagen-specific probe identifies fibrotic tissue in two rodent models of disease.

Background & Aims The gold standard in assessing liver fibrosis is biopsy despite limitations like invasiveness and sampling error and complications including morbidity and mortality. Therefore, there is a major unmet medical need to quantify fibrosis non-invasively to facilitate early diagnosis of chronic liver disease and provide a means to monitor disease progression. The goal of this study was to evaluate the ability of several magnetic resonance imaging (MRI) techniques to stage liver fibrosis.

Methods A gadolinium (Gd)-based MRI probe targeted to type I collagen (termed EP-3533) was utilized to non-invasively stage liver fibrosis in a carbon tetrachloride (CCl4) mouse model and the results were compared to other MRI techniques including relaxation times, diffusion, and magnetization transfer measurements.

Results The most sensitive MR biomarker was the change in liver:muscle contrast to noise ratio (ΔCNR) after EP-3533 injection. We observed a strong positive linear correlation between ΔCNR and liver hydroxyproline (i.e. collagen) levels (r = 0.89) as well as ΔCNR and conventional Ishak fibrosis scoring. In addition, the area under the receiver operating curve (AUR0C) for distinguishing early (Ishak ⩽3) from late (Ishak ⩾4) fibrosis was 0.942 ± 0.052 (p <0.001). By comparison, other MRI techniques were not as sensitive to changes in fibrosis in this model.

Conclusions We have developed an MRI technique using a collagen-specific probe for diagnosing and staging liver fibrosis, and validated it in the CCl4 mouse model. This approach should provide a better means to monitor disease progression in patients.

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.

Background: In the setting of chronic liver injury in humans, epidermal growth factor (EGF) and EGF receptor (EGFR) are up-regulated and have been proposed to have vital roles in both liver regeneration and development of hepatocellular carcinoma (HCC). Chronic liver injury also leads to hepatic stellate cell (HSC) differentiation and a novel subpopulation of HSCs which express CD133 and exhibit properties of progenitor cells has been described in rats. The carbon tetrachloride (CCl4)-induced mouse model has been historically relied upon to study liver injury and regeneration. We exposed mice to CCl4 to assess whether EGF and CD133+ HSCs are up-regulated in chronically injured liver. Methods: CCl4 in olive oil was administered to strain A/J mice three times per week by oral gavage. Results: Multiple well-differentiated HCCs were found in all livers after 15 weeks of CCl4 treatment. Notably, HCCs developed within the setting of fibrosis and not cirrhosis. CD133 was dramatically up-regulated after CCl4 treatment, and increased expression of desmin and glial fibrillary acidic protein, representative markers of HSCs, was also observed. EGF expression significantly decreased, contrary to observations in humans, whereas the expression of amphiregulin, another EGFR ligand, was significantly increased. Conclusions: Species-specific differences exist with respect to the histopathological and molecular pathogenesis of chronic liver disease. CCl4-induced chronic liver injury in A/J mice has important differences compared to human cirrhosis leading to HCC.