Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis in the absence of a history of significant alcohol use or other known liver disease. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. The Pathology Committee of the NASH Clinical Research Network designed and validated a histological feature scoring system that addresses the full spectrum of lesions of NAFLD and proposed a NAFLD activity score (NAS) for use in clinical trials. The scoring system comprised 14 histological features, 4 of which were evaluated semi-quantitatively: steatosis (0-3), lobular inflammation (0-2), hepatocellular ballooning (0-2), and fibrosis (0-4). Another nine features were recorded as present or absent. An anonymized study set of 50 cases (32 from adult hepatology services, 18 from pediatric hepatology services) was assembled, coded, and circulated. For the validation study, agreement on scoring and a diagnostic categorization ("NASH," "borderline," or "not NASH") were evaluated by using weighted kappa statistics. Inter-rater agreement on adult cases was: 0.84 for fibrosis, 0.79 for steatosis, 0.56 for injury, and 0.45 for lobular inflammation. Agreement on diagnostic category was 0.61. Using multiple logistic regression, five features were independently associated with the diagnosis of NASH in adult biopsies: steatosis (P ‫؍‬ .009), hepatocellular ballooning (P ‫؍‬ .0001), lobular inflammation (P ‫؍‬ .0001), fibrosis (P ‫؍‬ .0001), and the absence of lipogranulomas (P ‫؍‬ .001). The proposed NAS is the unweighted sum of steatosis, lobular inflammation, and hepatocellular ballooning scores. In conclusion, we present a strong scoring system and NAS for NAFLD and NASH with reasonable inter-rater reproducibility that should be useful for studies of both adults and children with any degree of NAFLD. NAS of >5 correlated with a diagnosis of NASH, and biopsies with scores of less than 3 were diagnosed as "not NASH." (HEPATOLOGY 2005;41:1313-1321

SUMMARY Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole exome sequencing and DNA copy number analyses, and 196 HCC also by DNA methylation, RNA, miRNA, and proteomic expression. DNA sequencing and mutation analysis identified significantly mutated genes including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or down-regulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.

Background & Aims: NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods: We fed foz/foz and wild-type mice an atherogenic diet for 16 weeks, gavaged MCC950 or vehicle until 24 weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6 weeks and determined the effects on liver fibrosis. Results: In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24 weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion: MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. Lay summary: Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

Serum ferritin (SF) levels are commonly elevated in patients with nonalcoholic fatty liver disease (NAFLD), due to systemic inflammation, increased iron stores or both. The aim of this study was to examine the relationship between elevated SF and NAFLD severity. Demographic, clinical, histologic, laboratory and anthropometric data were analyzed in 628 adult patients with NAFLD (age≥18 years) with biopsy-proven NAFLD and a serum ferritin measurement within six months of their liver biopsy. A threshold SF>1.5XULN (i.e. >300 ng/ml in women and >450 ng/ml in men) was significantly associated with male sex, elevated serum ALT, AST, iron, transferrin-iron saturation, iron stain grade and decreased platelets (p<0.01). Histologic features of NAFLD were more severe among patients with SF>1.5XULN including steatosis, fibrosis, hepatocellular ballooning and diagnosis of NASH (p<0.026). On multiple regression analysis, SF>1.5XULN was independently associated with advanced hepatic fibrosis (OR, 1.66, 95% CI, 1.05-2.62, p=0.028) and increased NAFLD Activity Score (NAS) (OR, 1.99, 95% CI, 1.06-3.75, p=0.033). Conclusions A SF >1.5XULN is associated with hepatic iron deposition, a diagnosis of NASH, and worsened histologic activity, and is an independent predictor of advanced hepatic fibrosis among patients with NAFLD. Furthermore, elevated SF is independently associated with higher NAS even among patients without hepatic iron deposition. We conclude that serum ferritin is useful to identify NAFLD patients at risk for NASH and advanced fibrosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of referral to liver clinics, and its progressive form, non-alcoholic steatohepatitis (NASH), can lead to cirrhosis and end-stage liver disease. The main risk factors for NAFLD/NASH are the metabolic abnormalities commonly observed in metabolic syndrome: insulin resistance, visceral obesity, dyslipidemia and altered adipokine profile. At present, the causes of progression from NAFLD to NASH remain poorly defined, and research in this area has been limited by the availability of suitable animal models of this disease. In the past, the main models used to investigate the pathogenesis of steatohepatitis have either failed to reproduce the full spectrum of liver pathology that characterizes human NASH, or the liver pathology has developed in a metabolic context that is not representative of the human condition. In the last few years, a number of models have been described in which the full spectrum of liver pathology develops in an appropriate metabolic context. In general, the underlying cause of metabolic defects in these models is chronic caloric overconsumption, also known as overnutrition. Overnutrition has been achieved in a number of different ways, including forced feeding, administration of high-fat diets, the use of genetically hyperphagic animals, or a combination of these approaches. The purpose of the present review is to critique the liver pathology and metabolic abnormalities present in currently available animal models of NASH, with particular focus on models described in approximately the last 5 years.