Environmental toxicants modulate disease severity in pediatric metabolic dysfunction-associated steatohepatitis.

OBJECTIVES: Metabolic dysfunction-associated steatotic liver disease (MASLD) is common in children. We hypothesized environmental toxins could drive progression to metabolic dysfunction-associated steatohepatitis (MASH), and assayed serum toxins and metabolites in children with histologically characterized MASLD/MASH. METHODS: Environmental chemicals, common in household items, perfluoroalkyl substances (PFAS), brominated flame retardants (PBDEs), and metabolic profiles were assayed in children enrolled in the multicenter NASH Clinical Research Network Pediatric Database 2. Mixture models, using repeated holdout weighted quantile sum regression (WQSrh) were run in addition to single chemical/metabolite logistic regression. For metabolomic analyses, random subset version of WQSrh was used for the large number of predictors versus participants. Nominal and false discovery rate (FDR) p-values (two-sided) were computed. RESULTS: Four hundred and thirty-five children distributed across MASH (n = 293) and MASLD (n = 142), with 304 (69.9%) males. Mean (standard deviation) for Nonalcoholic Steatohepatitis Score (NAS) and alanine aminotransferase (ALT) for MASLD were 3.1 (1.0), 67.9 (43.4), and for MASH 4.2 (1.4), 144 (121). There was an inverse association between PFAS/PBDE mixture and MASH versus MASLD, lobular inflammation (p = 0.026), NAS (p = 0.009, FDR p = 0.04), and log-transformed ALT (p = 0.005, FDR p = 0.025) driven by perfluorohexane sulfonate (PFHxS). Metabolites from positive hydrophilic interaction liquid chromatography mode, biliverdin (p = 0.002) and 1-methylhistidine (associated with meat ingestion, p = 0.02) and reverse phase negative mode, hippuric acid (solvent exposure, p = 0.022) significantly associated with MASH. CONCLUSIONS: Significant negative PFAS/PBDE mixture effect and odds of MASH were dominated by PHFXS. Several metabolites are significantly associated with MASH which inform mechanistic pathways and could drive key therapeutic and diagnostic strategies in children.

MASLD in people with HIV exhibits higher fibrosis stage despite lower disease activity than in matched controls.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) is common in people with HIV (PWH). The morphological spectrum of MASLD compared to matched controls and of the correlation between the NAFLD activity score (NAS) and fibrosis stage in PWH remains unknown. METHODS: Overall, 107 liver biopsies from PWH with MASLD (MASLD-PWH) were matched to 107 biopsies from individuals with MASLD and without HIV (MASLD controls) on age at biopsy, race/ethnicity, sex, type 2 diabetes, body mass index (BMI) and alanine aminotransferase (ALT) level. Biopsies were scored using NAS. RESULTS: Compared to MASLD-controls, MASLD-PWH had lower steatosis grade (OR: 0.65, 95% CI: (0.47-0.90), p = 0.01), lower lobular inflammation grade (OR: 0.55, 95% CI: (0.34-0.89), p = 0.02), less portal inflammation (OR: 0.42, 95% CI: (0.25-0.72), p = 0.002) and less ballooned hepatocytes (OR: 0.60, 95% CI: (0.41-0.88), p = 0.01). Thus, NAS was lower in MASLD-PWH (OR: 0.69, 95% CI: (0.56-0.85), p < 0.001) than in MASLD controls. There was a trend towards lower prevalence of steatohepatitis in MASLD-PWH (OR: 0.84, 95% CI: (0.68-1.03), p = 0.09). A multivariate analysis demonstrated that MASLD-PWH cases had significantly less steatosis (OR: 0.66, p = 0.03), portal inflammation (OR: 0.34, p = 0.001) and ballooned hepatocytes (OR: 0.55, p = 0.01), yet higher stage fibrosis (OR: 1.42, p = 0.03) compared to MASLD controls. CONCLUSION: The NAS and histological drivers of fibrosis (e.g. inflammation and hepatocyte ballooning) are less pronounced in MASLD-PWH, and yet fibrosis stage was generally higher when compared to matched controls with MASLD without HIV. This suggests HIV-specific factors beyond hepatic necroinflammation may contribute to fibrosis progression in MASLD-PWH.

Liver stiffness progression in biopsy-proven metabolic dysfunction-associated steatotic disease among people with diabetes versus people without diabetes: A prospective multicenter study.

BACKGROUND AND AIMS: There are limited data on the progression of liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in people with type 2 diabetes mellitus (T2DM) versus those without T2DM in biopsy-proven metabolic dysfunction-associated steatotic liver disease. We examined LSM progression in participants with T2DM versus those without T2DM in a large, prospective, multicenter cohort study. APPROACH AND RESULTS: This study included 1231 adult participants (62% female) with biopsy-proven metabolic dysfunction-associated steatotic liver disease who had VCTEs at least 1 year apart. LSM progression and regression were defined by a ≥20% increase and an upward or downward change, respectively, in the LSM category in the Baveno VII categories for compensated advanced chronic liver disease, compared between participants with T2DM (n = 680) versus no T2DM (n = 551) at baseline. The mean (±SD) age and body mass index were 51.8 (±12.0) years and 34.0 (±6.5) kg/m 2 , respectively. The median (IQR) time between the first and last VCTE measurements was 4.1 (2.5-6.5) years. Participants with T2DM had higher LSM progression at 4 years (12% vs. 10%), 6 years (23% vs. 16%), and 8 years (50% vs. 39%), p = 0.04. Using a multivariable Cox proportional hazards model adjusted for multiple confounders, the presence of T2DM remained an independent predictor of LSM progression (adjusted HR: 1.35, 95% CI: 1.01-1.81, p = 0.04). T2DM was not associated with LSM regression ( p = 0.71). Mean HbA1c was significantly associated with LSM progression ( p = 0.003) and regression ( p = 0.02). CONCLUSIONS: Using serial VCTE data from a multicenter study of participants with biopsy-proven metabolic dysfunction-associated steatotic liver disease, we demonstrate that T2DM and HbA1c are associated with LSM progression.

Tirzepatide for Metabolic Dysfunction-Associated Steatohepatitis with Liver Fibrosis.

BACKGROUND: Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease associated with liver-related complications and death. The efficacy and safety of tirzepatide, an agonist of the glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptors, in patients with MASH and moderate or severe fibrosis is unclear. METHODS: We conducted a phase 2, dose-finding, multicenter, double-blind, randomized, placebo-controlled trial involving participants with biopsy-confirmed MASH and stage F2 or F3 (moderate or severe) fibrosis. Participants were randomly assigned to receive once-weekly subcutaneous tirzepatide (5 mg, 10 mg, or 15 mg) or placebo for 52 weeks. The primary end point was resolution of MASH without worsening of fibrosis at 52 weeks. A key secondary end point was an improvement (decrease) of at least one fibrosis stage without worsening of MASH. RESULTS: Among 190 participants who had undergone randomization, 157 had liver-biopsy results at week 52 that could be evaluated, with missing values imputed under the assumption that they would follow the pattern of results in the placebo group. The percentage of participants who met the criteria for resolution of MASH without worsening of fibrosis was 10% in the placebo group, 44% in the 5-mg tirzepatide group (difference vs. placebo, 34 percentage points; 95% confidence interval [CI], 17 to 50), 56% in the 10-mg tirzepatide group (difference, 46 percentage points; 95% CI, 29 to 62), and 62% in the 15-mg tirzepatide group (difference, 53 percentage points; 95% CI, 37 to 69) (P<0.001 for all three comparisons). The percentage of participants who had an improvement of at least one fibrosis stage without worsening of MASH was 30% in the placebo group, 55% in the 5-mg tirzepatide group (difference vs. placebo, 25 percentage points; 95% CI, 5 to 46), 51% in the 10-mg tirzepatide group (difference, 22 percentage points; 95% CI, 1 to 42), and 51% in the 15-mg tirzepatide group (difference, 21 percentage points; 95% CI, 1 to 42). The most common adverse events in the tirzepatide groups were gastrointestinal events, and most were mild or moderate in severity. CONCLUSIONS: In this phase 2 trial involving participants with MASH and moderate or severe fibrosis, treatment with tirzepatide for 52 weeks was more effective than placebo with respect to resolution of MASH without worsening of fibrosis. Larger and longer trials are needed to further assess the efficacy and safety of tirzepatide for the treatment of MASH. (Funded by Eli Lilly; SYNERGY-NASH ClinicalTrials.gov number, NCT04166773.).

Aramchol improves hepatic fibrosis in metabolic dysfunction-associated steatohepatitis: Results of multimodality assessment using both conventional and digital pathology.

BACKGROUND AND AIMS: Antifibrotic trials rely on conventional pathology despite recognized limitations. We compared single-fiber digital image analysis with conventional pathology to quantify the antifibrotic effect of Aramchol, a stearoyl-CoA desaturase 1 inhibitor in development for metabolic dysfunction-associated steatohepatitis. APPROACH AND RESULTS: Fifty-one patients with metabolic dysfunction-associated steatohepatitis enrolled in the open-label part of the ARMOR trial received Aramchol 300 mg BID and had paired pre-post treatment liver biopsies scored by consensus among 3 hepatopathologists, and separately assessed by a digital image analysis platform (PharmaNest) that generates a continuous phenotypic Fibrosis Composite Severity (Ph-FCS) score. Fibrosis improvement was defined as: ≥1 NASH Clinical Research Network (NASH-CRN) stage reduction; "improved" by ranked pair assessment; reduction in Ph-FCS ("any" for ≥0.3 absolute reduction and "substantial" for ≥25% relative reduction). Fibrosis improved in 31% of patients (NASH-CRN), 51% (ranked pair assessment), 74.5% (any Ph-FCS reduction), and 41% (substantial Ph-FCS reduction). Most patients with stable fibrosis by NASH-CRN or ranked pair assessment had a Ph-FCS reduction (a third with substantial reduction). Fibrosis improvement increased with treatment duration: 25% for <48 weeks versus 39% for ≥48 weeks by NASH-CRN; 43% versus 61% by ranked pair assessment, mean Ph-FCS reduction -0.54 (SD: 1.22) versus -1.72 (SD: 1.02); Ph-FCS reduction (any in 54% vs. 100%, substantial in 21% vs. 65%). The antifibrotic effect of Aramchol was corroborated by reductions in liver stiffness, Pro-C3, and enhanced liver fibrosis. Changes in Ph-FCS were positively correlated with changes in liver stiffness. CONCLUSIONS: Continuous fibrosis scores generated in antifibrotic trials by digital image analysis quantify antifibrotic effects with greater sensitivity and a larger dynamic range than conventional pathology.

Investigating the Relationship Between Rare Genetic Variants and Fibrosis in Pediatric Nonalcoholic Fatty Liver Disease.

Background and Aims: Nonalcoholic Fatty Liver Disease (NAFLD) is a complex human disease. Common genetic variation in the patatin-like phospholipase domain containing 3 (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) genes have been associated with an increased risk of developing NAFLD, nonalcoholic steatohepatitis (NASH), and fibrosis in adults. The role of rare genetic variants in the development and progression of NAFLD in children is not well known. We aimed to explore the role of rare genetic variants in pediatric patients with advanced fibrosis. Methods: Whole exome sequencing data was generated for 229 pediatric patients diagnosed with NAFLD recruited from the NASH Clinical Research Network (NASH CRN). Case-control single variant and gene-based collapsing analyses were used to test for rare variants that were enriched or depleted within the pediatric NAFLD cohort specifically for advanced fibrosis (cases) versus those without fibrosis (controls) or six other histologic characteristics. Exome data from non-NAFLD population controls were also used for additional analyses. All results were adjusted for multiple testing using a Bonferroni correction. Results: No genome-wide significant associations were found between rare variation and presence of advanced fibrosis or NASH, nor the severity of steatosis, inflammation, or hepatocellular ballooning. Significantly, no enrichment of rare variants in PNPLA3 or TM6SF2 was observed across phenotypes. Conclusion: In a cohort of children with histologically proven NAFLD, no genome-wide significant associations were found between rare genetic variation and advanced fibrosis or six other histologic features. Of particular interest was the lack of association with genes of interest in adults: PNPLA3 and TM6SF2, though limitations in sample size may reduce the ability to detect associations, particularly with rare variation.

Review article: New developments in biomarkers and clinical drug development in alpha-1 antitrypsin deficiency-related liver disease.

BACKGROUND: Alpha-1 antitrypsin liver disease (AATLD) occurs in a subset of patients with alpha-1 antitrypsin deficiency. Risk factors for disease progression and specific pathophysiologic features are not well known and validated non-invasive assessments for disease severity are lacking. Currently, there are no approved treatments for AATLD. AIMS: To outline existing understanding of AATLD and to identify knowledge gaps critical to improving clinical trial design and development of new treatments. METHODS: This report was developed following a multi-stakeholder forum organised by the Alpha-1 Antitrypsin Deficiency Related Liver Disease Expert Panel in which experts presented an overview of the available literature on this topic. RESULTS: AATLD results from a 'gain of toxic function' and primarily manifests in those with the homozygous Pi*ZZ genotype. Accumulation of misfolded 'Z' AAT protein in liver cells triggers intracellular hepatocyte injury which may ultimately lead to hepatic fibrosis. Male gender, age over 50 years, persistently elevated liver tests, concomitant hepatitis B or C virus infection, and metabolic syndrome, including obesity and type 2 diabetes mellitus, are known risk factors for adult AATLD. While the gold standard for assessing AATLD disease activity is liver histology, less invasive measures with low intra- and inter-observer variability are needed. Measurement of liver stiffness shows promise; validated thresholds for staging AATLD are in development. Such advances will help patients by enabling risk stratification and personalised surveillance, along with streamlining the development process for novel therapies. CONCLUSIONS: This inaugural forum generated a list of recommendations to address unmet needs in the field of AATLD.

A serum-induced gene signature in hepatocytes is associated with pediatric nonalcoholic fatty liver disease.

OBJECTIVE: Pediatric nonalcoholic fatty liver disease (NAFLD) is a growing problem, but its underlying mechanisms are poorly understood. We used transcriptomic reporter cell assays to investigate differences in transcriptional signatures induced in hepatocyte reporter cells by the sera of children with and without NAFLD. METHODS: We studied serum samples from 45 children with NAFLD and 28 children without NAFLD. The sera were used to induce gene expression in cultured HepaRG cells and RNA-sequencing was used to determine gene expression. Computational techniques were used to compare gene expression patterns. RESULTS: Sera from children with NAFLD induced the expression of 195 genes that were significantly differentially expressed in hepatocytes compared to controls with obesity. NAFLD was associated with increased expression of genes promoting inflammation, collagen synthesis, and extracellular matrix remodeling. Additionally, there was lower expression of genes involved in endobiotic and xenobiotic metabolism, and downregulation of peroxisome function, oxidative phosphorylation, and xenobiotic, bile acid, and fatty acid metabolism. A 13-gene signature, including upregulation of TREM1 and MMP1 and downregulation of CYP2C9, was consistently associated with all diagnostic categories of pediatric NAFLD. CONCLUSION: The extracellular milieu of sera from children with NAFLD induced specific gene profiles distinguishable by a hepatocyte reporter system. Circulating factors may contribute to inflammation and extracellular matrix remodeling and impair xenobiotic and endobiotic metabolism in pediatric NAFLD.

MASH Resolution Index: development and validation of a non-invasive score to detect histological resolution of MASH.

BACKGROUND: Dynamic changes in non-invasive tests, such as changes in alanine aminotransferase (ALT) and MRI proton-density-fat-fraction (MRI-PDFF), may help to detect metabolic dysfunction-associated steatohepatitis (MASH) resolution, but a combination of non-invasive tests may be more accurate than either alone. We developed a novel non-invasive score, the MASH Resolution Index, to detect the histological resolution of MASH. METHODS: This study included a derivation cohort of 95 well-characterised adult participants (67% female) with biopsy-confirmed MASH who underwent contemporaneous laboratory testing, MRI-PDFF and liver biopsy at two time points. The primary objective was to develop a non-invasive score to detect MASH resolution with no worsening of fibrosis. The most predictive logistic regression model was selected based on the highest area under the receiver operating curve (AUC), and the lowest Akaike information criterion and Bayesian information criterion. The model was then externally validated in a distinct cohort of 163 participants with MASH from a clinical trial. RESULTS: The median (IQR) age and body mass index were 55 (45-62) years and 32.0 (30-37) kg/m2, respectively, in the derivation cohort. The most accurate model (MASH Resolution Index) included MRI-PDFF, ALT and aspartate aminotransferase. The index had an AUC of 0.81 (95% CI 0.69 to 0.93) for detecting MASH resolution in the derivation cohort. The score calibrated well and performed robustly in a distinct external validation cohort (AUC 0.83, 95% CI 0.76 to 0.91), and outperformed changes in ALT and MRI-PDFF. CONCLUSION: The MASH Resolution Index may be a useful score to non-invasively identify MASH resolution.

Artificial intelligence-assisted digital pathology for non-alcoholic steatohepatitis: current status and future directions.

The worldwide prevalence of non-alcoholic steatohepatitis (NASH) is increasing, causing a significant medical burden, but no approved therapeutics are currently available. NASH drug development requires histological analysis of liver biopsies by expert pathologists for trial enrolment and efficacy assessment, which can be hindered by multiple issues including sample heterogeneity, inter-reader and intra-reader variability, and ordinal scoring systems. Consequently, there is a high unmet need for accurate, reproducible, quantitative, and automated methods to assist pathologists with histological analysis to improve the precision around treatment and efficacy assessment. Digital pathology (DP) workflows in combination with artificial intelligence (AI) have been established in other areas of medicine and are being actively investigated in NASH to assist pathologists in the evaluation and scoring of NASH histology. DP/AI models can be used to automatically detect, localise, quantify, and score histological parameters and have the potential to reduce the impact of scoring variability in NASH clinical trials. This narrative review provides an overview of DP/AI tools in development for NASH, highlights key regulatory considerations, and discusses how these advances may impact the future of NASH clinical management and drug development. This should be a high priority in the NASH field, particularly to improve the development of safe and effective therapeutics.

Liver biopsy for assessment of suspected drug-induced liver injury in metabolic dysfunction-associated steatohepatitis clinical trials: Expert consensus from the Liver Forum.

BACKGROUND: Causality assessment of suspected drug-induced liver injury (DILI) during metabolic dysfunction-associated steatohepatitis (MASH) clinical trials can be challenging, and liver biopsies are not routinely performed as part of this evaluation. While the field is moving away from liver biopsy as a diagnostic and prognostic tool, information not identified by non-invasive testing may be provided on histology. AIM: To address the appropriate utilisation of liver biopsy as part of DILI causality assessment in this setting. METHODS: From 2020 to 2022, the Liver Forum convened a series of webinars on issues pertaining to liver biopsy during MASH trials. The Histology Working Group was formed to generate a series of consensus documents addressing these challenges. This manuscript focuses on liver biopsy as part of DILI causality assessment. RESULTS: Expert opinion, guidance and recommendations on the role of liver biopsy as part of causality assessment of suspected DILI occurring during clinical trials for a drug(s) being developed for MASH are provided. Lessons learned from prior MASH programs are reviewed and gaps identified. CONCLUSIONS: Although there are no pathognomonic features, histologic evaluation of suspected DILI during MASH clinical trials may alter patient management, define the pattern and severity of injury, detect findings that favour a diagnosis of DILI versus MASH progression, identify prognostic features, characterise the clinicopathological phenotype of DILI, and/or define lesions that influence decisions about trial discontinuation and further development of the drug.

Artificial intelligence scoring of liver biopsies in a phase II trial of semaglutide in nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Artificial intelligence-powered digital pathology offers the potential to quantify histological findings in a reproducible way. This analysis compares the evaluation of histological features of NASH between pathologists and a machine-learning (ML) pathology model. APPROACH AND RESULTS: This post hoc analysis included data from a subset of patients (n=251) with biopsy-confirmed NASH and fibrosis stage F1-F3 from a 72-week randomized placebo-controlled trial of once-daily subcutaneous semaglutide 0.1, 0.2, or 0.4 mg (NCT02970942). Biopsies at baseline and week 72 were read by 2 pathologists. Digitized biopsy slides were evaluated by PathAI's NASH ML models to quantify changes in fibrosis, steatosis, inflammation, and hepatocyte ballooning using categorical assessments and continuous scores. Pathologist and ML-derived categorical assessments detected a significantly greater percentage of patients achieving the primary endpoint of NASH resolution without worsening of fibrosis with semaglutide 0.4 mg versus placebo (pathologist 58.5% vs. 22.0%, p < 0.0001; ML 36.9% vs. 11.9%; p =0.0015). Both methods detected a higher but nonsignificant percentage of patients on semaglutide 0.4 mg versus placebo achieving the secondary endpoint of liver fibrosis improvement without NASH worsening. ML continuous scores detected significant treatment-induced responses in histological features, including a quantitative reduction in fibrosis with semaglutide 0.4 mg versus placebo ( p =0.0099) that could not be detected using pathologist or ML categorical assessment. CONCLUSIONS: ML categorical assessments reproduced pathologists' results of histological improvement with semaglutide for steatosis and disease activity. ML-based continuous scores demonstrated an antifibrotic effect not measured by conventional histopathology.

Nutrition assessment and MASH severity in children using the Healthy Eating Index.

BACKGROUND: Pediatric metabolic-associated fatty liver disease (MAFLD) is a global health problem, with lifestyle modification as its major therapeutic strategy. Rigorous characterization of dietary content on MAFLD in children is lacking. We hypothesized an objectively measured healthier diet would positively modulate MAFLD. METHODS: Diet was assessed using the Nutrition Data System for Research in children enrolled from 10 tertiary clinical centers to determine the Healthy Eating Index (HEI, 0-100) and individual food components. RESULTS: In all, 119 children were included (13.3 ± 2.7 y), 80 (67%) male, 67 (18%) White, and 90 (76%) Hispanic, with an average body mass index Z-score of 2.2 ± 0.5. Diet was classified as low HEI < 47.94 (n = 39), mid HEI ≥ 47.94 and < 58.89 (n = 41), or high HEI ≥ 58.89 (n=39). Children with high HEI (healthier diet) had lower body weight (p = 0.005) and more favorable lipids. Mean serum triglycerides for low, mid, and high HEI were 163, 148, and 120 mg/dL, respectively; p = 0.04 mid versus high, p = 0.01 low versus high. Mean HDL was 38, 41 and 43 mg/dL; p = 0.02 low vs high. Less severe steatosis was noted with added sugar ≤ 10% of calories (p = 0.03). Higher lobular inflammation is associated with a higher percentage of calories from fat (OR (95% CI) = 0.95 (0.91-1.00), p = 0.04). CONCLUSIONS: In children with MAFLD, high HEI is associated with lower body weight and more favorable lipids, while added sugar and fat intake has individual histologic features. Differential consumption of major dietary components may modify both metabolic risk factors and histologic liver injury, highlighting the importance of objective diet assessments in children with MAFLD.

Development of Fibro-PeN, a clinical prediction model for moderate-to-severe fibrosis in children with nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Liver fibrosis is common in children with NAFLD and is an important determinant of outcomes. High-performing noninvasive models to assess fibrosis in children are needed. The objectives of this study were to evaluate the performance of existing pediatric and adult fibrosis prediction models and to develop a clinical prediction rule for identifying moderate-to-severe fibrosis in children with NAFLD. APPROACH AND RESULTS: We enrolled children with biopsy-proven NAFLD in the Nonalcoholic Steatohepatitis Clinical Research Network within 90 days of liver biopsy. We staged liver fibrosis in consensus using the Nonalcoholic Steatohepatitis Clinical Research Network scoring system. We evaluated existing pediatric and adult models for fibrosis and developed a new pediatric model using the least absolute shrinkage and selection operator with linear and spline terms for discriminating moderate-to-severe fibrosis from none or mild fibrosis. The model was internally validated with 10-fold cross-validation. We evaluated 1055 children with NAFLD, of whom 26% had moderate-to-severe fibrosis. Existing models performed poorly in classifying fibrosis in children, with area under the receiver operator curves (AUC) ranging from 0.57 to 0.64. In contrast, our new model, fibrosis in pediatric NAFLD was derived from fourteen common clinical variables and had an AUC of 0.79 (95% CI: 0.77-0.81) with 72% sensitivity and 76% specificity for identifying moderate-to-severe fibrosis. CONCLUSION: Existing fibrosis prediction models have limited clinical utility in children with NAFLD. Fibrosis in pediatric NAFLD offers improved performance characteristics for risk stratification by identifying moderate-to-severe fibrosis in children with NAFLD.

Safety and efficacy of once-weekly efruxifermin versus placebo in non-alcoholic steatohepatitis (HARMONY): a multicentre, randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Fibroblast growth factor 21 (FGF21) regulates metabolism and protects cells against stress. Efruxifermin is a bivalent Fc-FGF21 analogue that replicates FGF21 agonism of fibroblast growth factor receptor 1c, 2c, or 3c. The aim of this phase 2b study was to assess its efficacy and safety in patients with non-alcoholic steatohepatitis (NASH) and moderate (F2) or severe (F3) fibrosis. METHODS: HARMONY is a multicentre, randomised, double-blind, placebo-controlled, 96-week, phase 2b trial that was initiated at 41 clinics in the USA. Adults with biopsy-confirmed NASH, defined by a non-alcoholic fatty liver disease activity score (NAS) of 4 or higher and scores of 1 or higher in each of steatosis, ballooning, and lobular inflammation, with histological stage F2 or F3 fibrosis, were randomly assigned (1:1:1), via an interactive response system, to receive placebo or efruxifermin (28 mg or 50 mg), subcutaneously once weekly. Patients, investigators, pathologists, site staff, and the sponsor were masked to group assignments during the study. The primary endpoint was the proportion of patients with improvement in fibrosis of at least 1 stage and no worsening of NASH, based on analyses of baseline and week 24 biopsies (liver biopsy analysis set [LBAS]). A sensitivity analysis evaluated the endpoint in the full analysis set (FAS), for which patients with missing biopsies were considered non-responders. This trial is registered with ClinicalTrials.gov, NCT04767529, and is ongoing. FINDINGS: Between March 22, 2021, and Feb 7, 2022, 747 patients were assessed for eligibility and 128 patients (mean age 54·7 years [SD 10·4]; 79 [62%] female and 49 male [38%]; 118 [92%] white; and 56 [41%] Hispanic or Latino) were enrolled and randomly assigned to receive placebo (n=43), efruxifermin 28 mg (n=42; two randomised patients were not dosed because of an administrative error), or efruxifermin 50 mg (n=43). In the LBAS (n=113), eight (20%) of 41 patients in the placebo group had an improvement in fibrosis of at least 1 stage and no worsening of NASH by week 24 versus 15 (39%) of 38 patients in the efruxifermin 28 mg group (risk ratio [RR] 2·3 [95% CI 1·1-4·8]; p=0·025) and 14 (41%) of 34 patients in the efruxifermin 50 mg group (2·2 [1·0-5·0]; p=0·036). Based on the FAS (n=128), eight (19%) of 43 patients in the placebo group met this endpoint versus 15 (36%) of 42 in the efruxifermin 28 mg group (RR 2·2 [95% CI 1·0-4·8]; p=0·033) and 14 (33%) of 43 in the efruxifermin 50 mg group (1·9 [0·8-4·3]; p=0·123). The most frequent efruxifermin-related adverse events were diarrhoea (16 [40%] of 40 patients in the efruxifermin 28 mg group and 17 [40%] of 43 patients in efruxifermin 50 mg group vs eight [19%] of 43 patients in the placebo group; all events except one were grade 1-2) and nausea (11 [28%] patients in the efruxifermin 28 mg group and 18 [42%] patients in the efruxifermin 50 mg group vs ten [23%] patients in the placebo group; all grade 1-2). Five patients (two in the 28 mg group and three in the 50 mg group) discontinued due to adverse events. Serious adverse events occurred in four patients in the 50 mg group; one was defined as drug related (ulcerative esophagitis in a participant with a history of gastro-oesophageal reflux disease). No deaths occurred. INTERPRETATION: Efruxifermin improved liver fibrosis and resolved NASH over 24 weeks in patients with F2 or F3 fibrosis, with acceptable tolerability, supporting further assessment in phase 3 trials. FUNDING: Akero Therapeutics.

Diagnostic performance of circulating biomarkers for non-alcoholic steatohepatitis.

There are no approved diagnostic biomarkers for at-risk non-alcoholic steatohepatitis (NASH), defined by the presence of NASH, high histological activity and fibrosis stage ≥2, which is associated with higher incidence of liver-related events and mortality. FNIH-NIMBLE is a multi-stakeholder project to support regulatory approval of NASH-related biomarkers. The diagnostic performance of five blood-based panels was evaluated in an observational (NASH CRN DB2) cohort (n = 1,073) with full spectrum of non-alcoholic fatty liver disease (NAFLD). The panels were intended to diagnose at-risk NASH (NIS4), presence of NASH (OWLiver) or fibrosis stages >2, >3 or 4 (enhanced liver fibrosis (ELF) test, PROC3 and FibroMeter VCTE). The prespecified performance metric was an area under the receiver operating characteristic curve (AUROC) ≥0.7 and superiority over alanine aminotransferase for disease activity and the FIB-4 test for fibrosis severity. Multiple biomarkers met these metrics. NIS4 had an AUROC of 0.81 (95% confidence interval: 0.78-0.84) for at-risk NASH. The AUROCs of the ELF test, PROC3 and FibroMeterVCTE for clinically significant fibrosis (≥stage 2), advanced fibrosis (≥stage 3) or cirrhosis (stage 4), respectively, were all ≥0.8. ELF and FibroMeter VCTE outperformed FIB-4 for all fibrosis endpoints. These data represent a milestone toward qualification of several biomarker panels for at-risk NASH and also fibrosis severity in individuals with NAFLD.

Clinical and histologic factors associated with discordance between steatosis grade derived from histology vs. MRI-PDFF in NAFLD.

BACKGROUND: Magnetic resonance imaging-proton density fat fraction (MRI-PDFF) is an excellent biomarker for the non-invasive quantification of hepatic steatosis. AIM: To examine clinical and histologic factors associated with discordance between steatosis grade determined by histology and MRI-PDFF in patients with non-alcoholic fatty liver disease (NAFLD) METHODS: We included 728 patients with biopsy-proven NAFLD from UC San Diego (n = 414) and Yokohama City University (n = 314) who underwent MRI-PDFF and liver biopsy. Patients were stratified by steatosis, and matched with MRI-PDFF cut-points for each steatosis grade: 0 (MRI-PDFF < 6.4%), 1 (MRI-PDFF: 6.4%-17.4%), 2 (MRI-PDFF: 17.4%-22.1%), 3 (MRI-PDFF ≥ 22.1%). Primary outcome was major discordance defined as ≥2 steatosis grade difference determined by histology and MRI-PDFF. RESULTS: Mean (±SD) age and BMI were 55.3 (±13.8) years and 29.9 (±4.9) kg/m2 , respectively. The distributions of histology and MRI-PDFF-determined steatosis were 5.5% grade 0 (n = 40), 44.8% 1 (n = 326, 44.8%), 33.9% 2 (n = 247), and 15.8% 3 (n = 115) vs. 23.5% grade 0 (n = 171), 49.7% 1 (n = 362), 12.9% 2 (n = 94), and 13.9% 3 (n = 101). Major discordance rate was 6.6% (n = 48). Most cases with major discordance had greater histology-determined steatosis grade (n = 40, 88.3%), higher serum AST and liver stiffness, and greater likelihood of fibrosis ≥2, ballooning ≥1 and lobular inflammation ≥2 (all p < 0.05). CONCLUSION: Histology overestimates steatosis grade compared to MRI-PDFF. Patients with advanced NASH are likely to be upgraded on steatosis grade by histology. These data have important implications for steatosis estimation and reporting on histology in clinical practice and trials, especially in patients with stage 2 fibrosis.

Fibrosis Progression Rate in Biopsy-Proven Nonalcoholic Fatty Liver Disease Among People With Diabetes Versus People Without Diabetes: A Multicenter Study.

BACKGROUND & AIMS: There are limited data regarding fibrosis progression in biopsy-proven nonalcoholic fatty liver disease (NAFLD) in people with type 2 diabetes mellitus (T2DM) compared with people without T2DM. We assessed the time to fibrosis progression in people with T2DM compared with people without T2DM in a large, multicenter, study of people with NAFLD who had paired liver biopsies. METHODS: This study included 447 adult participants (64% were female) with NAFLD who had paired liver biopsies more than 1 year apart. Liver histology was systematically assessed by a central pathology committee blinded to clinical data. The primary outcome was the cumulative incidence of a ≥1-stage increase in fibrosis in participants with T2DM compared with participants without T2DM. RESULTS: The mean (SD) age and body mass index (calculated as weight in kilograms divided by the square of the height in meters) were 50.9 (11.5) years and 34.7 (6.3), respectively. The median time between biopsies was 3.3 years (interquartile range, 1.8-6.1 years). Participants with T2DM had a significantly higher cumulative incidence of fibrosis progression at 4 years (24% vs 20%), 8 years (60% vs 50%), and 12 years (93% vs 76%) (P = .005). Using a multivariable Cox proportional hazards model adjusted for multiple confounders, T2DM remained an independent predictor of fibrosis progression (adjusted hazard ratio, 1.69; 95% CI, 1.17-2.43; P = .005). The cumulative incidence of fibrosis regression by ≥1 stage was similar in participants with T2DM compared with participants without T2DM (P = .24). CONCLUSIONS: In this large, multicenter cohort study of well-characterized participants with NAFLD and paired liver biopsies, we found that fibrosis progressed faster in participants with T2DM compared with participants without T2DM. These data have important implications for clinical practice and trial design.

The nonalcoholic steatohepatitis extended hepatocyte ballooning score: histologic classification and clinical significance.

BACKGROUND AND AIMS: The NAFLD activity score was developed to measure histologic changes in NAFLD during therapeutic trials. Hepatocyte ballooning (HB) is the most specific feature in steatohepatitis diagnosis, yet the impact of variations in HB has not been incorporated. APPROACH AND RESULTS: Liver biopsies from patients enrolled in the NASH Clinical Research Network with an initial diagnosis of NASH or NAFL (n=1688) were evaluated to distinguish classic hepatocyte ballooning (cHB) from smaller, nonclassic hepatocyte ballooning (nHB), and also to designate severe ballooning and assign an extended hepatocyte ballooning (eB) score [0 points, no ballooning (NB); 1 point, few or many nHB; 2 points, few cHB; 3 points, many cHB; 4 points, severe cHB] to the biopsy assessment. The eB score was reproducible among NASH CRN liver pathologists (weighted kappa 0.76) and was significantly associated with older age (mean 52.1 y, cHB; 48.5 y, nHB, p<0.001), gender (72.3% female, cHB; 54.5% female, nHB, p<0.001), diabetes (49.8% diabetes, cHB; 28.2% diabetes, nHB, p<0.001), metabolic syndrome (68.5% metabolic syndrome, nHB; 50.2% metabolic syndrome, NB, p<0.001), and body mass index [33.2, 34.2, 35 mean body mass index (kg/m2); NB, nHB, and cHB, respectively, p<0.05]. Finally, fibrosis stage, as a marker of disease severity, was significantly correlated with the eB score (p<0.001). CONCLUSIONS: The eB score allows for a reproducible and more precise delineation of the range of ballooned hepatocyte morphology and corresponds with both clinical features of NASH and fibrosis stage.

A randomized, double-blind, placebo-controlled phase IIa trial of efruxifermin for patients with compensated NASH cirrhosis.

Background & Aims: Efruxifermin has shown clinical efficacy in patients with non-alcoholic steatohepatitis (NASH) and F1-F3 fibrosis. The primary objective of the BALANCED Cohort C was to assess the safety and tolerability of efruxifermin in patients with compensated NASH cirrhosis. Methods: Patients with NASH and stage 4 fibrosis (n = 30) were randomized 2:1 to receive efruxifermin 50 mg (n = 20) or placebo (n = 10) once-weekly for 16 weeks. The primary endpoint was safety and tolerability of efruxifermin. Secondary and exploratory endpoints included evaluation of non-invasive markers of liver injury and fibrosis, glucose and lipid metabolism, and changes in histology in a subset of patients who consented to end-of-study liver biopsy. Results: Efruxifermin was safe and well-tolerated; most adverse events (AEs) were grade 1 (n = 7, 23.3%) or grade 2 (n = 19, 63.3%). The most frequent AEs were gastrointestinal, including transient, mild to moderate diarrhea, and/or nausea. Significant improvements were noted in key markers of liver injury (alanine aminotransferase) and glucose and lipid metabolism. Sixteen-week treatment with efruxifermin was associated with significant reductions in non-invasive markers of fibrosis including Pro-C3 (least squares mean change from baseline [LSMCFB] -9 µg/L efruxifermin vs. -3.4 µg/L placebo; p = 0.0130) and ELF score (-0.4 efruxifermin vs. +0.4 placebo; p = 0.0036), with a trend towards reduced liver stiffness (LSMCFB -5.7 kPa efruxifermin vs. -1.1 kPa placebo; n.s.). Of 12 efruxifermin-treated patients with liver biopsy after 16 weeks, 4 (33%) achieved fibrosis improvement of at least one stage without worsening of NASH, while an additional 3 (25%) achieved resolution of NASH, compared to 0 of 5 placebo-treated patients. Conclusions: Efruxifermin appeared safe and well-tolerated with encouraging improvements in markers of liver injury, fibrosis, and glucose and lipid metabolism following 16 weeks of treatment, warranting confirmation in larger and longer term studies. Lay summary: Cirrhosis resulting from non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease, represents a major unmet medical need. Currently there are no approved drugs for the treatment of NASH. This proof-of-concept randomized, double-blind clinical trial demonstrated the potential therapeutic benefit of efruxifermin treatment compared to placebo in patients with cirrhosis due to NASH. Clinical Trial Number: NCT03976401.