Severe acute liver disease in adults: Contemporary role of histopathology.

Liver biopsies have consistently contributed to our understanding of the pathogenesis and aetiologies of acute liver disease. As other diagnostic modalities have been developed and refined, the role of biopsy in the management of patients with acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and acute hepatitis, including acute liver injury (ALI), has changed. Liver biopsy remains particularly valuable when first-line diagnostic algorithms fail to determine aetiology. Despite not being identified as a mandatory diagnostic tool in recent clinical guidelines for the management of ALF or ACLF, many centres continue to undertake biopsies given the relative safety of transjugular biopsy in this setting. Several studies have demonstrated that liver biopsy can provide prognostic information, particularly in the context of so-called indeterminate hepatitis, and is extremely useful in excluding conditions such as metastatic tumours that would preclude transplantation. In addition, its widespread use of percutaneous biopsies in cases of less severe acute liver injury, for example in the establishment of a diagnosis of acute presentation of autoimmune hepatitis or confirmation of a probable or definite drug-induced liver injury (DILI), has meant that many centres have seen a shift in the ratio of specimens they are receiving from patients with chronic to acute liver disease. Histopathologists therefore need to be equipped to deal with these challenging specimens. This overview provides an insight into the contemporary role of biopsies (as well as explant and autopsy material) in diagnosing acute liver disease. It outlines up-to-date clinical definitions of liver injury and considers recent recommendations for the diagnosis of AIH and drug-induced, autoimmune-like hepatitis (DI-AIH).

Prospective head-to-head comparison of non-invasive scores for diagnosis of fibrotic MASH in patients with type 2 diabetes.

BACKGROUND: Non-invasive scores have been proposed to identify patients with fibrotic, metabolic dysfunction associated steatohepatitis (MASH), who are at the highest risk of progression to complications of cirrhosis and may benefit from pharmacologic treatments. However, data in type 2 diabetes (T2DM) patients are lacking. The aim of this multicenter prospective study was to compare head-to-head FAST (FibroScan-aspartate aminotransferase [AST]), MAST (magnetic resonance imaging [MRI]-AST), MEFIB (magnetic resonance elastography [MRE] plus FIB-4), and FNI (fibrotic NASH index) for detecting fibrotic MASH in T2DM patients. METHODS: 330 T2DM outpatients with biopsy-proven metabolic dysfunction associated steatotic liver disease (MASLD) from the QUID-NASH study (NCT03634098), who underwent FibroScan, MRI-PDFF and MRE at the time of liver biopsy (LB) were studied. The main outcome was fibrotic MASH, defined as NAS ≥ 4 (with at least one point each) and fibrosis stage ≥ 2 (centrally reviewed). RESULTS: 245 patients (median age 59 years, male 65%, BMI 31 kg/m2; fibrotic MASH 39%) had all data available for scores comparison. FAST and MAST had similar accuracy (AUROCs 0.81 vs. 0.79, p = 0.41) but outperformed FNI (0.74; p = 0.01) and MEFIB (0.68; p < 0.0001). When using original cutoffs, MAST outperformed FAST, MEFIB and FNI when comparing the percentage of correctly classified patients, in whom LB would be avoided (69% vs. 48%, 46%, 39%, respectively; p < 0.001). When using cutoffs specific to our population, FAST outperformed FNI and MAST (56% vs. 40%, and 38%, respectively; p < 0.001). CONCLUSION: Our findings show that FAST, MAST, MEFIB and FNI are accurate non-invasive tools to identify T2DM patients with fibrotic MASH in secondary/tertiary diabetes clinics. Cutoffs adapted to T2DM population should be considered. TRIAL REGISTRATION NUMBER: NCT03634098.

A Phase 3, Randomized, Controlled Trial of Resmetirom in NASH with Liver Fibrosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive liver disease with no approved treatment. Resmetirom is an oral, liver-directed, thyroid hormone receptor beta-selective agonist in development for the treatment of NASH with liver fibrosis. METHODS: We are conducting an ongoing phase 3 trial involving adults with biopsy-confirmed NASH and a fibrosis stage of F1B, F2, or F3 (stages range from F0 [no fibrosis] to F4 [cirrhosis]). Patients were randomly assigned in a 1:1:1 ratio to receive once-daily resmetirom at a dose of 80 mg or 100 mg or placebo. The two primary end points at week 52 were NASH resolution (including a reduction in the nonalcoholic fatty liver disease [NAFLD] activity score by ≥2 points; scores range from 0 to 8, with higher scores indicating more severe disease) with no worsening of fibrosis, and an improvement (reduction) in fibrosis by at least one stage with no worsening of the NAFLD activity score. RESULTS: Overall, 966 patients formed the primary analysis population (322 in the 80-mg resmetirom group, 323 in the 100-mg resmetirom group, and 321 in the placebo group). NASH resolution with no worsening of fibrosis was achieved in 25.9% of the patients in the 80-mg resmetirom group and 29.9% of those in the 100-mg resmetirom group, as compared with 9.7% of those in the placebo group (P<0.001 for both comparisons with placebo). Fibrosis improvement by at least one stage with no worsening of the NAFLD activity score was achieved in 24.2% of the patients in the 80-mg resmetirom group and 25.9% of those in the 100-mg resmetirom group, as compared with 14.2% of those in the placebo group (P<0.001 for both comparisons with placebo). The change in low-density lipoprotein cholesterol levels from baseline to week 24 was -13.6% in the 80-mg resmetirom group and -16.3% in the 100-mg resmetirom group, as compared with 0.1% in the placebo group (P<0.001 for both comparisons with placebo). Diarrhea and nausea were more frequent with resmetirom than with placebo. The incidence of serious adverse events was similar across trial groups: 10.9% in the 80-mg resmetirom group, 12.7% in the 100-mg resmetirom group, and 11.5% in the placebo group. CONCLUSIONS: Both the 80-mg dose and the 100-mg dose of resmetirom were superior to placebo with respect to NASH resolution and improvement in liver fibrosis by at least one stage. (Funded by Madrigal Pharmaceuticals; MAESTRO-NASH ClinicalTrials.gov number, NCT03900429.).

Cenicriviroc Lacked Efficacy to Treat Liver Fibrosis in Nonalcoholic Steatohepatitis: AURORA Phase III Randomized Study.

BACKGROUND AND AIMS: Cenicriviroc (CVC) is a novel, orally administered, chemokine receptor type 2 and 5 antagonist that showed antifibrotic potential in preclinical and phase IIb studies of nonalcoholic steatohepatitis (NASH). Herein, we report efficacy and safety results from the phase III study. METHODS: The AURORA (A Study for the Efficacy and Safety of CVC for the Treatment of Liver Fibrosis in Adults With NASH) study was a phase III, randomized, double-blind, placebo-controlled, 2-part study of patients with NASH and stage 2/3 liver fibrosis. Adults, 18-75 years of age, were randomized to CVC 150 mg or placebo once daily for 12 months (part 1) or 60 months (part 2). Liver biopsies were performed at screening, month 12, and early study discontinuation or termination. The primary efficacy endpoint was the proportion of patients with fibrosis improvement ≥1 stage without worsening of steatohepatitis at month 12 relative to screening. Adverse events were assessed throughout the study. RESULTS: A total of 1778 patients were randomized and discontinued (part 1: n = 1293; part 2: n = 485). In part 1, at month 12, a similar proportion of patients receiving CVC or placebo achieved the primary endpoint (22.3% vs 25.5%; odds ratio, 0.84; 95% confidence interval, 0.63-1.10; P = .21) and complete resolution of steatohepatitis without worsening of fibrosis (23.0% vs 27.2%; P = .21). The safety profile was generally comparable across treatment groups. CONCLUSIONS: This study did not demonstrate the efficacy of CVC for treating liver fibrosis assessed by histology in adults with NASH; however, CVC was safe and well tolerated in patients with NASH and liver fibrosis. (ClinicalTrials.gov, Number: NCT03028740).

Adipose tissue macrophage dysfunction is associated with a breach of vascular integrity in NASH.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. METHODS: Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. RESULTS: We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. CONCLUSIONS: NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. IMPACT AND IMPLICATIONS: Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.

Design of the phase 3 MAESTRO clinical program to evaluate resmetirom for the treatment of nonalcoholic steatohepatitis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) associated with steatosis, hepatocellular injury, inflammation and fibrosis. In a Phase 2 trial in adults with NASH (NCT02912260), resmetirom, an orally administered, liver-targeted thyroid hormone receptor-ß selective agonist, significantly reduced hepatic fat (via imaging) and resolved NASH without worsening fibrosis (via liver biopsy) in a significant number of patients compared with placebo. AIMS: To present the design of the Phase 3 MAESTRO clinical programme evaluating resmetirom for treatment of NASH (MAESTRO-NAFLD-1 [NCT04197479], MAESTRO-NAFLD-OLE [NCT04951219], MAESTRO-NASH [NCT03900429], MAESTRO-NASH-OUTCOMES [NCT05500222]). METHODS: MAESTRO-NASH is a pivotal serial biopsy trial in up to 2000 adults with biopsy-confirmed at-risk NASH. Patients are randomised to a once-daily oral placebo, 80 mg resmetirom, or 100 mg resmetirom. Liver biopsies are conducted at screening, week 52 and month 54. MAESTRO-NAFLD-1 is a 52-week safety trial in ~1400 adults with NAFLD/presumed NASH (based on non-invasive testing); ~700 patients from MAESTRO-NAFLD-1 are enrolled in MAESTRO-NAFLD-OLE, a 52-week active treatment extension to further evaluate safety. MAESTRO-NASH-OUTCOMES is enrolling 700 adults with well-compensated NASH cirrhosis to evaluate the potential for resmetirom to slow progression to hepatic decompensation events. Non-invasive tests (biomarkers, imaging) are assessed longitudinally throughout, in addition to validated patient-reported outcomes. CONCLUSION: The MAESTRO clinical programme was designed in conjunction with regulatory authorities to support approval of resmetirom for treatment of NASH. The surrogate endpoints, based on week 52 liver biopsy, serum biomarkers and imaging, are confirmed by long-term clinical liver-related outcomes in MAESTRO-NASH (month 54) and MAESTRO-NASH-OUTCOMES (time to event).

Prospective Comparison of Attenuation Imaging and Controlled Attenuation Parameter for Liver Steatosis Diagnosis in Patients With Nonalcoholic Fatty Liver Disease and Type 2 Diabetes.

BACKGROUND & AIMS: Similarly to the controlled attenuation parameter (CAP), the ultrasound-based attenuation imaging (ATI) can quantify hepatic steatosis. We prospectively compared the performance of ATI and CAP for the diagnosis of hepatic steatosis in patients with type 2 diabetes and nonalcoholic fatty liver disease using histology and magnetic resonance imaging-proton density fat fraction (MRI-PDFF) as references. METHODS: Patients underwent ATI and CAP measurement, MRI, and biopsy on the same day. Steatosis was classified as S0, S1, S2, and S3 on histology (<5%, 5%-33%, 33%-66%, and >66%, respectively) while the thresholds of 6.4%, 17.4%, and 22.1%, respectively, were used for MRI-PDFF. The area under the curve (AUC) of ATI and CAP was compared using a DeLong test. RESULTS: Steatosis could be evaluated in 191 and 187 patients with MRI-PDFF and liver biopsy, respectively. For MRI-PDFF steatosis, the AUC of ATI and CAP were 0.86 (95% confidence interval [CI], 0.81-0.91) vs 0.69 (95% CI, 0.62-0.75) for S0 vs S1-S3 (P = .02) and 0.71 (95% CI, 0.64-0.77) vs 0.69 (95% CI, 0.61-0.75) for S0-S1 vs S2-S3 (P = .60), respectively. For histological steatosis, the AUC of ATI and CAP were 0.92 (95% CI, 0.87-0.95) vs 0.95 (95% CI, 0.91-0.98) for S0 vs S1-S3 (P = .64) and 0.79 (95% CI, 0.72-0.84) vs 0.76 (95% CI, 0.69-0.82) for S0-S1 vs S2-S3 (P = .61), respectively. CONCLUSION: ATI may be used as an alternative to CAP for the diagnosis and quantification of steatosis, in patients with type 2 diabetes and nonalcoholic fatty liver disease.

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.

Practical diagnosis of cirrhosis in non-alcoholic fatty liver disease using currently available non-invasive fibrosis tests.

Unlike for advanced liver fibrosis, the practical rules for the early non-invasive diagnosis of cirrhosis in NAFLD remain not well defined. Here, we report the derivation and validation of a stepwise diagnostic algorithm in 1568 patients with NAFLD and liver biopsy coming from four independent cohorts. The study algorithm, using first the elastography-based tests Agile3+ and Agile4 and then the specialized blood tests FibroMeterV3G and CirrhoMeterV3G, provides stratification in four groups, the last of which is enriched in cirrhosis (71% prevalence in the validation set). A risk prediction chart is also derived to allow estimation of the individual probability of cirrhosis. The predicted risk shows excellent calibration in the validation set, and mean difference with perfect prediction is only -2.9%. These tools improve the personalized non-invasive diagnosis of cirrhosis in NAFLD.

Prospective direct comparison of non-invasive liver tests in outpatients with type 2 diabetes using intention-to-diagnose analysis.

BACKGROUND: No prospective diagnostic studies have directly compared widespread non-invasive liver tests in patients with type 2 diabetes (T2D) using the intention-to-diagnose method for each of the three main histological features of metabolic dysfunction associated steatotic liver disease - namely fibrosis, metabolic dysfunction-associated steatohepatitis (MASH), and steatosis. AIMS: To compare the performance of nine tests using the intention-to-diagnose rather than the standard method, which would exclude non-evaluable participants METHODS: Biopsy was used as the reference with predetermined cut-offs, advanced fibrosis being the main endpoint. The Nash-FibroTest panel including FibroTest-T2D, SteatoTest-T2D and MashTest-T2D was optimised for type 2 diabetes. FibroTest-T2D was compared to vibration-controlled transient elastography stiffness (VCTE), two-dimensional shear-wave elastography stiffness (TD-SWE), and Fibrosis-4 blood test. NashTest-T2D was compared to aspartate aminotransferase. SteatoTest-T2D was compared to the controlled attenuation parameter and the hepatorenal gradient. RESULTS: Among 402 cases, non-evaluable tests were 6.7% for VCTE, 4.0% for hepatorenal gradient, 3.2% for controlled attenuation parameter, 1.5% for TD-SWE, 1.2% for NashTest-T2D, and 0.02% for Fibrosis-4, aspartate aminotransferase and SteatoTest-T2D. The VCTE AUROC for advanced fibrosis was over-estimated by 6% (0.83 [95% CI: 0.78-0.87]) by standard analysis compared to intention-to-diagnose (0.77 [0.72-0.81] p = 0.008). The AUROCs for advanced fibrosis did not differ significantly in intention-to-diagnose between FibroTest-T2D (0.77; 95% CI: 0.73-0.82), VCTE (0.77; 95% CI: 0.72-0.81) and TD-SWE(0.78; 0.74-0.83) but were all higher than the Fibrosis-4 score (0.70; 95% CI all differences ≥7%; p ≤ 0.03). For MASH, MashTest-T2D had a higher AUROC (0.76; 95% CI: 0.70-0.80) than aspartate aminotransferase (0.72; 95% CI: 0.66-0.77; p = 0.035). For steatosis, AUROCs did not differ significantly between SteatoTest-T2D, controlled attenuation parameter and hepatorenal gradient. CONCLUSIONS: In intention-to-diagnose analysis, FibroTest-T2D, TD-SWE and VCTE performed similarly for staging fibrosis, and out-performed Fibrosis-4 in outpatients with type 2 diabetes. The standard analysis over-estimated VCTE performance. CLINICALTRIAL: gov: NCT03634098.

Randomized, Controlled Trial of the FGF21 Analogue Pegozafermin in NASH.

BACKGROUND: Pegozafermin is a long-acting glycopegylated (pegylated with the use of site-specific glycosyltransferases) fibroblast growth factor 21 (FGF21) analogue in development for the treatment of nonalcoholic steatohepatitis (NASH) and severe hypertriglyceridemia. The efficacy and safety of pegozafermin in patients with biopsy-proven noncirrhotic NASH are not well established. METHODS: In this phase 2b, multicenter, double-blind, 24-week, randomized, placebo-controlled trial, we randomly assigned patients with biopsy-confirmed NASH and stage F2 or F3 (moderate or severe) fibrosis to receive subcutaneous pegozafermin at a dose of 15 mg or 30 mg weekly or 44 mg once every 2 weeks or placebo weekly or every 2 weeks. The two primary end points were an improvement in fibrosis (defined as reduction by ≥1 stage, on a scale from 0 to 4, with higher stages indicating greater severity), with no worsening of NASH, at 24 weeks and NASH resolution without worsening of fibrosis at 24 weeks. Safety was also assessed. RESULTS: Among the 222 patients who underwent randomization, 219 received pegozafermin or placebo. The percentage of patients who met the criteria for fibrosis improvement was 7% in the pooled placebo group, 22% in the 15-mg pegozafermin group (difference vs. placebo, 14 percentage points; 95% confidence interval [CI], -9 to 38), 26% in the 30-mg pegozafermin group (difference, 19 percentage points; 95% CI, 5 to 32; P = 0.009), and 27% in the 44-mg pegozafermin group (difference, 20 percentage points; 95% CI, 5 to 35; P = 0.008). The percentage of patients who met the criteria for NASH resolution was 2% in the placebo group, 37% in the 15-mg pegozafermin group (difference vs. placebo, 35 percentage points; 95% CI, 10 to 59), 23% in the 30-mg pegozafermin group (difference, 21 percentage points; 95% CI, 9 to 33), and 26% in the 44-mg pegozafermin group (difference, 24 percentage points; 95% CI, 10 to 37). The most common adverse events associated with pegozafermin therapy were nausea and diarrhea. CONCLUSIONS: In this phase 2b trial, treatment with pegozafermin led to improvements in fibrosis. These results support the advancement of pegozafermin into phase 3 development. (Funded by 89bio; ENLIVEN ClinicalTrials.gov number, NCT04929483.).

High Prevalence of NASH and Advanced Fibrosis in Type 2 Diabetes: A Prospective Study of 330 Outpatients Undergoing Liver Biopsies for Elevated ALT, Using a Low Threshold.

OBJECTIVE: Most people with type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH) or advanced fibrosis (AF) remain undiagnosed, resulting in missed opportunities for early intervention. This multicenter, prospective study assessed the yield of using routinely available data to identify these patients. RESEARCH DESIGN AND METHODS: A total of 713 outpatients with T2DM, screened in four diabetology clinics for nonalcoholic fatty liver disease according to American Diabetes Association criteria, were referred to hepatologists for further work-up (Fibrosis-4 and vibration-controlled transient elastography [VCTE]). A liver biopsy was proposed when ALT levels were persistently >20 IU/L in female patients or >30 IU/L in male patients, in the absence of other liver disease. RESULTS: Liver biopsies were performed in 360 patients and considered adequate for reading after central review for 330 specimens (median patient age, 59 years; male patients, 63%; median BMI and HbA1c values, 32 and 7.5%, respectively). Prevalence of NASH, AF, and cirrhosis were 58%, 38%, and 10%, respectively. Liver lesions were independently associated with the components of metabolic syndrome but not with the micro- and macrovascular complications of T2DM. Models based on routinely available data with or without VCTE had good accuracy to predict AF (respectively: area under the receiver operating characteristic curve [AUROC], 0.84 and 0.77; and correctly classified 59% and 45%) and NASH (respectively: AUROC, 0.82 and 0.81; 44% and 42%). CONCLUSIONS: Despite the use of a low ALT threshold, prevalence of NASH (58%) or AF (38%) was high. Routinely available data had a high yield in identifying patients with T2DM with AF and/or NASH requiring further liver assessment.

Machine learning algorithm improves the detection of NASH (NAS-based) and at-risk NASH: A development and validation study.

BACKGROUND AND AIMS: Detecting NASH remains challenging, while at-risk NASH (steatohepatitis and F≥ 2) tends to progress and is of interest for drug development and clinical application. We developed prediction models by supervised machine learning techniques, with clinical data and biomarkers to stage and grade patients with NAFLD. APPROACH AND RESULTS: Learning data were collected in the Liver Investigation: Testing Marker Utility in Steatohepatitis metacohort (966 biopsy-proven NAFLD adults), staged and graded according to NASH CRN. Conditions of interest were the clinical trial definition of NASH (NAS ≥ 4;53%), at-risk NASH (NASH with F ≥ 2;35%), significant (F ≥ 2;47%), and advanced fibrosis (F ≥ 3;28%). Thirty-five predictors were included. Missing data were handled by multiple imputations. Data were randomly split into training/validation (75/25) sets. A gradient boosting machine was applied to develop 2 models for each condition: clinical versus extended (clinical and biomarkers). Two variants of the NASH and at-risk NASH models were constructed: direct and composite models.Clinical gradient boosting machine models for steatosis/inflammation/ballooning had AUCs of 0.94/0.79/0.72. There were no improvements when biomarkers were included. The direct NASH model produced AUCs (clinical/extended) of 0.61/0.65. The composite NASH model performed significantly better (0.71) for both variants. The composite at-risk NASH model had an AUC of 0.83 (clinical and extended), an improvement over the direct model. Significant fibrosis models had AUCs (clinical/extended) of 0.76/0.78. The extended advanced fibrosis model (0.86) performed significantly better than the clinical version (0.82). CONCLUSIONS: Detection of NASH and at-risk NASH can be improved by constructing independent machine learning models for each component, using only clinical predictors. Adding biomarkers only improved the accuracy of fibrosis.

Biomarkers for staging fibrosis and non-alcoholic steatohepatitis in non-alcoholic fatty liver disease (the LITMUS project): a comparative diagnostic accuracy study.

BACKGROUND: The reference standard for detecting non-alcoholic steatohepatitis (NASH) and staging fibrosis-liver biopsy-is invasive and resource intensive. Non-invasive biomarkers are urgently needed, but few studies have compared these biomarkers in a single cohort. As part of the Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) project, we aimed to evaluate the diagnostic accuracy of 17 biomarkers and multimarker scores in detecting NASH and clinically significant fibrosis in patients with non-alcoholic fatty liver disease (NAFLD) and identify their optimal cutoffs as screening tests in clinical trial recruitment. METHODS: This was a comparative diagnostic accuracy study in people with biopsy-confirmed NAFLD from 13 countries across Europe, recruited between Jan 6, 2010, and Dec 29, 2017, from the LITMUS metacohort of the prospective European NAFLD Registry. Adults (aged ≥18 years) with paired liver biopsy and serum samples were eligible; those with excessive alcohol consumption or evidence of other chronic liver diseases were excluded. The diagnostic accuracy of the biomarkers was expressed as the area under the receiver operating characteristic curve (AUC) with liver histology as the reference standard and compared with the Fibrosis-4 index for liver fibrosis (FIB-4) in the same subgroup. Target conditions were the presence of NASH with clinically significant fibrosis (ie, at-risk NASH; NAFLD Activity Score ≥4 and F≥2) or the presence of advanced fibrosis (F≥3), analysed in all participants with complete data. We identified thres holds for each biomarker for reducing the number of biopsy-based screen failures when recruiting people with both NASH and clinically significant fibrosis for future trials. FINDINGS: Of 1430 participants with NAFLD in the LITMUS metacohort with serum samples, 966 (403 women and 563 men) were included after all exclusion criteria had been applied. 335 (35%) of 966 participants had biopsy-confirmed NASH and clinically significant fibrosis and 271 (28%) had advanced fibrosis. For people with NASH and clinically significant fibrosis, no single biomarker or multimarker score significantly reached the predefined AUC 0·80 acceptability threshold (AUCs ranging from 0·61 [95% CI 0·54-0·67] for FibroScan controlled attenuation parameter to 0·81 [0·75-0·86] for SomaSignal), with accuracy mostly similar to FIB-4. Regarding detection of advanced fibrosis, SomaSignal (AUC 0·90 [95% CI 0·86-0·94]), ADAPT (0·85 [0·81-0·89]), and FibroScan liver stiffness measurement (0·83 [0·80-0·86]) reached acceptable accuracy. With 11 of 17 markers, histological screen failure rates could be reduced to 33% in trials if only people who were marker positive had a biopsy for evaluating eligibility. The best screening performance for NASH and clinically significant fibrosis was observed for SomaSignal (number needed to test [NNT] to find one true positive was four [95% CI 4-5]), then ADAPT (six [5-7]), MACK-3 (seven [6-8]), and PRO-C3 (nine [7-11]). INTERPRETATION: None of the single markers or multimarker scores achieved the predefined acceptable AUC for replacing biopsy in detecting people with both NASH and clinically significant fibrosis. However, several biomarkers could be applied in a prescreening strategy in clinical trial recruitment. The performance of promising markers will be further evaluated in the ongoing prospective LITMUS study cohort. FUNDING: The Innovative Medicines Initiative 2 Joint Undertaking.

Evaluation of PXL065 - deuterium-stabilized (R)-pioglitazone in patients with NASH: A phase II randomized placebo-controlled trial (DESTINY-1).

BACKGROUND & AIMS: Pioglitazone (Pio) is efficacious in NASH, but its utility is limited by PPARγ-driven side effects. Pio is a mixture of two enantiomers (R, S). PXL065, deuterium-stabilized R-Pio, lacks PPARγ activity but retains non-genomic activity. We tested the hypothesis that PXL065 would have similar efficacy but a better safety profile than Pio in patients with NASH. METHODS: Patients (≥8% liver fat, NAFLD activity score [NAS] ≥4, F1-F3) received daily doses of PXL065 (7.5, 15, 22.5 mg) or placebo 1:1:1:1 for 36 weeks. The primary endpoint was relative % change in liver fat content (LFC) on MRI-proton density fat fraction; liver histology, non-invasive tests, safety-tolerability, and pharmacokinetics were also assessed. RESULTS: One hundred and seventeen patients were evaluated. All PXL065 groups met the primary endpoint (-21 to -25% LFC, p = 0.008-0.02 vs. placebo); 40% (22.5 mg) achieved a ≥30% LFC reduction. Favorable trends in non-invasive tests including reductions in PIIINP (p = 0.02, 22.5 mg) and NAFLD fibrosis score (p = 0.04, 22.5 mg) were observed. On histology (n = 92), a ≥1 stage fibrosis improvement occurred in 40% (7.5 mg), 50% (15 mg, p = 0.06), and 35% (22.5 mg) vs. 17% for placebo; up to 50% of PXL065-treated patients achieved a ≥2 point NAS improvement without fibrosis worsening vs. 30% with placebo. Metabolic improvements included: HbA1c (-0.41% p = 0.003) and insulin sensitivity (HOMA-IR, p = 0.04; Adipo-IR, p = 0.002). Adiponectin increased (+114%, 22.5 mg, p <0.0001) vs. placebo. There was no dose-dependent effect on body weight or PXL065-related peripheral oedema signal. Overall, PXL065 was safe and well tolerated. Pharmacokinetics confirmed dose-proportional and higher steady state R- vs. S-Pio exposure. IMPACT AND IMPLICATIONS: Pioglitazone (Pio) is an approved diabetes medicine with proven efficacy in non-alcoholic steatohepatitis (NASH); PXL065 is a novel related oral agent which has been shown to retain Pio's efficacy in preclinical NASH models, with reduced potential for PPARγ-driven side effects. Results of this phase II study are important as PXL065 improved several key NASH disease features with a favorable safety profile - these findings can be applied by researchers seeking to understand pathophysiology and to develop new therapies. These results also indicate that PXL065 warrants further clinical testing in a pivotal NASH trial. Other implications include the potential future availability of a distinct oral therapy for NASH that may be relevant for patients, providers and caregivers seeking to prevent the progression and complications of this disease. CONCLUSIONS: PXL065 is a novel molecule which retains an efficacy profile in NASH similar to Pio with reduced potential for PPARγ-driven side effects. A pivotal clinical trial is warranted to confirm the histological benefits reported herein. IMPACT AND IMPLICATIONS: Pioglitazone (Pio) is an approved diabetes medicine with proven efficacy in non-alcoholic steatohepatitis (NASH); PXL065 is a novel related oral agent which has been shown to retain Pio's efficacy in preclinical NASH models, with reduced potential for PPARγ-driven side effects. Results of this phase II study are important as PXL065 improved several key NASH disease features with a favorable safety profile - these findings can be applied by researchers seeking to understand pathophysiology and to develop new therapies. These results also indicate that PXL065 warrants further clinical testing in a pivotal NASH trial. Other implications include the potential future availability of a distinct oral therapy for NASH that may be relevant for patients, providers and caregivers seeking to prevent the progression and complications of this disease.

Adipose Tissue Insulin Resistance Predicts the Severity of Liver Fibrosis in Patients With Type 2 Diabetes and NAFLD.

CONTEXT: Although type 2 diabetes (T2D) is a risk factor for liver fibrosis in nonalcoholic fatty liver disease (NAFLD), the specific contribution of insulin resistance (IR) relative to other factors is unknown. OBJECTIVE: Assess the impact on liver fibrosis in NAFLD of adipose tissue (adipose tissue insulin resistance index [adipo-IR]) and liver (Homeostatic Model Assessment of Insulin Resistance [HOMA-IR]) IR in people with T2D and NAFLD. DESIGN: Participants were screened by elastography in the outpatient clinics for hepatic steatosis and fibrosis, including routine metabolites, cytokeratin-18 (a marker of hepatocyte apoptosis/steatohepatitis), and HOMA-IR/adipo-IR. SETTING: University ambulatory care practice. PARTICIPANTS: A total of 483 participants with T2D. INTERVENTION: Screening for steatosis and fibrosis with elastography. MAIN OUTCOME MEASURES: Liver steatosis (controlled attenuation parameter), fibrosis (liver stiffness measurement), and measurements of IR (adipo-IR, HOMA-IR) and fibrosis (cytokeratin-18). RESULTS: Clinically significant liver fibrosis (stage F ≥ 2 = liver stiffness measurement ≥8.0 kPa) was found in 11%, having more features of the metabolic syndrome, lower adiponectin, and higher aspartate aminotransferase (AST), alanine aminotransferase, liver fat, and cytokeratin-18 (P < 0.05-0.01). In multivariable analysis including just clinical variables (model 1), obesity (body mass index [BMI]) had the strongest association with fibrosis (odds ratio, 2.56; CI, 1.87-3.50; P < 0.01). When metabolic measurements and cytokeratin-18 were included (model 2), only BMI, AST, and liver fat remained significant. When fibrosis stage was adjusted for BMI, AST, and steatosis (model 3), only Adipo-IR remained strongly associated with fibrosis (OR, 1.51; CI, 1.05-2.16; P = 0.03), but not BMI, hepatic IR, or steatosis. CONCLUSIONS: These findings pinpoint to the central role of dysfunctional, insulin-resistant adipose tissue to advanced fibrosis in T2D, beyond simply BMI or steatosis. The clinical implication is that targeting adipose tissue should be the priority of treatment in NAFLD.

Screening HIV Patients at Risk for NAFLD Using MRI-PDFF and Transient Elastography: A European Multicenter Prospective Study.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a growing concern in the aging population with human immunodeficiency virus (HIV). Screening for NAFLD is recommended in patients with metabolic risk factors or unexplained transaminitis. This study aimed to prospectively assess the prevalence and associated factors of liver steatosis and advanced fibrosis (AF) in HIV-monoinfected patients at risk of NAFLD. METHODS: We conducted a multicenter study in HIV-monoinfected patients, nonexcessive drinkers with metabolic syndrome, and/or persistently elevated liver enzymes, and/or clinical lipodystrophy. All participants had magnetic resonance imaging proton density fat fraction (MRI-PDFF), Fibroscan/controlled attenuation parameter (CAP), and cytokine and genetic analysis. RESULTS: From March 2014 to November 2015, we enrolled 442 participants and analyzed 402: male (85%); median age, 55 years (interquartile range [IQR], 50-61 years); body mass index, 27.0 kg/m2 (IQR, 23.6-28.7 kg/m2); metabolic syndrome (67%); and CD4 cell count, 630/mm3 (IQR, 510-832/mm3). Overall 257 of 402 (64%) had NAFLD (MRI-PDFF ≥5%). Among them, 11.3% had a liver stiffness ≥9.6 kPa, suggestive of AF. Multivariable analysis identified 7 factors of steatosis: high CD4-cell count (odds ratio [OR], 4.04; 95% confidence interval [CI], 1.92-8.51), high leptin level (OR, 2.12; 95% CI, 1.14-3.93), non-CC PNPLA3s738409 genetic polymorphism (OR, 1.92; 95% CI, 1.11-3.33), low high-density lipoprotein (OR, 1.83; 95% CI, 1.03-3.27), high triglycerides (OR, 1.48; 95% CI, 1.18-1.84), elevated alanine transaminase (OR, 1.23; 95% CI, 1.16-1.31), and hyper ferritinemia (OR, 1.05; 95% CI, 1.03-1.07). Two factors were associated with AF: high body mass index (OR, 1.23 ; 95% CI, 1.07-1.42 ; P = .005, and elevated aspartate aminotransferase (OR, 1.03; 95% CI, 1.01-1.05; P = .001). Using MRI-PDFF as a reference, CAP (best cutoff, 280 dB/m) had good accuracy (area under the receiver operating characteristic curve = 0.86; 95% CI, 0.82-0.90) for the diagnosis of moderate to severe steatosis. CONCLUSIONS: In a large cohort of HIV-moninfected patients at risk of NAFLD, steatosis is present in two-thirds of cases, and around 10% have AF. The CAP technique is accurate for screening steatosis in this population.

A new preclinical model of western diet-induced progression of non-alcoholic steatohepatitis to hepatocellular carcinoma.

Non-alcoholic steatohepatitis (NASH) results from the accumulation of excessive liver lipids leading to hepatocellular injury, inflammation, and fibrosis that greatly increase the risk for hepatocellular carcinoma (HCC). Despite the well-characterized clinical and histological pathology for NASH-driven HCC in humans, its etiology remains unclear and there is a deficiency in pre-clinical models that recapitulate the progression of the human disease. Therefore, we developed a new mouse model amenable to genetic manipulations and gene targeting that mimics the gradual NASH to HCC progression observed in humans. C57BL/6NJ mice were fed a Western high-fat diet and sugar water (HFD/SW) and monitored for effects on metabolism, liver histology, tumor development, and liver transcriptome for up to 54 weeks. Chronic HFD/SW feeding led to significantly increased weight gain, serum and liver lipid levels, liver injury, and glucose intolerance. Hepatic pathology progressed and mice developed hepatocellular ballooning, inflammation, and worse fibrosis was apparent at 16 weeks, greatly increased through 32 weeks, and remained elevated at 54 weeks. Importantly, hepatocellular cancer spontaneously developed in 75% of mice on HFD/SW, half of which were HCC, whereas none of the mice on the chow diet developed HCC. Chronic HFD/SW induced molecular markers of de novo lipogenesis, endoplasmic reticulum stress, inflammation, and accumulation of p62, all of which also participate in the human pathology. Moreover, transcriptome analysis revealed activation of HCC-related genes and signatures associated with poor prognosis of human HCC. Overall, we have identified a new preclinical model that recapitulates known hallmarks of NASH-driven HCC that can be utilized for future molecular mechanistic studies of this disease.

Clinical Interest of Serum Alpha-2 Macroglobulin, Apolipoprotein A1, and Haptoglobin in Patients with Non-Alcoholic Fatty Liver Disease, with and without Type 2 Diabetes, before or during COVID-19.

In patients with non-alcoholic fatty liver disease (NAFLD) with or without type 2 diabetes mellitus (T2DM), alpha-2 macroglobulin (A2M), apolipoprotein A1 (ApoA1), and haptoglobin are associated with the risk of liver fibrosis, inflammation (NASH), and COVID-19. We assessed if these associations were worsened by T2DM after adjustment by age, sex, obesity, and COVID-19. Three datasets were used: the "Control Population", which enabled standardization of protein serum levels according to age and sex (N = 27,382); the "NAFLD-Biopsy" cohort for associations with liver features (N = 926); and the USA "NAFLD-Serum" cohort for protein kinetics before and during COVID-19 (N = 421,021). The impact of T2DM was assessed by comparing regression curves adjusted by age, sex, and obesity for the liver features in "NAFLD-Biopsy", and before and during COVID-19 pandemic peaks in "NAFLD-Serum". Patients with NAFLD without T2DM, compared with the values of controls, had increased A2M, decreased ApoA1, and increased haptoglobin serum levels. In patients with both NAFLD and T2DM, these significant mean differences were magnified, and even more during the COVID-19 pandemic in comparison with the year 2019 (all p < 0.001), with a maximum ApoA1 decrease of 0.21 g/L in women, and a maximum haptoglobin increase of 0.17 g/L in men. In conclusion, T2DM is associated with abnormal levels of A2M, ApoA1, and haptoglobin independently of NAFLD, age, sex, obesity, and COVID-19.