Portal inflammation (PI) and ductular reaction (DR) in metabolic dysfunction-associated steatotic liver disease (MASLD) have shown associations with disease severity. We developed a histologic categorization of these features to correlate with known features of MASLD. This study proposes a scoring schema for PI, PP and DR, and relates them to histologic and clinical features in children and adults. This expanded scoring system was developed to identify clinically relevant categories and defined criteria for scoring biopsies. In adults (N:483), more severe PI, PP, and DR were associated with older age (p ≤ 0.002), and PP and DR were associated with increased alkaline phosphatase (ALP) (p ≤ 0.003), GGT (p ≤ 0.001), and total bilirubin (p ≤ 0.01). More severe PI, PP, and DR were associated with higher NAFLD activity score (NAS), fibrosis stage, and diagnosis of metabolic dysfunction-associated steatohepatitis (MASH) (p ≤ 0.05). In children (N:151), PP and DR were associated with younger age (p ≤ 0.0001), and elevated AST, ALT, and ALP (p ≤ 0.05). More severe PI, PP, and DR were associated with advanced fibrosis stage, and PP and DR were associated with diagnosis of borderline or definite MASH in children (p ≤ 0.05). From multivariable ordinal logistic regression analysis, a higher fibrosis stage was independently associated with more severe PI in both adults and children. Interobserver agreement was substantial for PI, PP and DR. The proposed scoring system demonstrated reproducibility and associations between more severe portal-based disease and advanced liver histology, age, and elevated liver enzymes in adults and children. Evaluation of portal disease could provide insight into therapeutic response and disease progression.

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.).

Background: NAFLD is highly prevalent with limited treatment options. Bile acids (BAs) increase in the systemic circulation and liver during NAFLD progression. Changes in plasma membrane localization and zonal distribution of BA transporters can influence transport function and BA homeostasis. However, a thorough characterization of how NAFLD influences these factors is currently lacking. This study aimed to evaluate the impact of NAFLD and the accompanying histologic features on the functional capacity of key hepatocyte BA transporters across zonal regions in human liver biopsies. Methods: A novel machine learning image classification approach was used to quantify relative zonal abundance and plasma membrane localization of BA transporters (bile salt export pump [BSEP], sodium-taurocholate cotransporting polypeptide, organic anion transporting polypeptide [OATP] 1B1 and OATP1B3) in non-diseased (n = 10), NAFL (n = 9), and NASH (n = 11) liver biopsies. Based on these data, membrane-localized zonal abundance (MZA) measures were developed to estimate transporter functional capacity. Results: NAFLD diagnosis and histologic scoring were associated with changes in transporter membrane localization and zonation. Increased periportal BSEP MZA (mean proportional difference compared to non-diseased liver of 0.090) and decreased pericentral BSEP MZA (−0.065) were observed with NASH and also in biopsies with higher histologic scores. Compared to Non-diseased Liver, periportal OATP1B3 MZA was increased in NAFL (0.041) and NASH (0.047). Grade 2 steatosis (mean proportional difference of 0.043 when compared to grade 0) and grade 1 lobular inflammation (0.043) were associated with increased periportal OATP1B3 MZA . Conclusions: These findings provide novel mechanistic insight into specific transporter alterations that impact BA homeostasis in NAFLD. Changes in BSEP MZA likely contribute to altered BA disposition and pericentral microcholestasis previously reported in some patients with NAFLD. BSEP MZA assessment could inform future development and optimization of NASH-related pharmacotherapies.

SummaryBackgroundMetabolic 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.MethodsOverall, 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.ResultsCompared 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.ConclusionThe 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.

Background and Aims: Senescent hepatocytes accumulate in parallel with fibrosis progression during NASH. The mechanisms that enable progressive expansion of nonreplicating cell populations and the significance of that process in determining NASH outcomes are unclear. Senescing cells upregulate thrombomodulin–protease-activated receptor-1 (THBD-PAR1) signaling to remain viable. Vorapaxar blocks the activity of that pathway. We used vorapaxar to determine if and how THBD-PAR1 signaling promotes fibrosis progression in NASH. Approach and Results: We evaluated the THBD-PAR1 pathway in liver biopsies from patients with NAFLD. Chow-fed mice were treated with viral vectors to overexpress p16 in hepatocytes and induce replicative senescence. Effects on the THBD-PAR1 axis and regenerative capacity were assessed; the transcriptome of p16-overexpressing hepatocytes was characterized, and we examined how conditioned medium from senescent but viable (dubbed “undead”) hepatocytes reprograms HSCs. Mouse models of NASH caused by genetic obesity or Western diet/CCl 4 were treated with vorapaxar to determine effects on hepatocyte senescence and liver damage. Inducing senescence upregulates the THBD-PAR1 signaling axis in hepatocytes and induces their expression of fibrogenic factors, including hedgehog ligands. Hepatocyte THBD-PAR1 signaling increases in NAFLD and supports sustained hepatocyte senescence that limits effective liver regeneration and promotes maladaptive repair. Inhibiting PAR1 signaling with vorapaxar interrupts this process, reduces the burden of ‘undead’ senescent cells, and safely improves NASH and fibrosis despite ongoing lipotoxic stress. Conclusion: The THBD-PAR1 signaling axis is a novel therapeutic target for NASH because blocking this pathway prevents accumulation of senescing but viable hepatocytes that generate factors that promote maladaptive liver repair.

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.

PDF file - 54K, Related primer sequences

Background Several early-phase clinical trials for the treatment of nonalcoholic steatohepatitis (NASH) use liver fat content as measured with the MRI-derived proton density fat fraction (PDFF) for a primary outcome. These trials have shown relative reductions in liver fat content with placebo treatment alone, a phenomenon termed "the placebo effect." This phenomenon confounds the results and limits generalizability to future trials. Purpose To quantify the effect of placebo treatment on change in the absolute PDFF value and to identify variables associated with this observed change. Materials and Methods This is a secondary analysis of prospectively collected data from seven early phase clinical trials that included participants with a diagnosis of NASH based on MRI and/or liver biopsy who received placebo treatment. The primary outcome was a greater than or equal to 30% relative reduction in PDFF after placebo treatment. Normalization of PDFF, relative change in alanine aminotransferase (ALT) level, and normalization of ALT level were also examined. An exploratory linear mixed-effects model was used to estimate an overall change in absolute PDFF and to explore parameters associated with this response. Results A total of 187 participants (median age, 52 years [IQR, 43-60 years]; 114 women) who received placebo treatment were evaluated. A greater than or equal to 30% relative reduction in baseline PDFF was seen in 20% of participants after 12 weeks of placebo treatment (10 of 49), 9% of participants after 16 weeks (two of 22), and 28% of participants after 24 weeks (34 of 122). A repeated-measures linear mixed-effects model estimated a decrease of 2.3 units (median relative reduction of 13%) in absolute PDFF values after 24 weeks of placebo treatment (95% CI: 3.2, 1.4; P < .001). Conclusion In this analysis of 187 participants, a clinically relevant decrease in PDFF was observed with placebo treatment. Based on the study model, assuming an absolute PDFF decrease of approximately 3 units (upper limit of 95% CI) to account for this "placebo effect" in sample size calculations for future clinical trials is suggested. Clinical trial registration nos. NCT01066364, NCT01766713, NCT01963845, NCT02443116, NCT02546609, NCT02316717, and NCT02442687 © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Yoon in this issue.

PDF file - 127K, S1. Panc 10.05 cells do not generate functional Hh ligands. S2. Other malignant hepatoma lines can increase MF Hh and glycolytic activity. S3. Summary of glycolytic metabolism. S4. MF-derived lactate provides energy source for lipogenesis in other malignant hepatoma lines. S5. Schematic of alterations in hepatoma cells due to release of metabolic end products by glycolytic MF