Hepatology

IntroductionMetabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) have risen substantially in prevalence over recent decades, driven by a growing global burden of obesity, diabetes mellitus, and other cardiometabolic risk factors. In response, researchers have intensified efforts to evaluate novel and re-purposed therapies that may prevent or reverse disease progression. Although several pharmacological therapies are under investigation, robust comparative evidence on their relative effectiveness and safety remains limited. We will therefore conduct a systematic review and network meta-analysis (SRNMA) of randomized controlled trials (RCTs) evaluating pharmacological therapies for adults with MASLD or MASH. MethodsWe will search four electronic databases (Ovid MEDLINE, Embase, CINAHL and Cochrane CENTRAL) from inception to August 2025 without language and other restrictions. Eligible studies will include parallel-arm RCTs enrolling [≥]10 adults per arm with MASLD or MASH; comparing any pharmacological therapy to standard care, no treatment, lifestyle modifications, placebo or alternative pharmacotherapies; and having a minimum follow-up duration of 12 weeks. Primary clinical outcomes are all-cause mortality, cardiovascular mortality, hospitalization, progression to cirrhosis, hepatic decompensation, hepatocellular carcinoma, and serious treatment-related adverse events. Surrogate outcomes include histological, imaging, biochemical, and metabolic markers of disease activity. Paired reviewers will independently screen identified hits for eligibility, extract data from eligible studies, and assess risk of bias using the Risk Of Bias instrument for Use in SysTematic reviews-for Randomised Controlled Trials (ROBUST-RCT). We will conduct separate NMAs for MASLD and MASH populations using a frequentist graph-theoretic random-effects model. Certainty of evidence will be assessed using GRADE. Subgroup and sensitivity analyses will explore effect modification by comorbidities and study quality. Ethics and DisseminationNo ethics approval is required. Results will be disseminated via peer-reviewed publication and conference presentations to inform clinicians, guideline developers, and health system decision-makers. PROSPERO Registration NumberCRD420251103235.

Background & AimsIron corrected T1 (cT1) is an MRI derived biomarker of liver disease activity. Emerging data suggest a change in cT1 of [≥] 80 ms reflects histological improvement. We aimed to validate the association between the [≥] 80 ms decline in cT1 and histological improvement, specifically the resolution of MASH. MethodsA retrospective analysis of study participants from three interventional clinical trials with histologically confirmed MASH (n = 150) who underwent multi-parametric MRI to measure cT1 (LiverMultiScan(R)) and biopsies at baseline and end of study. Histological responders were defined using the four criteria: (1) a decrease in NAFLD Activity score (NAS) [≥] 2 with no worsening in fibrosis, (2) a decrease in fibrosis [≥] 1 stage with no worsening in NAS, (3) both a NAS decrease [≥] 2 and a fibrosis decrease [≥] 1, and (4) MASH resolution with no worsening in fibrosis. Difference in the magnitude of change in cT1 between responders and non-responders was assessed. ResultsSignificant decreases in cT1 were observed in responders for all the histological criteria. The largest decrease was observed for those achieving MASH resolution, and was 119ms, compared to 43ms for non-responders. The optimal reduction in cT1 for separating responders from non-responders for MASH resolution was -74ms (64ms-73ms for the other criteria), in close agreement with the previously predefined threshold of -80ms. Those achieving an [≥] 80 ms reduction in cT1 were substantially more likely to achieve histological response with odds ratios ranging from 2.7 to 6.3. ConclusionsThese results demonstrate that a reduction in cT1 of 80 ms was associated with histological response supporting the utility of cT1 to predict clinical improvement in patients undergoing therapeutic intervention.

Background and aimsThe prevalence and etiology of liver fibrosis vary over time and impact racial/ethnic groups unevenly. This study measured time-trends and identified factors associated with advanced liver fibrosis in the U.S. MethodsStandardized methods were used to analyze data on 47,422 participants ([≥] 20 years) in the National Health and Nutrition Examination Survey (1999-2018). Advanced liver fibrosis was defined as Fibrosis-4 [≥]2.67 and/or Forns Index [≥]6.9 and elevated ALT. ResultsThe estimated number of people with advanced liver fibrosis increased from 1.3 million (95% CI, 0.8-1.9) to 3.5 million (95% CI, 2.8-4.2), a nearly 3-fold increase. Prevalence was higher in non-Hispanic Black and Mexican American persons than in non-Hispanic White persons. In multivariable logistic regression analysis, cadmium was an independent risk factor in all racial/ethnic groups. Smoking and current excessive alcohol use were risk factors in most. Importantly, non-Hispanic Black persons had a distinctive set of risk factors compared to non-Hispanic White persons that included poverty (OR = 2.09; 95%CI, 1.44-3.03), and susceptibility to lead exposure (OR = 3.25; 95%CI, 1.95-5.43), but did not include diabetes (OR = 0.88; 95% CI, 0.61-1.27, P =0.52). Non-Hispanic Black persons were more likely to have high exposure to lead, cadmium, polychlorinated biphenyls, and poverty than Non-Hispanic White persons. ConclusionsThe number of people with advanced liver fibrosis has increased, creating a need to expand the liver care workforce. The risk factors for advanced fibrosis varied by racial/ethnicity. These variations provide useful information for the design of screening programs. Poverty and toxic exposures were associated with the high prevalence of advanced liver fibrosis in non-Hispanic Black persons and need to be addressed. Lay summaryBecause liver disease often produces few warning signs, simple and inexpensive screening tests that can be performed by non-specialists are needed to allow timely detection and linkage to care. This study shows that non-Hispanic Black persons have a distinctive set of risk factors that need to be taken into account when designing liver disease screening tests. Exposure to exogenous toxins may be especially important risk factors for advanced liver fibrosis in non-Hispanic Black persons.

ObjectiveIn sub-Saharan Africa, hepatitis B is the principal cause of liver disease. Non-invasive biomarkers of liver fibrosis are needed to identify patients requiring antiviral treatment. We assessed aspartate aminotransferase-to-platelet ratio index (APRI), gamma-glutamyl transferase-to-platelet ratio (GPR) and FIB-4 to diagnose significant fibrosis and cirrhosis in an individual patient data (IPD) meta-analysis. DesignIn total, 3,549 patients from 12 cohorts of HBsAg positive individuals in 8 sub-Saharan African countries were included. Transient elastography was used as a reference test for cirrhosis (>12.2 kPa), excluding patients who were pregnant, had hepatitis C, D, or HIV co-infection, were on hepatitis B therapy, or had acute hepatitis. A bivariate Bayesian IPD model was fitted with patient-level covariates and study-level random effects. ResultsAPRI and GPR had the best discriminant performance (area under receiver operating curve 0.81 and 0.82) relative to FIB-4 (0.77) for cirrhosis. The World Health Organization (WHO) recommended APRI threshold of [≥]2.0 was associated with a sensitivity and specificity (95% credible interval) of 16.5% (12.5-20.5) and 99.5% (99.2-99.7) for cirrhosis. For APRI, we identified an optimised rule-in threshold for cirrhosis (cut-off 0.65) with a sensitivity and specificity of 56.2% (50.5-62.2) and 90.0% (89.0-91.0), and an optimised rule-out threshold (cut-off 0.36) with a sensitivity and specificity of 80.6% (76.1-85.1) and 64.3% (62.8-65.8). ConclusionsThe WHO recommended APRI threshold of 2.0 is too high to diagnose cirrhosis in sub-Saharan Africa. We identified new and optimised rule-in and rule-out thresholds for cirrhosis, with direct consequences for treatment guidelines in this setting.

Hepatocytes (HCs), which share a developmental origin with cholangiocytes (CCs), have the capacity to undergo reparative reprogramming into CCs in response to liver injury and, under specific conditions, can also transform malignantly into cholangiocarcinoma (CCA). However, the molecular mechanisms governing HC plasticity in liver diseases remain poorly understood. In this study, we investigated the role of Spalt Like Transcription Factor 4 (SALL4), an oncofetal transcription factor, in both malignant and regenerative HC fate transitions toward the biliary lineage. Using Sleeping Beauty hydrodynamic tail vein injection-mediated murine liver cancer models, we explored HC-to-CCA transformation, while the DDC diet-induced cholestasis model was used to investigate regenerative HC-to-CC reprogramming. Our findings reveal that SALL4 is specifically required for myristoylated Akt (myrAkt)-YAP1S127A (AY)-driven HC-to-CCA transformation, as its loss significantly suppressed malignant reprogramming and clonal expansion. Surprisingly, SALL4 overexpression also prevented AY-driven CCA development while promoting the expansion of liver progenitor cell (LPC)-like fatty HCs. Mechanistically, we propose Bmi1 as a key downstream effector of SALL4 in YAP1-dependent HC- to-CCA transformation. Additionally, in the DDC-fed cholestasis model, Sall4 deletion enhanced HC-to-LPC activation while impairing LPC differentiation into mature CCs. These findings establish SALL4 as a critical regulator of HC plasticity in both malignant and regenerative contexts and highlight its potential as a therapeutic target for specific liver cancer subtypes. SIGNIFICANCEHepatocyte plasticity supports repair but can drive malignancy, acting as a double-edged sword. We identify SALL4 as regulator of YAP1-driven hepatocyte-to-cholangiocyte reprogramming, revealing the YAP1-SALL4- BMI1 axis as a therapeutic target for cholangiocarcinoma.

Liver cirrhosis and chronic liver diseases impose a substantial and growing health burden globally, with sub-Saharan Africa (SSA) disproportionately affected. Leveraging Global Burden of Disease 2021 data and Bayesian hierarchical models, we quantified mortality and disability trends in SSA from 1990 to 2021 and projected disease burden through 2035, incorporating scenarios for hepatitis B vaccination scale-up. In 2021, liver cirrhosis accounted for an estimated 181,311 deaths in SSA, despite a 29% decline in age-standardized death rates (ASDR) since 1990. Absolute deaths increased by 65%, predominantly driven by hepatitis B (37%), hepatitis C (28%), and alcohol-related cirrhosis (17%). Disability-adjusted life years (DALYs) surged by 76%, from approximately 1.05 million in 1990 to 1.85 million in 2021, highlighting rising absolute disability alongside a 29% reduction in age-standardized DALYs. Mortality and disability burdens were highest in Somalia, Central African Republic, and Guinea-Bissau. Males bore nearly twice the burden of females. While death rates declined across all socio-demographic strata, absolute deaths rose by 55-86%. Projections to 2035 suggest further potential reductions in mortality from hepatitis B (up to 21.5%), hepatitis C (up to 18.7%), and alcohol-related cirrhosis, while the burden of non-alcoholic fatty liver disease is expected to remain stable or increase slightly. Scaling up hepatitis B vaccination could further avert 27% of related deaths by 2035. These findings reveal persistent and widening disparities in cirrhosis burden across SSA, underscoring the urgent need for integrated, context-specific interventions combining viral hepatitis control with metabolic liver disease management to improve equitable liver health outcomes.

Bile acids (BAs) are liver-synthesized steroids that facilitate lipid digestion and regulate diverse metabolic pathways. Because of their cytotoxicity, excessive hepatic BA accumulation has been implicated in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). Although circulating BAs are frequently elevated in MASLD, it is unclear whether similar alterations occur within the liver. Moreover, the strong overlap between MASLD and metabolic syndrome complicates efforts to distinguish BA changes driven by liver disease from those arising due to broader metabolic dysfunction. Here we show in a series of human studies that the BA dysmetabolism in MASLD originates from insulin resistance rather than hepatic steatosis. We found that circulating BA concentrations are twofold higher in patients with MASLD than in healthy controls, despite similar intrahepatic levels. Causal inference using a MASLD genetic risk score indicated that this elevation is not attributable to hepatic steatosis. Instead, circulating BAs, but not intrahepatic BAs, associate with glycemia and hepatic insulin sensitivity. We found reduced expression of the BA uptake transporter NTCP in insulin-resistant individuals, implicating impaired hepatic BA clearance. During hepatic vein catheterization, insulin acutely lowered conjugated BA concentrations in hepatic venous blood, consistent with diminished splanchnic BA spillover. Physiology-based simulations of impaired hepatic BA clearance recapitulated the human phenotype. Taken together, our findings argue against a major role for BAs in MASLD pathogenesis but reveal a previously unrecognized link between circulating BA dynamics and hepatic insulin action.

Background and AimsThe newly proposed nomenclature for steatotic liver diseases (SLD) aims to reduce the stigma associated with "non-alcoholic fatty liver disease" (NAFLD), increase awareness, and provide a framework for delineating pathogenic pathways. Approach and ResultsWe projected the new nomenclatures diagnostic scheme onto National Health and Nutrition Examination Survey (NHANES) data and determined SLD prevalence, fibrosis risk factors, subtypes, and consistency with previous classifications. Steatosis grade and fibrosis stage were estimated from vibration controlled transient elastography (VCTE). At a threshold of 240 dB/m, 62.1% [95% confidence interval (CI), 59.8-64.3%] of adults ([≥] 20 years) and 30.5% (95% CI, 27.1-34.0%) of adolescents (12-19 years) had SLD. By American Gastroenterological Association criteria, 19.3 million (95% CI, 15.8-22.8) adults with SLD qualify for hepatology referral. Over 98% of adults but only 85% of adolescents with NAFLD met criteria for definite MASLD. Significant fibrosis ([≥] 8.6 kPa) occurred in 13.5 million (95% CI, 10.9-16.2) adults with MASLD; risk factors varied by race and ethnicity. Significant fibrosis occurred in over 1.5 million adults without any identified LD and was associated with lead (Pb) exposure, odds ratio = 3.89 (95% CI, 2.00-7.56). ConclusionsThe overarching term, SLD, changes the diagnostic algorithm and creates an umbrella classification that highlights the extraordinary prevalence of liver steatosis. The more precise nomenclature establishes a valuable patient-centric platform for research and clinical care, clarifying risk groups and risk factors, including adolescents with NAFLD but without definite MASLD and adults without SLD in whom toxic exposures may increase fibrosis risk.

BackgroundMetabolic dysfunction-associated steatohepatitis (MASH) is a prevalent and progressive liver disease with limited pharmacologic treatment options. Pemvidutide, a GLP-1-glucagon dual receptor agonist, has shown promise in targeting both hepatic steatosis and fibrosis. This systematic review and meta-analysis aimed to evaluate the efficacy and safety of pemvidutide in adults with MASH. MethodsWe systematically searched PubMed, Scopus, Web of Science, and Cochrane CENTRAL through December 15, 2025, for randomized controlled trials (RCTs) comparing once-weekly subcutaneous pemvidutide (1.2 mg, 1.8 mg, or 2.4 mg) with placebo in adult MASH patients. Primary outcomes were changes in liver fat content (LFC, % via MRI-PDFF) and Enhanced Liver Fibrosis (ELF) score. Secondary outcomes included body weight, glycemic and lipid parameters, blood pressure, heart rate, and adverse gastrointestinal events. Dose-specific pairwise meta-analyses were performed using a random-effects model. ResultsThree RCTs encompassing 370 participants (195 pemvidutide, 175 placebo) were included. Pemvidutide significantly reduced LFC at all doses, with the greatest effect at 1.8 mg (MD = -21.63%, 95% CI: -27.23 to -16.02; p<0.0001). ELF score improvement was significant at 1.2 mg and 1.8 mg doses but not at 2.4 mg. Across all doses, body weight decreased significantly, while HbA1c remained unchanged. Pemvidutide also reduced systolic blood pressure and total cholesterol (1.8 mg and 2.4 mg), with modest HDL reduction at 2.4 mg. Mild-to-moderate gastrointestinal adverse events were observed, with nausea more frequent at 1.8 mg. ConclusionsPemvidutide is effective in reducing liver fat and improving cardiometabolic parameters in MASH, with a favorable safety profile. Dose-specific effects on fibrosis suggest potential early antifibrotic activity, highlighting its promise as a dual-targeted therapy for MASH. Further long-term studies are warranted to confirm sustained hepatic and metabolic benefits.

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) and hypertension frequently coexist in adults across the Americas, yet evidence to guide pharmacologic management in individuals with both conditions remains limited. Antihypertensive medications may influence liver outcomes, but comparative data across drug classes are sparse. MethodsWe performed a cross-sectional analysis using pooled data from the US National Health and Nutrition Examination Survey (1999 to 2018), linked to mortality records through 2019. Hepatic steatosis was assessed using the US Fatty Liver Index (US-FLI), Fatty Liver Index (FLI), and Hepatic Steatosis Index (HSI). Fibrosis was assessed using noninvasive scores. Antihypertensive exposure, identified through prescription records, included ACE inhibitors, ARBs, beta blockers, calcium channel blockers (CCBs), and diuretics. Associations with liver fibrosis were estimated using logistic regression. All cause and cardiovascular mortality were assessed using Cox proportional hazards models with inverse probability of treatment weighting. FindingsAmong 2,909 adults with MASLD receiving monotherapy antihypertensive treatment, use of ACEIs and ARBs was associated with lower odds of liver fibrosis compared with CCBs. In adjusted models, ACEIs were associated with reduced all-cause mortality (adjusted hazard ratio 0.30; 95% CI 0.11-0.82), as were ARBs (0.25; 95% CI 0.07-0.94). No significant differences were observed for cardiovascular mortality across medication classes. InterpretationUse of angiotensin converting enzyme inhibitors and angiotensin receptor blockers was associated with lower fibrosis burden and improved survival in individuals with metabolic dysfunction associated steatotic liver disease. These findings are hypothesis generating and warrant confirmation in prospective studies. FundingNo Funding.

Although viral hepatocellular carcinoma (HCC) is declining, non-viral HCC, which often is the end-stage of non-alcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICI) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a small portion of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell mediated tumor regression and postulated that anti-fibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1 induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with inhibition of TGF-{beta} receptor signaling, collagen deposition and depletion of immunosuppressive fibroblasts. SignificanceImmune checkpoint inhibitors are used in HCC treatment but overall response rates for single agent PD-1/PD-L1 blockers have remained stubbornly low. Using a mouse model of NASH-driven HCC, we show that co-treatment with the safe and inexpensive angiotensin II receptor inhibitor losartan substantially enhanced anti-PD-1 triggered HCC regression. Although losartan did not influence the reinvigoration of exhausted CD8+ T cells it considerably enhanced their intratumoral invasion, which we postulated to be compromised by peritumoral fibrosis. Indeed, the beneficial effect of losartan correlated with inhibition of TGF-{beta} signaling and collagen deposition, and depletion of immunosuppressive fibroblasts. Losartan should be evaluated for its adjuvant activity in HCC patients undergoing PD-1/PD-L1 blocking therapy.

BackgroundMetabolic-associated fatty liver disease (MAFLD) is a prevalent chronic liver condition with limited approved pharmacological treatments [1]. Ursodeoxycholic acid (UDCA) and ademetionine show variable efficacy, primarily on liver enzymes. This study presents a preliminary analysis comparing the efficacy of a novel dietary supplement, Gepaktiv, against these comparators in MAFLD patients. MethodsIn this open-label, randomized controlled trial (clinicaltrials.gov NCT07068191 and ITMCTR 2025001469), 19 patients with MAFLD, confirmed by hepatomegaly (liver size [&ge;]3 cm above normal by ultrasound), elevated alanine aminotransferase (ALT, 90-150 U/L), and FibroScan results (steatosis [&ge;]260 dB/m, fibrosis [&ge;]11 kPa), were allocated to Gepaktiv (n=6, 1500 mg/day), UDCA (n=7, 10 mg/kg/day), or Ademetionine (n=6, orally 400 mg 2 times a day) for 15 days. Patients with significant alcohol consumption (>20 g/day for women, >30 g/day for men) were excluded. Primary outcomes were median changes from baseline to day 15 in ALT, aspartate aminotransferase (AST), liver size (craniocaudal diameter, cm, via ultrasound), steatosis (controlled attenuation parameter, CAP, dB/m), and fibrosis (transient elastography, kPa). ResultsThe Gepaktiv group showed median [IQR] reductions of ALT -48.9 [-54.0 to -35.0] U/L, AST - 62.8 [-66.0 to -44.0] U/L, liver size -1.9 [-2.0 to -1.2] cm, and steatosis -32.5 [-45.0 to -30.0] dB/m. These reductions were significantly greater compared to both UDCA and Ademetionine groups (p < 0.01 for ALT, AST, and liver size; p < 0.05 for steatosis). Fibrosis reduction was minimal and not statistically significant between groups. ConclusionThe Gepaktiv group was associated with greater improvements in biochemical and imaging markers of MAFLD compared to UDCA and Ademetionine in this preliminary analysis. These findings warrant further investigation in larger, long-term trials. Note: These preliminary results have not been peer-reviewed and should not guide clinical practice.

Purpose Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major cause of chronic liver disease and liver-related morbidity worldwide. Although dietary factors may influence MASLD progression, the long-term liver-specific implications of artificially sweetened beverage (ASB) intake remain unclear. We aimed to examine the association between ASB intake and the risk of liver-related adverse events and liver-related death among individuals with MASLD. Methods This prospective cohort study included 50,562 participants with MASLD from the UK Biobank. ASB intake was assessed using 24-hour dietary recalls and categorized as 0, >0-1, and >1 serving/day. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for liver-related adverse events and liver-related death. Restricted cubic spline models were used to assess dose-response patterns, and competing-risk analyses were performed by treating liver-related death as a competing event for liver-related adverse events. Additional substitution, subgroup and sensitivity analyses were conducted to evaluate the robustness of the findings. Results During a median follow-up of 12.8 years, 292 liver-related adverse events and 91 liver-related deaths occurred. Compared with participants reporting no ASB intake, those consuming >1 serving/day had a higher risk of liver-related adverse events in the fully adjusted model (HR 1.40, 95% CI 1.02-1.93; P = 0.039), whereas the association for >0-1 serving/day was not statistically significant (HR 1.26, 95% CI 0.92-1.71; P = 0.149). The risk of liver-related adverse events increased across ASB intake categories (P for trend = 0.023). Restricted cubic spline analysis indicated a positive linear association between ASB intake and liver-related adverse events (P-overall <0.001; P-nonlinearity = 0.72). In competing-risk analysis, the association for >1 serving/day remained consistent after accounting for liver-related death as a competing event (sub-HR 1.40, 95% CI 1.02-1.93; P = 0.038; Gray test P = 0.006). The association was robust in sensitivity analyses. ASB intake was not significantly associated with liver-related death, and beverage substitution analyses showed no significant associations. Conclusion Among individuals with MASLD, high ASB intake, particularly >1 serving/day, was associated with an increased risk of liver-related adverse events, but not liver-related death. This association was consistent across dose-response, competing-risk, and sensitivity analyses, suggesting that high ASB intake may represent a potential dietary risk marker for adverse liver outcomes in MASLD.

Existing drug therapies for hepatocellular carcinoma (HCC), including sorafenib, extend patient survival by only three months. We sought to identify novel druggable targets for use in combination with sorafenib to increase its efficacy. We implemented an in vivo genetic screening paradigm utilizing a library of 43 genes-of-interest expressed in the context of repopulation of the injured livers of Fumarylacetoacetate Hydrolase-deficient (Fah-/-) mice, which led to highly penetrant HCC. We then treated mice with vehicle or sorafenib to discover genetic determinants of sensitivity and resistance. Liver X Receptor alpha (LXR) emerged as a potential target. To examine LXR agonism in combination with sorafenib treatment, we added varying concentrations of sorafenib and LXR agonist drugs to HCC cell lines. We performed transcriptomic analysis to elucidate the mechanisms of HCC death. Fah-/- mice injected with the screening library developed HCC tumor clones containing Myc cDNA plus various other cDNAs. Treatment with sorafenib resulted in sorafenib-resistant HCCs that were significantly depleted in Nr1h3 cDNA, encoding LXR, suggesting that LXR activation is incompatible with tumor growth in the presence of sorafenib treatment in vivo. The combination of sorafenib and LXR agonism led to enhanced cell death as compared to monotherapy in multiple HCC cell lines, due to reduced expression of cell cycle regulators and increased expression of genes associated with apoptosis. Combination therapy also enhanced cell death in a sorafenib-resistant primary human HCC cell line. Our novel in vivo screen led to the discovery that LXR agonist drugs potentiate the efficacy of sorafenib in treating HCC.

Bile represents a clinically accessible biological fluid that can mitigates major limitations associated with tissue-based sampling for the generation of organoid models to study hepatobiliary disease, including biliary tract cancers where tissue availability is often limited. Importantly, bile can also enable the generation of non-malignant cholangiocyte organoids that are otherwise difficult to obtain. Here, we describe an operator-oriented, step-by-step protocol to generate organoids from fresh bile collected during endoscopic retrograde cholangiopancreatography (ERCP), together with two complementary workflows for siRNA delivery in 3D cultures. We detail critical control points that are often under-reported, yet considerably influence success and reproducibility. The protocol was optimized and applied in a real-world cohort of 21 patients undergoing ERCP, including benign biliary obstruction due to choledocholithiasis (n=5) and malignant strictures (n=16: cholangiocarcinoma n=13, gallbladder adenocarcinoma n=1, ampullary tumors n=2). Expandable organoids were established in 17/21 cases (81%), with establishment rates of 60% for choledocholithiasis and 85-100% across malignant entities. Anticipated results include organoid outgrowth within [~]2-3 weeks and morphological heterogeneity in cultures derived from malignant strictures, where normal-like and tumor-like populations may initially coexist and can drift toward a cystic phenotype under routine expansion, motivating optional manual handpicking when tumor-enriched lines are required. As downstream readouts, we show feasibility of DNA-based profiling in selected paired bile-organoid samples (targeted sequencing and ULP-WGS copy-number analysis) and demonstrate proof-of-concept gene silencing via siRNA in both dissociated cells prior to re-embedding, and intact fully formed organoids while preserving 3D architecture. Collectively, this workflow provides a practical and reproducible framework to establish, expand, characterize and functionally perturb bile-derived organoids from routine clinical procedures, facilitating standardized implementation across laboratories.

Background & AimsA cholestatic pattern of liver enzymes is associated with progressive liver disease and major adverse liver-related outcomes (MALO) among patients with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). We aimed to authenticate the efficacy of a newly formulated liver function test (LFT) score for distinguishing patients with cholestatic vs. hepatocellular patterns and to evaluate its prognostic utility in MASLD patients. MethodsA retrospective longitudinal study on a dataset of over 250,000 individuals diagnosed with MASLD and/or obesity with cardiovascular risk factors. Patients were categorized into cholestatic (C), mixed (M), or hepatocellular (H) patterns according to the LFT score, or the well-known R score. Long-term MALO, major adverse cardiovascular events (MACE), and all-cause mortality were tracked. ResultsThe LFT score excelled in differentiating patients into C, M, or H groups accurately. While about two-thirds of our cohort initially showed a low FIB4 (<1.3), patients in the C category experienced a higher incidence of MALO and MACE compared to those in the H category (0.5% vs. 0.2% and 7.1% vs. 3.6%, respectively) over the span of 10 years post-diagnosis. Additionally, the 15-year overall survival rate was notably lower for C patients compared to their H counterparts (63% vs. 77%, p<0.0001). The LFT score was more effective than the R score in distinguishing between H and C patients for prognostic purposes, and a baseline cholestatic pattern indicates poorer outcomes regardless of subsequent LFT changes. ConclusionsThe LFT score accurately categorizes cholestatic MASLD patients and may serve as a useful prognostic tool.

Background and ObjectivesBariatric surgery is a highly effective obesity treatment, yet it may predispose individuals to alcohol-related liver injury. While altered ethanol metabolism following procedures like Roux-en-Y gastric bypass (RYGB) is well described, the long-term hepatic consequences, particularly the risk of portal hypertension in patients who develop alcohol-related hepatitis (AH,) remain poorly defined. MethodsUsing the TriNetX US Collaborative Network, we identified adult patients diagnosed with AH or alcohol-related cirrhosis. We compared outcomes between patients with a history of RYGB or sleeve gastrectomy (SG) who subsequently developed AH (Bariatric+AH group) and those with AH and no history of bariatric surgery (AH-only group). Propensity score matching was performed on over 44 demographic, clinical, and laboratory variables. Cox proportional hazards models and Kaplan-Meier survival curves were used to estimate the risk of clinically significant portal hypertension (PH) events, liver transplantation, and all-cause mortality at three-, five-, and seven-year follow-ups. ResultsAfter matching, 772 patients were included in each cohort. At 7 years post-index event, the Bariatric + AH group exhibited a significantly higher risk of PH-related complications compared to the AH-only group (HR 1.519; 95% CI, 1.15-2.005; p = 0.003). No significant differences were observed in liver transplantation (HR 1.412; 95% CI, 0.850-2.346; p = 0.181) or all-cause mortality (HR 1.085; 95% CI, 0.904-1.303; p = 0.381). These findings were consistent across all follow-up intervals. ConclusionBariatric surgery is associated with an increased long-term risk of portal hypertension in patients who develop alcohol-related hepatitis despite similar mortality and transplantation rates. These findings underscore the need for targeted postoperative counseling, liver-focused surveillance strategies, and integration of hepatologic risk assessment into metabolic surgery care pathways.

BackgroundHBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(-) treatment-naive CHB participants. MethodsLiver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed. ResultsIntrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(-) samples (p<0.0001). HBsAg staining, in contrast, was present in over 85% of both HBeAg(+) and (-) livers. 23 (95.8%) HBeAg(+) participants had [&le;]50% iDNA of total HBV DNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(-) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers. ConclusionSerum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(-) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(-) CHB.

Immune dysfunction, both depression and hyperactivation, in liver disease contributes to significant morbidities and mortalities, depending on liver damage severity and etiology. The underlying causes of immune dysfunction in advanced liver disease, whether pathogen or host-mediated, remain unclear. We reported lasting generalized CD8+ T cell hyperfunction in individuals with advanced liver fibrosis in chronic HCV infection. The separation of viral and fibrosis-driven effects or the association of this phenomenon with clinical outcomes of advanced liver fibrosis remains to be determined. Here, a hepatotoxic murine model of liver fibrosis was used to decouple liver fibrosis from viral infection. Carbon tetrachloride (CCl4)-treated mice presented progressive liver fibrosis within {approx}12 weeks, resulting in severe diffuse fibrosis, focal necrosis and surrounding mixed inflammation; pathology similar to that of chronic HCV infection. Taking advantage of this model, we investigated if liver fibrosis caused systemic CD8+ T cell hyperfunction and evaluated its impact on host immune response. At peak liver fibrosis, circulating CD8+ T cells presented increased expression of IFN-{gamma} and granzyme B (GrzB) in comparison to control animals. CD8+ T cell hyperfunction arose by 8 weeks of CCl4 treatment and was sustained with continued liver insult. As a result, fibrotic mice were unable to resist an ectopic tumour challenge and were less responsive to immunotherapy. Furthermore, CD8+ T cell dysfunction was observed in other contexts of chronic liver insult such as high fat diet-induced liver steatosis, even in the absence of significant fibrosis. Collectively, this study shows the impact of chronic liver insult on systemic CD8+ T cell function and its association with impaired immune response, such as tumour surveillance.

Liver cancer is a leading cause of cancer-related death world-wide in part due to the shortage of effective therapies, and MYC overexpression defines an aggressive and especially difficult to treat subset of patients. Given MYCs ability to reprogram cancer cell metabolism, and the livers role as a coordinator of systemic metabolism, we hypothesized that MYC induces metabolic dependencies that could be targeted to attenuate liver tumor growth. We discovered that MYC-driven liver cancers catabolize alanine in a GPT2-dependent manner to sustain their growth. GPT2 is the predominant alanine-catabolizing enzyme expressed in MYC-driven liver tumors and genetic ablation of GPT2 limited MYC-driven liver tumorigenesis. In vivo isotope tracing studies uncovered a role for alanine as a substrate for a repertoire of pathways including the tricarboxylic acid cycle, nucleotide production, and amino acid synthesis. Treating transgenic MYC-driven liver tumor mouse models with L-Cycloserine, a compound that inhibits GPT2, was sufficient to diminish the frequency of mouse tumor formation and attenuate growth of established human liver tumors. Thus, we identify a new targetable metabolic dependency that MYC-driven liver tumors usurp to ensure their survival.