BACKGROUND AIMS: Refractory pruritus and other complications of cholestasis are indications for liver transplantation (LT) in patients with Alagille syndrome (ALGS). We evaluated predictors of event-free survival (EFS) and transplant-free survival (TFS) in ALGS patients treated with maralixibat (MRX), an ileal bile acid transporter inhibitor. APPROACH RESULTS: We assessed ALGS patients from three clinical trials of MRX with up to 6 years of follow-up. EFS was defined as absence of LT, surgical biliary diversion (SBD), hepatic decompensation, or death; TFS was absence of LT or death. Forty-six potential predictors were evaluated, including age, pruritus (ItchRO[Obs] 0-4 scale), biochemistries, platelets, and serum bile acids (sBA). Harrell's concordance statistic assessed goodness-of-fit, then Cox proportional hazard models confirmed the statistical significance of the predictors identified. A further analysis was performed to identify cutoffs using a grid search. Seventy-six individuals met criteria of receiving MRX for ≥ 48 weeks with laboratory values available at Week 48 (W48). Median duration of MRX was 4.7 years (IQR: 1.6-5.8); 16 had events (10 LT, 3 decompensation, 2 death, 1 SBD). 6-year EFS improved with a clinically meaningful > 1-point ItchRO(Obs) reduction from baseline to W48 (88% vs. 57%; p = 0.005), W48 bilirubin < 6.5 mg/dL (90% vs. 43%; p < 0.0001), and W48 sBA < 200 µmol/L (85% vs. 49%; p = 0.001). These parameters were also predictive of 6-year TFS. CONCLUSIONS: Improvement in pruritus over 48 weeks and lower W48 bilirubin and sBA levels were associated with fewer events. These data may help identify potential markers of disease progression for MRX-treated ALGS patients.

BACKGROUND AND AIMS: Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection and affects the lives of millions of people worldwide by causing liver inflammation, cirrhosis and liver cancer. Interferon-alpha (IFN-α) therapy is a conventional immunotherapy that has been widely used in CHB treatment and achieved promising therapeutic outcomes by activating viral sensors and interferon-stimulated genes (ISGs) suppressed by HBV. However, the longitudinal landscape of immune cells of CHB patients and the effect of IFN-α on the immune system are not fully understood. APPROACH AND RESULTS: Here, we applied single-cell RNA sequencing (scRNA-seq) to delineate the transcriptomic landscape of peripheral immune cells in CHB patients before and after PegIFN-α therapy. Notably, we identified three CHB-specific cell subsets, Pro-infla CD14 + monocytes, Pro-infla CD16 + monocytes and IFN + CX3CR1- NK cells, which highly expressed pro-inflammatory genes and positively correlated with HBsAg. Furthermore, PegIFN-α treatment attenuated percentages of hyperactivated monocytes, increased ratios of long-lived naive/memory T cells and enhanced effector T cell cytotoxicity. Finally, PegIFN-α treatment switched the transcriptional profiles of entire immune cells from TNF-driven to IFN-α-driven pattern and enhanced innate antiviral response, including virus sensing and antigen presentation. CONCLUSIONS: Collectively, our study expands the understanding of the pathological characteristics of CHB and the immunoregulatory roles of PegIFN-α, which provides a new powerful reference for the clinical diagnosis and treatment of CHB.

The microbiome plays a crucial role in integrating environmental factors into host physiology, potentially linking it to autoimmune liver diseases, such as autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis. All autoimmune liver diseases are associated with reduced diversity of the gut microbiome and altered abundance of certain bacteria. However, the relationship between the microbiome and liver diseases is bidirectional and varies over the course of the disease. This makes it challenging to dissect whether such changes in the microbiome are initiating or driving factors in autoimmune liver diseases, secondary consequences of disease and/or pharmacological intervention, or alterations that modify the clinical course that patients experience. Potential mechanisms include the presence of pathobionts, disease-modifying microbial metabolites, and more nonspecific reduced gut barrier function, and it is highly likely that the effect of these changes is during the progression of the disease. Recurrent disease after liver transplantation is a major clinical challenge and a common denominator in these conditions, which could also represent a window to disease mechanisms of the gut-liver axis. Herein, we propose future research priorities, which should involve clinical trials, extensive molecular phenotyping at high resolution, and experimental studies in model systems. Overall, autoimmune liver diseases are characterized by an altered microbiome, and interventions targeting these changes hold promise for improving clinical care based on the emerging field of microbiota medicine.

Representatives from academia, industry, regulatory agencies, and patient advocacy groups convened under the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) in June 2022 with the primary goal of achieving consensus on chronic HBV and HDV treatment endpoints to guide clinical trials aiming to "cure" HBV and HDV. Conference participants reached an agreement on some key points. The preferred primary endpoint for phase II/III trials evaluating finite treatments for chronic hepatitis B (CHB) is a "functional" cure, defined as sustained HBsAg loss and HBV DNA less than the lower limit of quantitation (LLOQ) 24 weeks off-treatment. An alternate endpoint would be "partial cure" defined as sustained HBsAg level < 100 IU/mL and HBV DNA < LLOQ 24 weeks off-treatment. Clinical trials should initially focus on patients with HBeAg positive or negative CHB, who are treatment-naive or virally suppressed on nucleos(t)ide analogs. Hepatitis flares may occur during curative therapy and should be promptly investigated and outcomes reported. HBsAg loss would be the preferred endpoint for chronic hepatitis D, but HDV RNA < LLOQ 24 weeks off-treatment is a suitable alternate primary endpoint of phase II/III trials assessing finite strategies. For trials assessing maintenance therapy, the primary endpoint should be HDV RNA < LLOQ assessed at on-treatment week 48. An alternate endpoint would be ≥2 log reduction in HDV RNA combined with normalization of alanine aminotransferase level. Suitable candidates for phase II/III trials would be treatment-naiive or experienced patients with quantifiable HDV RNA. Novel biomarkers (hepatitis B core-related antigen [HBcrAg] and HBV RNA) remain exploratory, while nucleos(t)ide analogs and pegylated interferon still have a role in combination with novel agents. Importantly, patient input is encouraged early on in drug development under the FDA/EMA patient-focused drug development programs.

BACKGROUND AIMS: Chronic hepatitis E virus (HEV) infections remain a serious problem in immunocompromised patients, as specifically approved antiviral drugs are unavailable. In 2020, a 24-week multicenter phase II pilot trial was carried out, evaluating the nucleotide analog sofosbuvir by treating nine chronically HEV-infected patients with sofosbuvir [Trial Number NCT03282474]. During the study, antiviral therapy reduced virus RNA levels initially, but did not lead to a sustained virologic response (SVR). Here, we characterize the changes in HEV intra-host populations during sofosbuvir treatment to identify the emergence of treatment-associated variants. APPROACH RESULTS: We performed high-throughput sequencing on RNA-dependent RNA polymerase sequences to characterize viral population dynamics in study participants. Subsequently, we utilized an HEV-based reporter replicon system to investigate sofosbuvir sensitivity in high-frequency variants. Most patients had heterogenous HEV populations, suggesting high adaptability to treatment-related selection pressures. We identified numerous amino acid alterations emerging during treatment and found that the half-maximum effective concentration (EC50) of patient-derived replicon constructs was up to ~12-fold higher than the wild-type control, suggesting that variants associated with lower sensitivity were selected during sofosbuvir treatment. In particular, a single amino acid substitution (A1343V) in the finger domain of ORF1 could reduce susceptibility to sofosbuvir significantly in eight of nine patients. CONCLUSIONS: In conclusion, viral population dynamics played a critical role during antiviral treatment. High population diversity during sofosbuvir treatment led to the selection of variants (especially A1343V) with lower sensitivity to the drug, uncovering a novel mechanism of resistance-associated variants during sofosbuvir treatment.

Liver cancer, primarily hepatocellular carcinoma (HCC), exhibits highly heterogeneous histological and molecular aberrations across tumors and within individual tumor nodules. Such inter- and intra-tumor heterogeneity may lead to diversity in the natural history of disease progression and various clinical disparities across the patients. Recently developed multi-modality, single-cell, and spatial omics profiling technologies have enabled interrogation of the inter-/intra-tumor heterogeneity in the cancer cells as well as the tumor immune microenvironment. These features may influence the natural history and efficacy of emerging therapies targeting novel molecular and immune pathways, some of which had been deemed undruggable. Thus, comprehensive characterization of the heterogeneities at various levels may facilitate discovery of biomarkers that enable personalized and rational treatment decisions and optimize treatment efficacy while minimizing the risk of adverse effects. Such companion biomarkers will also refine HCC treatment algorithms across disease stages for cost-effective patient management by optimizing the allocation of limited medical resources. Despite this promise, the complexity of the inter-/intra-tumor heterogeneity and ever-expanding inventory of therapeutic agents and regimens have made clinical evaluation and translation of biomarkers increasingly challenging. To address this issue, novel clinical trial designs have been proposed and incorporated into recent studies. In this review, we discuss the latest findings in the molecular and immune landscape of HCC for their potential and utility as biomarkers, framework of evaluation and clinical application of predictive/prognostic biomarkers, and ongoing biomarker-guided therapeutic clinical trials. These new developments may revolutionize patient care and substantially impact the still dismal HCC mortality.

BACKGROUND AND AIMS: Liver fibrosis results from the accumulation of myofibroblasts (MFs) derived from quiescent HSCs, and yes-associated protein (YAP) controls this state transition. Although fibrosis is also influenced by HSC death and senescence, whether YAP regulates these processes and whether this could be leveraged to treat liver fibrosis are unknown. APPROACH AND RESULTS: YAP activity was manipulated in MF-HSCs to determine how YAP impacts susceptibility to pro-apoptotic senolytic agents or ferroptosis. Effects of senescence on YAP activity and susceptibility to apoptosis versus ferroptosis were also examined. CCl 4 -treated mice were treated with a ferroptosis inducer or pro-apoptotic senolytic to determine the effects on liver fibrosis. YAP was conditionally disrupted in MFs to determine how YAP activity in MF-HSC affects liver fibrosis in mouse models. Silencing YAP in cultured MF-HSCs induced HSC senescence and vulnerability to senolytics, and promoted ferroptosis resistance. Conversely, inducing HSC senescence suppressed YAP activity, increased sensitivity to senolytics, and decreased sensitivity to ferroptosis. Single-cell analysis of HSCs from fibrotic livers revealed heterogeneous sensitivity to ferroptosis, apoptosis, and senescence. In mice with chronic liver injury, neither the ferroptosis inducer nor senolytic improved fibrosis. However, selectively depleting YAP in MF-HSCs induced senescence and decreased liver injury and fibrosis. CONCLUSION: YAP determines whether MF-HSCs remain activated or become senescent. By regulating this state transition, Yap controls both HSC fibrogenic activity and susceptibility to distinct mechanisms for cell death. MF-HSC-specific YAP depletion induces senescence and protects injured livers from fibrosis. Clarifying determinants of HSC YAP activity may facilitate the development of novel anti-fibrotic therapies.

Comment in Hepatology. 2023 Jun 1;77(6):E176-E177.

Comment in Hepatology. 2023 Jun 1;77(6):E191.

Comment in Hepatology. 2023 Jun 1;77(6):E184-E185.

BACKGROUND AIMS: Immunotherapies have altered the treatment paradigm in hepatocellular carcinoma (HCC). Surrogate and modified endpoints are used to assess early success in clinical studies and to guide clinical practice. We sought to determine whether surrogate endpoints and modifications to the conventional criteria for tumor response (RECIST) including modified RECIST (mRECIST) and immune-modified RECIST (imRECIST) are valid measures to predict overall survival (OS) in HCC treated with immunotherapies. APPROACH RESULTS: We performed an individual-level post-hoc analysis of patients treated with atezolizumab and bevacizumab in the IMbrave150 trial (N = 279) and a cross-sectional analysis of a multi-center real-world patient cohort treated with immunotherapy (N = 328). Landmark analyses showed that objective response rates (ORR) by RECIST was associated with greater OS including among Child-Pugh A and B patients and among patients treated with immunotherapies in the 1st or 2nd line setting (IMbrave150: HR 0.24, 95% CI 0.17-0.33; RW: HR 0.25, 95% CI 0.15-0.43). ORR by mRECIST or imRECIST were not associated with greater predictive power of OS benefit (mRECIST: HR 0.30, 95% CI 0.22-0.42; imRECIST: HR 0.36, 95% CI 0.30-0.51). Progression-free survival determined by RECIST was only moderately correlated with OS and this association was not improved using mRECIST or imRECIST. CONCLUSIONS: Our results clarify the utility of surrogate and modified endpoints in HCC treated with immunotherapies, and support the use of RECIST ORR as an appropriate signal-finding measure for the evaluation of emerging treatments. Contrary to their intended purpose, mRECIST and imRECIST did not provide meaningful improvements in predicting OS benefit.

BACKGROUND AND AIMS: The Liver Imaging Reporting and Data System (LI-RADS) and European Association for the Study of the Liver (EASL) diagnostic criteria for noninvasive diagnosis of HCC can only be applied to patients at a high risk of HCC. This systematic review assesses adherence to the LI-RADS and EASL high-risk population criteria in published studies. APPROACH AND RESULTS: PubMed was searched for original research, published between January 2012 and December 2021, reporting LI-RADS and EASL diagnostic criteria on contrast-enhanced ultrasound, CT, or MRI. The algorithm version, publication year, risk status, and etiologies of chronic liver disease were recorded for each study. Adherence to high-risk population criteria was evaluated as optimal (unequivocal adherence), suboptimal (equivocal), or inadequate (clear violation). A total of 219 original studies were included, with 215 that used the LI-RADS criteria, 4 EASL only, and 15 that evaluated both LI-RADS and EASL criteria. Optimal, suboptimal, or inadequate adherence to high-risk population criteria was observed in 111/215 (51.6%), 86/215 (40.0%), and 18/215 (8.4%) LI-RADS and 6/19 (31.6%), 5/19 (26.3%), and 8/19 (42.1%) EASL studies ( p < 0.001) regardless of the imaging modality. Adherence to high-risk population criteria significantly improved according to the CT/MRI LI-RADS versions (optimal in v2018 in 64.5% of studies; v2017, 45.8%; v2014, 24.4%; v2013.1, 33.3%; p < 0.001) and the publication year (2020-2021, 62.5%; 2018-2019, 33.9%; 2014-2017, 39.3% of all LI-RADS studies; p = 0.002). No significant differences in adherence to high-risk population criteria were observed in the versions of contrast-enhanced ultrasound LI-RADS ( p = 0.388) or EASL ( p = 0.293). CONCLUSION: Adherence to high-risk population criteria was optimal or suboptimal in about 90% and 60% of LI-RADS and EASL studies, respectively.

Comment in Hepatology. 2023 Jun 1;77(6):E188-E189.

BACKGROUND AND AIMS: HDV infection leads to the most aggressive form of human viral hepatitis for which there is no FDA-approved therapy. PEG IFN-lambda-1a (Lambda) has previously demonstrated a good tolerability profile in HBV and HCV patients compared to PEG IFN-alfa. The goal of Phase 2 LIMT-1 trial was to evaluate the safety and efficacy of Lambda monotherapy in patients with HDV. APPROACH AND RESULTS: An open-label study of Lambda 120 or 180 mcg, administered once weekly by subcutaneous injections for 48 weeks, followed by 24 weeks of posttreatment follow-up. Thirty-three patients were allocated to Lambda 180 mcg (n=14) or 120 mcg (n=19). Baseline mean values: HDV RNA 4.1 log10 IU/mL (SD±1.4); ALT 106 IU/L (35-364); and bilirubin 0.5 mg/dL (0.2-1.2). Intention-to-treat rates of virologic response to Lambda 180 mcg and 120 mcg, 24 weeks following treatment cessation were 5 of 14(36%) and 3 of 19 (16%), respectively. The posttreatment response rate of 50% was seen in low BL viral load (≤4 log10) on 180 mcg. Common on-treatment adverse events included flu-like symptoms and elevated transaminase levels. Eight (24%) cases of hyperbilirubinemia with or without liver enzyme elevation, leading to drug discontinuation, were mainly observed in the Pakistani cohort. The clinical course was uneventful, and all responded favorably to dose reduction or discontinuation. CONCLUSIONS: Treatment with Lambda in patients with chronic HDV may result in virologic response during and following treatment cessation. Clinical phase 3 development of Lambda for this rare and serious disease is ongoing.

BACKGROUND AND AIMS: Patients with compensated cirrhosis with clinically significant portal hypertension (CSPH: HVPG > 10 mm Hg) have a high risk of decompensation. HVPG is, however, an invasive procedure not available in all centers. The present study aims to assess whether metabolomics can improve the capacity of clinical models in predicting clinical outcomes in these compensated patients. APPROACH AND RESULTS: This is a nested study from the PREDESCI cohort (an RCT of nonselective beta-blockers vs. placebo in 201 patients with compensated cirrhosis and CSPH), including 167 patients for whom a blood sample was collected. A targeted metabolomic serum analysis, using ultra-high-performance liquid chromatography-mass spectrometry, was performed. Metabolites underwent univariate time-to-event cox regression analysis. Top-ranked metabolites were selected using Log-Rank p -value to generate a stepwise cox model. Comparison between models was done using DeLong test. Eighty-two patients with CSPH were randomized to nonselective beta-blockers and 85 to placebo. Thirty-three patients developed the main endpoint (decompensation/liver-related death). The model, including HVPG, Child-Pugh, and treatment received ( HVPG/Clinical model ), had a C-index of 0.748 (CI95% 0.664-0.827). The addition of 2 metabolites, ceramide (d18:1/22:0) and methionine (HVPG/Clinical/Metabolite model), significantly improved the model's performance [C-index of 0.808 (CI95% 0.735-0.882); p =0.032]. The combination of these 2 metabolites together with Child-Pugh and the type of treatment received (Clinical/Metabolite model) had a C-index of 0.785 (CI95% 0.710-0.860), not significantly different from the HVPG-based models including or not metabolites. CONCLUSIONS: In patients with compensated cirrhosis and CSPH, metabolomics improves the capacity of clinical models and achieves similar predictive capacity than models including HVPG.

BACKGROUND AND AIMS: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. APPROACH AND RESULTS: In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-β1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-β1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. CONCLUSIONS: Modulating the SCT/SR axis may be critical for managing PSC.