Over the past years, there has been a remarkable advance in the systemic treatment options for advanced HCC. The overall survival has gradually increased over time, with larger benefits for patients with sensitive tumors and preserved liver function, the latter being an essential condition for the delivery of sequential lines of treatment and optimization of clinical outcomes. With the approval of new first-line agents and the introduction of immune checkpoint inhibitor-based therapies, the treatment landscape of advanced HCC is becoming wider than ever. Atezolizumab plus bevacizumab and, more recently, durvalumab plus tremelimumab have entered the clinical practice and are the current standard of care for treatment-naïve patients, surpassing sorafenib and lenvatinib monopoly. As no head-to-head comparisons are available among all the first-line treatment options, the recommendation for the most appropriate choice and sequence is patient-driven and integrates efficacy data with clinical comorbidities, background liver disease, and the safety profile of available drugs. In addition, predictive biomarkers for successful patients' stratification are yet to be available and constitute the focus of ongoing research. The treatment algorithm is likely to become even more complex since systemic therapeutic approaches are now being translated into earlier stages of the disease, with an impact on the evolution of the sequential treatment of HCC patients.

BACKGROUND AND AIMS: Immune cells play a crucial role in liver aging. However, the impact of dynamic changes in the local immune microenvironment on age-related liver injury remains poorly understood. We aimed to characterize intrahepatic immune cells at different ages to investigate key mechanisms associated with liver aging. APPROACH AND RESULTS: We carried out single-cell RNA sequencing (scRNA-seq) on mouse liver tissues at four different ages, namely, the newborn, suckling, young, and aged stages. The transcriptomic landscape, cellular classification, and intercellular communication were analyzed. We confirmed the findings by multiplex immunofluorescence staining, flow cytometry, in vitro functional experiments, and chimeric animal models. Nine subsets of 89,542 immune cells with unique properties were identified, of which Cxcl2+ macrophages within the monocyte/macrophage subset were preferentially enriched in the aged liver. Cxcl2+ macrophages presented a senescence-associated secretory phenotype (SASP) and recruited neutrophils to the aged liver through the CXCL2-CXCR2 axis. Through the secretion of IL-1β (interleukin-1 beta) and TNF-α (tumor necrosis factor alpha), Cxcl2+ macrophages stimulated neutrophil extracellular traps (NETs) formation. Targeting the CXCL2-CXCR2 axis limited the neutrophils migration toward the liver and attenuated age-related liver injury. Moreover, the relationship between Cxcl2+ macrophages and neutrophils in age-related liver injury was further validated by human liver transplantation samples. CONCLUSIONS: This in-depth study illustrates that the mechanism of Cxcl2+ macrophage-driven neutrophil activation involves the CXCL2-CXCR2 axis, and provide a potential therapeutic strategy for age-related liver injury.

Clinical trials have been a central driver of change and have provided the evidence base necessary to advance new therapies for liver diseases. This review provides a perspective on the status of trials in hepatology and a vantage point into the emerging capabilities and external forces that will shape the conduct of clinical trials in the future. The adaptations to clinical trial operations in response to the disruptions by the COVID-19 pandemic and opportunities for innovation in hepatology trials are emphasized. Future trials in hepatology will be driven by unmet therapeutic needs and fueled by technological advances incorporating digital capabilities with expanded participant-derived data collection, computing, and analytics. Their design will embrace innovative trial designs adapted to these advances and that emphasize broader and more inclusive participant engagement. Their conduct will be further shaped by evolving regulatory needs and the emergence of new stakeholders in the clinical trials ecosystem. The evolution of clinical trials will offer unique opportunities to advance new therapeutics that will ultimately improve the lives of patients with liver diseases.

Hepatitis B (HBV) is a major cause of global morbidity and mortality, and the leading cause of liver cancer worldwide. Significant advances have recently been made toward the development of a finite HBV treatment that achieves permanent loss of HBsAg and HBV DNA (so-called "HBV cure"), which could provide the means to eliminate HBV as a public health threat. However, the HBV cure is just one step toward achieving WHO HBV elimination targets by 2030, and much work must be done now to prepare for the successful implementation of the HBV cure. In this review, we describe the required steps to rapidly scale-up future HBV cure equitably. We present key actions required for successful HBV cure implementation, integrated within the World Health Organization (WHO) Global Health Sector Strategy (GHSS) 2022-2030 framework. Finally, we highlight what can be done now to progress toward the 2030 HBV elimination targets using available tools to ensure that we are preparing, but not waiting, for the cure.

Comment in Hepatology. 2023 Sep 1;78(3):E50-E51.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is an acute liver and multisystem failure in patients with previously stable cirrhosis. A common cause of ACLF is sepsis secondary to bacterial infection. Sepsis-associated ACLF involves a loss of differentiated liver function in the absence of direct liver injury, and its mechanism is unknown. We aimed to study the mechanism of sepsis-associated ACLF using a novel mouse model. APPROACH AND RESULTS: Sepsis-associated ACLF was induced by cecal ligation and puncture procedure (CLP) in mice treated with thioacetamide (TAA). The combination of TAA and CLP resulted in a significant decrease in liver synthetic function and high mortality. These changes were associated with reduced metabolic gene expression and increased CCAAT enhancer binding protein beta (C/EBPβ) transcriptional activity. We found that C/EBPβ binding to its target gene promoters was increased. In humans, C/EBPβ chromatin binding was similarly increased in the ACLF group compared with control cirrhosis. Hepatocyte-specific Cebpb knockout mice had reduced mortality and increased gene expression of hepatocyte differentiation markers in TAA/CLP mice, suggesting that C/EBPβ promotes liver failure in these mice. C/EBPβ activation was associated with endothelial dysfunction, characterized by reduced Angiopoietin-1/Angiopoietin-2 ratio and increased endothelial production of HGF. Angiopoietin-1 supplementation or Hgf knockdown reduced hepatocyte C/EBPβ accumulation, restored liver function, and reduced mortality, suggesting that endothelial dysfunction induced by sepsis drives ACLF through HGF-C/EBPβ pathway. CONCLUSIONS: The transcription factor C/EBPβ is activated in both mouse and human ACLF and is a potential therapeutic target to prevent liver failure in patients with sepsis and cirrhosis.

BACKGROUND AND AIMS: Hyperlipidemia has been extensively recognized as a high-risk factor for non-alcoholic steatohepatitis (NASH); however, clinical susceptibility to NASH is highly heterogeneous. The key controller(s) of NASH susceptibility in patients with hyperlipidemia have not yet been elucidated. Here, we aimed to reveal the key regulators of NASH in patients with hyperlipidemia and to explore its role and underlying mechanisms. APPROACH AND RESULTS: To identify the predominant suppressors of NASH in the setting of hyperlipidemia, we collected liver biopsy samples from patients with hyperlipidemia, with or without NASH, and performed RNA-seq analysis. Notably, decreased LIM domain only 7 (LMO7) expression robustly correlated with the occurrence and severity of NASH. Although overexpression of LMO7 effectively blocked hepatic lipid accumulation and inflammation, LMO7 deficiency in hepatocytes greatly exacerbated diet-induced NASH progression. Mechanistically, lysine 48 (K48)-linked ubiquitin-mediated proteasomal degradation of tripartite motif 47 (TRIM47) and subsequent inactivation of the JNK/p38 MAPK cascade are required for the protective function of LMO7 in NASH. CONCLUSIONS: These findings provide proof-of-concept evidence supporting LMO7 as a robust suppressor of NASH in the context of hyperlipidemia, indicating that targeting the LMO7-TRIM47 axis is a promising therapeutic strategy for NASH.

Comment in Hepatology. 2023 Sep 1;78(3):E61-E62.

BACKGROUND AND AIMS: Human innate lymphoid cells (ILCs) are critically involved in the modulation of homeostatic and inflammatory processes in various tissues. However, only little is known about the composition of the intrahepatic ILC pool and its potential role in chronic liver disease. Here, we performed a detailed characterization of intrahepatic ILCs in both healthy and fibrotic livers. APPROACH AND RESULTS: A total of 50 livers (nonfibrotic = 22, and fibrotic = 29) were analyzed and compared with colon and tonsil tissue (each N = 14) and peripheral blood (N = 32). Human intrahepatic ILCs were characterized ex vivo and on stimulation using flow cytometry and single-cell RNA sequencing. ILC differentiation and plasticity were analyzed by both bulk and clonal expansion experiments. Finally, the effects of ILC-derived cytokines on primary human HSteCs were studied. Unexpectedly, we found that an "unconventional" ILC3-like cell represented the major IL-13-producing liver ILC subset. IL-13 + ILC3-like cells were specifically enriched in the human liver, and increased frequencies of this cell type were found in fibrotic livers. ILC3-derived IL-13 production induced upregulation of proinflammatory genes in HSteCs, indicating a potential role in the regulation of hepatic fibrogenesis. Finally, we identified KLRG1-expressing ILC precursors as the potential progenitor of hepatic IL-13 + ILC3-like cells. CONCLUSIONS: We identified a formerly undescribed subset of IL-13-producing ILC3-like cells that is enriched in the human liver and may be involved in the modulation of chronic liver disease.

Pegylated-Interferon alpha (PegIFN-α) is a treatment option for chronic hepatitis B (CHB) but has limited efficacy. It has both immune modulatory and antiviral capacity, however the mechanisms of PegIFN-α in relation to CHB pathogenesis remain understudied. The current editorial reviews a recent study related to the in-vivo use of PegIFN-α using single-cell RNA sequencing (scRNA-seq) technology. PegIFN-α reverts the transcriptome profile of treated samples close to that of the healthy population, diminishing the pro-inflammatory genes and 'inflammatory scores' which will be important for the HBV-cure program.

Comment in Hepatology. 2023 Sep 1;78(3):E58.

Comment in Hepatology. 2023 Sep 1;78(3):E53-E54.

BACKGROUND AND AIMS: Early allograft dysfunction (EAD) is a severe event leading to graft failure after liver transplant (LT). Extracellular high-mobility group box-1 (HMGB1) is a damage-associated molecular pattern that contributes to hepatic ischemia-reperfusion injury (IRI). However, the contribution of intracellular HMGB1 to LT graft injury remains elusive. We hypothesized that intracellular neutrophil-derived HMGB1 from recipients protects from post-LT EAD. APPROACH AND RESULTS: We generated mice with conditional ablation or overexpression of Hmgb1 in hepatocytes, myeloid cells, or both. We performed LTs and injected lipopolysaccharide (LPS) to evaluate the effect of intracellular HMGB1 in EAD. Ablation of Hmgb1 in hepatocytes and myeloid cells of donors and recipients exacerbated early allograft injury after LT. Ablation of Hmgb1 from liver grafts did not affect graft injury; however, lack of Hmgb1 from recipient myeloid cells increased reactive oxygen species (ROS) and inflammation in liver grafts and exacerbated injury. Neutrophils lacking HMGB1 were more activated, showed enhanced pro-oxidant and pro-inflammatory signatures, and reduced biosynthesis and metabolism of inositol polyphosphates (InsPs). On LT reperfusion or LPS treatment, there was significant neutrophil mobilization and infiltration into the liver and enhanced production of ROS and pro-inflammatory cytokines when intracellular Hmgb1 was absent. Depletion of neutrophils using anti-Ly6G antibody attenuated graft injury in recipients with myeloid cell Hmgb1 ablation. CONCLUSIONS: Neutrophil HMGB1 derived from recipients is central to regulate their activation, limits the production of ROS and pro-inflammatory cytokines, and protects from early liver allograft injury.

BACKGROUND AND AIMS: Biliary tract cancers are rare, heterogeneous cancers with poor prognoses. Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-βRII (a TGF-β "trap") fused to a human IgG1 monoclonal antibody blocking programmed death ligand 1, was evaluated in patients with locally advanced/metastatic chemorefractory biliary tract cancers. APPROACH AND RESULTS: This multicenter, single-arm, open-label, phase 2 study (NCT03833661) enrolled adults with locally advanced or metastatic biliary tract cancer that was intolerant to or had failed first-line systemic platinum-based chemotherapy. Patients received 1200 mg bintrafusp alfa intravenously Q2W. The primary endpoint was confirmed objective response according to Response Evaluation Criteria in Solid Tumors 1.1 assessed by IRC. Secondary endpoints included duration of response, durable response rate, safety, progression-free survival, and overall survival.Between March 2019 and January 2020, 159 patients were enrolled. Median follow-up was 16.1 (range, 0.0-19.3) months; 17 patients (10.7%; 95% CI: 6.4%-16.6%) achieved an objective response. Median duration of response was 10.0 (range, 1.9-15.7) months; 10 patients (6.3%; 95% CI: 3.1%-11.3%) had a durable response (≥6 mo). Median progression-free survival was 1.8 months (95% CI: 1.7-1.8 mo); median overall survival was 7.6 months (95% CI: 5.8-9.7 mo). Overall survival rates were 57.9% (6 mo) and 38.8% (12 mo). Grade ≥3 adverse events occurred in 26.4% of patients, including one treatment-related death (hepatic failure). Frequent grade ≥3 adverse events included anemia (3.8%), pruritus (1.9%), and increased alanine aminotransferase (1.9%). CONCLUSIONS: Although this study did not meet its prespecified primary endpoint, bintrafusp alfa demonstrated clinical activity as second-line treatment in this hard-to-treat cancer, with durable responses and a manageable safety profile.

BACKGROUND AIMS: Cholangiocarcinoma(CCA) is a highly lethal malignancy originating from the biliary ducts. Current CCA diagnostic and prognostic assessments cannot satisfy the clinical requirement. Bile detection is rarely performed and herein we aim to estimate the clinical significance of bile liquid biopsy by assessing bile exosomal concentrations and components. APPROACH RESULTS: Exosomes in bile and sera from CCA, pancreatic cancer and common bile duct stone(CBDS) were identified and quantified by TEM, NTA and nanoFCM. Exosomal components were assessed by LC-MS/MS and miRNA-seq. Bile exosomal concentration in different diseases had no significant difference, but miR-182-5p and miR-183-5p were ectopically up-regulated in CCA bile exosomes. High miR-182/183-5p in both CCA tissues and bile indicates poor prognosis. Bile exosomal miR-182/183-5p is secreted by CCA cells, and can be absorbed by biliary epithelium or CCA cells. With xenografts in humanized mice, we showed that bile exosomal miR-182/183-5p promotes CCA proliferation, invasion and EMT by targeting HPGD in CCA cells and mast cells(MCs) and increasing PGE2 generation, which stimulates PTGER1 and increases CCA stemness. In scRNA-seq, HPGD is predominantly expressed in MCs. miR-182/183-5p prompts MC to release VEGF-A release from MC by increasing VEGF-A expression, which facilitates angiogenesis. CONCLUSIONS: CCA cells secret exosomal miR-182/183-5p into bile, which targets HPGD in CCA cells and MCs and increases PGE2 and VEGF-A release. PGE2 promotes stemness by activating PTGER1. Our results reveal a type of CCA self-driven progression dependent on bile exosomal miR-182/183-5p and MCs, which is a new interplay pattern of CCA and bile.

Wnt-β-catenin signaling has emerged as an important regulatory pathway in liver, playing key roles in zonation and mediating contextual hepatobiliary repair after injuries. In this review, we will address the major advances in understanding the role of Wnt signaling in hepatic zonation, regeneration, and cholestasis-induced injury. We will also touch upon some important unanswered questions, and discuss the relevance of modulating the pathway to provide therapies for complex liver pathologies that remain a continued unmet clinical need.

BACKGROUND AIMS: Lipid accumulation induced by alcohol consumption is not only an early pathophysiological response but also a prerequisite for the progression of alcohol-associated liver disease (ALD). Alternative splicing regulates gene expression and protein diversity; dysregulation of this process is implicated in human liver diseases. However, how the alternative splicing regulation of lipid metabolism contributes to the pathogenesis of ALD remains undefined. APPROACH RESULTS: Serine-arginine-rich protein kinase 2 (SRPK2), a key kinase controlling alternative splicing, is activated in hepatocytes in response to alcohol, in mice with chronic-plus-binge alcohol feeding, and in patients with ALD. Such induction activates sterol regulatory element binding transcription factor 1 (SREBP-1) and promotes lipogenesis in ALD. Overexpression of fibroblast growth factor 21 (FGF21) in transgenic mice abolishes alcohol-mediated induction of SRPK2 and its associated steatosis, lipotoxicity, and inflammation; these alcohol-induced pathologies are exacerbated in FGF21 knockout mice. Mechanistically, SRPK2 is required for alcohol-mediated impairment of serine-arginine splicing factor 10 (SRSF10), which generates exon 7 inclusion in lipin 1, and triggers concurrent induction of lipogenic regulators-lipin 1β and SREBP-1. FGF21 suppresses alcohol-induced SRPK2 accumulation through mTORC1 inhibition-dependent degradation of SRPK2. Silencing SRPK2 rescues alcohol-induced splicing dysregulation and liver injury in FGF21 knockout mice. CONCLUSIONS: These studies reveal that 1) The regulation of alternative splicing by SRPK2 is implicated in lipogenesis in humans with ALD; 2) FGF21 is a key hepatokine that ameliorates ALD pathologies largely by inhibiting SRPK2; and 3) Targeting SRPK2 signaling by FGF21 may offer potential therapeutic approaches to combat ALD.

There is great interest in identifying microbiome features as reliable noninvasive diagnostic and/or prognostic biomarkers for non-cirrhotic NASH fibrosis. Several cross-sectional studies have reported gut microbiome features associated with advanced NASH fibrosis and cirrhosis, where the most prominent features are associated with cirrhosis. However, no large, prospectively collected data exist establishing microbiome features that discern non-cirrhotic NASH fibrosis, integrate the fecal metabolome as disease biomarkers, and are unconfounded by BMI and age. Results from shotgun metagenomic sequencing performed on fecal samples prospectively collected from 279 US patients with biopsy-proven NASH (F1-F3 fibrosis) enrolled in the REGENERATE I303 study were compared to those from three healthy control cohorts and integrated with the absolute quantification of fecal bile acids. Microbiota beta-diversity was different, and BMI- and age-adjusted logistic regression identified 12 NASH-associated species. Random forest prediction models resulted in an AUC of 0.75 to 0.81 in a receiver operator characteristic analysis. Additionally, specific fecal bile acids were significantly lower in NASH and correlated with plasma C4 levels. Microbial gene abundance analysis revealed 127 genes increased in controls, many involving protein synthesis, whereas 362 genes were increased in NASH many involving bacterial environmental responses (FDR < 0.01). Finally, we provide evidence that fecal bile acid levels may be a better discriminator of non-cirrhotic NASH versus health than either plasma bile acids or gut microbiome features. These results may have value as a set of baseline characteristics of non-cirrhotic NASH against which therapeutic interventions to prevent cirrhosis can be compared and microbiome-based diagnostic biomarkers identified.

BACKGROUND AIMS: The Patient-Reported Outcomes Measurement Information System (PROMIS) is increasingly used to measure health-related quality of life (HRQOL), yet, it has not been well-studied in chronic liver disease (CLD). This study compares PROMIS Profile-29 to Short-Form Health Survey (SF-36) and Chronic Liver Disease Questionnaire (CLDQ) in patients with CLD. APPROACH RESULTS: 204 adult outpatients with CLD completed PROMIS-29, CLDQ, SF-36 and usability questionnaires. Mean scores were compared between groups, correlation between domain scores were assessed and floor/ceiling effects were calculated. Etiologies of CLD were NAFLD (44%), hepatitis C (16%) and alcohol (16%). 53% had cirrhosis and 33% were Child-Pugh B/C with a mean Model for End-stage Liver Disease Score of 12.0. In all 3 tools, the poorest scores were in physical function and fatigue. Presence of cirrhosis or complications was associated with worse scores in most PROMIS Profile-29 domains indicating known groups validity. Strong correlations (r ≥ 0.7) were present between Profile-29 and SF-36 or CLDQ domains measuring similar concepts, indicating strong convergent validity. Profile-29 was completed faster than SF-36 and CLDQ (5.4 ± 3.0, 6.7 ± 3.3, 6.5 ± 5.2 min, p = 0.003) and rated equally on usability. All CLDQ and SF-36 domains reached the floor or ceiling while none were noted for Profile-29. These floor/ceiling effects were magnified when assessed in those with and without cirrhosis indicating improved depth of measurement by Profile-29. CONCLUSIONS: Profile-29 is a valid, more efficient, well-received tool that provides improved depth of measurement when compared to SF-36 and CLDQ and, therefore, an ideal tool to measure general HRQOL in CLD.

Progression of chronic liver injury to fibrosis, abnormal liver regeneration and hepatocellular carcinoma (HCC) are driven by a dysregulated dialog between epithelial cells with their micro-environment, in particular immune, fibroblasts and endothelial cells. There is currently no antifibrogenic therapy and drug treatment of HCC is limited to tyrosine kinase inhibitors and immunotherapy targeting the tumor microenvironment. Metabolic reprogramming of epithelial and non-parenchymal cells is critical at each stage of disease progression, suggesting that targeting specific metabolic pathways could constitute an interesting therapeutic approach. In this review, we will discuss how modulating intrinsic metabolism of key effector liver cells might disrupt the pathogenic sequence from chronic liver injury to fibrosis/cirrhosis, regeneration and HCC.