Seminars in Liver Disease

Background: Liver fibrosis screening is recommended in at-risk groups, but a clear definition of “at risk” for entry criteria is lacking. We analyzed different combinations of established risk factors to define specific screening entry criteria with a prespecified sensitivity requirement. Methods: Data regarding individuals aged 40–70 years from Finnish health-examination surveys (FINRISK 2002–2012 and Health 2000, n=15,057) and the UK Biobank (n=454,990) were linked with healthcare registries for liver cirrhosis-related events (LREs; liver-related hospitalizations, cancer, or death). The predictive performance of 1919 combinations of risk factors, including alcohol consumption, metabolic disturbances, abnormal liver function tests, and Chronic Liver Disease risk score, was assessed for 10-year LRE risk requiring a minimum 90% sensitivity. Validations were performed using liver stiffness measurement (LSM) >12 kPa in the NHANES 2017–2020 sample (n=3367). Results: Optimal entry criteria for predicting 10-year LRE risk with >90% sensitivity included any one of: hazardous alcohol use, severe obesity, metabolic syndrome, an AST-to-ALT ratio >0.8 with elevated ALT, and an intermediate-to-high Chronic Liver Disease risk score. The sensitivity and specificity for this strategy were 91% and 51% for LREs, respectively, in the Finnish cohort, and 91% and 41% for LSM >12 kPa in the US sample. In the US sample, applying these entry criteria followed by fibrosis-4 ≥1.3 for predicting LSM >12 kPa reduced the sensitivity to 45% (specificity: 85%), which was attributed to the suboptimal sensitivity of fibrosis-4. Conclusions: This study identifies an inexpensive risk factor-based strategy with >90% sensitivity for predicting LRE and LSM >12 kPa, which is practical and scalable for targeted liver fibrosis screening to improve population outcomes. However, a more sensitive first-line noninvasive fibrosis test is needed.

HCC is the fourth leading cause of cancer-related mortality with increasing incidence worldwide. Historically, treatment for early disease includes liver transplantation, surgical resection, and/or other local therapies, such as thermal ablation. As a result of technical advances and high-quality prospective data, the use of definitive external beam radiotherapy with ablative doses has emerged. Intermediate-stage disease has been generally addressed with arterially directed therapies (eg, chemoembolization or radioembolization) and external beam radiotherapy, while advanced stages have been addressed by systemic therapy or best supportive care. The role of each local/locoregional therapy has rapidly evolved in the context of novel pharmacotherapies, including immunotherapies and antiangiogenic agents. The combinations, indications, and timing of treatments vary widely among specialties and geographies. Here, we aim to synthesize the best quality evidence available regarding the efficacy and safety of different liver-directed modalities, with a focus on recent prospective clinical data of external beam radiotherapy within the context of other available liver-directed therapies across Barcelona Liver Classification (BCLC) stages.

Background: Fibrosis drives liver-related mortality in metabolic dysfunction–associated steatohepatitis (MASH), yet we have limited medical therapies to target MASH-fibrosis progression. Here we report that mannose, a simple sugar, attenuates MASH steatosis and fibrosis in 2 robust murine models and human liver slices. Methods: The well-validated fat-and-tumor MASH murine model for liver steatosis and fibrosis was employed. Mannose was supplied in the drinking water at the start (“Prevention” group) or at week 6 of the 12-week MASH regimen (“Therapy” group). The in vivo antifibrotic effects of mannose supplementation were tested in a second model of carbon tetrachloride (CCl4)-induced liver fibrosis. A quantitative and automated digital pathology approach was used to comprehensively assess steatosis and fibrosis phenotypes. Mannose was also tested in vitro in human and primary mouse hepatocytes conditioned with free fatty acids alone or with fructose, and human precision-cut liver slices from patients with end-stage MASH cirrhosis. Results: Oral mannose supplementation improved liver fibrosis in vivo in both fat-and-tumor MASH and CCl4 mouse models, as well as in human precision-cut liver slice MASH samples. Mannose also reduced liver steatosis in fat-and-tumor MASH mice, and in human and mouse hepatocytes in vitro. Ketohexokinase, the main enzyme in fructolysis, was decreased with mannose in whole mouse liver, cultured hepatocytes, and human precision-cut liver slices. Removal of fructose or overexpression of ketohexokinase each abrogated the antisteatotic effects of mannose. Conclusions: This study identifies mannose as a novel therapeutic candidate for MASH that mitigates steatosis by dampening hepatocyte ketohexokinase expression and exerts independent antifibrotic effects in 2 mouse models and human liver tissue slices.

Background: To identify clinical characteristics and serological biomarkers that predicted subsequent nosocomial infection in ATTIRE trial patients. Methods: We identified 360 patients at hospitalization without infection and not prescribed antibiotics and compared clinical characteristics between those who subsequently developed a nosocomial infection and not. In a 68-patient subcohort, we compared plasma biomarkers of bacterial translocation, infection, and inflammation at hospitalization between those who developed a nosocomial infection and not. In a 56-patient subcohort, we investigated plasma lipidomic profiles in those who did and did not develop nosocomial infection using Lipotype Shotgun platform analysis and multivariate statistical techniques. To further investigate lipid pathways, we compared outcomes in patients taking statins or not at hospitalization. Results: Serum bilirubin >188 µmol/L at hospitalization predicted subsequent nosocomial infection in univariate and multivariate analyses, with 80% specificity. The most common nosocomial infections were respiratory tract (29%) and those developing infection had significantly greater 28 and 90-day mortality than those not (p=9.34E−05 and 0.014). Serological biomarkers of bacterial translocation, infection, and inflammation did not predict subsequent infection. Partial least squares discriminatory analyses identified cholesterol esters (CEs) (CE.18.1.2, CE.18.1.0, and CE.16.0.0) as important predictors of infection but provided only a small improvement in predictive ability over bilirubin alone. RNA-sequencing analyses suggest this is mediated by a downregulation of the cellular cholesterol esterification enzyme sterol O-acyltransferase 1. Statin use was not associated with nosocomial infection prevention. Conclusions: In ATTIRE, elevated serum bilirubin at hospitalization was the only clinical characteristic that predicted subsequent development of nosocomial infection. Considering the rising incidence of antimicrobial resistance, these data could be used to limit antibiotic prophylaxis or aid trial design for investigating use in high-risk patients.

Background: The absence of representative Epstein-Barr virus–associated intrahepatic cholangiocarcinoma (EBVaICC) cell lines has limited our understanding of the molecular and immunological characteristics of this cancer subtype. Methods: We reviewed patients with metastatic cholangiocarcinoma at Sun Yat-sen University Cancer Center from January 2015 to August 2023. Among them, 22 patients with EBVaICC and 66 patients with non-EBVaICC who received anti-PD1 treatment were included. Additionally, 2 EBV-positive ICC cell lines, RBE-EBV and HuH28-EBV, were developed through cell-to-cell infection. Stable EBV infection and responsiveness to viral reactivation were confirmed. Transcriptomic and bioinformatics analyses were performed, and in vitro experiments examined the immune effects of EBV-positive ICC. Key immune-related genes and cytokines were validated by reverse transcription quantitative polymerase chain reaction and ELISA in cell lines and patient plasma samples. Results: In this study, we found that patients with EBVaICC showed enhanced immune responses and improved overall and progression-free survival compared to patients with non-EBVaICC. We first successfully established and validated 2 EBV-positive ICC cell lines (RBE-EBV and HuH28-EBV). These cell lines were confirmed for stable EBV infection and displayed responsiveness to viral reactivation, making them suitable for future studies. Transcriptomic analyses and in vitro studies revealed that EBV activated the cGAS-STING pathway, resulting in MHC-I upregulation and CXCL10 secretion in ICC cells, which collectively enhanced CD8+ T cell chemotaxis and cytotoxicity. Furthermore, ELISA analysis showed higher plasma levels of CXCL10 and IFN-γ in patients with EBVaICC, suggesting a potential role for EBV in enhancing immunotherapy sensitivity in this subtype. Conclusions: The established EBV-positive ICC cell lines revealed enhanced immunogenicity driven by cGAS-STING pathway activation, providing valuable models for future research and insights into the mechanisms of improved immunotherapy sensitivity in EBVaICC.

Background: Autophagic and endosomal pathways coordinately contribute to HBV virions and subviral particles (SVPs) production. To date, limited evidence supports that HBV and exosomes have a common pathway for their biogenesis and secretion. The final steps of HBV production and release have not yet been well studied. Methods: We examined the production and release of HBV virions and SVPs by using GW4869 (N,N’-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3’-pht hal amide dihydrochloride), a small molecule inhibiting ceramide-mediated inward membrane budding. Neutral sphingomyelinase, the target of GW4869, and RAB27A and –B,  2 small GTPases involved in exosome release control, were silenced using gene silencing to confirm the results obtained. Western blot, immunofluorescence staining, and confocal microscopy were applied. Results: GW4869 inhibited HBV virion release, causing their accumulation along with SVPs in hepatocytes. This triggered cellular endoplasmic reticulum stress, leading to protein kinase B-mechanistic target of rapamycin kinase signaling pathway inactivation. GW4869 treatment increased autophagosome formation and impaired autophagic degradation by blocking autophagosome-lysosome fusion. Consequently, HBsAg is increasingly localized to autophagosomes and late endosomes/multivesicular bodies. Silencing neutral sphingomyelinase yielded consistent results. Similarly, RAB27A silencing inhibited HBV virion and SVP secretion, causing their accumulation within hepatoma cells. Notably, GW4869 treatment, as well as RAB27A and -B silencing, increased the presence of LC3+CD63+HBsAg+ complexes. Conclusions: Our results demonstrate the involvement of the autophagosome-late endosome/multivesicular bodies-exosome axis in regulating HBV production and release, highlighting amphisomes as a potential platform for HBV release.

Background: Postoperative liver failure due to insufficient liver cell quantity and function remains a major cause of mortality following surgery. Hence, additional investigation and elucidation are required concerning suitable surgeries for promoting in vivo regeneration. Methods: We established the portal vein ligation (PVL) and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) mouse models to compare their in vivo regeneration capacity. Then, RNA-seq and microRNA-seq were conducted on the livers from both mouse models. Weighted gene co-expression network analysis algorithm was leveraged to identify crucial gene modules. ScRNA-seq analysis was used to understand the distinctions between Signature30high hepatocytes and Signature30low hepatocytes. Moreover, in vivo, validation was performed in fumarylacetoacetate hydrolase knockout mice with gene editing using the CRISPR-cas9 system. A dual luciferase report system was carried out to further identify the regulatory mechanisms. Results: RNA-seq analysis revealed that ALPPS could better promote cell proliferation compared to the sham and portal vein ligation models. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state. ScRNA-seq analysis confirmed that signature30high hepatocytes had stronger proliferative ability than signature30low hepatocytes. Using microRNA-seq analysis, we identified 53 microRNAs that were time-dependently reduced after ALPPS. Finally, miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS. Conclusions: ALPPS could successfully promote liver regeneration by activating hepatocytes into a proliferative state. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state of hepatocytes. miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS.

Background: The incidence of alcohol-associated hepatitis (AH) is rising in women of reproductive age. While the adverse effects of alcohol on pregnancy are well documented, there is limited data on pregnancy in women with a history of AH. Methods: This study was completed by using the TriNetX Research Network. The primary objectives were to evaluate the incidence of pregnancy and related complications in pregnancies following an episode of AH (AH pregnancies) compared to pregnancies in healthy patients (control pregnancies). The secondary objective was to assess long-term liver-related complications and mortality in women with AH who experienced a pregnancy compared to no pregnancy. Propensity score matching was used for comparative analyses to balance cohorts by age, race, ethnicity, prior delivery, and obesity status. Results: The incidence of pregnancy was significantly lower in women with AH compared to controls (26 vs. 54 cases per 1000 person-years, p<0.001). AH pregnancies were associated with higher odds of spontaneous abortion (OR 2.0, 95% CI: 1.2 to 3.3, p =0.011), pre-eclampsia (OR 1.9, 95% CI: 1.1 to 3.0, p =0.002), peri-partum hemorrhage (OR 2.7, 95% CI: 1.3 to 5.6, p =0.007) and perinatal psychiatric disorders (OR 3.2, 95% CI: 1.6 to 6.2, p =0.001). The incidence of cirrhosis and hepatic decompensation were similar between women with AH who experienced a pregnancy compared to no pregnancy, but Kaplan Meier analysis revealed a significantly faster time to event in the no-pregnancy group. Conclusions: Pregnancies following AH diagnosis were associated with adverse pregnancy outcomes. Pregnancy after AH does not reduce the overall risk of developing advanced liver disease but may delay disease progression. These findings highlight the importance of tailored reproductive counseling and support for this population.

Background: Glutamine (Gln) is a critical amino acid for energy expenditure. It participates in extracellular matrix (ECM) formation and circulates in the hepatic parenchyma in a spatial-oriented manner. Posthepatectomy liver mass recovery poses a regenerative challenge. However, little is known about the role of Gln in liver regeneration, notably the spatial orientation in the remodeling process. This study aimed to elucidate Gln-potentiated liver regeneration and ECM remodeling after mass loss. Methods: We studied the regenerative process in hepatectomized mice supplemented with Gln. Second harmonic generation/two-photon excitation fluorescence microscopy, an artificial intelligence–assisted structure-based imaging, was used to demonstrate the spatial-oriented process in a hepatic acinus. Results: Gln promotes liver mass regrowth through the cell cycle, Gln metabolism, and adipogenesis pathways after hepatectomy. Ornithine transaminase, one of the upregulated enzymes, showed temporal, spatial, and functional correspondence with the regeneration process. Second harmonic generation/two-photon excitation fluorescence microscopy highlighted transient hepatic steatosis and ECM collagen synthesis, predominantly in the portal tract instead of the central vein area. Structural remodeling was also observed in the portal tract area. Conclusions: Gln promotes liver regeneration through cellular proliferation and metabolic reprogramming after hepatectomy. Using structure-based imaging, we found that Gln potentiated hepatic steatosis and ECM collagen deposition predominantly in the portal tract area. These results highlighted the spatial orientation and mechanistic implications of Gln in liver regeneration.

Background: Transarterial chemoembolization (TACE) is the primary treatment modality for advanced HCC, yet its efficacy assessment and prognosis prediction largely depend on imaging and serological markers that possess inherent limitations in terms of real-time capability, sensitivity, and specificity. Here, we explored whether multiple features of cell-free mitochondrial DNA (cf-mtDNA), including copy number, mutations, and fragmentomics, could be used to predict the response and prognosis of patients with HCC undergoing TACE treatment. Methods: A total of 60 plasma cell-free DNA samples were collected from 30 patients with HCC before and after the first TACE treatment and then subjected to capture-based mtDNA sequencing and whole-genome sequencing. Results: Comprehensive analyses revealed a clear association between cf-mtDNA multiple features and tumor characteristics. Based on cf-mtDNA multiple features, we also developed HCC death and progression risk prediction models. Kaplan-Meier curve analyses revealed that the high-death risk or high-progression–risk group had significantly shorter median overall survival (OS) and progression-free survival than the low-death risk or low-progression-risk group (all p<0.05). Moreover, the change in cf-mtDNA multiple features before and after TACE treatment exhibited an exceptional ability to predict the risk of death and progression in patients with HCC (log-rank test, all p<0.01; HRs: 0.36 and 0.33, respectively). Furthermore, we observed the consistency of change between the cf-mtDNA multiple features and copy number variant burden before and after TACE treatment in 40.00% (12/30) patients with HCC. Conclusions: Altogether, we developed a novel strategy based on profiling of cf-mtDNA multiple features for prognosis prediction and efficacy evaluation in patients with HCC undergoing TACE treatment.

Background: Patients with HCC face numerous barriers to curative therapies, particularly Black patients and those impacted by adverse social determinants of health (SDOH). This study aimed to identify patient-reported barriers and facilitators to curative therapies, to inform interventions that improve equitable access to care. Methods: We conducted 2 qualitative sessions with Black participants and participants experiencing adverse SDOH with HCC referred for liver transplant (LT) or resection. We also conducted one-on-one interviews with participants from sessions that underwent LT (n=2). Human-centered design methods, including journey mapping and group ideation, were used to identify challenges and solutions at various stages in the care pathway. Data were analyzed to identify key themes and to compare the experiences of Black patients with those experiencing adverse SDOH. Results: Both groups faced significant barriers, particularly related to information overload, communication gaps with health care providers, and the complexity of navigating the LT pathway. However, Black patients reported additional challenges related to the psychological burden of the diagnosis and distrust in the health care system, while those with adverse SDOH frequently cited financial instability, lack of social support, and challenges in coordinating care between multiple health systems. Despite these differences, common facilitators included compassionate health care teams and strong personal support networks. Both groups suggested solutions such as improvements in education timing and delivery, better communication pathways, and peer support groups to improve preparedness for treatment and recovery. Conclusions: While Black patients and those with adverse SDOH experience unique barriers, common threads—such as information gaps and desire for peer support suggest shared opportunities for interventions.

Background: Intrahepatic cholangiocarcinoma (ICC) is a poor prognosis of malignant cancer with high lymph node metastasis and resistance to systemic therapies. Recent studies suggested that the involvement of IL-8 could promote ICC metastasis through epithelial–mesenchymal transition while the ICC-ALDH1A1high subtype is clarified by multi-omics study. The correlation between ALDH1A1 and IL-8 in ICC remains elusive. This study aims to further explore the roles and regulatory mechanisms of ALDH1A1 and IL-8 in ICC. Methods: We analyzed IL-8 and ALDH1A1 expression in ICC patients and cells. CXCR2 inhibitor (SB225002) was applied to inhibit the function of IL-8, and JSH-23 was applied to inhibit the NF-κB signaling pathway. We examined the effects of IL-8 inhibition on NF-κB, ALDH1A1 expression, and cell growth, migration, invasion, and stemness. Moreover, we examined the effects of ALDH1A1 on NF-κB, IL-8 expression, and cell growth, migration, invasion, and stemness. The effects of IL-8 and ALDH1A1 on tumor growth and NF-κB expression were validated using subcutaneous tumors in nude mice. Results: IL-8-derived tumor cells could promote ICC progression. The high expression of IL-8 in serum was associated with lymph node metastasis. IL-8 could upregulate ALDH1A1 expression by activating the NF-κB signaling pathway, promoting tumor progression. Upregulation of ALDH1A1 could activate NF-κB to promote IL-8 secretion, forming a positive feedback loop to promote tumor invasiveness and cell stemness in ICC. Conclusions: IL-8-derived tumor cells could upregulate ALDH1A1 expression by activating the NF-κB signaling pathway, promoting tumor progression. Upregulation of ALDH1A1 could activate NF-κB to promote IL-8 secretion, forming a positive feedback loop to promote tumor invasiveness and cell stemness in ICC.