A human liver cell atlas reveals heterogeneity and epithelial progenitors.

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.

Tight Junction Proteins as Therapeutic Targets to Treat Liver Fibrosis and Hepatocellular Carcinoma.

In the last decade tight junction proteins exposed at the surface of liver or cancer cells have been uncovered as mediators of liver disease biology: Claudin-1 and Occludin are host factors for hepatitis C virus entry and Claudin-1 has been identified as a driver for liver fibrosis and hepatocellular carcinoma (HCC). Moreover, Claudins have emerged as therapeutic targets for liver disease and HCC. CLDN1 expression is upregulated in liver fibrosis and HCC. Monoclonal antibodies (mAbs) targeting Claudin-1 have completed preclinical proof-of-concept studies for treatment of liver fibrosis and HCC and are currently in clinical development for advanced liver fibrosis. Claudin-6 overexpression is associated with an HCC aggressive phenotype and treatment resistance. Claudin-6 mAbs or chimeric antigen receptor-T cells therapies are currently being clinically investigated for Claudin-6 overexpressing tumors. In conclusion, targeting Claudin proteins offers a novel clinical opportunity for the treatment of patients with advanced liver fibrosis and HCC.

Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

Liver Disease and Coronavirus Disease 2019: From Pathogenesis to Clinical Care.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus that emerged in late 2019, is posing an unprecedented challenge to global health. Coronavirus disease 2019 (COVID-19), the clinical disease caused by SARS-CoV-2, has a variable presentation ranging from asymptomatic infection to life-threatening acute respiratory distress syndrome and multiorgan failure. Liver involvement is common during COVID-19 and exhibits a spectrum of clinical manifestations from asymptomatic elevations of liver function tests to hepatic decompensation. The presence of abnormal liver tests has been associated with a more severe presentation of COVID-19 disease and overall mortality. Although SARS-CoV-2 RNA has been detected in the liver of patients with COVID-19, it remains unclear whether SARS-CoV-2 productively infects and replicates in liver cells and has a direct liver-pathogenic effect. The cause of liver injury in COVID-19 can be attributed to multiple factors, including virus-induced systemic inflammation, hypoxia, hepatic congestion, and drug-induced liver disease. Among patients with cirrhosis, COVID-19 has been associated with hepatic decompensation and liver-related mortality. Additionally, COVID-19's impact on health care resources can adversely affect delivery of care and outcomes of patients with chronic liver disease. Understanding the underlying mechanisms of liver injury during COVID-19 will be important in the management of patients with COVID-19, especially those with advanced liver disease. This review summarizes our current knowledge of SARS-CoV-2 virus-host interactions in the liver as well the clinical impact of liver disease in COVID-19.

Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Single-cell genomics and spatial transcriptomics: Discovery of novel cell states and cellular interactions in liver physiology and disease biology.

Transcriptome analysis enables the study of gene expression in human tissues and is a valuable tool to characterise liver function and gene expression dynamics during liver disease, as well as to identify prognostic markers or signatures, and to facilitate discovery of new therapeutic targets. In contrast to whole tissue RNA sequencing analysis, single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics enables the study of transcriptional activity at the single cell or spatial level. ScRNA-seq has paved the way for the discovery of previously unknown cell types and subtypes in normal and diseased liver, facilitating the study of rare cells (such as liver progenitor cells) and the functional roles of non-parenchymal cells in chronic liver disease and cancer. By adding spatial information to scRNA-seq data, spatial transcriptomics has transformed our understanding of tissue functional organisation and cell-to-cell interactions in situ. These approaches have recently been applied to investigate liver regeneration, organisation and function of hepatocytes and non-parenchymal cells, and to profile the single cell landscape of chronic liver diseases and cancer. Herein, we review the principles and technologies behind scRNA-seq and spatial transcriptomic approaches, highlighting the recent discoveries and novel insights these methodologies have yielded in both liver physiology and disease biology.

HCV-Induced Epigenetic Changes Associated With Liver Cancer Risk Persist After Sustained Virologic Response.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.

Tight Junction Proteins and the Biology of Hepatobiliary Disease.

Tight junctions (TJ) are intercellular adhesion complexes on epithelial cells and composed of integral membrane proteins as well as cytosolic adaptor proteins. Tight junction proteins have been recognized to play a key role in health and disease. In the liver, TJ proteins have several functions: they contribute as gatekeepers for paracellular diffusion between adherent hepatocytes or cholangiocytes to shape the blood-biliary barrier (BBIB) and maintain tissue homeostasis. At non-junctional localizations, TJ proteins are involved in key regulatory cell functions such as differentiation, proliferation, and migration by recruiting signaling proteins in response to extracellular stimuli. Moreover, TJ proteins are hepatocyte entry factors for the hepatitis C virus (HCV)-a major cause of liver disease and cancer worldwide. Perturbation of TJ protein expression has been reported in chronic HCV infection, cholestatic liver diseases as well as hepatobiliary carcinoma. Here we review the physiological function of TJ proteins in the liver and their implications in hepatobiliary diseases.

[Hepatitis D virus: viral cycle and new therapeutic approaches]. / Virus de l'hépatite D : cycle viral et nouvelles stratégies thérapeutiques.

An estimated 70 million people are chronically infected with hepatitis D (delta) virus (HDV) worldwide. HDV is a small satellite virus of hepatitis B virus (HBV) requiring HBV for the completion of its cycle. Hepatitis D is the most severe form of chronic viral hepatitis. It is responsible for an acceleration and an aggravation of chronic liver disease compared to HBV monoinfected patients. Current treatments based on pegylated interferon rarely allow viral clearance in chronically infected patients. For long time, the absence of easy-to-use models has limited the knowledge on virus-host interactions. Notably, hepatocyte host factors involved in the viral life cycle remain largely unknown. These host factors are potential therapeutic targets for novel treatment strategies, including molecules currently evaluated in clinical trials. This review summarizes our knowledge on HDV molecular virology and innovative therapeutic strategies targeting hepatocyte host factors.

Liver Resection versus Radiofrequency Ablation plus Transcatheter Arterial Chemoembolization in Cirrhotic Patients with Solitary Large Hepatocellular Carcinoma.

PURPOSE: To compare liver resection (LR) with single-step, balloon-occluded radiofrequency (RF) ablation plus drug-eluting embolics transarterial chemoembolization in cirrhotic patients with single hepatocellular carcinoma (HCC) ≥ 3 cm. MATERIALS AND METHODS: From 2010 to 2014, 25 patients with compensated cirrhosis and single HCC ≥ 3 cm (median size 4.5 cm; range, 3.0-6.8 cm) not suitable for LR or liver transplantation were treated with RF ablation plus transarterial chemoembolization in a prospective observational single-center pilot study; all patients had complete tumor necrosis after treatment. A retrospective control group included 29 patients (median HCC size 4.0 cm; range, 3.0-7.4 cm) who underwent LR. RF ablation plus transarterial chemoembolization group included more patients with severe portal hypertension (65.5% vs 35.0%, P = .017). Primary endpoints were overall survival (OS) and tumor recurrence (TR) rates. RESULTS: One death and 1 major complication (4%) were observed in LR group. No major complications were reported in RF ablation plus transarterial chemoembolization group (P = .463). OS rates at 1 and 3 years were 91.8% and 79.3% in LR group and 89.4% and 48.2% in RF ablation plus transarterial chemoembolization group (P = 0.117). TR rates at 1 and 3 years were 29.5% and 45.0% in LR group and 42.4% and 76.0% in RF ablation plus transarterial chemoembolization group (P = .034). Local tumor progression (LTP) rates at 3 years were significantly lower in LR group (21.8% vs 58.1%, P = .005). Similar results were found in patients with HCC ≤ 5 cm (TR rates 35.4% vs 75.1%, P = .016; LTP 16.0% vs 55.7%, P = .013). CONCLUSIONS: LR achieved lower TR and LTP rates than RF ablation plus transarterial chemoembolization, but 3-years OS rates were not statistically different between the 2 groups. RF ablation plus transarterial chemoembolization is an effective treatment option in patients with compensated cirrhosis and solitary HCC ≥ 3 cm unsuitable for LR.

HepPar1-Positive Circulating Microparticles Are Increased in Subjects with Hepatocellular Carcinoma and Predict Early Recurrence after Liver Resection.

Circulating microparticles (MPs) are novel potential biomarkers in cancer patients. Their role in hepatocellular carcinoma (HCC) is under intensive investigation. In this study, we tested the hypothesis that MPs expressing the antigen HepPar1 are increased in the blood of subjects with HCC and may serve as markers of early recurrence after liver resection (LR). We studied 15 patients affected by HCC undergoing LR, and used flow cytometry to assess the number of circulating HepPar1+ MPs. Ten subjects without HCC (five with liver cirrhosis and five with healthy livers) were used as controls. After LR, HCC patients underwent a follow-up to check for early recurrence, which occurred in seven cases. The number of circulating HepPar1+ MPs was significantly higher in subjects affected by HCC, compared to individuals without cancer (p < 0.01). We also found that, among HCC patients, the number of circulating HepPar1+ MPs, measured before LR, was significantly higher in those who displayed early recurrence compared to those without recurrence (p = 0.02). Of note, other types of circulating MPs, such as those derived from endothelial cells (CD144+) or those produced by the activated endothelium (CD144+/CD62+), were not associated with HCC, nor could they predict HCC recurrence. HepPar1+ MPs deserve further investigation as novel biomarkers of disease and prognosis in HCC patients.

Spontaneous idiopathic liver hemorrhage: a systematic review of a rare entity.

BACKGROUND: Spontaneous idiopathic liver hemorrhage (SILH) is a rare life-threatening condition occurring without a clear and specific etiology. A systematic review was performed to provide guidelines for the perioperative management of patients affected by SILH. A case report was also included. METHODS: A systematic search of the last 24-year literature was conducted and the manuscript was structured following point-by-point the PRISMA guidelines. RESULTS: After an initial selection of 6995 titles, 15 articles were considered for the final qualitative analysis (n = 22 patients, including the present report). Conservative treatment was chosen in 12 cases (54.5%) with stable clinical conditions, while 9 patients (40.9%) required a primary operative approach for emergency presentation at diagnosis. Direct liver resection was the preferred surgical treatment (n = 6), mostly major hepatectomies (n = 4). Hepatic arterial embolization was performed as the primary operative approach in three patients, followed by emergency laparotomy during the same hospitalization because of rebleeding in one case. Contrast-enhanced CT scan was the gold standard for diagnosis (n = 19). CONCLUSIONS: Conservative treatment of SILH is mainly based on stable clinical conditions and may be considered even in case of a limited arterial blush found on imaging. The absence of underlying hepatic or systemic disorders seems to correlate with favorable outcomes and no mortality.

Left-sided portal hypertension: Update and proposition of management algorithm.

Left-sided or segmental portal hypertension (SPHT) is a rare entity, most often associated with pancreatic disease or antecedent pancreatic surgery. The starting point is splenic vein obstruction secondary to local inflammation or, less often, extrinsic compression. SPHT leads to splenomegaly and development of collateral porto-systemic venous circulation. SPHT should be suspected in patients with pancreatic history who present with episodic upper gastrointestinal bleeding and splenomegaly with normal liver function tests. The most common clinical presentation is major upper gastrointestinal bleeding secondary to rupture of esophageal and/or gastric varices. At the present time, there are no management recommendations for SPHT, particularly when the patient is asymptomatic. In patients with upper gastro-intestinal bleeding, hemostasis can be obtained either by medical or interventional means according to patient status and available resources. For symptomatic patients, splenectomy is the reference treatment. Recently, less invasive, radiologic procedures, such as splenic artery embolization, have been developed as an alternative to surgery. Additionally, sonography-guided endoscopic hemostasis can also be envisioned, leading to the diagnosis and treatment of the lesion by elastic band ligation or by glue injection into the varices during the same procedure. The goal of this article is to describe the pathophysiological mechanisms behind SPHT and its clinical manifestations and treatment, based on a review of the literature. Because of the absence of recommendations for the management of SPHT, we propose a decisional algorithm for the management of SPHT based on the literature.

Single-cell RNA-sequencing of virus-specific cellular immune responses in chronic hepatitis B patients.

Chronic hepatitis B (CHB) is a major global health challenge. CHB can be controlled by antivirals but a therapeutic cure is lacking. CHB is characterized by limited HBV-specific T cell reactivity and functionality and expression of inhibitory receptors. The mechanisms driving these T cell phenotypes are only partially understood. Here, we created a single-cell RNA-sequencing dataset of HBV immune responses in patients to contribute to a better understanding of the dysregulated immunity. Blood samples of a well-defined cohort of 21 CHB and 10 healthy controls, including a subset of 5 matched liver biopsies, were collected. scRNA-seq data of total immune cells (55,825) plus sorted HBV-specific (1,963), non-naive (32,773) and PD1+ T cells (96,631) was generated using the 10X Genomics platform (186,123 cells) or the full-length Smart-seq2 protocol (1,069 cells). The shared transcript count matrices of single-cells serve as a valuable resource describing transcriptional changes underlying dysfunctional HBV-related T cell responses in blood and liver tissue and offers the opportunity to identify targets or biomarkers for HBV-related immune exhaustion.

Impact of anti-HDV reflex testing at HBs antigen positive discovery in a single center France: Support for primary HDV screening in France.

BACKGROUND: Hepatitis Delta virus (HDV) infection is a major cause of liver-related morbidity and mortality in patients infected with HBV, with a global HDV prevalence uncertain. In France, 2 to 5 % of HBs antigen (HBsAg) carriers present anti-HDV antibodies (anti-HDV). The EASL recommends testing for anti-HDV in all HBsAg-positive patients. Since January 2022, we have systematically carried out anti-HDV serology when a positive HBsAg is discovered (new HBsAg carriers). OBJECTIVES: We evaluated the benefit of anti-HDV reflex testing after one year of practice by comparing anti-HDV and HBsAg serology data over the last six years, among the new HBsAg carriers and all the HBsAg carriers. STUDY DESIGN: HBsAg and anti-HDV were screened using the Abbott Architect HBsAg quanti kit and the DIA.PRO HDVAb kit. Serological, demographic, virological, and clinical data were analyzed. RESULTS: Implementing anti-HDV reflex testing leads to more than a 2-fold increase in diagnoses of HDV infection among all HBsAg carriers. If the anti-HDV positive rate remains stable among the new HBsAg carriers, a significant increase in the anti-HDV positive rate from 6.8 % to 10.3 % was observed considering all HBsAg carriers. Interestingly, the discovery of anti-HDV carriage increased from 3.9 % to 6.5 % in 2022, allowing earlier identification of HBV-HDV-infected patients and a fast referral to hepatologists for adequate clinical management and, in some cases, the introduction of bulevirtide-based therapy. CONCLUSIONS: Our preliminary results at one year seem promising and evaluating the cost-effectiveness of reflex tests in real life with feedback would be helpful.

Long-term effectiveness of resection and radiofrequency ablation for single hepatocellular carcinoma ≤3 cm. Results of a multicenter Italian survey.

BACKGROUND & AIMS: The aim of this study was to compare liver resection and radiofrequency ablation in patients with single hepatocellular carcinoma ≤3 cm and compensated cirrhosis. METHODS: The study involved 544 Child-Pugh A cirrhotic patients (246 in the resection group and 298 in the radiofrequency group) observed in 15 Italian centers. Overall survival and tumor recurrence rates were analyzed using the Kaplan Meier method before and after propensity score matching. Cox regression models were used to identify factors associated with overall survival and tumor recurrence. RESULTS: Two cases of perioperative mortality were observed in the resection group and the rate of major complications was 4.5% in the resection group and 2.0% in the radiofrequency group (p=0.101). Four-year overall survival rates were 74.4% in the resection group and 66.2% in the radiofrequency group (p=0.353). Four-year cumulative HCC recurrence rates were 56% in the resection group and 57.1% in the radiofrequency group (p=0.765). Local tumor progression was detected in 20.5% of ablated patients and in one resected patient (p<0.001). After propensity score matching, both survival and tumor recurrence were still not significantly different although a trend towards lower recurrence was observed in resected patients. Older age and higher alpha-fetoprotein levels were independent predictors of poor overall survival while older age and higher alanine-aminotransferase levels resulted to be independent factors associated with higher recurrence rate. CONCLUSIONS: In spite of a higher rate of local tumor progression, radiofrequency ablation can provide results comparable to liver resection in the treatment of single hepatocellular carcinoma ≤3 cm occurring in compensated cirrhosis.

Differences between sporadic hyperplastic gastric polyps and portal hypertensive gastric polyps: a review.

Portal hypertension (PH) is one of the most severe complications of chronic liver diseases. It is defined as an increase in pressure in the portal venous system which results in a portosystemic gradient >5 mmHg. In the western world, cirrhosis is the most frequent cause of PH, mainly due to nonalcoholic fatty liver disease and alcoholic liver disease. Patients with PH have esophageal varices in 68-73% of cases, portal hypertensive gastropathy in 51-73% and hyperplastic polyps (HPs) in 0.9-2%. Recent studies have shown that HPs found in PH patients are different from classical HPs. They constitute a new entity called portal hypertensive polyps (PHPs). The main difference between sporadic HPs and PHP is the presence of larger and more numerous vascular capillaries in the lamina propria. The clinical course of PHPs is unknown. Their physiopathology seems different from HPs: the increased congestion caused by higher portal pressure in the stomach may induce capillaries proliferation and neoangiogenesis. PHPs may be responsible for symptoms, such as pyloric obstruction, iron deficiency and anemia. Their prevalence in portal hypertensive and cirrhotic patients is from 1% to 8%. PHPs can be single or numerous, in the antrum or the gastric corpus. Their size ranges from 2 to 3 cm. PHPs seem to disappear or shrink with the treatment of PH. They should be resected in case of symptom and if >10 mm, after Helicobacter pylori eradication if present. However, their recurrence is frequent (40-79%), thus surveillance endoscopy is mandatory, at the same time as esophageal varices.