Prominent role of gut dysbiosis in the pathogenesis of cystic fibrosis-related liver disease in mice.

BACKGROUND & AIMS: Cystic fibrosis-related liver disease (CFLD) is a chronic cholangiopathy that increases morbidity and mortality in patients with CF. Current treatments are unsatisfactory, and incomplete understanding of CFLD pathogenesis hampers therapeutic development. We have previously shown that mouse CF cholangiocytes respond with excessive inflammation to LPS. Thus, we investigated the role of the gut-liver axis in the pathogenesis of CFLD. METHODS: Wild-type (WT), whole-body CFTR knockout (CFTR-KO) and gut-corrected (CFTR-KO-GC) mice were studied. Liver changes were assessed by immunohistochemistry and single-cell transcriptomics (scRNAseq), inflammatory mediators were analyzed by proteome array, faecal microbiota by 16S rRNAseq and gut permeability by FITC-dextran assay. RESULTS: The livers of CFTR-KO mice showed ductular proliferation and periportal inflammation, whereas livers of CFTR-KO-GC mice had no evident pathology. scRNAseq analysis of periportal cells showed increased presence of neutrophils, macrophages and T-cells and activation of pro-inflammatory and pathogen-mediated immune pathways in CFTR-KO livers, consistent with a response to gut-derived stimuli. CFTR-KO mice exhibited gut dysbiosis with enrichment of Enterobacteriaceae and Enterococcus spp., which was associated with increased intestinal permeability and mucosal inflammation, whereas gut dysbiosis and inflammation were absent in CFTR-KO-GC mice. Treatment with nonabsorbable antibiotics ameliorated intestinal permeability and liver inflammation in CFTR-KO mice. Faecal microbiota transfer from CFTR-KO to germ-free WT mice did not result in dysbiosis nor liver pathology, indicating that defective intestinal CFTR is required to maintain dysbiosis. CONCLUSION: Defective CFTR in the gut sustains a pathogenic microbiota, creates an inflammatory milieu, and alters intestinal permeability. These changes are necessary for the development of cholangiopathy. Restoring CFTR in the intestine or modulating the microbiota could be a promising strategy to prevent or attenuate liver disease. IMPACT AND IMPLICATIONS: Severe cystic fibrosis-related liver disease (CFLD) affects 10% of the patients and contributes to increased morbidity and mortality of CF patients. Treatment options remain limited due to a lack of understanding of the disease pathophysiology. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mediates Cl- and HCO3- secretion in the biliary epithelium and its defective function is thought to cause cholestasis and excessive inflammatory responses in CF. However, our study in CFTR-knockout mice demonstrates that microbial dysbiosis, combined with increased intestinal permeability caused by defective CFTR in the intestinal mucosa, acts as a necessary co-factor for the development of CFLD-like liver pathology in mice. These findings uncover a major role for the gut microbiota in CFLD pathogenesis and call for further investigation and clinical validation to develop targeted therapeutic strategies acting on the gut-liver axis in CF.

Automated graded prognostic assessment for patients with hepatocellular carcinoma using machine learning.

BACKGROUND: Accurate mortality risk quantification is crucial for the management of hepatocellular carcinoma (HCC); however, most scoring systems are subjective. PURPOSE: To develop and independently validate a machine learning mortality risk quantification method for HCC patients using standard-of-care clinical data and liver radiomics on baseline magnetic resonance imaging (MRI). METHODS: This retrospective study included all patients with multiphasic contrast-enhanced MRI at the time of diagnosis treated at our institution. Patients were censored at their last date of follow-up, end-of-observation, or liver transplantation date. The data were randomly sampled into independent cohorts, with 85% for development and 15% for independent validation. An automated liver segmentation framework was adopted for radiomic feature extraction. A random survival forest combined clinical and radiomic variables to predict overall survival (OS), and performance was evaluated using Harrell's C-index. RESULTS: A total of 555 treatment-naïve HCC patients (mean age, 63.8 years ± 8.9 [standard deviation]; 118 females) with MRI at the time of diagnosis were included, of which 287 (51.7%) died after a median time of 14.40 (interquartile range, 22.23) months, and had median followed up of 32.47 (interquartile range, 61.5) months. The developed risk prediction framework required 1.11 min on average and yielded C-indices of 0.8503 and 0.8234 in the development and independent validation cohorts, respectively, outperforming conventional clinical staging systems. Predicted risk scores were significantly associated with OS (p < .00001 in both cohorts). CONCLUSIONS: Machine learning reliably, rapidly, and reproducibly predicts mortality risk in patients with hepatocellular carcinoma from data routinely acquired in clinical practice. CLINICAL RELEVANCE STATEMENT: Precision mortality risk prediction using routinely available standard-of-care clinical data and automated MRI radiomic features could enable personalized follow-up strategies, guide management decisions, and improve clinical workflow efficiency in tumor boards. KEY POINTS: • Machine learning enables hepatocellular carcinoma mortality risk prediction using standard-of-care clinical data and automated radiomic features from multiphasic contrast-enhanced MRI. • Automated mortality risk prediction achieved state-of-the-art performances for mortality risk quantification and outperformed conventional clinical staging systems. • Patients were stratified into low, intermediate, and high-risk groups with significantly different survival times, generalizable to an independent evaluation cohort.

Automated MRI liver segmentation for anatomical segmentation, liver volumetry, and the extraction of radiomics.

OBJECTIVES: To develop and evaluate a deep convolutional neural network (DCNN) for automated liver segmentation, volumetry, and radiomic feature extraction on contrast-enhanced portal venous phase magnetic resonance imaging (MRI). MATERIALS AND METHODS: This retrospective study included hepatocellular carcinoma patients from an institutional database with portal venous MRI. After manual segmentation, the data was randomly split into independent training, validation, and internal testing sets. From a collaborating institution, de-identified scans were used for external testing. The public LiverHccSeg dataset was used for further external validation. A 3D DCNN was trained to automatically segment the liver. Segmentation accuracy was quantified by the Dice similarity coefficient (DSC) with respect to manual segmentation. A Mann-Whitney U test was used to compare the internal and external test sets. Agreement of volumetry and radiomic features was assessed using the intraclass correlation coefficient (ICC). RESULTS: In total, 470 patients met the inclusion criteria (63.9±8.2 years; 376 males) and 20 patients were used for external validation (41±12 years; 13 males). DSC segmentation accuracy of the DCNN was similarly high between the internal (0.97±0.01) and external (0.96±0.03) test sets (p=0.28) and demonstrated robust segmentation performance on public testing (0.93±0.03). Agreement of liver volumetry was satisfactory in the internal (ICC, 0.99), external (ICC, 0.97), and public (ICC, 0.85) test sets. Radiomic features demonstrated excellent agreement in the internal (mean ICC, 0.98±0.04), external (mean ICC, 0.94±0.10), and public (mean ICC, 0.91±0.09) datasets. CONCLUSION: Automated liver segmentation yields robust and generalizable segmentation performance on MRI data and can be used for volumetry and radiomic feature extraction. CLINICAL RELEVANCE STATEMENT: Liver volumetry, anatomic localization, and extraction of quantitative imaging biomarkers require accurate segmentation, but manual segmentation is time-consuming. A deep convolutional neural network demonstrates fast and accurate segmentation performance on T1-weighted portal venous MRI. KEY POINTS: • This deep convolutional neural network yields robust and generalizable liver segmentation performance on internal, external, and public testing data. • Automated liver volumetry demonstrated excellent agreement with manual volumetry. • Automated liver segmentations can be used for robust and reproducible radiomic feature extraction.

Community-Level Factors Associated with Hepatocellular Carcinoma Incidence and Mortality: An Observational Registry Study.

BACKGROUND: Hepatocellular carcinoma (HCC) incidence and outcomes vary across populations in the United States, but few studies evaluate local drivers of observed disparities. We measured HCC incidence at the community level and assessed community-level HCC risk factors with the goal of informing resource allocation to improve early case detection, which is associated with improved outcomes. METHODS: Clinical and demographic data including census tract of residence for all adults diagnosed with HCC in the Connecticut Tumor Registry between 2008 and 2019 were combined with publicly available U.S. Census and Centers for Disease Control and Prevention (CDC) data at the ZIP Code tabulation area (ZCTA) level. The average annual incidence of HCC was calculated for each ZCTA and associations between community-level characteristics, HCC incidence, stage at diagnosis, and survival were evaluated. RESULTS: Average annual HCC incidence during the study period was 8.9/100,000 adults and varied from 0 to 97.7 per 100,000 adults by ZCTA. At the community level, lower rates of high school graduation, higher rates of poverty, and rural community type were associated with higher HCC incidence. Persons with HCC living in the highest incidence ZCTAs were diagnosed at a younger age and were less likely to be alive at 1, 2, and 5 years after diagnosis. CONCLUSIONS: Community-level socioeconomic factors are strongly associated with HCC incidence and survival in Connecticut. IMPACT: This reproducible geo-localization approach using cancer registry, Census, and CDC data can be used to identify communities most likely to benefit from health system investments to reduce disparities in HCC.

Adherence to Tumor Board Recommendations in the Treatment of Patients with Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a heterogeneous disease that typically arises in the setting of chronic liver disease, making treatment selection complex. Multidisciplinary liver tumor boards (MDLTB) have been shown to improve outcomes in patients with HCC. However, in many cases, patients evaluated by MDLTBs ultimately do not receive the board's recommended treatment. Purpose: This study aims to assess adherence to MDLTB recommendations for the treatment of HCC, the reasons for non-adherence, and the survival of Barcelona Clinic Liver Cancer (BCLC) Stage A patients treated with curative treatment compared to palliative locoregional therapy. Patients and Methods: A single-site, retrospective cohort study was conducted of all patients with treatment-naïve HCC who were evaluated by an MDLTB at a tertiary care center in Connecticut between 2013 and 2016, of which 225 patients met inclusion criteria. Investigators conducted a chart review and recorded adherence to the MDLTB's recommendations, and in cases of discordance, evaluated and recorded the underlying cause; investigators assessed MDLTB recommendations' compliance with BCLC guidelines. Survival data was accrued through February 1st of 2022 and analyzed via Kaplan-Meier analysis and multivariate Cox regression. Results: Treatment adherent to MDLTB recommendations occurred in 85.3% of patients (n=192). The majority of non-adherence occurred in the management of BCLC Stage A disease. In cases where adherence was possible but the recommendation was not followed, most discrepancies were whether to treat with curative or palliative intent (20/24), with almost all discrepancies occurring in patients (19/20) with BCLC Stage A disease. For patients with Stage A unifocal HCC, those who received curative therapy lived significantly longer than patients who received palliative locoregional therapy (5.55 years vs 4.26 years, p=0.037). Conclusion: Most forms of non-adherence to MDLTB recommendations were unavoidable; however, treatment discordance in the management of patients with BCLC Stage A unifocal disease may present an opportunity for clinically significant quality improvement.

Intrahepatic Cholangiocarcinoma Developing in Patients with Metabolic Syndrome Is Characterized by Osteopontin Overexpression in the Tumor Stroma.

Metabolic syndrome (MetS) is a common condition closely associated with non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). Recent meta-analyses show that MetS can be prodromal to intrahepatic cholangiocarcinoma (iCCA) development, a liver tumor with features of biliary differentiation characterized by dense extracellular matrix (ECM) deposition. Since ECM remodeling is a key event in the vascular complications of MetS, we aimed at evaluating whether MetS patients with iCCA present qualitative and quantitative changes in the ECM able to incite biliary tumorigenesis. In 22 iCCAs with MetS undergoing surgical resection, we found a significantly increased deposition of osteopontin (OPN), tenascin C (TnC), and periostin (POSTN) compared to the matched peritumoral areas. Moreover, OPN deposition in MetS iCCAs was also significantly increased when compared to iCCA samples without MetS (non-MetS iCCAs, n = 44). OPN, TnC, and POSTN significantly stimulated cell motility and the cancer-stem-cell-like phenotype in HuCCT-1 (human iCCA cell line). In MetS iCCAs, fibrosis distribution and components differed quantitatively and qualitatively from non-MetS iCCAs. We therefore propose overexpression of OPN as a distinctive trait of MetS iCCA. Since OPN stimulates malignant properties of iCCA cells, it may provide an interesting predictive biomarker and a putative therapeutic target in MetS patients with iCCA.

Cell-matrix interactions control biliary organoid polarity, architecture, and differentiation.

BACKGROUND AND AIMS: Cholangiopathies are an important cause of morbidity and mortality. Their pathogenesis and treatment remain unclear in part because of the lack of disease models relevant to humans. Three-dimensional biliary organoids hold great promise; however, the inaccessibility of their apical pole and the presence of extracellular matrix (ECM) limits their application. We hypothesized that signals coming from the extracellular matrix regulate organoids' 3-dimensional architecture and could be manipulated to generate novel organotypic culture systems. APPROACH AND RESULTS: Biliary organoids were generated from human livers and grown embedded into Culturex Basement Membrane Extract as spheroids around an internal lumen (EMB). When removed from the EMC, biliary organoids revert their polarity and expose the apical membrane on the outside (AOOs). Functional, immunohistochemical, and transmission electron microscope studies, along with bulk and single-cell transcriptomic, demonstrate that AOOs are less heterogeneous and show increased biliary differentiation and decreased expression of stem cell features. AOOs transport bile acids and have competent tight junctions. When cocultured with liver pathogenic bacteria (Enterococcus spp.), AOOs secrete a range of proinflammatory chemokines (ie, MCP1, IL8, CCL20, and IP-10). Transcriptomic analysis and treatment with a beta-1-integrin blocking antibody identified beta-1-integrin signaling as a sensor of the cell-extracellular matrix interaction and a determinant of organoid polarity. CONCLUSIONS: This novel organoid model can be used to study bile transport, interactions with pathobionts, epithelial permeability, cross talk with other liver and immune cell types, and the effect of matrix changes on the biliary epithelium and obtain key insights into the pathobiology of cholangiopathies.

Immune checkpoint inhibitors in malignancies after liver transplantation: A systematic review and pooled analysis.

BACKGROUND AND AIMS: Treatment of de novo malignancies and recurrent hepatocellular carcinoma with immune checkpoint inhibitors (ICI) in liver transplant recipients (LT) is an attractive strategy that is infrequently pursued because of the lack of strong evidence regarding their safety and efficacy. In this systematic review with pooled analysis, we aimed to assess safety and efficacy of ICI therapy following LT. METHODS: We performed a systematic search of case reports and series published until January 2022. We included 31 publications reporting a total of 52 patients treated with ICIs after LT and assessed in a pooled analysis the risk of graft rejection and the outcome of ICI therapy. RESULTS: Acute graft rejection occurred in 15 patients (28.8%) and 7 patients (13.4% of the total cohort) died because of graft loss. Rejection was associated with shorter overall survival (OS) (17.2 months, confidence interval [CI] 12.1-22.2 vs. 3.5 months, CI 1.6-5.4, p < 0.001). Disease control rate was 44.2% (n = 23), and in these patients, OS was longer than in non-responders (26.4 months, CI 20.8-32.0 vs. 3.4 months, CI 2.1-4.7, p < 0.001). CONCLUSIONS: Observational, off-label experience suggests that treatment with ICI for advanced malignancies in LT recipients might not be discarded a priori. This notwithstanding, ICI treatment in these patients is associated with a substantial risk of graft rejection and mortality. Prospective studies are needed to provide adequate safety and efficacy figures of ICI treatment in this fragile population.

European Society for Organ Transplantation (ESOT) Consensus Statement on Outcome Measures in Liver Transplantation According to Value-Based Health Care.

Liver transplantation is a highly complex, life-saving, treatment for many patients with advanced liver disease. Liver transplantation requires multidisciplinary teams, system-wide adaptations and significant investment, as well as being an expensive treatment. Several metrics have been proposed to monitor processes and outcomes, however these lack patient focus and do not capture all aspects of the process. Most of the reported outcomes do not capture those outcomes that matter to the patients. Adopting the principles of Value-Based Health Care (VBHC), may provide an opportunity to develop those metrics that matter to patients. In this article, we present a Consensus Statement on Outcome Measures in Liver Transplantation following the principles of VBHC, developed by a dedicated panel of experts under the auspices of the European Society of Organ Transplantation (ESOT) Guidelines' Taskforce. The overarching goal is to provide a framework to facilitate the development of outcome measures as an initial step to apply the VMC paradigm to liver transplantation.

ESOT Consensus Platform for Organ Transplantation: Setting the Stage for a Rigorous, Regularly Updated Development Process.

The European Society for Organ Transplantation (ESOT) has created a platform for the development of rigorous and regularly updated evidence based guidelines for clinical practice in the transplantation field. A dedicated Guideline Taskforce, including ESOT-council members, a representative from the Centre for Evidence in Transplantation, editors of the journal Transplant International has developed transparent procedures to guide the development of guidelines, recommendations, and consensus statements. During ESOT's first Consensus Conference in November 2022, leading experts will present in-depth evidence based reviews of nine themes and will propose recommendations aimed at reaching a consensus after public discussion and assessment by an independent jury. All recommendations and consensus statements produced for the nine selected topics will be published including the entire evidence-based consensus-finding process. An extensive literature review of each topic was conducted to provide final evidence and/or expert opinion.

Locoregional Therapy in the Management of Intrahepatic Cholangiocarcinoma: Is There Sufficient Evidence to Guide Current Clinical Practice?

PURPOSE OF REVIEW: Intrahepatic cholangiocarcinoma (iCCA) carries a dismal prognosis and, despite increasing incidence, still lacks effective treatments. In this scenario, locoregional therapies (LRT) are gaining interest as they may be effective at local tumor control and complementary to surgical and non-surgical approaches. In this article, we will review the evolving role of LRT performed by interventional radiologists in the management of iCCA. RECENT FINDINGS: Accumulating retrospective evidence indicates that ablative therapies and transarterial embolizations are of benefit for iCCA with unresectable disease, demonstrating promising safety profiles and prolonged or comparable survival outcomes compared to systemic therapy and surgery. Additionally, for surgical candidates, portal ± hepatic venous embolization can improve the safety of hepatectomy by inducing preoperative hypertrophy of the non-involved liver lobe. LRTs are playing an increasingly important role in the multimodal treatment of iCCA from various perspectives with reduced toxicity relative to traditional treatments. To expand the scope of applications for LRTs in this setting, future prospective randomized studies are needed to confirm their efficacy and advantage.

Holistic management of hepatocellular carcinoma: The hepatologist's comprehensive playbook.

Hepatocellular carcinoma (HCC) is a common complication in patients with chronic liver disease and leads to significant morbidity and mortality. Liver disease and liver cancer are preventable by mitigating and managing common risk factors, including chronic hepatitis B and C infection, alcohol use, diabetes, obesity and other components of the metabolic syndrome. The management of patients with HCC requires treatment of the malignancy and adequate control of the underlying liver disease, as preserving liver function is critical for successful cancer treatment and may have a relevant prognostic role independent of HCC management. Hepatologists are the ideal providers to guide the care of patients with HCC as they are trained to identify patients at risk, apply appropriate surveillance strategies, assess and improve residual liver function, evaluate candidacy for transplant, provide longitudinal care to optimize and preserve liver function during and after HCC treatment, survey for cancer recurrence and manage its risk factors, and prevent and treat decompensating events. We highlight the need for a team-based holistic approach to the patient with liver disease and HCC and identify necessary gaps in current care and knowledge.

Inflammatory pathways and cholangiocarcinoma risk mechanisms and prevention.

Cholangiocarcinoma (CCA), a neoplasm burdened by a poor prognosis and currently lacking adequate therapeutic treatments, can originate at different levels of the biliary tree, in the intrahepatic, hilar, or extrahepatic area. The main risk factors for the development of CCA are the presence of chronic cholangiopathies of various etiology. To date, the most studied prodromal diseases of CCA are primary sclerosing cholangitis, Caroli's disease and fluke infestations, but other conditions, such as metabolic syndrome, nonalcoholic fatty liver disease and obesity, are emerging as associated with an increased risk of CCA development. In this review, we focused on the analysis of the pro-inflammatory mechanisms that induce the development of CCA and on the role of cells of the immune response in cholangiocarcinogenesis. In very recent times, these cellular mechanisms have been the subject of emerging studies aimed at verifying how the modulation of the inflammatory and immunological responses can have a therapeutic significance and how these can be used as therapeutic targets.

Translational Value of Tumor-Associated Lymphangiogenesis in Cholangiocarcinoma.

The prognosis of cholangiocarcinoma remains poor in spite of the advances in immunotherapy and molecular profiling, which has led to the identification of several targetable genetic alterations. Surgical procedures, including both liver resection and liver transplantation, still represent the treatment with the best curative potential, though the outcomes are significantly compromised by the early development of lymph node metastases. Progression of lymphatic metastasis from the primary tumor to tumor-draining lymph nodes is mediated by tumor-associated lymphangiogenesis, a topic largely overlooked until recently. Recent findings highlight tumor-associated lymphangiogenesis as paradigmatic of the role played by the tumor microenvironment in sustaining cholangiocarcinoma invasiveness and progression. This study reviews the current knowledge about the intercellular signaling and molecular mechanism of tumor-associated lymphangiogenesis in cholangiocarcinoma in the hope of identifying novel therapeutic targets to halt a process that often limits the success of the few available treatments.

Molecular determinants of peri-apical targeting of inositol 1,4,5-trisphosphate receptor type 3 in cholangiocytes.

Fluid and bicarbonate secretion is a principal function of cholangiocytes, and impaired secretion results in cholestasis. Cholangiocyte secretion depends on peri-apical expression of the type 3 inositol trisphosphate receptor (ITPR3), and loss of this intracellular Ca2+ release channel is a final common event in most cholangiopathies. Here we investigated the mechanism by which ITPR3 localizes to the apical region to regulate secretion. Isolated bile duct units, primary mouse cholangiocytes, and polarized Madin-Darby canine kidney (MDCK) cells were examined using a combination of biochemical and fluorescence microscopy techniques to investigate the mechanism of ITPR3 targeting to the apical region. Apical localization of ITPR3 depended on the presence of intact lipid rafts as well as interactions with both caveolin 1 (CAV1) and myosin heavy chain 9 (MYH9). Chemical disruption of lipid rafts or knockdown of CAV1 or MYH9 redistributed ITPR3 away from the apical region. MYH9 interacted with the five c-terminal amino acids of the ITPR3 peptide. Disruption of lipid rafts impaired Ca2+ signaling, and absence of CAV1 impaired both Ca2+ signaling and fluid secretion. Conclusion: A cooperative mechanism involving MYH9, CAV1, and apical lipid rafts localize ITPR3 to the apical region to regulate Ca2+ signaling and secretion in cholangiocytes.