Liver Immune Profiling Reveals Pathogenesis and Therapeutics for Biliary Atresia.

Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.

Endocrine Autoimmune Disease as a Fragility of Immune Surveillance against Hypersecreting Mutants.

Some endocrine organs are frequent targets of autoimmune attack. Here, we addressed the origin of autoimmune disease from the viewpoint of feedback control. Endocrine tissues maintain mass through feedback loops that balance cell proliferation and removal according to hormone-driven regulatory signals. We hypothesized the existence of a dedicated mechanism that detects and removes mutant cells that missense the signal and therefore hyperproliferate and hypersecrete with potential to disrupt organismal homeostasis. In this mechanism, hypersecreting cells are preferentially eliminated by autoreactive T cells at the cost of a fragility to autoimmune disease. The "autoimmune surveillance of hypersecreting mutants" (ASHM) hypothesis predicts the presence of autoreactive T cells in healthy individuals and the nature of self-antigens as peptides from hormone secretion pathway. It explains why some tissues get prevalent autoimmune disease, whereas others do not and instead show prevalent mutant-expansion disease (e.g., hyperparathyroidism). The ASHM hypothesis is testable, and we discuss experimental follow-up.

LiverZap: a chemoptogenetic tool for global and locally restricted hepatocyte ablation to study cellular behaviours in liver regeneration.

The liver restores its mass and architecture after injury. Yet, investigating morphogenetic cell behaviours and signals that repair tissue architecture at high spatiotemporal resolution remains challenging. We developed LiverZap, a tuneable chemoptogenetic liver injury model in zebrafish. LiverZap employs the formation of a binary FAP-TAP photosensitiser followed by brief near-infrared illumination inducing hepatocyte-specific death and recapitulating mammalian liver injury types. The tool enables local hepatocyte ablation and extended live imaging capturing regenerative cell behaviours, which is crucial for studying cellular interactions at the interface of healthy and damaged tissue. Applying LiverZap, we show that targeted hepatocyte ablation in a small region of interest is sufficient to trigger local liver progenitor-like cell (LPC)-mediated regeneration, challenging the current understanding of liver regeneration. Surprisingly, the LPC response is also elicited in adjacent uninjured tissue, at up to 100 µm distance to the injury. Moreover, dynamic biliary network rearrangement suggests active cell movements from uninjured tissue in response to substantial hepatocyte loss as an integral step of LPC-mediated liver regeneration. This precisely targetable liver cell ablation tool will enable the discovery of key molecular and morphogenetic regeneration paradigms.

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Cholangiocytes Modulate CD100 Expression in the Liver and Facilitate Pathogenic T-Helper 17 Cell Differentiation.

BACKGROUND & AIMS: Chronic inflammation surrounding bile ducts contributes to the disease pathogenesis of most cholangiopathies. Poor efficacy of immunosuppression in these conditions suggests biliary-specific pathologic principles. Here we performed biliary niche specific functional interpretation of a causal mutation (CD100 K849T) of primary sclerosing cholangitis (PSC) to understand related pathogenic mechanisms. METHODS: Biopsy specimens of explanted livers and endoscopy-guided sampling were used to assess the CD100 expression by spatial transcriptomics, immune imaging, and high-dimensional flow cytometry. To model pathogenic cholangiocyte-immune cell interaction, splenocytes from mutation-specific mice were cocultured with cholangiocytes. Pathogenic pathways were pinpointed by RNA sequencing analysis of cocultured cells and cross-validated in patient materials. RESULTS: CD100 is mainly expressed by immune cells in the liver and shows a unique pattern around PSC bile ducts with RNA-level colocalization but poor detection at the protein level. This appears to be due to CD100 cleavage as soluble CD100 is increased. Immunophenotyping suggests biliary-infiltrating T cells as the major source of soluble CD100, which is further supported by reduced surface CD100 on T cells and increased metalloproteinases in cholangiocytes after coculturing. Pathogenic T cells that adhered to cholangiocytes up-regulated genes in the T-helper 17 cell differentiation pathway, and the CD100 mutation boosted this process. Consistently, T-helper 17 cells dominate biliary-resident CD4 T cells in patients. CONCLUSIONS: CD100 exerts its functional impact through cholangiocyte-immune cell cross talk and underscores an active, proinflammatory role of cholangiocytes that can be relevant to novel treatment approaches.

Clonal analysis of metachronous double biliary tract cancers.

The molecular mechanisms underpinning the development of metachronous tumors in the remnant bile duct following surgical resection of primary biliary tract carcinomas (BTCs) are unknown. This study aimed to elucidate these mechanisms by evaluating the clinicopathologic features of BTCs, the alterations to 31 BTC-related genes on targeted sequencing, and the aberrant expression of p53, p16, SMAD4, ARID1A and ß-catenin on immunohistochemistry. Twelve consecutive patients who underwent resection of metachronous BTCs following primary BTC resection with negative bile duct margins were enrolled. Among the 12 metachronous tumors, six exhibited anterograde growth in the lower portion and six exhibited retrograde growth in the upper portion of the biliary tree. Surgical resection of metachronous BTCs resulted in recurrence-free survival in seven, local recurrence in five, and death in two patients. Nine achieved 5-year overall survival after primary surgery. Molecular analyses revealed that recurrently altered genes were: TP53, SMAD4, CDKN2A, ELF3, ARID1A, GNAS, NF1, STK11, RNF43, KMT2D and ERBB3. Each of these was altered in at least three cases. A comparison of the molecular features between 12 paired primary and metachronous BTCs indicated that 10 (83%) metachronous tumors developed in clonal association with corresponding primary tumors either successionally or phylogenically. The remaining two (17%) developed distinctly. The successional tumors consisted of direct or evolved primary tumor clones that spread along the bile duct. The phylogenic tumors consisted of genetically unstable clones and conferred a poor prognosis. Metachronous tumors distinct from their primaries harbored fewer mutations than successional and phylogenic tumors. In conclusion, over 80% of metachronous BTCs that develop following primary BTC resection are probably molecularly associated with their primaries in either a successional or a phylogenetic manner. Comparison between the molecular features of a metachronous tumor and those of a preceding tumor may provide effective therapeutic clues for the treatment of metachronous BTC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Obliteration of portal venules contributes to portal hypertension in biliary cirrhosis.

The effects of the obliteration of portal venules (OPV) in cirrhotic portal hypertension are poorly understood. To investigate its contribution to portal hypertension in biliary cirrhosis and its underlying mechanism, we evaluated OPV using two-dimensional (2D) histopathology in liver explants from patients with biliary atresia (BA, n = 63), primary biliary cholangitis (PBC, n = 18), and hepatitis B-related cirrhosis (Hep-B-cirrhosis, n = 35). Then, three-dimensional (3D) OPV was measured by X-ray phase-contrast CT in two parallel models in rats following bile duct ligation (BDL) or carbon tetrachloride (CCl4) administration, representing biliary cirrhosis and post-necrotic cirrhosis, respectively. The portal pressure was also measured in the two models. Finally, the effects of proliferative bile ducts on OPV were investigated. We found that OPV was significantly more frequent in patients with biliary cirrhosis, including BA (78.57 ± 16.45%) and PBC (60.00 ± 17.15%), than that in Hep-B-cirrhotic patients (29.43 ± 14.94%, p < 0.001). OPV occurred earlier, evidenced by the paired liver biopsy at a Kasai procedure (KP), and was irreversible even after a successful KP in the patients with BA. OPV was also significantly more frequent in the BDL models than in the CCl4 models, as shown by 2D and 3D quantitative analysis. Portal pressure was significantly higher in the BDL model than that in the CCl4 model. With the proliferation of bile ducts, portal venules were compressed and irreversibly occluded, contributing to the earlier and higher portal pressure in biliary cirrhosis. OPV, as a pre-sinusoidal component, plays a key role in the pathogenesis of portal hypertension in biliary cirrhosis. The proliferated bile ducts and ductules gradually take up the 'territory' originally attributed to portal venules and compress the portal venules, which may lead to OPV in biliary cirrhosis. © 2024 The Pathological Society of Great Britain and Ireland.

HSCs-derived exosomes regulate the levels of inflammatory cytokines in HIBECs through miR-122-5p mediated p38 MAPK signaling pathway.

PBC is an autoimmune-mediated liver disease, and intrahepatic biliary epithelial cells (IBECs) are the target cells of early damage. Previous studies found that miRNAs and inflammation is closely related to PBC. In this study, we extracted exosomes from serum and human IBECs supernatant, and RNA-sequence analyzed the expression profiles of miRNAs. Elisa measured the levels of inflammatory cytokines. RT- qPCR and western blot detected the levels of miR-122-5p, p38 and p-p38. The results showed that 263 differentially expressed (DE) miRNAs were identified in serum exosomes of PBC patients. The levels of IL-1ß, IL-6, IL-12, IL-17 A, IFN-γ, TNF-α and TGF-ß1 in peripheral blood of PBC patients were higher than those of normal controls. According to the validation results and previous literature, exosomal miR-122-5p was finally selected as the study object, and correlated with inflammatory factors. In vitro experiments further found that exosomal miR-122-5p may derive from hepatic stellate cells (HSCs), and can be HIBECs intake, and influence HIBECs inflammatory factor levels though p38 MAPK signaling pathways. This may provide a new strategy for the treatment of PBC.

Clinical and Bacteriological Specificities of Escherichia coli Bloodstream Infections From Biliary Portal of Entries.

BACKGROUND: Escherichia coli is frequently responsible for bloodstream infections (BSIs). Among digestive BSIs, biliary infections appear to be less severe. Respective roles of host factors, bacterial determinants (phylogroups, virulence, and antibiotic resistance), and portal of entry on outcome are unknown. METHODS: Clinical characteristics and prognosis of 770 episodes of E coli BSI were analyzed and isolates sequenced (Illumina technology) comparing phylogroups, multilocus sequence type, virulence, and resistance gene content. BSI isolates were compared with 362 commensal E coli from healthy subjects. RESULTS: Among 770 episodes, 135 were biliary, 156 nonbiliary digestive, and 479 urinary. Compared to urinary infections, BSIs of digestive origin occurred significantly more in men, comorbid, and immunocompromised patients. Digestive portal of entry was significantly associated with septic shock and death. Among digestive infections, patients with biliary infections were less likely to die (P = .032), despite comparable initial severity. Biliary E coli resembled commensals (phylogroup distribution, sequence type, and few virulence-associated genes) whereas nonbiliary digestive and urinary strains carried many virulence-associated genes. CONCLUSIONS: Escherichia coli strains responsible for biliary infections exhibit commensal characteristics and are associated with lower mortality rates, despite similar initial severity, than other digestive BSIs. Biliary drainage in addition to antibiotics in the management of biliary infections may explain improved outcome.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

Proteomics Defines Plasma Biomarkers for the Early Diagnosis of Biliary Atresia.

Early diagnosis of biliary atresia (BA) is crucial for improving the chances of survival and preserving the liver function of pediatric patients with BA. Herein, we performed proteomics analysis using data-independent acquisition (DIA) and parallel reaction monitoring (PRM) to explore potential biomarkers for the early diagnosis of BA compared to other non-BA jaundice cases. Consequently, we detected and validated differential protein expression in the plasma of patients with BA compared to the plasma of patients with intrahepatic cholestasis. Bioinformatics analysis revealed the enriched biological processes characteristic of BA by identifying the differential expression of specific proteins. Signaling pathway analysis revealed changes in the expression levels of proteins associated with an alteration in immunoglobulin levels, which is indicative of immune dysfunction in BA. The combination of polymeric immunoglobulin receptor expression and immunoglobulin lambda variable chain (IGL c2225_light_IGLV1-47_IGLJ2), as revealed via machine learning, provided a useful early diagnostic model for BA, with a sensitivity of 0.8, specificity of 1, accuracy of 0.89, and area under the curve value of 0.944. Thus, our study identified a possible effective plasma biomarker for the early diagnosis of BA and could help elucidate the underlying mechanisms of BA.

Discrimination of pancreato-biliary cancer and pancreatitis patients by non-invasive liquid biopsy.

BACKGROUND: Current diagnostics for the detection of pancreato-biliary cancers (PBCs) need to be optimized. We therefore propose that methylated cell-free DNA (cfDNA) derived from non-invasive liquid biopsies serves as a novel biomarker with the ability to discriminate pancreato-biliary cancers from non-cancer pancreatitis patients. METHODS: Differentially methylated regions (DMRs) from plasma cfDNA between PBCs, pancreatitis and clinical control samples conditions were identified by next-generation sequencing after enrichment using methyl-binding domains and database searches to generate a discriminatory panel for a hybridization and capture assay with subsequent targeted high throughput sequencing. RESULTS: The hybridization and capture panel, covering around 74 kb in total, was applied to sequence a cohort of 25 PBCs, 25 pancreatitis patients, 25 clinical controls, and seven cases of Intraductal Papillary Mucinous Neoplasia (IPMN). An unbiased machine learning approach identified the 50 most discriminatory methylation markers for the discrimination of PBC from pancreatitis and controls resulting in an AUROC of 0.85 and 0.88 for a training (n = 45) and a validation (n = 37) data set, respectively. The panel was also able to distinguish high grade from low grade IPMN samples. CONCLUSIONS: We present a proof of concept for a methylation biomarker panel with better performance and improved discriminatory power than the current clinical marker CA19-9 for the discrimination of pancreato-biliary cancers from non-cancerous pancreatitis patients and clinical controls. This workflow might be used in future diagnostics for the detection of precancerous lesions, e.g. the identification of high grade IPMNs vs. low grade IPMNs.

Dietary intake, obesity, and physical activity in association with biliary tract cancer risk: Results from meta-analyses of individual-level data from prospective cohort studies of 723,326 adults.

Biliary tract cancer (BTC) is a rare and aggressive malignancy with increasing incidence. Most BTC cases are diagnosed with metastatic disease which carries a 5-year survival rate of <5%. Physical activity, diet, and obesity might be associated with BTC risk, but studies have been limited particularly in African descendants. We addressed this knowledge gap by evaluating associations of BTC risk with obesity, physical activity, and dietary intakes in 723,326 adult participants in four cohort studies conducted in China, the United Kingdom, and the United States. Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) in each cohort; results were combined using meta-analysis. All cohorts had ≥11 median follow-up years with 839 incident BTC cases combined. BTC risk was positively associated with body mass index (BMI) and waist-to-hip ratio (WHR) whereas physical activity, fruit intake, and fish intake were inversely associated. HR and (95% CI) comparing BMI >35.0 to 18.5-24.9: 1.71 (1.26, 2.31), p-trend <.0001; comparing BMI-adjusted WHR top to bottom quartile: 1.20 (0.94, 1.53), p-trend = .05; comparing ≥15-0 metabolic equivalent task-hours/week 0.76 (0.61, 0.94), p-trend = .009; comparing highest to lowest intake tertile for fruit and fish 0.79 (0.66, 0.95), p-trend = .01; 0.82 (0.68, 0.98), p-trend = .04, respectively. Associations were, in general, similar across ancestry groups. Our study provides strong evidence for important roles of obesity, diet, and physical activity in BTC etiology and stresses the need for lifestyle modification to combat the rising incidence of this fatal malignancy.

Obesity is associated with biliary tract cancer mortality and incidence: A pooled analysis of 21 cohort studies in the Asia Cohort Consortium.

Body fatness is considered a probable risk factor for biliary tract cancer (BTC), whereas cholelithiasis is an established factor. Nevertheless, although obesity is an established risk factor for cholelithiasis, previous studies of the association of body mass index (BMI) and BTC did not take the effect of cholelithiasis fully into account. To better understand the effect of BMI on BTC, we conducted a pooled analysis using population-based cohort studies in Asians. In total, 905 530 subjects from 21 cohort studies participating in the Asia Cohort Consortium were included. BMI was categorized into four groups: underweight (<18.5 kg/m2 ); normal (18.5-22.9 kg/m2 ); overweight (23-24.9 kg/m2 ); and obese (25+ kg/m2 ). The association between BMI and BTC incidence and mortality was assessed using hazard ratios (HR) and 95% confidence intervals (CIs) by Cox regression models with shared frailty. Mediation analysis was used to decompose the association into a direct and an indirect (mediated) effect. Compared to normal BMI, high BMI was associated with BTC mortality (HR 1.19 [CI 1.02-1.38] for males, HR 1.30 [1.14-1.49] for females). Cholelithiasis had significant interaction with BMI on BTC risk. BMI was associated with BTC risk directly and through cholelithiasis in females, whereas the association was unclear in males. When cholelithiasis was present, BMI was not associated with BTC death in either males or females. BMI was associated with BTC death among females without cholelithiasis. This study suggests BMI is associated with BTC mortality in Asians. Cholelithiasis appears to contribute to the association; and moreover, obesity appears to increase BTC risk without cholelithiasis.

Efficacy and biomarker analysis of second-line nab-paclitaxel plus sintilimab in patients with advanced biliary tract cancer.

Biliary tract cancer (BTC) is a highly aggressive malignancy with limited second-line therapy. We conducted this phase 2 trial to evaluate the efficacy and safety of second-line nab-paclitaxel plus sintilimab in advanced BTC. Histologically confirmed advanced BTC patients with documented disease progression after first-line chemotherapy were enrolled. Subjects received nab-paclitaxel 125 mg/m2 on days 1 and 8 plus sintilimab 200 mg on day 1, administered every 3 weeks. The primary end point was the objective response rate (ORR). The secondary end points were progression-free survival (PFS), overall survival (OS), and adverse reactions. Simultaneously, next-generation sequencing, programmed cell death ligand 1 immunohistochemistry and multiplex immunofluorescence of tumor-infiltrating lymphocytes were applied to explore potential biomarkers. Twenty-six subjects were consecutively enrolled. The ORR was 26.9% (7/26), including two complete responses and five partial responses, which met the primary end point. The disease control rate was 61.5% (16/26). The median PFS was 169 days (about 5.6 months, 95% confidence interval [CI] 60-278 days). The median OS was 442 days (about 14.7 months, 95% CI 298-586 days). Grade 3 treatment-related adverse events (TRAEs) were mainly anemia (27%), leukopenia (23%), neutropenia (19%), and peripheral sensory neuropathy (8%). No grade 4 or 5 TRAEs occurred. Biomarker analysis suggested that positive PD-L1 and high proportions of CD8+ T-cell infiltration were correlated with improved clinical outcome. Nab-paclitaxel plus sintilimab is a potentially effective and tolerable second-line regimen for advanced BTC that deserves to be studied in large-scale trials. PD-L1 status and CD8+ T cell infiltration might be promising biomarkers for efficacy prediction.

SWOG 1609 cohort 48: anti-CTLA-4 and anti-PD-1 for advanced gallbladder cancer.

INTRODUCTION: Most patients with advanced gallbladder cancer are treated with multiagent chemotherapy. Immune checkpoint inhibitors offer the possibility of a durable response with less toxicity. This prospective, multicenter, open-label study was designed to evaluate the anticancer activity of nivolumab plus ipilimumab in patients with advanced gallbladder cancer. METHODS: Nineteen patients with advanced gallbladder cancer refractory to ≥1 previous therapy received nivolumab 240 mg intravenously every 2 weeks and ipilimumab 1 mg/kg intravenously every 6 weeks until disease progression or unacceptable toxicity. The primary end point was confirmed radiographic overall response rate (ORR) (complete response [CR] + partial response [PR] confirmed on subsequent scan); secondary end points included unconfirmed overall response, clinical benefit rate (confirmed and unconfirmed responses + stable disease >6 months), progression-free survival, overall survival, and toxicity. RESULTS: The confirmed ORR was 16% (CR, n = 1 [5%]; PR, n = 2 [11%]); all were microsatellite stable, and the confirmed CR had undetectable programmed death-ligand 1 by immunohistochemistry. The unconfirmed ORR and clinical benefit rates were both 32%. The median duration of response was 14.8 months (range, 4-35.1+ months). The 6-month progression-free survival was 26% (95% CI, 12-55). The median overall survival was 7.0 months (95% CI, 3.9-19.1). The most common toxicities were fatigue (32%), anemia (26%), and anorexia (26%). Aspartate aminotransferase elevation was the most common grade 3/4 toxicity (11%). There was 1 possibly related death (sepsis with attendant hepatic failure). CONCLUSIONS: Ipilimumab plus nivolumab was well tolerated and showed modest efficacy with durable responses in previously treated patients with advanced gallbladder cancer. CLINICAL TRIAL REGISTRATION: NCT02834013 (ClincialTrials.gov). PLAIN LANGUAGE SUMMARY: This prospective study assessed the efficacy and safety of nivolumab plus ipilimumab in 19 patients with advanced gallbladder cancer refractory to previous therapy. The combination demonstrated modest efficacy with a 16% confirmed overall response rate, durable responses, and manageable toxicities, suggesting potential benefits for this challenging patient population.

Long Term Ex-Situ Normothermic Machine Perfusion Allows Regeneration of Human Livers with Severe Bile Duct Injury.

Bile duct regeneration is hypothesised to prevent biliary strictures, a leading cause of morbidity after liver transplantation. Assessing the capacity for biliary regeneration may identify grafts as suitable for transplantation that are currently declined, but has been unfeasible until now. This study used Long-Term Ex-Situ Normothermic Machine Perfusion (LT-NMP) to assess biliary regeneration. Human livers that were declined for transplantation were perfused at 36°C for up to 13.5 days. Bile duct biopsies, bile and perfusate were collected throughout perfusion, which were examined for features of injury and regeneration. Biliary regeneration was defined as new Ki-67 positive biliary epithelium following severe injury. Ten livers were perfused for a median duration of 7.5 days. Severe bile duct injury occurred in all grafts, and biliary regeneration occurred in 70% of grafts. Traditional biomarkers of biliary viability such as bile glucose improved during perfusion but this was not associated with biliary regeneration (p>0.05). In contrast, the maintenance of IL-6 and VEGF-A levels in bile were associated with biliary regeneration (p=0.017 for both cytokines). This is the first study to demonstrate biliary regeneration during LT-NMP and identify a cytokine signature in bile as a novel biomarker for biliary regeneration during LT-NMP.

Biliary complications after adult-to-adult living-donor liver transplantation: An international multicenter study of 3633 cases.

In living-donor liver transplantation, biliary complications including bile leaks and biliary anastomotic strictures remain significant challenges, with incidences varying across different centers. This multicentric retrospective study (2016-2020) included 3633 adult patients from 18 centers and aimed to identify risk factors for these biliary complications and their impact on patient survival. Incidences of bile leaks and biliary strictures were 11.4% and 20.6%, respectively. Key risk factors for bile leaks included multiple bile duct anastomoses (odds ratio, [OR] 1.8), Roux-en-Y hepaticojejunostomy (OR, 1.4), and a history of major abdominal surgery (OR, 1.4). For biliary anastomotic strictures, risk factors were ABO incompatibility (OR, 1.4), blood loss >1 L (OR, 1.4), and previous abdominal surgery (OR, 1.7). Patients experiencing biliary complications had extended hospital stays, increased incidence of major complications, and higher comprehensive complication index scores. The impact on graft survival became evident after accounting for immortal time bias using time-dependent covariate survival analysis. Bile leaks and biliary anastomotic strictures were associated with adjusted hazard ratios of 1.7 and 1.8 for graft survival, respectively. The study underscores the importance of minimizing these risks through careful donor selection and preoperative planning, as biliary complications significantly affect graft survival, despite the availability of effective treatments.

Primary cilia as a targetable node between biliary injury, senescence and regeneration in liver transplantation.

BACKGROUND & AIMS: Biliary complications are a major cause of morbidity and mortality in liver transplantation. Up to 25% of patients that develop biliary complications require additional surgical procedures, re-transplantation or die in the absence of a suitable regraft. Here, we investigate the role of the primary cilium, a highly-specialised sensory organelle, in biliary injury leading to post-transplant biliary complications. METHODS: Human biopsies were used to study the structure and function of primary cilia in liver transplant recipients that develop biliary complications (N=7) in comparison with recipients without biliary complications (N=12). To study the biological effects of the primary cilia during transplantation, we generated murine models that recapitulate liver procurement and cold storage, and assessed the elimination of the primary cilia in biliary epithelial cells in the K19CreERTKif3aflox/flox mouse model. To explore the molecular mechanisms responsible for the observed phenotypes we used in vitro models of ischemia, cellular senescence and primary cilia ablation. Finally, we used pharmacological and genetic approaches to target cellular senescence and the primary cilia, both in mouse models and discarded human donor livers. RESULTS: Prolonged ischemic periods before transplantation result in ciliary shortening and cellular senescence, an irreversible cell cycle arrest that blocks regeneration. Our results indicate that primary cilia damage results in biliary injury and a loss of regenerative potential. Senescence negatively impacts primary cilia structure and triggers a negative feedback loop that further impairs regeneration. Finally, we explore how targeted interventions for cellular senescence and/or the stabilisation of the primary cilia improve biliary regeneration following ischemic injury. CONCLUSIONS: Primary cilia play an essential role in biliary regeneration and we demonstrate that senolytics and cilia-stabilising treatments provide a potential therapeutic opportunity to reduce the rate of biliary complications and improve clinical outcomes in liver transplantation. IMPACT AND IMPLICATIONS: Up to 25% of liver transplants result in biliary complications, leading to additional surgery, retransplants, or death. We found that the incidence of biliary complications is increased by damage to the primary cilium, an antenna that protrudes from the cell and is key to regeneration. Here, we show that treatments that preserve the primary cilia during the transplant process provide a potential solution to reduce the rates of biliary complications.

PTEN deficiency induces an extrahepatic cholangitis-cholangiocarcinoma continuum via aurora kinase A in mice.

BACKGROUND & AIMS: The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aimed to evaluate the role of PTEN in the pathogenesis of eCCA and identify novel therapeutic targets for this disease. METHODS: The Pten gene was genetically deleted using the Cre-loxp system in biliary epithelial cells. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry, reverse-transcription PCR, cell culture, and RNA sequencing. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. We also tested the effectiveness of an Aurka inhibitor. RESULTS: We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as 1 month after birth. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated disease progression, potentially by downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expression of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS: Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum that was dependent on Aurka. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS: The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition, cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted disease progression. This model will be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.