Citations

Abstract Background and Aims Primary sclerosing cholangitis (PSC) increases the risk of developing cholangiocarcinoma (CCA), which is the leading cause of premature death in these patients. Current diagnostic methods for early detection of either condition remain largely suboptimal. This study investigates serum metabolites as potential non-invasive diagnostic biomarkers. Methods This multicenter international study analyzed 459 serum samples from 13 international centers, including patients with PSC (n = 216), PSC who developed CCA during follow-up (PSC to CCA; n = 24), PSC with concomitant CCA (PSC-CCA; n = 88), ulcerative colitis (UC; n = 14), and healthy individuals (n = 79). Serum metabolomics was evaluated by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) and the accuracy of the single candidate metabolite biomarkers was further assessed. Machine learning was used to generate the best diagnostic and predictive algorithms. Results Fifty-two serum metabolites were identified as markers of PSC, independent of age, biological sex, cirrhosis, UC, or ursodeoxycholic acid (UDCA) treatment. A diagnostic model based on only six lipids distinguished PSC patients from healthy controls with high accuracy (AUC 0.952 discovery, 0.918 validation), independently of the levels of transaminases or cholestasis markers (i.e., GGT, AP, bilirubin) in blood. Subgroup analyses were performed in PSC patients with and without inflammatory bowel disease (IBD), confirming the model’s consistent performance across both subgroups when compared with their respective control groups – UC and healthy controls (AUC 0.959 and 0.939, respectively). The PSC model also confirmed its diagnostic performance in patients with established PSC-CCA or progression to CCA within 2 years, when compared to UC and healthy controls, showing AUC values ranging from 0.911 to 0.976. Additionally, 20 metabolites were significantly altered in PSC patients with CCA (PSC-CCA), regardless of demographics, cirrhosis, UDCA treatment, IBD status, or CCA subtype, and independently of serum transaminases and cholestatic markers levels. A model incorporating six lipids accurately identified PSC-CCA cases compared to PSC patients (AUC = 0.912 discovery; 0.896 validation), showing excellent performance in early-stage tumor detection (AUC 0.921) and outperforming CA19-9 (AUC 0.708). Importantly, the model retained high accuracy even in CCA patients with low CA19-9 levels (AUC 0.905) and in PSC patients who developed CCA within a year of clinical diagnosis (AUC 0.792). Conclusion The PSC and PSC-CCA blood tests provide a non-invasive approach for diagnosing PSC and PSC-CCA, addressing an important need in this population. Their integration into clinical practice could enhance risk stratification, early detection, personalized surveillance, and treatment decision-making in PSC patients.

Abstract Background and Aims Cholangiocarcinoma (CCA) is a rare and aggressive malignancy originating from the bile ducts, characterized by a poor prognosis and limited treatment options. Potentially curative interventions include surgical resection with negative margins (R0) and liver transplantation. However, despite these treatments, the likelihood of post-surgical recurrence remains high, with 50–70% of patients experiencing disease recurrence within five years. This highlights the urgent need for reliable prognostic biomarkers to guide clinical decision-making and personalize postoperative care. Numerous prognostic biomarkers identified in tumor tissue for CCA have been proposed, but many studies suffer from limitations such as single-center designs, small patient cohorts, and semi-quantitative techniques such as immunohistochemistry. These methods often rely on observer interpretation and antibody specificity, which can compromise objectivity and reproducibility. To overcome these limitations, we aimed to identify and validate robust quantitative prognostic biomarkers for CCA by analyzing tumor mRNA expression, integrating them using machine learning (ML), and developing a web-based tool to predict tumor recurrence and overall survival (OS). Method A systematic literature review was conducted to identify candidate prognostic tissue biomarkers for CCA, which were further validated using Cox proportional hazards (PH) OS analyses in five independent transcriptomic cohorts. An international multicenter cohort of 221 patients undergoing tumor resection was assembled, and tumor mRNA expression was analyzed using high-throughput TaqMan OpenArray RT-qPCR. Associations between clinical and molecular features and recurrence-free survival (RFS) and OS were assessed using Cox PH analyses. Six ML survival models were trained and evaluated using nested cross-validation, followed by feature reduction. Shapley Additive exPlanations (SHAP) values were applied to assess feature importance and enhance model interpretability. Results Among 496 candidate CCA prognostic biomarkers identified from the literature, 52 showed prognostic value in Cox PH analyses across transcriptomic cohorts. In the multicenter validation cohort, integrative Cox PH analyses incorporating clinical variables and gene expression identified 11 genes significantly associated with RFS and 25 with OS. The 11 RFS-associated genes, combined with five clinical variables, were used to train six ML survival models, among which the random survival forest (RSF) model demonstrated the highest predictive accuracy. Following training of RSF models across all combinations of four genes and five clinical variables, and subsequent feature importance–based selection, a final model incorporating three genes (FSCN1, LDHA, and SLC2A1) and four clinical variables (CCA subtype, resection margin status, biological sex, and age at surgery) achieved a testing time-dependent AUC of 0.82. This model significantly stratified patients into high- and low recurrence-risk groups, with a median RFS (mRFS) of 8 months and 70 months, respectively. In addition, an OS prediction model using the same feature set achieved a testing AUC of 0.775 and significantly stratified patients according to 5-year OS risk, with a median OS (mOS) of 23 months in the high-risk group and 77 months in the low-risk group. Conclusion We identified and validated a robust panel of prognostic tissue biomarkers in patients with resected CCA. Integration of these biomarkers with clinical variables into ML-based prognostic models enabled accurate prediction of RFS and OS. The resulting web-based tool allows clinicians to input RT-qPCR results for the validated biomarkers and obtain precise, individualized prognostic predictions for postoperative recurrence and survival.

Background and Aims: Cholangiocarcinoma (CCA) is a rare but aggressive malignancy with poorly understood genetic susceptibility. To date, genome-wide association studies (GWAS) investigating germline variants associated with CCA risk remain limited. We aimed to identify genetic risk loci for CCA and its clinical subtypes through comprehensive GWAS and post-GWAS analyses. Approach and Results: We conducted a GWAS of 2366 CCA cases and 11,750 controls of European ancestry. Genome-wide significant loci ( p <5×10 −8 ) were identified and further examined through fine-mapping, functional annotation, and HLA imputation. Subgroup analyses were conducted by CCA subtypes and primary sclerosing cholangitis (PSC) status. Cross-trait linkage disequilibrium score regression and Mendelian randomization were employed to investigate the shared genetic architecture and potential causal relationships with a diverse range of traits. We identified 1 new genome-wide significant variant, rs535777 (OR=1.44), near HLA-DRB1/DQA1 associated with CCA, and 2 variants associated with extrahepatic CCA: rs116224263 (OR=0.17) in LINC02506 at 4p15.1 and rs6914950 (OR=1.63) near HLA-DRB1/DQB1 . Stratified analyses revealed rs2395184 (OR=3.51) near HLA-DRA/DRB5 associated with PSC-related CCA, and rs142674434 (OR=2.98) in THSD7A at 7p21.3 associated with non-PSC-related CCA. HLA imputation uncovered new amino acid residues associated with disease risk. Cross-trait analyses identified shared genetic signals between CCA and anthropometric, lipidemic, lifestyle, and medical traits. Mendelian randomization supported putative causal associations for 12 traits with CCA or its subtypes. Conclusions: Our large-scale GWAS highlights new genetic variants and HLA-linked mechanisms underlying CCA susceptibility. Integrating multi-step post-GWAS approaches enhances understanding of CCA pathogenesis and may facilitate the development of risk biomarkers for early detection and precision prevention strategies.

ABSTRACT Introduction Hepatocellular carcinoma (HCC) development in patients with metabolic dysfunction‐associated steatotic liver disease (MASLD) is a growing health concern, but the underlying mechanisms are not fully understood. Epigenetic aging biomarkers, reflecting cellular and tissue aging, have been linked to various age‐related pathologies, but their association with MASLD‐HCC is unknown. We investigated associations between five epigenetic aging biomarkers and MASLD‐HCC risk. Methods We performed whole blood DNA methylation assay (Infinium 850k array) and calculated principal components‐based (PC) versions of HorvathAge, HannumAge, PhenoAge and GrimAge and the DunedinPACE aging rate. We further calculated relative age accelerations for PCHorvathAge, PCHannumAge, PCPhenoAge and PCGrimAge. The aging biomarkers were modelled as continuous variables and categorised into tertiles based on distributions among controls. Associations between each aging biomarker and MASLD‐HCC were examined using logistic regression, calculating odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for covariates. Results Data on 272 MASLD‐HCC cases and 316 cancer‐free MASLD controls recruited from six sites and matched on chronological age, sex and study site were analysed. Higher relative age accelerations of PCPhenoAge (OR T3 vs. T1 = 2.25, 95% CI: 1.45–3.50; OR continuous = 1.04, 95% CI: 1.02–1.07, p = 0.009), PCGrimAge (OR T3 vs. T1 = 3.97, 95% CI: 2.41–6.64; OR continuous = 1.16, 95% CI: 1.10–1.24, p = 8.76 × 10 −07 ) and DunedinPACE (OR T3 vs. T1 = 3.45, 95% CI: 2.17–5.55; OR continuous = 1.72, 95% CI: 1.43–2.10, p = 2.58 × 10 −08 ) were associated with MASLD‐HCC, but not PCHorvathAge or PCHannumAge. Conclusion Higher relative age accelerations of PCPhenoAge, PCGrimAge and DunedinPACE aging rate are associated with risk of MASLD‐HCC. These aging biomarkers could improve HCC risk assessment and facilitate risk stratification in patients with MASLD.

Objective: To establish comprehensive recommendations on the management of perihilar cholangiocarcinoma (pCCA) and to identify areas in need of future investigation. Summary Background Data: The lack of consensus in management of pCCA across institutions worldwide impairs optimal patient care and best-achievable oncological outcome. A holistic and critical assessment of the body of evidence and collaborative guidance are required. Methods: The Consensus4pCCA conference developed unbiased consensus using the Zurich-Danish model. An impartial and multidisciplinary jury adopted comprehensive recommendations grounded on the work of eight expert panels answering predefined clinical questions and considering the best-quality evidence through a systematic review. The development of recommendations complied with GRADE and SIGN50 methodologies. Results: Seventy-one international experts considered 570 studies. Ten jury members concluded 71 recommendations covering definition and diagnosis, preoperative assessment and optimization, surgical resection, liver transplantation, and outcome reporting for pCCA patients. The consensus underscored that making patients amenable to high-quality curative resection and reducing its associated morbidity must be of highest priority. As such, multidisciplinary expertise and centralized care at all stages of disease management are critical to ensure both safety and oncological adequacy. To overcome the evidence-gap for rare diseases like pCCA, the jury urged large-scale registries to address open questions across the disease continuum and highlighted priorities for future investigation. Conclusion: The Consensus4pCCA represents an unprecedented commitment to collectively harmonize global treatment strategies for pCCA. This collective effort bridges the gap between expert opinions and clinical evidence and holds great promise to improve patient outcomes in the future.

Abstract Animal studies implicate one-carbon metabolism and DNA methylation genes in hepatocellular carcinoma (HCC) development in the setting of metabolic perturbations. Using human samples, we investigated the associations between common and rare variants in these closely related biochemical pathways and risk for metabolic HCC development in a multicenter international study. We performed targeted exome sequencing of 64 genes among 556 metabolic HCC cases and 643 cancer-free controls with metabolic conditions. Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for multiple comparisons. Gene-burden tests were used for rare variant associations. Analyses were performed in the overall sample and among non-Hispanic whites. The results show that among non-Hispanic whites, presence of rare functional variants in ABCC2 was associated with 7-fold higher risk of metabolic HCC (OR = 6.92, 95% CI: 2.38–20.15, P = 0.0004), and this association remained significant when analyses were restricted to functional rare variants observed in ≥2 participants (cases 3.2% versus controls 0.0%, P = 1.02 × 10−5). In the overall multiethnic sample, presence of rare functional variants in ABCC2 was nominally associated with metabolic HCC (OR = 3.60, 95% CI: 1.52–8.58, P = 0.004), with similar nominal association when analyses were restricted to functional rare variants observed in ≥2 participants (cases 2.9% versus controls 0.2%, P = 0.006). A common variant in PNPLA3 (rs738409[G]) was associated with higher HCC risk in the overall sample (P = 6.36 × 10−6) and in non-Hispanic whites (P = 0.0002). Our findings indicate that rare functional variants in ABCC2 are associated with susceptibility to metabolic HCC in non-Hispanic whites. PNPLA3-rs738409 is also associated with metabolic HCC risk.

Abstract Background & Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD‐causative genes, encoding for endoplasmic reticulum (ER)‐resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. Methods ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4‐phenylbutyric acid (4‐PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. Results The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4‐PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4‐PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. Conclusions Restoration of proteostasis in cystic cholangiocytes with 4‐PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

Cholangiocarcinoma (CCA) is the second most common malignancy arising in the liver. It carries a poor prognosis, in part because its pathogenesis is not well understood. The type 3 inositol 1,4,5‐trisphosphate receptor (ITPR3) is the principal intracellular calcium ion (Ca2+) release channel in cholangiocytes, and its increased expression has been related to the pathogenesis of malignancies in other types of tissues, so we investigated its role in CCA. ITPR3 expression was increased in both hilar and intrahepatic CCA samples as well as in CCA cell lines. Deletion of ITPR3 from CCA cells impaired proliferation and cell migration. A bioinformatic analysis suggested that overexpression of ITPR3 in CCA would have a mitochondrial phenotype, so this was also examined. ITPR3 normally is concentrated in a subapical region of endoplasmic reticulum (ER) in cholangiocytes, but both immunogold electron microscopy and super‐resolution microscopy showed that ITPR3 in CCA cells was also in regions of ER in close association with mitochondria. Deletion of ITPR3 from these cells impaired mitochondrial Ca2+ signaling and led to cell death. Conclusion: ITPR3 expression in cholangiocytes becomes enhanced in CCA. This contributes to malignant features, including cell proliferation and migration and enhanced mitochondrial Ca2+ signaling.

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6‐targeting miRNAs, miR‐433 and miR‐22, are down‐regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin‐5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin‐5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin‐5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. Conclusion: These data suggest that down‐regulated Exportin‐5 impairs the nuclear export of miR‐433 and miR‐22 precursor forms, causing a decrease in levels of mature miR‐433 and miR‐22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR‐506 is overexpressed in PBC cholangiocytes and directly targets both Cl – / anion exchanger 2 and type III inositol 1,4,5‐trisphosphate receptor, leading to cholestasis. Here, the regulation of miR‐506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin‐8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR‐506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR‐506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR‐506 (1) induced dedifferentiation with down‐regulation of biliary and epithelial markers together with up‐regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase‐3‐dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR‐506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. Conclusion : Different proinflammatory cytokines enhance the expression of miR‐506 in biliary epithelial cells; miR‐506 induces PBC‐like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (H epatology 2018;67:1420‐1440)