Molecular portraits of patients with intrahepatic cholangiocarcinoma who diverge as rapid progressors or long survivors on chemotherapy.

OBJECTIVE: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. DESIGN: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. RESULTS: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. CONCLUSIONS: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Immunogenomics of cholangiocarcinoma.

The development of cholangiocarcinoma spans years, if not decades, during which the immune system becomes corrupted and permissive to primary tumor development and metastasis. This involves subversion of local immunity at tumor sites, as well as systemic immunity and the wider host response. While immune dysfunction is a hallmark of all cholangiocarcinoma, the specific steps of the cancer-immunity cycle that are perturbed differ between patients. Heterogeneous immune functionality impacts the evolutionary development, pathobiological behavior, and therapeutic response of these tumors. Integrative genomic analyses of thousands of primary tumors have supported a biological rationale for immune-based stratification of patients, encompassing immune cell composition and functionality. However, discerning immune alterations responsible for promoting tumor initiation, maintenance, and progression from those present as bystander events remains challenging. Functionally uncoupling the tumor-promoting or tumor-suppressing roles of immune profiles will be critical for identifying new immunomodulatory treatment strategies and associated biomarkers for patient stratification. This review will discuss the immunogenomics of cholangiocarcinoma, including the impact of genomic alterations on immune functionality, subversion of the cancer-immunity cycle, as well as clinical implications for existing and novel treatment strategies.

miR-21-5p promotes NASH-related hepatocarcinogenesis.

BACKGROUND AND AIMS: The mechanisms governing the progression of non-alcoholic fatty liver disease (NAFLD) towards steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain elusive. Here, we evaluated the role of hsa-miRNA-21-5p in NASH-related hepatocarcinogenesis. METHODS: Hepatic hsa-miR-21-5p expression was evaluated in two cohorts of patients with biopsy-proven NAFLD (n = 199) or HCC (n = 366 HCC and n = 11 NAFLD-HCC). Serum/liver metabolomic profiles were correlated with hsa-miR-21-5p in NAFLD obese patients. Wild-type (WT) and Mir21 KO mice were fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks to induce NASH and NASH-HCC, respectively. RESULTS: In obese individuals, hsa-miR-21-5p expression increased with NAFLD severity and associated with a hepatic lipotoxic profile. CDAA-fed WT mice displayed increased hepatic mmu-miR-21-5p levels and progressively developed NASH and fibrosis, with livers presenting macroscopically discernible pre-neoplastic nodules, hyperplastic foci and deregulated cancer-related pathways. Mir21 KO mice exhibited peroxisome-proliferator-activated receptor α (PPARα) activation, augmented mitochondrial activity, reduced liver injury and NAS below the threshold for NASH diagnosis, with the pro-inflammatory/fibrogenic milieu reversing to baseline levels. In parallel, Mir21 KO mice displayed reduced number of pre-neoplastic nodules, hepatocyte proliferation and activation of oncogenic signalling, being protected from NASH-associated carcinogenesis. The hsa-miRNA-21-5p/PPARα pathway was similarly deregulated in patients with HCC- or NASH-related HCC, correlating with HCC markers and worse prognosis. CONCLUSIONS: Hsa-miR-21-5p is a key inducer of whole-spectrum NAFLD progression, from simple steatosis to NASH and NASH-associated carcinogenesis. The inhibition of hsa-miR-21-5p, leading to a pro-metabolic profile, might constitute an appealing therapeutic approach to ameliorate NASH and prevent progression towards HCC.

Preoperative immunological plasma markers TRAIL, CSF1 and TIE2 predict survival after resection for biliary tract cancer.

Introduction: Systemic inflammatory markers have been validated as prognostic factors for patients with biliary tract cancer (BTC). The aim of this study was to evaluate specific immunologic prognostic markers and immune responses by analyzing preoperative plasma samples from a large prospectively collected biobank. Methods: Expression of 92 proteins representing adaptive and innate immune responses was investigated in plasma from 102 patients undergoing resection for BTC 2009-2017 (perihilar cholangiocarcinoma n=46, intrahepatic cholangiocarcinoma n=27, gallbladder cancer n=29), by means of a high-throughput multiplexed immunoassay. Association with overall survival was analyzed by Cox regression, with internal validation and calibration. Tumor tissue bulk and single-cell gene expression of identified markers and receptors/ligands was analyzed in external cohorts. Results: Three preoperative plasma markers were independently associated with survival: TRAIL, TIE2 and CSF1, with hazard ratios (95% confidence intervals) 0.30 (0.16-0.56), 2.78 (1.20-6.48) and 4.02 (1.40-11.59) respectively. The discrimination of a preoperative prognostic model with the three plasma markers was assessed with concordance-index 0.70, while the concordance-index of a postoperative model with histopathological staging was 0.66. Accounting for subgroup differences, prognostic factors were assessed for each type of BTC. TRAIL and CSF1 were prognostic factors in intrahepatic cholangiocarcinoma. In independent cohorts, TRAIL-receptor expression was higher in tumor tissue and seen in malignant cells, with TRAIL and CSF1 expressed by intra- and peritumoral immune cells. Intratumoral TRAIL-activity was decreased compared to peritumoral immune cells, while CSF1-activity was increased. The highest CSF1 activity was seen in intratumoral macrophages, while the highest TRAIL-activity was seen in peritumoral T-cells. Discussion: In conclusion, three preoperative immunological plasma markers were prognostic for survival after surgery for BTC, providing good discrimination, even compared to postoperative pathology. TRAIL and CSF1, prognostic factors in intrahepatic cholangiocarcinoma, showed marked differences in expression and activity between intra- and peritumoral immune cells.

Alternative promoters in CpG depleted regions are prevalently associated with epigenetic misregulation of liver cancer transcriptomes.

Transcriptional regulation is commonly governed by alternative promoters. However, the regulatory architecture in alternative and reference promoters, and how they differ, remains elusive. In 100 CAGE-seq libraries from hepatocellular carcinoma patients, here we annotate 4083 alternative promoters in 2926 multi-promoter genes, which are largely undetected in normal livers. These genes are enriched in oncogenic processes and predominantly show association with overall survival. Alternative promoters are narrow nucleosome depleted regions, CpG island depleted, and enriched for tissue-specific transcription factors. Globally tumors lose DNA methylation. We show hierarchical retention of intragenic DNA methylation with CG-poor regions rapidly losing methylation, while CG-rich regions retain it, a process mediated by differential SETD2, H3K36me3, DNMT3B, and TET1 binding. This mechanism is validated in SETD2 knockdown cells and SETD2-mutated patients. Selective DNA methylation loss in CG-poor regions makes the chromatin accessible for alternative transcription. We show alternative promoters can control tumor transcriptomes and their regulatory architecture.

Serum lipidome unravels a diagnostic potential in bile acid diarrhoea.

OBJECTIVE: Bile acid diarrhoea (BAD) is debilitating yet treatable, but it remains underdiagnosed due to challenging diagnostics. We developed a blood test-based method to guide BAD diagnosis. DESIGN: We included serum from 50 treatment-naive patients with BAD diagnosed by gold standard 75selenium homotaurocholic acid test, 56 feature-matched controls and 37 patients with non-alcoholic fatty liver disease (NAFLD). Metabolomes were generated using mass spectrometry covering 1295 metabolites and compared between groups. Machine learning was used to develop a BAD Diagnostic Score (BDS). RESULTS: Metabolomes of patients with BAD significantly differed from controls and NAFLD. We detected 70 metabolites with a discriminatory performance in the discovery set with an area under receiver-operating curve metric above 0.80. Logistic regression modelling using concentrations of decanoylcarnitine, cholesterol ester (22:5), eicosatrienoic acid, L-alpha-lysophosphatidylinositol (18:0) and phosphatidylethanolamine (O-16:0/18:1) distinguished BAD from controls with a sensitivity of 0.78 (95% CI 0.64 to 0.89) and a specificity of 0.93 (95% CI 0.83 to 0.98). The model was independent of covariates (age, sex, body mass index) and distinguished BAD from NAFLD irrespective of fibrosis stage. BDS outperformed other blood test-based tests (7-alpha-hydroxy-4-cholesten-3-one and fibroblast growth factor 19) currently under development. CONCLUSIONS: BDS derived from serum metabolites in a single-blood sample showed robust identification of patients with BAD with superior specificity and sensitivity compared with current blood test-based diagnostics.

Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis, and prognostication of cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). METHODS: EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 44), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC aetiology (n = 56), and hepatocellular carcinoma (n = 34) and healthy individuals (n = 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC = 0.947; odds ratio [OR] =36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC = 0.992; OR = 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC = 0.941; OR = 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. CONCLUSIONS: Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour. METHODS: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts. RESULTS: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth. CONCLUSIONS: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA. IMPACT AND IMPLICATIONS: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Combined Inhibition of Smoothened and the DNA Damage Checkpoint WEE1 Exerts Antitumor Activity in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.

BACKGROUND: Hepatocellular adenoma (HCA) is a rare liver tumour, which can have atypical morphological features such as cytological atypia, pseudoglandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in hepatocellular carcinoma (HCC) or in HCA without atypical morphology. METHODS: We analysed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumour was subtyped by immunohistochemistry, sequenced by a targeted NGS panel, and analysed on a DNA methylation microarray. RESULTS: Subtyping of the five HCAs with atypical features revealed two ß-catenin mutated HCA (b-HCA), two ß-catenin mutated inflammatory HCA (b-IHCA), and one sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as, e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phyloepigenetic trees using the DNA methylation data reproducibly showed that HCAs with morphological atypia are much more similar to nonmalignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). CONCLUSION: In our series, mutational, DNA methylation, as well as CNV analyses, supported a relationship of atypical HCAs with nonatypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling.

Unraveling the actin cytoskeleton in the malignant transformation of cholangiocyte biology.

Correct actin cytoskeleton organization is vital in the liver organ homeostasis and disease control. Rearrangements of the actin cytoskeleton may play a vital role in the bile duct cells cholangiocytes. An abnormal actin network leads to aberrant cell morphology, deregulated signaling networks and ultimately triggering the development of cholangiocarcinoma (CCA) and paving the route for cancer cell dissemination (metastasis). In this review, we will outline alterations of the actin cytoskeleton and the potential role of this dynamic network in initiating CCA, as well as regulating the course of this malignancy. Actin rearrangements not only occur because of signaling pathways, but also regulate and modify cellular signaling. This emphasizes the importance of the actin cytoskeleton itself as cause for aberrant signaling and in promoting tumorigenic phenotypes. We will highlight the impact of aberrant signaling networks on the actin cytoskeleton and its rearrangement as potential cause for CCA. Often, these exact mechanisms in CCA are limited understood and still must be elucidated. Indeed, focusing future research on how actin affects and regulates other signaling pathways may provide more insights into the mechanisms of CCA development, progression, and metastasis. Moreover, manipulation of the actin cytoskeleton organization highlights the potential for a novel therapeutic area.

Molecular therapeutic targets for cholangiocarcinoma: Present challenges and future possibilities.

A diagnosis of cholangiocarcinoma (CCA) is implicit with poor prognosis and limited treatment options, underscoring the near equivalence of incidence and mortality rates in this disease. In less than 9years from genomic identification to FDA-approval of the corresponding inhibitors, fibroblast growth factor receptor 2 (FGFR2) rearrangements and isocitrate dehydrogenase 1 (IDH1) mutations became exemplary successes of precision oncology in subsets of patients with CCA. However, clinical trial results from multikinase inhibitors in unselected populations have been less successful, while the impact of immunotherapies are only beginning to impact this setting. Development of future therapeutics is incumbent with new challenges. Many driver alterations occur in tumor suppressor-like genes which are not directly druggable. Therapeutically, this will require identification of ensuant "non-oncogene addiction" involving genes which are not themselves oncogenes but become tumor survival dependencies when a specific driver alteration occurs. The low recurrence frequency of genomic alterations between CCA patients will require careful evaluation of targeted agents in biomarker-enrolled trials, including basket trial settings. Systematic expansion of candidate drug targets must integrate genes affected by non-genetic alterations which incorporates the fundamental contribution of the microenvironment and immune system to treatment response, disease facets which have been traditionally overlooked by DNA-centric analyses. As treatment resistance is an inevitability in advanced disease, resistance mechanisms require characterization to guide the development of combination therapies to increase the duration of clinical benefit. Patient-focused clinical, technological and analytical synergy is needed to deliver future solutions to these present therapeutic challenges.

Involvement of Epigenomic Factors in Bile Duct Cancer.

Cholangiocarcinoma (CCA) is the second most common type of primary liver cancer. Due to its often-silent manifestation, sporadic nature, and typically late clinical presentation, it remains difficult to diagnose and lacks effective nonsurgical therapeutic options. Extensive research aiming in understanding the mechanisms underlying this disease have provided strong evidence for the significance of epigenetics contributing to its onset, progression, and dissemination. This dysregulation in a myriad of signaling pathways, leading to malignancy, spans altered deoxyribonucleic acid and histone methylation, histone acetylation, and chromatin remodeling, as well as genetic modifications in essential genes controlling these epigenetic processes. An advantage to epigenetic modifications is that they, compared with mutations, are reversible and can partially be controlled by inhibiting the responsible enzymatic machinery. This opens novel possibilities for developing new treatment modalities with benefit for CCA patients.In this article, we have reviewed the current status of epigenome modifications described in CCA, including the role of posttranslational histone modifications and chromatin remodeling, as well as novel advances in treatment options.

Whole blood microRNAs capture systemic reprogramming and have diagnostic potential in patients with biliary tract cancer.

BACKGROUND & AIMS: Late diagnosis is a critical factor undermining clinical management of patients with biliary tract cancer (BTC). While biliary tumours display extensive inter-patient heterogeneity, the host immune response may be comparatively homogenous, providing diagnostic opportunities. Herein, we investigated whether cancer-associated systemic reprogramming could be detected non-invasively to improve diagnosis of BTC. METHODS: In this prospective Danish study, whole blood (WB) microRNA (miRNA) profiling was performed in samples from 218 patients with BTC, 99 healthy participants, and 69 patients with differential diagnoses split into discovery (small RNA-sequencing) and validation (RT-qPCR) cohorts. miRNA expression and activity were further investigated in 119 and 660 BTC tissues, respectively. RESULTS: Four WB miRNAs (let-7a-3p, miR-92b-5p, miR-145-3p, miR-582-3p) were identified and validated as diagnostic of BTC on univariable analysis. Two diagnostic miRNA indexes were subsequently identified that were elevated in patients with BTC and in patients with differential diagnoses, compared to healthy participants. The combination of these miRNA indexes with serum CA 19-9 significantly improved the diagnostic performance of CA 19-9 alone, consistently achieving superior AUC values irrespective of clinical setting (minimum AUC >0.84) or tumour location (minimum AUC >0.87). The diagnostic information captured by miRNA indexes was not recapitulated by routine clinical measurements. Index miRNA expression in BTC tissues was associated with distinct pathobiological and immune features. CONCLUSIONS: WB miRNA profiles are altered in patients with BTC. Quantification of miRNA indexes in combination with serum CA 19-9 has the potential to improve early diagnosis of BTC, pending further validation. LAY SUMMARY: Surgery is currently the only curative intervention for patients with biliary tract cancer (BTC). However, resection is not possible for most patients who are diagnosed with late-stage disease. With the aim of identifying new early diagnostic opportunities, we analysed circulating microRNAs (small non-coding RNAs whose role in cancer is being increasingly recognised) in whole blood samples. We identified a microRNA signature that could distinguish patients with BTC from healthy participants. These miRNAs significantly improved the diagnostic potential of the routinely measured biomarker, CA 19-9, and were implicated in distinct immune processes in tumour tissues.

Lipid alterations in chronic liver disease and liver cancer.

Lipids are a complex and diverse group of molecules with crucial roles in many physiological processes, as well as in the onset, progression, and maintenance of cancers. Fatty acids and cholesterol are the building blocks of lipids, orchestrating these crucial metabolic processes. In the liver, lipid alterations are prevalent as a cause and consequence of chronic hepatitis B and C virus infections, alcoholic hepatitis, and non-alcoholic fatty liver disease and steatohepatitis. Recent developments in lipidomics have also revealed that dynamic changes in triacylglycerols, phospholipids, sphingolipids, ceramides, fatty acids, and cholesterol are involved in the development and progression of primary liver cancer. Accordingly, the transcriptional landscape of lipid metabolism suggests a carcinogenic role of increasing fatty acids and sterol synthesis. However, limited mechanistic insights into the complex nature of the hepatic lipidome have so far hindered the development of effective therapies.

Mutational signatures and processes in hepatobiliary cancers.

The evolutionary history of hepatobiliary cancers is embedded in their genomes. By analysing their catalogue of somatic mutations and the DNA sequence context in which they occur, it is possible to infer the mechanisms underpinning tumorigenesis. These mutational signatures reflect the exogenous and endogenous origins of genetic damage as well as the capacity of hepatobiliary cells to repair and replicate DNA. Genomic analysis of thousands of patients with hepatobiliary cancers has highlighted the diversity of mutagenic processes active in these malignancies, highlighting a prominent source of the inter-cancer-type, inter-patient, intertumour and intratumoural heterogeneity that is observed clinically. However, a substantial proportion of mutational signatures detected in hepatocellular carcinoma and biliary tract cancer remain of unknown cause, emphasizing the important contribution of processes yet to be identified. Exploiting mutational signatures to retrospectively understand hepatobiliary carcinogenesis could advance preventative management of these aggressive tumours as well as potentially predict treatment response and guide the development of therapies targeting tumour evolution.

Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. METHODS: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. RESULTS: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. CONCLUSION: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. LAY SUMMARY: Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.