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Introduction: Intrahepatic cholangiocarcinoma (iCCA) is a primary liver malignancy with poor prognosis. Current prognostic methods are most accurate for patients with surgically resectable disease. However, a significant proportion of patients with iCCA are not surgical candidates. We aimed to develop a generalizable staging system based on clinical variables to determine prognosis of all patients with iCCA., Methods: The derivation cohort included 436 patients with iCCA seen between 2000 and 2011. For external validation, 249 patients with iCCA seen from 2000 to 2014 were enrolled. Survival analysis was performed to identify prognostic predictors. All-cause mortality was the primary end point., Results: Eastern Cooperative Oncology Group status, tumor number, tumor size, metastasis, albumin, and carbohydrate antigen 19-9 were incorporated into a 4-stage algorithm. Kaplan-Meier estimates for 1-year survival were 87.1% (95% confidence interval [CI] 76.1-99.7), 72.7% (95% CI 63.4-83.4), 48.0% (95% CI 41.2-56.0), and 16% (95% CI 11-23.5), respectively, for stages I, II, III, and IV. Univariate analysis yielded significant differences in risk of death for stages II (hazard ratio [HR] 1.71; 95% CI 1.0-2.8), III (HR 3.32; 95% CI 2.07-5.31), and IV (HR 7.44; 95% CI 4.61-12.01) compared with stage I (reference). Concordance indices showed the new staging system was superior to the TNM staging for predicting mortality in the derivation cohort, P < 0.0001. In the validation cohort, however, the difference between the 2 staging systems was not significant., Discussion: The proposed independently validated staging system uses nonhistopathologic data to successfully stratify patients into 4 stages. This staging system has better prognostic accuracy compared with the TNM staging and can assist physicians and patients in treatment of iCCA., (Copyright © 2023 by The American College of Gastroenterology.)

Background: Most patients with localized cholangiocarcinoma (CCA) endure cancer relapse after curative resection underscoring the importance of systemic therapy. The current study attempts to determine the impact of perioperative chemotherapy (PC) on survival in patients with CCA undergoing resection., Methods: Patients diagnosed with CCA undergoing curative-intent resection between January 1, 2000, and December 31, 2019, in a tertiary care center were included. Cox proportional hazard modeling was used to determine the impact of PC on disease-free survival (DFS) and overall survival (OS). In addition, a nomogram was constructed to estimate 3-year DFS., Results: Among the 182 patients included in the analysis, 102 underwent surgery alone, and 80 received surgery plus PC. Forty-two patients received neoadjuvant therapy, and 38 patients received adjuvant therapy. On multivariate analysis, PC was significantly associated with an improved DFS (HR, 95% CI: 0.63, 0.41-0.98; p = 0.04) and OS (HR, 95% CI: 0.46, 0.27-0.78; p < 0.01). In the interaction analysis, the survival benefit was especially seen in patients with positive resection margins and tumor size > 5 cm., Conclusion: In patients with CCA undergoing curative resection, receipt of PC was associated with improved DFS and OS. The nomogram constructed from this database provides an estimate of 3-year DFS after surgical resection. Randomized trials are needed to define the optimal regimen and sequence., Competing Interests: Declaration of competing interest None., (© 2023 Published by Elsevier Ltd.)

Purpose: Increased awareness of the distinct tumor biology for adolescents and young adults (AYAs) with cancer has led to improvement in outcomes for this population. However, in cholangiocarcinoma (CCA), a paucity of data exist on the AYA population. To our knowledge, we present the largest study to date on AYA disease biology, treatment patterns, and survival outcomes in CCA., Methods: A multi-institutional cohort of patients with CCA diagnosed with intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma (ECC) was used for analysis. Retrospective chart review was conducted on patients who were 50 years old and younger (young; n = 124) and older than 50 years (older; n = 723)., Results: Among 1,039 patients screened, 847 patients met eligibility (72% ICC, 28% ECC). Young patients had a larger median tumor size at resection compared with older patients (4.2 v 3.6 cm; P = .048), more commonly had N1 disease (65% v 43%; P = .040), and were more likely to receive adjuvant therapy (odds ratio, 4.0; 95% CI, 1.64 to 9.74). Tumors of young patients were more likely to harbor an FGFR2 fusion, BRAF mutation, or ATM mutation ( P < .05 for each). Young patients were more likely to receive palliative systemic therapy (96% v 69%; P < .001), targeted therapy (23% v 8%; P < .001), and treatment on a clinical trial (31% v 19%; P = .004). Among patients who presented with advanced disease, young patients had a higher median overall survival compared with their older counterparts (17.7 v 13.5 months; 95% CI, 12.6 to 22.6 v 11.4 to 14.8; P = .049)., Conclusion: Young patients with CCA had more advanced disease at resection, more commonly received both adjuvant and palliative therapies, and demonstrated improved survival compared with older patients. Given the low clinical trial enrollment and poor outcomes among some AYA cancer populations, data to the contrary in CCA are highly encouraging.

Background: Treatment patterns for intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) differ, but limited studies exist comparing them. This study examines differences in molecular profiling rates and treatment patterns in these populations, focusing on use of adjuvant, liver-directed, targeted, and investigational therapies., Methods: This multicenter collaboration included patients with ICC or ECC treated at 1 of 8 participating institutions. Retrospective data were collected on risk factors, pathology, treatments, and survival. Comparative statistical tests were 2-sided., Results: Among 1039 patients screened, 847 patients met eligibility (ICC = 611, ECC = 236). Patients with ECC were more likely than those with ICC to present with early stage disease (53.8% vs 28.0%), undergo surgical resection (55.1% vs 29.8%), and receive adjuvant chemoradiation (36.5% vs 4.2%) (all P < .00001). However, they were less likely to undergo molecular profiling (50.3% vs 64.3%) or receive liver-directed therapy (17.9% vs 35.7%), targeted therapy (4.7% vs 18.9%), and clinical trial therapy (10.6% vs 24.8%) (all P < .001). In patients with recurrent ECC after surgery, the molecular profiling rate was 64.5%. Patients with advanced ECC had a shorter median overall survival than those with advanced ICC (11.8 vs 15.1 months; P < .001)., Conclusions: Patients with advanced ECC have low rates of molecular profiling, possibly in part because of insufficient tissue. They also have low rates of targeted therapy use and clinical trial enrollment. While these rates are higher in advanced ICC, the prognosis for both subtypes of cholangiocarcinoma remains poor, and a pressing need exists for new effective targeted therapies and broader access to clinical trials., (© The Author(s) 2023. Published by Oxford University Press.)

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., (© 2023. Springer Nature Limited.)

There is an urgent need for the identification of reliable prognostic biomarkers for patients with intrahepatic cholangiocarcinoma (iCCA) and alterations in N-glycosylation have demonstrated an immense potential to be used as diagnostic strategies for many cancers, including hepatocellular carcinoma (HCC). N-glycosylation is one of the most common post-translational modifications known to be altered based on the status of the cell. N-glycan structures on glycoproteins can be modified based on the addition or removal of specific N-glycan residues, some of which have been linked to liver diseases. However, little is known concerning the N-glycan alterations that are associated with iCCA. We characterized the N-glycan modifications quantitatively and qualitatively in three cohorts, consisting of two tissue cohorts: a discovery cohort ( n = 104 cases) and a validation cohort ( n = 75), and one independent serum cohort consisting of patients with iCCA, HCC, or benign chronic liver disease ( n = 67). N-glycan analysis in situ was correlated to tumor regions annotated on histopathology and revealed that bisected fucosylated N-glycan structures were specific to iCCA tumor regions. These same N-glycan modifications were significantly upregulated in iCCA tissue and serum relative to HCC and bile duct disease, including primary sclerosing cholangitis (PSC) ( P < 0.0001). N-glycan modifications identified in iCCA tissue and serum were used to generate an algorithm that could be used as a biomarker of iCCA. We demonstrate that this biomarker algorithm quadrupled the sensitivity (at 90% specificity) of iCCA detection as compared with carbohydrate antigen 19-9, the current "gold standard" biomarker of CCA., Significance: This work elucidates the N-glycan alterations that occur directly in iCCA tissue and utilizes this information to discover serum biomarkers that can be used for the noninvasive detection of iCCA., (© 2023 The Authors; Published by the American Association for Cancer Research.)

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., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: We recently developed a fluorescence in situ hybridization probe set for evaluating suspicious biliary and pancreatic duct strictures (PB-FISH). We aimed to determine whether PB-FISH results in biliary brush cytology specimens are associated with outcomes of patients with cholangiocarcinoma (CCA)., Methods: We performed a retrospective study of patients with CCA tested by PB-FISH from January 2015 to August 2018. CCA was stratified by primary sclerosing cholangitis (PSC) into those with (PSC CCA) or without PSC ( de novo CCA). PB-FISH results were categorized as polysomy (gain of multiple loci), nonpolysomy (single locus gain, single locus gain with 9p21 loss, homozygous 9p21 loss, tetrasomy), and disomy (no abnormalities). Overall survival (OS) was estimated using Kaplan-Meier methods and compared between the PB-FISH results using log-rank tests. Cox models were adjusted for age, sex, CA 19-9, cytology results, source of brushing sample, and treatments., Results: Characteristics of 264 eligible patients (median age 60.4; range 18-92) were comparable for patients with PB-FISH polysomy vs nonpolysomy vs disomy. The median OS was similar between disomy, nonpolysomy, and polysomy in the overall population (22.7 vs 22.7 vs 20.3 months, respectively). For de novo CCA, both polysomy and nonpolysomy were associated with worse OS compared with disomy (polysomy: hazard ratio [HR] = 2.09, 95% confidence interval [CI] = 1.14-3.83; nonpolysomy: HR = 2.4, 95% CI = 0.54-2.46; P = 0.027). For PSC CCA, neither polysomy nor nonpolysomy were significantly associated with worse OS (polysomy: 0.90, 95% CI = 0.47-1.75; nonpolysomy: HR = 1.78, CI = 0.71-4.49; P = 0.27)., Discussion: PB-FISH alterations are associated with worse survival in de novo CCA, though statistical significance was lost when adjusting for confounding variables., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: Curative surgical treatments afford the best prognosis for patients with intrahepatic cholangiocarcinoma (iCCA); however, the comparative effectiveness of treatment options and factors associated with curative treatment receipt for early stage iCCA remain unknown., Methods: The authors identified patients who were diagnosed with early stage iCCA, defined as a unifocal tumor <3 cm, during 2004-2018 from the National Cancer Database. Multivariable logistic and Cox regression analyses were used to identify the factors associated with curative treatment and overall survival (OS), respectively., Results: The proportion of patients with early stage iCCA increased from 4.5% in 2004 to 7.3% in 2018, with the odds of early stage detection increasing by 3.1% per year (odds ratio [OR], 1.031; 95% CI, 1.015-1.049). Of 1093 patients who had early stage iCCA, 464 (42.5%) underwent resection, 113 (10.3%) underwent ablation, 62 (5.7%) underwent liver transplantation, and 454 (41.5%) received noncurative treatments. Hispanic patients (adjusted OR [aOR], 0.57; 95% CI, 0.33-0.97) and Black patients (aOR, 0.47; 95% CI, 0.28-0.77) were less likely to receive curative treatments than White patients. Compared with patients who underwent surgical resection, those who underwent liver transplantation had a trend toward improved OS (adjusted hazard ratio [aHR], 0.63; 95% CI, 0.37-1.08), whereas those who underwent local ablation (aHR, 1.39; 95% CI, 1.01-1.92) and noncurative treatments (aHR, 3.97; 95% CI, 3.24-4.88) experienced worse OS., Conclusions: More than one third of patients with early stage iCCA did not receive curative treatment, with Hispanic and Black patients being less likely to receive curative treatments than White patients. Surgical resection and liver transplantation were associated with improved survival compared with local ablation. Future studies should investigate disparities in curative treatment receipt and outcomes for early stage iCCA., (© 2022 American Cancer Society.)

Background and Aims: Malignant biliary strictures can be difficult to diagnose, with up to 20% considered indeterminate after initial tissue sampling. This study aimed to determine the performance characteristics of transpapillary biopsy sampling (TPB) and fluorescence in situ hybridization (FISH) in isolation or in combination with standard brush cytology (BC) in patients who received trimodality sampling for biliary strictures., Methods: This single-center retrospective cohort study included patients with biliary strictures undergoing ERCP with trimodality sampling between September 2014 and April 2019. Performance characteristics for each diagnostic test alone and in combination were calculated., Results: Two hundred four patients underwent trimodality biliary sampling, including 104 (51.0%) with malignancy. The diagnostic sensitivity for malignancy with BC (17.3%) significantly improved with dual modality (BC+FISH, 58.7%; BC+TPB, 40.4%) or trimodality sampling (68.3%; P < .001 for all comparisons). Trimodality sampling improved diagnostic sensitivity for malignancy compared with BC+FISH (P = .002) and BC+TPB (P < .001). There was no statistically significant difference in the sensitivity of trimodality sampling in detecting cholangiocarcinoma (79.7%) compared with pancreatic cancer (62.5%; P = .1). Among 57 patients with primary sclerosing cholangitis (PSC), the sensitivity of detecting biliary malignancy (n = 20) was 20% for BC and significantly improved with the addition of FISH (80%; P < .001) but not with TPB (35.0%; P = .25). Trimodality sampling did not further improve diagnostic sensitivity (85%) over BC+FISH (80%) for malignancy in the setting of PSC (P = 1)., Conclusions: Trimodality sampling improves the diagnostic sensitivity for the detection of malignant biliary strictures with no significant difference in sensitivity for cholangiocarcinoma compared with pancreatic cancer. However, in patients with PSC, trimodality sampling was not superior to BC+FISH., (Copyright © 2022 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Prostate-specific membrane antigen (PSMA) is a validated target for molecular diagnostics and targeted radionuclide therapy. Our purpose was to evaluate PSMA expression in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatic adenoma (HCA); investigate the genetic pathways in HCC associated with PSMA expression; and evaluate HCC detection rate with 68 Ga-PSMA-11 positron emission tomography (PET). In phase 1, PSMA immunohistochemistry (IHC) on HCC (n = 148), CCA (n = 111), and HCA (n = 78) was scored. In a subset (n = 30), messenger RNA (mRNA) data from the Cancer Genome Atlas HCC RNA sequencing were correlated with PSMA expression. In phase 2, 68 Ga-PSMA-11 PET was prospectively performed in patients with treatment-naïve HCC on a digital PET scanner using cyclotron-produced 68 Ga. Uptake was graded qualitatively and semi-quantitatively using standard metrics. On IHC, PSMA expression was significantly higher in HCC compared with CCA and HCA (P < 0.0001); 91% of HCCs (n = 134) expressed PSMA, which principally localized to tumor-associated neovasculature. Higher tumor grade was associated with PSMA expression (P = 0.012) but there was no association with tumor size (P = 0.14), fibrosis (P = 0.35), cirrhosis (P = 0.74), hepatitis B virus (P = 0.31), or hepatitis C virus (P = 0.15). Overall survival tended to be longer in patients without versus with PSMA expression (median overall survival: 4.2 vs. 1.9 years; P = 0.273). FGF14 (fibroblast growth factor 14) mRNA expression correlated positively (rho = 0.70; P = 1.70 × 10 -5 ) and MAD1L1 (Mitotic spindle assembly checkpoint protein MAD1) correlated negatively with PSMA expression (rho = -0.753; P = 1.58 × 10 -6 ). Of the 190 patients who met the eligibility criteria, 31 patients with 39 HCC lesions completed PET; 64% (n = 25) lesions had pronounced 68 Ga-PSMA-11 standardized uptake value: SUV max (median [range] 9.2 [4.9-28.4]), SUV mean 4.7 (2.4-12.7), and tumor-to-liver background ratio 2 (1.1-11). Conclusion: Ex vivo expression of PSMA in neovasculature of HCC translates to marked tumor avidity on 68 Ga-PSMA-11 PET, which suggests that PSMA has the potential as a theranostic target in patients with HCC., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background: Chemoprevention for biliary tract cancers (BTC), which comprise intrahepatic cholangiocarcinoma (iCCA), extrahepatic cholangiocarcinoma (eCCA), and gallbladder cancer, is controversial. We examined associations between low-dose aspirin, statins, NSAIDs, and metformin with BTC risk., Methods: We used a population-based cohort of 5.7 million persons over age 18 without personal history of cancer (except nonmelanoma skin cancer), receiving at least one commonly prescribed drug between July 1, 2005, and December 31, 2012, from the Swedish Prescribed Drug Registry. Hazard ratios (HR) were calculated using age-scaled multivariable-adjusted Cox models., Results: 2,160 individuals developed BTC. Low-dose aspirin was not associated with BTC risk [HR, 0.93; 95% confidence interval (CI), 0.81-1.07], iCCA (HR, 1.21; 95% CI, 0.93-1.57), eCCA (HR, 0.80; 95% CI, 0.60-1.07), or gallbladder cancer (HR, 0.87; 95% CI, 0.71-1.06). Statins were associated with lower risk of BTC (HR, 0.66; 95% CI, 0.56-0.78), iCCA (HR, 0.69; 95% CI, 0.50-0.95), eCCA (HR 0.54; 95% CI, 0.38-0.76), and gallbladder cancer (HR, 0.72; 95% CI, 0.57-0.91). For all BTC subtypes, combined low-dose aspirin and statins were not associated with lower risk than statins alone. NSAIDs were associated with higher risk of BTC and its subtypes. Metformin was not associated with BTC risk (HR, 0.98; 95% CI, 0.82-1.18), iCCA (HR, 1.06; 95% CI, 0.77-1.48), eCCA (HR, 1.15; 95% CI, 0.82-1.61), or gallbladder cancer (HR, 0.84; 95% CI, 0.63-1.11)., Conclusions: Statins were associated with a decreased risk of BTC and its subtypes. Low-dose aspirin alone was not associated with a decreased risk, and use of both was not associated with further decrease in risk beyond statins alone., Impact: Statins were most consistently associated with a decreased risk of BTC and its subtypes., (©2022 American Association for Cancer Research.)

Introduction: Cholangiocarcinoma (CCA) is a heterogeneous group of aggressive biliary malignancies. While surgery and liver transplantation are the only potentially curative modalities for early-stage disease, limited options are available for most patients with incurable-stage disease. Survival outcomes remain dismal. Recent molecular profiling efforts have led to improved understanding of the genomic landscape of CCA and to the identification of subgroups with distinct diagnostic, prognostic, and therapeutic implications., Areas Covered: : We provide an updated review and future perspectives on features of cholangiocarcinogenesis that can be translated into therapeutic biomarkers and targets. We highlight the critical studies that have established current systemic chemotherapy and targeted therapeutics, while elaborating on novel targeted and immunotherapeutic approaches in development. Relevant literature and clinical studies were identified by searching PubMed and www.ClinicalTrials.gov., Expert Opinion: : While therapies targeting the various molecular subgroups of CCA are rapidly emerging and changing treatment paradigms, their success has been limited by the genetic heterogeneity of CCA and the plasticity of the targets. Novel strategies aiming to combine immunotherapy, chemotherapy, and molecularly targeted therapeutics will be required to offer durable clinical benefit and maximize survival.

Cholangiocarcinoma is associated with several different risk factors, many of which have known genetic associations. Advances in our understanding of the human genome have translated to the development of gene specific and whole genome assays for identifying gene variants and other alterations associated with cancer development. An improved understanding of the inherited genetic variants associated with risk of cholangiocarcinoma has the potential to improve our understanding of the basic biology of cholangiocarcinoma, enhance the performance of risk stratification models for identifying individuals at highest risk for cholangiocarcinoma, and identifying genetic variants associated with predisposition to cholangiocarcinoma in families with multiple affected individuals. It is increasingly recognized that major cancer-causing mutations or other gene alterations associated with familial risk of multiple cancers can also occur as germline events in individuals with apparently sporadically occurring cancer. In this chapter we review the major risk factors for cholangiocarcinoma as well as known gene variants associated with these risk factors, gene variants that have been associated with cholangiocarcinoma as the result of interrogation of candidate genes known to be associated with putative cancer causing pathways in cholangiocarcinoma, as well as the prevalence of major cancer causing genetic aberrations shown to be inherited in the germline of patients with sporadically developed cholangiocarcinoma. There has not yet been any large-scale genome wide association study of cholangiocarcinoma, and the results from such a study are eagerly anticipated., Competing Interests: Conflict of interest statement M.A.S. has no financial or personal disclosures relevant to the contents of this manuscript. L.R.R. reports grants from NIH (CA186566 and CA210964), Mayo Clinic, The Cholangiocarcinoma Foundation, Bayer, Boston Scientific, Exact Sciences, Fujifilm Medical Sciences, Gilead Sciences, Glycotest Inc., PSC Partners Seeking a Cure, Redhill Biopharma, and TARGET PharmaSolutions during the conduct of the study; other support from MedEd Design LLC, Pontifax, Global Life Science Consulting, The Lynx Group, AstraZeneca, Bayer, Eisai, Exact Sciences, and GRAIL Inc., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background and Aims: Sirtuin 1 (SIRT1) is a complex NAD + -dependent protein deacetylase known to act as a tumor promoter or suppressor in different cancers. Here, we describe a mechanism of SIRT1-induced destabilization of primary cilia in cholangiocarcinoma (CCA)., Approach and Results: A significant overexpression of SIRT1 was detected in human CCA specimens and CCA cells including HuCCT1, KMCH, and WITT1 as compared with normal cholangiocytes (H69 and NHC). Small interfering RNA (siRNA)-mediated knockdown of SIRT1 in HuCCT1 cells induced cilia formation, whereas overexpression of SIRT1 in normal cholangiocytes suppressed ciliary expression. Activity of SIRT1 was regulated by presence of NAD+ in CCA cells. Inhibition of NAD -producing enzyme nicotinamide phosphoribosyl transferase increased ciliary length and frequency in CCA cells and in SIRT1-overexpressed H69 cells. Furthermore, we also noted that SIRT1 induces the proteasomal mediated degradation of ciliary proteins, including α-tubulin, ARL13B, and KIF3A. Moreover, overexpression of SIRT1 in H69 and NHC cells significantly induced cell proliferation and, conversely, SIRT1 inhibition in HuCCT1 and KMCH cells using siRNA or sirtinol reduced cell proliferation. In an orthotopic transplantation rat CCA model, the SIRT1 inhibitor sirtinol reduced tumor size and tumorigenic proteins (glioma-associated oncogene 1, phosphorylated extracellular signal-regulated kinase, and IL-6) expression., Conclusions: In conclusion, these results reveal the tumorigenic role of SIRT1 through modulation of primary cilia formation and provide the rationale for developing therapeutic approaches for CCA using SIRT1 as a target., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: Intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) are the most common primary liver cancers (PLCs). Differences in their clinical features and outcomes are open for investigation in a large-scale study. We aim to investigate the differences in clinical features and outcomes between iCCA and HCC., Approach and Results: The Surveillance, Epidemiology, and End Results Program 18 Database (2000-2017) was used to extract demographic and clinical features of HCC and iCCA patients. Logistic regression analysis was performed to identify factors associated with iCCA diagnosis versus HCC. Cox regression analysis was used to assess factors affecting overall survival (OS). There were 13,611 iCCA and 96,151 HCC patients. Half of iCCA (50.7%) and three quarters of HCC (76.3%) patients were male. Diagnosis in recent year, age (<50 or ≥65), female sex, non-Hispanic White race, higher income, rural area, and higher tumor burden were independently associated with iCCA diagnosis versus HCC. Patients with iCCA had worse OS than those with HCC (9 vs. 13 months; P < 0.001). However, OS was comparable between iCCA and HCC in multivariable analysis (adjusted hazard ratio [aHR] = 1.02; 95% CI = 0.99-1.05). In subgroup analyses, iCCA was associated with better OS than HCC in patients with tumor ≥5 cm (aHR = 0.83; 95% CI = 0.80-0.86), lymph node involvement (aHR = 0.76; 95% CI = 0.72-0.81), distant metastasis (aHR = 0.76; 95% CI = 0.73-0.79), poorly/undifferentiated tumors (aHR = 0.88; 95% CI = 0.83-0.94), and those receiving noncurative treatment (aHR = 0.96; 95% CI = 0.93-0.98)., Conclusions: We identified the demographic, socioeconomic, and clinical features associated with iCCA diagnosis over HCC among patients with PLC. Although iCCA patients presented at an advanced stage, OS was similar between iCCA and HCC in multivariable analysis. iCCA was associated with longer OS for subgroups with poor prognostic features., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: Existing therapeutic approaches to treat cholangiocarcinoma (CCA) have limited effectiveness, prompting further study to develop therapies for CCA. We report a mechanistic role for the heparan sulfate editing enzyme sulfatase 2 (SULF2) in CCA pathogenesis., Approach and Results: In silico analysis revealed elevated SULF2 expression in human CCA samples, occurring partly through gain of SULF2 copy number. We examined the effects of knockdown or overexpression of SULF2 on tumor growth, chemoresistance, and signaling pathway activity in human CCA cell lines in vitro. Up-regulation of SULF2 in CCA leads to increased platelet-derived growth factor receptor beta (PDGFRβ)-Yes-associated protein (YAP) signaling activity, promoting tumor growth and chemotherapy resistance. To explore the utility of targeting SULF2 in the tumor microenvironment for CCA treatment, we tested an anti-SULF2 mouse monoclonal antibody, 5D5, in a mouse CCA xenograft model. Targeting SULF2 by monoclonal antibody 5D5 inhibited PDGFRβ-YAP signaling and tumor growth in the mouse xenograft model., Conclusions: These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA., (© 2021 by the American Association for the Study of Liver Diseases.)

Cholangiocarcinomas (CCA) are a group of heterogeneous tumors arising from the biliary epithelia. Significant sequencing efforts have provided further insights into the molecular mechanisms of this disease including fibroblast growth factor receptor ( FGFR ) alterations, which occurs in approximately 15%-20% of intrahepatic CCAs. Herein, we describe the FGFR inhibitor (FGFRi)-associated treatment toxicity and cancer-specific outcomes from a multicenter single-institution cohort., Methods: This is a retrospective study of patients with CCA and known FGFR alterations treated with FGFRi. We describe the toxicity and efficacy in patients treated at Mayo Clinic between January 2010 and December 2020., Results: Our group identified 61 patients with advanced or metastatic CCA, 19 males (31%) and 42 females (69%), harboring FGFR alterations who received FGFRi. The most common grade 1 or higher adverse events for all patients included fatigue (92%), AST elevations (78%), anemia (80%), decreased platelet count (63%), and hyperphosphatemia (74%). Median progression-free survival on FGFRi was 5.8 months for all patients (95% CI, 4.9 to 9.0). Females had significantly longer progression-free survival at 6.9 months (95% CI, 5.2 to 11.8) on FGFRi compared with males at 4.9 months (95% CI, 2.8 to not estimable; P = .038)., Conclusion: FGFRi are well tolerated with clinical efficacy. With the recent approval of FGFRi by the US Food and Drug Administration and ongoing clinical trials for new FGFRi, understanding outcomes and toxicity associated with these medications is important for precision oncology., Competing Interests: Nguyen H. Tran Honoraria: QED Therapeutics Consulting or Advisory Role: QED Therapeutics No other potential conflicts of interest were reported. Nguyen H. Tran Honoraria: QED Therapeutics Consulting or Advisory Role: QED Therapeutics No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Background and Aims: Statins have been proven to be cytotoxic to human cholangiocarcinoma cells by inhibiting cell division and inducing apoptosis. We aimed to determine the effect of statin use on the risk of cancer development and survival in patients with extrahepatic cholangiocarcinoma (ECC), including perihilar cholangiocarcinoma (pCCA) and distal cholangiocarcinoma (dCCA)., Approach and Results: A total of 394 patients with ECC and hyperlipidemia who received care at Mayo Clinic Rochester between 2005 and 2015 were matched by age, sex, race, ethnicity, and residency to 788 controls with hyperlipidemia. Clinical and outcome data were abstracted. The odds ratios (ORs) for risk and hazard ratios for outcomes were calculated. The mean age and standard deviation (SD) for cases and controls was 65.6 years (13.8). The number of statin users in cases and controls was 73 (19%) and 403 (51%), respectively. Hepatitis C virus infection (OR, 15.84; 95% confidence interval [CI], 4.06-61.87; P < 0.001) was the most significant risk factor for pCCA followed by inflammatory bowel disease and cirrhosis, whereas other liver disease, including biliary stone disease (OR, 4.06; CI, 2.24-7.36; P < 0.001), was the only significant risk factor for dCCA. Statin use was associated with significantly reduced risk for all ECC (OR, 0.22; CI, 0.16-0.29) as well as for the subtypes pCCA (OR, 0.3; CI, 0.21-0.41) and dCCA (OR, 0.06; CI, 0.03-0.14), all P < 0.0001. Moderate-intensity dosage was found to decrease the risk of ECC (OR, 0.48; CI, 0.34-0.67; P < 0.001). Comparing statin ever users to nonusers, patients with dCCA who used statins had significantly overall better survival (hazard ratio = 0.53; CI, 0.29-0.97; P = 0.04)., Conclusions: This case-control study suggests that statins decrease the risk of ECC and may improve survival in patients with dCCA. Additional validation studies are warranted., (© 2020 by the American Association for the Study of Liver Diseases.)

Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non-invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlighted.

Background and Aims: Presence of malignant regional lymph nodes (MRLNs) precludes curative oncological resection or liver transplantation for cholangiocarcinoma (CCA). Limited data support the utility of endoscopic ultrasound (EUS)/fine needle aspiration (FNA) for detection of MRLNs in extrahepatic CCA, but there are no data for its role in intrahepatic CCA (iCCA). The aim of this study is to evaluate the staging impact of EUS for CCA, including analysis by subtype., Approach and Results: We identified consecutive patients with CCA who underwent EUS staging at a single tertiary care center from October 2014 to April 2018. Among this cohort, we abstracted clinical demographic, radiographical, procedural, cytopathological, and surgical data. STATA 15 software was used for comparative analysis calculations (StataCorp LP, College Station, TX). The study cohort included 157 patients; 24 (15%), 124 (79%), and 9 (6%) with intrahepatic, perihilar, and distal CCA, respectively. EUS was able to identify regional lymph nodes (RLNs) in a higher percentage of patients compared to cross-sectional imaging (86% vs. 47%; P < 0.001). FNA was performed in 133 (98.5%) patients with RLNs, with a median of three passes per node. EUS-FNA identified MRLN in 27 of 31 (87.1%) patients ultimately found to have MRLNs. For iCCA, EUS detected a higher percentage of RLN compared to cross-sectional imaging (83% vs. 50%; P = 0.048), with MRLNs identified in 4 (17%) patients. Among the entire cohort, identification of at least one MRLN by EUS was associated with lower median survival (353 vs. 1,050 days; P < 0.001) and increased risk of death (hazard ratio = 4.1; P < 0.001)., Conclusions: EUS-FNA is effective for identifying MRLN in patients with CCA, and should be routinely incorporated into staging of all CCA subtypes given the impact of MRLN on prognosis and management decisions., (© 2020 by the American Association for the Study of Liver Diseases.)

Background & Aims: Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies., Methods: An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC., Results: KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with ∼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFβ signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors., Conclusion: An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches., Lay Summary: Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications., Competing Interests: Conflict of interest J.M.L. is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer-Ingelheim and Ipsen, and consulting fees from Bayer HealthCare Pharmaceuticals, Merck, Eisai Inc, Bristol-Myers Squibb, Celsion Corporation, Eli Lilly, Roche, Genentech, Glycotest, Nucleix, Can-Fite Biopharma, AstraZeneca, and Exelixis. A.V. reports personal fees from NGM Pharmaceuticals, Gilead, Nucleix, Fuji Wako, Guidepoint and Exact Sciences. L.R.R. is receiving research support from Ariad Pharmaceuticals, Bayer, BTG International, Exact Sciences, Gilead Sciences, GRAIL Inc., RedHill Biopharma Ltd., TARGET PharmaSolutions and Wako Diagnostics, and is advisory board member of Bayer, Exact Sciences, Gilead Sciences, GRAIL, Inc., QED Therapeutics, Inc. and TAVEC. B.M. reports personal fees from Bayer and Gilead. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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 (Ca 2+ ) 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 Ca 2+ 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 Ca 2+ signaling., (© 2019 by the American Association for the Study of Liver Diseases.)

Background: Chemotherapy is frequently used in cholangiocarcinoma as an adjunct to surgical resection, but the appropriate sequence of chemotherapy with surgery is unclear., Patients and Methods: Using the National Cancer Database, we identified patients who underwent surgery and chemotherapy for stage I-III cholangiocarcinoma between 2006 and 2014. The propensity score reflecting the probability of receiving neoadjuvant chemotherapy was estimated by multivariate logistic regression method. Patients in the neoadjuvant and adjuvant chemotherapy study arms were then propensity-matched in 1:3 ratios using the nearest neighbor method. Overall Survival (OS) in the matched data set was estimated using the Kaplan-Meier method. Hazard ratios (HRs) were calculated using Cox proportional hazard regression model., Results: Of the 1450 patients who met our inclusion criteria, 299 (20.6%) received neoadjuvant chemotherapy while 1151 (79.3%) received adjuvant chemotherapy. The median age at diagnosis was 63 years. 278 patients in the neoadjuvant group were matched to 700 patients in the adjuvant group. In the matched cohort, patients who received neoadjuvant chemotherapy had a superior OS compared to those who received adjuvant chemotherapy (Median OS: 40.3 vs. 32.8 months; HR: 0.78; 95% CI: 0.64-0.94, p = 0.01). The 1- and 5-year OS rates for the neoadjuvant chemotherapy group were 85.8% and 42.5% respectively compared to 84.6% and 31.7% for the adjuvant chemotherapy group., Conclusion: In this large national database study, neoadjuvant chemotherapy was associated with a longer OS in a select group of patients with cholangiocarcinoma compared to those who underwent upfront surgical resection followed by adjuvant chemotherapy., (Copyright © 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Background: Although molecular characterization of iCCA has been studied recently, integrative analysis of molecular and clinical characterization has not been fully established. If molecular features of iCCA can be predicted based on clinical findings, we can approach to distinguish targeted treatment. We analyzed RNA sequencing data annotated with clinicopathologic data to clarify molecular-specific clinical features and to evaluate potential therapies for molecular subtypes., Methods: We performed next-generation RNA sequencing of 30 surgically resected iCCA from Korean patients and the clinicopathologic features were analyzed. The RNA sequences from 32 iCCA resected from US patients were used for validation., Results: Patients were grouped into two subclasses on the basis of unsupervised clustering, which showed a difference in 5-year survival rates (48.5% vs 14.2%, p = 0.007) and similar survival outcome in the US samples. In subclass B (poor prognosis), both data sets were similar in higher carcinoembryonic antigen and cancer antigen 19-9 levels, underlying cholangitis, and bile duct-type pathology; in subclass A (better prognosis), there was more frequent viral hepatitis and cholangiolar-type pathology. On pathway analysis, subclass A had enriched liver-related signatures. Subclass B had enriched inflammation-related and TP53 pathways, with more frequent KRAS mutations. CCA cell lines with similar gene expression patterns of subclass A were sensitive to gemcitabine., Conclusions: Two molecular subtypes of iCCA with distinct clinicopathological differences were identified. Knowledge of clinical and pathologic characteristics can predict molecular subtypes, and knowledge of different subtype signaling pathways may lead to more rational, targeted approaches to treatment.

Background: To determine the association between the number of patients with intra-hepatic cholangiocarcinoma (IHCC) treated annually at a treatment facility (volume) and overall survival (outcome)., Methods: Patients with IHCC reported to the National Cancer Database (years 2004-2015) were included. We classified facilities by tertiles (T; mean IHCC patients treated/year): T1: <2.56; T2: 2.57-5.39 and T3: ≥5.40. Volume-outcome relationship was determined by using Cox regression adjusting for patient demographics, comorbidities, tumor characteristics, insurance type and therapy received., Results: There were 11,344 IHCC patients treated at 1106 facilities. On multivariable analysis, facility volume was independently associated with all-cause mortality (p < 0.001). The unadjusted median OS by facility volume was: T1: 5 months (m), T2: 8.1 m, and T3: 13.1 m (p < 0.001). Compared with patients treated at T3 facilities, patients treated at lower-tertile facilities had significantly higher risk of death [T2 hazard ratio (HR), 1.12 [95% CI, 1.05-1.23]; T1 HR, 1.21 [95% CI, 1.11-1.33]. Patients treated at high-volume centers were more likely to get surgery (34.6 vs 13.1%) and adjuvant therapy., Conclusion: IHCC patients treated at high-volume facilities had a significant improvement in OS and were more likely to receive surgery and adjuvant therapy as compared to that of patients at low-volume facilities., (Copyright © 2018 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Intrahepatic cholangiocarcinoma remains a highly heterogeneous malignancy that has eluded effective patient stratification to date. The extent to which such heterogeneity can be influenced by individual driver mutations remains to be evaluated. Here, we analyzed genomic (whole-exome sequencing, targeted exome sequencing) and epigenomic data from 496 patients and used the three most recurrently mutated genes to stratify patients (IDH, KRAS, TP53, "undetermined"). Using this molecular dissection approach, each subgroup was determined to possess unique mutational signature preferences, comutation profiles, and enriched pathways. High-throughput drug repositioning in seven patient-matched cell lines, chosen to reflect the genetic alterations specific for each patient group, confirmed in silico predictions of subgroup-specific vulnerabilities linked to enriched pathways. Intriguingly, patients lacking all three mutations ("undetermined") harbored the most extensive structural alterations, while isocitrate dehydrogenase mutant tumors displayed the most extensive DNA methylome dysregulation, consistent with previous findings., Conclusion: Stratification of intrahepatic cholangiocarcinoma patients based on occurrence of mutations in three classifier genes (IDH, KRAS, TP53) revealed unique oncogenic programs (mutational, structural, epimutational) that influence pharmacologic response in drug repositioning protocols; this genome dissection approach highlights the potential of individual mutations to induce extensive molecular heterogeneity and could facilitate advancement of therapeutic response in this dismal disease. (Hepatology 2018)., (© 2017 by the American Association for the Study of Liver Diseases.)

Purpose: Cholangiocarcinoma (CCA) is a malignancy of the bile ducts. The purpose of this discovery study was to identify effective serum markers for surveillance of cholangiocarcinoma., Experimental Design: Using a glycomic method, patients with CCA were determined to have increased levels of alpha-1,3 and alpha-1,6 linked fucosylated glycan. Proteomic analysis of the serum fucosylated proteome identified proteins such as alpha-2-macroglobulin, kininogen, hemopexin, fetuin-A, alpha-1 anti-trypsin, and ceruloplasmin as being hyperfucosylated in HCC. The levels of these glycoproteins in 109 patients with CCA, primary sclerosing cholangitis (PSC), and control patients were compared to the performance of CA-19-9, the current "gold standard" assay for cholangiocarcinoma., Results: Fucosylated Fetuin-A (fc-Fetuin-A) had the best ability to differentiate CCA from PSC, with an AUROC of 0.812 or 0.8665 at differentiating CCA from those with PSC or other liver disease. CA-19-9 had poor ability to differentiate PSC from cholangiocarcinoma (AUROC of 0.625)., Conclusion and Clinical Relevance: Using glycomic and proteomic methods we identified a set of proteins that contain altered glycan in the sera of those with CCA. One of these proteins, fucosylated Fetuin-A may have value in the surveillance of people at risk for the development of cholangiocarcinoma., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Cholangiocarcinoma is a highly lethal cancer with limited therapeutic options. Recent genomic analysis of cholangiocarcinoma has revealed the presence of fibroblast growth factor receptor 2 (FGFR2) fusion proteins in up to 13% of intrahepatic cholangiocarcinoma (iCCA). FGFR fusions have been identified as a novel oncogenic and druggable target in a number of cancers. In this study, we established a novel cholangiocarcinoma patient derived xenograft (PDX) mouse model bearing an FGFR2-CCDC6 fusion protein from a metastatic lung nodule of an iCCA patient. Using this PDX model, we confirmed the ability of the FGFR inhibitors, ponatinib, dovitinib and BGJ398, to modulate FGFR signaling, inhibit cell proliferation and induce cell apoptosis in cholangiocarcinoma tumors harboring FGFR2 fusions. In addition, BGJ398 appeared to be superior in potency to ponatinib and dovitinib in this model. Our findings provide a strong rationale for the investigation of FGFR inhibitors, particularly BGJ398, as a therapeutic option for cholangiocarcinoma patients harboring FGFR2 fusions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Optimal management of patients with intrahepatic cholangiocarcinoma (ICCA) and elevated CA19-9 remains undefined. We hypothesized CA19-9 elevation above normal indicates aggressive biology and that inclusion of CA19-9 would improve staging discrimination., Methods: The National Cancer Data Base (NCDB-2010-2012) was reviewed for patients with ICCA and reported CA19-9. Patients were stratified by CA19-9 above/below normal reference range. Unadjusted Kaplan-Meier and adjusted Cox-proportional-hazards analysis of overall survival (OS) were performed., Results: A total of 2,816 patients were included: 938 (33.3%) normal; 1,878 (66.7%) elevated CA19-9 levels. Demographic/pathologic and chemotherapy/radiation were similar between groups, but patients with elevated CA19-9 had more nodal metastases and less likely to undergo resection. Among elevated-CA19-9 patients, stage-specific survival was decreased in all stages. Resected patients with CA19-9 elevation had similar peri-operative outcomes but decreased long-term survival. In adjusted analysis, CA19-9 elevation independently predicted increased mortality with impact similar to node-positivity, positive-margin resection, and non-receipt of chemotherapy. Proposed staging system including CA19-9 improved survival discrimination over AJCC 7th edition., Conclusion: Elevated CA19-9 is an independent risk factor for mortality in ICCA similar in impact to nodal metastases and positive resection margins. Inclusion of CA19-9 in a proposed staging system increases discrimination. Multi-disciplinary therapy should be considered in patients with ICCA and CA19-9 elevation. J. Surg. Oncol. 2016;114:475-482. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Unlabelled: Whether aspirin use is protective against cholangiocarcinoma (CCA) remains unclear. We determined the association between aspirin use and other risk factors for each CCA subtype individually. In a hospital-based case-control study, 2395 CCA cases (1169 intrahepatic, 995 perihilar, and 231 distal) seen at the Mayo Clinic, Rochester, MN, from 2000 through 2014 were enrolled. Controls selected from the Mayo Clinic Biobank were matched two to one with cases by age, sex, race, and residence (n = 4769). Associations between aspirin use, other risk factors, and CCA risk were determined. Aspirin was used by 591 (24.7%) CCA cases and 2129 (44.6%) controls. There was a significant inverse association of aspirin use with all CCA subtypes, with adjusted odds ratios (AORs) of 0.35 (95% confidence interval [CI], 0.29-0.42), 0.34 (95% CI 0.27-0.42), and 0.29 (95% CI 0.19-0.44) for intrahepatic, perihilar, and distal CCA, respectively (P < 0.001 for all). Primary sclerosing cholangitis was more strongly associated with perihilar (AOR = 453, 95% CI 104-999) than intrahepatic (AOR = 93.4, 95% CI 27.1-322) or distal (AOR = 34.0, 95% CI 3.6-323) CCA, whereas diabetes was more associated with distal (AOR = 4.2, 95% CI 2.5-7.0) than perihilar (AOR = 2.9, 95% CI 2.2-3.8) or intrahepatic (AOR = 2.5, 95% CI 2.0-3.2) CCA. Cirrhosis not related to primary sclerosing cholangitis was associated with both intrahepatic and perihilar CCA, with similar AORs of 14. Isolated inflammatory bowel disease without primary sclerosing cholangitis was not associated with any CCA subtype., Conclusions: Aspirin use was significantly associated with a 2.7-fold to 3.6-fold decreased risk for the three CCA subtypes; our study demonstrates that individual risk factors confer risk of different CCA subtypes to different extents. (Hepatology 2016;64:785-796)., (© 2016 by the American Association for the Study of Liver Diseases.)

Background and Aims: Fluorescence in situ hybridization (FISH) has improved the diagnostic performance of cytology for the evaluation of malignant biliary strictures in the United States and Europe. The utility of FISH for the diagnosis of biliary strictures in Asia is currently unknown. We aimed to compare the sensitivity of FISH and conventional cytology for the diagnosis of malignant biliary strictures in Thai patients., Methods: A prospective study was performed at 2 university hospitals between 2010 and 2013. Patients being evaluated for malignant-appearing biliary strictures were included (N = 99). Bile duct brushings were collected and assessed by cytology and FISH. Sensitivities with 95% confidence intervals of cytology and FISH were the main outcome measures., Results: The overall sensitivities of cytology and FISH were 38% and 55%, respectively (P = .001). For those with a diagnosis of cancer based on clinical evidence without biopsy confirmation (n = 44), the sensitivities of cytology and FISH were 43% and 57%, respectively (P = .06). For the 49 patients for whom a cancer diagnosis was confirmed by pathology, FISH had a significantly higher sensitivity than cytology, with a sensitivity of 53% versus 33%, respectively (P = .008)., Conclusions: FISH improves the diagnostic performance of cytology and can be used as a complementary tool to bile duct brushing and biopsy for the evaluation of malignancy in biliary strictures in Asian populations., (Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Sphingosine kinase 2 (Sphk2) has an oncogenic role in cancer. A recently developed first-in-class Sphk2 specific inhibitor ABC294640 displays antitumor activity in many cancer models. However, the role of Sphk2 and the antitumor activity of its inhibitor ABC294640 are not known in cholangiocarcinoma. We investigated the potential of targeting Sphk2 for the treatment of cholangiocarcinoma. We found that Sphk2 is overexpressed in five established human cholangiocarcinoma cell lines (WITT, HuCCT1, EGI-1, OZ and HuH28) and a new patient-derived cholangiocarcinoma cell line (LIV27) compared to H69 normal cholangiocytes. Inhibition of Sphk2 by ABC294640 inhibited proliferation and induced caspase-dependent apoptosis. Furthermore, we found that ABC294640 inhibited STAT3 phosphorylation, one of the key signaling pathways regulating cholangiocarcinoma cell proliferation and survival. ABC294640 also induced autophagy. Inhibition of autophagy by bafilomycin A1 or chloroquine potentiated ABC294640-induced cytotoxicity and apoptosis. In addition, ABC294640 in combination with sorafenib synergistically inhibited cell proliferation of cholangiocarcinoma cells. Strong decreases in STAT3 phosphorylation were observed in WITT and HuCCT1 cells exposed to the ABC294640 and sorafenib combination. These findings provide novel evidence that Sphk2 may be a rational therapeutic target in cholangiocarcinoma. Combinations of ABC294640 with sorafenib and/or autophagy inhibitors may provide novel strategies for the treatment of cholangiocarcinoma.

Unlabelled: Circulating tumor cells (CTCs) in blood are associated with poor survival of patients with breast, prostate, or colon cancer. We hypothesized that CTCs are associated with poor survival of patients with cholangiocarcinoma (CCA). Eighty-eight patients with CCA were prospectively enrolled at Mayo Clinic Rochester between June 2010 and September 2014. The CellSearch system by Veridex was used for detection of CTCs in peripheral blood. Associations between CTC, patient and tumor characteristics, and survival were examined using the Cox's proportional hazards model. Fifteen patients (17%) were positive for CTC ≥2 and 8 patients (9%) for CTC ≥5. CTCs were associated with tumor extent. CTC ≥2 (hazard ratio [HR]: 2.5; 95% confidence interval [CI]: 1.1-5.4; P = 0.02) and CTC ≥5 (HR, 4.1; 95% CI: 1.4-10.8; P = 0.01) were both independent predictors of survival. In subgroup analyses, CTC ≥2 (HR, 8.2; 95% CI: 1.8-57.5; P < 0.01) and CTC ≥5 (HR, 7.7; 95% CI: 1.4-42.9; P = 0.02) were both associated with shorter survival among patients with metastasis. There was a trend toward association of CTC ≥5 with shorter survival in patients with nonmetastatic CCA (HR, 4.3; 95% CI: 1.0-13.8; P = 0.06). CTC ≥2 (HR, 10.5; 95% CI: 2.2-40.1; P < 0.01) and CTC ≥5 (HR, 10.2; 95% CI: 1.5-42.3; P = 0.02) were both associated with shorter survival among patients with perihilar/distal CCA. CTC ≥5 was associated with shorter survival of patients with intrahepatic CCA (HR, 4.2; 95% CI: 1.1-14.1; P = 0.04)., Conclusion: CTCs were associated with more-aggressive tumor characteristics and independently associated with survival in patients with CCA. Assessment of CTCs may be useful for identifying CCA patients at risk of early mortality., (© 2015 by the American Association for the Study of Liver Diseases.)

Background & Aims: Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis., Methods: We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH., Results: The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P < .001) or routine cytology analysis (18.8%) (P < .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P < .001), a mass by cross-sectional imaging (P < .001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis (P = .011)., Conclusions: We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Genetic risk factors for cholangiocarcinoma (CCA) remain poorly understood. We assessed the effect of single-nucleotide polymorphisms (SNPs) of genes modulating inflammation or carcinogenesis on CCA risk and survival. We conducted a case-control, candidate gene association study of 370 CCA patients and 740 age-, sex-, and residential area-matched healthy controls. Eighteen functional or putatively functional SNPs in nine genes were genotyped. The log-additive genotype effects of SNPs on CCA risk and survival were determined using logistic regression and the log-rank test, respectively. Initial analysis identified significant associations between SNP rs2143417 and rs689466 in cyclooxygenase 2 (COX-2) and CCA risk, after adjusting for multiple comparisons (cutoff of P = 0.0028). However, these findings were not replicated in another independent cohort of 212 CCA cases and 424 matched controls. No significant association was found between any SNP and survival of CCA patients. Although COX-2 has been shown to contribute to cholangiocarcinogenesis, the COX-2 SNPs tested were not associated with risk of CCA. This study shows a lack of association between variants of genes related to inflammation and carcinogenesis and CCA risk and survival. Other factors than these genetic variants may play more important roles in CCA risk and survival., (© 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose of Review: This review will cover the role of the fibroblast growth factor pathway in the pathogenesis, targeted therapy potential and prognostic value in patients with cholangiocarcinoma (CCA)., Recent Findings: Recent studies that have identified fibroblast growth factor receptor 2 (FGFR2) fusions, prognostic implications of FGFR2 fusions, treatment strategies that target FGFR2 in CCA and future directions for understanding and targeting the FGFR2 pathway in this disease, will be discussed., Summary: Understanding the role of the FGFR2 pathway as a disease pathogenetic mechanism and the ability to develop targeted therapies and diagnostics surrounding this concept are critical elements toward developing novel targeted approaches in CCA.

Objectives: Current staging systems for perihilar cholangiocarcinoma (pCCA) are inadequate, as they are based on surgical pathology and therefore not relevant to unresectable patients. Clinical trials for potential targeted therapies for pCCA are hampered by the lack of an accurate, nonoperative staging system for predicting survival. We aimed at developing a clinical staging system for pCCA, which would be of prognostic relevance for all pCCA patients and help stratify patients for clinical trials., Methods: Clinical information at the time of pCCA diagnosis of 413 patients seen at Mayo Clinic, Rochester, MN between 2002 and 2010 was retrospectively analyzed. A survival predictive model was developed using Cox proportional hazards analysis. The performance of the staging system was compared with the current AJCC/UICC (the American Joint Committee on Cancer/the Union for International Cancer Control) 7th tumor-node-metastasis (TNM) staging system., Results: Eastern Cooperative Oncology Group (ECOG) status, tumor size and number, vascular encasement, lymph node and peritoneal metastasis and CA 19-9 level were grouped into a four-tier staging system. The median survivals of stages I, II, III, and IV patients were 48.6, 21.8, 8.6, and 2.8 months, with hazard ratios (95% confidence interval) of 1.0 (reference), 1.7 (1.1-2.6), 3.1 (2.0-4.7), and 8.7 (5.2-14.5), respectively (P<0.0001). This staging system had greater concordance statistics (standard error) than the TNM staging system (0.725 (0.018) vs. 0.614 (0.017)), indicating better performance in predicting survival., Conclusions: This staging system, based on nonoperative information at the time of pCCA diagnosis, has excellent discriminatory power to classify patients into four prognostic stages. It could be useful to clinicians and for the design of clinical trials.

Desmoplastic malignancies such as cholangiocarcinoma (CCA) are characterized by a dense stroma containing an abundance of myofibroblasts termed cancer-associated fibroblasts (CAF). The CAF phenotype represents an "activated state" in which cells are primed for cell death triggered by BH3 mimetics. Accordingly, this primed state may be therapeutically exploited. To elucidate the mechanisms underlying this poorly understood apoptotic priming, we examined the role of platelet-derived growth factor (PDGF) in CAF priming for cell death given its prominent role in CAF activation. PDGF isomers PDGF-B and PDGF-D are abundantly expressed in CCA cells derived from human specimens. Either isomer sensitizes myofibroblasts to cell death triggered by BH3 mimetics. Similar apoptotic sensitization was observed with co-culture of myofibroblasts and CCA cells. Profiling of Bcl-2 proteins expressed by PDGF-primed myofibroblasts demonstrated an increase in cellular levels of Puma. PDGF-mediated increases in cellular Puma levels induced proapoptotic changes in Bak, which resulted in its binding to Bcl-2. Short hairpin RNA-mediated down-regulation of Puma conferred resistance to PDGF-mediated apoptotic priming. Conversely, the BH3 mimetic navitoclax disrupted Bcl-2/Bak heterodimers, allowing Bak to execute the cell death program. Treatment with a Bcl-2-specific BH3 mimetic, ABT-199, reduced tumor formation and tumor burden in a murine model of cholangiocarcinoma. Collectively, these findings indicate that apoptotic priming of CAF by PDGF occurs via Puma-mediated Bak activation, which can be converted to active full-blown apoptosis by navitoclax or ABT-199 for therapeutic benefit., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Patients with cholangiocarcinoma often present with locally advanced or metastatic disease. There is a need for effective therapeutic strategies for advanced stage cholangiocarcinoma. Recently, FGFR2 translocations have been identified as a potential target for tyrosine kinase inhibitor therapies. This study evaluated 152 cholangiocarcinomas and 4 intraductal papillary biliary neoplasms of the bile duct for presence of FGFR2 translocations by fluorescence in situ hybridization and characterized the clinicopathologic features of cases with FGFR2 translocations. Thirteen (10 women, 3 men; 8%) of 156 biliary tumors harbored FGFR2 translocations, including 12 intrahepatic cholangiocarcinomas (12/96; 13%) and 1 intraductal papillary neoplasm of the bile duct. Histologically, cholangiocarcinomas with FGFR2 translocations displayed prominent intraductal growth (62%) or anastomosing tubular glands with desmoplasia (38%). Immunohistochemically, the tumors with FGFR2 translocations frequently showed weak and patchy expression of CK19 (77%). Markers of the stem cell phenotype in cholangiocarcinoma, HepPar1 and CK20, were negative in all cases. The median cancer-specific survival for patients whose tumors harbored FGFR2 translocations was 123 months compared to 37 months for cases without FGFR2 translocations (P = .039). This study also assessed 100 cholangiocarcinomas for ERBB2 amplification and ROS1 translocations. Of the cases tested, 3% and 1% were positive for ERBB2 amplification and ROS1 translocation, respectively. These results confirm that FGFR2, ERRB2, and ROS1 alterations are potential therapeutic targets for intrahepatic cholangiocarcinoma., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Patients diagnosed with primary sclerosing cholangitis (PSC) and dominant strictures often undergo endoscopic retrograde cholangiopancreatography with brush cytology to exclude or confirm the development of malignancy. Equivocal (atypical or suspicious) routine cytologic results may confound patient management decisions, especially in the absence of a mass on imaging. The objective of the current study was to identify independent predictors of malignancy in patients with PSC with an equivocal cytology diagnosis., Methods: Patients with PSC underwent brush cytology for routine cytology and fluorescence in situ hybridization (FISH) during endoscopy as per standard care. FISH slides were classified as polysomy if at least 5 cells displayed a gain of ≥ 2 probes. A retrospective search identified 102 patients without a mass lesion noted on initial imaging studies, an equivocal routine cytology, and ≥ 2 years of follow-up., Results: Of 102 patients, 30 (29%) with an equivocal cytology result developed cancer within 2 years. Serum carbohydrate antigen 19-9 (CA 19-9) levels ≥ 129 U/mL (hazard ratio [HR] 3.19; P = .001) and polysomy (HR 8.70; P < .001) were each found to be predictive of cancer. Of 10 patients who had elevated CA 19-9 levels and polysomy, all went on to develop cancer (9 within 2 years). Although only 10 patients were included in this subset, the combination of elevated CA 19-9 and polysomy was found to be predictive of cancer (HR 10.92; P < .001)., Conclusions: Polysomy by FISH identified those patients most likely to have or develop malignancy in the challenging clinical scenario of PSC with no mass at baseline and equivocal cytology. The combination of an elevated serum CA 19-9 level with polysomy is highly suspicious for the presence of malignancy., (© 2013 American Cancer Society.)

Cholangiocarcinoma is a highly lethal cancer of the biliary tract. The intrahepatic subtype of cholangiocarcinoma is increasing in incidence globally. Despite technologic advancements over the past decade, little is known about the somatic changes that occur in these tumors. The goal of this study was to determine the frequency of common oncogenes in resected cholangiocarcinoma specimens that could provide potential therapeutic targets for patients diagnosed with cholangiocarcinoma. Formalin-fixed, paraffin-embedded tissue blocks from 94 resected cholangiocarcinomas were used to extract DNA from areas comprising more than 20% tumor. Specimens were evaluated using the Sequenom MassARRAY OncoCarta Mutation Profiler Panel (San Diego, CA). This matrix-assisted laser desorption/ionization-time of flight mass spectrometry single genotyping panel evaluates 19 oncogenes for 238 somatic mutations. Twenty-five mutations were identified in 23 of the 94 cholangiocarcinomas within the following oncogenes: KRAS (n = 12), PIK3CA (n = 5), MET (n = 4), EGFR (n = 1), BRAF (n = 2), and NRAS (n = 1). Mutations were identified in 7 (26%) of 27 extrahepatic cholangiocarcinomas and 16 (24%) of 67 intrahepatic cholangiocarcinomas. When combined with IDH1/2 testing, 40 (43%) of the 94 cholangiocarcinomas had a detectable mutation. MassARRAY technology can be used to detect mutations in a wide variety of oncogenes using paraffin-embedded tissue. Clinical testing for somatic mutations may drive personalized therapy selection for cholangiocarcinomas in the future. The variety of mutations detected suggests that a multiplexed mutation detection approach may be necessary for managing patients with biliary tract malignancy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cancer-associated fibroblasts (CAF) are abundant in the stroma of desmoplastic cancers where they promote tumor progression. CAFs are "activated" and as such may be uniquely susceptible to apoptosis. Using cholangiocarcinoma as a desmoplastic tumor model, we investigated the sensitivity of liver CAFs to the cytotoxic drug navitoclax, a BH3 mimetic. Navitoclax induced apoptosis in CAF and in myofibroblastic human hepatic stellate cells but lacked similar effects in quiescent fibroblasts or cholangiocarcinoma cells. Unlike cholangiocarcinoma cells, neither CAF nor quiescent fibroblasts expressed Mcl-1, a known resistance factor for navitoclax cytotoxicity. Explaining this paradox, we found that mitochondria isolated from CAFs or cells treated with navitoclax both released the apoptogenic factors Smac and cytochrome c, suggesting that they are primed for cell death. Such death priming in CAFs appeared to be due, in part, to upregulation of the proapoptotic protein Bax. Short hairpin RNA-mediated attenuation of Bax repressed navitoclax-mediated mitochondrial dysfunction, release of apoptogenic factors, and apoptotic cell death. In a syngeneic rat model of cholangiocarcinoma, navitoclax treatment triggered CAF apoptosis, diminishing expression of the desmoplastic extracellular matrix protein tenascin C, suppressing tumor outgrowth, and improving host survival. Together, our findings argue that navitoclax may be useful for destroying CAFs in the tumor microenvironment as a general strategy to attack solid tumors.

Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background & Aims: Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy., Methods: We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib., Results: Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab., Conclusions: We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Unlabelled: It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR-25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR-25 expression, suggesting Hedgehog signaling stimulates miR-25 production. Functionally, miR-25 was shown to protect cells against TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Correspondingly, antagonism of miR-25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor-4 (DR4) as a potential novel miR-25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays., Conclusion: These data implicate elevated miR-25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR-25 target DR4 provides a mechanism by which miR-25 contributes to evasion of TRAIL-induced cholangiocarcinoma apoptosis., (Copyright © 2011 American Association for the Study of Liver Diseases.)