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    Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells. Carotenuto Pietro,Hedayat Somaieh,Fassan Matteo,Cardinale Vincenzo,Lampis Andrea,Guzzardo Vincenza,Vicentini Caterina,Scarpa Aldo,Cascione Luciano,Costantini Daniele,Carpino Guido,Alvaro Domenico,Ghidini Michele,Trevisani Francesco,Te Poele Robert,Salati Massimiliano,Ventura Sofia,Vlachogiannis Georgios,Hahne Jens C,Boulter Luke,Forbes Stuart J,Guest Rachel V,Cillo Umberto,Said-Huntingford Ian,Begum Ruwaida,Smyth Elizabeth,Michalarea Vasiliki,Cunningham David,Rimassa Lorenza,Santoro Armando,Roncalli Massimo,Kirkin Vladimir,Clarke Paul,Workman Paul,Valeri Nicola,Braconi Chiara Hepatology (Baltimore, Md.) BACKGROUND AND AIMS:Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS:High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS:MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics. 10.1002/hep.31094
    Genomic and Transcriptomic Profiling of Combined Hepatocellular and Intrahepatic Cholangiocarcinoma Reveals Distinct Molecular Subtypes. Xue Ruidong,Chen Lu,Zhang Chong,Fujita Masashi,Li Ruoyan,Yan Shu-Mei,Ong Choon Kiat,Liao Xiwen,Gao Qiang,Sasagawa Shota,Li Yanmeng,Wang Jincheng,Guo Hua,Huang Qi-Tao,Zhong Qian,Tan Jing,Qi Lisha,Gong Wenchen,Hong Zhixian,Li Meng,Zhao Jingmin,Peng Tao,Lu Yinying,Lim Kiat Hon Tony,Boot Arnoud,Ono Atushi,Chayama Kazuaki,Zhang Zemin,Rozen Steve George,Teh Bin Tean,Wang Xin Wei,Nakagawa Hidewaki,Zeng Mu-Sheng,Bai Fan,Zhang Ning Cancer cell We performed genomic and transcriptomic sequencing of 133 combined hepatocellular and intrahepatic cholangiocarcinoma (cHCC-ICC) cases, including separate, combined, and mixed subtypes. Integrative comparison of cHCC-ICC with hepatocellular carcinoma and intrahepatic cholangiocarcinoma revealed that combined and mixed type cHCC-ICCs are distinct subtypes with different clinical and molecular features. Integrating laser microdissection, cancer cell fraction analysis, and single nucleus sequencing, we revealed both mono- and multiclonal origins in the separate type cHCC-ICCs, whereas combined and mixed type cHCC-ICCs were all monoclonal origin. Notably, cHCC-ICCs showed significantly higher expression of Nestin, suggesting Nestin may serve as a biomarker for diagnosing cHCC-ICC. Our results provide important biological and clinical insights into cHCC-ICC. 10.1016/j.ccell.2019.04.007
    Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma. Arbelaiz Ander,Azkargorta Mikel,Krawczyk Marcin,Santos-Laso Alvaro,Lapitz Ainhoa,Perugorria Maria J,Erice Oihane,Gonzalez Esperanza,Jimenez-Agüero Raul,Lacasta Adelaida,Ibarra Cesar,Sanchez-Campos Alberto,Jimeno Juan P,Lammert Frank,Milkiewicz Piotr,Marzioni Marco,Macias Rocio I R,Marin Jose J G,Patel Tushar,Gores Gregory J,Martinez Ibon,Elortza Félix,Falcon-Perez Juan M,Bujanda Luis,Banales Jesus M Hepatology (Baltimore, Md.) Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with poor prognosis. Several conditions, such as primary sclerosing cholangitis (PSC), are risk factors. Noninvasive differential diagnosis between intrahepatic CCA and hepatocellular carcinoma (HCC) is sometimes difficult. Accurate noninvasive biomarkers for PSC, CCA, and HCC are not available. In the search for novel biomarkers, serum extracellular vesicles (EV) were isolated from CCA (n = 43), PSC (n = 30), or HCC (n = 29) patients and healthy individuals (control, n = 32); and their protein content was characterized. By using nanoparticle tracking analysis, serum EV concentration was found to be higher in HCC than in all the other groups. Round morphology (by transmission electron microscopy), size (∼180 nm diameter by nanoparticle tracking analysis), and markers (clusters of differentiation 9, 63, and 81 by immunoblot) indicated that most serum EV were exosomes. Proteome profiles (by mass spectrometry) revealed multiple differentially expressed proteins among groups. Several of these proteins showed high diagnostic values with maximum area under the receiver operating characteristic curve of 0.878 for CCA versus control, 0.905 for CCA stage I-II versus control, 0.789 for PSC versus control, 0.806 for noncirhottic PSC versus control, 0.796 for CCA versus PSC, 0.956 for CCA stage I-II versus PSC, 0.904 for HCC versus control, and 0.894 for intrahepatic CCA versus HCC. Proteomic analysis of EV derived from CCA human cells in vitro revealed higher abundance of oncogenic proteins compared to EV released by normal human cholangiocytes. Orthotopic implant of CCA human cells in the liver of immunodeficient mice resulted in the release to serum of EV containing some similar human oncogenic proteins. CONCLUSION:Proteomic signatures found in serum EV of CCA, PSC, and HCC patients show potential usefulness as diagnostic tools. (Hepatology 2017;66:1125-1143). 10.1002/hep.29291
    Serum Metabolites as Diagnostic Biomarkers for Cholangiocarcinoma, Hepatocellular Carcinoma, and Primary Sclerosing Cholangitis. Banales Jesus M,Iñarrairaegui Mercedes,Arbelaiz Ander,Milkiewicz Piotr,Muntané Jordi,Muñoz-Bellvis Luis,La Casta Adelaida,Gonzalez Luis M,Arretxe Enara,Alonso Cristina,Martínez-Arranz Ibon,Lapitz Ainhoa,Santos-Laso Alvaro,Avila Matias A,Martínez-Chantar Maria L,Bujanda Luis,Marin Jose J G,Sangro Bruno,Macias Rocio I R Hepatology (Baltimore, Md.) Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of low-molecular-weight metabolites by new high-throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19-9 (CA 19-9) and alpha-fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy-proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14-15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases. 10.1002/hep.30319
    Molecular diagnosis of intrahepatic cholangiocarcinoma. Haga Hiroaki,Patel Tushar Journal of hepato-biliary-pancreatic sciences Intrahepatic cholangiocarcinomas (iCCA) are primary intrahepatic malignancies originating from biliary epithelia. While both hepatocellular cancer and iCCA can present as mass lesions within the liver, these cancers are distinct in their morphology, etiology, pathology, natural history and response to therapy. There is a need for accurate and sensitive molecular markers for the diagnosis of iCCA. Recent advances in elucidating molecular and genetic characteristics of iCCA offer the potential of molecular-based diagnosis of iCCA. Specific genetic mutations of IDH1/2, BAP1, p53, and KRAS, FGFR gene fusions and alterations in microRNA have all been described in iCCA. Although there are no accurate serum or biliary biomarkers currently available for diagnosis of iCCA, several potential candidates have been identified. Knowledge of specific genetic or molecular abnormalities offers potential for individualized approaches for the treatment of patients with iCCA in the future. 10.1002/jhbp.156
    Intrahepatic cholangiocarcinoma: current management and emerging therapies. Rahnemai-Azar Amir A,Weisbrod Allison B,Dillhoff Mary,Schmidt Carl,Pawlik Timothy M Expert review of gastroenterology & hepatology INTRODUCTION:Intrahepatic cholangiocarcinoma (iCCA) is a malignancy with an increasing incidence and a high-case fatality. While surgery offers the best hope at long-term survival, only one-third of tumors are amenable to surgical resection at the time of the diagnosis. Unfortunately, conventional chemotherapy offers limited survival benefit in the management of unresectable or metastatic disease. Recent advances in understanding the molecular pathogenesis of iCCA and the use of next-generation sequencing techniques have provided a chance to identify 'target-able' molecular aberrations. These novel molecular therapies offer the promise to personalize therapy for patients with iCCA and, in turn, improve the outcomes of patients. Area covered: We herein review the current management options for iCCA with a focus on defining both established and emerging therapies. Expert commentary: Surgical resection remains as an only hope for cure in iCCA patients. However, frequently the diagnosis is delayed till advanced stages when surgery cannot be offered; signifying the urge for specific diagnostic tumor biomarkers and targeted therapies. New advances in genomic profiling have contributed to a better understanding of the landscape of molecular alterations in iCCA and offer hope for the development of novel diagnostic biomarkers and targeted therapies. 10.1080/17474124.2017.1309290
    Intrahepatic Cholangiocarcinoma: Continuing Challenges and Translational Advances. Sirica Alphonse E,Gores Gregory J,Groopman John D,Selaru Florin M,Strazzabosco Mario,Wei Wang Xin,Zhu Andrew X Hepatology (Baltimore, Md.) Intrahepatic cholangiocarcinoma (iCCA) has over the last 10-20 years become the focus of increasing concern, largely due to its rising incidence and high mortality rates worldwide. The significant increase in mortality rates from this primary hepatobiliary cancer, particularly over the past decade, has coincided with a rapidly growing interest among clinicians, investigators, and patient advocates to seek greater mechanistic insights and more effective biomarker-driven targeted approaches for managing and/or preventing this challenging liver cancer. In addition to discussing challenges posed by this aggressive cancer, this review will emphasize recent epidemiological, basic, and translational research findings for iCCA. In particular, we will highlight emerging demographic changes and evolving risk factors, the critical role of the tumor microenvironment, extracellular vesicle biomarkers and therapeutics, intertumoral and intratumoral heterogeneity, and current and emerging targeted therapies regarding iCCA. Specifically, recent evidence linking non-bile duct medical conditions, such as nonalcoholic fatty liver disease and nonspecific cirrhosis, to intrahepatic cholangiocarcinogenesis together with geographic and ethnic variation will be assessed. Recent developments concerning the roles played by transforming growth factor-β and platelet-derived growth factor D in driving the recruitment and expansion of cancer-associated myofibroblasts within cholangiocarcinoma (CCA) stroma as well as their therapeutic implications will also be discussed. In addition, the potential significance of extracellular vesicles as bile and serum biomarkers and therapeutic delivery systems for iCCA will be described. An integrated systems approach to classifying heterogeneous iCCA subtypes will be further highlighted, and recent clinical trials and emerging targeted therapies will be reviewed, along with recommendations for future translational research opportunities. Established international CCA networks are now facilitating collaborations aimed at advancing iCCA translational and clinical research. 10.1002/hep.30289