Emerging Technologies for the Diagnosis of Perihilar Cholangiocarcinoma.

The diagnosis of malignant biliary strictures remains problematic, especially in the perihilar region and in primary sclerosing cholangitis (PSC). Conventional cytology obtained during endoscopic retrograde cholangiography (ERC)-guided brushings of biliary strictures is suboptimal due to limited sensitivity, albeit it remains the gold standard with a high specificity. Emerging technologies are being developed and validated to address this pressing unmet patient need. Such technologies include enhanced visualization of the biliary tree by cholangioscopy, intraductal ultrasound, and confocal laser endomicroscopy. Conventional cytology can be aided by employing complementary and advanced cytologic techniques such as fluorescent in situ hybridization (FISH), and this technique should be widely adapted. Interrogation of bile and serum by examining extracellular vesicle number and cargo, and exploiting next-generation sequencing and proteomic technologies, is also being explored. Examination of circulating cell-free deoxyribonucleic acid (cfDNA) for differentially methylated regions is a promising test which is being rigorously validated. The special expertise required for these analyses has to date hampered their validation and adaptation. Herein, we will review these emerging technologies to inform the reader of the progress made and encourage further studies, as well as adaptation of validated approaches.

Fibroblast growth factor receptor inhibition induces loss of matrix MCL1 and necrosis in cholangiocarcinoma.

BACKGROUND & AIMS: Myeloid cell leukemia 1 (MCL1), a prosurvival member of the BCL2 protein family, has a pivotal role in human cholangiocarcinoma (CCA) cell survival. We previously reported that fibroblast growth factor receptor (FGFR) signalling mediates MCL1-dependent survival of CCA cells in vitro and in vivo. However, the mode and mechanisms of cell death in this model were not delineated. METHODS: Human CCA cell lines were treated with the pan-FGFR inhibitor LY2874455 and the mode of cell death examined by several complementary assays. Mitochondrial oxidative metabolism was examined using a XF24 extracellular flux analyser. The efficiency of FGFR inhibition in patient-derived xenografts (PDX) was also assessed. RESULTS: CCA cells expressed two species of MCL1, a full-length form localised to the outer mitochondrial membrane, and an N terminus-truncated species compartmentalised within the mitochondrial matrix. The pan-FGFR inhibitor LY2874455 induced non-apoptotic cell death in the CCA cell lines associated with cellular depletion of both MCL1 species. The cell death was accompanied by failure of mitochondrial oxidative metabolism and was most consistent with necrosis. Enforced expression of N terminus-truncated MCL1 targeted to the mitochondrial matrix, but not full-length MCL1 targeted to the outer mitochondrial membrane, rescued cell death and mitochondrial function. LY2874455 treatment of PDX-bearing mice was associated with tumour cell loss of MCL1 and cell necrosis. CONCLUSIONS: FGFR inhibition induces loss of matrix MCL1, resulting in cell necrosis. These observations support a heretofore unidentified, alternative MCL1 survival function, namely prevention of cell necrosis, and have implications for treatment of human CCA. LAY SUMMARY: Herein, we report that therapeutic inhibition of a cell receptor expressed by bile duct cancer cells resulted in the loss of a critical survival protein termed MCL1. Cellular depletion of MCL1 resulted in the death of the cancer cells by a process characterised by cell rupture. Cell death by this process can stimulate the immune system and has implications for combination therapy using receptor inhibition with immunotherapy.

Targeting cholangiocarcinoma.

Cholangiocarcinoma (CCA) represents a diverse group of epithelial cancers associated with the biliary tract, and can best be stratified anatomically into intrahepatic (iCCA), perihilar (pCCA) and distal (dCCA) subsets. Molecular profiling has identified genetic aberrations associated with these anatomic subsets. For example, IDH catalytic site mutations and constitutively active FGFR2 fusion genes are predominantly identified in iCCA, whereas KRAS mutations and PRKACB fusions genes are identified in pCCA and dCCA. Clinical trials targeting these specific driver mutations are in progress. However, The Tumor Genome Atlas (TCGA) marker analysis of CCA also highlights the tremendous molecular heterogeneity of this cancer rendering comprehensive employment of targeted therapies challenging. CCA also display a rich tumor microenvironment which may be easier to target. For example, targeting cancer associated fibroblasts for apoptosis with BH3-mimetics and/or and reversing T-cell exhaustion with immune check point inhibitors may help aid in the treatment of this otherwise devastating malignancy. Combinatorial therapy attacking the tumor microenvironment plus targeted therapy may help advance treatment for CCA. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma.

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.

Emerging molecular therapeutic targets for cholangiocarcinoma.

Cholangiocarcinomas (CCAs) are diverse epithelial tumors arising from the liver or large bile ducts with features of cholangiocyte differentiation. CCAs are classified anatomically into intrahepatic (iCCA), perihilar (pCCA), and distal CCA (dCCA). Each subtype has distinct risk factors, molecular pathogenesis, therapeutic options, and prognosis. CCA is an aggressive malignancy with a poor overall prognosis and median survival of less than 2years in patients with advanced disease. Potentially curative surgical treatment options are limited to the subset of patients with early-stage disease. Presently, the available systemic medical therapies for advanced or metastatic CCA have limited therapeutic efficacy. Molecular alterations define the differences in biological behavior of each CCA subtype. Recent comprehensive genetic analysis has better characterized the genomic and transcriptomic landscape of each CCA subtype. Promising candidates for targeted, personalized therapy have emerged, including potential driver fibroblast growth factor receptor (FGFR) gene fusions and somatic mutations in isocitrate dehydrogenase (IDH)1/2 in iCCA, protein kinase cAMP-activated catalytic subunit alpha (PRKACA) or beta (PRKACB) gene fusions in pCCA, and ELF3 mutations in dCCA/ampullary carcinoma. A precision genomic medicine approach is dependent on an enhanced understanding of driver mutations in each subtype and stratification of patients according to their genetic drivers. We review the current genomic landscape of CCA, the potentially actionable molecular aberrations in each CCA subtype, and the role of immunotherapy in CCA.

IL-33 facilitates oncogene-induced cholangiocarcinoma in mice by an interleukin-6-sensitive mechanism.

UNLABELLED: Cholangiocarcinoma (CCA) is a lethal hepatobiliary neoplasm originating from the biliary apparatus. In humans, CCA risk factors include hepatobiliary inflammation and fibrosis. The recently identified interleukin (IL)-1 family member, IL-33, has been shown to be a biliary mitogen which also promotes liver inflammation and fibrosis. Our aim was to generate a mouse model of CCA mimicking the human disease. Ectopic oncogene expression in the biliary tract was accomplished by the Sleeping Beauty transposon transfection system with transduction of constitutively active AKT (myr-AKT) and Yes-associated protein. Intrabiliary instillation of the transposon-transposase complex was coupled with lobar bile duct ligation in C57BL/6 mice, followed by administration of IL-33 for 3 consecutive days. Tumors developed in 72% of the male mice receiving both oncogenes plus IL-33 by 10 weeks but in only 20% of the male mice transduced with the oncogenes alone. Tumors expressed SOX9 and pancytokeratin (features of CCA) but were negative for HepPar1 (a marker of hepatocellular carcinoma). Substantive overlap with human CCA specimens was revealed by RNA profiling. Not only did IL-33 induce IL-6 expression by human cholangiocytes but it likely facilitated tumor development in vivo by an IL-6-sensitive process as tumor development was significantly attenuated in Il-6(-/-) male animals. Furthermore, tumor formation occurred at a similar rate when IL-6 was substituted for IL-33 in this model. CONCLUSION: The transposase-mediated transduction of constitutively active AKT and Yes-associated protein in the biliary epithelium coupled with lobar obstruction and IL-33 administration results in the development of CCA with morphological and biochemical features of the human disease; this model highlights the role of inflammatory cytokines in CCA oncogenesis.

Platelet-derived growth factor primes cancer-associated fibroblasts for apoptosis.

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.

Primary Sclerosing Cholangitis as a Premalignant Biliary Tract Disease: Surveillance and Management.

Primary sclerosing cholangitis (PSC) is a premalignant biliary tract disease that confers a significant risk for the development of cholangiocarcinoma (CCA). The chronic biliary tract inflammation of PSC promotes pro-oncogenic processes such as cellular proliferation, induction of DNA damage, alterations of the extracellular matrix, and cholestasis. The diagnosis of malignancy in PSC can be challenging because inflammation-related changes in PSC may produce dominant biliary tract strictures mimicking CCA. Biomarkers such as detection of methylated genes in biliary specimens represent noninvasive techniques that may discriminate malignant biliary ductal changes from PSC strictures. However, conventional cytology and advanced cytologic techniques such as fluorescence in situ hybridization for polysomy remain the practice standard for diagnosing CCA in PSC. Curative treatment options of malignancy arising in PSC are limited. For a subset of patients selected by using stringent criteria, liver transplantation after neoadjuvant chemoradiation is a potential curative therapy. However, most patients have advanced malignancy at the time of diagnosis. Advances directed at identifying high-risk patients, early cancer detection, and development of chemopreventive strategies will be essential to better manage the cancer risk in this premalignant disease. A better understanding of dysplasia definition and especially its natural history is also needed in this disease. Herein, we review recent developments in our understanding of the risk factors, pathogenic mechanisms of PSC associated with CCA, as well as advances in early detection and therapies.

Molecular Profiling and Research of Therapeutic Targets.

The heterogeneous nature of hepatocellular carcinoma (HCC) poses a challenge to effective therapy. Significant strides have been made in molecular profiling. Telomerase promoter mutations are now known to represent early events in linear carcinogenesis. However, the translation of these advances into clinical practice has been limited. Although further work is needed to extrapolate genetic information to patient care, progress has been made in using genetics and stratification for HCC therapy. Targeted therapies for patients with c-MET overexpression, VEGF-A amplification and Wnt ß-catenin-driven tumors offer promise.

Cholangiocarcinoma: molecular pathways and therapeutic opportunities.

Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. Currently, there are no curative medical therapies for CCA. Recent advances have enhanced our understanding of the genetic basis of this disease, and elucidated therapeutically relevant targets. Therapeutic efforts in development are directed at several key pathways due to genetic aberrations including receptor tyrosine kinase pathways, mutant IDH enzymes, the PI3K-AKT-mTOR pathway, and chromatin remodeling networks. A highly desmoplastic, hypovascular stroma is characteristic of CCAs and recent work has highlighted the importance of targeting this pathway via stromal myofibroblast depletion. Future efforts should concentrate on combination therapies with action against the cancer cell and the surrounding tumor stroma. As the mutational landscape of CCA is being illuminated, molecular profiling of patient tumors will enable identification of specific mutations and the opportunity to offer directed, personalized treatment options.

Pathogenesis, diagnosis, and management of cholangiocarcinoma.

Cholangiocarcinomas (CCAs) are hepatobiliary cancers with features of cholangiocyte differentiation; they can be classified anatomically as intrahepatic CCA (iCCA), perihilar CCA (pCCA), or distal CCA. These subtypes differ not only in their anatomic location, but in epidemiology, origin, etiology, pathogenesis, and treatment. The incidence and mortality of iCCA has been increasing over the past 3 decades, and only a low percentage of patients survive until 5 years after diagnosis. Geographic variations in the incidence of CCA are related to variations in risk factors. Changes in oncogene and inflammatory signaling pathways, as well as genetic and epigenetic alterations and chromosome aberrations, have been shown to contribute to the development of CCA. Furthermore, CCAs are surrounded by a dense stroma that contains many cancer-associated fibroblasts, which promotes their progression. We have gained a better understanding of the imaging characteristics of iCCAs and have developed advanced cytologic techniques to detect pCCAs. Patients with iCCAs usually are treated surgically, whereas liver transplantation after neoadjuvant chemoradiation is an option for a subset of patients with pCCAs. We review recent developments in our understanding of the epidemiology and pathogenesis of CCA, along with advances in classification, diagnosis, and treatment.

Polo-like kinase 2 is a mediator of hedgehog survival signaling in cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and thus rely on potent survival signals to circumvent cell death by TRAIL. Hedgehog (Hh) signaling is an important survival pathway in CCA. Herein, we further examine the mechanisms whereby Hh signaling mediates apoptosis resistance in CCA, revealing a pivotal role for the cell division regulating serine/threonine kinase polo-like kinase 2 (PLK2). We employed 50 human CCA samples (25 intrahepatic and 25 extrahepatic CCA) as well as human KMCH-1, Mz-CHA-1, and HUCCT-1 CCA cells for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. In human samples, polo-like kinase (PLK)1/2/3-immunoreactive cancer cells were present in the preponderance of intra- and extrahepatic CCA specimens. Inhibition of Hh signaling by cyclopamine reduced PLK2, but not PLK1 or PLK3, messenger RNA and protein expression in vehicle-treated and sonic Hh-treated CCA cells, confirming our previous microarray study. PLK2 regulation by Hh signaling appears to be direct, because the Hh transcription factors, glioma-associated oncogene 1 and 2, bind to the PLK2 promotor. Moreover, inhibition of PLK2 by the PLK inhibitor, BI 6727 (volasertib), or PLK2 knockdown was proapoptotic in CCA cells. BI 6727 administration or PLK2 knockdown decreased cellular protein levels of antiapoptotic myeloid cell leukemia 1 (Mcl-1), an effect reversed by the proteasome inhibitor, MG-132. Finally, BI 6727 administration reduced Mcl-1 protein expression in CCA cells, resulting in CCA cell apoptosis and tumor suppression in vivo. CONCLUSION: PLK2 appears to be an important mediator of Hh survival signaling. These results suggest PLK inhibitors to be of therapeutic value for treatment of human CCA.

Molecular pathogenesis of cholangiocarcinoma.

It has become increasingly apparent of late that inflammation plays an integral role in a spectrum of malignancies including cholangiocarcinoma (CCA). Primary sclerosing cholangitis with chronic inflammation is the most common risk factor for CCA in the Western world. Recent work has highlighted that inflammatory pathways are essential in carcinogenesis and tissue invasion and migration. Inflammation advances carcinogenesis by induction of DNA damage, evasion of apoptosis, promotion of cell proliferation, and neoangiogenesis. CCA is characterized by the presence of a desmoplastic stroma consisting of cancer-associated fibroblasts, tumor-associated macrophages, and tumor-infiltrating lymphocytes. This rich inflammatory milieu is vital to the cancer ecosystem, and targeting its components represents an attractive therapeutic option.

Current diagnostic and management options in perihilar cholangiocarcinoma.

Cholangiocarcinomas (CCAs) are heterogeneous biliary tract tumors with dismal prognosis. Perihilar CCA (pCCA) involves the large bile ducts of the hepatic hilum, and is the most common type of CCA. Primary sclerosing cholangitis (PSC) is an established risk factor for pCCA. Although the diagnosis of pCCA is challenging, recent advances have been made including cytologic techniques such as fluorescence in situ hybridization. Endoscopic ultrasound with sampling of regional lymph nodes is emerging as a valuable diagnostic modality in the diagnosis and staging of pCCA. Curative treatment options are limited to early stage disease, and include surgical resection and liver transplantation after neoadjuvant therapy. This underscores the importance of early detection, and the need for development of innovative diagnostic tools such as biomarkers. A dense desmoplastic tumor stroma plays an integral role in pCCA progression. The tumor stroma represents an additional target for development of new therapies. Herein, we discuss these advances in the diagnosis and treatment of pCCA.

Sequence-specific recruitment of heterochromatin protein 1 via interaction with Krüppel-like factor 11, a human transcription factor involved in tumor suppression and metabolic diseases.

Heterochromatin protein 1 (HP1) proteins are "gatekeepers" of epigenetic gene silencing that is mediated by lysine 9 of histone H3 methylation (H3K9me). Current knowledge supports a paradigm whereby HP1 proteins achieve repression by binding to H3K9me marks and interacting to H3K9 histone methyltransferases (HMTs), such as SUV39H1, which methylate this residue on adjacent nucleosomes thereby compacting chromatin and silencing gene expression. However, the mechanism underlying the recruitment of this epigenetic regulator to target gene promoters remains poorly characterized. In the current study, we reveal for the first time a mechanism whereby HP1 is recruited to promoters by a well characterized Krüppel-like transcription factor (KLF), in a sequence-specific manner, to mediate complex biological phenomena. A PXVXL HP1-interacting domain identified at position 487-491 of KLF11 mediates the binding of HP1α and KLF11 in vitro and in cultured cells. KLF11 also recruits HP1α and its histone methyltransferase, SUV39H1, to promoters to limit KLF11-mediated gene activation. Indeed, a KLF11ΔHP1 mutant derepresses KLF11-regulated cancer genes, by inhibiting HP1-SUV39H1 recruitment, decreasing H3K9me3, while increasing activation-associated marks. Biologically, impairment of KLF11-mediated HP1-HMT recruitment abolishes tumor suppression, providing direct evidence that HP1-HMTs act in a sequence-specific manner to achieve this function rather than its well characterized binding to methylated chromatin without intermediary. Collectively, these studies reveal a novel role for HP1 as a cofactor in tumor suppression, expand our mechanistic understanding of a KLF associated to human disease, and outline cellular and biochemical mechanisms underlying this phenomenon, increasing the specificity of targeting HP1-HMT complexes to gene promoters.