Molecular Pathogenesis and Targeted Therapies for Intrahepatic

Published OnlineFirst September 24, 2015; DOI: 10.1158/1078-0432.CCR-14-3296

Review Clinical Cancer Research Molecular Pathogenesis and Targeted Therapies for Intrahepatic Cholangiocarcinoma Agrin Moeini1,2, Daniela Sia1,2,3, Nabeel Bardeesy4, Vincenzo Mazzaferro3, and Josep M. Llovet1,2,5

Abstract

Intrahepatic cholangiocarcinoma (iCCA) is a molecularly of recurrent novel fusion events (FGFR2 and ROS1 fusions) heterogeneous hepatobiliary neoplasm with poor prognosis and somatic mutations in metabolic (IDH1/2) and chromatin- and limited therapeutic options. The incidence of this neoplasm remodeling genes (ARID1A, BAP1). These latest advancements is growing globally. One third of iCCA tumors are amenable to along with known mutations in KRAS/BRAF/EGFR and 11q13 surgical resection, but most cases are diagnosed at advanced high-level amplification have contributed to a better under- stages with chemotherapy as the only established standard of standing of the landscape of molecular alterations in iCCA. practice. No molecular therapies are currently available for the More than 100 clinical trials testing molecular therapies alone treatment of this neoplasm. The poor understanding of the or in combination with chemotherapy including iCCA patients biology of iCCA and the lack of known oncogenic addiction have not reported conclusive clinical benefits. Recent discover- loops has hindered the development of effective targeted ies have shown that up to 70% of iCCA patients harbor therapies. Studies with sophisticated animal models defined potential actionable alterations that are amenable to therapeu- IDH mutation as the first gatekeeper in the carcinogenic process tic targeting in early clinical trials. Thus, the first biomarker- and led to the discovery of striking alternative cellular origins. driven trials are currently underway. Clin Cancer Res; 22(2); 1–10. RNA- and exome-sequencing technologies revealed the presence 2015 AACR.

Introduction During the past decade a growing interest has been expressed in iCCA due to a marked increase in both incidence and mortality Intrahepatic cholangiocarcinoma (iCCA) is the second most rates (1, 4). Currently, surgical resection represents the sole common liver cancer following hepatocellular carcinoma (HCC), curative treatment option in 30% to 40% of patients with 5-year accounting for 5% to 10% of all primary liver malignancies with survival of 20% to 40% (1, 5). The majority of iCCA patients have an annual incidence of 2 cases per 100,000 in Western countries no underlying liver disease or known risk factors, which further (1, 2). At present, it is widely accepted that iCCA arises from the hinders the development of screening strategies for early detec- malignant transformation of the intrahepatic cholangiocytes and tion. In patients with advanced disease, the combination of is anatomically distinguished from the extrahepatic biliary tract gemcitabine and cisplatin has been shown to confer a survival cancers (eCCA), which are known as perihilar (pCCA) and distal advantage over gemcitabine alone and is currently proposed as (dCCA), with the second-order bile ducts acting as the separation the standard of practice (6). As opposed to HCC, to date there is no point (3). approved targeted molecular therapy for iCCA, and the identification of a first-line conclusive treatment remains an unmet need. Recently, the use of next-generation sequencing technologies has enabled the identification of recurrent actionable molecular 1Liver Cancer Translational Research Laboratory, Liver Unit, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Hospi- alterations that hold the promise of improving the management tal Clínic, CIBERehd, Universitat de Barcelona, Barcelona, Catalonia, of advanced iCCA patients. Herein, we provide an overview of the Spain. 2Liver Cancer Program, Division of Liver Diseases, Depart- recent discoveries of new molecular targets that should ultimately ment of Medicine, Tisch Cancer Institute, Icahn School of Medicine at lead to the development of more personalized therapeutic Mount Sinai, New York, New York. 3Gastrointestinal Surgery and Liver Transplantation Unit, Department of Surgery, National Cancer Insti- approaches. tute IRCCS Foundation, Milan, Italy. 4Cancer Center, Center for Regenerative Medicine, and Department of Molecular Biology, Mas- sachusetts General Hospital, Harvard University, Boston, Massachu- Epidemiology and Risk Factors 5 setts. Institucio Catalana de Recerca i Estudis Avancats,¸ Barcelona, iCCA is a devastating disease with poor prognosis. Several Catalonia, Spain. studies have reported global trends of increasing incidence and Note: V. Mazzaferro and J.M. Llovet share senior authorship. mortality for iCCA in contrast with decreasing rates for eCCA (7– Corresponding Author: Josep M. Llovet, BCLC Group, Liver Unit, IDIBAPS, 10). iCCA presents more commonly at older age with a slight CIBERehd, Hospital Clínic, University of Barcelona, Rosello 149, Barcelona predominance in men (male to female ratio 1.2–1.5:1; ref. 1). 08036, Catalonia, Spain. Phone: 349-3227-9156; Fax: 349-3227-5792; E-mail: There is a considerable geographic and demographic variation in [email protected] the epidemiology of iCCA, which likely reflects distinct environ- doi: 10.1158/1078-0432.CCR-14-3296 mental and genetic predispositions. The incidence of iCCA is the 2015 American Association for Cancer Research. highest in Southeast Asia and more specifically in Thailand (>80

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Table 1. Main epidemiologic and molecular differences between iCCA and extrahepatic subtypes (pCCA-dCCA) Gene or molecule iCCA pCCA-dCAA References Proportion of CCA cases 5%–20% pCCA (50%–70%), dCCA (15%–20%) (12–15) Incidence rate Increasing Stable or slightly decreasing (7–10) Anatomic location Intrahepatic biliary tract Extrahepatic biliary tract (3) pCCA (near origin of cystic duct) dCCA (lower half of large duct) Differenctial risk factors (n ¼ positive cases/total, % casesa) Biliary lithiasisb 377/1,539 (24%) 289/549 (52%) (17, 18, 20–23) Cirrhosis 161/1,622 (10%) 23/712 (3%) (17–23) HCV 61/1,522 (4%) 11/712 (1.5%) (17–21, 23) HBV 129/1,411 (9%) 4/712 (0.6%) (17–22) Alcoholc 158/1,524 (10%) 37/712 (5%) (17–22) Molecular alterations (n ¼ positive cases/total, % casesa) Somatic mutations TP53 99/606 (16%) 36/137 (26%) (50–53, 56–62) KRAS 165/885 (19%) 29/152 (19%) (50–53, 56–62) IDH1/2 143/951 (15%) 3/164 (2%) (51–54, 56–62) ARID1A 50/390 (13%) 20/137 (14%) (51–54, 56–57, 59, 61–62) BAP1 45/443 (11%) 3/164 (2%) (51–54, 56–57, 59, 61–62) BRAF 28/574 (5%) 0/137 (0%) (50–51, 53–54, 55–59, 61) EGFR 14/545 (3%) 3/151 (2%) (50–51, 53–54, 55–59, 61) Fusion proteins FGFR2 fusions 71/307 (23%) 0/36 (0%) (51, 56, 57, 72, 73, 75) Chromosomal abberations (ampifications)d 17q11 (ERBB2) 0/170 (0%) 10/55 (18%) (31, 66) 11q13 (FGF19, CCDN1, ORAOV1) 5/128 (4%) NA (31) NOTE: Frequencies in iCCA have been calculated only in non–liver fluke cases. Abbreviations: dCCA, distal cholangiocarcinoma; HBV, hepatitis B virus infection; HCV, hepatitis C virus infection; iCCA, intrahepatic cholangiocarcinoma; NA, not applicable; pCCA, perihilar cholangiocarcinoma. aThe percentage of cases has been calculated by considering the number of samples presenting the molecular alteration over the total number of samples analyzed in all cohorts (discovery and validation set of samples). bBiliary lithiasis includes patients with hepatolithiasis, cholelithiasis, and choledocholithiasis. cPatients with heavy alcohol consumption or alcoholic liver disease. dGenomic amplifications evaluated by FISH assay or copy number alteration by SNP array.

cases per 100,000) and can be as low as 0.2 per 100,000 in some cell of origin. Thus, iCCA is currently believed to derive from Western countries (1, 11). Even though the vast majority of biliary epithelial cells (cholangiocytes) of the intrahepatic biliary iCCAs are sporadic, several risk factors have been identified. tract, hepatic progenitor cells (HPC), or even mature hepatocytes. Historically, most of these risk factors have been established All liver cells share a common embryonic origin, arising from for CCA without distinguishing between iCCA and eCCA, despite bipotential progenitors known as hepatoblasts (26). However, the fact that increasing evidence supports the hypothesis that in the adult liver, normal tissue turnover is mainly sustained by they represent distinct entities with marked differences in their differentiated hepatocytes and cholangiocytes. Nevertheless, genomic features and epidemiology (Table 1; refs. 3, 12–15). The upon major injury, there is an expansion of cells in the region most prevalent risk factors for HCC have also been significantly of the canals of Hering that have been proposed to be bipotent associated with iCCA but not with eCCA (Table 1), including HPCs capable of differentiating into hepatocyte or cholangio- cirrhosis and chronic hepatitis B and C infections (1, 11, 16–23). cyte lineages (Fig. 1; ref. 27). Alternatively, hepatocytes can Other risk factors for iCCA include primary sclerosing cholangitis dedifferentiate into progenitor-like cells in response to acute (PSC), biliary duct cysts, hepatolithiasis, and hepatobiliary flukes. injury (28, 29). Hepatolithiasis has been defined as a well-known risk factor for With this backdrop, the hypothesis that iCCA and HCC may iCCA (up to 20%) in Asian countries but not in Western countries share a common ancestor such as the HPCs has been an important (11). Less-established risk factors with modest associations subject of discussion during the past decade. Notably, emerging include inflammatory bowel disease, obesity, diabetes, and alco- data point to an overlapping molecular profile between specific hol abuse (1, 11). subclasses of iCCA and HCC tumors. Two independent studies (30, 31) have demonstrated that a subset of iCCA tumors are enriched with liver-specific stem cell gene signatures (30, 32, 33) Cells of Origin and molecular subclasses of poor prognosis and aggressive phe- iCCA includes a group of histologically heterogeneous tumors notype of HCC (proliferation; ref. 34; and S2 subclass; ref. 35). with diverse cellular phenotypes and cell markers, which suggests Reciprocally, a subset of HCC samples expressing biliary cell the possible existence of multiple cells of origin (Fig. 1; ref. 24). In markers (i.e., CK19 and CK7; ref. 36) or enriched by iCCA-like addition, the existence of mixed hepatocellular cholangiocarci- gene expression signatures (37) show overall survival rates similar noma (HCC-iCCA) tumors (25), a subtype with predominance of to those for iCCA patients. In addition, cholangiolocellular car- stem cell features, points out the presence of a possible common cinoma (CLC), a stem cell featured mixed HCC-iCCA tumor,

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Hepatocyte Cholangiocyte Portal triad Hepatic progenitor Vein cells Injury and/or oncogenic hit

BD CoH Ductule Artery

Renewal Renewal ALB+/AFP+ EpCAM+ NCAM+ CK7+/CK19+ CD133+ WNT and NOTCH signaling activation IDH1/2 mutations HPCs

ALB+/AFP+ ALB–/AFP– Progenitor– EpCAM+ Mixed EpCAM+ CLC like HCC NCAM– HCC–iCCA NCAM+ CK7+/CK19+ CK7+/CK19+ Hepatocyte- ALB+/AFP+ Hep-markers+/– Cholangiocyte- EpCAM+ committed EpCAM+ EpCAM+ committed NCAM+ precursor CK19+ NCAM+/– precursor CK7+/CK19+ CK7+/CK19+ CD133+ ?

ALB+/AFP+ ALB–/AFP– EpCAM– EpCAM+ HCC iCCA NCAM– NCAM– CK7–/CK19– CK7+/CK19+ NOTCH signaling activation ALB+/AFP+ Mature Mature EpCAM+ hepatocyte cholangiocyte CK7+/CK19+

Figure 1. Schematic representation of multiple cells of origin in primary liver cancers. Hepatic progenitor cells (HPC) are located at canals of Hering (CoH) near the portal triads and are thought to have the potential to differentiate into hepatocytes and cholangiocytes. There is evidence that the differentiated hepatocytes can give rise to such cells. HCC and iCCA can develop from the neoplastic transformation of mature hepatocytes and cholangiocytes, respectively. In addition, HPC and its intermediate states are thought to be the common cell of origin for hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (iCCA), and mixed HCC-iCCA tumors [i.e., cholangiolocellular carcinoma (CLC)]. Furthermore, recent evidence supports the hypothesis that mature hepatocytes can transdifferentiate to cholangiocytes, leading to the development of iCCA. shares similar histopathologic features with iCCA and CK19- ogenesis. The expression of gain-of-function IDH mutations, positive HCC (12, 38). These data suggest HPC as a possible commonly reported in iCCA, led to the inhibition of hepato- common ancestor for a subset of primary liver cancers. Alterna- cyte differentiation both in vitro and in vivo and caused the tively, the mutations associated with these tumors may "repro- expansion of HPCs (39). In turn, combined IDH and KRAS gram" differentiated liver cells toward a progenitor-like state. mutations in GEMMs showed pronounced oncogenic cooper- Recently, several studies using genetically engineered mouse ation, leading to the development of premalignant biliary models (GEMM) and primary progenitor cell models have shed lesions and subsequent progression to iCCA. These data impli- light on the link between cell differentiation and iCCA path- cate mutant IDH in the subversion of liver differentiation states

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Table 2. Potential molecular alterations amenable for targeted therapies in iCCA No. of positive/total Gene or molecule Type of alteration samples (frequency)a References Somatic mutations Metabolic enzymes IDH1/2 Activating mutations 143/951 (15%) (51–54, 56–62) Tyrosine kinase signaling KRAS Activating mutations 165/885 (19%) (50–53, 56–62) BRAF Activating mutations 28/574 (5%) (50–51, 53–54, 55–59, 61) EGFR Activating mutations 14/545 (3%) (50–51, 53–54, 55–59, 61) Chromatin-remodeling genes ARID1A Inactivating mutations 50/390 (13%) (51–54, 56–57, 59, 61–62) BAP1 Inactivating mutations 45/443 (11%) (51–54, 56–57, 59, 61–62) PBRM1 Inactivating mutations 34/443 (8%) (51–54, 56–57, 59, 61) Tyrosine kinase (TK) fusion proteins FGFR2 fusions FGFR2–BICC1 TK fusion protein 46/211 (22%) (51, 56, 57, 72, 73, 75) FGFR2–PPHLN1 TK fusion protein 17/153 (11%) (51, 56, 57, 72, 73, 75) FGFR2–AHCYL1 TK fusion protein 7/111 (6%) (51, 56, 57, 72, 73, 75) FGFR2–MGEA5 TK fusion protein 1/53 (2%) (51, 56, 57, 72, 73, 75) FGFR2–TACC3 TK fusion protein 2/53 (4%) (51, 56, 57, 72, 73, 75) FGFR2–KIAA1598 TK fusion protein 1/53 (2%) (51, 56, 57, 72, 73, 75) ROS fusions ROS1 fusions TK fusion protein 2/23 (9%) (77) Chromosomal aberrations 11q13 (FGF19, CCND1, ORAOV1) High-level amplification 5/128 (4%) (32) aThe frequency in iCCA has been calculated by considering the number of samples presenting the molecular alteration over the total number of samples for which the specific alteration has been evaluated (discovery and validation set of samples) in different studies. Frequencies in iCCA have been calculated only in non–liver fluke cases.

and in the persistence of HPCs that are susceptible to the the striking survival benefits obtained in BRAF-mutated melano- accumulation of additional oncogenic hits (Fig. 1). While these mas treated with vemurafenib (48) or in lung cancer harboring studies did not directly determine the origin of HPCs, they did ALK rearrangements and treated with crizotinib (49). Unfortu- point to expansion of progenitor-like cells as a key mechanism nately, to date, no oncogene addiction loop has been reported in contributing to liver carcinogenesis. Similarly, mice with genet- iCCA. ic alterations in Hippo pathway components in the liver (i.e., The molecular pathogenesis of iCCA is a complex process YAP, SAV1, MST1/2) show expansion of progenitor-like cells, involving multiple genomic alterations and signaling pathway followed by the development of both HCC and iCCA (40–42). deregulations. Before the implementation of next-generation In parallel, two independent studies demonstrated that differ- sequencing technologies, our knowledge of the role of muta- entiated hepatocytes have the potential to give rise to iCCA tions in iCCA was limited, encompassing recurrent activating through the activation of NOTCH signaling (43, 44). Aberrant mutations in KRAS (19%), low frequency mutations in BRAF activation of NOTCH signaling has been described in both (5%), and EGFR (3%), and widely varying reports of loss-of- iCCA (60%) and HCC (30%) tumors (45, 46). Interestingly, in function mutations in the tumor suppressor TP53 (16%, range a GEMM with constitutive overexpression of NOTCH1,asubset 1%–38%; Tables 1 and 2; refs. 31, 50–64). While KRAS and of the HCC tumors presented progenitor-like cell features with TP53 mutations are relatively common in all CCA, mutations in a mixed biliary and hepatocytic phenotype (45). In contrast, a IDH1/2 and BRAF are considerably more prevalent in iCCA recent study revealed that iCCA originates from the transfor- (Table 1). Epigenetic alterations through promoter hyper- mation of biliary epithelial cells in the context of chronic injury methylation have also been described, and the most recurrent and p53 inactivation (47). Collectively, it appears that iCCA (>25%) affects p16INK4A/CDKN2, p14ARF, RASSF1A, APC, can emerge from different liver cell types depending on the GSTP,andSOCS-3 (58). Inflammation-related signaling path- initial triggering mutation and/or environmental insult. Future ways, such as JAK–STAT3, and proliferation-related pathways, studies are needed to fully define these routes to iCCA, and to such as EGFR and HGF–MET signaling, show profound dereg- understand their molecular underpinnings as well their rele- ulation in iCCA (58). In addition, recent studies have proposed vance to different iCCA subtypes. emerging roles for NOTCH and WNT signaling in iCCA pathogenesis. Furthermore, two independent whole-transcriptome analyses discerned the existence of two distinct molecular Molecular Pathogenesis subclasses of iCCA (31, 50). Both studies identified a prolifer- Over the past 15 years, major scientific breakthroughs that have ation molecular subclass that definestumorswithactivationof significantly changed the management of human cancers have oncogenic signaling pathways, including RAS–MAPK, MET, and been driven by the discovery and successful therapeutic targeting EGFR, and poor prognosis. In addition, approximately 40% of of the so-called "oncogenic addiction loops." The term "oncogene patients belong to the Inflammation subclass, characterized by addiction" is used to define the dependency status of cancer cells enrichment of cytokine related pathways, constitutive activa- on the activation or loss of specific genes. Several examples exist of tion of STAT3, and better prognosis (31).

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Emerging signaling pathways of the TK included in the rearrangement and involves enforced NOTCH signaling. The NOTCH signaling pathway is known to dimerization, subsequent transautophosphorylation, and acti- play an important role during embryonic development and is vation of downstream signaling pathways (57, 72, 73). Trans- essential for a proper maturation of the liver architecture. Recent- forming and oncogenic potential of FGFR2 fusions (FGFR2– ly, NOTCH pathway deregulation has been implicated in induc- BICC1, FGFR2–PPHLN1, FGFR2–AHCYL1, FGFR2–TACC3) has tion of inflammation (65) and the development and progression been proven in vitro (57, 72, 73, 76) and in vivo (72). Furthermore, of iCCA (66, 67). In human CCAs, upregulation of NOTCH1 and the presence of FGFR2 fusions seems to predict higher sensitivity NOTCH4 has been reported in 82.9% and 56.1%, respectively, to selective FGFR2 inhibitors (57, 72, 73, 76). However, the (46). In preclinical studies, liver-induced expression of NOTCH1 relative oncogenic potential of the different FGFR2 fusions or intracellular domain in mice resulted in the formation of iCCAs their sensitivity to specific FGFR2 inhibitors remains unknown (67). Considering that a number of NOTCH inhibitors are cur- and should be extensively investigated in future studies. rently under development, the NOTCH pathway may represent a Screening of FGFR2 fusions in multiple studies by massive novel amenable target in iCCA (Fig. 2). However, a recent study parallel sequencing technologies or FISH-based assay has reported different effects of targeting NOTCH receptors in a revealed striking differences in the incidence of the FGFR2 mouse model of primary liver cancer driven by v-akt viral onco- fusion events with a range between 3% and 50% of iCCA gene homolog (AKT) and neuroblastoma RAS viral oncogene patients (51, 56, 57, 72, 73, 75). FGFR2 fusions were found to homolog (NRAS; ref. 68). Interestingly, while the inhibition of be rare in mixed HCC-iCCA and mostly absent in HCC and NOTCH2 reduced tumor burden, NOTCH1 inhibition altered the eCCA (Table 1; refs. 57, 72). Thus, FGFR2 fusions are a novel relative proportion of tumor types, reducing HCC-like tumors but hallmark of iCCA. dramatically increasing CCA-like tumors (68). Thus, further stud- A significant association has been observed between the pres- ies are needed to understand the complex role of NOTCH sig- ence of FGFR2 fusions (FGFR2–PPHLN1, FGFR2–BICC1) and naling in primary liver cancer. KRAS mutations and signaling pathway activation, suggesting a possible cooperative role in driving iCCA pathogenesis (57). Even WNT signaling. The WNT pathway is highly activated in the tumor though no clear association between presence of FGFR2 fusions epithelium of human CCAs and is often characterized by over- and clinicopathologic parameters (e.g., gender, age, stage, and expression of the ligands WNT7B and WNT10A along with several prognosis) has been identified across the multiple datasets, a large target genes (69, 70). It has been demonstrated that inflammatory study conducted in Japan has suggested a significant association macrophages in the stroma surrounding the tumor are required with viral hepatitis (72), and a female predominance was for the maintenance of this highly activated WNT signaling status observed in a North American cohort (75). Larger epidemiologic (69, 71). As recently demonstrated in two rodent models mim- studies need to be conducted to clarify such discrepancies. FGFR2 icking human iCCA, the WNT pathway was progressively activat- Besides fusions, ROS1 kinase fusion proteins have been ed during the course of iCCA development, and treatment in vitro identified in 8.7% (2/23) of CCAs (77). Expression of FIG–ROS1 in vitro in and in vivo with WNT inhibitors (ICG001 and C59) successfully in NIH3T3 cells conferred transforming ability both and inhibited tumor growth (69). Considering the recent develop- vivo, which could be inhibited by specific targeting (77). Further- FIG–ROS ment of several pharmacologic WNT inhibitors and the absence of more, the oncogenic potential of has been recently APC and CTNNB1 mutations in iCCA, the WNT pathway may validated in an orthotopic allograft mouse iCCA model harboring KRAS TP53 FIG–ROS represent another important clinical opportunity (Fig. 2). and mutations (78). alone was also able to promote tumorigenesis, although with reduced penetrance and Identification of Novel Drivers longer latency. Notably, preliminary data support the efficacy of therapeutic targeting of ROS1 kinase in vitro and in vivo with small Recent technological advancements have led to a better under- ATP-competitive inhibitors (e.g., foretinib, crizotinib). Further standing of the genetic and molecular forces that drive human investigation will be required to establish the frequency of ROS fi cancers. Signi cant progress has been made also in iCCA, where fusions across different iCCA patient populations and to evaluate deep-sequencing studies have unveiled novel mutations (i.e., the potential benefit of such therapies for patients with these IDH1/2 ARID1A ROS1 FGFR2 , ) and oncogenic fusion genes ( and translocated alleles. fusions). In the following section, we highlight the most promising discoveries, with particular emphasis on those potentially New somatic alterations amenable to targeted therapies (Table 2; Fig. 2). The application of exome-sequencing technologies has led to the discovery of novel somatic mutations in the protein-coding Tyrosine kinase fusion genes region of several genes and has defined a mutational landscape of FGFR2 is a tyrosine kinase (TK) protein that acts as cell-surface the disease. Interestingly, emerging data supports a different receptor for fibroblast growth factors and plays an essential role genetic profile between liver fluke–related and non–liver fluke in the regulation of cell proliferation, differentiation, migration, related CCAs in terms of gene expression (79) and mutation and apoptosis. Recently, several FGFR2 chromosomal fusions profiles (80). Exome sequencing of 8 cases of liver fluke-related with multiple genomic partners have been identified in several CCAs identified 10 novel mutated genes involved in histone cancers, including iCCA (Table 2; refs. 51, 56, 57, 72–75). All modification, genomic instability, and G protein signaling of these fusions contain the same portion of the FGFR2 recep- (e.g., KMT2C, ROBO2, PEG3, and GNAS) and confirmed muta- tor (exons 1–19) and are fused to different partners through tions in already known genes (TP53 and KRAS; ref. 80). A follow- genomic breakpoints within the same intronic region (e.g., up study was later conducted by the same group and profiled BICC1, PPHLN1, CCDC6, MGEA5, TACC3). The oncogenic acti- 209 CCAs collected from Asia and Europe, associated with vation of these FGFR2 fusion proteins relies on the activation Opisthorchis viverrini (n ¼ 108) and non–O. viverrini–related

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Cetuximab Bevacizumab mAbs antiligand JAG1–2 Panitumumab (e.g., OMP-21M18) DLL1–4 WNT inhibitors (e.g., XAV-939, ICG-001, IWP-2, IWR-1-endo) NOTCH1–4

VEGF FGFR2 fusions EGF mAbs antireceptors (e.g., OMP-52M51, OMP-59R5) WNT FZD Cell Tumor membrane cell FGFR2 FGFR2 PDGFR PDGFR

VEGFR VEGFR EGFR

EGFR P P P P ERBB2 P P ERBB2 P P P P TK TK TK TK TK TK TK TK γ BICC1 -secretase TK TK PPHLN1 DVL AXIN MGEA5 BGJ398 NCID γ-secretase AZD4547 TACC3 Cediranib inhibitors Erlotinib LY2874455 KIAA1598 Lapatinib Sunitinib (e.g., RO4929097, Vandetanib Ponatinib MK0752)

GSK3 inhibitors GSK3 (e.g., CHIR-99021, TWS119, tidelglusib) RAS–MAPK PIK3–AKT NCID pathway pathway PP β-catenin P AXIN β APC β-catenin β-catenin -catenin Ub Ub KRAS PIK3 Ub

PTEN WNT/β-catenin inhibitors Sorafenib BRAF (e.g., XAV-939, ICG-001, Degradation Regorafenib IWP-2, IWR-1-endo) AKT Refametinib Selumetinib MEK Trametinib Citrate

ERK MTOR Everolimus Mitochondria Isocitrate

AG-120 IDH1 Citrate TCA Nucleus Target gene α-KG expression Isocitrate cycle β-catenin IDH1 IDH2 mut α-KG HDAC inhibitors TCF/LEF IDH2 BAP1 AG-221 (e.g., vorinostat, 2-HG mut panabinostat) ARID1A 2-HG TET1/2

Histone demethylation

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etiologies (n ¼ 101; ref. 52). In summary, these studies reveal that Management and Molecular Targeted TP53 SMAD4 KMT2C GNAS (i) , , , and are more commonly Therapies mutated in O. viverrini–infected CCA cases; (ii) IDH1/IDH2 mutations are almost exclusive for non–O. viverrini–related iCCA; At present, the treatment of choice for iCCA when feasible is and (iii) fluke-related CCAs present a mean of 26 somatic muta- surgical resection (1), whereas liver transplantation remains con- tions per tumor, compared with a mean of 16 mutations per troversial. Upon resection, the median overall survival is of tumor in CCA with other etiologies. In addition, whole-exome around 3 years and recurrence occurs in up to 60% of patients, sequencing (WES) studies have led to the identification of somatic depending on several prognostic factors, among which tumor mutations in chromatin-remodeling genes, BAP1, ARID1A, and burden and lymphonodal status appear to be the most relevant PBRM1—in iCCA (52, 54). Functional studies have revealed (1, 16). The prognosis for patients diagnosed with unresectable tumor-suppressive activity of BAP1 and ARID1A, further support- disease is even more dismal, with a life expectancy around 1 year ing the potential role of chromatin modulators in iCCA devel- and actuarial probability of survival of 5% at 5 years (1, 58). opment (52). In particular, ARID1A encodes an accessory subunit The lack of clinical trials conducted specifically in iCCA pati- of the SWI/SNF chromatin-remodeling complex and mutations in ents as opposed to all biliary tract cancers (BTC) and the limited this gene have recently been identified in a wide variety of cancers. number of patients studied are among the challenges that preclude Silencing of ARID1A in CCA cell lines (including non–O. viver- clinical practice guidelines in establishing a standard of care for rini–associated and O. viverrini–associated) resulted in a signifi- patients with advanced iCCA (1). Among the 112 clinical trials cant increase of cell proliferation. Conversely, overexpression of reported in advanced BTCs testing systemic therapies (81), the wild-type ARID1A led to retarded cell proliferation confirming the majority are single-arm phase II studies with low statistical power tumor-suppressive role of this gene (52). The possibility that iCCA and unclear impact on overall survival. The current standard of patients harboring mutations in these genes may benefit from practice for advanced-stage iCCA is represented by systemic che- treatment with histone deacetylase (HDAC) inhibitors, such as motherapy with gemcitabine and cisplatin (6). Survival benefits vorinostat or panobinostat, remains unclear and needs to be favoring the combination arm as opposed to gemcitabine alone further explored. (11.7 vs. 8 months; ref. 6) were demonstrated in a subgroup IDH1 and IDH2 mutations have been reported in approximately analysis of patients with iCCA (n ¼ 80) included in a large 14% of iCCAs (Table 2). In a large cohort of iCCA cases (n ¼ 326), randomized phase III trial (n ¼ 410, ABC-02) of patients with IDH1/2 mutations were associated with better overall survival (60). advanced and metastatic BTCs. In contrast, in a recent WES-based study, patients with IDH1 or On the other hand, so far no molecular targeted therapy has IDH2 mutations had shorter survival compared with patients with been proven effective for iCCA or other biliary tract cancers. wild-type IDH genes (3-year survival of 33% in IDH mutants vs. The results of few trials with targeted therapies as monotherapy 81% in IDH wild-type; ref. 54). IDH1 and IDH2 mutations in iCCA (i.e., selumitinib) or in combination with chemotherapy (i.e., and other cancer types cluster at the hotspots codons 132 and 172, sorafenib plus gemcitabine, cetuximab plus gemcitabine–oxali- respectively. IDH1 and IDH2 encode metabolic enzymes whose platin) have been discouraging with limited effects on overall normal function is to interconvert the metabolic intermediate survival (1). In this sense, patient stratification based on molec- isocitrate to a-ketoglutarate (a-KG) in conjunction with the gen- ular biomarkers (Table 2) may be essential for clinical success in eration of NADPH. Mutations in IDH1 and IDH2 are always treating iCCA patients. Toward this direction, the first clinical present in a heterozygous state with the wild-type allele and they trials driven by biomarkers (e.g., FGFR2 aberrations and IDH1/2 result in the acquisition of an abnormal enzymatic activity, the mutations) in BTCs, including iCCA, are currently ongoing and reduction of a-KG to 2-hydroxyglutarate (2-HG). 2-HG has been their results are anxiously awaited (Fig. 2, Table 3). BGJ398, a designated as an "oncometabolite" that contributes to cancer selective FGFR inhibitor, has shown efficacy in vitro by blocking formation by inhibiting multiple dioxygenase enzymes that the neoplastic transformation and growth of cell lines expressing require a-KG for their activity, resulting in altered cell differenti- FGFR2 fusions (57). Clinical efficacy of BGJ398 is currently being ation, survival, and extracellular matrix maturation (Fig. 2). Abnor- investigated in a phase II multicenter single-arm study in adult mal DNA methylation and increased protein levels of TP53 are patients with advanced or metastatic CCA harboring FGFR2 gene common features of tumors with IDH1 and IDH2 mutations (60). fusions or other FGFR genetic alterations who have failed che- Furthermore, using in vitro stem cell systems and GEMMs, it has motherapy (NCT02150967). Furthermore, promising prelimi- been demonstrated that mutant IDH mutations are able to pro- nary data have been reported following treatment with ponatinib, mote iCCA formation by blocking hepatocyte differentiation and a multikinase inhibitor, in 2 iCCA patients harboring FGFR2 inducing proliferation of hepatic progenitors (39). fusions (FGFR2–TACC3, FGFR2–MGEA5), resulting in tumor size

Figure 2. Current and potential targeted therapies in intrahepatic cholangiocarcinoma. Tyrosine kinase receptor signaling: several growth factor signaling pathways (i.e., EGF/EGFR) have been reported to be aberrantly activated in iCCA. The specify binding of growth factors results in oligomerization and autophosphorylation of their receptors, followed by signaling through the RAS–MAPK and PI3K–AKT effector cascades. FGFR2 fusions: The presence of fusion partners in the cytoplasmic domain of FGFR2 results in constitutively active receptors that induce signaling through downstream signaling pathways. NOTCH signaling: Binding of ligands on the surface of neighboring cells to the extracellular domain of NOTCH receptors (NOTCH-R) induces proteolytic cleavage of the receptor, releasing its intracellular domain (NICD), which then translocates to the nucleus and regulates expression of target genes. WNT/b-catenin signaling: activation of frizzled (FZD) receptors by WNT ligands triggers the displacement of the regulatory APC/Axin/GSK3-complex, accumulation of b-catenin and induction of target genes. IDH signaling: Mutated IDH enzymes acquire the capacity to synthesize 2-hydroxygluterate (2-HG) from a-ketoglutarate (a-KG). 2-HG alters the activity of a-KG–dependent dioxygenase enzymes involved in multiple cellular processes, including cell differentiation, survival, and DNA methylation. Molecular targeted therapies have also been highlighted; drugs currently assessed in phase II clinical trials (red) and those evaluated in early clinical trials or preclinical studies (brown) are shown.

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Table 3. Ongoing clinical trials using targeted therapiesa Treatment Targets Clinical trial phase Number of trials Biomarker driven BGJ398 FGFR, ABL, FYN, KIT, LCK, LYN, YES II 1 Ponatinib hydrocloride BCR-ABL, VEGFR, PDGFR, FGFR, EPH, SRC, KIT, RET, TIE2, FLT3 II 1 AG-221 Mutated IDH2 I/II 1 AG-120 Mutated IDH1 I 1 Monotherapy Cabozantinib (XL-184) MET, VEGFR2, RET, c-KIT, FLT1/3/4, TIE2 II 1 Everolimus mTOR II 2 Sunitinib VEGFR, PDGFR, KIT, FLT3, RET II 1 Regorafenib RET, RAF-1, VEGFR, KIT, BRAF (V600E), PDGFRB, FGFR1, TIE2 II 2 Celecoxib COX IV 1c Trastuzumab HER2-neu II 1 LY2801653 c-MET, MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 I 1 BKM120 VPS34/mTOR/DNAPK/PI4Kb II 1 Lapatinib ErbB2-4/EGFR/SRC II 2 Selumetinib MEK1/2 II 1 MK2206 AKT1-3 II 1 RAV12 RAAG12 I 1 PLX8394 BRAF I/II 1 Combination Selumetinib þ MK-2206 MEK1 þ AKT1-3 II 1 Bosutinib þ capecitabine ABL/SRC/c-KIT I 1 AZD2171 þ AZD0530 VEGFR/PDGFR/FGFR1/c-KIT þ SRC/ABL/LCK/YES/EGFR/LYN I 1 Pazopanib þ GSK1120212 VEGFR/PDGFR/FGFR/KIT þ MEK1/2 I 1 Cetuximab þ erlotinib EGFR I/II 2c Trastuzumab þ tipifarnib HER2-neu þ FTI I 1 Erlotinib þ bevacizumab EGFR þ VEGFA II 2 Combination with chemotherapy Radiotherapy þ bevacizumab VEGFA I 1 Chemotherapyb þ veliparib PARP1/2 I 1 Chemotherapy þ bevacizumab VEGFA II 2c Chemotherapy panitumumab EGFR II 5c Chemotherapy vandetanib (ZD6474) VEGFR, EGFR I, II 2c Chemotherapy þ cediranib VEGFR II 1 Chemotherpy sorafenib BRAF, VEGFR, PDGFR I/II 2 Chemotherapyb cetuximab EGFR II 2c Chemotherpyb þ selumetinib MEK1/2 I/II 1 Chemotherpy trametinib MEK1/2 II 1 Chemotherapyb þ sirolimus mTOR I 1 Chemotherapy þ pazopanib VEGFR/PDGFR/FGFR/KIT II 1 Chemotherapy þ AZD2171 VEGFR/PDGFR/FGFR1/c-KIT II 1 Chemotherapyb CX-4945 CX2 I/II 1c Chemotherapy þ erlotinib EGFR I/II 3 Abbreviations: FGFR, ﬁbroblast growth factor; KIT, c-kit proto-oncogene receptor tyrosine kinase; PDGFR, platelet-derived growth factor receptor. aInformation acquired from clinicaltrials.gov. bChemotherapy (standard of practice: gemcitabine and cisplatin). cRandomized controlled clinical trials.

reduction (51). Currently, a pilot study with ponatinib is ongoing great promise for improving the future management and treat- in BTC patients with FGFR2 fusions (NCT02265341). At the same ment of iCCA patients through the first biomarker-driven clinical time, based on demonstrated efficacy in preclinical studies, spe- studies currently ongoing. Whether FGFR2 aberrations may rep- cific inhibitors for IDH1 (AG-120) and IDH2 (AG-221) are resent a novel oncogene addiction loop in iCCA still remains an currently being investigated in phase I (NCT02073994) and phase unanswered question. Nevertheless, FGFR2 fusions have the I/II (NCT02273739) clinical trials, respectively (Table 3). In potential to represent a new avenue of research for basic inves- parallel, considering the emerging roles of NOTCH and WNT tigators and clinicians. Finally, the intriguing possibility of mul- pathway activation in the pathogenesis of iCCA, the first clinical tiple cells of origin in iCCA deserves further investigation as a trials targeting these pathways using available specific inhibitors means to understand the mechanisms underlying the carcino- are expected to move forward (Fig. 2). genesis process and to determine whether this can be of relevance in clinical application. Future Perspectives Disclosure of Potential Conflicts of Interest The application of new technologies has led to a more accurate V. Mazzaferro reports receiving speakers bureau honoraria from Bayer and mapping of the genomic landscape of iCCA, a devastating disease BTG. J.M. Llovet reports receiving commercial research grants from Bayer, with limited treatment options. Among the newly discovered Blueprint Medicines, and Boehringer Ingelheim; other commercial research molecular alterations, FGFR2 fusions and IDH1/2 mutations hold support from Bayer, Boehringer Ingelheim, and Bristol-Myers Squibb; and is

OF8 Clin Cancer Res; 22(2) January 15, 2016 Clinical Cancer Research

Pathogenesis and Targeted Therapies in iCCA

a consultant/advisory board member for Bayer, Biocompatibles, Blueprint dation, and the NIH under award numbers R01CA136567-02 and Medicines, Boehringer Ingelheim, Bristol-Myers Squibb, Celsion, Eli Lilly, P50CA1270003. V. Mazzaferro is partially supported by the AIRC (Italian GlaxoSmithKline, and Novartis. No potential conﬂicts of interest were disclosed Association for Cancer Research) and a 51000 Milan-INT institutional grant by the other authors. in hepato-oncology. J.M Llovet is supported by grants from the Samuel Waxman Cancer Research Foundation, Asociacion Espanola~ Contra el Cancer, Spanish Grant Support National Health Institute (SAF-2013-41027), and a European Commission A. Moeini is supported by a fellowship from Spanish National Health HEP-CAR grant (667273-2). Institute (FPI program, BES-2011-046915). D. Sia is supported by the ILCA-Bayer Fellowship. N. Bardeesy holds the Gallagher Endowed Chair in Gastrointestinal Cancer Research at Massachusetts General Hospital and Received June 5, 2015; revised August 6, 2015; accepted August 6, 2015; is supported by a V Foundation Translational Award, the TargetCancer Foun- published OnlineFirst September 24, 2015.

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OF10 Clin Cancer Res; 22(2) January 15, 2016 Clinical Cancer Research

Molecular Pathogenesis and Targeted Therapies for Intrahepatic Cholangiocarcinoma

Agrin Moeini, Daniela Sia, Nabeel Bardeesy, et al.

Clin Cancer Res Published OnlineFirst September 24, 2015.

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