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Molecular Pathogenesis and Targeted Therapiesfor Intrahepatic CholangiocarcinomaAgrin Moeini1,2, Daniela Sia1,2,3, Nabeel Bardeesy4, Vincenzo Mazzaferro3, andJosep M. Llovet1,2,5
Abstract
Intrahepatic cholangiocarcinoma (iCCA) is a molecularlyheterogeneous hepatobiliary neoplasm with poor prognosisand limited therapeutic options. The incidence of this neoplasmis growing globally. One third of iCCA tumors are amenable tosurgical resection, but most cases are diagnosed at advancedstages with chemotherapy as the only established standard ofpractice. No molecular therapies are currently available for thetreatment of this neoplasm. The poor understanding of thebiology of iCCA and the lack of known oncogenic addictionloops has hindered the development of effective targetedtherapies. Studies with sophisticated animal models definedIDHmutation as the first gatekeeper in the carcinogenic processand led to the discovery of striking alternative cellular origins.RNA- and exome-sequencing technologies revealed the presence
of recurrent novel fusion events (FGFR2 and ROS1 fusions)and somatic mutations in metabolic (IDH1/2) and chromatin-remodeling genes (ARID1A, BAP1). These latest advancementsalong with known mutations in KRAS/BRAF/EGFR and 11q13high-level amplification have contributed to a better under-standing of the landscape of molecular alterations in iCCA.More than 100 clinical trials testing molecular therapies aloneor in combination with chemotherapy including iCCA patientshave not reported conclusive clinical benefits. Recent discover-ies have shown that up to 70% of iCCA patients harborpotential actionable alterations that are amenable to therapeu-tic targeting in early clinical trials. Thus, the first biomarker-driven trials are currently underway. Clin Cancer Res; 22(2);291–300. �2015 AACR.
IntroductionIntrahepatic cholangiocarcinoma (iCCA) is the second most
common liver cancer following hepatocellular carcinoma (HCC),accounting for 5% to 10% of all primary liver malignancies withan annual incidence of 2 cases per 100,000 in Western countries(1, 2). At present, it is widely accepted that iCCA arises from themalignant transformation of the intrahepatic cholangiocytes andis anatomically distinguished from the extrahepatic biliary tractcancers (eCCA), which are known as perihilar (pCCA) and distal(dCCA), with the second-order bile ducts acting as the separationpoint (3).
During the past decade a growing interest has been expressed iniCCA due to a marked increase in both incidence and mortalityrates (1, 4). Currently, surgical resection represents the solecurative treatment option in 30% to 40% of patients with 5-yearsurvival of 20% to 40% (1, 5). Themajority of iCCA patients haveno underlying liver disease or known risk factors, which furtherhinders the development of screening strategies for early detec-tion. In patients with advanced disease, the combination ofgemcitabine and cisplatin has been shown to confer a survivaladvantage over gemcitabine alone and is currently proposed asthe standardof practice (6). Asopposed toHCC, todate there is noapproved targeted molecular therapy for iCCA, and the identifi-cation of a first-line conclusive treatment remains an unmet need.Recently, the use of next-generation sequencing technologies hasenabled the identification of recurrent actionable molecularalterations that hold the promise of improving the managementof advanced iCCA patients. Herein, we provide an overview of therecent discoveries of newmolecular targets that should ultimatelylead to the development of more personalized therapeuticapproaches.
Epidemiology and Risk FactorsiCCA is a devastating disease with poor prognosis. Several
studies have reported global trends of increasing incidence andmortality for iCCA in contrast with decreasing rates for eCCA (7–10). iCCA presents more commonly at older age with a slightpredominance in men (male to female ratio 1.2–1.5:1; ref. 1).There is a considerable geographic and demographic variation inthe epidemiology of iCCA, which likely reflects distinct environ-mental and genetic predispositions. The incidence of iCCA is thehighest in Southeast Asia and more specifically in Thailand (>80
1Liver Cancer Translational Research Laboratory, Liver Unit, Institutd'Investigacions Biom�ediques August Pi i Sunyer (IDIBAPS), Hospi-tal Clínic, CIBERehd, Universitat de Barcelona, Barcelona, Catalonia,Spain. 2Liver Cancer Program, Division of Liver Diseases, Depart-ment of Medicine,Tisch Cancer Institute, Icahn School of Medicine atMount Sinai, NewYork, NewYork. 3Gastrointestinal Surgeryand LiverTransplantation Unit, Department of Surgery, National Cancer Insti-tute IRCCS Foundation, Milan, Italy. 4Cancer Center, Center forRegenerative Medicine, and Department of Molecular Biology, Mas-sachusetts General Hospital, Harvard University, Boston, Massachu-setts. 5Instituci�o Catalana de Recerca i Estudis Avancats, Barcelona,Catalonia, Spain.
Note: V. Mazzaferro and J.M. Llovet share senior authorship.
Corresponding Author: Josep M. Llovet, BCLC Group, Liver Unit, IDIBAPS,CIBERehd, Hospital Clínic, University of Barcelona, Rosell�o 149, Barcelona08036, Catalonia, Spain. Phone: 349-3227-9156; Fax: 349-3227-5792; E-mail:[email protected]
doi: 10.1158/1078-0432.CCR-14-3296
�2015 American Association for Cancer Research.
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cases per 100,000) and can be as low as 0.2 per 100,000 in someWestern countries (1, 11). Even though the vast majority ofiCCAs are sporadic, several risk factors have been identified.Historically, most of these risk factors have been establishedfor CCAwithout distinguishing between iCCA and eCCA, despitethe fact that increasing evidence supports the hypothesis thatthey represent distinct entities with marked differences in theirgenomic features and epidemiology (Table 1; refs. 3, 12–15). Themost prevalent risk factors for HCC have also been significantlyassociated with iCCA but not with eCCA (Table 1), includingcirrhosis and chronic hepatitis B and C infections (1, 11, 16–23).Other risk factors for iCCA include primary sclerosing cholangitis(PSC), biliary duct cysts, hepatolithiasis, and hepatobiliary flukes.Hepatolithiasis has been defined as a well-known risk factor foriCCA (up to 20%) in Asian countries but not inWestern countries(11). Less-established risk factors with modest associationsinclude inflammatory bowel disease, obesity, diabetes, and alco-hol abuse (1, 11).
Cells of OriginiCCA includes a group of histologically heterogeneous tumors
with diverse cellular phenotypes and cell markers, which suggeststhe possible existence ofmultiple cells of origin (Fig. 1; ref. 24). Inaddition, the existence of mixed hepatocellular cholangiocarci-noma (HCC-iCCA) tumors (25), a subtypewith predominance ofstem cell features, points out the presence of a possible common
cell of origin. Thus, iCCA is currently believed to derive frombiliary epithelial cells (cholangiocytes) of the intrahepatic biliarytract, hepatic progenitor cells (HPC), or evenmature hepatocytes.
All liver cells share a common embryonic origin, arising frombipotential progenitors known as hepatoblasts (26). However,in the adult liver, normal tissue turnover is mainly sustained bydifferentiated hepatocytes and cholangiocytes. Nevertheless,upon major injury, there is an expansion of cells in the regionof the canals of Hering that have been proposed to be bipotentHPCs capable of differentiating into hepatocyte or cholangio-cyte lineages (Fig. 1; ref. 27). Alternatively, hepatocytes candedifferentiate into progenitor-like cells in response to acuteinjury (28, 29).
With this backdrop, the hypothesis that iCCA and HCC mayshare a commonancestor such as theHPCs has been an importantsubject of discussion during the past decade. Notably, emergingdata point to an overlapping molecular profile between specificsubclasses of iCCA and HCC tumors. Two independent studies(30, 31) have demonstrated that a subset of iCCA tumors areenriched with liver-specific stem cell gene signatures (30, 32, 33)and molecular subclasses of poor prognosis and aggressive phe-notype of HCC (proliferation; ref. 34; and S2 subclass; ref. 35).Reciprocally, a subset of HCC samples expressing biliary cellmarkers (i.e., CK19 and CK7; ref. 36) or enriched by iCCA-likegene expression signatures (37) showoverall survival rates similarto those for iCCA patients. In addition, cholangiolocellular car-cinoma (CLC), a stem cell featured mixed HCC-iCCA tumor,
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 mutationsTP53 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 proteinsFGFR2 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, notapplicable; pCCA, perihilar cholangiocarcinoma.aThe percentage of cases has been calculated by considering the number of samples presenting themolecular alteration over the total number of samples analyzed inall 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.
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shares similar histopathologic features with iCCA and CK19-positive HCC (12, 38). These data suggest HPC as a possiblecommon ancestor for a subset of primary liver cancers. Alterna-tively, the mutations associated with these tumors may "repro-gram" differentiated liver cells toward a progenitor-like state.
Recently, several studies using genetically engineered mousemodels (GEMM) and primary progenitor cell models have shedlight on the link between cell differentiation and iCCA path-
ogenesis. The expression of gain-of-function IDH mutations,commonly reported in iCCA, led to the inhibition of hepato-cyte differentiation both in vitro and in vivo and caused theexpansion of HPCs (39). In turn, combined IDH and KRASmutations in GEMMs showed pronounced oncogenic cooper-ation, leading to the development of premalignant biliarylesions and subsequent progression to iCCA. These data impli-cate mutant IDH in the subversion of liver differentiation states
© 2015 American Association for Cancer Research
HepatocyteHepatic
progenitorcells
Portal triad
Artery
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ALB+/AFP+
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?
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CLCProgenitor–like HCC
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 portaltriads and are thought to have the potential to differentiate into hepatocytes and cholangiocytes. There is evidence that the differentiated hepatocytes cangive 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), andmixed HCC-iCCA tumors [i.e., cholangiolocellular carcinoma (CLC)]. Furthermore, recent evidence supports the hypothesis that mature hepatocytes cantransdifferentiate to cholangiocytes, leading to the development of iCCA.
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and in the persistence of HPCs that are susceptible to theaccumulation of additional oncogenic hits (Fig. 1). While thesestudies did not directly determine the origin of HPCs, they didpoint to expansion of progenitor-like cells as a key mechanismcontributing to liver carcinogenesis. Similarly, mice with genet-ic alterations in Hippo pathway components in the liver (i.e.,YAP, SAV1, MST1/2) show expansion of progenitor-like cells,followed by the development of both HCC and iCCA (40–42).In parallel, two independent studies demonstrated that differ-entiated hepatocytes have the potential to give rise to iCCAthrough the activation of NOTCH signaling (43, 44). Aberrantactivation of NOTCH signaling has been described in bothiCCA (60%) and HCC (30%) tumors (45, 46). Interestingly, ina GEMM with constitutive overexpression of NOTCH1, a subsetof the HCC tumors presented progenitor-like cell features witha mixed biliary and hepatocytic phenotype (45). In contrast, arecent study revealed that iCCA originates from the transfor-mation of biliary epithelial cells in the context of chronic injuryand p53 inactivation (47). Collectively, it appears that iCCAcan emerge from different liver cell types depending on theinitial triggering mutation and/or environmental insult. Futurestudies are needed to fully define these routes to iCCA, and tounderstand their molecular underpinnings as well their rele-vance to different iCCA subtypes.
Molecular PathogenesisOver the past 15 years,major scientific breakthroughs that have
significantly changed the management of human cancers havebeen driven by the discovery and successful therapeutic targetingof the so-called "oncogenic addiction loops." The term "oncogeneaddiction" is used to define the dependency status of cancer cellson the activation or loss of specific genes. Several examples exist of
the striking survival benefits obtained in BRAF-mutated melano-mas treated with vemurafenib (48) or in lung cancer harboringALK rearrangements and treated with crizotinib (49). Unfortu-nately, to date, no oncogene addiction loop has been reported iniCCA.
The molecular pathogenesis of iCCA is a complex processinvolving multiple genomic alterations and signaling pathwayderegulations. Before the implementation of next-generationsequencing technologies, our knowledge of the role of muta-tions in iCCA was limited, encompassing recurrent activatingmutations in KRAS (19%), low frequency mutations in BRAF(5%), and EGFR (3%), and widely varying reports of loss-of-function mutations in the tumor suppressor TP53 (16%, range1%–38%; Tables 1 and 2; refs. 31, 50–64). While KRAS andTP53mutations are relatively common in all CCA, mutations inIDH1/2 and BRAF are considerably more prevalent in iCCA(Table 1). Epigenetic alterations through promoter hyper-methylation have also been described, and the most recurrent(>25%) affects p16INK4A/CDKN2, p14ARF, RASSF1A, APC,GSTP, and SOCS-3 (58). Inflammation-related signaling path-ways, such as JAK–STAT3, and proliferation-related pathways,such as EGFR and HGF–MET signaling, show profound dereg-ulation in iCCA (58). In addition, recent studies have proposedemerging roles for NOTCH and WNT signaling in iCCA path-ogenesis. Furthermore, two independent whole-transcriptomeanalyses discerned the existence of two distinct molecularsubclasses of iCCA (31, 50). Both studies identified a prolifer-ation molecular subclass that defines tumors with activation ofoncogenic signaling pathways, including RAS–MAPK, MET, andEGFR, and poor prognosis. In addition, approximately 40% ofpatients belong to the Inflammation subclass, characterized byenrichment of cytokine related pathways, constitutive activa-tion of STAT3, and better prognosis (31).
Table 2. Potential molecular alterations amenable for targeted therapies in iCCA
Gene or molecule Type of alterationNo. of positive/totalsamples (frequency)a References
Somatic mutationsMetabolic enzymesIDH1/2 Activating mutations 143/951 (15%) (51–54, 56–62)
Tyrosine kinase signalingKRAS 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 genesARID1A 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 proteinsFGFR2 fusionsFGFR2–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 fusionsROS1 fusions TK fusion protein 2/23 (9%) (77)
Chromosomal aberrations11q13 (FGF19, CCND1, ORAOV1) High-level amplification 5/128 (4%) (32)
aThe frequency in iCCAhas been calculated by considering the number of samples presenting themolecular alteration over the total number of samples for which thespecific alteration has been evaluated (discovery and validation set of samples) in different studies. Frequencies in iCCA have been calculated only in non–liver flukecases.
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Emerging signaling pathwaysNOTCH signaling. The NOTCH signaling pathway is known toplay an important role during embryonic development and isessential for a proper maturation of the liver architecture. Recent-ly, NOTCH pathway deregulation has been implicated in induc-tion of inflammation (65) and the development and progressionof iCCA (66, 67). In human CCAs, upregulation ofNOTCH1 andNOTCH4 has been reported in 82.9% and 56.1%, respectively,(46). In preclinical studies, liver-induced expression of NOTCH1intracellular domain in mice resulted in the formation of iCCAs(67). Considering that a number of NOTCH inhibitors are cur-rently under development, the NOTCH pathway may represent anovel amenable target in iCCA (Fig. 2). However, a recent studyreported different effects of targeting NOTCH receptors in amouse model of primary liver cancer driven by v-akt viral onco-gene homolog (AKT) and neuroblastoma RAS viral oncogenehomolog (NRAS; ref. 68). Interestingly, while the inhibition ofNOTCH2 reduced tumor burden,NOTCH1 inhibition altered therelative proportion of tumor types, reducingHCC-like tumors butdramatically increasing CCA-like tumors (68). Thus, further stud-ies are needed to understand the complex role of NOTCH sig-naling in primary liver cancer.
WNT signaling. TheWNTpathway is highly activated in the tumorepithelium of human CCAs and is often characterized by over-expression of the ligandsWNT7B andWNT10A alongwith severaltarget genes (69, 70). It has been demonstrated that inflammatorymacrophages in the stroma surrounding the tumor are requiredfor the maintenance of this highly activated WNT signaling status(69, 71). As recently demonstrated in two rodent models mim-icking human iCCA, the WNT pathway was progressively activat-ed during the course of iCCA development, and treatment in vitroand in vivo with WNT inhibitors (ICG001 and C59) successfullyinhibited tumor growth (69). Considering the recent develop-ment of several pharmacologicWNT inhibitors and the absence ofAPC and CTNNB1 mutations in iCCA, the WNT pathway mayrepresent another important clinical opportunity (Fig. 2).
Identification of Novel DriversRecent technological advancements have led to a better under-
standing of the genetic and molecular forces that drive humancancers. Significant progress has been made also in iCCA, wheredeep-sequencing studies have unveiled novel mutations (i.e.,IDH1/2, ARID1A) and oncogenic fusion genes (ROS1 and FGFR2fusions). In the following section, we highlight the most prom-ising discoveries, with particular emphasis on those potentiallyamenable to targeted therapies (Table 2; Fig. 2).
Tyrosine kinase fusion genesFGFR2 is a tyrosine kinase (TK) protein that acts as cell-surface
receptor for fibroblast growth factors and plays an essential rolein the regulation of cell proliferation, differentiation, migration,and apoptosis. Recently, several FGFR2 chromosomal fusionswith multiple genomic partners have been identified in severalcancers, including iCCA (Table 2; refs. 51, 56, 57, 72–75). Allof these fusions contain the same portion of the FGFR2 recep-tor (exons 1–19) and are fused to different partners throughgenomic breakpoints within the same intronic region (e.g.,BICC1, PPHLN1, CCDC6, MGEA5, TACC3). The oncogenic acti-vation of these FGFR2 fusion proteins relies on the activation
of the TK included in the rearrangement and involves enforceddimerization, subsequent transautophosphorylation, and acti-vation of downstream signaling pathways (57, 72, 73). Trans-forming and oncogenic potential of FGFR2 fusions (FGFR2–BICC1, FGFR2–PPHLN1, FGFR2–AHCYL1, FGFR2–TACC3) hasbeen proven in vitro (57, 72, 73, 76) and in vivo (72). Furthermore,the presence of FGFR2 fusions seems to predict higher sensitivityto selective FGFR2 inhibitors (57, 72, 73, 76). However, therelative oncogenic potential of the different FGFR2 fusions ortheir sensitivity to specific FGFR2 inhibitors remains unknownand should be extensively investigated in future studies.Screening of FGFR2 fusions in multiple studies by massiveparallel sequencing technologies or FISH-based assay hasrevealed striking differences in the incidence of the FGFR2fusion events with a range between 3% and 50% of iCCApatients (51, 56, 57, 72, 73, 75). FGFR2 fusions were found tobe rare in mixed HCC-iCCA and mostly absent in HCC andeCCA (Table 1; refs. 57, 72). Thus, FGFR2 fusions are a novelhallmark of iCCA.
A significant association has been observed between the pres-ence of FGFR2 fusions (FGFR2–PPHLN1, FGFR2–BICC1) andKRAS mutations and signaling pathway activation, suggesting apossible cooperative role in driving iCCApathogenesis (57). Eventhough no clear association between presence of FGFR2 fusionsand clinicopathologic parameters (e.g., gender, age, stage, andprognosis) has been identified across themultiple datasets, a largestudy conducted in Japan has suggested a significant associationwith viral hepatitis (72), and a female predominance wasobserved in a North American cohort (75). Larger epidemiologicstudies need to be conducted to clarify such discrepancies.
Besides FGFR2 fusions, ROS1 kinase fusion proteins have beenidentified in 8.7% (2/23) of CCAs (77). Expression of FIG–ROS1in NIH3T3 cells conferred transforming ability both in vitro and invivo, which could be inhibited by specific targeting (77). Further-more, the oncogenic potential of FIG–ROS has been recentlyvalidated in an orthotopic allograftmouse iCCAmodel harboringKRAS and TP53 mutations (78). FIG–ROS alone was also able topromote tumorigenesis, although with reduced penetrance andlonger latency. Notably, preliminary data support the efficacy oftherapeutic targeting of ROS1 kinase in vitro and in vivowith smallATP-competitive inhibitors (e.g., foretinib, crizotinib). Furtherinvestigation will be required to establish the frequency of ROSfusions across different iCCA patient populations and to evaluatethe potential benefit of such therapies for patients with thesetranslocated alleles.
New somatic alterationsThe application of exome-sequencing technologies has led to
the discovery of novel somatic mutations in the protein-codingregion of several genes and has defined amutational landscape ofthe disease. Interestingly, emerging data supports a differentgenetic profile between liver fluke–related and non–liver flukerelated CCAs in terms of gene expression (79) and mutationprofiles (80). Exome sequencing of 8 cases of liver fluke-relatedCCAs identified 10 novel mutated genes involved in histonemodification, genomic instability, and G protein signaling(e.g., KMT2C, ROBO2, PEG3, and GNAS) and confirmed muta-tions in already known genes (TP53 and KRAS; ref. 80). A follow-up study was later conducted by the same group and profiled209 CCAs collected from Asia and Europe, associated withOpisthorchis viverrini (n ¼ 108) and non–O. viverrini–related
Pathogenesis and Targeted Therapies in iCCA
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© 2015 American Association for Cancer Research
CetuximabPanitumumab Bevacizumab Antiligand mAbs
(e.g., OMP-21M18)JAG1–2DLL1–4
NOTCH1–4
Antireceptor mAbs(e.g., OMP-52M51,
OMP-59R5)
WNT inhibitors(e.g., XAV-939, ICG-001,
IWP-2, IWR-1-endo)
WNTFZD
Tumorcell
DVL
GSK3
APCUb
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P PP P
P P P P P P
GSK3 inhibitors(e.g., CHIR-99021,
TWS119, tideglusib)
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α-KG
Degradation
γ-secretaseinhibitors
(e.g., RO4929097,MK0752)
BICC1PPHLN1MGEA5TACC3KIAA1598
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Nucleus
LapatinibErlotinib
Vandetanib
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2 PDG
FR
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EGFR
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CediranibSunitinib
RefametinibSelumetinibTrametinib
HDAC inhibitors(e.g., vorinostat,panabinostat)
SorafenibRegorafenib
PTEN
ARID1A
BAP1
TET1/2
TCF/LEF
Target geneexpression
Histonedemethylation
Everolimus
AG-221
TCAcycle
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PIK3–AKTpathway
MTOR
AKT
PIK3
IDH1
Mitochondria
AG-120
Isocitrate
Citrate
WNT/β-catenin inhibitors(e.g., XAV-939, ICG-001,
IWP-2, IWR-1-endo)
β-catenin
β-catenin
β-catenin β-catenin β-catenin
NC
ID
NC
ID
2-HGα-KG
IDH2Isocitrate
Citrate
2-HG
IDH1mut
IDH2mut
ERK
MEK
BRAF
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BGJ398AZD4547
LY2874455Ponatinib
AXIN
AXIN
FGFR2 fusionsEGF VEGF
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etiologies (n¼ 101; ref. 52). In summary, these studies reveal that(i) TP53, SMAD4, KMT2C, and GNAS are more commonlymutated in O. viverrini–infected CCA cases; (ii) IDH1/IDH2mutations are almost exclusive for non–O. viverrini–related iCCA;and (iii) fluke-related CCAs present a mean of 26 somatic muta-tions per tumor, compared with a mean of 16 mutations pertumor in CCA with other etiologies. In addition, whole-exomesequencing (WES) studies have led to the identificationof somaticmutations in chromatin-remodeling genes, BAP1, ARID1A, andPBRM1—in iCCA (52, 54). Functional studies have revealedtumor-suppressive activity of BAP1 and ARID1A, further support-ing the potential role of chromatin modulators in iCCA devel-opment (52). In particular, ARID1A encodes an accessory subunitof the SWI/SNF chromatin-remodeling complex andmutations inthis gene have recently been identified in awide variety of cancers.Silencing of ARID1A in CCA cell lines (including non–O. viver-rini–associated and O. viverrini–associated) resulted in a signifi-cant increase of cell proliferation. Conversely, overexpression ofwild-typeARID1A led to retarded cell proliferation confirming thetumor-suppressive role of this gene (52). Thepossibility that iCCApatients harboring mutations in these genes may benefit fromtreatment with histone deacetylase (HDAC) inhibitors, such asvorinostat or panobinostat, remains unclear and needs to befurther explored.
IDH1 and IDH2mutations havebeen reported in approximately14% of iCCAs (Table 2). In a large cohort of iCCA cases (n¼ 326),IDH1/2mutationswere associatedwithbetter overall survival (60).In contrast, in a recent WES-based study, patients with IDH1 orIDH2mutations had shorter survival compared with patients withwild-type IDH genes (3-year survival of 33% in IDH mutants vs.81% in IDHwild-type; ref. 54). IDH1 and IDH2mutations in iCCAand other cancer types cluster at the hotspots codons 132 and 172,respectively. IDH1 and IDH2 encode metabolic enzymes whosenormal function is to interconvert the metabolic intermediateisocitrate to a-ketoglutarate (a-KG) in conjunction with the gen-eration of NADPH. Mutations in IDH1 and IDH2 are alwayspresent in a heterozygous state with the wild-type allele and theyresult in the acquisition of an abnormal enzymatic activity, thereduction of a-KG to 2-hydroxyglutarate (2-HG). 2-HG has beendesignated as an "oncometabolite" that contributes to cancerformation by inhibiting multiple dioxygenase enzymes thatrequire a-KG for their activity, resulting in altered cell differenti-ation, survival, and extracellularmatrixmaturation (Fig. 2). Abnor-mal DNA methylation and increased protein levels of TP53 arecommon features of tumors with IDH1 and IDH2mutations (60).Furthermore, using in vitro stem cell systems and GEMMs, it hasbeen demonstrated that mutant IDH mutations are able to pro-mote iCCA formation by blocking hepatocyte differentiation andinducing proliferation of hepatic progenitors (39).
Management and Molecular TargetedTherapies
At present, the treatment of choice for iCCA when feasible issurgical resection (1), whereas liver transplantation remains con-troversial. Upon resection, the median overall survival is ofaround 3 years and recurrence occurs in up to 60% of patients,depending on several prognostic factors, among which tumorburden and lymphonodal status appear to be the most relevant(1, 16). The prognosis for patients diagnosed with unresectabledisease is even more dismal, with a life expectancy around 1 yearand actuarial probability of survival of 5% at 5 years (1, 58).
The lack of clinical trials conducted specifically in iCCA pati-ents as opposed to all biliary tract cancers (BTC) and the limitednumber of patients studied are among the challenges that precludeclinical practice guidelines in establishing a standard of care forpatients with advanced iCCA (1). Among the 112 clinical trialsreported in advanced BTCs testing systemic therapies (81), themajority are single-arm phase II studies with low statistical powerand unclear impact on overall survival. The current standard ofpractice for advanced-stage iCCA is represented by systemic che-motherapy with gemcitabine and cisplatin (6). Survival benefitsfavoring the combination arm as opposed to gemcitabine alone(11.7 vs. 8 months; ref. 6) were demonstrated in a subgroupanalysis of patients with iCCA (n ¼ 80) included in a largerandomized phase III trial (n ¼ 410, ABC-02) of patients withadvanced and metastatic BTCs.
On the other hand, so far no molecular targeted therapy hasbeen proven effective for iCCA or other biliary tract cancers.The results of few trials with targeted therapies as monotherapy(i.e., selumitinib) or in combination with chemotherapy (i.e.,sorafenib plus gemcitabine, cetuximab plus gemcitabine–oxali-platin) have been discouraging with limited effects on overallsurvival (1). In this sense, patient stratification based on molec-ular biomarkers (Table 2) may be essential for clinical success intreating iCCA patients. Toward this direction, the first clinicaltrials driven by biomarkers (e.g., FGFR2 aberrations and IDH1/2mutations) in BTCs, including iCCA, are currently ongoing andtheir results are anxiously awaited (Fig. 2, Table 3). BGJ398, aselective FGFR inhibitor, has shown efficacy in vitro by blockingthe neoplastic transformation and growth of cell lines expressingFGFR2 fusions (57). Clinical efficacy of BGJ398 is currently beinginvestigated in a phase II multicenter single-arm study in adultpatients with advanced or metastatic CCA harboring FGFR2 genefusions or other FGFR genetic alterations who have failed che-motherapy (NCT02150967). Furthermore, promising prelimi-nary data have been reported following treatmentwith ponatinib,a multikinase inhibitor, in 2 iCCA patients harboring FGFR2fusions (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 autophosphorylationof their receptors, followed by signaling through the RAS–MAPK and PI3K–AKT effector cascades. FGFR2 fusions: The presence of fusion partners in thecytoplasmic domain of FGFR2 results in constitutively active receptors that induce signaling through downstream signaling pathways. NOTCH signaling: Bindingof 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 offrizzled (FZD) receptors by WNT ligands triggers the displacement of the regulatory APC/Axin/GSK3-complex, accumulation of b-catenin and induction oftarget genes. IDH signaling: Mutated IDH enzymes acquire the capacity to synthesize 2-hydroxygluterate (2-HG) from a-ketoglutarate (a-KG). 2-HG alters theactivity of a-KG–dependent dioxygenase enzymes involved in multiple cellular processes, including cell differentiation, survival, and DNA methylation.Molecular targeted therapies havealsobeenhighlighted; drugs currently assessed in phase II clinical trials (red) and thoseevaluated in early clinical trials or preclinicalstudies (brown) are shown.
Pathogenesis and Targeted Therapies in iCCA
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reduction (51). Currently, a pilot studywith ponatinib is ongoingin BTC patients with FGFR2 fusions (NCT02265341). At the sametime, based on demonstrated efficacy in preclinical studies, spe-cific inhibitors for IDH1 (AG-120) and IDH2 (AG-221) arecurrently being investigated inphase I (NCT02073994) andphaseI/II (NCT02273739) clinical trials, respectively (Table 3). Inparallel, considering the emerging roles of NOTCH and WNTpathway activation in the pathogenesis of iCCA, the first clinicaltrials targeting these pathways using available specific inhibitorsare expected to move forward (Fig. 2).
Future PerspectivesThe application of new technologies has led to a more accurate
mapping of the genomic landscape of iCCA, a devastating diseasewith limited treatment options. Among the newly discoveredmolecular alterations, FGFR2 fusions and IDH1/2mutations hold
great promise for improving the future management and treat-ment of iCCA patients through the first biomarker-driven clinicalstudies currently ongoing. Whether FGFR2 aberrations may rep-resent a novel oncogene addiction loop in iCCA still remains anunanswered question. Nevertheless, FGFR2 fusions have thepotential to represent a new avenue of research for basic inves-tigators and clinicians. Finally, the intriguing possibility of mul-tiple cells of origin in iCCA deserves further investigation as ameans to understand the mechanisms underlying the carcino-genesis process and to determine whether this can be of relevancein clinical application.
Disclosure of Potential Conflicts of InterestV. Mazzaferro reports receiving speakers bureau honoraria from Bayer and
BTG. J.M. Llovet reports receiving commercial research grants from Bayer,Blueprint Medicines, and Boehringer Ingelheim; other commercial researchsupport from Bayer, Boehringer Ingelheim, and Bristol-Myers Squibb; and is
Table 3. Ongoing clinical trials using targeted therapiesa
Treatment Targets Clinical trial phase Number of trials
Biomarker drivenBGJ398 FGFR, ABL, FYN, KIT, LCK, LYN, YES II 1Ponatinib hydrocloride BCR-ABL, VEGFR, PDGFR, FGFR, EPH, SRC, KIT, RET, TIE2, FLT3 II 1AG-221 Mutated IDH2 I/II 1AG-120 Mutated IDH1 I 1
MonotherapyCabozantinib (XL-184) MET, VEGFR2, RET, c-KIT, FLT1/3/4, TIE2 II 1Everolimus mTOR II 2Sunitinib VEGFR, PDGFR, KIT, FLT3, RET II 1Regorafenib RET, RAF-1, VEGFR, KIT, BRAF (V600E), PDGFRB, FGFR1, TIE2 II 2Celecoxib COX IV 1c
Trastuzumab HER2-neu II 1LY2801653 c-MET, MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 I 1BKM120 VPS34/mTOR/DNAPK/PI4Kb II 1Lapatinib ErbB2-4/EGFR/SRC II 2Selumetinib MEK1/2 II 1MK2206 AKT1-3 II 1RAV12 RAAG12 I 1PLX8394 BRAF I/II 1
CombinationSelumetinib þ MK-2206 MEK1 þ AKT1-3 II 1Bosutinib þ capecitabine ABL/SRC/c-KIT I 1AZD2171 þ AZD0530 VEGFR/PDGFR/FGFR1/c-KIT þ SRC/ABL/LCK/YES/EGFR/LYN I 1Pazopanib þ GSK1120212 VEGFR/PDGFR/FGFR/KIT þ MEK1/2 I 1Cetuximab þ erlotinib EGFR I/II 2c
Trastuzumab þ tipifarnib HER2-neu þ FTI I 1Erlotinib þ bevacizumab EGFR þ VEGFA II 2
Combination with chemotherapyRadiotherapy þ bevacizumab VEGFA I 1Chemotherapyb þ veliparib PARP1/2 I 1Chemotherapy þ bevacizumab VEGFA II 2c
Chemotherapy � panitumumab EGFR II 5c
Chemotherapy � vandetanib (ZD6474) VEGFR, EGFR I, II 2c
Chemotherapy þ cediranib VEGFR II 1Chemotherpy � sorafenib BRAF, VEGFR, PDGFR I/II 2Chemotherapyb � cetuximab EGFR II 2c
Chemotherpyb þ selumetinib MEK1/2 I/II 1Chemotherpy � trametinib MEK1/2 II 1Chemotherapyb þ sirolimus mTOR I 1Chemotherapy þ pazopanib VEGFR/PDGFR/FGFR/KIT II 1Chemotherapy þ AZD2171 VEGFR/PDGFR/FGFR1/c-KIT II 1Chemotherapyb � 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.
Moeini et al.
Clin Cancer Res; 22(2) January 15, 2016 Clinical Cancer Research298
on April 27, 2018. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from
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a consultant/advisory board member for Bayer, Biocompatibles, BlueprintMedicines, Boehringer Ingelheim, Bristol-Myers Squibb, Celsion, Eli Lilly,GlaxoSmithKline, andNovartis. No potential conflicts of interest were disclosedby the other authors.
Grant SupportA. Moeini is supported by a fellowship from Spanish National Health
Institute (FPI program, BES-2011-046915). D. Sia is supported by theILCA-Bayer Fellowship. N. Bardeesy holds the Gallagher Endowed Chairin Gastrointestinal Cancer Research at Massachusetts General Hospital andis supported by a V Foundation Translational Award, the TargetCancer Foun-
dation, and the NIH under award numbers R01CA136567-02 andP50CA1270003. V. Mazzaferro is partially supported by the AIRC (ItalianAssociation for Cancer Research) and a 5�1000 Milan-INT institutional grantin hepato-oncology. J.MLlovet is supported by grants from the SamuelWaxmanCancer Research Foundation, Asociaci�on Espa~nola Contra el C�ancer, SpanishNational Health Institute (SAF-2013-41027), and a European CommissionHEP-CAR grant (667273-2).
Received June 5, 2015; revised August 6, 2015; accepted August 6, 2015;published OnlineFirst September 24, 2015.
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