Pathogenesis, Diagnosis, and Management of Cholangiocarcinoma SUMERA RIZVI and GREGORY J

GASTROENTEROLOGY 2013;145:1215–1229

REVIEWS IN BASIC AND CLINICAL AND GASTROENTEROLOGY AND HEPATOLOGY REVIEWS PERSPECTIVES

Robert F. Schwabe and John W. Wiley, Section Editors

Pathogenesis, Diagnosis, and Management of Cholangiocarcinoma SUMERA RIZVI and GREGORY J. GORES

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota

Cholangiocarcinomas (CCAs) are hepatobiliary can- encasement.3 pCCA is the most common type of CCA. In a cers with features of cholangiocyte differentiation; large series of patients with bile duct cancer, 8% had iCCA, they can be classified anatomically as intrahepatic 50% had pCCA, and 42% had distal CCA.4 CCA has a poor CCA (iCCA), perihilar CCA (pCCA), or distal CCA. prognosis; patients have a median survival of 24 months These subtypes differ not only in their anatomic after diagnosis. The only curative treatment option is sur- location, but in epidemiology, origin, etiology, path- gery, for early stage disease.5 ogenesis, and treatment. The incidence and mortality of iCCA has been increasing over the past 3 decades, Epidemiology and only a low percentage of patients survive until 5 years after diagnosis. Geographic variations in the inci- Cholangiocarcinoma accounts for 3% of all gastrointestinal tumors. Over the past 3 decades, the overall dence of CCA are related to variations in risk factors. 6 Changes in oncogene and inflammatory signaling path- incidence of CCA appears to have increased. The percentage of patients who survive 5 years after diagnosis has not ways, as well as genetic and epigenetic alterations and 7,8 chromosome aberrations, have been shown to contribute increased during this time period, remaining at 10%. to the development of CCA. Furthermore, CCAs are In the United States, Hispanics and Asians have the surrounded by a dense stroma that contains many highest incidence of CCA (2.8 per 100,000 and 3.3 per cancer-associated fibroblasts, which promotes their 100,000, respectively), whereas African Americans have the progression. We have gained a better understanding of lowest incidence of CCA (2.1 per 100,000). African Amer- the imaging characteristics of iCCAs and have developed icans also have lower age-adjusted mortality rates advanced cytologic techniques to detect pCCAs. Patients compared with whites (1.4 per 100,000 vs 1.7 per 100,000). Men have a slightly higher incidence of CCA and mortality with iCCAs usually are treated surgically, whereas liver 7 transplantation after neoadjuvant chemoradiation is an from cancer than women. With the exception of patients option for a subset of patients with pCCAs. We review with primary sclerosing cholangitis (PSC), a diagnosis of recent developments in our understanding of the epide- CCA is uncommon before age 40 years. miology and pathogenesis of CCA, along with advances fi in classi cation, diagnosis, and treatment. Abbreviations used in this paper: a-SMA, a-smooth muscle actin; CA19-9, carbohydrate antigen 19-9; CAF, cancer-associated fibroblast; Keywords: Cancer-Associated Fibroblasts; Distal Chol- CCA, cholangiocarcinoma; CT, computed tomography; CXCR4, chemo- angiocarcinoma; Intrahepatic Cholangiocarcinoma; kine (C-X-C motif) receptor 4; dCCA, distal cholangiocarcinoma; ECM, Molecular Pathogenesis. extracellular matrix; EGFP, enhanced green fluorescent protein; EGFR, epidermal growth factor–receptor; EMT, epithelial–mesenchymal transition; ERBB2, v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2; ERC, endoscopic retrograde cholangiography; ERK, extra- holangiocarcinoma (CCA) is the most common cellular signal regulated kinase; FGFR, fibroblast growth factor receptor; C biliary malignancy and the second most common FISH, fluorescence in situ hybridization; HGF, hepatocyte growth factor; hepatic malignancy after hepatocellular carcinoma (HCC).1 HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C CCAs are epithelial tumors with features of cholangiocyte virus; iCCA, intrahepatic cholangiocarcinoma; IDH, isocitrate dehydro- differentiation. Intrahepatic cholangiocarcinomas (iCCAs) genase; IL 6, interleukin 6; KRAS, Kirsten rat sarcoma viral oncogene homolog; MAPK, mitogen-activated protein kinase; miR, microRNA; are located within the hepatic parenchyma. The second- MCL1, myeloid cell leukemia sequence 1; MET, met proto-oncogene; order bile ducts serve as the point of separation between MMP, matrix metalloproteinase; MRI, magnetic resonance imaging; OR, iCCAs and perihilar CCAs (pCCAs) or distal CCAs odds ratio; pCCA, perihilar cholangiocarcinoma; PDGF, platelet-derived (dCCAs)—the cystic duct is the anatomic boundary between growth factor; PI, phosphatidyl inositol; PI3K, phosphatidylinositol-4,5- these latter 2 subtypes (Figure 1A).2 The Bismuth–Corlette bisphosphate 3-kinase; PSC, primary sclerosing cholangitis; STAT, signal fi fi transducer and activator of transcription; TP53, tumor protein 53. classi cation strati es perihilar tumors on the basis of © 2013 by the AGA Institute biliary involvement. This classification recently was 0016-5085/$36.00 extended to also take into account arterial and venous http://dx.doi.org/10.1053/j.gastro.2013.10.013 1216 RIZVI AND GORES GASTROENTEROLOGY Vol. 145, No. 6

PERSPECTIVES Globally, hepatobiliary malignancies account for 13% of In the West, PSC is the most common predisposing

REVIEWS cancer-related deaths; 10%–20% of these are attributable to condition for CCA. Among patients with PSC, the annual AND CCA. The mean age at diagnosis of CCA is 50 years. The risk of development of CCA is 0.5%–1.5%, with a lifetime global incidence of iCCA varies widely, from rates of 113 prevalence of 5%–10%17; CCA is diagnosed within 2 years per 100,000 in Thailand to 0.1 per 100,000 in Australia.9,10 of PSC in most of these patients. A number of potential Differences in the prevalence of genetic and other risk risk factors for CCA in patients with PSC have been factors presumably account for this extensive variation. studied, including smoking and alcohol, although defin- Epidemiologic studies have indicated that the age-adjusted itive data are lacking.8 mortality rate for iCCA is increasing, whereas the mortality Hepatitis B virus (HBV) or hepatitis C virus (HCV) infec- – rate from pCCA and dCCA could be decreasing.9 14 Astudy tion and cirrhosis have been proposed as potential etiologies – of a World Health Organization database reported a sub- of iCCA.23 25 A recent meta-analysis of 11 studies found that stantial global increase in iCCA mortality, with a decreasing cirrhosis, HBV, and HCV were major risk factors for iCCA, trend in mortality from pCCA plus dCCA.15 Although this with odds ratios (ORs) of 22.92, 5.1, and 4.8, respectively.26 A observed increase in the incidence of CCA over the past 30 case-control study from Korea found a significant associa- years has been recorded as an increase in iCCA, it could result tion between HBV (OR, 2.3) and CCA, but not HCV and from misclassification of perihilar tumors as iCCAs.16 Ac- CCA. Cirrhosis also was found to be a significant risk factor cording to the US Surveillance, Epidemiology, and End Re- for CCA, with an OR of 13.6. HCV and cirrhosis were asso- sults database, the age-adjusted incidence rate for iCCA ciated with iCCA in a US case-control study. Compared with increased from 0.59 per 100,000 in 1990 to 0.91 per 100,000 controls, patients with iCCA had a higher prevalence of in 2001. It subsequently decreased to 0.6 per 100,000 by 2007. anti-HCV antibodies, with an OR of 7.9.24 Conversely, the incidence rate for pCCA plus dCCA remained CCA development has been associated with other risk around 0.8 per 100,000 until 2001, and then gradually factors, including inflammatory bowel disease indepen- increased to 0.97 per 100,000 by 2007. Perihilar tumors were dent of PSC, alcohol, smoking, fatty liver disease, diabetes, – coded as iCCAs before 2001 and subsequently were coded as cholelithiasis, and choledocholithiasis.8,27 29 Additional pCCAs after implementation of the third edition of the In- studies have associated variants of genes that regulate ternational Classification of Disease for Oncology. This up- DNA repair, inflammation, and carcinogen metabolism date likely influenced the aforementioned changes in with CCA development.8 Further studies are necessary to incidence rates of both CCA subtypes. Similar trends in the verify these potential associations. incidence of CCA subtypes were noted in the United Kingdom after the change to the third edition of the Inter- national Classification of Disease for Oncology in 2008.6,16 Cells of Origin iCCA is a histologically diverse hepatobiliary malignancy considered to develop from biliary epithelial cells Risk Factors or hepatic progenitor cells (Figure 1B). A recently pro- There are several established risk factors for CCA, posed classification of iCCAs subdivided these tumors and most cases are sporadic.6,8,17 Geographic variations in into the conventional, bile ductular, or intraductal incidence rates of CCA are related in part to variations in neoplasm type, or rare variants (combined hepatocellular risk factors. For example, in Southeast Asia, which has one CCA, undifferentiated type, squamous/adenosquamous of the highest incidence rates of CCA, infection with the type). The conventional type includes small-duct or pe- hepatobiliary flukes Opisthorchis viverrini and Clonorchis ripheral type and large-duct or perihilar type.30 Neural cell sinensis has been associated with the development of CCA. adhesion molecule, a marker of hepatic progenitor cells, Both parasites cause chronic inflammation and are has been detected in the bile ductular and combined he- considered carcinogens.8,18 Hepatolithiasis is another risk patocellular CCA types, so these might have originated – factor for CCA (mainly iCCA) in Asian countries.8 Chronic from hepatic progenitor cells.30 32 biliary inflammation secondary to calculi has been pro- Distal and pCCA have been proposed to arise from the posed to increase the risk of malignancy. Moreover, biliary epithelium and peribiliary glands.33 Extrahepatic infestation with hepatobiliary flukes has been shown to be bile ducts and large intrahepatic bile ducts are lined by more common in patients with hepatolithiasis.8,19 The mucin-producing cuboidal cholangiocytes. A recent study incidence and prevalence of CCA in patients with bile duct showed that mucin-producing iCCAs and hilar CCAs had (choledochal) cysts are also higher in Asian than in gene expression and immunohistochemical profiles similar Western countries.20,21 Choledochal cystic diseases, to those of the cylindric, mucin-producing cholangiocytes including Caroli’s disease, are rare congenital abnormal- that line hilar and intrahepatic large bile ducts.34 ities of the pancreatic and biliary ducts. Choledochal cysts A model in which iCCAs arise from transdifferentiation can be intrahepatic or extrahepatic, and are diagnosed in and subsequent neoplastic conversion of normal hepato- patients at an average age of 32 years old.8,17 Thorotrast, a cytes into malignant cholangiocytes has been proposed. previously used contrast agent that is now banned, was Fan et al35 showed in mice that overexpression of Notch1 found to increase risk for CCA by 300-fold in a Japanese and AKT resulted in the development of invasive cys- study.22 tadenocarcinomas via conversion of hepatocytes into December 2013 CHOLANGIOCARCINOMA 1217

cytokines activate inducible nitric oxide synthase, leading to excess nitric oxide with resultant single-stranded, dou- ble-stranded, and oxidative DNA lesions, as well as inhi- 38 bition of DNA repair enzymes. Interleukin (IL)-6, an AND inflammatory mediator secreted by CCA and stromal in- REVIEWS PERSPECTIVES flammatory cells, can function in an autocrine or paracrine manner to promote cell survival and provide mitogenic signals.39,40 Myeloid cell leukemia sequence 1 (MCL1) is an anti-apoptotic BCL2 family member that mediates tumor necrosis factor–related resistance to apoptosis-inducing ligands in CCAs.41 IL-6 increases the expression of MCL1 via constitutive activation of signal transducer and activator of transcription (STAT) signaling and protein kinase B (Akt).40,42 MCL1 transcription is activated by IL-6 via a p38 mitogen-activated protein kinase (MAPK)-dependent pathway.43 IL-6 binds to the gp130 receptor, leading to its subsequent dimerization and activation of the gp130-associated janus kinases, including janus kinase 1 and janus kinase 2, which leads to STAT3 activation.44,45 Epigenetic silencing of suppressor of cytokine signaling 3 results in sustained IL-6 signaling via STAT3.46 Inflammatory signaling pathways therefore appear to promote the development of CCA by causing DNA damage and blocking the apoptosis normally induced by the DNA damage response. These cytokines also promote cell proliferation. The combination of DNA damage, evasion of apoptosis, and cell proliferation are all components of cell transformation. Epidermal growth factor–receptor (EGFR) signaling also contributes to cholangiocarcinogenesis and CCA progression. Activation of EGFR leads to activation of extracellular-signal regulated kinases (ERKs) 1 and 2 (also Figure 1. Anatomic localization of CCA and cells of origin in CCA. (A) known as p44/42 MAPK). EGFR inhibitors decrease Anatomic localization of CCA. CCA is divided into 3 subtypes, based expression of cyclooxygenase-2 by CCA cells.47 V-erb-b2 fi on anatomic location. Modi ed with permission from Elsevier and avian erythroblastic leukemia viral oncogene homolog 2 Razumilava et al.17 (B) Cells of origin in CCA. (ERBB2) is another member of the EGFR family that cholangiocyte precursors of iCCA.35 Sekiya and Suzuki36 contributes to CCA development. In mice, overexpression of ERBB2 led to formation of tumors along the biliary also showed that in mice, Notch-mediated conversion of 48 hepatocytes into biliary cells leads to macronodular epithelium. Hepatocyte growth factor (hepapoietin A; scatter factor) (HGF) is a stromal paracrine mediator that cirrhosis and iCCAs. Therefore, iCCAs may not have a 49–51 single lineage, but instead derive from different cells of regulates tumor invasiveness and metastasis. Activa- origin. In support of this theory, a recent study showed tion of MET, the receptor for HGF, up-regulates several that transformed hepatocytes, hepatoblasts, and hepatic signaling pathways, including those involving phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)–AKT, progenitor cells can give rise to a broad spectrum of liver 52 37 STAT3, and MAPK. CCAs express higher levels of MET tumors, ranging from CCA to HCC. These studies 53,54 indicate that multiple cell types, rather than only chol- and HGF than nontumor tissues. MET overexpression was associated with activation of members of the EGFR angiocytes, transform and develop into CCAs. Additional 54,55 animal models of CCA and lineage tracing studies are family, particularly of ERBB2. necessary to help identify the cells of origin for CCA. Cholestasis also contributes to the development of CCA, and bile acids have important roles in this process, activating growth factors that mediate proliferation. Bile fl In ammation acids activate EGFR and increase expression of CCAs frequently arises under conditions of cyclooxygenase-2 via a MAPK cascade.56 In addition to bile inflammation, which is believed to contribute to patho- acids, cyclooxygenase-2 overexpression is induced by oxy- genesis. A variety of cytokines, growth factors, tyrosine sterols and inducible nitric oxide synthase.57 Oxysterols kinases, and bile acids can contribute to alterations in are overlooked in the pathogenesis of CCA.58 These proliferation, apoptosis, senescence, and cell-cycle regula- oxidative degradation products of cholesterol are abun- tion required for cholangiocarcinogenesis.5 Inflammatory dant in bile. They are endogenous ligands for the 1218 RIZVI AND GORES GASTROENTEROLOGY Vol. 145, No. 6

59— 71 fi PERSPECTIVES hedgehog signaling pathway a developmental pathway compared with 17% in iCCAs. In a transcriptome pro le 60 REVIEWS implicated in CCA progression. analysis of 104 CCAs and 59 matched nontumor samples AND (controls), patients could be categorized based on overall survival time, early recurrence, and presence or absence of Genetics KRAS mutations; a detailed class comparison identified 4 A few studies have assessed the roles of genetic factors, subclasses of patients. Those with CCAs with altered such as chromosome aberrations or genetic and epigenetic expression of genes that regulate proteasome activity; with alterations in tumor suppressor genes and oncogenes, in the dysregulation of ERBB2; and with overexpression of EGFR, pathogenesis of human CCA. However, these studies have MET, and Ki67 had the worst outcomes.71 produced no definitive results because they analyzed a limited Inactivation of TP53, which regulates the cell cycle, is number of genes in combined CCA specimens, without one of the most common genetic abnormalities in cancer separate analyses of different subtypes.61 A comparative ge- cells and also has been detected during chol- nomics hybridization analysis of 32 CCA samples from pa- angiocarcinogenesis. A review of 10 studies, comprising tients (7 iCCA, 13 pCCA, and 12 dCCA) showed that they all 229 patients with CCA from Europe, Asia, and the United contained gains at 16q, 17p, 17q, 19p, and 19q, which States, reported TP53 mutations in 21% of CCAs.73 Mu- included regions encoding ERBB2, mitogen-activated pro- tations in other genes, including EGFR, neuroblastoma tein kinase kinase 2 (MEK2), and platelet-derived growth RAS viral (v-ras) oncogene homolog (NRAS), PI3K, and factor- beta (PDGFB).62 A meta-analysis of 5 studies that used APC, have been less frequently described.44 comparative genomics hybridization to analyze 98 iCCAs There has been growing interest in the effects of somatic found copy number losses at 1p, 4q, 8p, 9p, 17p, and 18q, and mutations in genes encoding isocitrate dehydrogenases gains at 1q, 5p, 7p, 8q, 17q, and 20q.61 In this meta-analysis, (IDH) 1 and 2. IDH1 and IDH2 mutations frequently have there was considerable variation among the 4 studies that been detected in gliomas, but rarely have been observed in – were performed in Asia63 66 and the 1 study from Europe.67 other solid tumors. IDH mutations were detected in 22% of This variation could have resulted from differences in CCA specimens—more frequently in iCCAs (28%) than ethnicity and etiologic associations among the studies. pCCAs and dCCAs (7%).74 Recurrent mutations in IDH1 Whole-exome sequencing analyses of 8 liver fluke-related were observed in a subset of biliary tract tumor samples in a CCAs identified 206 somatic mutations in 187 genes.68 The recent broad-based mutation profile analysis of gastroin- frequency of these mutations was validated in an addi- testinal tumors.75 A subsequent analysis of 62 CCAs tional 46 liver fluke-related CCAs. Mutations frequently detected IDH1 mutations in only iCCAs.75 IDH1 and IDH2 were detected in oncogenes and tumor suppressor genes mutations were associated significantly with increased such as those encoding tumor protein 53 (TP53) (muta- levels of p53 and DNA hypermethylation.76 Epigenetic tions in 44.4% of CCAs), Kirsten rat sarcoma viral oncogene changes associated with IDH mutations likely mediate their homolog (KRAS) (16.7%), and SMAD family member 4 oncogenic effects. The product of the enzymatic activity of (16.7%). Mutations also were found in myeloid/lymphoid mutant IDH1 and IDH2 is 2-hydroxyglutarate (Figure 2A). or mixed-lineage leukemia 3 (MLL3) (14.8% of cases), ring This metabolite therefore might serve as a biomarker for finger protein 43 (RNF43) (9.3%), paternally expressed 3 IDH1 and IDH2 mutations, and for a subset of patients who – (PEG3) (5.6%), and roundabout, axon guidance receptor, might be treated with IDH inhibitors77 79 (Figure 2B). homolog 2 (ROBO2) (9.3%). These genes are involved in A number of epigenetic alterations, such as promoter deactivation of histone modifiers, activation of G-proteins, hypermethylation and microRNA dysregulation, have and loss of genomic stability.68 This study, performed in been associated with the development of CCA. However, Asia, has been the only whole-exome sequence analysis of whole-epigenome analysis has not been conducted and CCAs. Further whole-genome sequencing studies are microRNA (miR) profiling is possible with only small needed to evaluate CCAs from Western patients. numbers of tumor samples.80,81 Promoter hyper- A recent study comprising single-nucleotide poly- methylation has been reported to silence tumor suppres- morphism array, gene expression profile, and mutation sor genes including CDKN2 (observed in 17%–83% of analyses of 149 iCCAs identified inflammation and prolif- CCAs), suppressor of cytokine signaling 3 (in 62%46), Ras as- eration classes of this tumor.45 Several copy number alter- sociation (RalGDS/AF-6) domain family member 1 ations were identified, including losses at 3p, 4q, 6q, 9p, 9q, (RASSF1A) (in 31%–69%), and APC (in 27%–47%).45,61 13q, 14q, 8p, 17p, and 21q, and gains at 1q and 7p.45 Fea- Gene fusions, such as the BCR-ABL gene in chronic tures of the inflammation class included activation of in- myeloid leukemia, are driver mutations in cancer, which play flammatory pathways, overexpression of cytokines, and a role in certain cancers.82 Fibroblast growth factor receptor activation of STAT3. The proliferation class was character- (FGFR) fusions are active kinases. A recent study identified ized by activation of oncogene signaling pathways involving novel FGFR2 gene fusions in CCA.82 Cells with these FGFR RAS, MAPK, and MET. Activating mutations in KRAS have fusions were susceptible to FGFR inhibitors, signifying that – been detected frequently in CCAs.69 71 At least 2 studies FGFR kinase inhibition may be a valid therapeutic strategy have reported a higher incidence of activating mutations in in CCA patients harboring these gene fusions.82 KRAS in pCCAs compared with iCCAs.71,72 In one cohort, miRs are noncoding RNAs that function in post- the incidence of these mutations was 53% in pCCAs transcriptional regulation of gene expression. A cluster December 2013 CHOLANGIOCARCINOMA 1219 AND REVIEWS PERSPECTIVES

Figure 2. IDH mutations. (A) Function of wild-type and mutant IDH (mIDH). Wild-type enzymes catalyze a reaction that converts isocitrate to a-ketoglutarate and reduces NADP to NADPH. The mutant enzymes acquire a neomorphic activity that converts the normal metabolite a-KG to 2-HG, and consumption rather than production of NADPH. 2-HG leads to inhibition of certain dioxygenases, which has been postulated to result in cancer-promoting events. (B) Potential of personalized medicine for CCA, using mIDH inhibitors, as an example. a-KG, a-ketoglutarate; 2-HG, 2-hydroxyglutarate; NADPH, nicotinamide adenine dinucleo- tide phosphate. of 38 miRs was expressed differentially in 27 iCCA sam- Developmental Pathways ples, compared with nontumor tissues. miR21 is overex- The Notch signaling pathway regulates embryonic pressed in CCAs and could have oncogenic effects, partly development and proliferation of the biliary tree.85 Not by inhibiting programmed cell death 4 and tissue inhibi- 83 surprisingly, therefore, Notch dysregulation also has tor of matrix metalloproteinase (MMP)3. miR21 also been implicated in cholangiocarcinogenesis. Two recent was found to regulate phosphatase and tensin homolog studies in mice have shown that Notch activation is deleted on chromosome ten–dependent activation of PI3K 84 required for conversion of normal adult hepatocytes to signaling in CCAs, to affect chemosensitivity. miR200C biliary cells that are precursors of iCCA.35,36 Over- prevents the epithelial–mesenchymal transition (EMT); 80 expression of intracellular domain of the Notch 1 re- changes in its level might be used as a prognostic factor. ceptor in liver cells of mice resulted in formation of Further studies are needed to determine how alterations in iCCAs.86 In this model, an inhibitor of g-secretase, an miRs contribute to the development of CCA, and how enzyme necessary for Notch signaling, suppressed tumor ’ these changes might be used to determine patients formation. prognoses. 1220 RIZVI AND GORES GASTROENTEROLOGY Vol. 145, No. 6

PERSPECTIVES Another evolutionary conserved, developmental insulin-like growth factor binding proteins. PDGF-

REVIEWS pathway is the Hedgehog signaling pathway. Hedgehog mediated interactions between CAFs and tumor cells AND signaling is deregulated in many types of tumors, have been observed, such as recruitment of CAFs by including CCAs. Inhibition of hedgehog signaling with PDGF-D secreted by CCA cells.60,100,101 PDGF-D stimu- cyclopamine impedes CCA cell migration, proliferation, lates CAF migration via its receptor platelet-derived and invasion.87,88 Hedgehog signaling also has been growth factor receptor (PDGFR), which is highly implicated in survival signaling by myofibroblast-derived expressed on CAFs, and activation of small Rho guanosine CCAs. PDGF-b protects CCA cells and promotes tumor triphosphatases and the JNK signaling pathway.100 survival in mice with CCAs, but cyclopamine reverses these Activated CAFs also secrete paracrine factors that pro- effects.60 mote initiation and progression of cancer. These include Wnt signaling also is required for intrahepatic bile duct matricellular proteins, growth factors, chemokines, and development and proliferation.89 Wnt-inducible signaling ECM proteases. Periostin is a matricellular protein that is pathway protein 1v is overexpressed in stroma nests overexpressed by CAFs compared with normal fibroblasts; around CCAs, and levels of Wnt-inducible signaling its presence correlates with shorter survival times of pa- pathway protein 1v are associated with reduced survival tients. Knockdown of the periostin receptor, the a5 sub- times of patients. Wnt-inducible signaling pathway pro- unit of integrin, with small interfering RNA, reduced tein 1v stimulated the invasive activity of CCA cell lines by stimulation of tumor proliferation and invasion by peri- activating MAPK1 and MAPK3.90 ostin.102 The ECM that surrounds pancreatic tumors also has been shown to overexpress periostin, which promotes tumor invasiveness.103 Tenascin-C, another ECM protein Tumor Microenvironment produced by CAFs, also promotes tumor migration and Carcinogenesis in CCA includes alterations in the invasiveness.92 In CCA cell lines, HGF promoted inva- stroma, recruitment of fibroblasts, remodeling of the siveness and motility by inducing phosphorylation of Akt extracellular matrix (ECM), changing patterns of immune and ERK 1/2.104 Similarly, stromal cell–derived factor-1, cell migration, and promotion of angiogenesis through activation of its receptor chemokine (C-X-C (Figure 3A).91 iCCAs and pCCAs are characterized by a motif) receptor 4 (CXCR4), induced CCA cell invasion via dense and reactive desmoplastic stroma (Figure 3B) that ERK 1/2 and Akt.105,106 This process was disrupted by the contains many a-smooth muscle actin (a-SMA)–positive CXCR4 inhibitor AMD3100.106 myofibroblasts, also known as cancer-associated fibro- ECM degradation and remodeling is required for tumor blasts (CAFs). The tumor stroma surrounds the malignant progression. MMPs degrade and remodel the ECM during ducts and glands and comprises most of the tumor fibrogenesis and carcinogenesis. MMP1, MMP2, MMP3, mass.92,93 The stroma promotes tumor progression via and MMP9 are strongly expressed in CCAs and are asso- reciprocal communication between the stromal cells and ciated with invasive tumors.107,108 Fibroblast activation cancer cells.92 protein is a stromal protein; its high expression by CAFs The precise origin of CAFs is unclear, although several has been associated with tumors with an aggressive cell types, including hepatic stellate cells, portal fibro- phenotype.109 blasts, and bone marrow–derived precursor cells, have The exact mechanisms by which tumor and stroma – been proposed as candidates.92,94 96 The EMT also has communicate are not clear. However, the importance of been proposed to produce CAFs.93 During tumorigen- the desmoplastic stroma in CCA progression indicates esis, the EMT is characterized by the presence of tumor that it could be a new therapeutic target, perhaps via se- cells that express mesenchymal markers such as vimen- lective targeting of CAFs.110 tin, tenascin, fibronectin, and the zinc finger protein Snail.92 Immunohistochemical studies have shown the expression of these markers by human CCA cell Animal Models – lines.97 99 In mice, xenograft tumors grown from Animal models are essential for the development of enhanced green fluorescent protein (EGFP)-expressing new therapeutic strategies and diagnostic tools.111 Animal human CCA cells were found to be surrounded and models of CCA (Table 1) include mice with xenograft – infiltrated by a-SMA–expressing CAFs. Interestingly, tumors,43,112 119 mice with genetic changes that lead to – EGFP was not co-expressed with a-SMA, indicating that CCA formation,86,120 124 rats with orthotopic tu- the EMT does not produce CAFs in CCAs.100 Based on mors,125,126 and animals that develop CCAs after exposure – combined evidence, a-SMA–expressing CAFs appear to to carcinogens.55,127 129 Although these models offer an be a heterogeneous population of cells that originate opportunity to bridge the chasm between in vitro findings from several cell lineages, but not from epithelial cancer and clinical applicability, they have limitations. The tumor cells. microenvironment is an important feature in CCA devel- CAFs produce factors that stimulate ECM production, opment. It sometimes can be a challenge to study in- leading to a fibrogenic response (Figure 3C).92 Factors teractions between cancer cells and the stroma in mice produced by CAFs include transforming growth factor-b, with xenograft tumors because the tumor is not growing PDGF isomers, connective tissue growth factor, and in the same microenvironment as it does in human beings. December 2013 CHOLANGIOCARCINOMA 1221 AND REVIEWS PERSPECTIVES

Figure 3. Microenvironment of cholangiocarcinoma. (A) Components of the tumor microenvironment in CCA. (B) Micrograph of a stromal CCA. (C) Factors secreted by cancer-associated ﬁbroblasts. CTGF, connective tissue growth factor; SDF-1, stromal cell–derived factor 1; TGF-b, transforming growth factor-b.

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AND REVIEWS

Table 1. Animal Models of Cholangiocarcinoma GORES AND RIZVI 1222 Experimental approach Key features Study Mice with xenograft tumors Injection of 3 Â 106 Mz-ChA-1 cells Tumor development in 3 weeks Fava et al112 Injection of 5 Â 106 Sk-ChA-1 cells Æ intratumoral tamoxifen injections Significantly decreased CCA development with intratumoral Pawar et al113 tamoxifen injections Injection of 2 Â 106 QBC939 cells Æ magnetic nanoparticle injections via tail vein CCA tumor growth inhibition with magnetic nanoparticles Tang et al114 Injection of IL-6 overexpressed Mz-ChA-1 stable cell line (Mz-ChA-IL6) vs Overexpression of IL-6 increased growth of xenograft tumors Meng et al43 control vector Mz-ChA-1 cell line Injection of miR26a overexpressed CCLP1 cell line vs scramble control Overexpression of miR26a increased growth of xenograft tumors Zhang et al115 CCLP1 cell line Injection of miR494 overexpressed stable HuCCT1 cell line vs control vector Overexpression of miR494 increased growth of xenograft tumors Olaru et al116 HuCCT1 cell line Injection of stable QBC939 cell line transfected with Slug siRNA vs control Slug silencing suppressed growth of xenograft tumors Zhang et al117 vector QBC939 cell line Injection of CypA silenced stable M139 cell line vs control vector M139 cell line CypA silencing decreased growth of xenograft tumors Obchoei et al118 Injection of stable QBC939 cell line transfected with Beclin-1 siRNA vs Beclin-1 silencing decreased growth of xenograft tumors Hou et al119 control vector QBC939 cell line Genetically engineered mouse models Liver-specific inactivation of SMAD4 and PTEN Tumor formation in all animals at 4–7 months of age Xu et al120 Chronic carbon tetrachloride exposure in TP53-deficient mice Development of tumors with dense peritumoral fibrosis and other histologic Farazi et al121 and genetic features of human iCCA Liver-specific inactivation of macrophage stimulating factor 1 and 2 Tumor development (HCC or CCA) in all mice by 6 months of age Song et al122 Liver-specific ablation of WW45, a homolog of Drosophila Salvador and Development of tumors with mixed histologic features of HCC and CCA Lee et al123 adaptor for the Hippo kinase Liver-specific activation of KRAS and deletion of TP53 Development of stroma-rich tumors; shortened time to tumor development O’Dell et al124 and increased metastasis with the combination of KRAS activation and TP53 deletion Overexpression of intracellular domain of Notch1 in livers of transgenic mice Formation of tumors with features characteristic of iCCA Zender et al86 Orthotopic rat models Inoculation of BDEneu cells into bile duct of isogenic rats Rapid (21–26 days) development of cholangiocarcinoma characterized by biliary Sirica et al125 obstruction and gross peritoneal metastasis; origin of tumor stroma and tumor tissue from same species (rat) Three-dimensional organotypic culture model of CCA in rats Stromal microenvironment, gene expression profile, and pathophysiologic Campbell et al126 characteristics that mimic desmoplastic iCCA in vivo Carcinogen-induced models Furan-induced cholangiocarcinogenesis in rat liver Formation of mucin-producing CCA tumors; overexpression of C-NEU and MET Radaeva et al55 Chronic administration of thioacetamide in lean rats and rats with Increased development and growth of CCA tumors in Fava et al127 6 No. 145, Vol. GASTROENTEROLOGY faulty leptin receptors lean rats treated with thioacetamide Administration of DEN Æ LMBDL to induce chronic cholestasis Increased CCA progression in mice with LMBDL given DEN compared with Yang et al128 and CCA development mice without LMBDL given DEN Inoculation with O viverrini and administration of Development of pus and tumor in liver starting at 20 weeks after O viverrini Plengsuriyakarn dimethylnitrosamine in hamsters infection/DEN administration; all hamsters in experimental group et al129 were dead by 28 weeks BDEneu, highly malignant cholangiocarcinoma cell line; CCLP1, cholangiocarcinoma cell line; C-NEU, rat homologue of human ERBB2; CypA, cyclophilin A; DEN, diethylnitrosamine; HuCCT1, cholangiocarcinoma cell line; KRAS, Kirsten rat sarcoma viral oncogene homolog; LMBDL, left and median bile duct ligation; M139, cholangiocarcinoma cell line; Mz-ChA-1, cholangiocarcinoma cell line; PTEN, phosphatase and tensin homolog deleted on chromosome ten; QBC939, human hilar bile duct carcinoma cell line; Sk-ChA-1, cholangiocarcinoma cell line; siRNA, small interfering RNA; SMAD4, SMAD family member 4. December 2013 CHOLANGIOCARCINOMA 1223

A model described by Sirica et al,125 in which rat CCA candidates. After surgical resection, the median time of cells were injected into rat biliary trees, is unique in disease-free survival is 26 months; reported rates of recur- that the stroma and epithelial cells were derived from the rence are 60%–65%.136,137 Approximately 60% of patients same species. These animals allow for investigations of survive for 5 years after resection. Factors associated with AND tumor–stroma interactions that more closely resemble recurrence and reduced survival time after resection include REVIEWS PERSPECTIVES those of patients. Although transgenic models do allow vascular invasion, lymph node metastasis, multiple tumors, for study of the tumor microenvironment, they tend to be and cirrhosis.4,138 Nuclear expression of S100A4, a member technically challenging and expensive. Animals with ge- of the S100 family of calcium-binding proteins, in netic alterations that lead to production of CCAs that neoplastic ducts was associated with metastasis and resemble human tumors are needed. reduced time of survival after surgical resection in a subset of patients with CCA.139 Liver transplantation as a curative option for iCCA is Diagnosis and Management highly controversial. iCCA was reported to recur in 70% of It can be a challenge to diagnose CCA because of patients within 5 years of liver transplantation, and the its paucicellular nature, anatomic location, and silent median disease-free survival time was 8 months in a series clinical character. Diagnosis requires a high index of sus- of 14 patients with iCCA or mixed HCC-iCCA.135 Patients picion and a multidisciplinary approach that involves with very small iCCAs (<2 cm) in the context of cirrhosis, clinical, laboratory, endoscopic, and radiographic analyses. however, do as well as patients undergoing liver transplantation for HCC. Locoregional therapy, including iCCA transarterial chemoembolization and radiofrequency iCCA is divided into mass-forming, periductal ablation, has garnered interest as a therapeutic option for infiltrating, and intraductal growth types.130 The clinical patients with unresectable iCCA.140 The standard practice manifestations of iCCA include nonspecific symptoms of care for advanced-stage iCCA is systemic chemotherapy such as abdominal pain, cachexia, malaise, fatigue, and with gemcitabine and cisplatin.141 night sweats.2 iCCA frequently presents as an intrahepatic mass lesion; imaging modalities including computed to- pCCA mography (CT) and magnetic resonance imaging (MRI) pCCAs can have exophytic or intraductal macro- aid in the diagnosis. The use of contrast enhancement scopic growth patterns. The exophytic or mass-forming improves the sensitivity of MRI for detection of iCCA type can be of the nodular subtype or the periductal sub- because these tumors typically have progressive uptake of type (the most common subtype).142 There are also sub- contrast during the venous phase. HCCs, on the other types of intraductal patterns, including the intraductal hand, are characterized by rapid contrast uptake during growing type, mucin-producing type, papilloma type, and the arterial phase, followed by a delayed venous washout cystic type.17 Patients with pCCA can present with phase.131 CT and MRI have similar utility in the evalua- nonspecific symptoms including abdominal discomfort, tion of tumor size and detection of satellite lesions. cachexia, weight loss, and malaise. However, their presen- However, CT may be better for assessment of vascular tation typically is consistent with biliary obstruction pre- encasement, identification of extrahepatic metastasis, and senting with jaundice, and less commonly cholangitis.17 determination of resectability.17,132 Hypertrophy–atrophy complex, a phenomenon character- Serum levels of carbohydrate antigen 19-9 (CA19-9), a ized by hypertrophy of the unaffected liver lobe and atro- tumor biomarker, can aid in diagnosis, but this assay phy of the affected lobe, presents as unilobar palpable detects iCCA with only 62% sensitivity and 63% speci- prominence on physical examination.2 Laboratory analyses, ficity.133 Moreover, increased levels of CA19-9 also have including measurements of alkaline phosphatase and bili- been observed in patients with benign diseases such as rubin levels, do not provide specific information because bacterial cholangitis or choledocholithiasis.5 Nonetheless, they typically reflect concomitant cholestasis and chol- very high levels of CA19-9 (1000 U/mL) have been angitis. For the same reason, serum levels of CA19-9 are less associated with metastatic iCCA, so this assay might be specific in detecting pCCA than iCCA. IgG4 disease can used in disease staging rather than diagnosis.134 Mixed present in a similar manner, so its presence should be tumors are characterized by histologic and imaging fea- excluded by evaluation for serum levels of IgG4.2 tures of HCC and iCCA. In these cases, immunohisto- In addition to MRI and CT, magnetic resonance chol- chemical analysis for cytokeratins 7 and 19 can be angiopancreatography, endoscopic retrograde cholangi- useful—tumors positive for cytokeratins can be considered ography (ERC), and endoscopic ultrasound are used in the to be mixed hepatocellular CCA.17,135 Adefinitive diag- diagnosis of pCCA (Figure 4). Of these, MRI plus mag- nosis of iCCA requires liver biopsy analysis. According to netic resonance cholangiopancreatography is the preferred the World Health Organization classification criteria, imaging modality because it can assess resectability and iCCAs can be adenocarcinomas or mucinous carcinomas.2 tumor extent with an accuracy of up to 95%.2 Endoscopic The treatment of choice for iCCA is surgical resection. ultrasound aids in evaluation for the presence of regional Patients should undergo surgery only if they have lymphadenopathy and omental metastasis via fine-needle potentially resectable tumors and are appropriate surgical aspiration. However, fine-needle aspiration should not 1224 RIZVI AND GORES GASTROENTEROLOGY Vol. 145, No. 6 PERSPECTIVES REVIEWS AND

Figure 4. Diagnostic modalities used for cholangiocarcinoma. (A)MRIofapCCAmass(out- lined in circle). (B) CT image of a pCCA mass with right portal vein encasement (black arrow). (C) Magnetic resonance cholangiopancreatography image of common hepatic duct involvement by tumor (white arrow). (D) ERC image showing excluded segmental ducts (white arrows) in a patient with a hilar biliary stricture extending into the right main hepatic duct.

be performed on the primary tumor because it can account vascular encasement and parenchymal value of disseminate the tumor.143 ERC serves a diagnostic and the potential remnant lobe.3 Surgical resection entails therapeutic purpose—it is used to assess and sample the lobar hepatic and bile duct resection, regional lymphade- biliary tree via brush cytology and endoscopic biopsy, as nectomy, and Roux-en-Y hepaticojejunostomy. Potential well as dilatation and stent placement in cases of biliary contraindications to curative surgical resection include obstruction. contralateral or bilateral vascular encasement and pCCA Fluorescence in situ hybridization (FISH) analysis in- extension bilaterally to the level of the secondary biliary creases the sensitivity of cytology in diagnosing pCCA.144 branches. The presence of regional lymphadenopathy does FISH can detect polysomy or amplification of at least 2 chromosomes: tetrasomy and trisomy 7. Of these, polysomy in the presence of a dominant stricture is considered sufficient for the diagnosis of pCCA, especially if the polysomy can be confirmed over time.145 Tetrasomy can be seen during the M phase of mitosis and should be interpreted with caution.5 Trisomy 7 often is observed with inflammation of the biliary tree. Detection of polysomy by FISH also has been shown to predict the development of malignancies in patients with PSC with no mass and equivocal cytology. In a recent study, patients with PSC who had polysomy and levels of CA19-9 greater than 129 U/mL all went on to develop cancer, mainly within 2 years (Figure 5).146 The only curative options for pCCA are surgical resection and neoadjuvant chemoradiation followed by liver transplantation. The Bismuth–Corlette staging classification is based on the anatomic location of the CCA within Figure 5. Time to diagnosis of a cholangiocarcinoma based on FISH the biliary tree and is meant to help guide decision mak- analysis and CA19-9 levels. Reprinted with permission from Wiley ing. Recently, this classification was expanded to take into InterScience and Barr et al.146 December 2013 CHOLANGIOCARCINOMA 1225

Table 2. Criteria for Liver Transplantation in pCCA Diagnosis is made on the basis of the presence of a Diagnosis of cholangiocarcinoma dominant stricture and positive cytology and/or detection 2 Positive transluminal biopsy of polysomy by FISH. Surgical treatment of dCCA typi-

Positive biliary brush cytology cally entails a Whipple procedure. Only 27% of patients AND Malignant-appearing stricture on ERC with a CA 19-9 level > 100 U/mL survive for 5 years after surgical resection that attains REVIEWS PERSPECTIVES and/or FISH polysomy negative margins.4 The role of neoadjuvant chemo- Mass lesion on cross-sectional imaging and malignant-appearing stricture on ERC/MRCP radiation is limited. For patients who are not candidates 17 Tumor size for surgical resection, chemotherapy may be considered. Radial tumor diameter of 3cm Tumor confined to biliary tree Absence of intrahepatic or extrahepatic metastasis Future Directions Unresectability Treatment options for CCA are limited and overall Unresectable hilar tumor (above the cystic duct) survival rates are low. Earlier detection of CCA increases CCA in a PSC patient (owing to skip lesions, the field defect, and parenchymal liver disease) the chance of having curative treatment options. However, despite recent advances in diagnosis, such as improved MRCP, magnetic resonance cholangiopancreatography. imaging and cytology techniques, including FISH, further work is necessary to overcome the challenge of diagnosing not necessarily preclude surgery.147 Occasionally, a tumor CCA at an earlier stage. CCA often still is diagnosed based may be resectable but the remnant lobe has limited vol- on clinical criteria, such as a malignant-appearing bile ume. In such cases, resectability can be achieved by pre- duct stricture, increased serum levels of CA19-9, appear- operative relief of biliary obstruction and portal vein ance of a mass during MRI, normal serum levels of IgG4 embolization of the affected lobe with resultant compen- level, and so forth. satory hyperplasia of the contralateral unaffected liver There are significant geographic and ethnic variations lobe.147 Rates of 5-year survival after surgical resection in the incidence of CCA, so genetic factors are likely to with negative margins range from 11% to 41%.147 contribute to its pathogenesis. Inflammatory and onco- With the advent of new liver transplantation protocols, genic signaling pathways also are involved in chol- neoadjuvant chemoradiation followed by transplantation angiocarcinogenesis, and are potential therapeutic targets. has become an appealing option for patients selected care- Further studies are necessary to elucidate the role of ge- fully using stringent criteria (Table 2). Sixty-five percent of netic aberrations, particularly in regions encoding key patients who were treated with neoadjuvant therapy followed components of signaling pathways. In addition, the role of by liver transplantation at 12 large-volume transplant centers miRs as biomarkers remains to be fully elucidated. CCAs survived for 5 years.148 Rigorous selection is imperative for are heterogeneous; treatments are likely to be designed successful outcomes. Eligibility criteria include radial diam- based on features of each individual tumor.150 Potential eter of tumor of less than 3 cm, absence of intrahepatic or therapeutic targets could include the MET tyrosine re- extrahepatic metastasis, and, in the case of patients without ceptor kinase, FGFR2, the PI3K–Akt–mTOR pathway, and PSC, unresectability.149 Because of the presence of paren- IDH mutations. Molecular profiling of tumors, to identify chymal liver disease, patients with PSC typically require liver their specific mutations, could make it possible to offer transplantation rather than surgical resection. targeted therapies in personalized treatments (Figure 2B). For patients who are not candidates for surgical resection Although cancer cells contain many genetic and functional or liver transplantation, systemic chemotherapy with gem- aberrations, the tumor stroma appears to be more uniform citabine and cisplatin is recommended. 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