Cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is an epithelial cancer originating from the bile ducts with features of cholangiocyte differentiation.1 CCA is the second most common primary hepatic malignancy, and epidemiologic studies suggest its incidence is increasing in Western countries.2 Advanced CCA has a devastating prognosis, with a median survival of <24 months.3 The only curative therapy is surgical extirpation or liver transplantation, but unfortunately the majority of patients present with advanced stage disease, which is not amenable to surgical therapies. Anatomically, CCA is classified into extrahepatic and intrahepatic forms of the disease. The extrahepatic form is more common, accounting for 80% to 90% of CCAs. It is further divided into proximal or perihilar and distal subsets depending on the location of the cancer within the extrahepatic biliary system. Perihilar disease is also frequently referred to as a Klatskin tumor. Three different growth patterns of extrahepatic CCA can be observed: (1) periductal infiltrating, (2) papillary or intraductal, and (3) mass forming.4 Intrahepatic CCA typically presents as an intrahepatic mass. In addition to their distinct morphology and clinical presentations, intrahepatic and extrahepatic CCAs differ in etiopathogenesis, molecular signatures, and management. In the last several years there have been significant new insights into the molecular pathogenesis of CCA. New diagnostic and therapeutic modalities have also been developed, resulting in improved detection rates and outcomes. In addition, we have now entered the era of targeted therapies for human cancers. Therefore, it is timely and topical to review these advances with a focus on promising targeted therapies for this disease. An additional goal is to stimulate further interest in this disease with the hope of improving outcomes for this still highly lethal malignancy. Hepatobiliary malignancies account for 13% of the 7.6 million annual cancer-related deaths worldwide and for 3% of the 560,000 annual cancer-related deaths in the United States. CCA accounts for 10% to 20% of the deaths from hepatobiliary malignancies. The prevalence of CCA shows a wide geographic variability, with the highest rates in Asia and the lowest in Australia.5 In the United States, the incidence of CCA has been reported to be 0.95/100,000 for intrahepatic forms and 0.82/100,000 for extrahepatic forms of the disease.5 Its prevalence in different racial and ethnic groups is heterogeneously distributed, with the highest age-adjusted prevalence in Hispanics (1.22/100,000) and the lowest in African Americans (0.17-0.5/100,000).6 In the last 4 decades, United States incidence rates of intrahepatic CCA have increased by 165%, whereas the extrahepatic CCA incidence has remained stable.7, 8 The significant increase in age-adjusted incidence of intrahepatic CCA was confirmed after correction for a prior misclassification of hilar CCA as intrahepatic CCA.2 Similarly, increasing incidence rates of intrahepatic CCA have also been reported in Western Europe and Japan.9, 10 The cause for the increasing incidence has not been identified. We speculate that increased lipid mediators such as oxysterols may contribute to the current increased incidence in Western societies.11 In Western nations, the median age at presentation is >65 years, and it is only rarely diagnosed in patients <40 years of age except in patients with primary sclerosing cholangitis (PSC).5 There is a slight male predominance for CCA. CCA, cholangiocarcinoma; CT, computed tomography; DIA, digital image analysis; EGFR, epidermal growth factor receptor; ERCP, endoscopic retrograde cholangiopancreatography; FISH, fluorescence in situ hybridization; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MRCP, magnetic resonance cholangiopancreatography; MRI, magnetic resonance imaging; PDT, photodynamic therapy; PET, positron emission tomography; PSC, primary sclerosing cholangitis; PTC, percutaneous transhepatic cholangiography; RNOS, reactive nitrogen oxide species; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. In the majority of cases, the etiology of CCA remains obscure. However, several conditions associated with inflammation and cholestasis have been identified as risk factors for CCA (Table 1). PSC is a common risk factor. The prevalence of CCA in this condition is 5% to 15%, and the annual incidence rate is 0.6% to 1.5%.12, 13 The majority of PSC patients who develop CCA do so within the first 2.5 years following the diagnosis of PSC.12, 13 Thus, the symptomatic patient who presents with their first diagnosis of PSC should be carefully screened for CCA. Hepatobiliary flukes—especially the species Opisthorchis viverrini and Clonorchis sinenesis—are risk factors for CCA.14 They are endemic in portions of East Asia, where ingesting undercooked fish is common. Several case–control studies as well as animal models have confirmed the correlation between liver fluke infection and CCA.15-17 Another risk factor more commonly found in Asia than in Western countries is hepatolithiasis, for which an incidence rate of 10% for CCA has been described.18-20 Biliary malformations such as Caroli's disease and choledochal cysts carry a 10% to 15% risk for developing CCA.21-23 Hepatitis C and cirrhosis have also been reported as possible risk factors for CCA.24 Biliary–enteric drainage procedures are associated with CCA in the presence of recurrent cholangitis.25, 26 Finally, compounds such as thorotrast and dioxins have been correlated with an increased risk for CCA.27 Although most patients have no identifiable overt risk factors for CCA, it remains possible that subclinical biliary tract inflammation underlies the pathogenesis of CCA in most patients. CCA likely results from malignant transformation of cholangiocytes, although transformation of epithelial cells within peribiliary glands and/or biliary stem cells may also contribute to its development. There is also evidence that subsets of CCA and mixed hepatocellular carcinoma/CCA originate from hepatic stem/progenitor cells.28, 29 Etiologic and experimental evidence implicates inflammation and cholestasis as key factors in the pathogenesis of CCA. They create an environment that promotes damage in DNA mismatch repair genes/proteins, proto-oncogenes, and tumor suppressor genes.30 Cytokines, growth factors, and bile acids, found in increased concentrations in inflammation and cholestasis, contribute to these molecular changes and augment the growth and survival of altered cells. Cytokines stimulate expression of inducible nitric oxide synthase (iNOS) expression in epithelial cells, and iNOS up-regulation is present in inflammatory cholangiopathies and CCA.31 Increased iNOS activity results in generation of nitric oxide and reactive nitrogen oxide species (RNOS) known to interact with cellular DNA and proteins. The interaction between RNOS and the cellular genome results in mutations and DNA strand breaks. Mutagenesis is further promoted by interaction between nitric oxide and RNOS with DNA repair enzymes such as human 8-oxoguanine glycosylase, which is directly inactivated by S-nitrosylation of its active site cysteine residues.32 A variety of oncogenic mutations have been identified in human CCA tissues. Their frequency depends on tumor stage, tumor type, anatomical location, etiology, and ethnic population. Although dysregulation of the proto-oncogene k-ras and the tumor suppressor gene p53 is commonly observed in malignancies, mutations of k-ras have only been described in 20% to 54% of intrahepatic CCA. This is in sharp distinction to pancreatic ductal carcinoma where k-ras mutations are present in >90% of cancers.33, 34 Thus, despite shared developmental ontology between the pancreatic ducts and the biliary tree, their adult cancers are different. Nuclear accumulation of p53 and up-regulation of the related protein mdm-2 and WAF-1 have been reported in 21.7% to 76% of CCAs.35-42 Other inactivated tumor suppressor genes include p16INK4a, DPC4/Smad4, and APC.43-45 Correlation between these markers and prognosis varies among studies. Other dysregulated genes/factors involved in cell cycle regulation and found in CCA are listed in Table 2. The majority of these genetic changes were described in intrahepatic CCA. Given the paucicellular, desmoplastic nature of extrahepatic bile ducts, genetic analysis of these tumors will require careful laser capture microdissection of the CCA cellular elements—a tedious process that has seldom been applied to this tumor. Interleukin-6 (IL-6) appears to be a critical signaling molecule in the pathogenesis of human cancers.46 For example, IL-6 has recently been reported to promote cancer stem cell survival in human breast cancer by up-regulating expression of the stem cell survival regulator Notch-3.47 In human lung cancer, epidermal growth factor receptor (EGFR)-activating mutations enhance IL-6 expression, promoting its autocrine/paracrine growth-promoting and survival properties.48 Thus, IL-6 can be upstream or downstream of other potent oncogenes. IL-6 is also a key cytokine in the pathogenesis of CCA. It is a known mitogen, and its proliferative effect has been confirmed in CCA.49 IL-6 is produced at high levels by CCA cells, and elevated IL-6 serum concentrations have been reported in CCA patients.50, 51 IL-6 secretion by CCA cells is further enhanced by other inflammatory cytokines.52 In addition to autocrine and paracrine IL-6 stimulation, CCA cells overexpress the IL-6 receptor subunit gp130.51 The usual negative feedback regulation of IL-6 signaling is blocked by epigenetic silencing of suppressors of cytokine signaling 3 (SOCS-3).53 Uninhibited IL-6 stimulation results in up-regulation of the antiapoptotic Bcl-2 protein Mcl-1, rendering CCA resistant to cytotoxic therapies.53-55 IL-6 has also been shown to increase telomerase activity in CCA resulting in inhibition of telomere shortening and thereby evasion of cell senescence.56-61 In CCA cells, IL-6 activates p44/p42 and p38 mitogen-activated protein kinases (MAPKs), both shown to be critical for CCA cell proliferation.52 Activated p38 MAPK decreases cyclin-dependent kinase inhibitor p21WAF1/CIP1, a known negative cell cycle regulator.62 There is also cross-communication between IL-6 and other pathways (for example, IL-6–mediated overexpression of EGFR).63 Mechanisms of IL-6 signaling in human CCA are depicted in Fig. 1. IL-6 signaling and therapeutic targets in CCA. A schematic overview of IL-6 signaling and its downstream effectors as well as examples of potential therapeutic interventions is depicted. IL-6 binds to its receptor, which consists of the common receptor unit gp130 and the IL-6–specific subunit gp80. Activation of this receptor complex results in downstream activation of the JAK/STAT, PI3K/Akt, and MAPK signaling pathways. Activation of the JAK/STAT pathway results in nuclear translocation of phosphorylated STAT3 and transcriptional up-regulation of target genes, including the antiapoptotic Mcl-1. In addition, IL-6 increases EGFR expression by decreasing its promoter methylation. Activation of phosphoinositide 3-kinase (PI3K) results in phosphorylation and activation of Akt kinase, which in turn inhibits proapoptotic factors and facilitates cell growth. Activation of p38 MAPK decreases negative cell cycle regulators and is critical for CCA proliferation. Inhibitors of IL-6 receptor, Akt, MAPK, or JAK signaling, as well as bcl-2 protein inhibitors, have the potential of inhibiting these pathways. Receptor tyrosine kinases, which can be targeted pharmaceutically, are overexpressed in many cancers and modulate cancer biology. For example, inhibition of EGFR signaling has been shown to significantly suppress CCA cell growth.64 EGFR can directly be activated by bile acids and promote CCA cell proliferation, a potential explanation for the tropism exerted by CCA for the biliary tree.65, 66 EGFR activation is sustained in CCA by failure to internalize the ligand–receptor complex, a homeostatic mechanism essential for receptor inactivation.64 EGFR phosphorylation results in activation of the downstream kinases p42/44 MAPK and p38 MAPK, which in turn increase cyclooxygenase 2 (COX-2) expression in CCA cells.66 COX-2 plays an important role in CCA carcinogenesis through inhibition of apoptosis and growth stimulation.67-72 Additional induction of COX-2 is mediated by bile acids, oxysterols, and iNOS.11, 66, 70 Other COX-2–inducing molecules include the tyrosine kinase ErbB-2, which is overexpressed in CCA and involved in CCA carcinogenesis and progression.73, 74 It is an EGFR homologue and is able to homodimerize or heterodimerize with other members of the EGF superfamily, resulting in activation or the Raf/MAPK-pathway. Also, hepatocyte growth factor and its receptor c-met are frequently overexpressed in CCA.51, 74, 75 Hepatocyte growth factor is mitogenic, and its increased secretion by CCA cells together with the overexpression of its receptor represents an autocrine mechanism for sustained growth stimulation by CCA.76 In addition to the enhancement of these growth-promoting pathways, loss of growth inhibition has been demonstrated in CCA. Response to transforming growth factor-β1 is aberrant in CCA, resulting in increased proliferative rates. In the presence of IL-6, CCA cells are also resistant to activin-mediated growth inhibition.51 In summary, there is a complex net of different factors and pathways involved in CCA development, growth, and propagation. In the majority of cases, CCA is clinically silent, with symptoms only developing at an advanced stage. Once symptomatic, the clinical presentation depends on tumor location and growth pattern. Ninety percent of patients with extrahepatic ductal CCA present with painless jaundice, and 10% of patients present with cholangitis.77, 78 Unilobar biliary obstruction with ipsilateral vascular encasement results in atrophy of the affected lobe and hypertrophy of the unaffected lobe.79 Upon physical examination, this "atrophy–hypertrophy complex" phenomenon presents as palpable prominence of one hepatic lobe. Intrahepatic mass-forming CCA presents with symptoms typical for hepatic masses, including abdominal pain, malaise, night sweats, and cachexia. The tumor markers CA-125 and CEA can be elevated in CCA; however, they are nonspecific and can be increased in other gastrointestinal or gynecologic malignancies or cholangiopathies.80 CA 19-9 is the most commonly used tumor marker for CCA.81 Its sensitivity and specificity for detection of CCA in PSC are 79% and 98%, respectively, at a cutoff value of 129 U/mL. Other investigators have identified a higher cutoff of >180 U/mL to achieve this degree of specificity.82 A change from baseline of >63 U/L has a sensitivity of 90% and specificity of 98% for CCA.83 In patients without PSC, its sensitivity is 53% at a cutoff of >100 U/L and its negative predictive value is 76% to 92%.84 CA 19-9 can also be elevated in bacterial cholangitis and other gastrointestinal and gynecologic neoplasias; patients lacking the blood type Lewis antigen (10% of individuals) do not produce this tumor marker.85-88 Ultrasound and computed tomography (CT) are only of limited value for detection of intrahepatic and extrahepatic CCA due to their low sensitivity and specificity, as well as their low accuracy in estimating tumor extent of intrahepatic and extrahepatic CCA.78, 89-91 Their main role in CCA is detection of bile duct obstruction, vascular compression or encasement, tumor staging, and preoperative planning. For evaluation of tumor location and intraductal extent, cholangiography is the most important diagnostic modality, especially for extrahepatic CCA.92 Endoscopic retrograde cholangiopancreatography (ERCP), magnetic resonance cholangiopancreatography (MRCP), or percutaneous transhepatic cholangiography (PTC) can be used for this purpose.78 ERCP and PTC allow therapeutic interventions (for example, placement of biliary stents) as well as collection of tissue samples for pathologic and cytologic analysis. MRCP/magnetic resonance imaging (MRI) provides information about intrahepatic location and tumor dimensions of intrahepatic CCA, ductal as well as periductal tumor extent of extrahepatic CCA, vascular involvement, and metastases (Fig. 2). Its sensitivity and imaging quality of tumor tissue can be increased significantly with ferumoxide enhancement.93, 94 The most sensitive method for evaluation of regional lymphadenopathy is endosonography. Biopsy of lymph nodes via fine needle aspiration for further pathologic analysis can also be performed during the endosonographic procedure.95 However, biopsy of hilar lesions during is it can in tumor In cases, of a diagnosis can be with positron emission tomography with and specificity of in the of primary lesions has been reported as and 80% for intrahepatic CCA and and for extrahepatic For regional lymph the sensitivity of was and the specificity was have been reported in the of A that in patients can change and is in of CCA. A of the liver with ferumoxide of a patient with hilar CCA is depicted. The into the the biliary tumor. The pathologic diagnosis of CCA can be due to the desmoplastic nature of the periductal form of CCA. a cellular diagnosis of CCA is by common reactive changes in PSC, resulting in highly sensitivity and specificity of this in this patient population. a may increase the diagnostic of with a Also, of bile growth factor can malignant from The of digital image analysis and fluorescence in situ have significantly increased the diagnostic of on the of The sensitivity and specificity of for extrahepatic CCA are and with and 98% with In PSC the sensitivity and specificity of for CCA are and has a sensitivity of and a specificity of for detection of CCA in However, analysis is and subsets of (1) (2) or of and (3) or of at can in inflammatory of the biliary tree, especially This is a of EGFR, which is also on this is We do not to be diagnostic of CCA, although it likely the patient at risk for the of CCA. EGFR can this or CCA in PSC patients remains to be be with high rates will during the of the cell remains diagnostic of cancer in the clinical (for example, a biliary diagnostic modalities such as intraductal and imaging have However, they are not of the diagnostic and should be for in which other have to CCA but the of is In the role of will to be and diagnosis will still the In summary, the diagnosis of CCA is and should be in a that clinical and as well as and pathologic analysis. A diagnostic for ductal CCA is in Fig. evaluation of hilar CCA. In patients with clinical of hilar CCA, CA 19-9 serum endoscopic retrograde and as well as molecular cytologic analysis of biliary of should be In where these are is In in which a is 19-9 serum levels are are for carcinoma and/or is for CCA should be In cases, of the liver with ferumoxide should be a mass and/or vascular encasement are of CCA should be the is negative but there is significant for CCA, can be are further should be CCA. the is careful is In with a negative and for CCA, patients can be CCA has been classified the on (Table This is a pathologic and surgical of the An should information about disease extent, vascular involvement, and metastases without the patient to surgical It should also into account and age and with clinical outcomes. There is an for such a in hilar CCA. a of patients for clinical is survival in patients with hilar CCA depends on tumor with negative tumor Therefore, evaluation for of these tumors a including of biliary disease extent, vascular encasement, and hepatic atrophy in addition to the information by a clinical system. a has been by (Table of curative or to lymph and survival correlated with the tumor stage of this is the only curative therapy for CCA and is the of intrahepatic CCAs are by or survival rates are to and with of lymph and vascular rates after surgical of intrahepatic as well as extrahepatic CCA have significantly improved in the last a more careful patient thereby higher rates of is also the of for extrahepatic CCA in the of However, should only be with curative there is no significant survival of or with patients not for surgical of hilar CCA are in Table lymph metastases are not an to surgical they do not significantly outcomes in hilar survival rates after for hilar CCA are to and for distal extrahepatic CCA are to However, rates are rates may be higher for however, this is not for cancers originating from or with significant of the hepatic In PSC, outcomes of surgical are by advanced liver disease in the majority of these recurrent cholangitis with a the nature of the cancer, and their increased risk for further CCA. a is a risk factor for 26 a in a PSC patient should be with and the potential of additional CCA should be with PSC CCA may be as potential liver following of hilar CCA is to and is 5% to 10% at Several have been for their potential to increase including preoperative The goal of is to of the lobe increasing the of the liver following an This increased in patients with hilar CCA and liver and for extrahepatic and photodynamic be studies to significant or were or to of liver for intrahepatic CCA are with survival rates of to and be of liver for extrahepatic CCA were with survival rates of to However, the of new liver for extrahepatic CCA at the and the of highly promising on their as well as on analysis of outcomes and correlated risk factors, have been (Table and has been to its current This therapy with therapy with by and with to transplantation, patients for analysis of patients to the has and survival rates of and for tumor include CA 19-9 >100 U/mL on the of transplantation, prior mass on tumor in tumor and in For highly patients with perihilar CCA, CCA, and CCA on PSC, liver can be curative and is the of CCA significant related to cholestasis and its abdominal pain, and bacterial Therefore, therapies are important in the of this disease. Endoscopic placement is as as surgical or for of biliary drainage and of hepatic duct placement has been shown to be to hepatic duct for biliary have higher rates and are more for with an survival of require 2 to 3 due to or cholangitis and are in patients who are for surgical or is In where endoscopic placement is not PTC can be for biliary therapy and therapy have been as therapies. results have been reported with therapy in a on the of this is also associated with significant including gastrointestinal biliary obstruction, and hepatic Therefore, therapy be for or therapy of intrahepatic and extrahepatic CCA. In PDT, a as is by at a to the of the resulting in reactive cell and can cholestasis and quality of rates are low and include to and Several studies including a survival with in hilar In summary, is a and for of hilar as an have been a for in this There are no curative therapies for CCA. The most are and the was for CCA in by the and have been in with a variety of other including and However, of the studies was and most studies were on or demonstrated rates. In there is no a survival for a the of targeted it is that survival is a more than rates. For example, rates with for cell carcinoma are although is Given this will to have a are The of the molecular pathogenesis of CCA new therapeutic for molecular The majority of these target antiapoptotic and pathways. The of is able to expression, thereby protein expression of the bcl-2 protein and CCA cells to tumor necrosis factor–related apoptosis-inducing Other for inhibiting expression include the of the inhibitor and overexpression of molecules such as are also in to of CCA cells to was also through the of a inhibitor to kinase have been used to target EGFR signaling and tumor cell of IL-6 pathways by or MAPK results in growth inhibition of human CCA cell Other inhibitors, hepatocyte growth factor and telomerase growth inhibition and/or induction or to apoptosis in examples the promising potential of these new therapeutic However, the majority of these studies were in the in results are the majority of in studies have on tumor inhibition than on of in models of CCA will be to targeted In evaluation of therapeutic compounds is an essential in the of therapies. Several have been described for However, from these only with clinical resulting in an increasing the of in cancer hepatobiliary CCA models were to and which develop tumors after with or infection with several new genetic CCA models have been of the tumor suppressor genes results in of CCA in Another of intrahepatic mass-forming CCA is by with and models of CCA in which malignant transformation of by biliary of these cells in CCA in to of In summary, new models of CCA have been that human CCA in many The majority of these models intrahepatic CCA, however, and genetic models of hilar CCA still to be The of CCA is not There is evidence that expression of genes as well as up-regulation of antiapoptotic bcl-2 are involved in the rates of CCA to New targeted therapies may these Several essential pathways in CCA are by the and and are in clinical for other cancer include EGFR and and and vascular growth inhibition and The of several clinical the COX-2 inhibitor and the receptor tyrosine kinase and as or in with other in Other potential that have not been include inhibiting or antiapoptotic inhibitor the of and in and enhanced the tumor potential in a CCA Another to apoptosis by of (for example, by with are a but they the for molecular pathways in human CCA. have been in the diagnosis and of CCA. the clinical advanced cytologic for the diagnosis of CCA have increased the of for the diagnosis of this surgical and liver have advanced the surgical of this disease, and has as an important The of in animal models for CCA will more of targeted therapies for this disease. A clinical is prior to the of Although on CCA has been more is to and this disease.