Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis Redha Al-Bahrani1, Yasser Abuetabh1, Nikolas Zeitouni1, and Consolato Sergi1,2

Available online at www.annclinlabsci.org

Annals of Clinical & Laboratory Science, vol. 43, no. 2, 2013 195 Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis Redha Al-Bahrani1*, Yasser Abuetabh1*, Nikolas Zeitouni1, and Consolato Sergi1,2

1Department of Laboratory Medicine and Pathology, University of Alberta; 2Stollery Children’s Hospital, University of Alberta Hospital, Edmonton, Alberta, Canada

Abstract. Cholangiocarcinoma (CCA) is one of the most frequent malignant epithelial liver tumors after hepatocellular carcinoma (HCC). Its incidence seems to be increasing worldwide, although risk factors are heterogeneous and differ globally. Although diagnostic and therapeutic medicine have advanced in several countries, tackling this tumor remains a challenge. The causes of CCA’s increasing incidence are likely a differential increment of some factors according to the geographical area, which will be considered in this review. Environment-linked risk factors may play a critical role in the carcinogenesis. Liver flukes may play a major role in East Asia, while exposure to chemical compounds, such as naphthenic acids, has been postulated as a source of the rate increase in Western countries. Carcinogenesis is variable and confounding factors also need to be taken into account. Carcinogenesis depends on a sequential process and most probably involves both cholestasis and chronic inflammation as promoting steps after induction. The release and interaction of interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), and platelet-derived growth factor (PDGF) are at the basis of the proliferation of biliary epithelial cells or cholangiocytes. Additional steps for the final development of CCA may also involve an increase of the mutation rate of tumor suppressor genes, such as TP53, and the evasion of apoptosis.

Introduction To understand the relationship between environ- Cholangiocarcinoma (CCA) is a malignant neo- ment and tumor, it is important to consider habi- plasm of the biliary system of the liver, which can tat. The environment in which an animal lives is be localized in the extra- and/or intrahepatic biliary referred to as its habitat, which includes both biotic system. Unlike lung and colon carcinomas, CCA (living) and abiotic (non-living) components. The has not been extensively investigated, possibly be- abiotic category includes a huge range of features, cause of its presumptive rarity and worrisome prog- such as temperature, humidity and oxygen, while nosis at time of diagnosis. Lifestyle-associated some biotic components are predators, competi- changes and environmental pollutants have been tors, and individuals of the same species. Each spe- associated with the rise of neoplastic diseases. Thus, cies lives within a certain environmental ‘niche’, a review of carcinogenesis factors is an important including the substratum, temperature, and hydro- object of investigation in order to lay a foundation dynamic conditions which maintain the habitat. for studies involving several interacting molecular Factors disturbing the balance of this ‘niche’, such pathways. CCA has been described as a “silent kill- as environmental changes, may have dramatic con- er” due to its relatively silent clinical progression sequences. Both temporal and spatial domains may and the consequent difficulty in diagnosis before an have a major impact. These influences clearly result advanced stage [1, 2]. CCA is the second most in interactions between species. In some geographic common malignant liver tumor after hepatocellular areas of East Asia (Japan, Korea, Cambodia, and carcinoma (HCC) worldwide. Northeast Thailand), CCA seems to be mainly liver fluke-related, establishing a peculiar connection be- *These two authors contributed equally to this work. Address tween humans and worms. CCA was contained for correspondence to Consolato Maria Sergi, MD, PhD; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, a long period, but its rate now seems to be increas- Alberta, Canada, 8440-112 Street, Edmonton, T6G 2B7, Alberta, ing. Additional factors (endemic and/or epidemic) Canada; phone: 780 407 7951; fax: 780 407 3009; e mail: sergi@ ualberta.ca may be at the basis to this increase. CCA represents

Figure 1. (a) Gross photograph of an intrahepatic cholangiocarcinoma showing almost diffuse involvement of the liver. No evidence of cirrhosis in the non-tumorous hepatic tissue. (b) Microphotograph of an intrahepatic cholangiocarcinoma with atypical proliferating glands and prominent desmoplasia (Hematoxylin-Eosin staining, original magnification x 100). (c) Microphotograph of another intrahepatic cholangiocarcinoma with atypical proliferating glands in a nodular shape (Hematoxylin-Eosin staining, original magnification x 40). (d) Microphotograph of a hilar cholangiocarcinoma or Klatsckin tumor (Hematoxylin-Eosin staining, original magnification x 100). (e) Microphotograph of a intrahepatic cholangiocarcinoma in a liver needle biopsy showing cytokeratin or keratin 7 expression (Anti-CK 7 monoconal antibody immunohistochemistry, Avidin-Biotin Complex detection method, original magnification x 100). (f) Microphotograph of a intrahepatic cholangiocarcinoma in a liver needle biopsy showing cytokeratin or keratin 19 expression (Anti-CK 19 monoconal antibody immunohistochemistry, Avidin-Biotin Complex detection method, original magnification x 100). a serious complication of primary sclerosing chol- Extrahepatic CCA is further classified into three angitis (PSC), which is a devastating chronic in- types according to the location of the tumor with flammation of the biliary system, with possibility of respect to the hilum of the liver: perihilar tumor recurrence after liver transplantation [1, 2]. Genetic (Klatskin tumor), middle, and distal extrahepatic factors may also play a critical role in the develop- [5-8]. Most bile duct cancers are adenocarcinomas. ment of CCA. Mutations in tumor suppressor Rare CCA variants include adenosquamous and genes (e.g. TP53) have also been found in CCA [3, squamous carcinoma, cholangiolocellular carcino- 4]. In this review, we focus on risk factors, environ- ma, mucinous carcinoma, signet-ring-cell carcino- mental factors, and the oncogenesis of this intrigu- ma, sarcomatous carcinoma, lymphoepithelioma- ing liver tumor. We illustrate the possible effect of like carcinoma, clear-cell carcinoma, these factors and how they may contribute to the mucoepidermoid carcinoma, bile duct cystoadeno- development of CCA, although their specific inter- carcinoma, and combined HCC-CCA [5] (Figure actions remain unknown. 1). Additional classifications of CCA introduce different categories, such as nodular, papillary, diffuse, Classification sclerosing, and infiltrating. However, no clear data on CCA prognosis seem to be available due to their In the World Health Organization (WHO), CCA rarity of diagnosis [9]. is classified as extra-hepatic or intrahepatic. Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis 197

Table 1. Known Risk Factors for CCA

Risk factors Category Geographic Region Age Symptoms References Prevalence

Opisthorchis Parasitic Thailand 40-60 Acute cases: Abdominal 14,17 viverrini infection pain, flatulence, fatigue. Chronic cases: hepatomegaly, cholangitis and jaundice. Hepato-lithiasis Gallstone Taiwan, Japan >40 Abdominal pain and 141,142 formation jaundice Anastomosis Surgical procedure ______50-70 ______50,52,53 Thorium dioxide Radioactive Germany, United States, >45 Loss of appetite, nausea, 54,62 Japan diarrhea, abdominal pain, jaundice, liver enlargement, and prolonged bleeding Clonorchis Parasitic Asia (Korea, Taiwan, 30-60 Loss of appetite, nausea, 23,24 sinensis infection Japan, China) diarrhea, abdominal pain, jaundice, and liver enlargement Plutonium Radioactive Russia _ 143

Hepatitis B virus Viral infection Worldwide _ Abdominal pain, dark urine, 45 Hepatitis C virus jaundice, vomiting, and weight loss Ascaris Parasitic India, Latin America, _ Abdominal pain, jaundice, 34,35 lumbricoides infection China and weight loss. Primary Chronic Worldwide 30-60 Abdominal pain, dark Sclerosis 1 Inflammation urine, jaundice, and weight Cholangitis loss.

Note: There is no substantial difference between genders. Since a risk has not been clarified or standardized yet, the table does not contemplate the abnormal differentiation and/or maturation of the biliary tract, including ductal plate malformation (see text for completeness).

Epidemiology lowest incidence of CCA is in Western countries, whereas the highest is still within Southeast Asia, CCA has been described as the second most com- where it is officially recognized as a major health mon primary hepatic cancer after HCC. Patients issue [10,14]. CCA has dramatically increased from tend to develop this neoplastic disease between the 1975-2000 in Western countries and its incidence 5th and 6th decades of life, although patients with has also been found to be very high in Asians who underlying disorders may be younger. This finding have emigrated to North America [9]. Two of the has recently been emphasized in pediatric gastroen- primary risk factors for CCA (Opisthorchis viverrini terology conferences and scientific journals related and Clonorchis sinensis) have been found to have in- to youth health. It seems that males are altogether creased in recent years among Asian immigrants in more prone to CCA than females [10, 11]. Montreal, Quebec, Canada and San Francisco, However, Welzel et al., who found no significant California, USA, where two large Chinese immi- differences [12], have debated this finding. Most grant communities have settled since the beginning likely, the incidence of CCA may vary between gen- of the 20th century [15]. These two epidemiological ders according to the geographical region [13]. The aspects involving major health issues in North 198 Annals of Clinical & Laboratory Science, vol. 43, no. 2, 2013

America may be considered as stimuli to emphasize finely chopped raw fish in spicy salad dressing [19]. and promote investigation of this uncommon neo- The consumption of Koi pla in communities in plasm. We reviewed scientific articles that were ob- Thailand accounts for up to 80% of weekly meals, tained from PubMed and Medline, as well as WHO and it is often made in traditional restaurants using reports and several internet databases between imported fish. Koi hoi, another variety of the dish, 1965/01 and 2012/06. The search was restricted to contains raw snail meat and has been associated English language articles. Table 1 summarizes epi- with infections in humans by the rat lungworm demiological data that we were able to collect. Angiostrongylus cantonensis, though not with O. viverrini. O. viverrini is a member of the Environmental factors Opisthorchiidae family and is endemic in Thailand (20); another member of this family is C. sinensis, Environmental factors can be described as a trigger which is endemic in Japan, southern China, Korea, in epidemiological studies and may be unrelated to and Taiwan [21-24]. The life cycle ofO. viverrini is an “a priori” abnormal genetic background. These illustrated in Figure 2. O. viverrini has two inter- factors may include physical stress, diet, or expo- mediate hosts, including the freshwater snail and sure to chemicals and radiation. However, CCA the freshwater fish. In both species the metacercari- due to such environmental factors may play a criti- ae are embedded inside the muscle of the fish. cal role in triggering the gain-of-function or loss-of- Mammals represent the final host for this parasite, heterozygosity of genes within the liver and/or bili- including humans eating the infected uncooked ary system [4]. Most cases have shown that the fish. Following ingestion, the metacercaria enter the incidence of CCA is strongly related to chronic in- duodenum and migrate through papilla of Vater flammation of the biliary epithelium as a result of into the common bile duct and then the intrahe- parasitic infections such as Opisthorchis viverrini patic biliary system. In this location, the metacer- and Clonorchis sinensis, which are probably the most cariae mature and may remain in situ for several common risk factors for CCA worldwide [16, 17]. years [25]. O. viverrini infection is traditionally Microscopic demonstration of C. sinensis in stools considered the primary cause of CCA. However, or in duodenal aspirate, and eggs of O. viverrini are Satoshi et al. argued that this pathogen may not be the most practical diagnostic methods, respectively. the single factor for the development of CCA. It Morphological comparison with other GI parasites has been suggested that a certain promoter other is usually performed. An antigen 89 kDa of O. vi- than the cholangitis itself must be present, such as verrini can also be detected by ELISA test and a smoking or alcohol consumption; both tobacco PCR test capable of amplifying a segment of the smoke and alcohol are rich in nitrosamine [26, 27]. internal transcribed spacer region of ribosomal Interestingly, the administration of nitrosamine to DNA for the fluke’s eggs may also be performed. an animal model infected with O. viverrini deter- The highest infection rate of O. viverrini in the mines the development of biliary cancer [28]. The world has been found in northwestern Thailand, finding in some populations of an abnormal genetic where patients with this infection have developed background may support this hypothesis. In fact, CCA as a ‘neoplastic’ parasitic complication [18]. different polymorphisms of the Glutathione Remarkably, O. viverrini is also associated with S-transferase Mu 1 (GSTM1) and the Glutathione non-neoplastic hepatobiliary diseases, including S-transferase (GST), which are active in carcinogen- cholangitis or inflammation of the biliary tract, ic detoxification, have been found in some individ- hepatomegaly, and biliary lithiasis. The main cause uals with CCA. In target populations, patients’ his- of this infection is the consumption of raw fish, tories have been investigated for heavy alcohol which contains the metacercariae [14]. Raw fish consumption, smoking, and type of water and food dishes are a traditional meal in East Asia and have intake. Heavy alcohol consumption, smoking, and become quite diffuse in other countries in the last type of water intake have been emphasized to be decade as well. Koi pla is the most popular dish in mainly responsible for the rate increase of CCA the Northeast Thailand and consists of minced or [26]. The development of CCA is also a major issue Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis 199 now. Since the first case of infection reported in 1911 in Chiang Mai [29], the prevalence of O. viverrini in Thailand showed a steady increase to over ten million of the Thai population [30]. A further type of parasitic infection has been associated with the development of CCA, namely Ascaris lumbricoides (A. lumbricoides), which is also one of the most common parasitic infection worldwide, affecting more than 1.5 billion people [31]. The adultA. lumbricoides normally lives in the small intestine for 6-10 months and then migrates to different regions in the body, including the lungs, uri- Figure 2. Life cycle of Opisthorics vivverrini: Humans are infected with the O. viver- nary bladder, and biliary rini by eating the raw fish which has the encysted resting or maturing stage of a tree [32,33]. Biliary asca- trematode parasite in the tissues of an intermediate host a metacercaria. The metacercariae enter into the duodenum and migrate to the biliary tree, where they mature riasis has the highest inci- locally. The eggs are discharged into the intestine by the biliary fluid and exit with the dence in China, India, stool. In the freshwater the eggs get eaten by freshwater snails where the eggs hatched and areas of South and develop to cercariae. The cercariae invade the cyprinid fish (second intermediate host) by penetrating its skin. Human is the final host where the metacercariae grow America [34,35]. and continue the cycle by exiting through stools. The encysted maturing stage of a Migration of the parasite trematode in its intermediate host prior to transfer to the definitive host, usually from the small intestine represents the organism's infectious stage. to the intrahepatic biliary system may cause serious complication such as pan- hepatitis). Hepatitis viruses can be classified into creatitis and cholangitis [36]. Most cases of biliary seven types (A, B, C, D, E, F, G) with variable risks ascariasis are reported during cholecystectomy or of acute, chronic, or fulminant infection [38-39]. post-mortem. Unlike O. viverrini, biliary ascariasis Among them, hepatitis B and C viruses are the seems to be more frequent in females than males most prevalent worldwide and can have a fatal out- [37]. Aflatoxin exposure, which is quite frequent in come in infected patients. Hepatitis B virus (HBV) Asia, and has been considered a co-factor for some is the most common viral illness, infecting millions liver cirrhosis, is not strongly associated with CCA. of people worldwide [40]. One of the most worrisome complications of HBV infection is hepatocel- Viral infection lular carcinoma (HCC). More than 80% of individuals worldwide develop HCC following HBV Hepatitis is a general term referring to liver inflam- infection [41]. Hepatitis C virus (HCV) is the sec- mation with activity present in the portal triads, ond most common viral health issue, with 3% of liver lobulus, or periportal areas (interface people infected worldwide according to the WHO 200 Annals of Clinical & Laboratory Science, vol. 43, no. 2, 2013

[42]. It has been reported that both hepatitis B and Choledocho-Enteric Anastomosis C viruses may be associated with biliary carcinogenesis, although their role is still unclear [43,44]. Iatrogenic, post-traumatic or primitive anastomo- Lee et al reported that 1.9% and 13.8% of patients sis, or general communication between the biliary suffering from CCA have positive results for hepa- tree and the intestinal tract, may be an additional titis C and hepatitis B infection, respectively, al- underlying condition predisposing certain individ- though the rate of HBV is significantly higher than uals to the development of neoplasms in the biliary that of HCV [45]. The incidence of CCA increased tract. The inflammation and abnormalities of the dramatically between the 1970s and 80s. This in- enteric luminal content may play a role in inducing crease seems to mirror rates revealed by epidemio- the neoplastic transformation of the biliary epithe- logical studies involving infection with hepato- lium. The reflux of the small intestine contents into tropic viruses and showing a correlation in particular the biliary tree may offer an environment favorable between chronic infection of HBV and intra-hepat- to carcinogenesis in the biliary epithelium [50-52]. ic CCA. Torbenson et al epidemiologically investi- Although Strong does not consider the anastomosis gated liver explants from 1995 through 2005 [46]. procedure per se a cause of CCA and argues that These authors considered factors such as patient environmental changes may constitute an impor- history involving alcohol consumption, hepato- tant underlying factor, the reflux hypothesis is still tropic virus infection, and non-viral infection. They fascinating, considering the chemical interaction concluded that 2% of 511 patients were infected involving several molecules present in the pancre- with HCV without alcohol consumption as addi- atic juice. After exposure to the exocrine pancreatic tional lifestyle confounding factor. Conversely, secretion, bacterial flora may cause chronic cholan- HCV with alcohol consumption was seen in 5% of gitis, which may be a pre-neoplastic condition for 112 cases, while no CCA was found in 67 cases in- the development of CCA [53]. fected with HBV (46). These findings are noteworthy, but worldwide, there is clearly still much to Chemicals and Radiations Exposure investigate in this direction. Thorium (Th) is a natural radioactive chemical ele- Gallstone formation ment, discovered in 1828 by the Swedish chemist Jons Jakob Berzelius and named after Thor (the Hepatolithiasis is the presence of gallstones (calculi) Norse god of thunder). In nature, Th is found as in the biliary system and is a common disease in thorium-232, which decays by emitting an alpha East Asia, especially in Taiwan, where it is consid- particle and has a half-life of many billions of years. ered an endemic disease by local health authorities. Th was initially used as light source in gas mantles Hepatolithiasis is, indeed, described as ‘oriental and as an alloying material, but these applications cholangiohepatitis’ due to the prevalence of this dis- declined soon after health concerns were raised. ease in East Asia [47]. Hepatolithiasis has become a Thorotrast is a 25% of colloid solution of dextrin serious issue in the East Asia, because of its co-oc- and thorium dioxide (ThO2) that was used mostly currence with CCA in some patients. Hepatolithiasis in Germany and in the United States of America in occurs in the setting of several factors including the 1930s and ‘40s as a versatile medical radiogra- parasitic infestation, which may become a con- phy contrast agent [54,55]. In this period, thorotrast founding factor, and/or bacterial infection that pro- was considered to be harmless to the body and was duces enzymes such as beta-glucuronidase, which a perfect contrast agent that could be injected both may play a role in the lithiasis process of the biliary intra-arterially and intravenously with no apparent system [48]. The incidence of hepatolithiasis is high side effects. A posteriori, Thorotrast appeared, un- in East Asia, but is also high in Western countries, fortunately, to be carcinogenic. In fact, it contains especially in individuals who have lived in the East 90% alpha particles and 9% beta particles as well as for some time. This has been considered an impor- 1% of gamma rays emission [56,57]. It is estimated tant factor for populations migrating to Western that 100,000 patients worldwide were injected with countries [49]. thorotrast for X-ray imaging between 1930 and Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis 201

1964, when it was definitively discontinued inflammation of the biliary system with progressive [58,59]. The dose that was used radiographically destruction and fibrosis of extra- and intrahepatic was essentially individual-dependent, and different bile ducts. Conversely, primary biliary cirrhosis tumors have been associated with thorotrast expo- (PBC) is a disease of the intrahepatic biliary system sure. Historically, the first victim of thorotrast-re- only [71]. Familial cases, and haplotypes, including lated neoplasm was reported in 1947 with the pa- HLA-B8, DR3, DRw52a, DR2, DR4, and TNF- tient suffering from angiosarcoma of the liver SNPs, have been associated with PBC, and 95% of [60,61]. The most common tumors associated with patients have at least one auto-antibody [72-74]. thorotrast toxicity are CCA, HCC, and angiosar- Remarkably, 7-40% of patients affected with PSC coma. It has been estimated that the period for the develop CCA along the course of this disease. development of hepatic neoplasm is between 15 Patients with PSC are more susceptible to develop- and 45 years. Cumulative doses of thorotrast in the ing CCA when they are 30-50 years old [1, 75-77]. liver possibly cause dysfunction of the hepatocytes The inflammation may play a major role in the pro- at the intracytoplasmic level (detoxification organ- motion of carcinoma, and the induction is likely elles), leading to oncogenesis [62]. more prone to occur in patients with an abnormal genetic background. Oil sands carcinogenicity Biliary System Development Abnormalities Some informal reports seem to point to an increase of CCA in Northern Alberta, Canada [http://envi- In the liver, the ductal plate is the protostructure of ronmentalgeographies.wordpress.com/tag/huse- the intrahepatic biliary system and consists of a fe- man-short-2012/]. Exposure to toxins from the oil tal double-layered cylinder of biliary-type cells with extraction process may increase the risk of develop- a slit-like lumen forming around the portal vein ing liver cancers such as CCA. In the Athabasca re- and its surrounding mesenchyme (so-called stage of gion, part of the province of Alberta, Canada, tar ductal plate of liver development). The remodeling sands are the primary source of oil. Oil is extracted of the ductal plate is characterized by two simulta- from the sands by the “hot alkaline water process”, neous phases. It includes the incorporation of a few which separates the oil from the sand but also cre- ductal plate cells into the mesenchyme surrounding ates tailing water, which is stored in ponds that can the portal vein to form the interlobular biliary potentially leak, contaminating rivers and lakes structures (interlobular bile ducts) as well as by the [63,64]. There are several toxins in the wastewater: disappearance of nonmigrating ductal plate cells phenols, cresol, and naphthenic acids, all toxic and (so-called stage of remodeling ductal plate and stage metabolized in liver cells [65]. The naphthenic ac- of remodeled bile ducts, when the biliary structures ids seem to be the most dangerous toxin, because of are finally remodeled). The development of intrahe- both high toxicity and potential carcinogenicity. patic bile ducts proceeds from the hilar to periph- Past studies using animal models have already re- eral portions. Two or more of these developmental vealed morbid effects on fish, amphibian, birds, stages may be present in the same liver specimen and mammals. Naphthenic acids cause inflamma- and this should be taken into account in evaluating tion by increasing factors such as CYP3A4, MT, the maturation of the intrahepatic biliary duct sys- DNA ligase, and growth factors, all of which are tem. The complete or partial persistence of the tumorigenic inducers [66-70]. At this point, how- primitive double-layered cylinder of biliary-type ever, there are no detailed studies investigating and cells in the developing liver gives rise to portal tracts measuring the effects of naphthenic acids on liver with an increased number of biliary duct structures. cells. Further studies are evidently warranted to ex- The term “ductal plate malformation of the liver” plore these issues. was coined to label this complex biliary plexus with an excess of primitive biliary structures. Some pa- Chronic Inflammation tients harboring a ductal malformation of the liver may develop CCA [78-80]. The patterns of keratin Primary Sclerosing Cholangitis (PSC) is a rare 7 (K7 or CK7) expressing biliary structures of liver chronic cholestatic liver disease causing diffuse biopsies of infants aged less than one year have been 202 Annals of Clinical & Laboratory Science, vol. 43, no. 2, 2013 investigated retrospectively and 1-year follow-up sometimes observed teenagers with biliary cancer studies have been added [81]. There are specific [83]. The overall incidence of CCA in patients with patterns in biliary atresia, neonatal hepatitis (a cat- untreated cysts is up to ¼ of individuals harboring egory of inflammation of the liver probably includ- this developmental anomaly [84,85]. The mecha- ing several conditions showing lobular disarray and nism of carcinogenesis is still unclear, but could be giant cell transformation of the hepatocytes as well related to biliary stasis, reflux of pancreatic juice as the presence of extramedullary hematopoiesis), causing chronic inflammation, activation of bile ac- and paucity of the intrahepatic bile duct system ids, and deconjugation of carcinogens [86]. Two (PIBD). Keratins are intermediate filaments of the pre-neoplastic conditions have also been found and cytoskeleton and ductal plate remnants have been include bile duct adenomas and biliary papilloma- demonstrated to be present, recapitulating the tosis, but the scarcity of detailed information is a primitive stages of the IBDS. The lack of intrahe- major obstacle to definitive statements. Strikingly, patic interlobular bile ducts in infants aged less single nucleotide polymorphisms of the bile salt than one year is an adverse prognostic factor, which transporter protein in BSEP, FIC1, and MDR3 was independent from the etiology of neonatal liver genes can lead to unstable bile content and to de- disease [81]. This data has been supported by the conjugation of xenobiotics, which are previously expression of polyductin or fibrocystin, the gene conjugated in the liver [87-89]. It is important to product of the autosomal recessive polycystic kid- emphasize that in addition to congenital bile duct ney disease [82]. Ductal plate malformation may be abnormalities, a genetic background can lead to the quite variable, but represents altogether a common development of the neoplasm at an early stage in way to show a disorder in the correct development life [90]. In fact, individuals who are heterozygous of the intrahepatic biliary system in which apopto- for bile salt transporter polymorphisms have an in- sis may play a major role [78]. There are many re- creased predisposition to CCA as adults. The inter- modeling bile ducts in surgically correctable chol- acting exposure to cofactors that result in chronic angiopathies. The ductular proliferation in infants inflammation of the biliary tree remains unknown. with biliary atresia (BA) is frequently observed. In fact, BA shows an ongoing bile duct destruction Weak Association Factors that takes place in the liver. BA affects the development of the intra- and extrahepatic biliary system Based on case reports, weak associated risk factors and results in the progressive fibrotic obstruction of for CCA include polychlorinated biphenyls (PCBs), the pre-formed bile ducts. The rapid advances in oral contraceptives, and cigarette smoking [91-93]. the understanding of the cellular and molecular These factors have been recognized to have a sub- physiology of bile secretion have led to better stantial and well-delineated role in other tumors. knowledge of the pathophysiology of cholangiopa- Particular mention should be made of polychlori- thy and structural cell damage caused by various nated biphenyl (PCB), which is one of the 209 con- hereditary and acquired cholestatic disorders. By figurations of organochlorides with two to ten chlo- studying the models of response to liver injury, a rine atoms attached to biphenyl, which is a molecule unimodal distribution of the developmental stages composed of two benzene rings. PCBs were widely was found, but long-term follow-up studies are used as dielectric and coolant fluids (e.g. in trans- necessary for definitive statements. Abnormalities formers, capacitors, and electric motors). In 1979, of the intrahepatic biliary system have been associ- PCBs’ toxicity and classification as a persistent or- ated with the development of CCA in youth and ganic pollutant prompted a ban on their use in the adolescence. These abnormalities include chole- U.S., and twenty years later, this prohibition was dochal cysts, hepato-renal fibropolycystic disease, ratified in Europe by the Stockholm Convention and Caroli disease and syndrome. There is a 15% on Persistent Organic Pollutants [94]. The toxicity risk of malignant change approximately after the of PCBs had been known since the 1930s, although 2nd decade of life, at an average age of 34 years, al- these conclusions were often dismissed as negligi- though pediatric gastroenterologists have ble. In particular, the toxicity of PCBs to animals Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis 203 was first noticed and scientifically proved in the since there are four types of bases in DNA, in which 1970s, when emaciated seabird corpses with very transitions and transversions are possible. However, high PCB body burdens washed up dramatically on the majority of SNPs have two alleles. The number open beaches. It was also found that the toxicity of of SNPs is more than one and a half million, but PCBs varied in a relevant manner among matches. relatively few of these transform the gene product Singularly, PCBs also have shown toxic and muta- as there is no amino acid change (“silent” changes). genic effects by interfering with hormones in the In a previous study, we found that BRAF gene vari- body. In fact, PCBs both inhibit and imitate estra- ations might account for a proportion attributable diol. Estrogenic molecular imitation can feed estro- risk of developing malignant melanoma of 4% in a gen-dependent breast cancer cells, and possibly German population [99]. Genetic polymorphisms cause other cancers, such as uterine, cervical, and in the cytochrome P450 enzymes or in the bile salt liver cancer [95-97]. transporter proteins might lead to alterations in the efficiency with which environmental toxins (xeno- Oncogenesis biotics) are handled by the hepatocytes. The development of CCA is probably subject to a “second CCA development, as with most tumors, is proba- hit” to deconjugate such xenobiotics and to expose bly a multi-step process dependent on an interac- the bile duct epithelium to damage. Such second- tion between host genetic factors and environmen- ary hits may include nonspecific chronic inflamma- tal factors. A “two hit” hypothesis is considered tion, worm infections, and recurrent cholangitis valid for many tumors and may be substantiated for among others. A number of mutations in onco- CCA as well. The discovery that DNA sequence genes and tumor suppressor genes have been identi- variations (mutations and polymorphisms) can in- fied in CCA, suggesting this neoplasm may arise fluence the response of an individual to a drug or secondary to cellular and consequent DNA injury. predict the development of a disease has added a Several studies have shown an abnormal expression new dimension to evidence-based medicine. The of the K-RAS oncogene in up to 100% of cases and relevance of identifying individuals at increased risk the TP53 tumor suppressor gene in about 1/3 of of adverse drug reactions, the application of ge- formalin-fixed and paraffin-embedded specimens nomic technologies to drug development, and the with CCA [100-102]. It seems that these genetic clarification of the mechanisms of drug action on alterations are associated with a more aggressive cells are important targets in the therapeutic ap- phenotype of this neoplasm. An increase in the ex- proach to medicine in the 21st century [98]. DNA pression of C-MET and C-ERB-2 proto-oncogenes genome variations include mutations and polymor- has been shown, which is noteworthy as they have phisms that may easily be distinguished by frequen- been suggested as participants in the metastatic cy in addition to their association with disease. In transformation of the intrahepatic tumor [103]. other words, at a position in the genome where Moreover, the overexpression of the BCL-2 gene 93% of people have cytosine as a nucleotide, if the has been reported to reduce apoptosis in CCA cell- remaining 7% have a guanine as nucleotide, this lines [104]. Other anti-apoptotic proteins, mcl-1 genome variation is called “polymorphism”. and bcl-xl, are often expressed [105]. In addition to However, if one of the possible sequences is present the expression of the anti-apoptotic proteins, there in less than 1% of the population (e.g., 99.9% of are gene mutations involving p16INK4a and people have a C and 0.1% have a G), then the vari- p14ARF, which are cell cycle regulators and their ation is called a “mutation”. Single Nucleotide mutations lead to promoter methylation [106]. In Polymorphisms (SNPs) are DNA genome varia- fact, this phenomenon has been observed in the tions that involve only one nucleotide or base. Any CCA of patients affected with PSC. The list of oth- one of the four DNA bases may be substituted for er genetic variations includes growth factors, DNA any other – an A instead of a T, a T instead of a C, aneuploidy, and loss of heterozygosity of microsat- etc. It has been suggested that, theoretically, an ellite markers. Parasitic infestation, such as O. vi- SNP could have four possible forms, or alleles, verrini, can ultimately also co-contribute to CCA 204 Annals of Clinical & Laboratory Science, vol. 43, no. 2, 2013

Figure 3. oncogenesis, because of the host-parasite interac- increase CCA oncogenesis. When overexpressed, tion. The host response is mediated by Metastasis- the nuclear factor-kappaB (NFkB) can interact associated Protein 1 (MTA1), which has inflamma- with various genes involved in inﬂammatory or car- tory and carcinogenic activity, using cytokines such cinogenic responses, such as COX-2, iNOS, cas- as (cyto)-keratins K-18 and 19, and is overexpressed pases, bcl-2 and bcl-x, c-myc, cyclin D1, MMP-9 during parasitic infestations. MTA1 is also found to and ICAM-1, all of which are partially or fully re- be overexpressed in both HCC and CCA [107]. O. sponsible for cell proliferation, apoptosis, and se- viverrini infection up-regulates the C-SKI proto- nescence [110]. In addition, deregulation of K-ras oncogene and down-regulates genes of the TGF-β and BRAF (which causes overexpression of cyclo- pathway. Interestingly, C-SKI, which plays a role in oxygenase-2, interleukin-6 and c-erbB-2 or disrup- the terminal differentiation of skeletal muscle cells, tion of SMAD4 and PTEN pathways, inasmuch as but not in the determination of cells to the myo- SPP1, EFNB2, and E2F, IRX3, PTTG1 and genic lineage, may be mutated in CCA [108]. PPARg genes) are also commonly up-regulated in Platelet-Derived Growth Factor Alpha (PDGFA) CCA [111]. Thus far, carcinogenesis seems to fol- can also be deregulated by O. viverrini infection low a sequential process starting with inducers, and promote carcinogenesis down-regulating many which may be variable, and continuing with pro- anti-proliferative and angiogenesis pathways [109]. moting steps including cholestasis and chronic in- Several other genetic/transcriptional factors can flammation (Figure 3). The release of growth Cholangiocarcinoma: Risk Factors, Environmental Influences and Oncogenesis 205 interleukin-6 (IL-6), transforming growth factor cells and play an essential role in morphogenesis. beta (TGF-beta), tumor necrosis factor alpha (1) Gap Junctions (GJ), (2) Tight Junctions (TJ), (TNF-alpha), and PDGF are at the basis of cholan- (3) Adherent Junctions (AJ), and (4) desmosomes giocyte proliferation and are therefore under in- are the categories of cell-cell junctions, which are tense investigation worldwide. Enhanced prolifera- clearly distinguished from each other on the basis tive signaling, an increased mutation rate of tumor of their differing molecular compositions and ultra- suppressor genes, and the evasion of apoptosis may structural appearances [119,120]. AJs form the ma- constitute additional steps for the development of jor strength between adjacent cells. The cadherin CCA. Further studies are clearly warranted. family associated with the catenin family is the main component. Cadherins constitute a large Most recently, genotyping of CCA identified an in- family of membranous glycoproteins and both E- teresting subtype of tumors, likely with better prog- and N-cadherins (E-cad and N-cad) are widely noses. Wang et al. found isocitrate dehydrogenase 1 known to be the most crucial cadherin proteins in (IDH1) and isocitrate dehydrogenase 2 (IDH2) the cell-cell interaction. Extra-cytoplasmic domain mutations in about 10% of intrahepatic CCA (112) and cytoplasmic tail are two important domains of Nicotinamide adenine dinucleotide phosphate, ab- these molecules. The cytoplasmic tail of a cadherin breviated NADP+, is a coenzyme used in anabolic molecule binds with catenin molecules including, reactions, e.g. lipid and nucleic acid synthesis. β-catenin (β-cat) and Plakoglobin (Pg), which These reactions require NADPH as a reducing builds the cadherin-catenin unit [121,122]. The agent. IDH1 and IDH2 encode the NADP+- cadherin-catenin unit binds to α-catenin mole- dependent IDH, localizing to the cytoplasm and cules, which eventually bind to the actin of the cy- mitochondria, respectively. In the mitochondrial toskeleton [123,124]. Cell-to-cell interactions me- metabolism, IDH1 and IDH2 specifically catalyze diated by cadherins and catenins play a paramount the oxidative decarboxylation of isocitrate to pro- role in the regulation of cell motility, proliferation, duce α-ketoglutarate. These authors found that and differentiation and account for the inhibition CCA harboring IDH1 and IDH2 mutations had of cell proliferation. Therefore, destruction of cad- lower 5-hydroxymethylcytosine and higher 5-meth- herin-catenin units could lead to abnormal mor- ylcytosine levels, as well as increased dimethylation phology and cell dissociations. Tumor development of histone H3 lysine 79. Most strikingly, tumors and progression have been greatly associated with with IDH1 or IDH2 mutations were associated cadherin dysfunction. It has been well established with longer overall survival and longer recurrence- that the E-CAD gene is frequently down-regulated free periods. IDH1 and IDH2 mutations were also in cancer, and this finding is often considered a associated with increased levels of p53, but no mu- hallmark of cancer metastasis and progression tations in the p53 gene were found. These data sug- [125,126]. The expression of N-cad has been great- gest that mutations in IDH1 and IDH2 may cause ly associated with tumor prognosis and invasiveness stress that leads to p53 activation. IDH1 and IDH2 [127]. β-cat has been described as a multifunction- are not restricted to CCA, but have also been found al protein with many important structural and in several other cancers, including low-grade glio- transcriptional roles [128]. Pg is the homologue of mas, secondary glioblastoma multiforme, acute β-cat, and both have an associated role in Wnt sig- myeloid leukemia, chondrosarcomas, Ollier disease naling pathway. In the nucleus, β-cat binds mostly and Maffucci syndrome, and melanoma [113-118] with T-cell factor (TCF)/ lymphoid enhancer factor-1 transcription factor (LEF-1), which results in E-Cadherin and Beta-Catenin Expression in the activation of a wide range of genes involved in CCA and HCC Cell Lines cell proliferation [129]. Down-regulation or an increase in β-cat transcriptional activity has been In addition to their important functions in main- linked to malignant transformation and tumor pro- taining the rigidity and integrity of solid tissues, gression [130]. Lastly, Pg has also been described as cell-cell junctions are crucial units that connect performing tumor-suppressing activities [131,132]. 206 Annals of Clinical & Laboratory Science, vol. 43, no. 2, 2013

Since Pg is structurally homologous to β-cat, and expansion. Many inflammatory mediators such as has the ability to bind with TCF/LFF, which can- cytokines, chemokines, and eicanosoids seem to be not bind with DNA, Pg may negatively regulate at the basis of this stimulation and proliferation of β-cat transcription activity [133,134]. We investi- both untransformed and tumor cells. Further stud- gated cell-cell junctions by studying mainly the ex- ies will doubtless improve our understanding of pression and localization of AJ components repre- this complex system. As progression encompasses a sented by E-cad/β-cat signaling. We found that two substantial growth in tumor size, new therapeutic CCA cell lines (OZ and HuCCT-1) express E-cad/ strategies targeting not only initiation but also pro- β-cat, but with different localization patterns. In gression may be the promise of the future. HuCCT-1, E-cad and β-cat were localized in the cytoplasm, while in OZ, E-cad and β-cat were lo- Acknowledgements calized in the cytoplasmic membrane only [135]. We are grateful to the following funding agencies and institu- E-cad and β-cat expressions were mostly investi- tions: Tyrolean Cancer Research, Tyrol, Austria and University gated immunohistochemically and have been dem- of Alberta Internal Funds (Canada). The Saudi Cultural onstrated to be down-regulated and significantly Bureau, Ottawa, ON, Canada, supported RA-B and YA (MSc correlated to a high grade and poor differentiation Lab Med & Pathology graduate students). The sponsors had no role in study design, data collection, data analysis, data inter- of CCA [136-139]. Similarly, Ku et al (2002) have pretation or in the writing of this report. CS is the B105 lab studied the expression of E-cad and β-cat and their director and principal investigator, designed the project, and genetic alterations in six CCA cell lines [140]. They was responsible for interpreting data, obtaining sponsorship demonstrated that two CCA cell lines showed hy- funding, and coordinating the study. RA-B and YA wrote the permethylation of the E-CAD gene. However, most ﬁrst draft of the manuscript. RA-B and YA were responsible for data gathering and interpretation. NZ contributed to data in- studies examining genetic predisposition to CCA terpretation related to the environment. This study is part of a to date seem to be heterogeneous, thus warranting project that was approved by the institutional Human Research further research on the genetic background of AJ. Ethics Board (University of Alberta, Edmonton, Canada; HREB approval #20274) and operationally by the provincial Summary Health Care Provider (Alberta Health Services). References In summary, CCA is a one of the most frequent malignant epithelial liver tumors after hepatocellu- 1. Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD. Cholangiocarcinoma. 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