Open Access
Review

New genomic landscapes and therapeutic targets for biliary tract cancers

Michele Simbolo1,Matteo Fassan1,*,Andrea Mafficini1,Rita T. Lawlor2,Andrea Ruzzenente3,Aldo Scarpa2
1
ARC-Net Research Centre, University and Hospital Trust of Verona, Piazzale L. Scuro 2, 37134 Verona, Italy
2
ARC-Net Research Centre and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Piazzale L. Scuro 2, 37134 Verona, Italy
3
Department of Surgery and Oncology, University of Verona, Piazzale L. Scuro 2, 37134 Verona, Italy
DOI: 10.2741/4416 Volume 21 Issue 4, pp.707-718
Published: 01 January 2016
(This article belongs to the Special Issue The post-genomic revolution in diagnostic pathology)
*Corresponding Author(s):  
Matteo Fassan
E-mail:  
matteo.fassan@gmail.com
Abstract

Biliary tract cancers (BTCs) are a heterogeneous group of neoplasms characterized by a dismal prognosis. At variance with most solid tumors, no effective molecular targeted agent has been currently approved for BTCs treatment and their molecular landscape has only been recently investigated. Comprehensive mutational profiling studies identified IDH1/2 and BAP1 as characteristic of intrahepatic cholangiocarcinomas, while extrahepatic cholangiocarcinomas and gallbladder carcinomas were characterized by frequent KRAS and TP53 alterations. Moreover, targeted next-generation sequencing has uncovered alterations in several key cellular pathways. BTC-specific alterations include disorders of major regulators of cell cycle and chromatin remodeling processes, as well as deregulation of the mTOR-, TGF-beta/Smad- and receptor tyrosine kinases signaling. The next step will be the correlation of these findings with clinical trials to identify predictive biomarkers for the development of personalized therapies. This will permit early access for BTC patients to innovative drugs.

Key words

Cholangiocarcinoma, Molecular Profiling, Targeted Therapy, Review

2. Introduction

Biliary tract cancers (BTCs) are a heterogeneous group of cancers, representing only about 3% of all gastrointestinal malignancies (1).They may arise from the gallbladder (i.e. gallbladder carcinomas; GBC) or the biliary tree. These latter can be separated according to their anatomical origin into intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) (2, 3).

The mortality rate for these neoplasms is increasing worldwide (4). Surgery represents the only curative option at early stages of the disease, which is, however, affected by a high rate of tumor recurrence (5).

Remarkably, early diagnosis has been demonstrated to increase the survival rate of only 5% (6), and by the clinical point of view, a definite diagnosis of cholangiocarcinoma can be significantly delayed also in advanced stages because of the wide range of alternative diagnoses, including benign biliary tree strictures (i.e. choledocholithiasis and iatrogenic bile duct injuries), primary sclerosing cholangitis and other primary and metastatic carcinomas (7).

At variance with most solid tumors, no effective molecular targeted agent has been approved for BTCs, and consequently BTC patients have limited access to innovative clinical trials. It has to be underlined that the most important issue hampering the introduction of new therapeutic options for BTCs’ patients is represented by the limited information available on their molecular background (Table 1 and Figure 1).

Table 1. Most frequently mutated genes in cholangiocarcinomas by subtype (numbers represent percentage of mutated cases)
GeneICCECCGBCRef
ARID1A19NA065
NANA039
11.4.12.3.11.5.22
205NA75
BAP125NA065
NANA039
14.3.03.8.22
9.1.10NA75
BRAF0NA065
NANA5.9.39
4.3.0022
5.3.0NA75
CDKN2A3NA065
NANA5.9.39
1.4.03.8.22
5.3.10NA75
EGFR0NA12.5.65
NANA3.9.39
01.7.3.8.22
1.8.5NA75
ERBB20NA065
NANA9.8.39
003.8.22
1.8.20NA75
ERBB30NA065
NANA11.8.39
NANANA22
00NA75
FBXW70NA12.5.65
NANA5.9.39
1.4.3.5.022
5.5.15NA75
IDH1/219NA065
NANA039
200022
23.6.0NA75
KRAS3NA065
NANA7.8.39
15.7.47.4.19.2.22
23.6.40NA75
NRAS3NA065
NANA039
9.3.1.7.022
05NA75
PBRM117NA2565
NANA039
14.3.3.5.7.7.22
10.9.5NA75
PIK3CA6.2.NA12.5.65
NANA5.9.39
5.7.8.7.7.7.22
5.3.10NA75
PTEN6.2.NA065
NANA039
1.4.3.5.3.8.22
5.3.5NA75
SMAD40NA12.5.65
NANA3.9.39
1.4.10.5.7.7.22
3.6.20NA75
SMARCB10NA065
NANA3.9.39
007.7.22
00NA75
TP536.2.NA6365
NANA47.1.39
8.6.17.5.46.2.22
29.1.45NA75

Figure 1. Pathways most frequently altered in biliary tree tumors: inflammation related cytokines (IL-6), chromatin remodeling (PBRM1, and BAP1), mTOR and TGF-beta/Smad signaling, and receptor tyrosine kinases (EGF and FGF). IDH1/2 and BAP1 are characteristic of intrahepatic cholangiocarcinomas, while KRAS and TP53 genes alterations are more frequent in extrahepatic cholangiocarcinomas and gallbladder carcinomas.

This minireview summarizes the current evidence on the most frequently deregulated pathways in BTCs, focusing on their potential as novel therapeutic targets.

3. Role of chronic inflammation and related pathways

Major BTC risk factors are primary sclerosing cholangitis (PSC), long-standing intraductal gallstone disease, and infestation by the liver flukes Clonorchis sinensis and Opisthorchis viverrini (8). These data demonstrate an important role for chronic inflammation on biliary tree carcinogenesis.

Similar to what observed in the hepatitis-associated multistep liver carcinogenesis (9), several studies pinpointed a key role of inflammation-related cytokines on the induction of the malignant transformation of cholangiocytes. Among the others, interleukin 6 (IL-6) and its associated pathways have been demonstrated to be an important proinflammatory signaling in the biliary tree chronic inflammatory setting (10). Increased levels of IL-6 have been demonstrated in serum from cholangiocarcinoma (CCA) patients (10). Moreover, IL-6 levels significantly correlated with tumor burden and were significantly decreased two weeks after therapeutic surgery. Of note, IL-6 levels were able to discriminate between CCA and benign biliary disease with a sensitivity and specificity of 71% and 90%, respectively (11).

IL-6 has an autocrine role on CCA cell survival. Through the activation of the p38 and p44/p42 MAPK signaling (12), IL-6 promotes telomerase activity (13), up-regulation of Mcl-1 (14), and induces STAT3 activation (15). Notably, STAT3 expression is significantly up-regulated in dedifferentiated CCAs, and is associated with shorter survival of CCA patients (16).

Another important player in the promotion of malignant transformation of cholangiocytes is COX-2. Cholestasis and the presence of oxysterols, oxidized forms of cholesterol, can significantly induce the overexpression of COX-2, which subsequently promotes cell growth (17). Moreover, inflammatory cytokines significantly up-regulate the inducible nitric oxide synthase (iNOS); iNOS directly binds to COX-2 and enhances COX-2 activity (18). It has been demonstrated a significant overexpression of both iNOS and COX-2 in reactive biliary epithelia and biliary intraepithelial neoplastic lesions (19).

A recent whole-exome sequencing study investigated the impact of O. viverrini infection on CCA mutational profile (20). Of interest, BAP1 (10.5.% versus 2.8.%) and IDH1/IDH2 (9.3.% versus 2.8.%) mutation rates were significantly higher in non–O. viverrini CCAs. On the other hand, TP53 (9.3.% versus 39.8.%), SMAD4 (5.8.% versus 19.4.%), MLL3 (3.5.% versus 13.0.%) and GNAS (0% versus 5.6.%) were more frequently involved in O. viverrini-related tumors (20). Overall, these data strongly suggest distinct mutational profiles based on the different etiologies.

4. Cell survival pathways activation

mTOR signaling is one of the most involved pathway in the maintenance of cell survival and inhibition of apoptosis in solid tumors. Aberrant expressions of activated forms of mTOR (phospho-mTOR) and of its downstream effectors (ph-AKT1, ph-p70S6K and/or ph-4EBP1) is a common finding in BTCs (21, 22). Moreover, the overexpression of ph-mTOR has been associated to well-to-moderately differentiated tumors, and to tumors without metastatic spread (21). Elevated activated mTOR levels have been also associated to a significantly shorter overall survival for BTCs’ patients (23).

Another important mTOR-related gene is the phosphatase and tensin homologue (PTEN) which is a key tumor suppressor frequently altered in BTCs. Low PTEN expression and decreased PTEN/ph-AKT1 and PTEN/ph-mTOR expression ratios are poor prognostic factors in ECCs (24).

The role of PIK3CA alterations in BTCs is still under study. In a Chinese series, 32% of CCAs showed a mutation in the PIK3CA gene (25); another study found only one mutated case out of 11 (26).

Our group recently further supported mTOR pathway relevance in BTCs by demonstrating a significant up-regulation of ph-mTOR and of its downstream effectors in 51% of a large series of BTCs (22). This altered activation could be partly explained by the concomitant presence of a mutation in one of the genes involved in the mTOR pathway (i.e., AKT, FBXW7, PIK3CA, PIK3C2A, PIK3C2G, PTEN).

5. Tyrosine kinases receptors

The ErbB family of class I receptor tyrosine kinases comprises four distinct receptors: ErbB1 (EGFR), ErbB2 (HER2), ErbB3 and ErbB4 (27, 28).

Aberrant EGFR and/or HER2 expression and deregulation of their downstream signaling are frequently observed in ICCs. Different independent studies demonstrated a significant up-regulation of EGFR in ICC (29-32). Among these, Ito et al. (30) further observed that 39.5.% and 10.5.% of their ICCs showed also an overexpression of ErbB3 and ErbB4, respectively.

HER2 immunohistochemical overexpression has been demonstrated in variable percentages of noncancerous biliary proliferative disorders associated to CCA, including choledocholithiasis and PSC (33-35). Overall, these findings support an early involvement of HER2 deregulation in biliary carcinogenesis. Interestingly, Su et al. (36) demonstrated a higher level of HER2 in the bile collected from CCA patients than in the bile collected from patients with biliary tract infection, biliary stone disease, or normal controls.

Somatic mutations in the tyrosine kinase domain of EGFR have been described in a subgroup of patients with either cholangiocarcinoma or gallbladder carcinoma (37, 38). However, the clinical impact of these mutations should be further investigated. More recently, a whole-exome sequencing study identified recurrent mutations in the ErbB signaling (including EGFR, ERBB2, ERBB3, ERBB4 and their downstream genes) in GBCs (39).

It is well known that c-Met plays an important role in carcinogenesis in many cancer types, including hepatic tumors (34, 40). Terada et al. reported a significant c-Met overexpression in 81% of proliferative lesions associated to choledocolithiasis (41). Of note, c-Met was also significantly overexpressed in 58% of ICCs and correlated with tumor differentiation (41).

Also defects in the TGF-beta signaling play a significant role in many malignancies, including BTCs. Transforming growth factor signaling pathway disturbance in biliary cancers results from intragenic mutations in either the TGF-beta type I and II receptor genes, or the TGF-beta gene itself (42, 43). Moreover, homozygous or heterozygous deletions of the TGF-beta type I receptor gene has also been reported in biliary carcinomas (42, 43). Of note, deregulation of the TGF-beta signaling is associated to an increased production of peritumoral fibrosis (44), which is a hallmark of extrahepatic BTCs. In fact most EECs are often accompanied by abundant fibrous stroma (5).

Members of the FGFR family (i.e. FGFR1-4) have been associated with mutations, amplifications and translocation events with oncogenic potential (45). As observed in other solid tumors, the presence of FGFR gene fusions have been recently demonstrated also in CCAs (46, 47); several ICC cases characterized by FGFR2-BICC1 fusions and a single case with FGFR2-AHCYL1.

6. Intracellular signaling

Several studies associated KRAS gene alterations to GBCs. However, the reported frequency of gene mutations (in codons 12, 13, and 61) varies from 0% to 80% (22, 48). Of interest, in Western countries, where GBC is most related to chronic cholecystitis and cholelithiasis, KRAS mutations are rarely encountered (0-10%). In contrast, in Japanese series, where GBCs may often be associated with a congenital abnormality, the frequency of KRAS mutations is markedly higher.

Activation of the KRAS oncogenes has been described also for other BTCs (49). The variable incidence of KRAS mutations reflects the anatomical location and the type of duct on which CCAs develop. In fact, KRAS mutations are more frequently observed in ECCs and ICCs of the periductal-spreading type, whereas are almost absent in the ICCs mass-forming type (50, 51). These different rates may also reflect different etiologies; the prevalence ranges from 58% in sporadic CCAs to 8% in CCAs related to O. viverrini in Thailand (52).

BRAF mutations have been observed mainly in ICCs. In our series all three BRAF mutations observed clustered in ICC tumor subtype and were mutually exclusive with mutations in KRAS and/or NRAS (22). Similarly, Goeppert et al. observed BRAF mutations only in ICCs and this specific alteration affected 3% of the series. Tannapfel et al. characterized a large series of CCAs for BRAF mutations, and found activating BRAF missense mutations (V599E and also V599D) in 15/69 (22%) cases (53). Saetta et al. observed a high incidence of BRAF exon 15 mutations in GBCs (33%); of note, BRAF and KRAS mutations were mutually exclusive (54).

SMAD4 tumor suppressor gene is an important downstream component of TGF-beta signaling (55). Mutations determining loss of expression of SMAD4 gene have been described in biliary malignancies and in particular in ECCs (56). Of interest, the liver-specific targeted simultaneous disruption of SMAD4 and PTEN tumor suppressors generated a mouse model that develops biliary malignancies (57).

7. Cell cycle checkpoint

The p16/cyclin D1/Rb pathway is one of the most important cell-cycle regulator in many different types of cancers (58). Evidence for a role in biliary tract carcinogenesis derives from the demonstration of frequent cyclin D1 overexpression and inactivation of CDKN2A (p16) by a variety of mechanisms, including mutation, deletion, and abnormal hypermethylation (59). Ishikawa et al. reported a significant reduction of p16 expression due to promoter methylation in intraductal papillary neoplasms of livers with hepatolithiasis; intraductal papillary neoplasms are identified as precursor lesions of ICC (60). Methylation of the CDKN2A promoter and homozygous deletion of the gene has also been frequently observed in ICCs (61, 62).

Several studies pinpointed a central role for TP53 gene alterations in BTCs (63). Also MDM2 gene amplification has been demonstrated as an important TP53 master deregulator in CCAs (64). In our mono-institutional series of 153 CCAs, the presence of TP53 mutations was the only significant independent prognostic factor (22). It has to be noted that although TP53 mutations are frequent in ICCs, their prevalence is significantly higher in ECCs and GBCs. In fact, somatic mutations of TP53 occur in almost half of GBCs (65).

8. Metabolic related factors

Somatic mutations in the cytoplasmic and peroxisomal isocitrate dehydrogenase gene IDH1 and its mitochondrial counterpart IDH2 have been identified through large genomic screenings of human tumors (66). These mutations cause a single amino acid change at a conserved arginine residue within the isocitrate binding site of IDH1 (R132) or IDH2 (R172, R140) resulting in decreased enzymatic activity for oxidative decarboxylation of isocitrate to alfa-ketoglutarate (67).

A seminal study on the mutational profiling of 287 gastrointestinal cancers identified IDH1 mutations in a significant subset of patients affected by ICC (68). In the same study it was shown that the prevalenceof IDH1 and IDH2 gene mutations was greater than the combined prevalence of activating mutations in AKT1, KRAS, NRAS, and BRAF in ICC, making it the most prevalent drug targetable gene alteration in this cancer type (68). This finding was further supported by deep sequencing studies, which identified IDH1 and IDH2 gene mutations in almost 20% of ICCs (22, 65, 68).

The presence of IDH1/2 mutations in a significant proportion of ICCs may offer novel insights on how to develop targeted approaches against this treatment-refractory disease. The physiological IDH1/2 function is the catalyzation of the oxidative carboxylation of isocitrate to alpha-ketoglutarate. IDH1/2 mutations confer neomorphic activity through the reduction of alpha-ketoglutarate to the metabolite R(–)-2-hydroxyglutarate (2HG), resulting in the accumulation of 2HG in tumor tissues (67, 69). A recent study suggests that circulating 2HG may serve as a surrogate biomarker of IDH1 or IDH2 mutation status in ICC, and its levels are related to tumor burden (70).

9. Chromatin remodeling genes

Genome-wide sequencing has identified frequent mutations in chromatin remodeling genes across different tumor types. These discoveries have profound implications in understanding the mechanistic basis of epigenetic changes that have recently been demonstrated as widespread in most tumors (71).

The seminal report of Jiao et al. highlighted the key role of mutations in chromatin-remodeling genes in ICC with frequent inactivating mutations in three different genes (ARID1A, BAP1, PBRM1) affecting almost half of the tumors sequenced. These studies point to chromatin remodeling as a crucial area of investigation for innovative therapeutic strategies. Moreover, comparison of somatic mutation data for cholangiocarcinoma and gallbladder carcinoma suggests that, while both tumor types arise from biliary epithelium, they are genetically distinct (65).

Mutations in PBRM1 (which encodes BAF180) were identified in 41% of renal cell carcinomas, making PBRM1 the second most frequently mutated gene in these cancers after VHL (72). BAF180 contains six tandem bromodomains that bind acetylated histones, two bromo-adjacent homology (BAH) domains that mediate protein–protein interactions and a high-mobility group (HMG) domain that binds nucleosomal DNA. Given the function of these domains, BAF180 may recruit PBAF complexes to specific loci (73). BAF180 binds preferentially to different acetylated lysine configurations of histone tails, indicating that it may contribute to the recognition of the histone code (74). The frequency of mutations of this gene in CCAs was attested at between 5 and 20% depending on histopathological subtype (Table 1), further underlying its importance in human cancers (22, 39, 65, 75).

The ARID1A subunit of SWI/SNF complexes was also recently found to be specifically mutated in primary human cancers. The ARID1A subunit contains an ARID domain that binds DNA in a sequence-nonspecific manner (76). However, the role of this domain during the engagement and repositioning of a nucleosome is still not fully understood as PBRM1, ARID1A mutation frequencies significantly differ among CCA subtypes. Most ICC and ECC studies reported a mutation frequency of 5-20% (22, 65, 75). In GBCs, ARID1A seems to be infrequently altered (22, 39).

Protein ubiquitination was initially seen as a mechanism to label proteins for degradation; however, this idea has evolved as we have come to understand that ubiquitination and deubiquitinating enzymes (DUBs) regulate various cellular processes, including DNA repair, gene transcription, cell membrane trafficking, cell cycle progression, stress response, cell communication, differentiation and apoptosis, and they also have a role in cancer (77). BAP1 was discovered in a yeast two-hybrid screen owing to its interaction with the RING finger domain of the tumor suppressor BRCA1. BRCA1 forms a heterodimer through its RING domain with BRCA1-associated RING domain 1 (BARD1), and this BRCA1–BARD1 tumor suppressor complex has E3 ubiquitin ligase activity that regulates the DNA damage response (DDR) (78). BAP1 binds and deubiquitylates BARD1, thus modulating the E3 ligase activity of BRCA1-BARD1 (79). Mutations in the BAP1 gene seem to be specific of ICC tumors. Over 20% of ICC cases showed mutation in BAP1 in exome sequencing studies (20, 39, 65). Differently studies performed on ECCs and GBCs showed no or low mutation frequency in BAP1 gene (20, 22, 39, 75).

10. Conclusions

BTCs are characterized by an unpredictable clinical behaviour. A significantly poorer prognosis is usually observed for ECCs and GBCs in comparison to mass-forming ICCs; however, significant clinical differences are observed also among tumors characterized by similar clinico-pathological features. As such, no biomarker prognostically stratify BTCs’ patients, so far. However, the remarkable progress in molecular biology has led to a better understanding of the complex molecular landscape of BTCs’. Of note, particular mutational profiles have been identified according to tumor location through targeted next generation sequencing. Mutations in IDH1/2 and BAP1 have been recognized as characteristic of ICCs, while ECCs and GBCs are characterized by frequent KRAS and TP53 alterations. Alterations of the TP53 gene have also been associated with a worse patient prognosis.

Several other key cellular pathways, including disorders of major regulators of cell cycle and chromatin remodeling processes, as well as deregulation of the mTOR-, TGF-beta/Smad- and receptor tyrosine kinases signaling, have recently been shown to be altered in these tumors. This emerging knowledge is of utmost importance for cancer research and may be the breakthrough to novel innovative, targeted therapies.

The next step will be the correlation of these findings with clinical trials to identify predictive biomarkers for the development of personalized tailored therapies. This will also permit early access for BTCs patients to innovative drugs.

11. Acknowledgments

Current address of Matteo Fassan is Department of Medicine (DIMED), University of Padua, via Gabelli 61, 35121 Padua, Italy. M. Simbolo and M. Fassan equally contributed to this article. All authors participated in writing and approved the final, submitted manuscript. No conflict of interest to be declared. This work was partly supported by theItalian Cancer Genome Project grants from the Italian Ministry of University and Research (FIRB - RBAP10AHJB) and Fondazione Italiana Malattie Pancreas – Ministry of Health (J33G13000210001), AIRC grant n. 12182, and FP7 EU project CAM-PaC no: 602783.

Abbreviations: ICC, intrahepatic cholangiocarcinoma, ECC, extrahepatic cholangiocarcinoma, GBC, gallbladder carcinoma

References

    1. Y Shaib, HB El-Serag: The epidemiology of cholangiocarcinoma. Semin Liver Dis 24, 115-125 (2004)

    2. T Patel: Cholangiocarcinoma--controversies and challenges. Nat Rev Gastroenterol Hepatol 8, 189-200 (2011)

    3. AE Sirica: Cholangiocarcinoma: molecular targeting strategies for chemoprevention and therapy. Hepatology 41, 5-15 (2005)

    4. T Patel: Worldwide trends in mortality from biliary tract malignancies. BMC Cancer 2, 10 (2002)

    5. KN Lazaridis, GJ Gores: Cholangiocarcinoma. Gastroenterology 128, 1655-1667 (2005)

    6. SA Khan, HC Thomas, BR Davidson, SD Taylor-Robinson: Cholangiocarcinoma. Lancet 366, 1303-1314 (2005)

    7. DJ van Leeuwen, JW Reeders: Primary sclerosing cholangitis and cholangiocarcinoma as a diagnostic and therapeutic dilemma. Ann Oncol 10 Suppl 4, 89-93 (1999)

    8. PC de Groen, GJ Gores, NF LaRusso, LL Gunderson, DM Nagorney: Biliary tract cancers. NEJM 341, 1368-1378 (1999)

    9. M Jaiswal, NF LaRusso, LJ Burgart, GJ Gores GJ: Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Res 60, 184-190(2000)

    10. JS Goydos, AM Brumfield, E Frezza, A Booth, MT Lotze, SE Carty. Marked elevation of serum interleukin-6 in patients with cholangiocarcinoma: validation of utility as a clinical marker. Ann Surg 227, 398-404 (1998)

    11. P Tangkijvanich, D Thong-ngam, A Theamboonlers, O Hanvivatvong, P Kullavanijaya, Y Poovorawan: Diagnostic role of serum interleukin 6 and CA 19-9 in patients with cholangiocarcinoma. Hepatogastroenterology 51, 15-19 (2004)

    12. K Okada, Y Shimizu, S Nambu, K Higuchi, A Watanabe: Interleukin-6 functions as an autocrine growth factor in a cholangiocarcinoma cell line. J Gastroenterol Hepatol 9, 462-467 (1994)

    13. F Meng, Y Yamagiwa, Y Ueno, T Patel: Over-expression of interleukin-6 enhances cell survival and transformed cell growth in human malignant cholangiocytes. J Hepatol 44, 1055-1065 (2006)

    14. Y Yamagiwa, F Meng, T Patel: Interleukin-6 decreases senescence and increases telomerase activity in malignant human cholangiocytes. Life Sci 78, 2494-2502 (2006)

    15. H Isomoto, S Kobayashi, NW Werneburg, SF Bronk, ME Guicciardi, DA Frank, GJ Gores: Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells. Hepatology 42, 1329-1338 (2005)

    16. H Dokduang, A Techasen, N Namwat, N Khuntikeo, C Pairojkul, Y Murakami, W Loilome, P Yongvanit: STATs profiling reveals predominantly-activated STAT3 in cholangiocarcinoma genesis and progression. J Hepatobiliary Pancreat Sci 21, 767-776 (2014)

    17. JH Yoon, AE Canbay, NW Werneburg, SP Lee, GJ Gores: Oxysterols induce cyclooxygenase-2 expression in cholangiocytes: implications for biliary tract carcinogenesis. Hepatology 39, 732-738 (2004)

    18. N Ishimura, SF Bronk, GJ Gores: Inducible nitric oxide synthase upregulates cyclooxygenase-2 in mouse cholangiocytes promoting cell growth. Am J Physiol Gastrointest Liver Physiol 287, G88-95 (2004)

    19. S Aishima, Y Mano, Y Tanaka, Y Kubo, K Shirabe, Y Maehara, Y Oda: Different roles of inducible nitric oxide synthase and cyclooxygenase-2 in carcinogenesis and metastasis of intrahepatic cholangiocarcinoma. Hum Pathol 44, 1031-1037 (2013)

    20. W Chan-On, ML Nairismagi, CK Ong, WK Lim, S Dima, C Pairojkul, KH Lim, JR McPherson, I Cutcutache, HL Heng, L Ooi, A Chung, P Chow, PC Cheow, SY Lee, SP Choo, IB Tan, D Duda, A Nastase, SS Myint, BH Wong, A Gan, V Rajasegaran, CC Ng, S Nagarajan, A Jusakul, S Zhang, P Vohra, W Yu, D Huang, P Sithithaworn, P Yongvanit, S Wongkham, N Khuntikeo, V Bhudhisawasdi, I Popescu, SG Rozen, P Tan, BT The: Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers. Nat Genet 45, 1474-1478 (2013)

    21. D Lee, IG Do, K Choi, CO Sung, KT Jang, D Choi, JS Heo, SH Choi, J Kim, JY Park, HJ Cha, JW Joh, KY Choi, DS Kim: The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas. Mod Pathol 25, 131-139 (2012)

    22. M Simbolo, M Fassan, A Ruzzenente, A Mafficini, LD Wood, V Corbo, D Melisi, G Malleo, C Vicentini, G Malpeli, D Antonello, N Sperandio, P Capelli, A Tomezzoli, C Iacono, RT Lawlor, C Bassi, RH Hruban, A Guglielmi, G Tortora, F de Braud, A Scarpa A. Multigene mutational profiling of cholangiocarcinomas identifies actionable molecular subgroups. Oncotarget 5, 2839-2852 (2014)

    23. B Herberger, H Puhalla, M Lehnert, F Wrba, S Novak, A Brandstetter, B Gruenberger, T Gruenberger, R Pirker, M Filipits: Activated mammalian target of rapamycin is an adverse prognostic factor in patients with biliary tract adenocarcinoma. Clin Cancer Res 13, 4795-4799 (2007)

    24. JY Chung, SM Hong, BY Choi, H Cho, E Yu, SM Hewitt: The expression of phospho-AKT, phospho-mTOR, and PTEN in extrahepatic cholangiocarcinoma. Clin Cancer Res 15, 660-667 (2009)

    25. RF Xu, JP Sun, SR Zhang, GS Zhu, LB Li, YL Liao, JM Xie, WJ Liao: KRAS and PIK3CA but not BRAF genes are frequently mutated in Chinese cholangiocarcinoma patients. Biomed Pharmacother 65, 22-26 (2011)

    26. MO Riener, M Bawohl, PA Clavien, W Jochum: Rare PIK3CA hotspot mutations in carcinomas of the biliary tract. Genes Chromosomes Cancer 47, 363-367 (2008)

    27. R Roskoski Jr: The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys Res Commun 319, 1-11 (2004)

    28. N Normanno, C Bianco, L Strizzi, M Mancino, MR Maiello, A De Luca, F Caponigro, DS Salomon: The ErbB receptors and their ligands in cancer: an overview. Curr Drug Targets 6, 243-257 (2005)

    29. K Nakazawa, Y Dobashi, S Suzuki, H Fujii, Y Takeda, A Ooi: Amplification and overexpression of c-erbB-2, epidermal growth factor receptor, and c-met in biliary tract cancers. J Pathol 206, 356-365 (2005)

    30. Y Ito, T Takeda, Y Sasaki, M Sakon, T Yamada, S Ishiguro, S Imaoka, M Tsujimoto, S Higashiyama, M Monden, N Matsuura: Expression and clinical significance of the erbB family in intrahepatic cholangiocellular carcinoma. Pathol Res Pract 197, 95-100 (2001)

    31. A Nonomura, G Ohta, Y Nakanuma, R Izumi, Y Mizukami, F Matsubara, M Hayashi, K Watanabe, N Takayanagi: Simultaneous detection of epidermal growth factor receptor (EGF-R), epidermal growth factor (EGF) and ras p21 in cholangiocarcinoma by an immunocytochemical method. Liver 8, 157-166 (1988)

    32. A Altimari, M Fiorentino, E Gabusi, E Gruppioni, B Corti, A D’Errico, WF Grigioni: Investigation of ErbB1 and ErbB2 expression for therapeutic targeting in primary liver tumours. Dig LiverDis 35, 332-338 (2003)

    33. EM Brunt, PE Swanson: Immunoreactivity for c-erbB-2 oncopeptide in benign and malignant diseases of the liver. Am J Clin Pathol 97, S53-61 (1992)

    34. SI Aishima, KI Taguchi, K Sugimachi, M Shimada, M Tsuneyoshi: c-erbB-2 and c-Met expression relates to cholangiocarcinogenesis and progression of intrahepatic cholangiocarcinoma. Histopathology 40, 269-278 (2002)

    35. K Endo, BI Yoon, C Pairojkul, AJ Demetris, AE Sirica: ERBB-2 overexpression and cyclooxygenase-2 up-regulation in human cholangiocarcinoma and risk conditions. Hepatology 36, 439-450 (2002)

    36. WC Su, SC Shiesh, HS Liu, CY Chen, NH Chow, XZ Lin: Expression of oncogene products HER2/Neu and Ras and fibrosis-related growth factors bFGF, TGF-beta, and PDGF in bile from biliary malignancies and inflammatory disorders. Dig Dis Sci 46, 1387-1392 (2001)

    37. F Leone, G Cavalloni, Y Pignochino, I Sarotto, R Ferraris, W Piacibello, T Venesio, L Capussotti, M Risio, M Aglietta: Somatic mutations of epidermal growth factor receptor in bile duct and gallbladder carcinoma. Clin Cancer Res 12, 1680-1685 (2006)

    38. GY Gwak, JH Yoon, CM Shin, YJ Ahn, JK Chung, YA Kim, TY Kim, HS Lee: Detection of response-predicting mutations in the kinase domain of the epidermal growth factor receptor gene in cholangiocarcinomas. J Cancer Res Clin Oncol 131, 649-652 (2005)

    39. M Li, Z Zhang, X Li, J Ye, X Wu, Z Tan, C Liu, B Shen, XA Wang, W Wu, D Zhou, D Zhang, T Wang, B Liu, K Qu, Q Ding, H Weng, J Mu, Y Shu, R Bao, Y Cao, P Chen, T Liu, L Jiang, Y Hu, P Dong, J Gu, W Lu, W Shi, J Lu, W Gong, Z Tang, Y Zhang, X Wang, YE Chin, X Weng, H Zhang, W Tang, Y Zheng, L He, H Wang, Y Liu: Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway. Nat Genet 46, 872-876 (2014)

    40. T Ueki, J Fujimoto, T Suzuki, H Yamamoto, E Okamoto: Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma. Hepatology 25, 862-866 (1997)

    41. T Terada, Y Nakanuma, AE Sirica: Immunohistochemical demonstration of MET overexpression in human intrahepatic cholangiocarcinoma and in hepatolithiasis. Hum Pathol 29, 175-180 (1998)

    42. M Goggins, M Shekher, K Turnacioglu, CJ Yeo, RH Hruban, SE Kern: Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas. Cancer Res 58, 5329-5332 (1998)

    43. M Nagai, Y Kawarada, M Watanabe, T Iwase, T Muneyuki, K Yamao, K Fukutome, R Yatani: Analysis of microsatellite instability, TGF-beta type II receptor gene mutations and hMSH2 and hMLH1 allele losses in pancreaticobiliary maljunction-associated biliary tract tumors. Anticancer Res 19, 1765-1768 (1999)

    44. S Yazumi, K Ko, N Watanabe, H Shinohara, K Yoshikawa, T Chiba, R Takahashi: Disrupted transforming growth factor-beta signaling and deregulated growth in human biliary tract cancer cells. Int J Cancer 86, 782-789 (2000)

    45. N Turner, R Grose. Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 10, 116-129 (2010)

    46. Y Arai, Y Totoki, F Hosoda, T Shirota, N Hama, H Nakamura, H Ojima, K Furuta, K Shimada, T Okusaka, T Kosuge, T Shibata: Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma. Hepatology 59, 1427-1434 (2014)

    47. YM Wu, F Su, S Kalyana-Sundaram, N Khazanov, B Ateeq, X Cao, RJ Lonigro, P Vats, R Wang, SF Lin, AJ Cheng, LP Kunju, J Siddiqui, SA Tomlins, P Wyngaard, S Sadis, S Roychowdhury, MH Hussain, FY Feng, MM Zalupski, M Talpaz, KJ Pienta, DR Rhodes, DR Robinson, AM Chinnaiyan: Identification of targetable FGFR gene fusions in diverse cancers. Cancer Discov 3, 636-647 (2013)

    48. II Wistuba, AF Gazdar: Gallbladder cancer: lessons from a rare tumour. Nat Rev Cancer 4, 695-706 (2004)

    49. M Watanabe, M Asaka, J Tanaka, M Kurosawa, M Kasai, T Miyazaki: Point mutation of K-ras gene codon 12 in biliary tract tumors. Gastroenterology 107, 1147-1153 (1994)

    50. K Ohashi, Y Nakajima, H Kanehiro, M Tsutsumi, J Taki, Y Aomatsu, A Yoshimura, S Ko, T Kin, K Yagura K: Ki-ras mutations and p53 protein expressions in intrahepatic cholangiocarcinomas: relation to gross tumor morphology. Gastroenterology 109, 1612-1617 (1995)

    51. YK Kang, WH Kim, HW Lee, HK Lee, YI Kim: Mutation of p53 and K-ras, and loss of heterozygosity of APC in intrahepatic cholangiocarcinoma. Lab Invest 79, 477-483 (1999)

    52. J Albores-Saavedra, NV Adsay, JM Crawford JM, et al. Carcinoma of the gallbladder and extrahepatic bile ducts. In: World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Digestive System. Eds: FT Bosman, F Carneiro, RH Hruban, ND Theise. IARC Press: Lyon (2010)

    53. A Tannapfel, F Sommerer, M Benicke, A Katalinic, D Uhlmann, H Witzigmann, J Hauss, C Wittekind: Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut 52, 706-712 (2003)

    54. AA Saetta, P Papanastasiou, NV Michalopoulos, F Gigelou, P Korkolopoulou, T Bei, E Patsouris: Mutational analysis of BRAF in gallbladder carcinomas in association with K-ras and p53 mutations and microsatellite instability. Virchows Arch 445, 179-182 (2004)

    55. SC Chuang, KT Lee, KB Tsai, PC Sheen, E Nagai, K Mizumoto, M Tanaka: Immunohistochemical study of DPC4 and p53 proteins in gallbladder and bile duct cancers. World J Surg 28, 995-1000 (2004)

    56. SA Hahn, D Bartsch, A Schroers, H Galehdari, M Becker, A Ramaswamy, I Schwarte-Waldhoff, H Maschek, W Schmiegel: Mutations of the DPC4/Smad4 gene in biliary tract carcinoma. Cancer Res 58, 1124-1126 (1998)

    57. X Xu, S Kobayashi, W Qiao, C Li, C Xiao, S Radaeva, B Stiles, RH Wang, N Ohara, T Yoshino, D LeRoith, MS Torbenson, GJ Gores, H Wu, B Gao, CX Deng: Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice. J Clin Invest 116, 1843-1852 (2006)

    58. CJ Sherr: Cancer cell cycles. Science 274, 1672-1677 (1996)

    59. WR Jarnagin, DS Klimstra, M Hezel, M Gonen, Y Fong, K Roggin, K Cymes, RP DeMatteo, M D’Angelica, LH Blumgart, B Singh: Differential cell cycle-regulatory protein expression in biliary tract adenocarcinoma: correlation with anatomic site, pathologic variables, and clinical outcome. J Clin Oncol 24, 1152-1160 (2006)

    60. A Ishikawa, M Sasaki, Y Sato, S Ohira, MF Chen, SF Huang, K Oda, Y Nimura, Y Nakanuma: Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis. Hum Pathol 35, 1505-1514 (2004)

    61. AK Virmani, C Muller, A Rathi, S Zoechbauer-Mueller, M Mathis, AF Gazdar: Aberrant methylation during cervical carcinogenesis. Clin Cancer Res 7, 584-589 (2001)

    62. A Tannapfel, M Benicke, A Katalinic, D Uhlmann, F Kockerling, J Hauss, C Wittekind: Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver. Gut 47, 721-727 (2000)

    63. EP Berthiaume, J Wands: The molecular pathogenesis of cholangiocarcinoma. Semin Liver Dis 24, 127-137 (2004)

    64. S Furubo, K Harada, T Shimonishi, K Katayanagi, W Tsui, Y Nakanuma: Protein expression and genetic alterations of p53 and ras in intrahepatic cholangiocarcinoma. Histopathology 35, 230-240 (1999)

    65. Y Jiao, TM Pawlik, RA Anders, FM Selaru, MM Streppel, DJ Lucas, N Niknafs, VB Guthrie, A Maitra, P Argani, GJ Offerhaus, JC Roa, LR Roberts, GJ Gores, I Popescu, ST Alexandrescu, S Dima, M Fassan, M Simbolo, A Mafficini, P Capelli, RT Lawlor, A Ruzzenente, A Guglielmi, G Tortora, F de Braud, A Scarpa, W Jarnagin, D Klimstra, R Karchin, VE Velculescu, RH Hruban, B Vogelstein, KW Kinzler, N Papadopoulos, LD Wood: Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 45, 1470-1473 (2013)

    66. S Zhao, Y Lin, W Xu, W Jiang, Z Zha, P Wang, W Yu, Z Li, L Gong, Y Peng, J Ding, Q Lei, KL Guan, Y Xiong: Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. Science 324, 261-265 (2009)

    67. L Dang, DW White, S Gross, BD Bennett, MA Bittinger, EM Driggers, VR Fantin, HG Jang, S Jin, MC Keenan, KM Marks, RM Prins, PS Ward, KE Yen, LM Liau, JD Rabinowitz, LC Cantley, CB Thompson, MG Vander Heiden, SM Su: Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462, 739-744 (2009)

    68. DR Borger, KK Tanabe, KC Fan, HU Lopez, VR Fantin, KS Straley, DP Schenkein, AF Hezel, M Ancukiewicz, HM Liebman, EL Kwak, JW Clark, DP Ryan, V Deshpande, D Dias-Santagata, LW Ellisen, AX Zhu, AJ Iafrate: Frequent mutation of isocitrate dehydrogenase (IDH)1 and IDH2 in cholangiocarcinoma identified through broad-based tumor genotyping. Oncologist 17, 72-79 (2012)

    69. PS Ward, J Patel, DR Wise, O Abdel-Wahab, BD Bennett, HA Coller, JR Cross, VR Fantin, CV Hedvat, AE Perl, JD Rabinowitz, M Carroll, SM Su, KA Sharp, RL Levine, CB Thompson: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17, 225-234 (2010)

    70. DR Borger, L Goyal, T Yau, RT Poon, M Ancukiewicz, V Deshpande, DC Christiani, HM Liebman, H Yang, H Kim, K Yen, JE Faris, AJ Iafrate, EL Kwak, JW Clark, JN Allen, LS Blaszkowsky, JE Murphy, SK Saha, TS Hong, JY Wo, CR Ferrone, KK Tanabe, N Bardeesy, KS Straley, S Agresta, DP Schenkein, LW Ellisen, DP Ryan, AX Zhu: Circulating oncometabolite 2-hydroxyglutarate is a potential surrogate biomarker in patients with isocitrate dehydrogenase-mutant intrahepatic cholangiocarcinoma. Clin Cancer Res 20, 1884-1890 (2014)

    71. ML Suva, N Riggi, BE Bernstein: Epigenetic reprogramming in cancer. Science 339, 1567-1570 (2013)

    72. I Varela, P Tarpey, K Raine, D Huang, CK Ong, P Stephens, H Davies, D Jones, ML Lin, J Teague, G Bignell, A Butler, J Cho, GL Dalgliesh, D Galappaththige, C Greenman, C Hardy, M Jia, C Latimer, KW Lau, J Marshall, S McLaren, A Menzies, L Mudie, L Stebbings, DA Largaespada, LF Wessels, S Richard, RJ Kahnoski, J Anema, DA Tuveson, PA Perez-Mancera, V Mustonen, A Fischer, DJ Adams, A Rust, W Chan-on, C Subimerb, K Dykema, K Furge, PJ Campbell, BT Teh, MR Stratton, PA Futreal. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539-542 (2011)

    73. M Thompson: Polybromo-1: the chromatin targeting subunit of the PBAF complex. Biochimie 91, 309-319 (2009)

    74. R Chandrasekaran, M Thompson: Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions. Biochem Biophys Res Commun 355, 661-666 (2007)

    75. CR Churi, R Shroff, Y Wang, A Rashid, HC Kang, J Weatherly, M Zuo, R Zinner, D Hong, F Meric-Bernstam, F Janku, CH Crane, L Mishra, JN Vauthey, RA Wolff, G Mills, M Javle: Mutation profiling in cholangiocarcinoma: prognostic and therapeutic implications. PloS one 9, e115383 (2014)

    76. D Wilsker, A Patsialou, SD Zumbrun, S Kim, Y Chen, PB Dallas, E Moran: The DNA-binding properties of the ARID-containing subunits of yeast and mammalian SWI/SNF complexes. Nucleic Acids Res 32, 1345-1353 (2004)

    77. A Bheda, J Shackelford, JS Pagano: Expression and functional studies of ubiquitin C-terminal hydrolase L1 regulated genes. PloS one 4, e6764 (2009)

    78. RA Greenberg, B Sobhian, S Pathania, SB Cantor, Y Nakatani, DM Livingston: Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes. Genes Dev 20, 34-46 (2006)

    79. H Nishikawa, W Wu, A Koike, R Kojima, H Gomi, M Fukuda, T Ohta: BRCA1-associated protein 1 interferes with BRCA1/BARD1 RING heterodimer activity. Cancer Res 69, 111-119 (2009)

Share and Cite
Michele Simbolo, Matteo Fassan, Andrea Mafficini, Rita T. Lawlor, Andrea Ruzzenente, Aldo Scarpa. New genomic landscapes and therapeutic targets for biliary tract cancers. Frontiers in Bioscience-Landmark. 2016. 21(4); 707-718.