Integrative genomic analyses of European intrahepatic cholangiocarcinoma: Novel ROS1 fusion gene and PBX1 as prognostic marker

Abstract

Background: Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease.

Methods: We describe an integrated whole-exome sequencing and transcriptomic study of 37 iCCAs patients in Germany.

Results: We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke-associated Asian iCCAs.

Conclusions: By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

Keywords

fusion genes / genomics / intrahepatic cholangiocarcinoma / PBX1 / transcriptomics

References

[1] Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: gLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209-249.

[2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11-30.

[3] Ioka T, Kanai M, Kobayashi S, et al. Randomized phase III study of gemcitabine, cisplatin plus S-1 versus gemcitabine, cisplatin for advanced biliary tract cancer (KHBO1401- MITSUBA). J Hepatobiliary Pancreat Sci. 2023;30:102-110.

[4] Miyazaki M, Shimizu H, Yoshitomi H, et al. Clinical implication of surgical resection for recurrent biliary tract cancer: does it work or not? Ann Gastroenterol Surg. 2017;1:164-170.

[5] Rizvi S, Gores GJ. Pathogenesis, diagnosis, and management of cholangiocarcinoma. Gastroenterology. 2013;145:1215-1229.

[6] Khan SA, Toledano MB, Taylor-Robinson SD. Epidemiology, risk factor, and pathogenesis of cholangiocarcinoma. HPB. 2008;10:77-82.

[7] Banales JM, Marin JJG, Lamarca A, et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol. 2020;17:557-588.

[8] Bridgewater J, Galle PR, Khan SA, et al. Guidelines for the diagnosis and management of intrahepatic cholangiocarcinoma. J Hepatol. 2014;60:1268-1289.

[9] van Vugt JLA, Gaspersz MP, Coelen RJS, et al. The prognostic value of portal vein and hepatic artery involvement in patients with perihilar cholangiocarcinoma. HPB (Oxford). 2018;20:83-92.

[10] Forner A, Vidili G, Rengo M, et al. Clinical presentation, diagnosis and staging of cholangiocarcinoma. Liver Int. 2019;39(Suppl. 1):98-107.

[11] Endo I, Gonen M, Yopp AC, et al. Intrahepatic cholangiocarcinoma: rising frequency, improved survival, and determinants of outcome after resection. Ann Surg. 2008;248:84-96.

[12] Kelley RK, Ueno M, Yoo C, et al. Pembrolizumab in combination with gemcitabine and cisplatin compared with gemcitabine and cisplatin alone for patients with advanced biliary tract cancer (KEYNOTE-966): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2023;401:1853-1865.

[13] Ebata T, Hirano S, Konishi M, et al. Randomized clinical trial of adjuvant gemcitabine chemotherapy versus observation in resected bile duct cancer. Br J Surg. 2018;105:192-202.

[14] Edeline J, Benabdelghani M, Bertaut A, et al. Gemcitabine and oxaliplatin chemotherapy or surveillance in resected biliary tract cancer (PRODIGE 12-ACCORD 18-UNICANCER GI): a randomized phase III study. J Clin Oncol. 2019;37:658-667.

[15] Primrose JN, Neoptolemos J, Palmer DH, et al. Capecitabine compared with observation in resected biliary tract cancer (BILCAP): a randomised, controlled, multicentre, phase 3 study. Lancet Oncol. 2019;20:663-673.

[16] Valle J, Wasan H, Palmer DH, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273-1281.

[17] Okusaka T, Nakachi K, Fukutomi A, et al. Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan. Br J Cancer. 2010;103:469-474.

[18] Blechacz B. Cholangiocarcinoma: current knowledge and new developments. Gut Liver. 2017;11:13-26.

[19] Tyson GL, El-Serag HB. Risk factors for cholangiocarcinoma. Hepatology. 2011;54:173-184.

[20] Sripa B, Kaewkes S, Sithithaworn P, et al. Liver fluke induces cholangiocarcinoma. PLoS Med. 2007;4:1148-1155.

[21] Razumilava N, Gores GJ. Cholangiocarcinoma. Lancet. 2014;383:2168-2179.

[22] Kaewpitoon N, Kaewpitoon S-J, Pengsaa P, Sripa B. Opisthorchis viverrini: the carcinogenic human liver fluke. World J Gastroenterol. 2008;14:666-674.

[23] Zou S, Li J, Zhou H, et al. Mutational landscape of intrahepatic cholangiocarcinoma. Nat Commun. 2014;5:5696.

[24] Nakamura H, Arai Y, Totoki Y, et al. Genomic spectra of biliary tract cancer. Nat Genet. 2015;47:1003-1010.

[25] Jusakul A, Cutcutache I, Yong CH, et al. Whole-genome and epigenomic landscapes of etiologically distinct subtypes of cholangiocarcinoma. Cancer Discov. 2017;7:1116-1135.

[26] Chan-On W, Nairismägi ML, Ong CK, et al. Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers. Nat Genet. 2013;45:1474-1478.

[27] Jiao Y, Pawlik TM, Anders RA, et al. Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet. 2013;45:1470-1473.

[28] Sia D, Losic B, Moeini A, et al. Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma. Nat Commun. 2015;6:6087.

[29] Farshidfar F, Zheng S, Gingras M-C, et al. Integrative genomic analysis of cholangiocarcinoma identifies distinct IDH-mutant molecular profiles. Cell Rep. 2017;18:2780-2794.

[30] Boerner T, Drill E, Pak LM, et al. Genetic determinants of outcome in intrahepatic cholangiocarcinoma. Hepatology. 2021;74:1429-1444.

[31] Borad MJ, Champion MD, Egan JB, et al. Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma. PLoS Genet. 2014;10:e1004135.

[32] Churi CR, Shroff R, Wang Y, et al. Mutation profiling in cholangiocarcinoma: prognostic and therapeutic implications. PLoS One. 2014;9:e115383.

[33] Zhu AX, Macarulla T, Javle MM, et al. Final overall survival efficacy results of Ivosidenib for patients with advanced cholangiocarcinoma with IDH1 mutation: the phase 3 randomized clinical ClarIDHy trial. JAMA Oncol. 2021;7:1669-1677.

[34] Arai Y, Totoki Y, Hosoda F, et al. Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma. Hepatology. 2014;59:1427-1434.

[35] Graham RP, Barr Fritcher EG, Pestova E, et al. Fibroblast growth factor receptor 2 translocations in intrahepatic cholangiocarcinoma. Hum Pathol. 2014;45:1630-1638.

[36] Goyal L, Meric-Bernstam F, Hollebecque A, et al. Futibatinib for FGFR2-rearranged intrahepatic cholangiocarcinoma. N Engl J Med. 2023;388:228-239.

[37] Javle M, Lowery M, Shroff RT, et al. Phase II study of BGJ398 in patients with FGFR-Altered advanced cholangiocarcinoma. J Clin Oncol. 2018;36:276-282.

[38] Javle M, Roychowdhury S, Kelley RK, et al. Infigratinib (BGJ398) in previously treated patients with advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements: mature results from a multicentre, open-label, single-arm, phase 2 study. lancet Gastroenterol Hepatol. 2021;6:803-815.

[39] Ritchie ME, Phipson B, Wu Det al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47.

[40] Mootha VK, Lindgren CM, Eriksson K-F, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267-273.

[41] Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545-15550.

[42] Mayakonda A, Lin D-C, Assenov Y, et al. Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Res. 2018;28:1747-1756.

[43] Maruyama M, Kobayashi N, Westerman KA, et al. Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT. Transplantation. 2004;77:446-451.

[44] Kusaka Y, Tokiwa T, Sato J. Establishment and characterization of a cell line from a human cholangiocellular carcinoma. Res Exp Med (Berl). 1988;188:367-375.

[45] Miyagiwa M, Ichida T, Tokiwa T, et al. A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium. In Vitro Cell Dev Biol. 1989;25:503-510.

[46] Goeppert B, Frauenschuh L, Zucknick M, et al. Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer. Br J Cancer. 2013;109:2665-2674.

[47] Javle M, Bekaii-Saab T, Jain A, et al. Biliary cancer: utility of next-generation sequencing for clinical management. Cancer. 2016;122:3838-3847.

[48] Kendre G, Murugesan K, Brummer T, et al. Charting co-mutation patterns associated with actionable drivers in intrahepatic cholangiocarcinoma. J Hepatol. 2023;78:614-626.

[49] Sia D, Hoshida Y, Villanueva A, et al. Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes. Gastroenterology. 2013;144:829-840.

[50] Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science (80-). 2013;340:1546-1558.

[51] Cristinziano G, Porru M, Lamberti D, et al. FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma. J Hepatol. 2021;75:351-362.

[52] Kendall T, Verheij J, Gaudio E, et al. Anatomical, histomorphological and molecular classification of cholangiocarcinoma. Liver Int. 2019;39:7-18.

[53] Rahi H, Olave MC, Fritchie KJ, et al. Gene fusions in gastrointestinal tract cancers. Genes Chromosom Cancer. 2022;61:285-297.

[54] Zhang K, Chu K, Wu X, et al. Amplification of FRS2 and activation of FGFR/FRS2 signaling pathway in high-grade liposarcoma. Cancer Res. 2013;73:1298-1307.

[55] Pu X, Ye Q, Cai J, et al. Typing FGFR2 translocation determines the response to targeted therapy of intrahepatic cholangiocarcinomas. Cell Death Dis. 2021;12:256.

[56] Jain A, Borad MJ, Kelley RK, et al. Cholangiocarcinoma with FGFR genetic aberrations: a unique clinical phenotype. JCO Precis Oncol. 2018;2:1-12.

[57] Kendre G, Marhenke S, Lorz G, et al. The co-mutational spectrum determines the therapeutic response in murine FGFR2 fusion-driven cholangiocarcinoma. Hepatology. 2021;74:1357-1370.

[58] Neumann O, Burn TC, Allgäuer M, et al. Genomic architecture of FGFR2 fusions in cholangiocarcinoma and its implication for molecular testing. Br J Cancer. 2022;127:1540-1549.

[59] Ou S-HI, Chalmers ZR, Azada MC, et al. Identification of a novel TMEM106B-ROS1 fusion variant in lung adenocarcinoma by comprehensive genomic profiling. Lung Cancer. 2015;88:352-354.

[60] Drilon A, Jenkins C, Iyer S, et al. ROS1-dependent cancers—biology, diagnostics and therapeutics. Nat Rev Clin Oncol. 2021;18:35-55.

[61] Qin A, Johnson A, Ross JS, et al. Detection of known and novel FGFR fusions in non-small cell lung cancer by comprehensive genomic profiling. J Thorac Oncol. 2019;14:54-62.

[62] Lim SM, Yoo JE, Lim KH, et al. Rare incidence of ROS1 rearrangement in cholangiocarcinoma. Cancer Res Treat. 2017;49:185-192.

[63] Berasain C, Fernández-Barrena MG, Avila MA. New molecular interactions of c-Myc in cholangiocarcinoma may open new therapeutic opportunities. Hepatology. 2016;64:336-339.

[64] Vaquero J, Guedj N, Clapéron A, et al. Epithelial-mesenchymal transition in cholangiocarcinoma: from clinical evidence to regulatory networks. J Hepatol. 2017;66:424-441.

[65] Bertrand D, Chng KR, Sherbaf FG, et al. Patient-specific driver gene prediction and risk assessment through integrated network analysis of cancer omics profiles. Nucleic Acids Res. 2015;43:e44.

[66] Chaisaingmongkol J, Budhu A, Dang H, et al. Common molecular subtypes among asian hepatocellular carcinoma and cholangiocarcinoma. Cancer Cell. 2017;32:57-70. e3.

[67] Montal R, Sia D, Montironi C, et al. Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma. J Hepatol. 2020;73:315-327.

[68] Wagner BJ, Plum PS, Apel K, et al. Protein-loss of SWI/SNF-complex core subunits influences prognosis dependent on histological subtypes of intra- and extrahepatic cholangiocarcinoma. Oncol Lett. 2021;21:349.

[69] Takada K, Kubo T, Kikuchi J, et al. Effect of comprehensive cancer genomic profiling on therapeutic strategies and clinical outcomes in patients with advanced biliary tract cancer: a prospective multicenter study. Front Oncol. 2022;12:988527.

[70] Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49-63.

[71] Zou X, Tang X-Y, Qu Z-Y, et al. Targeting the PDGF/PDGFR signaling pathway for cancer therapy: a review. Int J Biol Macromol. 2022;202:539-557.

[72] Cadamuro M, Nardo G, Indraccolo S, et al. Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma. Hepatology. 2013;58:1042-1053.

[73] Yamashita S, Morine Y, Imura S, et al. A new pathological classification of intrahepatic cholangiocarcinoma according to protein expression of SSTR2 and Bcl2. World J Surg Oncol. 2021;19:142.

[74] Fingas CD, Mertens JC, Razumilava N, et al. Targeting PDGFR-β in Cholangiocarcinoma. Liver Int. 2012;32:400-409.

[75] DiMartino JF, Selleri L, Traver D, et al. The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver. Blood. 2001;98:618-626.

[76] Nourse J, Mellentin JD, Galili N, et al. Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor. Cell. 1990;60:535-545.

[77] Kamps MP, Murre C, Sun XH, Baltimore D. A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL. Cell. 1990;60:547-555.

[78] Veiga RN, de Oliveira JC, Gradia DF. PBX1: a key character of the hallmarks of cancer. J Mol Med (Berl). 2021;99:1667-1680.

[79] Gingras M-C, Covington KR, Chang DK, et al. Ampullary cancers harbor ELF3 tumor suppressor gene mutations and exhibit frequent WNT dysregulation. Cell Rep. 2016;14:907-919.

[80] Yachida S, Wood LD, Suzuki M, et al. Genomic sequencing identifies ELF3 as a driver of ampullary carcinoma. Cancer Cell. 2016;29:229-240.

[81] Saleh MM, Scheffler M, Merkelbach-Bruse S, et al. Comprehensive analysis of TP53 and KEAP1 mutations and their impact on survival in localized- and advanced-stage NSCLC. J Thorac Oncol. 2022;17:76-88.

[82] Subbiah V, Lassen U, Élez E, et al. Dabrafenib plus trametinib in patients with BRAFV600E-mutated biliary tract cancer (ROAR): a phase 2, open-label, single-arm, multicentre basket trial. Lancet Oncol. 2020;21:1234-1243.

[83] Ye Z, Zhang Y, Chen J, et al. First-line PD-1 inhibitors combination therapy for patients with advanced cholangiocarcinoma: a retrospective real-world study. Int Immunopharmacol. 2023;120:110344.

[84] Zhu C, Li H, Yang X, et al. Efficacy, safety, and prognostic factors of PD-1 inhibitors combined with lenvatinib and Gemox chemotherapy as first-line treatment in advanced intrahepatic cholangiocarcinoma: a multicenter real-world study. Cancer Immunol Immunother. 2023;72(9):2949-2960.

[85] Gupta A, Dixon E. Epidemiology and risk factors: intrahepatic cholangiocarcinoma. Hepatobiliary Surg Nutr. 2017;6:101-104.

[86] Nagtegaal ID, Odze RD, Klimstra D, et al. The 2019 WHO classification of tumours of the digestive system. Histopathology. 2020;76:182-188.

[87] Liau JY, Tsai JH, Yuan RH, et al. Morphological subclassification of intrahepatic cholangiocarcinoma: etiological, clinicopathological, and molecular features. Mod Pathol. 2014;27:1163-1173.

[88] Akita M, Fujikura K, Ajiki T, et al. Dichotomy in intrahepatic cholangiocarcinomas based on histologic similarities to hilar cholangiocarcinomas. Mod Pathol. 2017;30:986-997.

[89] Komuta M, Govaere O, Vandecaveye V, et al. Histological diversity in cholangiocellular carcinoma reflects the different cholangiocyte phenotypes. Hepatology. 2012;55:1876-1888.

[90] Aishima S, Oda Y. Pathogenesis and classification of intrahepatic cholangiocarcinoma: different characters of perihilar large duct type versus peripheral small duct type. J Hepatobiliary Pancreat Sci. 2015;22:94-100.

[91] Moeini A, Sia D, Bardeesy N, et al. Molecular pathogenesis and targeted therapies for intrahepatic cholangiocarcinoma. Clin Cancer Res. 2016;22:291-300.

[92] Jakubowski CD, Mohan AA, Kamel IR, Yarchoan M. Response to Crizotinib in ROS1 fusion-positive intrahepatic cholangiocarcinoma. JCO Precis Oncol. 2020;4:825-828.

[93] Gu T-L, Deng X, Huang F, et al. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma. PLoS One. 2011;6:e15640.

[94] Di Rocco G, Mavilio F, Zappavigna V. Functional dissection of a transcriptionally active, target-specific Hox-Pbx complex. EMBO J. 1997;16:3644-3654.

[95] Saleh M, Huang H, Green NC, Featherstone MS. A conformational change in PBX1A is necessary for its nuclear localization. Exp Cell Res. 2000;260:105-115.

[96] Asahara H, Dutta S, Kao H-Y, et al. Pbx-Hox heterodimers recruit coactivator-corepressor complexes in an isoform-specific manner. Mol Cell Biol. 1999;19:8219-8225.

[97] Xu Y, Zhao W, Olson SD, et al. Alternative splicing links histone modifications to stem cell fate decision. Genome Biol. 2018;19:133.

[98] Schnabel CA, Selleri L, Jacobs Y, et al. Expression of Pbx1b during mammalian organogenesis. Mech Dev. 2001;100:131-135.

[99] Liu Y, Zhai E, Chen J, et al. m6A-mediated regulation of PBX1-GCH1 axis promotes gastric cancer proliferation and metastasis by elevating tetrahydrobiopterin levels. Cancer Commun. 2022;42:327-344.

[100] Lin Z, Li Y, Han X, et al. Targeting SPHK1/PBX1 axis induced cell cycle arrest in non-small cell lung cancer. Int J Mol Sci. 2022;23:12741.

[101] Nagel S, Pommerenke C, Meyer C, et al. Establishment of the TALE-code reveals aberrantly activated homeobox gene PBX1 in Hodgkin lymphoma. PLoS One. 2021;16:1-23.

[102] Huang T-W, Cheng FHC, Yan C-CS, et al. Interplay between ceRNA and epigenetic control of microRNA: modelling approaches with application to the role of estrogen in ovarian cancer. Int J Mol Sci. 2022;23:2277.

[103] Zhu X, Wei L, Bai Y, et al. FoxC1 promotes epithelial-mesenchymal transition through PBX1 dependent transactivation of ZEB2 in esophageal cancer. Am J Cancer Res. 2017;7:1642-1653.

[104] Magnani L, Ballantyne EB, Zhang X, Lupien M. PBX1 genomic pioneer function drives ERα signaling underlying progression in breast cancer. PLoS Genet. 2011;7:e1002368.

[105] Jozwik KM, Carroll JS. Pioneer factors in hormone-dependent cancers. Nat Rev Cancer. 2012;12:381-385.

[106] Magnani L, Patten DK, Nguyen VTM, et al. The pioneer factor PBX1 is a novel driver of metastatic progression in ER’-positive breast cancer. Oncotarget. 2015;6:21878-21891.

[107] Grebbin BM, Schulte D. PBX1 as pioneer factor: a case still open. Front Cell Dev Biol. 2017;5:9.

[108] Depreter B, De Moerloose B, Vandepoele K, et al. Deciphering molecular heterogeneity in pediatric AML using a cancer vs. normal transcriptomic approach. Pediatr Res. 2021;89:1695-1705.

RIGHTS & PERMISSIONS

2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.