Exome sequencing of liver fluke–associated cholangiocarcinoma

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Opisthorchis viverrini–related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini–related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8–3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini–related CCA.

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Figure 1: MLL3 somatic mutations.

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  1. 1

    McLean, L. & Patel, T. Racial and ethnic variations in the epidemiology of intrahepatic cholangiocarcinoma in the United States. Liver Int. 26, 1047–1053 (2006).

  2. 2

    Sripa, B. et al. Liver fluke induces cholangiocarcinoma. PLoS Med. 4, e201 (2007).

  3. 3

    Vatanasapt, V. et al. Cancer incidence in Thailand, 1988–1991. Cancer Epidemiol. Biomarkers Prev. 4, 475–483 (1995).

  4. 4

    Valle, J. et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N. Engl. J. Med. 362, 1273–1281 (2010).

  5. 5

    Parsons, D.W. et al. The genetic landscape of the childhood cancer medulloblastoma. Science 331, 435–439 (2011).

  6. 6

    Greenman, C. et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153–158 (2007).

  7. 7

    The Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615 (2011).

  8. 8

    van Oijen, M.G. & Slootweg, P.J. Gain-of-function mutations in the tumor suppressor gene p53. Clin. Cancer Res. 6, 2138–2145 (2000).

  9. 9

    Rashid, A. et al. K-ras mutation, p53 overexpression, and microsatellite instability in biliary tract cancers: a population-based study in China. Clin. Cancer Res. 8, 3156–3163 (2002).

  10. 10

    Xu, X. et al. Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice. J. Clin. Invest. 116, 1843–1852 (2006).

  11. 11

    Jones, S. et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321, 1801–1806 (2008).

  12. 12

    Morin, R.D. et al. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476, 298–303 (2011).

  13. 13

    Shinada, K. et al. RNF43 interacts with NEDL1 and regulates p53-mediated transcription. Biochem. Biophys. Res. Commun. 404, 143–147 (2011).

  14. 14

    Low, S.K. et al. Genome-wide association study of pancreatic cancer in Japanese population. PLoS ONE 5, e11824 (2010).

  15. 15

    Deng, Y. & Wu, X. Peg3/Pw1 promotes p53-mediated apoptosis by inducing Bax translocation from cytosol to mitochondria. Proc. Natl. Acad. Sci. USA 97, 12050–12055 (2000).

  16. 16

    Aoki, K. et al. Chromosomal instability by β-catenin/TCF transcription in APC or β-catenin mutant cells. Oncogene 26, 3511–3520 (2007).

  17. 17

    Jiang, X. et al. The imprinted gene PEG3 inhibits Wnt signaling and regulates glioma growth. J. Biol. Chem. 285, 8472–8480 (2010).

  18. 18

    Idziaszczyk, S., Wilson, C.H., Smith, C.G., Adams, D.J. & Cheadle, J.P. Analysis of the frequency of GNAS codon 201 mutations in advanced colorectal cancer. Cancer Genet. Cytogenet. 202, 67–69 (2010).

  19. 19

    Wu, J. et al. Recurrent GNAS nutations define an unexpected pathway for pancreatic cyst development. Sci. Transl. Med. 3, 92ra66 (2011).

  20. 20

    Wang, B. et al. Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell 4, 19–29 (2003).

  21. 21

    Homayounfar, K. et al. Pattern of chromosomal aberrations in primary liver cancers identified by comparative genomic hybridization. Hum. Pathol. 40, 834–842 (2009).

  22. 22

    Wong, N. et al. Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma. J. Hepatol. 37, 633–639 (2002).

  23. 23

    Van Loo, P. et al. Allele-specific copy number analysis of tumors. Proc. Natl. Acad. Sci. USA 107, 16910–16915 (2010).

  24. 24

    Wong, K. et al. Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107, 209–221 (2001).

  25. 25

    Roskams, T. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma. Oncogene 25, 3818–3822 (2006).

  26. 26

    Cardinale, V. et al. Multipotent stem cells in the biliary tree. Ital. J. Anat. Embryol. 115, 85–90 (2010).

  27. 27

    Nakanuma, Y. A novel approach to biliary tract pathology based on similarities to pancreatic counterparts: is the biliary tract an incomplete pancreas? Pathol. Int. 60, 419–429 (2010).

  28. 28

    Gilbert, S.F. Lateral plate mesoderm and endoderm: the specification of liver, pancreas and gallbladder. in Developmental Biology 6th edn, 494–498 (Sinauer Associates, Sunderland, Massachusetts, 2000).

  29. 29

    Li, M. et al. Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma. Nat. Genet. 43, 828–829 (2011).

  30. 30

    Bengtsson, H. et al. A single-array preprocessing method for estimating full-resolution raw copy numbers from all Affymetrix genotyping arrays including GenomeWide SNP 5 & 6. Bioinformatics 25, 2149–2156 (2009).

  31. 31

    Bengtsson, H. et al. TumorBoost: normalization of allele-specific tumor copy numbers from a single pair of tumor-normal genotyping microarrays. BMC Bioinformatics 11, 245 (2010).

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We thank L. Farber for editing the manuscript. This work is supported in part by funding from the Singapore National Medical Research Council (NMRC/STAR/0006/2009), The Singapore Millennium Foundation, The Lee Foundation, the Singapore National Cancer Centre Research Fund, the Duke-NUS Graduate Medical School, the Cancer Science Institute, Singapore, the Research Team Strengthening Grant, the National Genetic Engineering and Biotechnology Center and the National Science and Technology Development Agency, Thailand. W.Y. is the recipient of the NUS Graduate School for Integrative Sciences and Engineering Scholarship, Singapore.

Author information

C.K.O., C.S., C.P., S.W., V.B., S.R., P.T. and B.T.T. conceived the study. V.B., S.R., P.T. and B.T.T. directed the study. C.P., S.W., Y.C., C.-N.Q., K.H.L., V.K., B.S., C.W., P.Y. and V.B. involved in procurement and histopathological review of samples. I.C., W.Y., J.R.M., G.E.A., Y.W., A.O., K.D., K.F., P.A.F. and S.R. performed the bioinformatics data analysis. C.K.O., C.S., C.C.Y.N., B.H.W., S.S.M., V.R., H.L.H., A.G., Z.J.Z., J.W., M.H.L., D.H., P.O. and W.C. performed the experiment on sequencing and the SNP array study. C.K.O. and B.T.T. wrote the manuscript, with the assistance and final approval of all authors.

Correspondence to Vajarabhongsa Bhudhisawasdi or Steve Rozen or Patrick Tan or Bin Tean Teh.

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The authors declare no competing financial interests.

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Supplementary Tables 1–12, Supplementary Figures 1–5 and Supplementary Note (PDF 2125 kb)

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Ong, C., Subimerb, C., Pairojkul, C. et al. Exome sequencing of liver fluke–associated cholangiocarcinoma. Nat Genet 44, 690–693 (2012) doi:10.1038/ng.2273

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