Novel target genes and a valid biomarker panel identified for cholangiocarcinoma

Cholangiocarcinoma is notoriously difficult to diagnose, and the mortality rate is high due to late clinical presentation. CpG island promoter methylation is frequently seen in cancer development. In the present study, we aimed at identifying novel epigenetic biomarkers with the potential to improve the diagnostic accuracy of cholangiocarcinoma. Microarray data analyses of cholangiocarcinoma cell lines treated with epigenetic drugs and their untreated counterparts were compared with previously published gene expression profiles of primary tumors and with non-malignant controls. Genes responding to the epigenetic treatment that were simultaneously downregulated in primary cholangiocarcinoma compared with controls (n = 43) were investigated for their promoter methylation status in cancer cell lines from the gastrointestinal tract. Genes commonly methylated in cholangiocarcinoma cell lines were subjected to quantitative methylation-specific polymerase chain reaction in a total of 93 clinical samples (cholangiocarcinomas and non-malignant controls). CDO1, DCLK1, SFRP1 and ZSCAN18, displayed high methylation frequencies in primary tumors and were unmethylated in controls. At least one of these four biomarkers was positive in 87% of the tumor samples, with a specificity of 100%. In conclusion, the novel methylation-based biomarker panel showed high sensitivity and specificity for cholangiocarcinoma. The potential of these markers in early diagnosis of this cancer type should be further explored.

[1]  R. Scott,et al.  Long Term Transcriptional Reactivation of Epigenetically Silenced Genes in Colorectal Cancer Cells Requires DNA Hypomethylation and Histone Acetylation , 2011, PloS one.

[2]  K. D. Sørensen,et al.  Identification and validation of highly frequent CpG island hypermethylation in colorectal adenomas and carcinomas , 2011, International journal of cancer.

[3]  J. Foekens,et al.  CDO1 Promoter Methylation is a Biomarker for Outcome Prediction of Anthracycline Treated, Estrogen Receptor-Positive, Lymph Node-Positive Breast Cancer Patients , 2010, BMC Cancer.

[4]  Y. Lee,et al.  Aberrant promoter CpG islands methylation of tumor suppressor genes in cholangiocarcinoma. , 2008, Oncology Research.

[5]  J. Lozano,et al.  Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer , 2010, PloS one.

[6]  S. Dışıbeyaz,et al.  Extraordinarily Elevated Ca19-9 in Benign Conditions: A Case Report and Review of the Literature , 2001, Tumori.

[7]  Yusuke Nakamura,et al.  Genome‐wide analysis of gene expression in human intrahepatic cholangiocarcinoma , 2005, Hepatology.

[8]  Mirang Kim,et al.  CpG methylation in exon 1 of transcription factor 4 increases with age in normal gastric mucosa and is associated with gene silencing in intestinal-type gastric cancers , 2008, Carcinogenesis.

[9]  Hwoong-Yong Jung,et al.  Aberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinoma. , 2002, The American journal of pathology.

[10]  M. Lerner,et al.  DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism. , 2011, Cancer research.

[11]  H. Soejima,et al.  Tumor Progression Through Epigenetic Gene Silencing of O6−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers , 2005, Annals of Surgical Oncology.

[12]  M. Bertagnolli,et al.  Molecular origins of cancer: Molecular basis of colorectal cancer. , 2009, The New England journal of medicine.

[13]  D. Iliopoulos,et al.  Epigenetic aberrations during oncogenesis , 2011, Cellular and Molecular Life Sciences.

[14]  R. Lothe,et al.  ADAMTS1, CRABP1, and NR3C1 Identified as Epigenetically Deregulated Genes in Colorectal Tumorigenesis , 2006, Cellular oncology : the official journal of the International Society for Cellular Oncology.

[15]  P. Selby,et al.  Circulating markers of biliary malignancy: opportunities in proteomics? , 2008, The Lancet. Oncology.

[16]  In Ah Kim,et al.  Promoter CpG island hypermethylation during breast cancer progression , 2010, Virchows Archiv.

[17]  N. Tsuboyama-Kasaoka,et al.  Human cysteine dioxygenase gene: structural organization, tissue-specific expression and downregulation by phorbol 12-myristate 13-acetate. , 1999, Bioscience, biotechnology, and biochemistry.

[18]  Jennifer D. Brooks,et al.  Promoter methylation and the detection of breast cancer , 2009, Cancer Causes & Control.

[19]  J. Herman,et al.  Promoter methylation profiles of tumor suppressor genes in intrahepatic and extrahepatic cholangiocarcinoma , 2005, Modern Pathology.

[20]  J. Herman,et al.  Alterations in DNA methylation: a fundamental aspect of neoplasia. , 1998, Advances in cancer research.

[21]  N. Cho,et al.  Methylation profiles of multiple CpG island loci in extrahepatic cholangiocarcinoma versus those of intrahepatic cholangiocarcinomas. , 2007, Archives of pathology & laboratory medicine.

[22]  Keitaro Matsuo,et al.  Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[23]  D. Hua,et al.  Quantitative analysis of multiple methylated genes in plasma for the diagnosis and prognosis of hepatocellular carcinoma. , 2011, Experimental and molecular pathology.

[24]  J. Issa,et al.  CpG island methylation in aberrant crypt foci of the colorectum. , 2002, The American journal of pathology.

[25]  Joachim Diebold,et al.  Functional epigenomics identifies genes frequently silenced in prostate cancer. , 2005, Cancer research.

[26]  M. Goggins,et al.  Epigenetic Down-Regulation of CDKN1C/p57KIP2 in Pancreatic Ductal Neoplasms Identified by Gene Expression Profiling , 2005, Clinical Cancer Research.

[27]  Christian B. Woods,et al.  Analysis of repetitive element DNA methylation by MethyLight , 2005, Nucleic acids research.

[28]  N. Wong,et al.  Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma. , 2002, Journal of hepatology.

[29]  A. Pollett,et al.  Promoter methylation of Wnt antagonists DKK1 and SFRP1 is associated with opposing tumor subtypes in two large populations of colorectal cancer patients. , 2011, Carcinogenesis.

[30]  R. Lothe,et al.  DNA hypermethylation of MAL: a promising diagnostic biomarker for colorectal tumors. , 2007, Gastroenterology.

[31]  M. Toyota,et al.  DNA methylation and cancer pathways in gastrointestinal tumors. , 2008, Pharmacogenomics.

[32]  T. Ørntoft,et al.  Reference values and biological variation for tumor marker CA 19-9 in serum for different Lewis and secretor genotypes and evaluation of secretor and Lewis genotyping in a Caucasian population. , 1999, Clinical chemistry.

[33]  N. Cho,et al.  Bile-based detection of extrahepatic cholangiocarcinoma with quantitative DNA methylation markers and its high sensitivity. , 2012, The Journal of molecular diagnostics : JMD.

[34]  R. Lothe,et al.  Three Epigenetic Biomarkers, GDF15, TMEFF2, and VIM, Accurately Predict Bladder Cancer from DNA-Based Analyses of Urine Samples , 2010, Clinical Cancer Research.

[35]  Jinfeng Sun,et al.  Methylation status of NEUROG2 and NID2 improves the diagnosis of stage I NSCLC. , 2012, Oncology letters.

[36]  Anurag Srivastava,et al.  Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients. , 2007, Life sciences.

[37]  N Clarke,et al.  Genome-wide methylation analysis identifies epigenetically inactivated candidate tumour suppressor genes in renal cell carcinoma , 2011, Oncogene.

[38]  K. Lindor,et al.  Incidence and Risk Factors for Cholangiocarcinoma in Primary Sclerosing Cholangitis , 2004, American Journal of Gastroenterology.

[39]  Young-Joon Kim,et al.  Genome-Wide Analysis of DNA Methylation and the Gene Expression Change in Lung Cancer , 2012, Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer.

[40]  Yoshiaki Kawano,et al.  Secreted antagonists of the Wnt signalling pathway , 2003, Journal of Cell Science.

[41]  Michael Gregor,et al.  Serum and bile markers for cholangiocarcinoma. , 2004, Seminars in liver disease.

[42]  K. McGlynn,et al.  Rising incidence of intrahepatic cholangiocarcinoma in the United States: a true increase? , 2004, Journal of hepatology.

[43]  Robert Brown,et al.  CpG-island methylation study of liver fluke-related cholangiocarcinoma , 2011, British Journal of Cancer.

[44]  S. Spechler,et al.  Acid, bile, and CDX: the ABCs of making Barrett's metaplasia. , 2008, American journal of physiology. Gastrointestinal and liver physiology.

[45]  K. Boberg,et al.  Cholangiocarcinoma in Primary Sclerosing Cholangitis: Risk Factors and Clinical Presentation , 2002, Scandinavian journal of gastroenterology.

[46]  Yuman Fong,et al.  Genome wide analysis and clinical correlation of chromosomal and transcriptional mutations in cancers of the biliary tract , 2008, Journal of experimental & clinical cancer research : CR.

[47]  G. Gores,et al.  Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. , 2007, Gastroenterology.

[48]  R. Wiesner,et al.  Cholangiocarcinoma complicating primary sclerosing cholangitis. , 1991, Annals of surgery.

[49]  G. Gores,et al.  Advances in the diagnosis of cholangiocarcinoma in patients with primary sclerosing cholangitis , 2006, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society.

[50]  R. Lothe,et al.  Identification of an epigenetic biomarker panel with high sensitivity and specificity for colorectal cancer and adenomas , 2011, Molecular Cancer.

[51]  P. Grundy,et al.  Gene expression analysis of blastemal component reveals genes associated with relapse mechanism in Wilms tumour. , 2011, European journal of cancer.

[52]  Daiya Takai,et al.  The CpG Island Searcher: A new WWW resource , 2003, Silico Biol..

[53]  Peter A. Jones,et al.  The fundamental role of epigenetic events in cancer , 2002, Nature Reviews Genetics.

[54]  A. Tannapfel,et al.  Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors , 2005, International journal of cancer.

[55]  W. Steinberg.  The clinical utility of the CA 19-9 tumor-associated antigen. , 1990, The American journal of gastroenterology.

[56]  C. Pairojkul,et al.  Cholangiocarcinoma: lessons from Thailand , 2008, Current opinion in gastroenterology.

[57]  A. Bird.  DNA methylation patterns and epigenetic memory. , 2002, Genes & development.

[58]  Daiya Takai,et al.  Comprehensive analysis of CpG islands in human chromosomes 21 and 22 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[59]  D. Hanahan,et al.  Hallmarks of Cancer: The Next Generation , 2011, Cell.

[60]  F. L. Thaete,et al.  Biliary tract carcinoma complicating primary sclerosing cholangitis: evaluation with CT, cholangiography, US, and MR imaging. , 1998, Radiology.

[61]  S. Belinsky,et al.  Gene-promoter hypermethylation as a biomarker in lung cancer , 2004, Nature Reviews Cancer.