Genome‐wide DNA methylation profiles in hepatocellular carcinoma

Alterations in DNA methylation frequently occur in hepatocellular cancer (HCC). We have previously demonstrated that hypermethylation in candidate genes can be detected in plasma DNA before HCC diagnosis. To identify, with a genome‐wide approach, additional genes hypermethylated in HCC that could be used for more accurate analysis of plasma DNA for early diagnosis, we analyzed tumor and adjacent nontumor tissues from 62 Taiwanese HCC cases using Illumina methylation arrays (Illumina, Inc., San Diego, CA) that screen 26,486 autosomal CpG sites. After Bonferroni adjustment, a total of 2,324 CpG sites significantly differed in methylation level, with 684 CpG sites significantly hypermethylated and 1,640 hypomethylated in tumor, compared to nontumor tissues. Array data were validated with pyrosequencing in a subset of five of these genes; correlation coefficients ranged from 0.92 to 0.97. Analysis of plasma DNA from 38 cases demonstrated that 37%‐63% of cases had detectable hypermethylated DNA (≥5% methylation) for these five genes individually. At least one of these genes was hypermethylated in 87% of the cases, suggesting that measurement of DNA methylation in plasma samples is feasible. Conclusion: The panel of methylated genes indentified in the current study will be further tested in a large cohort of prospectively collected samples to determine their utility as early biomarkers of HCC. (HEPATOLOGY 2012;55:1799–1810)

[1]  Wei Jiang,et al.  High-throughput DNA methylation profiling using universal bead arrays. , 2006, Genome research.

[2]  I. Tischoff,et al.  DNA methylation in hepatocellular carcinoma. , 2008, World journal of gastroenterology.

[3]  S. Hirohashi,et al.  Inactivation of p16INK4 in hepatocellular carcinoma , 1996, Hepatology.

[4]  R. Weksberg,et al.  Assessment of methylation level prediction accuracy in methyl-DNA immunoprecipitation and sodium bisulfite based microarray platforms , 2011, Epigenetics.

[5]  F. Jasmine,et al.  A genome-wide DNA methylation study in colorectal carcinoma , 2011, BMC Medical Genomics.

[6]  Chien-Jen Chen,et al.  High frequency of promoter hypermethylation of RASSF1A and p16 and its relationship to aflatoxin B1–DNA adduct levels in human hepatocellular carcinoma , 2002, Molecular carcinogenesis.

[7]  W. Yeo,et al.  Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[8]  H. Asakura,et al.  p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. , 1999, Gastroenterology.

[9]  S. Thorgeirsson,et al.  Exploring genomic profiles of hepatocellular carcinoma , 2011, Molecular carcinogenesis.

[10]  S. Thorgeirsson,et al.  A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells , 2006, Nature Medicine.

[11]  C. Boland,et al.  Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma , 2008, Hepatology.

[12]  Nita Ahuja,et al.  DNA methylation and environmental exposures in human hepatocellular carcinoma. , 2002, Journal of the National Cancer Institute.

[13]  R. Yuen,et al.  DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia , 2010, European Journal of Human Genetics.

[14]  T K Lau,et al.  Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. , 1998, American journal of human genetics.

[15]  Chien-Jen Chen,et al.  Silencing of glutathione S-transferase P1 by promoter hypermethylation and its relationship to environmental chemical carcinogens in hepatocellular carcinoma. , 2005, Cancer letters.

[16]  H. Sugimoto,et al.  Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma , 2007, British Journal of Cancer.

[17]  R. Siebert,et al.  Distinct DNA methylation patterns in cirrhotic liver and hepatocellular carcinoma , 2012, International journal of cancer.

[18]  S. Nomoto,et al.  Quantitative promoter methylation analysis of hepatocellular carcinoma, cirrhotic and normal liver , 2008, International journal of cancer.

[19]  A. Dobrovic,et al.  Analysing DNA methylation using bisulphite pyrosequencing. , 2011, Methods in molecular biology.

[20]  S. Imbeaud,et al.  Identification of Novel Oncogenes and Tumor Suppressors in Hepatocellular Carcinoma , 2010, Seminars in liver disease.

[21]  W. Tsai,et al.  Predicting Hepatocellular Carcinoma by Detection of Aberrant Promoter Methylation in Serum DNA , 2007, Clinical Cancer Research.

[22]  Chien-Jen Chen,et al.  Inactivation of the DNA repair gene O6‐methylguanine‐DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1‐DNA adducts and p53 mutation in hepatocellular carcinoma , 2003, International journal of cancer.

[23]  C. J. Chen,et al.  p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. , 1997, Cancer research.

[24]  Sean V. Tavtigian,et al.  Hepatocellular Carcinoma Displays Distinct DNA Methylation Signatures with Potential as Clinical Predictors , 2010, PloS one.

[25]  Lixin Wei,et al.  CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma , 2008, International journal of cancer.

[26]  Hideaki Kato,et al.  Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomas , 2007, Hepatology research : the official journal of the Japan Society of Hepatology.

[27]  H. Kim,et al.  Epigenomic Analysis of Aberrantly Methylated Genes in Colorectal Cancer Identifies Genes Commonly Affected by Epigenetic Alterations , 2011, Annals of Surgical Oncology.

[28]  C. J. Chen,et al.  Aflatoxin B1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and non-tumorous liver tissue. , 1991, Carcinogenesis.

[29]  H. Asakura,et al.  p16INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma , 1999 .

[30]  S. Thorgeirsson,et al.  Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. , 2007, The Journal of clinical investigation.

[31]  Anushya Muruganujan,et al.  PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium , 2009, Nucleic Acids Res..

[32]  Jing Zhao,et al.  Large-scale analysis of the genetic and epigenetic alterations in hepatocellular carcinoma from Southeast China. , 2008, Mutation research.

[33]  J. Issa,et al.  Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. , 2008, Carcinogenesis.

[34]  J. Herman,et al.  Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. , 2003, The American journal of pathology.

[35]  W. Yeo,et al.  High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma , 2005, Liver international : official journal of the International Association for the Study of the Liver.

[36]  Y. Jeng,et al.  Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma , 2007, International journal of cancer.

[37]  H. Ahsan,et al.  Aflatoxin B1 and polycyclic aromatic hydrocarbon adducts, p53 mutations and p16 methylation in liver tissue and plasma of hepatocellular carcinoma patients , 2006, International journal of cancer.