Purpose: To identify epigenetic molecular makers in plasma for the early detection of colorectal cancer. Experimental Design: We retrospectively analyzed the methylation status of 10 genes in fresh-frozen tissues and corresponding plasma samples from 243 patients with stage I and II sporadic colorectal cancer, 276 healthy individuals, and plasma from 64 colorectal adenoma patients using methylation-specific PCR. The methylation score (M score) was used to find molecular markers with high sensitivity and specificity. Results: Of the 243 colorectal cancer tissues, methylation was detected in 18% for p14, 34% for p16, 27% for APC, 34% for DAPK, 32% for HLTF, 21% for hMLH1, 39% for MGMT, 24% for RARβ2, 58% for RASSF2A, and 74% for Wif-1. Receiver operator characteristic curve analysis in plasma from 243 patients with cancer and 276 healthy individuals showed that the M score of any single gene had a sensitivity of <40% after controlling for age, sex, and tumor location. The specificity of the M score was not different between multigene and single gene analyses, but the sensitivity of the M score was significantly increased by multigene analysis. For all patients, the M score in a model including APC, MGMT, RASSF2A, and Wif-1 genes had a sensitivity of 86.5% and a specificity of 92.1% when 1.6 was used as a cutoff. In this model, the M score had a positive predictive value of 90.6% and a negative predictive value of 88.8%. Conclusion: The present study suggests that tumor-specific methylation of APC, MGMT, RASSF2A, and Wif-1 genes might be a valuable biomarker in plasma for the early detection of colorectal cancer. (Clin Cancer Res 2009;15(19):6185-91)

BACKGROUNDCpG island hypermethylation has been reported at the promoter region of many tumor suppressor genes in colorectal cancers. However, there are significant interindividual differences in the degree of DNA methylation in colorectal cancers. The objective of the current study was to understand whether single nucleotide polymorphisms (SNPs) around the promoter of a gene are implicated in the interindividual differences of CpG island hypermethylation.METHODSPromoter methylation of the p14ARF gene and messenger RNA (mRNA) expression levels of p14ARF, DNA methyltransferase 1 (DNMT1), and DNMT3b were investigated by using methylation‐specific polymerase chain reaction (PCR) analysis (MSP) and quantitative real‐time PCR analysis in fresh tissues from 188 patients with colorectal cancer. SNPs around the p14ARF promoter were genotyped in DNA from peripheral blood lymphocytes in 300 healthy individuals and in 188 patients with colorectal cancer by using matrix‐assisted laser desorption/ionization mass spectrometry.RESULTSp14ARF methylation was present in 61 of 188 colorectal cancers (32%). Fourteen SNPs among the 20 candidate SNPs were identified as monomorphic in the Korean population studied. Two individual SNPs (−4256 thymine to cytosine [T→C] and −1477 guanine to adenine [G→A]), which were in strong linkage disequilibrium (|D′| = 0.99; correlation coefficient [r2] = 0.95), were associated significantly with p14ARF methylation. Patients who had the CC variant at the−4256 locus or the AA variant at the −1477 locus had 2.42 times (95% confidence interval [95% CI], 1.07–5.46; P = .03) and 2.47 times (95% CI, 1.09–5.56; P = .03) greater risk of p14ARF methylation than patients who had the TT or GG homozygote, respectively, after adjusting for mRNA levels of DNMTs. Four major haplotypes were identified within a block (−4256 T→C, −3631 T→C, −1477 G→A, and +20,188 T→C). p14ARF promoter methylation also was associated significantly with the CCAT haplotype (odds ratio [OR], 8.31; 95% CI, 2.43–28.41; P = .0007) and the CTAC haplotype (OR, 9.71; 95% CI, 1.09–86.24; P = .04).CONCLUSIONSThe current results suggested that SNPs around the p14ARF promoter region may be responsible for the interindividual susceptibility to p14ARF promoter methylation among individuals with colorectal cancer. Cancer 2008. © 2008 American Cancer Society.

BACKGROUND: The objective of this was to identify functional single nucleotide polymorphisms (SNPs) in cyclin-dependent kinases (CDKs) and cyclins that are associated with risk of human cancer. METHODS: First, 45 SNPs in CDKs and cyclins were analyzed in 106 lung cancers and 108 controls for a pilot study. One SNP (reference SNP [rs] 769236, þ1 guanine to adenine [G!A]) at the promoter region of cyclin A2 (CCNA2) also was analyzed in 1989 cancers (300 breast cancers, 450 colorectal cancers, 450 gastric cancers, 367 hepatocellular carcinomas, and 422 lung cancers) and in 1096 controls. Genotyping was performed using matrix-assisted laser desorption-ionization/time-offlight mass spectrometry. Transcriptional activity of the SNP according to the cell cycle was analyzed by using a luciferase reporter assay and fluorescence-activated cell sorting analysis in NIH3T3 cells. RESULTS: In the pilot study, the SNP (rs769236) was associated significantly with the risk of lung cancer. In the expanded study, multivariate logistic regression indicated that the AA homozygous variant of the SNP was associated significantly with the development of lung cancer (P < .0001; codominant model), colorectal cancer (P < .0001), and hepatocellular carcinoma (P ¼ .02) but not with breast cancer or gastric cancer. The luciferase activity of a 300-base pair construct that contained the A allele was 1.5-fold greater than the activity of a construct with the G allele in NIH3T3 cells. The high luciferase activity of constructs that contained the A allele did not change with cell cycle progression. CONCLUSIONS: The current results suggested that an SNP (rs769236) at the promoter of CCNA2 may be associated significantly with increased risk of colon, liver, and lung cancers. Cancer 2011;117:4080-