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)

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrenceassociated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif-1, sFRP1, integrin a4, CDH1, DAP kinase and RARb2 genes in 251 ESCCs. The methylation status was determined by methylation-specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARb2, 43% for CDH1, 21% for integrin a4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif-1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR 5 5.26, 95% CI 5 1.48-18.67; p 5 0.01) and a poor recurrence-free survival after surgery (HR 5 3.13, 95% CI 5 1.21-8.09; p 5 0.02). The hazard of failure after recurrence was about 13.17 (95% CI 5 2.46-70.41; p 5 0.003) times higher in patients with Wif-1 methylation than in those without. For stage II cancers, integrin a4 methylation was associated with an increased risk of recurrence (OR 5 3.03, 95% CI 5 1.09-8.37; p 5 0.03) and a poor recurrence-free survival (HR 5 2.12, 95% CI 5 1.13-3.98; p 5 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin a4 genes may be used as recurrence-associated prognostic indicators in stage I and stage II ESCCs, respectively.

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This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non-small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation-specific PCR in 271 formalin-fixed paraffin-embedded tissues and 27 fresh-frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence-specific siRNA-mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh-frozen tumor tissues were significantly lower than in matched normal tissues (P<0.0001; Wilcoxon signed-rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (P=0.12) and Ki-67 proliferation index (P=0.15). However, patients with HOXA9 hypermethylation had poor recurrence-free survival (hazard ratio=3.98, 95% confidence interval = 1.07-17.09, P=0.01) in never-smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence-free survival in never-smokers with NSCLC.