A recent study reported that 5-fluorouracil (5-FU)-based chemotherapy is less effective in treating patients with advanced colorectal cancer demonstrating hypermethylation of the gene. The aim of our study was to confirm and validate these findings in large, uniformly treated, well-characterized patient cohorts. Two cohorts of 783 patients with colorectal cancer: 532 from a population-based, multicenter cohort (EPICOLON I) and 251 patients from a clinic-based trial were used to study the effectiveness of methylation and expression as a predictor of response of colorectal cancer patients to 5-FU-based chemotherapy. DNA methylation status of the gene in patients with colorectal cancer was assessed by quantitative bisulfite pyrosequencing analysis. IHC analysis of the TFAP2E protein expression was also performed. Correlation between TFAP2E methylation status and IHC staining was performed in 607 colorectal cancer samples. Among 357 hypermethylated tumors, only 141 (39.6%) exhibited loss of protein expression. Survival was not affected by hypermethylation in stage IV patients HR, 1.21; 95% confidence interval (CI), 0.79-1.87; log-rank = 0.6. In stage II-III cases, disease-free survival was not influenced by TFAP2E hypermethylation status in 5-FU-treated (HR, 0.91; 95% CI, 0.52-1.59; log-rank = 0.9) as well as in nontreated patients (HR, 0.88; 95% CI, 0.5-1.54; log-rank = 0.7). hypermethylation does not correlate with loss of its protein expression. Our large, systematic, and comprehensive study indicates that methylation and expression may not play a major role in predicting response to 5-FU-based chemotherapy in patients with colorectal cancer. .