Aberrant methylation of CpG islands is a common alteration in human colon cancer. Methylation of only a limited number of loci has been studied in aberrant crypt foci (ACF), the earliest identified premalignant lesions in the colon, and in only a limited number of lesions from the same patients. Methylation-specific PCR was used to analyze 35 ACF, samples of normal crypts with the same number of crypts as were in each ACF and 22 cancers from the same patients. Aberrant methylation in cellular retinol-binding protein 1 (CRBP1), MINT31 or H-cadherin (CDH13) was identified in 19 of 35 (54%) ACF. Hypermethylation of CRBP1, MINT31 or CDH13 was more frequent (15 of 22, 68%) in cancers. DNA methylation of CRBP1, MINT31 or CDH13 was not correlated between cancers and ACF from the same patients. Aberrant methylation was not correlated with patient age, number of crypts in the ACF or cancer stage. These results suggest that hypermethylation in the promoter region of some genes is an independent event, occurs early in human colon tumorigenesis and may play an important role during the transformation and progression of some lesions to colon cancers. ' 2005 Wiley-Liss, Inc.Key words: premalignant lesion; epigenetic alteration; hypermethylation; colon cancer Colon cancer is a malignant disease that develops stepwise through pathologically defined stages of tumor progression. Aberrant crypt foci (ACF) are the earliest neoplastic lesions identified microscopically in human colonic mucosa. 1,2 Multiple genetic alterations found in ACF, such as activation of protooncogenes, 3,4 inactivation of tumor-suppressor genes, 4 chromosomal instability 5 and microsatellite instability, 6,7 suggest that ACF are putative precursors of colorectal cancer. Methylation of CpG islands is a molecular alteration common in many cancers. 8 DNA methylation at cytosines located 5 0 to guanosines of CpG dinucleotides in the promoter region inhibits the transcription of genes and causes loss of gene expression. 8 While there have been many studies on the methylation of single or multiple genes in colon cancers 9-13 or polyps, 9,14,15 only a few have looked at methylation in ACF. [16][17][18] CpG island methylator phenotype (CIMP), 9 in which multiple loci are methylated in the same lesion but not in the normal mucosa, has been identified as a novel pathway of colon tumorigenesis. While concordant CIMP was reported for small numbers of polyps and cancers in the same patients 9 and for colonic lesions in patients with hyperplastic polyposis, 15 a lack of concordant methylation was reported for multiple polyps in patients without cancer. 14 ACF studies to date have looked at only a limited number of different samples from the same patient, and none has looked at cancers in the same patients. In our study, altered DNA methylation of the cellular retinol-binding protein 1 (CRBP1), H-cadherin (CDH13) and MINT31 was evaluated in multiple ACF and cancers from the same patients. The frequency of DNA methylation for these 3 loci was lower in ACF than in cancers, ...