Discriminant markers are required for accurate
cancer screening. We evaluated genes frequently methylated in colorectal
neoplasia to identify the most discriminant ones. Four genes specifically methylated in
colorectal cancer [
bone morphogenetic protein 3 (BMP3), EYA2, aristaless-like homeobox-4 (ALX4), and
vimentin] were selected from 41 candidate genes and evaluated on 74
cancers, 62
adenomas, and 70 normal epithelia. Methylation status was analyzed qualitatively and quantitatively and confirmed by
bisulfite genomic sequencing. Effect of methylation on gene expression was evaluated in five
colon cancer cell lines. K-ras and BRAF mutations were detected by sequencing. Methylation of BMP3, EYA2, ALX4, or
vimentin was detected respectively in 66%, 66%, 68%, and 72% of
cancers; 74%, 48%, 89%, and 84% of
adenomas; and 7%, 5%, 11%, and 11% of normal epithelia (P < 0.01,
cancer or
adenoma versus normal). Based on area under the curve analyses, discrimination was not significantly improved by combining markers. Comethylation was frequent (two genes or more in 72% of
cancers and 84% of
adenomas), associated with proximal
neoplasm site (P < 0.001), and linked with both BRAF and K-ras mutations (P < 0.01). Cell line experiments supported silencing of expression by methylation in all four study genes. This study shows BMP3, EYA2, ALX4, and
vimentin genes are methylated in most
colorectal neoplasms but rarely in normal epithelia. Comethylation of these genes is common, and pursuit of complementary markers for methylation-negative
neoplasms is a rational strategy to optimize screening sensitivity.