Identifizierung und Validierung von spezifischen Methylierungsmarkern zur Klassifizierung von kolorektalen Karzinomen und normalem Kolongewebe

Abstract

Colorectal cancer is the second most common cause of malignant death in industrialized countries. More accurate detection markers are needed to improve the effectiveness and efficiency of both the screening and surveillance of colorectal neoplasia. Aberrant DNA methylation occurs early in oncogenesis, is stable once initiated, and can be assayed in both primary tissues and remote clinical samples. While DNA methylation markers are attractive candidates for colorectal cancer screening, robust searches are needed to discover DNA methylation sequences that optimally discriminate colorectal neoplasia from other diseased and histologically normal tissues for accurate testing in body fluid samples.

Using well-characterized normal and neoplastic specimens, a candidate methylation-based gene microarray of oligonucleotides and a genome-wide methylation discovery process was utilized to identify markers for colorectal neoplasia. To identify markers highly specific for colorectal neoplasia, 12 genes selected from the candidate gene array and 30 fragments from the discovery process were validated on a second methylation-based microarray using a larger, more diverse sample set. Sequences were probed with DNA extracted from 89 colorectal adenocarcinomas, 56 colorectal polyps, 31 inflammatory bowel disease samples, 123 extracolonic cancers, and 67 endoscopically proven normal tissues.

We found markers highly methylated in colorectal neoplasia. The 16 most discriminating methylated sequences comprise both known and predicted genes including EYA4, N33, BCL6, FCGR2A, SIX6, NGFR, Q96PX8, DLX5, C14orf59, PCDH17, HVIAAT, SMAD7, CSPG2, GSK3B, CD44, and TMEFF2. Normal epithelium and extracolonic cancers revealed significantly lower or no methylation.

Individual markers displayed sensitivities > 80% and specificities > 85% indicating the DNA methylation markers identified in this study can correctly classify colorectal neoplasia from other normal and diseased tissues and are attractive marker candidates for clinical testing.