Identification and Validation of Colorectal Neoplasia–Specific Methylation Markers for Accurate Classification of Disease

Abstract

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis. (Mol Cancer Res 2007;5(2):153 -63)

DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening

“…We analyzed microarray and qPCR tissue data by using log 10 -transformed percent-methylation rates (PMRs) (12 ). PCR analyses of plasma samples were based on log 10 -transformed DNA concentrations (as measured by the respective methylation-marker, bisDNA, or CFF1 assay) normalized to the plasma volume.…”

“…We used microarrays as previously described to study diseased and healthy colon-derived tissues, other cancer tissues, and samples of healthy tissue from the same organs (12 ). Probes were designed for the region identified in the discovery process (typically a fragment of 250 -500 bp), and up to 1000 bp of flanking sequence was included if it was within a CpG island.…”

“…We assessed Ͼ600 marker candidates identified in genome-wide discovery experiments with a previously described scoring system that accounts for the number of independent identifications of that marker during discovery, the location of the given marker within a CpG island or the promoter region of a gene, identification in multiple experiments, and other criteria (12 ). Only markers with a score of at least 2 were advanced for further analysis.…”

Screening for Colorectal Cancer Based on the Promoter Methylation Status of the Septin 9 Gene in Plasma Cell Free DNA

“…Different strategies have been used to identify and characterize these changes, ranging from analysis of specific candidate gene sequences to genome wide approaches. The discovery process to identify methylation markers for colorectal cancer included both, using arbitrarily primed PCR methods as well as methylation hybridization arrays, and methylation specific PCR [21]. It is clear from this and other studies that the challenge in the approach is not finding methylation differences, of which there are hundreds, but in filtering these differences to identify target sequences that have practical value for the intended diagnostic purpose [22].…”

Electronic DNA detection and diagnostics

“…104 The promoter CPI hyper-methylation in different human cancers has been comprehensively analyzed using bisulphite sequencing, 75 which, to date, has been considered the gold standard technique for directly studying DNA methylation and for validating results obtained using other approaches. 105 The disadvantage of the bisulfite treatment is that it causes DNA degradation, incomplete conversion, low DNA yields, labor intensive and tedious. The bisulfite treatment of DNA analysis is not suitable for large-scale population screening studies of methylation.…”