Development of a Multiplex MethyLight Assay for the Detection of Multigene Methylation in Human Colorectal Cancer

In peripheral blood, cell-free methylated DNA has been reported to be a useful biomarker of noninvasive blood screening for the detection of colorectal cancer (CRC), including the genes ALX homeobox 4 (ALX4), septin 9 (SEPT9), or transmembrane protein with EGF-like, and two follistatin-like domains 2 (TMEFF2). Here we report a multiplex MethyLight polymerase chain reaction (PCR) assay that simultaneously detected the methylation status of ALX4, SEPT9, and TMEFF2, as well as quantifying methylation level of these genes in a total of 127 fresh tissue samples and 182 peripheral blood samples from CRC patients. Using the multiplex MethyLight assay, methylated ALX4, SEPT9, and TMEFF2 occurred in 56, 78, and 75% of CRC tissue samples and in 48, 75, and 71% of peripheral blood samples from CRC patients. The sensitivities of the combined study using the three genes as biomarkers for the detection of CRC in primary tissues and peripheral blood samples were 84 and 81%, with specificities of 87 and 90%, respectively. Combining the specificity of real-time PCR, the high throughput of multiplex PCR, and the high sensitivity of multigene detection, this multiplex MethyLight PCR assay may allow for future screening programs with large-scale noninvasive blood testing for early-stage CRC.