The samples were assessed using 2 triplex assays, with ACTB, BMP3, and TFPI2 in the first assay and ACTB, NDRG4, and VIM in the second assay.
The QuARTS reaction was further validated by quantifying methylated genes BMP3, NDRG4, VIM, and TFPI2 in colorectal tissue samples.
Comparing cancer to healthy epithelia, AUC values were 0.95, 0.96, 0.93, and 0.99 for BMP3, NDRG4, VIM, and TFPI2, respectively.
Linear regression analysis of percent methylation and age yielded statistically significant slope in the cancer group for markers BMP3 (P = 0.001), NDRG4 (P < 0.001), VIM (P < 0.001) and TFPI2 (P = 0.01) and in the healthy group for marker TFPI2 (P = 0.03).
We tested the reproducibility of the QuARTS assay by quantifying methylated BMP3, TFPI2, NDRG4, and VIM and the reference gene ACTB in 1 known positive control sample in triplicate 8 times.
In the present study, methylated markers BMP3, NDRG4, VIM, TFPI2, and their combination detected most colorectal cancers and adenomas > 1 cm at high diagnostic specificity when assayed with QuARTS, which is consistent with previous reports by us and others (6, 7, 13-15).