Fig. 3

Detection of colon adenocarcinoma using cumulative methylation of 13 methylated markers. Fresh frozen colon carcinoma and adjacent normal (Adj. Normal) tissues were analyzed for cumulative methylation using QM-MSP. A panel of 13 genes, a subset of the 22 CpG loci (18 genes) indicated in Fig. 1, were evaluated. The percent methylation (%M) of each gene and the cumulative methylation index (CMI; the sum of %M for all genes in the panel) were determined for each sample. Box–whiskers plots. In training set (A) and test set (D) significantly higher methylation was observed in carcinoma compared to adjacent normal tissues (P < 0.0001; Mann–Whitney). Histogram plots. Data from samples in the training set (B) and test set (E) are plotted. For each sample (X-axis), the height of the histogram bar indicates the level of cumulative methylation (Y-axis), each colored segment represents an individual gene, and the size of the segment is proportional to the %M of that gene. Microsatellite instability (MSI) status for each carcinoma is indicated by the bar map below the X- axis: blue (microsatellite unstable), black (microsatellite stable), and gray (unknown). Receiver operating characteristic (ROC) curves. The ROC curve from the training set (C), carcinoma versus normal control, identified a laboratory threshold maximized for sensitivity for detecting carcinoma at a specificity > 90%. This threshold (CMI = 88.5) was locked and used to determine the assay sensitivity and specificity for the test set (F)