A tissue-based comparative effectiveness analysis of biomarkers for early detection of colorectal tumors

Abstract

Objectives: We recently identified a six-gene methylation-based biomarker panel suitable for early detection of colorectal cancer (CRC). In this study, we compared the performance of this novel epi-panel with that of previously identified DNA methylation markers in the same clinical tissue sample sets.

Methods: Quantitative methylation-specific PCR was used to analyze the promoter region of SEPT9 and VIM in a total of 485 tissue samples, divided into test and validation sets. ITGA4, NTRK2, OSMR, and TUBG2 were also included in the analyses. Receiver operating characteristic (ROC) curves were used to compare the performances of the individual biomarkers with that of the novel epi-panel.

Results: SEPT9 and VIM were methylated in 82 and 67% of CRCs (n=169) and in 88 and 54% of the adenomas (n=104). Only 3% of the normal mucosa samples (n=107) were methylated for these genes, confirming that the methylation was highly cancer-specific. Areas under the ROC curve (AUC), distinguishing CRCs from normal mucosa, were 0.94 for SEPT9 and 0.81 for VIM. AUC values for separating adenomas from normal mucosa samples were 0.96 and 0.81 for the same genes. In comparison, the novel epi-panel achieved an AUC of 0.98 (CRC) and 0.97 (adenomas).ITGA4, OSMR, NTRK2, and TUBG2 were methylated in 90, 78, 7, and 1% of the CRCs, and in 76, 77, 3, and 0% of the adenomas. Between 0 and 2% of the normal mucosa samples were methylated for the same genes. ITGA4 and OSMR achieved an AUC of 0.96 and 0.92 (CRC vs. normal mucosa), and 0.93 and 0.92 (adenomas vs. normal mucosa).

Conclusions: We have confirmed the high performance of some of the previously identified DNA methylation markers. Furthermore, we showed that a recently reported epi-panel performed better than the individual DNA methylation biomarkers when analyzed in the same tissue samples. This observation was also true for VIM and SEPT9, which are included in commercially available noninvasive tests for CRC. These results further underscore the value of combining a manageable number of individual markers into a panel, which in addition to having a higher sensitivity and specificity might provide a more profound robustness to a noninvasive test compared with single markers.

Figure 1

Promoter methylation status of the previously identified DNA methylation markers in normal and tumor tissue samples. The figure illustrates the methylation status of the previously identified DNA methylation biomarkers across test and validation sets of colorectal carcinomas (n=169), adenomas (n=104), normal mucosa samples from colorectal cancer (CRC)-free individuals (n=107), and normal mucosa from CRC patients (n=105). For comparison the methylation status of the novel epi-panel for early detection of CRC is included. Green color indicates unmethylated sample, whereas red color indicates methylated sample.

Figure 2

Receiver operating characteristic (ROC) curves for the novel epi-panel and the previously identified DNA methylation markers. The area under the curve conveys the accuracy of the biomarkers in distinguishing colorectal carcinoma (a) or adenoma (b) from normal mucosa.

Figure 3

Bisulfite sequencing of the NTRK2 promoter confirmed the methylation status as assessed by quantitative methylation-specific PCR (qMSP). The figure illustrates the bisulfite sequencing results for the NTRK2 promoter in four colorectal cancer samples and two colon cancer cell lines. Rightmost column reflects the percent of methylated reference (PMR) values for the same samples assessed by qMSP. Red square illustrates methylated CpG site, blue square represents unmethylated site, whereas gray squares represent unknown or undetermined CpG sites.