Identification and validation of colorectal neoplasia-specific methylation biomarkers based on CTCF-binding sites

Abstract

To date, the sensitivity of currently available biomarkers based on the methylation of gene promoters is suboptimal for detecting adenomas and early-stage colorectal cancer (CRC). We aimed to develop biomarkers with methylated DNA binding sites of the multifunctional transcriptional factor CTCF for early detection of CRC. Using combined analyses of genome-wide occupation and the methylation profile of CTCF-binding sites, we identified candidate CTCF-binding sites. Then, we applied methylation-sensitive high-resolution melting (MS-HRM) and mass spectrometry analysis to screen and validate these candidate sites in diverse sample sets. We identified a set of colorectal neoplasia-specific biomarkers with robust performance. The top five biomarkers were selected and recommended for early detection of colorectal neoplasia. All of the five novel biomarkers exhibited a more robust discriminatory performance than that by BMP3 and NDRG4, two currently acknowledged robust methylation biomarkers. When the five new biomarkers were considered as a marker panel and tumor-positive was defined as having two or more (of the five) positive biomarkers, the marker panel could achieve a sensitivity of 91.67% for adenomas, 97.44% for Stage I CRC, 94.06% for Stage II CRC, 93.62% for Stage III CRC, and 93.54% for total colorectal tumors with a specificity of 94.05%. To our knowledge, this is the first study for colorectal neoplasia-specific methylation biomarkers based on CTCF-binding sites. Using a similar strategy, CTCF-binding sites could be potentially developed into biomarkers for other tumors. In summary, this study opens a new area in developing biomarkers for tumor prevention and treatment.

Keywords: CTCF-binding site; DNA methylation; biomarker; colorectal cancer; early-detection.

Figure 1

(A) Normalized melting profile. (B) Difference plots. The HRM standard melting curve was derived from five samples, with the following ratios of methylated DNA: 0, 1, 10, and 100% methylation. At the annealing temperature of 58°C, methylation levels as low as 1% could be easily detected. As shown in the figure, the methylation level of a representative CTCF-binding site in a tumor tissue is between 0–1%, and its methylation level in the matched normal counterpart is between 10–100%, indicating that the methylation level of this CTCF-binding site is significantly lower in tumor tissues than that in matched normal counterparts.

Figure 2. Hierarchical clustering of all 40 colorectal tissue samples and 23 tumor-specific CTCF-binding sites identified by MS-HRM analysis

Columns, patient samples; rows, tumor-specific CTCF-binding sites. Column annotations include the names of the CTCF-binding sites, row annotations include the names of the sample (N represents normal tissue; T represents tumor tissue) and tumor stages for tumor tissues (0 represents adenomas; I represents Stage I; II represents Stage II). The class information was unknown to the clustering algorithm. The average degree of methylation of each CTCF-binding site in each sample is represented by the decadic logarithm of the methylation proportion ranging from 0% methylated alleles (green) to methylation of all alleles (red). There are two main tissue clusters labeled as N and T. Cluster N consists of 18 normal tissues and two tumor tissues. Cluster T is composed of the remaining 18 tumor tissues.