Long-range epigenetic silencing at 2q14.2 affects most human colorectal cancers and may have application as a non-invasive biomarker of disease

Abstract

Large chromosomal regions can be suppressed in cancer cells as denoted by hypermethylation of neighbouring CpG islands and downregulation of most genes within the region. We have analysed the extent and prevalence of long-range epigenetic silencing at 2q14.2 (the first and best characterised example of coordinated epigenetic remodelling) and investigated its possible applicability as a non-invasive diagnostic marker of human colorectal cancer using different approaches and biological samples. Hypermethylation of at least one of the CpG islands analysed (EN1, SCTR, INHBB) occurred in most carcinomas (90%), with EN1 methylated in 73 and 40% of carcinomas and adenomas, respectively. Gene suppression was a common phenomenon in all the tumours analysed and affected both methylated and unmethylated genes. Detection of methylated EN1 using bisulfite treatment and melting curve (MC) analysis from stool DNA in patients and controls resulted in a predictive capacity of, 44% sensitivity in positive patients (27% of overall sensitivity) and 97% specificity. We conclude that epigenetic suppression along 2q14.2 is common to most colorectal cancers and the presence of a methylated EN1 CpG island in stool DNA might be used as biomarker of neoplastic disease.

Figure 1

DNA methylation across 2q14.2 in colorectal cancer. (A) Frequencies of the methylation in EN1, SCTR and INHBB CpG islands in normal colonic mucosa, and colorectal adenomas and carcinomas. Y-axis indicates the percentage of samples with methylation for each gene. (B) Incidence of methylation in colorectal carcinomas according Dukes' stage. Y-axis indicates the number of tumours exhibiting methylation in 0, 1, 2 or 3 of the CpG islands analysed (EN1, SCTR and INHBB).

Figure 2

Relative expression levels of the eight genes analysed across the 2q14.2 chromosome region in 17 colorectal tumour tissues and their paired normal mucosa. The log2 of the tumour/normal ratio is represented. Negative values indicate downregulation in the tumour as compared with the respective normal tissue. Expression levels were normalised using the 18S rRNA expression as control. Methylation status was determined using melting curve (MC) analysis and is depicted in column filling as indicated. Grey column indicates genes without promoter CpG island (MARCO, GLI2) or not analysed (TSN).

Figure 3

Overall survival in colorectal cancer patients according to the methylation status of INHBB and EN1. Tumours with methylated INHBB or EN1 showed a reduced survival rate, but differences reached only statistical significance for INHBB or the combination of both, INHBB and EN1.

Figure 4

Detection of EN1 methylation in stool and serum DNA as a diagnostic marker of colorectal cancer. ‘True positive’ indicates the percentage of all colorectal cancer patients with a methylated marker in both the tumour and the test sample (stool or serum DNA). ‘False positive’ corresponds to the percentage of healthy individuals with a methylated result in the test sample. The best score was obtained when melting curve (MC) analysis was applied to detect EN1 methylation in stool DNA (27% of overall sensitivity, 44% sensitivity in positive tumours, and 97% specificity).