Identification of an epigenetic biomarker panel with high sensitivity and specificity for colorectal cancer and adenomas

Abstract

Background: The presence of cancer-specific DNA methylation patterns in epithelial colorectal cells in human feces provides the prospect of a simple, non-invasive screening test for colorectal cancer and its precursor, the adenoma. This study investigates a panel of epigenetic markers for the detection of colorectal cancer and adenomas.

Methods: Candidate biomarkers were subjected to quantitative methylation analysis in test sets of tissue samples from colorectal cancers, adenomas, and normal colonic mucosa. All findings were verified in independent clinical validation series. A total of 523 human samples were included in the study. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the biomarker panel.

Results: Promoter hypermethylation of the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 was frequent in both colorectal cancers (65-94%) and adenomas (35-91%), whereas normal mucosa samples were rarely (0-5%) methylated. The combined sensitivity of at least two positives among the six markers was 94% for colorectal cancers and 93% for adenoma samples, with a specificity of 98%. The resulting areas under the ROC curve were 0.984 for cancers and 0.968 for adenomas versus normal mucosa.

Conclusions: The novel epigenetic marker panel shows very high sensitivity and specificity for both colorectal cancers and adenomas. Our findings suggest this biomarker panel to be highly suitable for early tumor detection.

Figure 1

DNA promoter hypermethylation status of 13 candidate genes in colon cancer cell lines. Colon cancer cell lines were used as in vitro models to explore the DNA promoter methylation frequency of 13 candidate biomarkers. Only candidates with methylation frequencies equal to or higher than 80% (CNRIP1, INA, BEX1, FBN1, SNCA, and C3orf14) were subjected to methylation analysis in tissue samples.

Figure 2

Summary of promoter methylation status in test sets of colorectal cancer and normal colorectal tissue. Red color indicates methylated sample, green color indicates unmethylated sample, and grey color indicates samples that were not successfully amplified and thereby not scorable. Gene promoters in the upper panel (CNRIP1, FBN1, INA and SNCA) were methylated in 10% or less of the normal tissue samples tested and represent biomarkers with potentially high specificities, and thus suitable for a future diagnostic test. Gene promoters in the lower panel (C3orf14 and BEX1) were methylated more frequently than 10% in normal tissue samples, which might limit the specificity in a test situation, and were excluded from further analyses.

Figure 3

Percent methylated reference (PMR) values of all biomarkers in combined test and validation sets of normal mucosa, adenomas, and colorectal cancer. Note that for SPG20 two outliers (PMR > 150) are excluded from the graph. The SPG20 promoter methylation status has previously been published in the same sample series [9].

Figure 4

Summary of genetic and epigenetic findings in colorectal cancers (test and validation sets). Red color: methylated (CNRIP1, FBN1, INA, MAL, SNCA, and/or SPG20), mutated (BRAF), and/or MSI positive samples. Green color: unmethylated, wt (BRAF), and/or MSS/MSI-low samples. Biomarker panel positive samples have co-methylation of two or more of the six biomarkers. The SPG20 promoter methylation status has previously been published in the same sample series [9].

Figure 5

Receiver Operating Characteristics (ROC) curves for methylation of individual and combined biomarkers in colorectal cancers and adenomas versus normal mucosa (test and validation sets). The area under the ROC curve (AUC) signifies the accuracy of the individual and combined biomarkers for distinguishing colorectal cancers (A and C) and adenomas (B and D) from normal colorectal tissue samples. A) Colorectal cancers versus controls for individual biomarkers. B) Adenomas versus controls for individual biomarkers. All six biomarkers are combined in C) Colorectal cancers versus controls and D) adenomas versus controls. Asymptotic significance, standard error and 95% confidence interval measurements for all values can be found in Additional file 1, Table S3. The ROC curves for the SPG20 biomarker has been published previously in the same sample series [9].

Figure 6

Direct bisulfite sequencing of CNRIP1, FBN1, INA, and SNCA confirmed methylation status as assessed by methylation-specific polymerase chain reaction (MSP). A) CNRIP1. B) FBN1. C) INA. D) SNCA. For all panels, the upper part is a schematic presentation of the CpG sites (vertical bars) amplified by the bisulfite sequencing primers (110 to 470, NM_015463; -85 to 325, NM_000138; -110 to 71, NM_032727; and -169 to 91, NM_000345, respectively). The transcription start site is represented by +1 and the arrows indicate the location of the MSP and qMSP primers. For the lower part of the panels, black circles represent methylated CpGs (the presence of more than 80% cytosine); white circles represent unmethylated CpGs (0 to 20% cytosine); and gray circles represent partially methylated sites (20-80% cytosine). The column of U, M, and U/M at the right side of this lower part lists the methylation status of the respective cell lines as assessed by us using MSP analyses. The transcription start point of CNRIP1 is here according to hg17 NM_015463. When the gene was annotated the transcription start point was moved 340 bases upstream of the indicated position. Abbreviations: MSP, methylation-specific polymerase chain reaction; s, sense; as, antisense; p, probe; U, unmethylated; M, methylated; U/M, presence of both unmethylated and methylated band.

Figure 7

Real-time PCR analysis of SNCA in cancer cell lines with known methylation status. A) Gene expression and DNA promoter methylation status of SNCA in cancer cell lines. The quantitative gene expression levels are displayed as ratios between the median of SNCA and the average of two endogenous controls, GUSB and ACTB. *Partially methylated signifies cell lines with the presence of both a methylated and unmethylated band for the MSP analysis, which most likely reflect monoallelic or heterogeneous methylation. B) Relative gene expression of SNCA in six colon cancer cell lines treated with 1 μM of 5-aza-2'deoxycytidine for 72 h (AZA), 0.5 μM of trichostatin A for 12 h (TSA), and a combination of both drugs (AZA+TSA).