Abstract
BackgroundColon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA.MethodsHierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann–Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy.ResultsIn the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue.ConclusionsWe identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.
Highlights
Colon cancer (CC) is treatable if detected in its early stages
In silico study of 22 CpG loci hypermethylated in colon cancer and breast cancer Based on previous studies showing high performance of methylated markers in detecting breast cancer [26], proven cancer markers were investigated for detection of CC
Individual gene methylation of the 13 candidate genes identified by in silico analysis was quantified in the training set (Table 1, Fig. 2) of fresh frozen colon tissue samples using quantitative multiplex methyl‐ ation-specific PCR (QM-MSP) [30]
Summary
Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. CRC incidence and mortality rates have continued to rise in many low- and middle-income countries such as some Eastern European countries and diverse populations in Latin America and Asia [5,6,7]. Limited resources in these countries and as a result, less accessible and effective screening programs, lead to later stage at diagnosis rendering treatment more extensive and less successful [8]. The global challenge is to establish a novel, easy, quick and low-cost CRC detection method tailored especially for low resource countries facing rising trends in CRC incidence and mortality, as populations in these countries adopt a more western lifestyle [9]
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