Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer

Abstract

BackgroundColorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. Frequent aberrant DNA methylation is known to occur in selected genes and early during CRC development, which has emerged as a new epigenetic biomarker for early detection of CRC. Previously, we reported that we identified that CpG sites of SDC2 were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of SDC2 methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying SDC2 methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of SDC2 methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for SDC2 methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC.MethodsBisulfite-pyrosequencing assay was performed to measure the SDC2 methylation status in tissue samples. For methylation analysis in stool DNA, a highly sensitive and accurate method was applied which implements consecutive two rounds of PCR consisting of unidirectional linear target enrichment (LTE) of SDC2 and quantitative methylation-specific real time PCR (qMSP) for SDC2, named as meSDC2 LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies).ResultsPositive SDC2 methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. SDC2 methylation level also significantly (P < 0.01) increased according to the severity of lesions. In stool DNA test for SDC2 methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (n = 50) and precancerous lesions (n = 21) with healthy subjects (n = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%.ConclusionsTaken together, our result indicates that stool DNA-based SDC2 methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.

Multiplex methylated DNA testing in plasma with high sensitivity and specificity for colorectal cancer screening

“…Such a difference could be due to lower ctDNA level in plasma than tumor DNA in stool and the 2/3 scoring algorithm used by plasma methylated SDC2 test. On the contrary, the 47.8% and 88.9% sensitivity of the low‐cost ColoDefense test for AA and CRC detection and its 92.8% specificity were quite comparable to those of Cologuard and stool SDC2 tests, thus providing a viable alternative for CRC screening and prevention for developing countries.…”

“…However, the positive detection rate for ColoDefense in 12.5 pg fully methylated genomic DNA was apparently higher than that for methylated SDC2 (45.8% vs 83.3%) or methylated SEPT9 (62.5% vs 83.3%) alone. For the analytical sensitivity of the assay, LoD is defined as the target concentration that produces positive result in more than 95% of replicate experiments . As such, the LoD of methylated SDC2 qPCR alone, methylated SEPT9 qPCR alone, and ColoDefense test were 100 pg, 50 pg, and 25pg, respectively, equivalent to ~33.3, ~15.2, and ~7.6 copies of human genome, indicating that the combination of methylated SDC2 and methylated SEPT9 could achieve higher sensitivity than either single biomarker alone.…”

“…Methylated SDC2 has been reported as a blood‐based biomarker for CRC in recent years. Several studies have reported high sensitivity and specificity of methylated SDC2 for CRC screening in serum, plasma or stool samples . Moreover, sensitivities of methylated SDC2 from these studies could be improved by nested PCR or normalization of the level of methylated SDC2 against ACTB level.…”

“…Meanwhile, several stool methylated SDC2 tests for CRC screening were reported in recently years. Oh et al showed that stool methylated SDC2 alone had a 90.0% sensitivity for detecting CRC and a 33.3% sensitivity for detecting NAA with a specificity of 90.9% . Niu et al also reported a stool methylated SDC2 test that detected 81.1% of CRC and 58.2% of AA with a specificity of 93.3% .…”

“…Several studies have reported high sensitivity and specificity of methylated SDC2 for CRC screening in serum, plasma or stool samples. 10,15,20 Moreover, sensitivities of methylated SDC2 from these studies could be improved by nested PCR or normalization of the level of methylated SDC2 against ACTB level. Oh et al showed the LoD of methylated SDC2 alone without nested PCR was 100 pg genomic DNA, consistent with our result ( Table 2).…”