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

Abstract

Background: Colorectal 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.

Methods: Bisulfite-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).

Results: Positive 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%.

Conclusions: Taken 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.

Keywords: Colorectal cancer; Early detection; Methylation; Precancerous lesion; SDC2; Stool DNA.

Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of College of Medicine of Soonchunhyang University, Yonsei University College of Medicine and Dongguk University Ilsan Hospital in South Korea.

Consent for publication

Written informed consent was obtained from all study participants according to institutional guidelines.

Competing interests

TaeJeong Oh, HyunIl Oh, YangYei Seo, and Sungwhan An are employees of Genomictree, Inc. TaeJeong Oh and Sungwhan An are shareholders of Genomictree, Inc. The other authors declare that they have no competing interests.

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Figures

Fig. 1

Assessment of methylation levels of the SDC2 gene in colorectal tissues by bisulfite-pyrosequencing. Methylation level of the SDC2 gene was evaluated in normal mucosa (N), hyperplastic (HP) and adenomatous polyps (Ade), and CRC tissues. The MtIs of each sample are represented by box and whisker plots. The difference in MtI of SDC2 is statistically significant at *P < 0.01 calculated by Kruskal-Wallis test in adenomatous polyps vs. normal controls, hyperplastic polyps vs. normal controls, CRC vs. normal controls, and CRC vs. adenomatous polyps

Fig. 2

Schematic diagram and analytic performance of meSDC2 LTE-qMSP. a Outline of meSDC2 LTE-qMSP. LTE was first performed using two primers: SDC2 and COL2A1 antisense primers attached to a universal primer to enrich methylated SDC2 DNA, and consecutive duplex real-time PCR was carried out using methylation-specific SDC2 sense primer, COL2A1-specific sense primer, universal primer, and probes. b The LoD of meSDC2 LTE-qMSP was compared with that of meSDC2-qMSP. Different amounts of HCT116 genomic DNA were diluted in unmethylated genomic DNA in total 20 ng of genomic DNA

Fig. 3

Methylation status of SDC2 in stool DNA by meSDC2 LTE-qMSP test. a meSDC2 LTE-qMSP test was performed in stool DNA from CRC patients in varying stages, adenoma patients (Ade), and healthy normal subjects (N). Distribution of the relative level of SDC2 methylation was expressed in CT values as 40-CT for each sample. A higher 40-CT represents a higher methylation level of SDC2. It is represented as 0, if the SDC2 CT was not detected. Methylation status of the SDC2 gene is plotted as box and whisker plots. The difference in methylation level of SDC2 was statistically significant at **P < 0.01 and *P < 0.05 calculated by Kruskal-Wallis test in CRC patients vs. healthy normal subjects and adenoma patients vs. healthy normal subjects, respectively. b ROC curve was plotted for CRC patients vs. healthy normal subjects. The cutoff value for methylation-positive and AUC are indicated in the plot