Aberrant DNA Methylation of SEPT9 and SDC2 in Stool Specimens as an Integrated Biomarker for Colorectal Cancer Early Detection

Abstract

Colorectal cancer (CRC) has become the second leading cause of new cancer cases and the fifth of cancer deaths in China, and early detection is the most effective way to reduce the incidence and mortality of CRC. A number of methylated DNA biomarkers have been found to associate with CRC and precancerous lesions in stool samples, indicating stool methylated DNA biomarkers are potential tools for CRC early detection. In this study, approximately 5 g of stool specimen was collected from 230 subjects (124 in the training set and 106 in the validation set). Stool DNA was extracted and bisulfiteconverted, followed by ColoDefense test, a multiplex qPCR assay, that simultaneously detects methylated SEPT9 (mSEPT9) and methylated SDC2 (mSDC2). Youden index was employed to determine the cut-off value of ColoDefense test for stool specimens. In the training set, the optimized cut-off value of stool ColoDefense test was: mSEPT9 analyzed with 3/3 algorithm and mean mSEPT9 Ct values of <38, or mSDC2 with 2/3 algorithm. Stool ColoDefense test achieved Youden indexes of 79.9 and 57.4% in detecting CRC and advanced adenomas (AA), respectively. Its sensitivities in the training set for AA and CRC were 66.7% (95% CI: 24.1-94.0%) and 89.1% (95% CI: 77.1-95.5%) with a 90.8% (95% CI: 80.3-96.2%) specificity, and AUC was 0.956 (95% CI: 0.924-0.988). In the validation set, its sensitivities for AA and CRC were 66.7% (95% CI: 24.1-94.0%) and 92.3% (95% CI: 78.0-98.0%) with a 93.2% (95% CI: 82.7-97.8%) specificity, and AUC was 0.977 (95% CI: 0.952-1.000). Positive detection rate of stool ColoDefense test has been found to be independent of age, gender, tumor location, and tumor size. In conclusion, stool ColoDefense test demonstrated high sensitivities and specificity for the detection of AA and CRC. Therefore, it has the potential to become a low-cost, convenient, and highly effective tool for CRC early detection.

Methylated SFRP2 and SDC2 in stool specimens for Colorectal Cancer early detection: A cost-effective strategy for Chinese population

“…The diagnoses of the patients were histologically confirmed by the pathologist (Table 1 ). Stools were collected prior to bowel preparation or colonoscopy in single-use disposable sampling boxes (Suzhou VersaBio Technologies Co. Ltd., Kunshan, Jiangsu, China) mounted on toilet seats, and approximately 5 g of each fecal specimen was transferred into a 50 mL centrifuge tube containing 25 mL of preservative buffer (Suzhou VersaBio Technologies Co. Ltd.) to stabilize human genomic DNA in the stool (Figure 1 ) 18 . All stool samples were stored at room temperature for no more than 7 days and transferred to -80 °C for long term storage before usage.…”

“…Using a fast bisulfite conversion kit (Suzhou VersaBio Technologies Co. Ltd.), eluted DNA was converted and purified by adding 150 μL conversion buffer and 25 μL protection buffer, followed by incubation at 80 °C for 45 min. Finally, two washing steps were performed, and the column was air dried and DNA was eluted with 100 μL elution buffer 18 .…”

Feasibility of Methylated CLIP4 in Stool for Early Detection of Colorectal Cancer: A Training Study in Chinese Population

“…It demonstrated sensitivities of 42.4% for AA and 92.3% for CRC with 86.6% specificity in an extensive study ( 11 ). Our previous study also evaluated a combined assay of m SEPT9 and m SDC2 , ColoDefence test, resulting in sensitivities of 66.7% for AA and 92.3% for CRC with 93.2% specificity ( 20 ). Compared to these two multiplex tests, m CLIP4 test achieved an even higher sensitivity for AA by an increase of 12% to 36% with a similarly high specificity.…”

“…Bisulfite conversion of the extracted DNA and purification of the converted DNA were performed with a bisulfite conversion kit (Suzhou VersaBio Technologies Co., Ltd.). Both kits were used according to previously published protocols ( 20 ).…”

Combining Serum DNA Methylation Biomarkers and Protein Tumor Markers Improved Clinical Sensitivity for Early Detection of Colorectal Cancer

“…They were also higher than those of mSEPT9 or methylated SDC2 ( mSDC2 ) alone. In addition, when stool samples were used instead, the sensitivity of the ColoDefense test was further improved to 66.7% for AA detection without any significant impact on its specificity of 91.9%, an enhanced advantage for CRC prevention [ 8 ].…”