Blood leukocytes methylation levels analysis indicate methylated plasma test is a promising tool for colorectal cancer early detection

Abstract

Background: A number of plasma methylated DNA biomarkers related to colorectal cancer (CRC) have been identified. However, the effect of methylation level in leukocytes on plasma-based methylation test was rarely reported. Methods: Blood samples from 213 individuals including 91 CRC patients were collected and separated into 3.5 mL of plasma and paired leukocyte fractions. DNA were extracted from plasma and leukocytes and bisulfite converted, followed by ColoDefense test that detects methylated SEPT9 (mSEPT9) and methylated SDC2 (mSDC2) simultaneously in a single qPCR reaction. Results: Both mSEPT9 and mSDC2 levels in leukocytes exhibited no significant difference among CRC, benign tumors and healthy controls. However, mSEPT9 and mSDC2 levels in plasma were significantly higher in CRC group than those in other groups. The sensitivities of mSEPT9 and mSDC2 alone for detecting CRC with plasma samples were 75.8% and 60.4% with specificities of 94.7% and 86.8%, respectively. These two markers in combination exhibited an improved sensitivity of 85.7% for CRC detection with a specificity of 86.8%, mostly attributable to increased sensitivity of 81.8% for detecting stage 0-II CRC. AUC values for mSEPT9 and mSDC2 alone were 0.864 (95% CI: 0.798 -0.929) and 0.796 (95% CI: 0.719 -0.874), respectively, but improved to 0.972 (95% CI: 0.949 -0.996) when combined for ColoDefense test. Conclusions: The leukocytes gDNA will not affect the performance of plasma ColoDefense test, and plasma ColoDefense test exhibited high sensitivity and specificity in a validation set, demonstrating its potential as a non-invasive and cost-effective method for CRC early detection.

Promising Epigenetic Biomarkers for the Early Detection of Colorectal Cancer: A Systematic Review

“…With our specific filters, we found six studies dedicated to mSDC2 [ 33 , 34 , 35 , 36 , 37 , 38 ] and seven studies for mSEPT9 [ 39 , 40 , 41 , 42 , 43 , 44 , 45 ]; two described mBMP3 performance [ 46 , 47 ], while no study evaluated mNDRG4 alone. A multi-target approach evaluating these biomarkers and others was employed in 16 studies, 5 of them deciphering mSDC2 and mSEPT9 performance [ 48 , 49 , 50 , 51 , 52 ].…”

“…Comparing sensitivities for methylated genes vs. methylated genes plus tumor-specific protein markers (CEA, AFP, CA19-9), an increase was observed (from 35.3 to 47.1% for stage 0+I, 48.6 to 74.3% for stage II, 64.0 to 80% for stage III, and 89.7 to 96.6% for stage IV) [ 51 ]. As for point (b), when mSEPT9 and mSDC2 were assessed from blood leukocytes, no significant difference was noticed between normal subjects vs. individuals with colorectal cancer (stages I-IV) [ 52 ]. At the same time, the ColoDefense test could differentiate CRC cases from precancerous lesions or healthy individuals [ 52 ].…”

“…As for point (b), when mSEPT9 and mSDC2 were assessed from blood leukocytes, no significant difference was noticed between normal subjects vs. individuals with colorectal cancer (stages I-IV) [ 52 ]. At the same time, the ColoDefense test could differentiate CRC cases from precancerous lesions or healthy individuals [ 52 ].…”

“…We considered a biomarker panel any evaluation of two or more methylated genes. Most studies described ColoDefense ® , a yet unapproved test for clinical diagnosis, which combines mSDC2 and mSEPT9 detection [48][49][50][51][52] (Table S3). For stage I CRC, the ColoDefense ® test showed a sensitivity between 69.2-81.8%, stage II CRC 85.7-100%, stage III 88.9-89.7%, and stage IV 75-100%.…”

“…The maximum specificity was 93.2%. Besides the performance evaluation of the blood ColoDefense ® test, two articles had a more particular approach: (a) testing protein biomarkers concomitant with methylated SDC2 and SEPT9 [51] or (b) evaluating a possible false-positive result based on leukocyte genomic DNA [52]. Comparing sensitivities for methylated genes vs. methylated genes plus tumor-specific protein markers (CEA, AFP, CA19-9), an increase was observed (from 35.3 to 47.1% for stage 0+I, 48.6 to 74.3% for stage II, 64.0 to 80% for stage III, and 89.7 to 96.6% for stage IV) [51].…”