Detection of Circulating Tumor DNA Methylation in Diagnosis of Colorectal Cancer.

INTRODUCTION: Emerging evidence has demonstrated the potential of the circulating tumor DNA (ctDNA) methylation in the application of cancer diagnosis. METHODS: Three genes including Septin9, Syndecan-2 (SDC2), and branched-chain amino acid transaminase 1 (BCAT1), which have been well demonstrated to have aberrant expression in colorectal cancer (CRC) as tumor suppressors, were selected for detection. A total of 234 peripheral plasma samples from 104 patients with CRC and 130 patients with colorectal polyps, and 60 plasma samples from healthy controls, were collected before any treatment. A real-time polymerase chain reaction-based gene panel was used to detect the methylation of Septin9, SDC2, and BCAT1. The composite score (P) was calculated according to the cycle threshold values of the 3 methylated genes using the logistic regression equation. RESULTS: The ctDNA methylation of the 3 genes had a significantly higher level in patients with CRC, compared with patients with colorectal polyps and healthy controls. The composite score (P) showed association with tumor stages in CRC but not with the tumor location (colon or rectum). In addition, BCAT1 and Septin9 showed better performance for CRC diagnosis, by which CRC was able to distinguish from polyps with sensitivity of 83.7%, specificity of 93.9%, and area under the curve of 0.908. The diagnostic efficiency was significantly improved by combining composite score (P), carcinoembryonic antigen, and fecal immunochemical test for hemoglobin (area under the curve = 0.962). DISCUSSION: The composite score (P) derived from the ctDNA methylation levels of Septin9, SDC2, and BCAT1 can be used for CRC diagnosis with high sensitivity and high specificity. A combination of ctDNA methylation, carcinoembryonic antigen, and fecal immunochemical test for hemoglobin was proved to be the most effective approach to diagnose CRC.

A novel plasma based early colorectal cancer screening assay base on methylated SDC2 and SFRP2.

BACKGROUND: Methylated SFRP2 was previously reported as a non-invasive biomarker for colorectal cancer (CRC) detection with a relatively low sensitivity for early stage CRC. The purpose of this study was to evaluate the performance of a new plasma based CRC screening assay, SpecColon test, which tested methylated SFRP2 and SDC2 simultaneously in a single qPCR reaction, in detecting CRC and advanced adenomas (AA). METHOD: One milliliter plasma of 122 CRC patients, 12 AA patients, 93 patients with benign polyps, and 91 normal individuals were collected from the Affiliated Hospital of Xuzhou Medical University, and all samples were examined by SpecColon test. RESULTS: The sensitivities for detecting AA and CRC by methylated SFRP2 alone were 50.0% (95% CI: 22.2-77.7%) and 63.1% (95% CI: 53.9-71.5%) with a specificity of 90.1% (95% CI: 81.6-95.1%). The sensitivities by methylated SDC2 alone were 33.3% (95% CI: 11.3-64.6%) and 56.6% (95% CI: 47.3-65.4%) with a specificity of 95.6% (95% CI: 88.5-98.6%). However, when methylated SFRP2 and methylated SDC2 were combined, the sensitivities for AA and CRC detection improved to 58.3% (95% CI: 28.6-83.5%) and 76.2% (95% CI: 67.5-83.3%) with a specificity of 87.9% (95% CI: 79.0-93.5%). The positive detection rates of benign polyp group and normal control group showed no significant difference (p > 0.01), whereas AA and CRC groups had significantly higher positive detection rates than normal individual group (p < 0.001). CONCLUSION: The sensitivities for AA and early stage CRC by combined test of methylated SFRP2 and methylated SDC2, the so called SpecColon test, improved upon those by either biomarker alone without significant impact on the specificity. It has the potential to become a powerful, convenient and highly effective screening tool for early CRC screening.

Performance of a Novel Blood-Based Early Colorectal Cancer Screening Assay in Remaining Serum after the Blood Biochemical Test.

BACKGROUND: Combination of multiple biomarkers was an effective strategy to improve sensitivity in cancer diagnosis and screening. However, the performance of the combination of methylated SEPT9 and SDC2 for detection of colorectal cancer (CRC) has yet to be reported. METHODS: A new qPCR-based assay combining the detection of methylated SEPT9 and SDC2 was used. Methylation statuses of SEPT9 and SDC2 were examined in 19 sets of cancer tissues and paired adjacent tissues and further evaluated with 225 serum samples, including 111 CRC patients and 114 no evidence of disease individuals. RESULTS: SEPT9 and SDC2 methylation levels were higher in 94.7% and 100.0% of cancer tissues than in their paired adjacent tissues. The sensitivities for detecting CRC by SEPT9 methylation alone and SDC2 methylation alone were 73.0% (95% CI: 63.6-80.8%) and 71.2% (95% CI: 61.8-79.2%), respectively, with the same specificity of 95.6% (95% CI: 89.6-98.4%). However, when SEPT9 methylation was combined with SDC2 methylation to detect CRC, the sensitivity was improved to 86.5% (95% CI: 78.4-92.0%) with a specificity of 92.1% (95% CI: 85.1-96.1%). CONCLUSION: The combination of methylated SEPT9 and SDC2 detection in serum has the potential to be a noninvasive strategy for CRC screening.

Plasma Angiopoietin-2/-1 Ratio is Elevated and Angiopoietin-2 Levels Correlate With Plasma Syndecan-1 Following Pediatric Trauma.

BACKGROUND: Angiopoietin-1 (Agpt-1) and Agpt-2 are cytokine regulators of vascular endothelial integrity. Elevated plasma Agpt-2 levels and ratios of Agpt-2:Agpt-1 are associated with adverse outcomes in adult trauma and pediatric sepsis populations. However, the behavior of the angiopoietins after pediatric trauma has not been characterized, and their relationship to endothelial glycocalyx damage, indicated by plasma syndecan-1 (Syn-1) levels, has not been established. METHODS: We performed a secondary analysis of prospectively collected data from 52 pediatric trauma patients and 12 control patients at a level one pediatric trauma center from 2013 to 2016. We measured Agpt-1, Agpt-2, and Syn-1 levels from plasma taken upon hospital arrival and 24 h after admission. Angiopoietin levels were compared to controls, and the correlation between Agpt-2 and Syn-1 was assessed. RESULTS: Plasma Agpt-1 and Agpt-2 levels are elevated immediately after pediatric trauma compared with controls. At 24 h, trauma patients demonstrated significantly elevated plasma Agpt-2:Agpt-1 ratios relative to controls due to decline of Agpt-1 levels to near that of controls. Higher 24-h Agpt-2 levels are associated with more hypoperfusion, and elevated 24-h Agpt-2:Agpt-1 ratios are associated with adverse clinical outcomes. Significant positive correlations between Agpt-2 and Syn-1 upon admission and at 24 h after injury were identified. CONCLUSION: Our findings suggest dysregulation of circulating angiopoietins after pediatric trauma that may be linked to endothelial glycocalyx injury. Larger prospective studies are needed to validate these findings and determine the relationship of Agpt-2 with other markers of endotheliopathy.

Systematic review of blood diagnostic markers in colorectal cancer.

The purpose of this systematic review was to compare the diagnostic ability of blood markers for colorectal cancer (CRC). A systematic review of the literature for diagnostic blood markers for primary human colorectal cancer over the last 5 years was performed. The primary outcome was to assess the diagnostic ability of these markers in diagnosing colorectal cancer. The secondary outcome was to see whether the marker was compared to other markers. The tertiary outcome was to assess diagnostic ability in early versus late CRC, including stage IV disease. We identified 51 studies (29 prospective, 14 retrospective, and 8 meta-analyses). The markers were divided in broadly four groups: nucleic acids (RNA/DNA/messenger RNA/microRNAs), cytokines, antibodies, and proteins. The most promising circulating markers identified among the nucleid acids were NEAT_v2 non-coding RNA, SDC2 methylated DNA, and SEPT9 methylated DNA. The most promising cytokine to detect CRC was interleukin 8, and the most promising circulating proteins were CA11-19 glycoprotein and DC-SIGN/DC-SIGNR. Sensitivities of these markers for detecting primary colorectal carcinoma ranged from 70 to 98% and specificities from 84 to 98.7%. The best studied blood marker was SEPT9 methylated DNA, which showed great variability with sensitivities ranging from 48.2 to 95.6% and specificities from 80 to 98.9%, making its clinical applicability challenging. If combined with fecal immunochemical test (FIT), the sensitivity improved from 78 to 94% in detecting CRC. Methylated SEPT9, methylated SDC2, and -SIGN/DC-SIGNR protein had better sensitivity and specificity than CEA or CA 19-9. With the exception of SEPT9 which is currently being implemented as a screening test for CRC all other markers lacked reproducibility and standardization and were studied in relatively small population samples.

Genome-wide identification and validation of a novel methylation biomarker, SDC2, for blood-based detection of colorectal cancer.

Aberrant DNA methylation has shown promise as a biomarker for the early detection of cancer. To discover novel genes frequently methylated at an early stage in colorectal cancer (CRC), DNA microarray analysis coupled with enriched methylated DNA was performed in primary tumors and compared with adjacent nontumor tissues of 12 patients with CRC at stages I to IV. Stepwise filtering for candidate selection in microarray data analysis yielded a set of genes that are highly methylated across all CRC tumors and that can be used as a composite biomarker for CRC detection. Verification assay identified the SDC2 gene as a potential methylation biomarker for early CRC detection. In clinical validation in tissues from 139 CRC patients, a much higher level of aberrant SDC2 methylation was measured in most primary tumors (97.8%), compared with corresponding nontumor tissue of CRC patients, irrespective of clinical stage. Clinical validation of SDC2 methylation in serum DNA from CRC patients (n = 131) at stages I to IV and from healthy individuals (n = 125) by quantitative methylation-specific PCR demonstrated a high sensitivity of 87.0% (95% CI, 80.0% to 92.3%) in detecting cancers, with a specificity of 95.2% (95% CI, 89.8% to 98.2%). Importantly, sensitivity at stage I was 92.3%, indicating the potential of SDC2 methylation as a blood-based DNA test for early detection of CRC.