A novel DNA methylation panel accurately detects colorectal cancer independently of molecular pathway

Abstract

Background: Colorectal cancer (CRC) is one of the most incident cancers, associated with significant morbidity and mortality, and usually classified into three main molecular pathways: chromosomal instability, microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Currently, available screening methods are either costly or of limited specificity, impairing global implementation. More cost-effective strategies, including DNA methylation-based tests, might prove advantageous. Although some are already available, its performance is suboptimal, entailing the need for better candidate biomarkers. Herein, we tested whether combined use of APC, IGF2, MGMT, RASSF1A, and SEPT9 promoter methylation might accurately detect CRC irrespective of molecular subtype.Methods: Selected genes were validated using formalin-fixed paraffin-embedded tissues from 214 CRC and 50 non-malignant colorectal mucosae (CRN). Promoter methylation levels were assessed using real-time quantitative methylation-specific PCR. MSI and CIMP status were determined. Molecular data were correlated with standard clinicopathological features. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.Results: Except for IGF2, promoter methylation levels were significantly higher in CRC compared to CRN. A threegene panel (MGMT, RASSF1A, SEPT9) identified malignancy with 96.6% sensitivity, 74.0% specificity and 91.5 positive predictive value (area under the curve: 0.97), independently of tumor location, stage, and molecular pathway. Conclusions:Combined promoter methylation analysis of MGMT/RASSF1A/SEPT9 displays a better performance than currently available epigenetic-based biomarkers for CRC, providing the basis for the development of a non-invasive assay to detect CRC irrespective of the molecular pathway.

Cell-Free DNA Methylation of Selected Genes Allows for Early Detection of the Major Cancers in Women

“…Furthermore, SOX17 promoter has been reported as aberrantly methylated in ccfDNA and CTCs from BrC patients’ [ 35 ], albeit its BrC biomarker potential requires further investigation. As for CRC, the significantly higher APC , FOXA1 , RARβ2 , RASSF1A , SCGB3A1 , SEPT9 and SOX17 methylation levels in cancer patients are in line with previous publications [ 16 , 27 , 36 , 37 , 38 , 39 ], although divergent results have been reported for MGMT [ 16 , 27 ]. Concerning LC, and except for SHOX2, our results are in accordance with previous studies [ 17 , 28 , 29 , 40 , 41 ].…”

“…Candidate genes were selected based on an extensive and critical literature review [ 15 , 17 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ], including our previously published results [ 16 , 18 ], and, globally, our findings are mostly in line with previous publications. For BrC, we confirmed APC , FOXA1 , RASSF1A and SCGB3A1 hypermethylation in ccfDNA, in accordance with published studies [ 15 , 23 , 24 ], whereas RARβ2 methylation findings paralleled some previous studies [ 15 , 32 ], but not others, either in tissue [ 33 ], fine-needle washings [ 18 ] or serum [ 23 , 34 ].…”

“…This is a stable genomic alteration which might be detected in serum/plasma circulating cell-free DNA (ccfDNA) [ 13 ], that might even portray tumor heterogeneity better than tissue biopsies [ 14 ]. Several methylated genes have been proposed as tumor biomarkers for BrC, CRC or LC detection, including APC , RARβ2 and RASSF1A [ 15 , 16 , 17 , 18 ]. Although SEPT9 and PTGER4 / SHOX2 methylation-based non- or minimally invasive tests are already commercially available for CRC and LC detection, respectively [ 19 , 20 ], they have limited sensitivity.…”

“…Thus, we aimed to develop a sensitive and specific methylation-based test enabling the simultaneous detection of BrC, CRC and LC in women using ccfDNA. For that purpose, promoter methylation levels of 9 genes ( APC , FOXA1 , MGMT , RARβ2 , RASSF1A , SCGB3A1 , SEPT9 , SHOX2 and SOX17 ), selected based on our previous experience [ 16 , 18 ] and extensive literature review [ 15 , 17 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ], were assessed by multiplex quantitative specific PCR (qMSP) in ccfDNA extracted from plasma samples of female subjects.…”

Stage-differentiated modelling of methylation patterns uncovers salient biomarkers and prognostic signatures in colorectal cancer progression

“…It is recognized that DNA methylation patterns could serve as valid biomarker candidates [70,71]. Freitas et al, have validated the performance of a 3-gene biomarker panel for the detection of colorectal cancer irrespective of the molecular subtype [72]. However optimal stage-salient epigenetic biomarkers have not yet been reported.…”