DNA methylation patterns play a crucial role in establishing a stable gene expression profile [28]. It is recognized as a significant biomarker for early colorectal cancer (CRC) detection, offering promise in reducing CRC mortality rates [29]. While numerous DNA methylation biomarkers for CRC detection have been identified, the precise selection of CpG sites has not been thoroughly considered, with only a few DNA methylation panels being tested in fecal samples. Our study involving 236 CRC patients and healthy controls, DNA methylation testing and model construction were conducted on fecal and tissue samples. A panel of 29 CpG sites in genes SCD2, SEPT9, and VIM from fecal samples was identified, and a model was developed to demonstrate its potential as a biomarker for early CRC screening. This research serves as a valuable resource for identifying DNA methylated biomarkers in fecal samples for CRC screening and highlights their potential clinical application.
The results demonstrate that our model effectively distinguishes CRC from normal individuals. In the verification cohort, the model showed an overall sensitivity of 91.43% and specificity of 100% in detecting CRC. These results was superior to the detection of SDC2 gene methylation in feces alone (sensitivity 77.0%, specificity 98.1%)[30], VIM gene methylation in feces (sensitivity 72.5%, specificity 86.9%)[31], and the combined detection of SEPT9 and SDC2 gene methylation (sensitivity 89.1%, specificity 90.8%)[14]. The excellent performance of this model can be attributed to the combined methylation assessment of the SCD2, SEPT9, and VIM genes, which enhances the labeling signal and improves sensitivity and specificity. Hypermethylation of any of these 3 genes was considered indicative of increased CRC risk during model development. Previous studies have also suggested that incorporating multiple gene methylation markers can enhance detection accuracy[32]. In addition, one-to-one screening for CRC hypermethylation sites using tissue and stool samples is more compelling. This screening method must collect both tumor tissue and stool samples from CRC patients. Difficulty in sample collection makes this method rarely used. Even so this method overcomes the effect of tumor molecular heterogeneity to some extent, and is able to accurately screen for CpG sites with the highest predictive value among hotspot genes. Thus, the process reduces the weakened of the sensitivity and specificity of the assay by sites of no predictive value.
In this study, most of the methylated sites of genes SCD2, SEPT9 and VIM were hypermethylated in tumor tissues and CRC feces, while they were hypomethylated in control fecal samples (Fig. 3). With AUC value of 99.31% (Fig. 4), the model constructed from their gene combinations was well discriminated between CRC patients and healthy subjects (Fig. 5). SEPT9 gene is an tumor suppressor gene. The aberrant DNA methylation reduces its transcriptional activity, which in turn leads to dysregulation of gene expression and aberrant physiological functions, which may cause cancer. SEPT9 promoter V2 region is methylated in CRC tissues and rarely methylated in normal colonic mucosal[33]. SDC2 encodes a membrane protein that plays a role in cell proliferation and migration. Oh et al[34] examined the methylation of the SDC2 gene in CRC tissues and normal tissues and found that hypermethylation was present only in tumor tissues with a probability of 100%. Cytoskeletal proteins encoded by the VIM gene are thought to be involved in cancer invasion and metastasis. Methylation of the VIM promoter is detectable in CRC but rarely in normal individuals. VIM promoter methylation in fecal samples has high sensitivity and specificity for CRC screening[35]. The methylation of the three selected genes is rarely detected in normal individuals and they have great potential to detect CRC. Although WNT2, BMP3 and NDRG4 genes showed hypermethylation in CRC tissues in this study, they were poorly discriminated between CRC fecal samples and healthy controls (Fig. 2). Previous studies have found NDRG4 methylation to have high sensitivity and specificity in stool and urine samples (positivity rates of 72.6 and 76.2%, respectively[36]) while the average methylation rate of this gene in feces was slightly lower in this study. Decrease in test results may be related to differences in assay methods and patient group. The genes associated with prognosis in this study, SFRP2 and P16, were methylated at approximately 10% in CRC tissues, not well distinct in both CRC feces and healthy controls (Fig. 2). It is hypothesized that these two genes have poor potential for prognostic testing in CRC using feces.
CRC tumors grow in specific locations and CRC cells (includes tumor cells from early stage carcinomas) are constantly shed into the lumen of the colon and released directly into the feces, which lays the foundation for the use of fecal DNA as a screening method for CRC[37]. Blood tests are also important for early screening of CRC. Since apoptotic and necrotic CRC cells release free DNA into the bloodstream, blood testing is feasible. However, DNA testing of serum samples tends to exhibit high background noise due to other diseases. Some biomarkers (e.g., methylation of the SFRP2 gene) can not only indicate CRC but also mammary cancer which will affect the specificity of the test. Human DNA extracted from feces, the other hand, originates directly from tubular lumen shedding is more likely to be derived from CRC than from metastases or other primary tumors. It has also been found that markers released from cell shedding of cancer cells in the intestines may invade the bloodstream before blood vessels[38]. Therefore, feces testing is more accurate for early screening of CRC. In addition, the modeling method of logistic regression has good performance[39]. Previous studies have shown that logistic regression modeling methods can improve the potential for CRC risk prediction[40], and thus the choice of modeling method may also be responsible for the better performance of the selected gene methylation sites and models in CRC detection.
SCD2, SEPT9, and VIM are emerging as key candidate methylation mutation biomarkers in the field of CRC research[41]. While these gene locus show promise in early detection, it is important to note that our study sample size was limited. Further research with a larger and more diverse sample pool is needed to validate their clinical significance as biomarkers. Despite this, the model presented in this study shows potential as a non-invasive screening tool for detecting colorectal cancer in its early stages, emphasizing the importance of additional validation in a real-world clinical setting.