The clinical significance of preoperative fecal sample SEPT9 methylation in predicting recurrence after endoscopic resection of stage pT1 colorectal cancer

SHENG Xin; LI Li; GU Fahui

doi:10.3969/j.issn.1009-0460.2025.12.004

Abstract：Objective To investigate the prognostic role of tumor suppressor gene septal protein 9 gene methylation(mSEPT9)detection in preoperative fecal samples in predicting early recurrence after endoscopic resection of pT1 colorectal cancer.Methods From March 2021 to June 2023,plasma and fecal samples of 106 patients with pT1 colorectal cancer who underwent endoscopic resection in Baishan Central Hospital were prospectively collected(preoperative baseline and 1 week postoperatively),genome-wide DNA was extracted,and the DNA methylation of SEPT9 target genes was evaluated by bisulfite next-generation directed sequencing,to screen the SEPT9-specific methylation sites.Based on binary Cox proportional hazards regression analysis,CpG methylation sites associated with 2-year postoperative recurrence were identified,and a prognostic model based on mSEPT9 levels in fecal samples was developed to predict recurrence free survival(RFS).All patients will be followed up for at least 2 years.Results In preoperative baseline samples,a consistent trend in methylation rates for all selected CpG sites between plasma and feces was observed,and the average methylation rates were(0.14%-32.75%),which was higher in fecal samples than in plasma samples for almost all selected sites(0.09%-12.93%).At 1 week postoperatively,the methylation rate of 9 CpG sites in the SEPT9 gene was found to be significantly lower compared with preoperative rates(P<0.05).The 2-year RFS rate was 89.62%(95/106).After correcting for some major objective confounders,a higher methylation rate of 15 CpG sites in the SEPT9 gene was associated with a 2-year postoperative recurrence risk(P<0.05)by binary Cox risk-proportional regression analysis.A prognostic model was developed based on these 15 methylated loci.The formula for the regression model was π(x)=1/11+e-ωt+b(π:risk score;x:methylation rate at the locus;ω:coefficient;11+e-ωt+b 1 b:intercept).Based on the Receiver Operating Characteristic curve,the area under the curve of the model predicting 2-year RFS was 0.826(95%CI:0.748-0.903)and had a higher predictive value(P<0.05)relative to conventional tumor markers(0.773).Stratified according to the cut-off value(0.353),40 cases were in the high-risk group and 66 cases were in the low-risk group.The RFS rate of high-risk group patients was significantly lower than that of low-risk group patients(72.50%vs.100.0%,P<0.001).After univariate and multivariate Cox risk proportional regression modeling,fecal mSEPT9 status(HR=3.931,P<0.001)was an independent factor for postoperative colorectal cancer recurrence.According to the Cancer Genome Atlas database and Gene Expression Omnibus data,there was a higher rate of SEPT9 methylation in colon adenocarcinoma(COAD)patients compared with normative colon tissues(P<0.05);furthermore,disease-free survival was shorter in COAD patients with SEPT9 mRNA expression below the median value(P=0.011).Conclusion In this study,a colorectal cancer prognostic model containing 15 SEPT9 gene promoter CpG mutation sites was successfully developed.The model was able to successfully predict the risk of recurrence in the early(within 2 years)after endoscopic resection in patients with pT1 colorectal cancer,thus highlighting the potential of fecal mSEPT9 as a biomarker for colorectal cancer.