Exploration And Clinical Value Of DNA Methylation Markers In Lymph Node Metastasis Of Colorectal Cancer

Background:Colorectal Cancer(CRC)is a malignant tumor with the third highest incidence and the second highest mortality in the world.Lymph Node Metastasis(LNM)is an important factor affecting the recurrence,prognosis and high mortality of CRC patients.Because the diagnosis of CRC LNM is still not perfect in clinical practice,some CRC patients suffer from the consequences of overtreatment or undertreatment.This study aims to develop and validate novel specific DNA methylation markers for CRC LNM to predict the individual risk of preoperative CRC LNM and to assist and improve CRC clinical treatment protocols.Method:In this study,145 paraffin-embedded tissue samples(79 LNM negative and 66 LNM positive)from Nanfang Hospital and 76 fresh frozen tissue samples(39 LNM negative and 37 LNM positive)from Zhujiang Hospital were collected and their related clinical data were collected.The 30 fresh frozen samples were libraryed and sequenced using the illumina TruSeq-Methyl capture EPIC platform to screen for CRC LNM differential methylation markers.Methylight qPCR was used to verify the differentially methylated markers in 221 samples.The diagnostic efficacy of differentially methylated markers for CRC LNM was analyzed and then compared with imaging and serologic tumor indices as well as clinicopathologic indices.Tissue sections from 56 randomly selected samples were used to validate the diagnostic efficacy of LBX2 antibodies for CRC LNM using immunohistochemical methods.Results:A total of 221 CRC samples were included,including 103 LNM positive cases and 118 LNM egative cases.Twelve differential methylation markers were initially identified by library construction and sequencing comparison.LBX2,STMN3 and SS18L1 were selected to continue to complete the verification after removing the markers with poor performance in the Methylight verification process.The differential methylation marker LBX2 with the best diagnostic efficacy for CRC LNM was finally selected.The AUC of LBX2 was 0.87(95%CI:0.82-0.92,P<0.001),specificity was 87.3%,sensitivity was 75.7%,accuracy was 81.9%,NPV was 80.5%,PPV was 83.9%.Multivariate logistic regression analysis showed that T stage(OR 1.65,95%CI 1.10-2.46,P<0.05)and lymphatic vessel invasion(OR 6.22,95%CI 1.1334.31,P<0.05)were independent risk factors for CRC LNM.Compared with pathological indicators,the diagnostic efficacy of LBX2 for CRC LNM was significantly better than that of T-stage(AUC=0.61,95%CI:0.53-0.68,P<0.001)and lymphatic vessel invasion(AUC=0.63,95%CI:0.56-0.70,P<0.001),Compared with imaging and serological indicators,the diagnostic efficacy of LBX2 for CRC LNM was significantly better than that of imaging(AUC=0.52,95%CI:0.44-0.60,P=0.59),CA199(AUC=0.58,95%CI:0.51-0.66,P<0.05),CEA(AUC=0.56,95%CI:0.48-0.64,P=0.13).Finally,the AUC of LBX2 antibody in the diagnosis of CRC LNM was 0.84(95%CI:0.73-0.94,P<0.001)in the immunohistochemical experiment.Conclusion:In this study,a DNA methylation marker,LBX2,was screened and validated for significant differentiation of CRC LNM status based on NGS technology,and LBX2 was found to be a superior diagnostic marker for CRC LNM compared to traditional clinical indicators.The LBX2 antibody also has good diagnostic efficacy for CRC LNM at the protein level.Therefore,LBX2 is expected to be a novel clinical indicator for predicting preoperative CRC LNM status.