DNA Methylation Marker To Identify Lymph Node Metastasis Of Colorectal Cancer

BackgroundColorectal cancer(CRC)is the third largest malignant tumor in the world.Currently,the incidence and mortality of CRC are increasing.Until now,the incidence and mortality have risen to the third and second place respectively.At present,imaging(such as CT,MRI,PET-CT,etc.)is the main method to diagnose CRC with lymph node metastasis,but the diagnostic accuracy is not high.Therefore,whether CRC with lymph node metastasis or not,the current clinical treatment guidelines for CRC suggest lymph node dissection during surgery.However,lymph node dissection could result in many serious complications such as intestinal adhesion,intestinal obstruction,lymphatic leakage,peritonitis.Therefore,the CRC patients without lymph node metastasis might experience transition therapy.It is well known that DNA methylation is a major epigenetic modification and it has been proved to be closely related to the occurrence and development of cancer.This study aims to identify CRC lymph node metastasis status on the molecular level based on DNA methylation sites found in CRC tumor tissue obtained before operation,it could more accurately serve the clinical diagnosis and treatment of CRC.MethodFirstly,DNA was extracted from fresh frozen tissues of 30 CRC patients(11 lymph node negative patients and 19 lymph node positive patients).After transformation,genome-wide methylation sequencing was performed.The difference sites of CRC lymph node metastasis were screened by difference analysis.Secondly,primers and probes matching the selected DNA methylation sites were designed to verify 221 CRC samples(118 lymph node negative patients and 103 lymph node positive patients)using Methylight real-time quantitative PCR.Thirdly,the screening DNA methylation sites were compared with imaging,serum carcinoembryonic antigen(CEA)and Carbohydrate antigen 199(CA199)and clinicopathological indicators in the identification of lymph node metastasis.F inally,the corresponding antibody was designed according to the selected DNA methylation sites,and its efficacy in detecting CRC lymph node metastasis would be verified on 56 randomly selected CRC tissue sections(28 lymph node negative patients and 28 lymph node positive patients)using immunohistochemistry technology.ResultsAccording to differential methylation region(DMR)analysis,a total of 3 methylation sites with DMR were screened out,namely LBX2,STMN3 and SS18L1.After validation of 221 CRC samples,the best DNA methylation site LBX2 was selected,with specificity of 0.87,sensitivity of 0.76,and accuracy of 0.82.The area under curve(AUC)was 0.87(95%CI 0.82-0.92,P<0.0001).However,the AUC of imaging was 0.52(95%CI 0.45-0.59);The AUC of CA199 was 0.58(95%CI 0.510.66),the AUC of CEA was 0.56(95%CI 0.48-0.64),the AUC of depth of invasion was 0.61(95%CI 0.54-0.67,P<0.01)and the AUC of lymphatic invasion was 0.63(95%CI 0.57-0.69,P<0.001).these indicate that LBX2 is significantly superior to the current clinical traditional diagnostic methods.Moreover,the AUC of LBX2 antibody was 0.84(95%CI 0.77-0.90,P<0.0001)in immunohistochemical validation.indicating that LBX2 antibody also has excellent efficacy in distinguishing CRC lymph node metastasis at protein level.ConclusionThis study demonstrates that DNA methylation site LBX2 could be used as a more reliable diagnostic method for CRC lymph node metastasis compared to traditional methods,so it is expected to provide better clinical decision making for CRC precision surgical treatment and prognostic assessment indicators.