Metabolomics And DNA Methylation For Early Warning And Early Diagnosis Of Colorectal Cancer

Backgrounds and Objectives1.Familial adenomatous polyposis(FAP)is an autosomal dominant intestinal polyp syndrome caused by mutations in the APC gene,accounting for approximately 1%of colorectal cancer(CRC).In recent years,there have been more and more studies on the molecular mechanisms of CRC pathogenesis.However,the metabolic pathways and changes that may be involved in the development of CRC are still unclear.This study was intended to perform a metabolomics study of familial adenomatous polyposis by chromatographic mass spectrometry,to find out the difference in serum metabolic profiles between familial adenomatous polyposis and normal controls,and to clarify the metabolomic changes that may result from APC mutation and possible mechanisms for the development of colorectal cancer.2.The pathway of polyp-Adenoma-Cancer is currently recognized as a model for the development of colorectal cancer.Increasing the detection rate of adenoma can significantly reduce the incidence of post-colonoscopy CRC,and effective intervention can significantly reduce the prevalence and mortality of colorectal cancer and improve the survival rate of patients.Therefore,the prevention strategy of colorectal cancer should be transferred from cancer to precancerous lesions,and in future advanced adenomas will play a more important role colorectal cancer screening programs.However,early detection of precancerous lesions and early CRC is still an important challenge to date.A large number of cells are shed from the colonic epithelium in malignant colorectal tumors,so colorectal cancer and precancerous lesions can be non-invasively detected by analyzing DNA markers in fecal exfoliated cells.This study was designed to investigate the feasibility of detecting methylated SEPT9(mSEPT9)as a non-invasive biomarker for early CRC in fecal exfoliated cells.Methods1.Serum metabolites from FAP patients(n=30)and healthy individuals(n=34)were detected and qualified using Ultra Performance Liquid Chromatography and Tandem Mass Spectrometry(UPLC-MS/MS).118 metabolites were identified with statistical tests of orthogonal partial least-squares-discriminant analysis(OPLS-DA),with the conditions of variable importance in projection(VIP)>1,p<0.05 using the Mann-Whitney U test,and fold change(FC)?2 or<0.52.The study is divided into two independent sample sets.Firstly,mSEPT9 was detected in fecal exfoliated cells and corresponding tissues in the training study consisted of 10 advanced adenomas without high-grade dysplasia(AA),15 AA with HGD,49 CRCs and 20 healthy controls.Then it was validated in fecal exfoliated cells in an independent study of 185 samples(71 CRC,15 AA,16 HGD,83 healthy controls).Plasma and fecal samples were also collected for Fecal Immunochemical Test(FIT)and tumor markers detection,such as carcinoembryonic antigen(CEA)tests in both studies.By analyzing the methylation of SEPT9 from colorectal tumors and normal tissue specimens to determine whether it can be used as a biomarker for disease staging,and then using improved fecal exfoliated cell enrichment methods to increase cell enrichment,then polymerase chain reaction(PCR)was performed for mSEPT9 detection in fecal exfoliated cell DNA,and each marker was then combined to find the optimal marker combinationResults1.A total of 118 differential metabolites were identified by statistical tests using orthogonal partial least squares discriminant analysis.Therefore,the OPLS-DA model can distinguish the serum metabolic profiles of FAP patients and healthy controls well.FAP patients have unique metabolic characteristics including tricarboxylic acid(TCA)circulation,amino acid metabolism,vitamin D,fatty acid metabolism,and bile acid(BAs)metabolism.2.The methylation of SEPT9 can be detected in both the patient's tumor tissue and fecal exfoliated cells.In addition,the results of mSEPT9 in fecal exfoliated cells were very consistent with that in tumor tissues(kappa=0.907).In the training set,the positive detection rate of mSEPT9 was 31.25%in AA,65.31%in HGD,and 77.46%in all CRC.In the validation set,56/71 CRC(78.87%)and 10/16 HGD(62.50%)were positive for mSEPT9.The sensitivity and specificity of mSEPT9 for CRC and HGD reached 75.86%[95%confidence interval(CI):65.90-83.64%]and 93.88%(87.28-97.16%),respectively,and the sensitivity was better than that of FIT.The combination of mSEPT9 and FIT further increased the sensitivity to HGD and CRC by 88.51%(80.12-93.64%)and specificity 88.78%(81.01-93.62%).Conclusions1.The level of metabolites in FAP patients is significantly different from that in normal subjects.The characteristics of metabolites in FAP patients are helpful to further analyze the metabolomic changes induced by APC gene mutations,which may be related to the occurrence of colorectal cancer.2.mSEPT9 demonstrated distinguished diagnostic ability in early stage CRC detection.Rather than combination of mSEPT9 with FIT and CEA,the combination of mSEPT9 and FIT further improved diagnostic sensitivity especially for early stage CRC,which is potential to provide a novel approach for early CRC screening.