| BackgroundBreast nodules are very common in women and can be divided into benign and malignant nodules.Most benign breast nodules are proliferative diseases,such as breast fibroadenoma and lobulate tumors.The incidence rate of malignant breast nodules,namely breast cancer(BCa),has ranked first among all cancers,which seriously threatens women’s health.The treatment methods for breast nodules depend on the nature of breast nodules in clinical practice.Therefore,the accurate differentiation of benign and malignant nodules is the key to improving the survival rate of breast tumor patients and an urgent clinical problem to be solved.Currently,the main methods for identifying BCa patients from benign breast nodule patients are ultrasound and X-ray photography.The above methods highly rely on the subjective judgment of clinicians,easily leading to missed diagnosis or misdiagnosis,and the diagnostic accuracy is not satisfactory.Therefore,it is important to find and identify effective molecular markers for the differential diagnosis of benign and malignant nodules.DNA methylation is an important and most studied form of epigenetic modification.Methylated CpG can affect the expression of genes by changing the structure of chromatin,DNA conformation,DNA stability,and the interaction between DNA and protein.DNA methylation plays an important role in carcinogenesis and development,and it has been widely recognized as a biomarker for early tumor diagnosis.Peripheral blood mononuclear cells(PBMCs)are composed of lymphocytes and monocytes,and its methylation status can specifically reflect the characteristics of tumor immunity in the tumor microenvironment during the transformation of benign nodules to malignant nodules,which is an early event in tumorigenesis.It is a potential differential diagnostic marker of benign and malignant breast nodules,and the content of PBMCs is significantly higher than that of ctDNA or cfDNA,which greatly reduces the difficulty of detection.However,there is still a lack of relevant researches on the clinical value of DNA methylation changes of PBMCs in the differential diagnosis of benign and malignant breast nodules.ObjectiveThis study aims at analyzing the DNA methylation level of PBMCs in patients with benign and malignant breast nodules,screening and identifying the DMPs(Differential Methylation Sites)for the differential diagnosis of benign and malignant breast nodules,constructing the DNA methylation diagnosis model,and exploring its value as a non-invasive biomarker in the differential diagnosis of benign and malignant breast nodules.Methods1.In the discovery stage,the peripheral venous blood samples of 55 BCa patients and 30 patients with benign nodules were collected,and PBMCs were isolated using the density gradient centrifugation method.Following the above,DNA was extracted from PBMCs,and quality control was performed.After quality control,the qualified DNA from each sample was bisulfite-modified.The whole-genome methylation changes of bisulfite transformed DNA was detected by Infinium MethylationEPIC BeadChip(850K)chip assay.According to the screening principle,|Δβ|>0.1 and adjust.P<0.05,DMPs between benign nodule patients and malignant nodule patients were obtained,and the distribution and potential biological functions of DMPs were clarified.In order to obtain DMPs that are more conducive for the differential diagnosis of benign and malignant breast nodules,we upgraded the principle to adjust.P≤1.0E-18,|Δβ|>0.15,and Random Forest was used for feature screening to obtain candidate DMPs.2.In the training stage,pyrosequencing was used to evaluate the methylation level of DMPs in the training cohort composed of 30 BCa patients and 30 patients with benign breast nodules.DMPs whose DNA methylation level changes in the training set are consistent with that in the discovery set were regarded as key DMPs,and the AUC(Area Under Curve)of the ROC(Receiver Operating Characteristic)curve was calculated.Subsequently,we constructed a differential diagnosis model to discriminate malignant breast nodules from benign breastnodules based on Logistic Regression analysis.In addition,the differential diagnosis efficiency of this model with common tumor markers CEA,CA125,and CA153 was compared.3.In the validation stage,the methylation level of DMPs in an independent validation cohort composed of 46 BCa patients and 46 benign nodule patients was evaluated by Targeted Bisulfite Sequencing Assay,and the differential diagnostic value of 3 DMPs and the diagnostic model was evaluated by ROC curve analysis.Besides,the differential diagnosis efficiency of this model with common tumor markers CEA,CA 125,and CA 153 were compared.4.The correlation between the model and clinical parameters in the training cohort and the validation cohort was statistically analyzed by non-parametric Mann Whitney U test or t test.Results1.In the discovery phase,based on |Δβ|>0.1 and adjust.P<0.05,a total of 5118 DMPs were found between patients with BCa and patients with benign breast nodules,and the methylation level of most DMPs(4640/5118)was downregulated in BCa group.DMPs are mainly distributed on chromosome 1,S shore,and Gene Body.The distribution of hypermethylated and hypomethylated DMPs was not exactly the same.KEGG and GO analysis suggested that DMPs were not only involved in the regulation of immune function,but also involved in many signal pathways such as Notch signal pathway.Further based on adjust.P≤1.0E-18,|Δβ|>0.15 and Random Forest feature screening,10 DMPs(cg21958871,cg26977936,cg18487946,cg14435695,cg23351954,cg04732548,cg27209741,cg13165070,cg01243072,cg10928544)were obtained,of which the methylation level of cg21958871,cg26977936,cg18487946,cg14435695,cg2335554 in BCa group was higher than that in benign nodule group and the methylation level of cg04732548,cg27209741,cg13165070,cg01243072,cg10928544 was lower.2.In the training phase,10 DMPs obtained in the discovery phase were further verified by pyrosequencing.Compared with benign nodule group,the methylation level of cg26977936 and cg23351954 in BCa group was upregulated,and the methylation level of cg27209741 was downregulated.The AUC of cg26977936,cg23351954,and cg27209741 for the differential diagnosis of benign and malignant nodules were 0.732(95%CI=0.602-0.838,sensitivity=90.00%,specificity=53.30%),0.660(95%CI=0.526-0.777,sensitivity=43.30%,specificity=90.00%)and 0.707(95%CI=0.576-0.818,sensitivity=90.00%,specificity=43.30%),respectively.Then,we established a differential diagnosis model based on the above 3 DMPs by Logistic Regression analysis:Logit(P)=-1.7787+24.3617*cg26977936+11.1975*cg23351954-13.6692*cg27209741,with the AUC of 0.837(95%CI=0.719-0.919,sensitivity=80.00%,specificity=80.00%).The diagnostic efficacy was significantly higher than that of common tumor markers CEA(AUC=0.718,95%CI=0.571-0.837,sensitivity=93.10%,specificity=50%),CA125(AUC=0.754,95%CI=0.610-0.866,sensitivity=62.10%,specificity=85.00%),and CA153(AUC=0.706,95%CI=0.559-0.827,sensitivity=72.40%,specificity=65.00%).3.In the validation stage,the differential diagnostic value of DMPs and this model were further verified by Targeted Bisulfite Sequencing Assay(MethylTarget)in another independent validation cohort.It was confirmed that the methylation level of cg26977936,cg23351954 and cg27209741 was differentially expressed among patients with benign and malignant nodules,with the AUC of 0.727(0.624-0.815,sensitivity=93.50%,specificity=45.70%),0.681(0.576-0.774,sensitivity=76.10%,specificity=54.30%)and 0.640(0.533-0.737,sensitivity=34.80%,specificity=89.10%).The diagnostic efficiency of the model was 0.827(95%CI=0.734-0.898,sensitivity=89.10%,specificity=63.00%),which was significantly higher than that of common tumor markers CEA(AUC=0.667,95%CI=0.541-0.777,sensitivity=97.50%,specificity=33.30%),CA125(AUC=0.540,95%CI=0.414-0.633,sensitivity=57.50%,specificity=55.60%),and CA153(AUC=0.737,95%CI=0.615-0.837,sensitivity=72.50%,specificity=66.70%).4.In the training cohort,there was no association between this model and age,BMI,menstrual status,ER,PR,HER2,molecular stage,histological grade,TNM stage,tumor size,and lymph node metastasis(P>0.05).In the validation cohort,there was no association between this model and BMI,ER,PR,HER2,molecular stage,histological grade,TNM stage,tumor size,and lymph node metastasis(P>0.05),but the methylation score of this model increased in BCa patients with age≥50 years old or in the postmenopausal state.Conclusion1.The DMPs map of benign and malignant breast nodules was described by 850K methylation chip,and the key candidate DMPs were identified in the progression of breast benign and malignant nodules,so as to provide data support for finding biomarkers for the differential diagnosis of breast benign and malignant nodules.2.The methylation level of candidate DMPs was verified by pyrosequencing and Targeted Bisulfite Sequencing Assay,and the differential diagnosis model of benign and malignant nodules:Logit(P)=-1.7787+24.3617*cg26977936+11.1975*cg23351954-13.6692*cg27209741 wasestablished.The model had excellent differential diagnosis value for benign and malignant nodules and can be used as a potential biomarker for the differential diagnosis of breast benign and malignant nodules... |
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