Value of abnormal methylation of cg08721802 locus in differential diagnosis of benign and malignant pulmonary nodules

Abstract:

Objective To investigate the value of DNA differential methylation modification sites based on peripheral blood mononuclear cells（PBMCs） in the differential diagnosis of benign and malignant pulmonary nodules.Methods The genome-wide methylation status of 20 cases of benign pulmonary nodules and 34 cases of malignant pulmonary nodules were detected by 850k methylation bead chip. The specific methylation maps of benign and malignant pulmonary nodules derived from PBMCs were preliminarily constructed to obtain differentially methylated positions（DMPs）. Gene ontology（GO） analysis and Kyoto encyclopedia of genes and genomes（KEGG） analysis were performed to analyze the genes where DMPs were located. LASSO regression analysis was used to screen DMPs. The 36 patients with benign pulmonary nodules and 84 patients with malignant lung nodules were randomly divided into two groups of cohorts（test group and validation group）. The selected sites were analyzed and validated through the pyrophosphate sequencing method，the receiver operating characteristic（ROC） curve analysis was adopted，and the area under the ROC curve（AUC） was calculated to evaluate its prediction effectiveness of benign and malignant lung nodules.Results The PBMCs differential methylation profiles of patients with benign and malignant pulmonary nodules were significantly different in the cohort. A total of 421 DPMs were obtained with Δβ>0.06 and adjust P<0.01 as pre-screening condition. Six CpG loci were further screened by LASSO regression model. Two loci，cg05460181（AUC=0.793，P<0.0001）and cg08721802（AUC=0.893，P<0.0001），were obtained based on Pearson correlation analysis and further removal of loci with primer mismatch risk and primer synthesis difficulty. In the test phase，the methylation levels of the two sites in 16 benign pulmonary nodules and 34 malignant pulmonary nodules were tested by pyrosequencing，and it was found that cg08721802 had high diagnostic value for benign and malignant pulmonary nodules（AUC=0.778，sensitivity=85.29%，specificity=56.25%，P<0.01）. In the validation phase，the methylation level of cg08721802 was detected in an independent sample set of 22 patients with benign pulmonary nodules and 51 patients with malignant pulmonary nodules. It was found that cg08721802 still had diagnostic value in differentiating benign and malignant pulmonary nodules（AUC=0.768，sensitivity=84%，specificity=50%，P<0.01）. The diagnostic efficiency of cg08721802 locus was significantly higher than that of traditional tumor markers，and the combination of cg08721802 locus with CEA could significantly improve the diagnostic value of nodules with a size of 6-20 mm.Conclusion The methylation level of cg08721802 is significantly different in patients with benign and malignant pulmonary nodules，which can be used as a new molecular marker for the differential diagnosis of benign and malignant pulmonary nodules.