cg08721802位点异常甲基化对良恶性肺结节鉴别诊断的价值探讨

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

摘要:

目的 探讨基于外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）的DNA差异甲基化修饰位点在肺良恶性结节临床鉴别诊断中的价值。方法 采用850k甲基化芯片检测20例良性肺结节和34例恶性肺结节患者PBMCs的全基因组甲基化状态，初步构建PBMCs来源的良恶性肺结节特异性甲基化图谱并获得差异甲基化位点（differentially methylated positions，DMPs），对DMPs所在的基因进行基因本体（gene ontology，GO）分析及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）分析。利用LASSO回归分析方法对DMPs进行统计筛选。将36例良性肺结节患者和84例恶性肺结节患者随机分成2组队列（测试队列和验证队列），通过焦磷酸测序方法对所选的位点进行分析验证，并采用受试者工作特征（receiver operating characteristic，ROC）曲线进行分析，计算ROC曲线下面积（area under the curve，AUC），评估其对肺良恶性结节的预测效能。结果 发现队列的良恶性肺结节患者PBMCs差异甲基化图谱具有明显差异。以Δβ>0.06，adjust P<0.01作为预筛选条件共得到421个DPMs。使用LASSO回归分析进一步筛选得到6个CpG位点。根据Pearson 相关分析，以及进一步去除具有引物错配风险及引物合成困难的位点，得到cg05460181（AUC=0.793，P<0.0001）和cg08721802（AUC= 0.893，P<0.0001）2个位点。测试阶段，通过焦磷酸测序测试2个位点在16例良性肺结节患者及34例恶性肺结节患者中的甲基化水平，发现cg08721802位点对肺良恶性结节具有较高的诊断价值（AUC=0.778，敏感度=85.29%，特异度=56.25%，P<0.01）。验证阶段，在另一个包含20例良性肺结节患者及50例恶性肺结节患者的独立样本集中对cg08721802的甲基化水平进行检测，发现其对区分良恶性肺结节仍具有临床价值（AUC=0.768，敏感度=84%，特异度=50%，P<0.01），cg08721802位点诊断效能明显高于传统肿瘤标志物，cg08721802位点与CEA结合明显提高了对大小为6~20 mm的结节诊断价值。结论 cg08721802甲基化水平在良恶性结节患者中具有明显差异，可以作为良恶性肺结节鉴别诊断的新型分子标志物。

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.