The application of DNA methylation detection in plasma and tumor tissues in patients with esophageal squamous cell carcinoma / 中华检验医学杂志

ABSTRACT

ObjectiveTo investigate the methylation status of multiple genes in plasma and tumor tissues and its application in molecular diagnosis of esophageal squamous cell carcinoma (ESCC).Methods Methylation specific polymerase chain reaction (MSP) was used to detect methylation status of 5 tumor suppressor genes,such as adenomatous polyposis coli ( APC ),retinoic acid receptor-beta2 ( RARβ2 ),E-cadherin (CDH1),cyclin-dependent kinase inhibitor4A (p16INK4α) and ras association domain family member 1 A (RASSF1A) in tumour tissues,adjacent normal tissues and plasma which obtained 1 d preoperative and 7 d postoperative in 76 cases with ESCC.60 healthy volunteers were randomly selected as a control which were age-matched and sex-matched.Chi square test was used to analyze DNA methylation rates of 5 genes in various groups of tissue and plasma samples; Kappa test was used to compare the consistency of DNA methylation in the plasma samples and tissue samples,and their correlation was analyzed by Spearman correlation test; Receiver operating characteristic curve (ROC) was used to evaluate the sensitivity and specificity for single gene detection and 5 genes joint detection for diagnosis of esophageal squamous cell carcinoma.ResultsThe methylation rates of APC,RARβ2,CDH1,p16INK4α and RASSF1A in tumour tissues of patients with ESCC were 44.7％ ( 34/76),72.4％ ( 55/76 ),72.4％ (55/76),86.8％ ( 66/76 ),55.3％ (42/76),respectively,which were significantly higher than that in the corresponding adjacent normal tissues [ 6.6％ ( 5/76 ),3.9％ ( 3/76 ),3.9％ ( 3/76 ),3.9％ ( 3/76 ),2.6％ ( 2/76 ),x2 =29.01,75.39,75.39,105.34,57.18,all P ＜ 0.001 ].The methylation rates of above 5 genes in patients' plasma were 42.1％ ( 32/76 ),63.2％ ( 48/76 ),63.2％ ( 48/76 ),71.1％ ( 54/76 ),50.0％ ( 38/76 ),respectively,which were significantly higher than that of control group [3.3％ (2/60),3.3％ (2/60),1.7％ ( 1/60),3.3％ (2/60),1.7％ (1/60),x2 =26.88,51.62,55.01,63.48,38.30,all P ＜ 0.001 ].The methylation consistency was favorable or well between plasma and tumour tissues in patients with ESCC ( Kappa value was 0.679,0.791,0.791,0.542 and 0.895,respectively.all P ＜0.001 ).In single-gene detection for patients' plasma,methylation,the sensitivity of 5 genes was 42.1％ ( 32/76 ),63.2％ ( 48/76 ),63.2％ ( 48/76 ),71.1％ ( 54/76 ),50.0％ ( 38/76 ),respectively.The specificity was 96.7％ ( 58/60 ),96.7％ ( 58/60 ),98.3％ (59/60),96.7％ (58/60),98.3％ (59/60),respectively.The area under curve (AUC) of ROC was 0.694 [95％ confidence interval( CI)0.606 - 0.782 ) ],0.799 ( 95％ CI 0.723 - 0.875 ),0.807 ( 95％CI 0.733 - 0.882),0.839 ( 95 ％ CI 0.769 - 0.908 ) and 0.742 ( 95 ％ CI 0.659 - 0.824 ),respectively.In united testing of 5 genes,the sensitivity was 80.3％ and the specificity was 88.3％,AUC was 0.843 (95％CI 0.773 -0.913 ).The sensitivity of united testing was significantly higher than that of single-gene detection of APC and RASSF1A(x2 =23.30,15.33 ; P ＜ 0.001 ),except RARβ2,CDH1 and p16INK4α (x2 =5.48,5.48,1.75; P =0.019,0.019,0.186);There was no significant differences in specificity between united testing and single-gene detection (x2 =1.922,1.922,3.348,1.922,3.348,all P ＞ 0.05 ).Conclusions The methylation consistency is favorable or well between tumour tissues and plasma in patients with ESCC.There is no significant superior in diagnosing ESCC with united testing of multiple tumor suppressor genes methylation in plasma than with single-gene detection.But the sensitivity of the former is better than the latter.