The Application Value Of Detecting Tumor Suppressor Genes Methylation In CtDNA By Using Next Generation Sequencing In The Diagnosis And Treatment Of Pancreatic Cancer

Objective: To investigate the clinical application value of detecting tumor suppressor genes methylation in circulating tumor DNA(ctDNA)in the diagnosis and treatment of pancreatic cancer.Methods: Next generation sequencing(NGS)was used to quantitatively detect the methylation levels of 5 tumor suppressor genes(NPTX2、RASSF1A、EYA2、p16 and pp ENK)in plasma ctDNA in 43 cases of pancreatic cancer(pancreatic cancer group),39 cases of benign pancreatic diseases(pancreatic benign disease group)and 20 healthy control subjects(healthy control group).The diagnostic abilities of ctDNA methylation were compared with those of CA19-9,and the relationship between the methylation levels and clinical pathological factors were also analyzed.Meanwhile,the dynamic changes of methylation levels of ctDNA before and one week after radical resection were compared in 23 pancreatic cancer patients.Results:(1)The methylation levels of ctDNA RASSF1 A,EYA2,pp ENK and p16 genes in pancreatic cancer group were significantly higher than those in healthy control group(P(27)0.05).The methylation levels of ctDNA EYA2,p16 and pp ENK genes in pancreatic cancer group were significantly higher than those in pancreatic benign diseases group(P(27)0.05).The methylation levels of ctDNA NPTX2,RASSF1 A,EYA2,p16 and pp ENK genes in pancreatic benign diseases group were significantly higher than those in healthy control group(P(27)0.05).The methylation levels of ctDNA NPTX2,RASSF1 A,EYA2,pp ENK and p16 genes were all not correlated with the tumor size,nerve invasion,lymph node metastasis and TNM stage of pancreatic cancer(P>0.05).(2)The sensitivity,negative predictive value,area under the curve of ROC(AUC)and Youden’s index of combined detection of ctDNA p16,RASSF1 A,pp ENK and EYA2 methylation for diagnosis of pancreatic cancer were 94.1%,88.0%,0.90 and 0.80,respectively,which were all higher than those of CA19-9(70.63%,58.37%,0.82 and 0.65),while its specifity,positive predictive value(77.81% and 89.0%)were inferior to CA19-9(94.4% and 94.0%).The sensitivity and AUC of combined detection of ctDNA p16,RASSF1 A,pp ENK and EYA2 methylation with CA19-9 were 100% and 0.96,which were higher than those of single marker detection,while its specificity was lower than that of single marker detection.The sensitivity,negative predictive value,AUC and Youden’s index of combined detection of ctDNA EYA2,p16 and pp ENK methylation for differential diagnosis of pancreatic cancer and pancreatic benign diseases were84.80%,82.74%,0.90 and 0.71,respectively,which were all higher than those of CA19-9(72.1%,74.52%,0.82 and 0.62),while its specifity and positive predictive value(85.72% and 87.52%)were inferior to CA19-9(89.72% and 88.63%).(3)The methylation levels of ctDNA NPTX2,EYA2 and pp ENK genes were significantly decreased at one week after radical resection compared with those before surgery(P(27)0.05).Conclusion: The methylation levels of RASSF1 A,p16,EYA2 and pp ENK genes in ctDNA were increased in pancreatic cancer,and were not correlated with tumor size,perineural invasion,regional lymph node metastasis and TNM stage.Combined detection of ctDNA RASSF1 A,p16,EYA2 and pp ENK genes methylation is useful to improve the detection rate of pancreatic cancer,and combined detection of ctDNA EYA2,p16 and pp ENK genes methylation is helpful to distinguish pancreatic cancer from pancreatic benign diseases.The methylation levels of ctDNA NPTX2,EYA2,pp ENK genes were significantly decreased after radical resection of pancreatic cancer.Detection of ctDNA tumor suppressor genes methylation is expected to become a novelty tumor biomarker for the prognosis and therapeutic effect evaluation of pancreatic cancer.