The Establishment And Application Of Detection Techniques For Gene Mutations In Circulating Free DNA Of Glioma

Glioma is the most common malignant tumor of central nervous system,with high recurrence rate and fatality rate and short survival time.In recent years,the development and application of molecular biology and high-throughput sequencing technology have enabled people to have a comprehensive understanding of the genomic changes of glioma,and the discovery of many molecular markers has promoted the pace of individualized treatment of glioma based on molecular typing.However,current molecular tests rely on tumor tissue derived from intracranial surgical resection or puncture biopsy.The limitations of tissue specimen sampling(invasive,high-risk,non-repeatable)and molecular heterogeneity make the current follow-up diagnosis and treatment plan based on the detection results of tumor tissue specimens at specific sites widely available in clinical practice questionable.As the main means of liquid biopsy,circulating tumor DNA(ct DNA)can be obtained repeatedly without invasion,tumor heterogeneity can be overcome and tumor changes can be monitored in real time,which provides methods and tools for solving the above problems.However,the content of free DNA in body fluids(blood or cerebrospinal fluid)is very small,and it is easy to be interfered by wild-type template signals.General detection techniques cannot be accurately detected,and high-sensitivity detection methods are needed.Therefore,for the purpose of developing sensitive and accurate detection technology for glioma free DNA molecular markers,this study established a digital PCR method for detecting glioma free DNA gene mutations for IDH1 and TERT mutations,which are important molecular markers of glioma.In addition,the digital PCR detection method established in this study was used to detect IDH1 and TERT promoter mutations in tumor tissue,blood and CEREBROspinal fluid samples of 101 patients with glioma and to detect and analyze their conditions.The results of this study showed that the established digital PCR method for IDH1 R132 H mutation and TERT promoter mutation could detect tumor gene mutations as low as0.01-0.1% in the background of wild-type DNA template.Compared with the fluorescence PCR method,the sensitivity was 100% and the specificity was between 97.9% and 100%,confirming the accuracy and reliability of the established method.Pair of IDH1 mutations in tumor tissues,plasma and cerebrospinal fluid samples,TERT mutation detection was analyzed,and the consistency of the cerebrospinal fluid and the accuracy of tumor gene mutations(80% 90%)is significantly higher than the plasma and the accuracy of tumor gene mutations(70% 85%),although cerebrospinal fluid free DNA concentration below the content of free DNA in tumor tissues,but more can reflect the change of tumor tissue in patients with genomic DNA.The distribution characteristics of IDH1 mutation and TERT mutation in 101 clinical samples were analyzed.It was found that IDH1 mutation was mainly found in low-grade glioma,while TERT mutation was mainly found in high-grade glioma,and the two were generally mutually exclusive.The relationships between IDH1,TERT promoter,IDH1 mutation and TERT promoter and progression-free survival(PFS)and total survival(OS)in 69 glioma patients were analyzed.It was found that IDH1 R132 H mutation was significantly associated with PFS and OS elongation in both tissue and blood samples,while TERT mutation was only significantly associated with PFS shortening.IDH1 and TERT double mutant groups had longer OS and PFS than IDH1 mutation group alone.The real-time detection of tumor mutation changes in blood and CEREBROspinal fluid samples of 3 patients at different treatment stages showed that the ct DNA mutation content in blood was significantly lower than that in CSF,and the IDH1 or TERT mutation content in blood or CSF was negatively correlated with the disease progression and treatment effect of the patients.In conclusion,this study confirmed the effectiveness of the glioma circulating free DNA gene mutation detection technology based on digital PCR technology in the clinical diagnosis and treatment of glioma,and preliminarily confirmed the application value of blood and CEREBROspinal fluid ct DNA molecular variation in the prognosis and efficacy evaluation of clinical patients.