A Novel Method For Detection Of PIK3CA Mutations In Breast Cancer Using DDPCR

Breast cancer is the first cancer in female cancer.PIK3 CA gene mutation is one of the common mutations of breast cancer except HER2 gene increase and mutation of P53 gene,and its mutation rate in breast cancer is about 8% to 40%.PIK3 CA gene mutation through PI3 K / AKT pathway induced AKT sustained activation,leading to fibroblasts and mammary epithelial cell growth and transformation,inhibition of apoptosis,and the occurrence and development of breast cancer are closely related.A large number of clinical research and practical experience has shown that PIK3 CA gene specific mutation in patients with EGFR,HER2 targeted drug treatment resistance,tumor patients accept the EGFR or HER2 target drug treatment before,they should get on PIK3 CA gene mutation detection,according to the test results Assess whether the patient is suitable for the use of the corresponding targeted drug as a clinical treatment.Droplet Digital PCR(DDPCR)is a sensitive method for quantitative detection that requires only a very small number of samples to be tested.This method does not require reference to standard or endogenous controls,high sensitivity and required sample size Less,greatly satisfying the clinical need for rare samples such as puncture samples,pleural effusion,and peripheral blood target sequences.Objective: develop a mutant gene detection method based on the Digital PCR Platform,which can not only monitor the mutation rate of PIK3 CA gene sensitive mutation,but also detect the emergence of drug resistance mutations,which is important for the treatment and prognosis of breast cancer patients Guiding significance.Methods: 10 pairs of primers and 3 pairs of probes were designed for E9 E545 K,E542K and E20 H1047 R sites of PIK3 CA gene of breast cancer,and the DNA of MCF 10 A and positive standard samples will used to test.The DDPCR method and the NGS method will be compared with the results of the clinical samples of 202 cases of breast cancer.Results: 10 pairs of primers and 3 pairs of probes were tested with DNA of MCF 10 A and positive standard samples.8 pairs of primers and 3 pairs of probes were finally established.The clinical data of breast cancer showed that,65 samples were used to detect E545 K mutation of PIK3 CA gene E9,the sensitivity was 100%,the specificity was 100% and the consistency was 100%.58 samples were used to detect E542 K mutation of PIK3 CA gene E9,the sensitivity was 93.9%,the specificity was 100%,the consistency was 96.5%.79 samples were used to detect H1047 R mutation of PIK3 CA gene E20,the sensitivity was 97.6%,the specificity was 94.6%,the consistency was 96.2%;the sensitivity and the specificity of the above three mutation detection are more than 93%,and the consistency are more than 96%,it proved that DDPCR detection method has high accuracy and specificity.In summary,this study successfully developed a breast cancer detection method for PIK3 CA gene E9 E545 K,E542K and E20 H1047 R mutation using DDPCR.