A Research On Drug-resistant Mutations In HER2-positive Breast Cancer Based On Circulating Tumor DNA And CRISPR/cas9 Technique

Objectives Small molecular tyrosine kinase inhibitor (TKI) has significantly improved the prognosis of patients with human epidermal growth factor receptor 2 positive [HER2+) metastatic breast cancer, however, the acquisition of resistance is inevitable. At present, TKI resistance mechanisms have become one of the great challenges of breast cancer research. This study aimed to explore the possiblemutations which were associated with resistance to TKI via analyzing clinical data, and verify whether these mutations were really correlated to TKI resistance in vitro study. At the same time, we explored a simple and quick mew method to vertify the point mutation resistance. Methods Firstly, we intended to obtain mutatons that may be associated with resistance by analysis of circulating tumor DNA (ctDNA) data in 18 HER2+metastatic breast cancer patients who received TKI treatment We then studied the relationship between mutations and drug resistance in vitro experiments. Molecular biology techniques and methods used in this study included western blot, realtime quantitative polymerase chain reaction (realtime-qPCR), molecular cloning technique, CRISPR (clustered regularly interspaced short palindromic repeat sequences)/cas9 gene editing technique, etc. Results We picked PIK3CA c.[3140A>G] and ERBB2c.[2264T>C] to be investigated based on the analysis of clinical data, and we chose BT474 breast cancer cell line based on the basic information of available cell lines. Stable mutant cells were acquired utilizing CRISPR/cas9 technique, andwe found that the sensitivity of mutant cells towards lapatinib and neratinib was significantly lower than primary cells. The mutant and primary cell mixtures were treated with lapatinib, neratinib, and cell culture medium respectively for 3 days, followed by real-time quantitative PCR. The relative level of target mutant gene in cells treated with either TKI or cell culture medium was higher than the cells before giving any treatment, and the relative level of target mutant genes in cells treated with TKI was higher than those treated with cell culture medium. The relative level of ERBB2c.[2264T>C] in cells treated with lapatinib was higher than neratinib, while the situation of PIK3CA c.[3140A>G] was just the opposite. Conclusions Mutations correlated to resistance could be identified by ctDNA detection. PIK3CA c.[3140A>G] and ERBB2c.[2264T>C] could improve the growth rate of HER2+breast cancer cells, and were both related to lapatinib and neratinib resistance. For cells harboring ERBB2c.[2264T>C], the degree of lapatinib resistance was higher than that of neratinib resistance, while the situation of cells with PIK3CA c.[3140A>G] was just the opposite. The procedure of ctDNA detection and analysis, CRISPR/cas9 gene editing, establishing cell mixtures, real-time quantitative PCR may become a novel strategy that is simple and convenient for high-throughput screening of drug-resistant mutations.