Background:The addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs,such as trastuzumab,lapatinib,and trastuzumab emtansine (T-DM 1),to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients.However,it was confused that metastatic patients vary in the response of targeted drug.Therefore,methods of accurately predicting drug response were really needed.To overcome the spatial and temporal limitations of biopsies,we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).Methods:From March 6,2014 to December 10,2014,24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible.We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes,and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes.Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group.The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.Results:A total of 486 single-nucleotide variants from 46 genes were detected.Of these 46 genes,phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA),proto-oncogene c-Kit (KIT),and tumor protein p53 (TP53) were the most common mutated genes.Seven genes,including epidermal growth factor receptor (EGFR),G protein subunit alpha S (GNAS),HRas proto-oncogene (HRAS),mutL homolog 1 (MLH1),cadherin 1 (CDH1),neuroblastoma RAS viral oncogene homolog (NRAS),and NOTCH1,that only occurred mutations in the resistant group were associated with the resistance of targeted therapy.In addition,we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.Conclusion:Targeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.