RT Journal Article SR Electronic T1 Coupling multiplex pre-amplification and droplet digital PCR for longitudinal monitoring of ESR1 and PIK3CA mutations from plasma cell-free DNA JF bioRxiv FD Cold Spring Harbor Laboratory SP 598847 DO 10.1101/598847 A1 Huilan Yao A1 Grant Wu A1 Subhasree Das A1 Crystal MacKenzie A1 Hua Gao A1 Victoria Rimkunas A1 Zhaojie Zhang A1 Stephanie Ferro A1 Amy Roden A1 Manav Korpal A1 Joanne Schindler A1 Peter G. Smith A1 Lihua Yu A1 Ping Zhu A1 Pavan Kumar YR 2019 UL http://biorxiv.org/content/early/2019/04/05/598847.abstract AB Here we report on the development of a sensitive and cost-effective method to longitudinally track ESR1 and PIK3CA mutations from cfDNA in patients with metastatic breast cancer (MBC) using a streamlined and de-centralized workflow. Hotspot mutations in ESR1 have been shown to cause resistance to aromatase inhibitor–based and anti-estrogenic therapies, while PIK3CA mutations have high prevalence in MBC. As a result, their utility as circulating biomarkers to predict or monitor response in the clinical development of investigational compounds has been the focus of many studies. Six regions in ESR1 and PIK3CA genes containing 20 hotspot mutations were pre-amplified, followed by optimized singleplex ddPCR assays to detect allele frequencies of individual mutations. Without pre-amplification, the limit of detection (LOD) and limit of linearity (LOL) of individual ddPCR assays were at 0.05-0.1% and 0.25% level, respectively. With pre-amplification, the LOD and LOL were slightly elevated at 0.1-0.25% and 0.25-0.5% levels, respectively. High concordance was achieved to the BEAMing assay (Sysmex Inostics) for mutation positive assays (r=0.98, P<0.0001). In conclusion, coupling pre-amplification and ddPCR assays allowed us for the detection of up to 20 hot spot mutations in ESR1 and PIK3CA with high sensitivity and reproducibility.