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.