Sensitive quantitation of ESR1 mutations in cell-free DNA from breast cancer patients using base-specific invasive reaction assisted qPCR

Acquired estrogen receptor 1 (ESR1) mutation is being promoted as a key mechanism of resistance to endocrine therapies in breast cancers. It is significative to monitor ESR1 mutations in real time, which provide an opportunity to alter therapy as these mutations emerge. Previous assays based on next-generation sequencing (NGS) and digital PCR (dPCR) usually due to high costs and complicated workflows hampered their clinical adoption in general medical institutions. Here, we proposed a new strategy using base-specific invasive reaction assisted qPCR measure for ESR1 mutations in cfDNA. Two pivotal steps involved in this strategy are target-specific signal generation and the quantification without adding any internal reference or making standard calibration curves. The strategy enabled a high specificity of 0.1% (better than traditional NGS-based method) and a minimum sensitivity of 0.1 copies mu L-1. As validation, with the strategy, cfDNA from endocrine therapy-resistant breast cancers and untreated ones were successfully analyzed (20% mutation rate (2/10) with mutation abundance of 0.54-1.65% vs. 0% mutation rate (0/5)). By virtue of cost-effective, highly flexible and precise, the strategy could be readily implemented in general laboratory, showing promising application perspectives in analysis of other types of mutations. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

Acquired ESR1 mutation is a key mechanism of resistance to endocrine therapies in breast cancers. A new strategy using base-specific invasive reaction assisted qPCR for measuring ESR1 mutations in cfDNA showed high specificity and sensitivity, with successful validation in endocrine therapy-resistant breast cancers.