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Sensitive quantitation of ESR1 mutations in cell-free DNA from breast cancer patients using base-specific invasive reaction assisted qPCR.
Journal of Pharmaceutical and Biomedical Analysis 2021 April 16
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 μL-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.
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