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

• Published in: Journal of pharmaceutical and biomedical analysis 2021-04, Vol.197, p.113959-113959, Article 113959
• Main Authors: Wang, Chen, Zeng, Huijuan, Zhang, Luning, Shen, Yiyun, Zou, Bingjie, Wang, Shaohua, Song, Qinxin, Zhou, Guohua
• Format: Article
• Language: English
• Subjects: Breast cancer; Breast Neoplasms - drug therapy; Breast Neoplasms - genetics; Cell-Free Nucleic Acids - genetics; cfDNA; Endocrine therapy; ESR1 mutation; Estrogen Receptor alpha - genetics; Female; Humans; Mutation; Real-Time Polymerase Chain Reaction; Resistance
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.113959

[Display omitted] •A new strategy for rapid quantitation of ESR1 mutations in breast cancer patients.•A next-generation sequencing (NGS)- and digital PCR (dPCR)-free method was proposed.•Unlike conventional methods, the strategy works without adding additional internal reference or making standard curves.•The detection results can be obtained in few hours from sampling to report. 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.