A Novel Sensitive Technique to Detect <i>ESR1</i> Hotspot Mutations in Liquid Biopsy Using Switch‐Blocker–Enhanced Targeted Amplification Coupled With Pyrosequencing

Background: ) ligand-binding domain mutations in circulating tumor DNA (ctDNA) is crucial for guiding therapy in estrogen receptor-positive metastatic breast cancer. However, widespread clinical adoption of approaches for monitoring drug resistance and guiding treatment decisions is hindered by limitations of current methods regarding sensitivity, cost, and multiplexing capability. Methods: targets various variants at positions 536, 537, and 538). Targeted enrichment was achieved by quantitative polymerase chain reaction, followed by pyrosequencing to confirm mutation components. Next-generation sequencing and Sanger sequencing served as supplementary methods for the verification of results. Results: plasmid templates, the proportion of L536H mutant copies increased from 0.0015% to 16.89% after targeted amplification, while the proportion of E380Q mutant copies increased from 0.0015% to 1.35%. In ctDNA samples previously analyzed by next-generation sequencing, the switch-blocker considerably enriched other mutant copies within the coverage range of the switch element. Conclusions: hotspot mutations in liquid biopsies. This assay has potential for dynamic monitoring of therapeutic resistance, facilitating timely treatment decisions in advanced breast cancer management.