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

The detection of estrogen receptor 1 (ESR1) 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.

The application of switch‐blocker technology, which has been patented for detecting ESR1 hotspot mutations (Y537S/C, D538G, E380Q, and L536H/P), suppresses the amplification of wild‐type alleles while allowing specific amplification of low‐frequency mutant alleles. We used a switch‐blocker to inhibit the amplification of a DNA target approximately 10 base pairs in length (e.g., the switch‐blocker covering codon 536 of ESR1 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.

The ESR1‐targeted DNA assay was validated for feasibility on plasmid circular templates and ctDNA linear templates. In tests using gradient‐diluted ESR1 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.

This switch‐blocker–enhanced pyrosequencing assay presents a targeted, multiplexed, and accessible approach for detecting ESR1 hotspot mutations in liquid biopsies. This assay has potential for dynamic monitoring of therapeutic resistance, facilitating timely treatment decisions in advanced breast cancer management.