Background: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,widespr...Background: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.Methods: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.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.展开更多
基金The Noncommunicable Chronic Diseases-National Science and Technology Major Project,Grant/Award Number:2023ZD0502200National Natural Science Foundation of China,Grant/Award Numbers:92459304,82230058,82303912,82403982+1 种基金National High Level Hospital Clinical Research Funding,Grant/Award Number:2025-LYZX-D-A02CAMS Innovation Fund for Medical Sciences,Grant/Award Numbers:2025-I2M-TS-02,2025-I2M-KJ-006。
文摘Background: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.Methods: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.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.
