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Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues
Published on: August 23, 2024
A Novel Sensitive Technique to Detect ESR1 Hotspot Mutations in Liquid Biopsy Using Switch-Blocker-Enhanced Targeted
Yantong Zhou1, Wenna Wang2, Bo Lan2
1State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital Chinese Academy of Medical Sciences and Peking Union Medical College Beijing China.
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.
Conclusions:
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.
Insights
A novel switch-blocker assay enhances detection of estrogen receptor 1 (ESR1) mutations in circulating tumor DNA. This sensitive method aids in monitoring drug resistance and guiding treatment for metastatic breast cancer patients.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Estrogen receptor 1 (ESR1) mutations in circulating tumor DNA (ctDNA) are key for treating estrogen receptor-positive metastatic breast cancer.
- Current methods for detecting ESR1 mutations face limitations in sensitivity, cost, and multiplexing, hindering clinical application.
Purpose of the Study:
- To develop and validate a sensitive, multiplexed assay for detecting ESR1 hotspot mutations in ctDNA.
- To overcome limitations of existing methods for monitoring drug resistance and guiding treatment decisions.
Main Methods:
- Utilized switch-blocker technology to specifically amplify low-frequency mutant ESR1 alleles while suppressing wild-type amplification.
- Employed quantitative PCR for targeted enrichment, followed by pyrosequencing for mutation confirmation.
- Validated the assay using plasmid and ctDNA templates, with NGS and Sanger sequencing for verification.
Main Results:
- The switch-blocker assay demonstrated significant enrichment of ESR1 mutant alleles (e.g., L536H from 0.0015% to 16.89%, E380Q from 0.0015% to 1.35%) in plasmid and ctDNA samples.
- The assay proved feasible for both circular plasmid and linear ctDNA templates.
- Enrichment of mutant copies was observed in ctDNA samples previously analyzed by next-generation sequencing.
Conclusions:
- The developed switch-blocker-enhanced pyrosequencing assay is a targeted, multiplexed, and accessible method for detecting ESR1 hotspot mutations in liquid biopsies.
- This assay holds potential for dynamic monitoring of therapeutic resistance in advanced breast cancer.
- Facilitates timely treatment adjustments for improved patient management.
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