A Novel Sensitive Technique to Detect ESR1 Hotspot Mutations in Liquid Biopsy Using Switch-Blocker-Enhanced Targeted...

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.