Highly sensitive detection of EGFR L858R mutation at the mRNA level
Mai Pham1, Quynh Pham2, Ung Nguyen2
1Department of Oncology, 103 Military Hospital, Vietnam Military Medical University, 100000, Hanoi, Viet Nam; Department of Oncology, Hanoi Medical University, 100000, Hanoi, Viet Nam.
Analytical Biochemistry
|July 5, 2022
Summary
A new RNA-based assay detects the EGFR L858R mutation at the mRNA level, offering higher sensitivity than DNA tests. This method reveals mutation expression and is detectable in patient plasma, aiding non-small cell lung cancer (NSCLC) treatment.
Area of Science:
- Molecular Biology
- Oncology
- Biomarker Discovery
Background:
- The EGFR L858R missense mutation is crucial for EGFR tyrosine kinase inhibitor (TKI) therapy response in non-small cell lung cancer (NSCLC).
- Current DNA-based detection methods obscure the clinical relevance of allele-specific expression levels of the EGFR L858R mutation.
- Understanding mRNA expression is vital for optimizing TKI therapy and patient stratification.
Purpose of the Study:
- To develop and validate a novel RNA-based assay for detecting the EGFR L858R mutation at the mRNA level.
- To assess the analytical sensitivity and clinical utility of this RNA-based assay compared to DNA-based methods.
- To investigate the expression variability of EGFR L858R mRNA in tumor tissues and its presence in patient plasma.
Main Methods:
- Development of a one-step real-time RT-PCR assay utilizing extendable blocking probes and a hot-start protocol.
- Validation of the assay's analytical sensitivity, capable of detecting the EGFR L858R mutation in a 10,000-fold excess of wild-type EGFR.
- Comparative analysis of the RNA-based assay against a reference DNA-based assay using formalin-fixed paraffin-embedded (FFPE) samples and patient plasma.
Main Results:
- The novel RNA-based assay demonstrated high analytical sensitivity (0.01%) for detecting the EGFR L858R mutation.
- The RNA-based assay identified significantly more EGFR L858R-positive FFPE samples (19.2%) compared to the DNA-based assay (15.0%).
- Significant variation in EGFR L858R mRNA levels was observed in tumor tissues, and circulating EGFR L858R mRNA was detected in the plasma of NSCLC patients.
Conclusions:
- The developed RNA-based real-time RT-PCR assay is a sensitive and specific tool for detecting EGFR L858R mutations at the mRNA level.
- This assay provides insights into allele-specific expression, overcoming limitations of DNA-based methods and potentially improving NSCLC patient management.
- The detection of circulating tumor mRNA offers a promising non-invasive biomarker for NSCLC diagnosis and treatment monitoring.
Keywords:
Allele-specific gene expressionCancer biomarkerCirculating mRNAEGFR L858R mutationLiquid biopsyRNA-based mutation analysis
