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.
Abstract:
The missense mutation EGFR L858R implies increased sensitivity to EGFR tyrosine kinase inhibitor (TKIs) therapy, despite a significant non-response rate. Currently, detection of EGFR L858R mutation is mostly DNA based, therefore, the allele-specific expression level of the mutated gene and its clinical relevance is hidden. Based on the extendable blocking probes and hot-start protocol for reverse transcription, we have developed and validated a novel one-step realtime RT-PCR assay that enables detection of EGFR L858R mutation at the mRNA level. This RNA-based assay was able to detect the EGFR L858R mutation in a 10,000-fold excess of its wildtype counterpart, indicating an analytical sensitivity of 0.01%. In comparison to the reference DNA-based assay, the RNA-based assay further detected the EGFR L858R mutation in significantly additional formalin-fixed paraffin-embedded (FFPE) samples (19.2% vs 15.0%). Interestingly, our data showed that the relative mRNA levels of EGFR L858R mutation varied greatly in tumor tissues (∼4 logs); and the circulating mRNA of EGFR L858R mutation was detectable in plasma of NSCLC patients. This novel RNA-based PCR assay provides a simple and ultrasensitive tool for detection of EGFR L858R mutation at the mRNA level as a new class of biomarkers.
Insights
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.
