Highly sensitive detection of EGFR L858R mutation at the mRNA level

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. [Display omitted] •Current DNA-based mutation assays unable to reveal RNA expression of mutant genes.•Novel assay enables highly sensitive detection of EGFR L858R mutation at mRNA level.•Relative mRNA expression levels of the EGFR L858R mutation was uncovered in NSCLC.•Promotes further studies on diagnostic/prognostic relevance of EGFR L858R mRNA.•Basis for detection of other clinically actionable gene mutations at mRNA levels.