An allele-specific quantitative PCR strategy for detection of EGFR T790M mutations by liquid biopsy in non-small cell lung cancer patients

Detection of mutations conferring resistance to tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor (EGFR), such as T790M, is critical for guiding targeted therapies in non-small cell lung cancer (NSCLC) patients. However, limitations in accessing tumor tissue have given rise to liquid biopsy approaches necessitating ultrasensitive techniques to identify tumor mutations in a large background of wild-type alleles. We developed a sensitive and cost-effective technique utilizing a 2′3’-dideoxy nucleotide (ddNTP) blocker that selectively blocks wild-type allele amplification, allowing specific detection of mutant alleles in circulating free DNA (cfDNA) from plasma. EGFR genomic sequences encompassing T790M were cloned into pMD20-T plasmids and titrated in a background of genomic DNA to test the sensitivity. The assay detected 10 copies of the mutant allele in the presence of 10,000 copies of wild-type alleles, indicating a sensitivity of 0.001 %. Among 74 NSCLC patients, 34 received TKI treatment and were screened for the T790M mutation. The current method would be a sensitive and cost-effective liquid biopsy tool for detecting EGFR T790M mutations in NSCLC cfDNA samples, with strong potential for identifying actionable mutations for targeted therapy, especially in low-resource settings.