Establishment of multiplex allele-specific blocker PCR for enrichment and detection of 4 common EGFR mutations in non-small cell lung cancer

BACKGROUND: Lung cancer is one of the most severe cancers and the majority of patients miss the best timing for surgery when diagnosed, thus having to rely on radiotherapy, chemotherapy or target therapy. Epidermal growth factor receptor (EGFR) upregulation occurs in a large percentage of patients, who can then benefit from tyrosine kinase inhibitors (TKI). However, the EGFR mutations they carry will vary the effectiveness of TKI. Circulating tumor DNA (ctDNA) contains genetic information from cancer tissue that can be used as a liquid biopsy by non-invasive sampling. This study aimed to provide a solution for minor allele detection from ctDNA. METHODS: Our novel method, named multiplex allele-specific blocker PCR (MAB PCR), combines amplification refractory mutation system (ARMS), blocker PCR and fluorescent-labeled probes for better discrimination and higher throughput. MAB PCR was specially designed for low-quality samples such as ctDNA. A sensitive assay based on MAB PCR was developed for enriching and detecting four common EGFR mutations. This assay was optimized and evaluated with manufactured plasmids, and validated with 34 tissue samples and 94 plasma samples. RESULTS: The limit of detection of this assay was 10(2) copies and the detection sensitivity reached 0.1% mutant allele fraction (MAF). The results of clinical sample testing had 100% accordance with sequencing, which proved that this assay was accurate and applicable in clinical settings. CONCLUSIONS: This assay could accomplish low-cost and rapid detection of 4 common EGFR mutations sensitively and accurately, which has huge potential in clinical usage for guiding medication. Furthermore, this design could be used to detect other mutations.