Exploring the impact of EGFR T790M neighboring SNPs on ARMS-based T790M mutation assay

The present study aimed to explore the influence of T790M neighboring single nucleotide polymorphism (SNP) on the sensitivity of amplification refractory mutation system (ARMS)-based T790M mutation assay. Three ARMS-quantitative polymerase chain reaction (qPCR) systems (system 1 had a forward ARMS primer without rs1050171, system 2 included a forward ARMS primer with rs1050171 and system 3 contained the above two forward ARMS primers) were used to detect the T790M mutation in two series plasmid samples and genomic DNA (gDNA) of the cell line H1975. A total of 670 formalin-fixed paraffin-embedded (FFPE) tumor samples from non-small cell lung cancer patients were used to detect the epidermal growth factor receptor (EGFR) gene T790M mutation by direct sequencing and ARMS-qPCR. The ARMS-qPCR system 1 effectively detected samples with as low as 1% T790M mutant plasmid 1 (without rs1050171) and with 50% T790M mutant plasmid 2 (with rs1050171), while the ARMS-qPCR system 2 detected samples with 20 and 50% T790M mutant plasmid 1, in addition to samples with 1% T790M mutant plasmid 2. For the ARMS-qPCR system 3, samples with as low as 1% T790M mutant plasmids 1 or 2 were effectively detected. For gDNA analysis of the cell line H1975, the T790M mutation was effectively detected by the ARMS-qPCR systems 2 and 3 (~50% mutation rate), but was detected with a low mutation abundance by the ARMS-qPCR system 1 (~1% mutation rate). Of the 670 FFPE samples, 5 cases were identified to have the T790M mutation by sequencing and by the ARMS-qPCR system 1. One sample (named N067), which was considered as T790M-negative by sequencing, was demonstrated to have the T790M mutation using the ARMS-qPCR system 1. Sample N094, which was variant homozygous for rs1050171 and was indicated to be T790M-negative by sequencing and by the ARMS-qPCR system 1, was identified to have the T790M mutation with the ARMS-qPCR system 3. The A-variant allele frequency of rs1050171 was observed to be 28.2% in the 670 FFPE tumor samples, while the presence of rs148188503 (c. C2355T, p. T785T) was observed in sample N558, and a novel SNP with a base substitution (c. T2375C) at position 792 (p. L792P) in exon 20 of the EGFR gene was observed in sample N310. rs1050171 is a high-frequency SNP located near T790M, and the mutation statuses of rs1050171 appear to influence the sensitivity of the ARMS-based T790M detection system, thus generating a 14.3% false-negative rate (1/7). The present study proposes the risk that target neighboring SNPs (as far as 8 bp away in the present study) may exert on the sensitivity of ARMS-based detection methods.