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. 2016 Nov;12(5):4238-4244.
doi: 10.3892/ol.2016.5184. Epub 2016 Sep 26.

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

Sanpeng Xu  1 , Yaqi Duan  1 , Liping Lou  1 , Fengjuan Tang  1 , Juan Shou  1 , Guoping Wang  1
Affiliations

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

Sanpeng Xu et al. Oncol Lett. 2016 Nov.

Abstract

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.

Keywords: amplification refractory mutation system; epidermal growth factor receptor gene T790M mutation; non-small cell lung cancer; rs1050171; single nucleotide polymorphism.

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Figures

Figure 1.
Figure 1.
Schematic diagram of the three T790M amplification refractory mutation system-polymerase chain reaction systems. The diagram was drawn to scale, with the proportional scale in the bottom right corner representing a length of 10 bp.
Figure 2.
Figure 2.
Detection results of cell line H1975 and mixed samples by the three T790M ARMS-qPCR systems. (A) The cell line H1975 has an apparent T790M mutation (~50% mutation rate) and is homozygous mutant genotype of rs1050171. The nucleotide location of the single nucleotide polymorphism is indicated by the arrows (↓). (B) The cell line H1975 was detected to have a low abundance of the T790M mutation by the ARMS-qPCR system 1 (~1% mutation rate), but was identified to have an obvious T790M mutation by the ARMS-qPCR systems 2 and 3 (~50% mutation rate). (C) Samples with 1–50% T790M mutant plasmid 1 could be detected by the ARMS-qPCR system 1. (D) Samples with 50% T790M mutant plasmid 2 could be detected by the ARMS-qPCR system 1, but not those with 1–20% T790M mutant plasmid 2. (E) Samples with 20 and 50% T790M mutant plasmid 1 could be detected by the ARMS-qPCR system 2, but not those with 1–10% mutant plasmid 1. (F) Samples with 1–50% T790M mutant plasmid 2 could be detected by the ARMS-qPCR system 2. (G) Samples with 1–50% T790M mutant plasmid 1 could be detected by the ARMS-qPCR system 3. (H) Samples with 1–50% T790M mutant plasmid 2 could be detected by the ARMS-qPCR system 3. ARMS, amplification refractory mutation system; qPCR, quantitative polymerase chain reaction; RF, reference.
Figure 3.
Figure 3.
Sequencing results of samples (A) N558 and (B) N310. (A) Sample N558 was observed to be homozygous mutant genotype of rs148188503. (B) A new SNP was detected in sample N310. The nucleotide location of the SNP is indicated by the arrows (↓). SNP, single nucleotide polymorphism.
Figure 4.
Figure 4.
T790M detection results of sample N094 by different methods. (A) The sample N094 was detected as T790M-negative by sequencing, and is homozygous mutant genotype of rs1050171. The nucleotide location of the single nucleotide polymorphism is indicated by the arrows (↓). (B) The sample N094 was identified as T790M-negative by the ARMS-qPCR system 1 (∆Cq value >7.5). (C) The sample N094 was demonstrated to have the T790M mutation by the ARMS-qPCR system 3 (∆Cq value <7.5). RF, reference; ARMS, amplification refractory mutation system; qPCR, quantitative polymerase chain reaction.
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