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

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.

SUBMITTER: Xu S

PROVIDER: S-EPMC5104263 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Exploring the impact of <i>EGFR</i> T790M neighboring SNPs on ARMS-based T790M mutation assay.

Xu Sanpeng S Duan Yaqi Y Lou Liping L Tang Fengjuan F Shou Juan J Wang Guoping G

Oncology letters 20160926 5

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 ...[more]

PMID: 27895798

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Project description:IntroductionMechanisms of resistance to EGFR exon 20 insertion mutation active inhibitors have not been extensively studied in either robust preclinical models or patient-derived rebiopsy specimens. We sought to characterize on-target resistance mutations identified in EGFR exon 20 insertion-mutated lung cancers treated with mobocertinib or poziotinib and evaluate whether these mutations would or would not have cross-resistance to next-generation inhibitors zipalertinib, furmonertinib, and sunvozertinib.MethodsWe identified mechanisms of resistance to EGFR exon 20 insertion mutation active inhibitors and then used preclinical models of EGFR exon 20 insertion mutations (A767_V769dupASV, D770_N771insSVD, V773_C774insH) plus common EGFR mutants to probe inhibitors in the absence/presence of EGFR-T790M or EGFR-C797S.ResultsMobocertinib had a favorable therapeutic window in relation to EGFR wild type for EGFR exon 20 insertion mutants, but the addition of EGFR-T790M or EGFR-C797S negated the observed window. Zipalertinib had a favorable therapeutic window for cells driven by EGFR-A767_V769dupASV or EGFR-D770_N771insSVD in the presence or absence of EGFR-T790M. Furmonertinib and sunvozertinib had the most favorable therapeutic windows in the presence or absence of EGFR-T790M in all cells tested. EGFR-C797S in cis to all EGFR mutations evaluated generated dependent cells that were resistant to the covalent EGFR tyrosine kinase inhibitors mobocertinib, zipalertinib, furmonertinib, sunvozertinib, poziotinib, and osimertinib.ConclusionsThis report highlights that poziotinib and mobocertinib are susceptible to on-target resistance mediated by EGFR-T790M or -C797S in the background of the most prevalent EGFR exon 20 insertion mutations. Furmonertinib, sunvozertinib, and to a less extent zipalertinib can overcome EGFR-T790M compound mutants, whereas EGFR-C797S leads to covalent inhibitor cross-resistance-robust data that support the limitations of mobocertinib and should further spawn the development of next-generation covalent and reversible EGFR exon 20 insertion mutation active inhibitors with favorable therapeutic windows that are less vulnerable to on-target resistance.

Dataset Information

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

Publications

Exploring the impact of <i>EGFR</i> T790M neighboring SNPs on ARMS-based T790M mutation assay.

Similar Datasets

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