A mutation-sensitive switch assay to detect five clinically significant epidermal growth factor receptor mutations

Abstract

Epidermal growth factor receptor (EGFR) mutations can affect the therapeutic efficacy of drugs used to treat nonsmall-cell lung cancer (NSCLC). We aimed to develop methods to detect five common EGFR somatic mutations in tumor tissues from NSCLC patients by using a nanoscale mutation-sensitive switch consisting of a high-fidelity polymerase and phosphorothioate-modified allele-specific primers. The five clinically significant EGFR mutations examined here are S768I, T790M, L858R, and 15- and 18-bp deletion mutations in exon 19. Our assays showed sensitivities of 100 copies and specificities of more than three log scales for matched templates relative to mismatched templates by routine polymerase chain reaction (PCR), real-time PCR, and multiplex PCR. This assay would be superior to DNA sequencing in situations where mutant DNA is not abundant.

FIG. 1.

Sequencing results for S768I, T790M, and L858R. Sequencing chromatography illustrates that the wild-type and mutant template sequences prepared in vitro are correct. The rectangular box base is the mutant site.

FIG. 2.

Sequencing results for the 15- and 18-bp deletions in exon 19. Sequencing chromatography illustrates that the wild-type and mutant template sequences prepared in vitro are correct. The rectangular box base is the mutant site.

FIG. 3.

On/off switch sensitivity and specificity. Representative results showing the relatively high sensitivity and specificity of the on/off switch for detecting five different EGFR mutations. (1) AGC/ATC codon of S768I; (2) ACG/ATG codon of T790M; (3) CTG/CGG codon of L858R; (4) 15-bp deletion of E746–A750; and (5) 18-bp deletion of L747–S752. The upper section of each panel shows results from the matched amplicons, while the lower sections show results from the mismatched amplicons where there are no amplified products until the template level is ≥10⁶ copies. The first lane is a bp marker. EGFR, epidermal growth factor receptor.

FIG. 4.

Amplification curves for S768I mutation detection. The x-axis shows the amplification curves, in which the matched amplicons amplified well from 10² to 10⁷ template copies (in dark gray), whereas the mismatched amplicons amplified only when the templates were present at ≥10⁵ copies (in light gray).

FIG. 5.

Multiplex PCR detection of four mutations. Multiplex PCR detection of four EGFR mutations in plasmid samples. PCR, polymerase chain reaction.

FIG. 6.

Multiplex PCR detection of EGFR L858R in tumor samples. No product was obtained from samples from a healthy subject (Lane 1). Lanes 2 and 3 show results from lung tissue samples from patients carrying the L858R mutation in EGFR.

FIG. 7.

Sequencing chromatography illustrated the L858R mutations in tumor samples. Sequencing chromatography illustrated the correctness of two L858R mutations in tumor samples. The rectangular box base is the mutant site.

FIG. 8.

PCR detection of the 15-bp deletion EGFR in exon 19 in plasmid samples. PCR detection of the 15-bp deletion in EGFR exon 19 in plasmid samples. Lane 1: No product was present at 10⁴ copies of wild-type template. Lanes 2 and 3: 10⁴ copies of wild-type template were mixed with 10¹ copies of mutant template. Lane 4 is a negative control.