Highly sensitive detection of a HER2 12-base pair duplicated insertion mutation in lung cancer using the Eprobe-PCR method

Abstract

Somatic mutation in human epidermal growth factor receptor-related 2 gene (HER2) is one of the driver mutations in lung cancer. HER2 mutations are found in about 2% of lung adenocarcinomas (ADCs). Previous reports have been based mainly on diagnostic screening by Sanger sequencing or next-generation sequencing (NGS); however, these methods are time-consuming and complicated. We developed a rapid, simple, sensitive mutation detection assay for detecting HER2 12 base pair-duplicated insertion mutation based on the Eprobe-mediated PCR method (Eprobe-PCR) and validated the sensitivity of this assay system for clinical diagnostics. We examined 635 tumor samples and analyzed HER2 mutations using the Eprobe-PCR method, NGS, and Sanger sequencing. In a serial dilution study, the Eprobe-PCR was able to detect mutant plasmid DNA when its concentration was reduced to 0.1% by mixing with wild-type DNA. We also confirmed amplification of the mutated plasmid DNA with only 10 copies per reaction. In ADCs, Eprobe-PCR detected the HER2 mutation in 2.02% (9/446), while Sanger sequencing detected it in 1.57% (7/446). Eprobe-PCR was able to detect the mutation in two samples that were undetectable by Sanger sequencing. All non-ADC samples were wild-type. There were no discrepancies between frozen and formalin-fixed paraffin-embedded tissues in the nine samples. HER2 mutations detected by NGS data validated the high sensitivity of the method. Therefore, this new technique can lead to precise molecular-targeted therapies.

Fig 1. Primer sets and Eprobe design for HER2 mutation detection.

Schematic diagram of primers for the detection of the HER2 12-bp duplicated insertion by Eprobe-PCR. The orange box is the duplicated insertion. The forward primer for detection of the mutant-type allele contains the full sequence of HER2 across the region known to be a frequent insertion site. The green bar is the Eprobe. The 3’ end-filled circle Eprobe shows the blocker that prevents primer extension during PCR.

Fig 2. Sensitivity of Eprobe-PCR for detecting HER2 12-bp duplicated insertion.

MT: HER2 12-bp duplicated insertion mutation type, WT: HER2 wild type, NTC: No template control (diluted water). (a) Evaluation of mutated genome amplification. The blue line indicates MT only plasmid DNA at 10,000 copies per reaction, red: 1,000, green: 100, purple: 10, light blue: 1, orange: WT plasmid DNA, black: NTC. The light blue line shows no amplification. It overlaps with WT and NTC lines. (b) Sensitivity of 12-bp duplicated insertion detection in heterogenetic conditions. The blue line indicates MT only plasmid DNA at 10,000 copies per reaction, red: 1,000, green: 100, purple: 10, light blue: 1, orange: WT plasmid DNA, black: NTC (diluted water). The total copy number for each was adjusted to 10,000 copies per reaction. The light blue line shows no amplification. It overlaps WT and NTC lines. (c) Cp (crossing point) values of two experiments (a) and (b) were calculated by the second derivative maximum method in the LightCycler480. The data were then transferred to Microsoft Excel (Microsoft, Redmond, WA, USA) and Cp values were evaluated.

Fig 3. Comparison of Eprobe-PCR and Sanger methods.

The left half of Fig 3 shows the amplification curves of Eprobe-PCR, and the right half shows the electrogram of Sanger sequencing. “4Peaks” was used to view and edit the sequence trace files (http://nucleobytes.com/4peaks/).