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

<div><p>Somatic mutation in human epidermal growth factor receptor-related 2 gene (<i>HER2</i>) is one of the driver mutations in lung cancer. <i>HER2</i> 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 <i>HER2</i> 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 <i>HER2</i> 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 <i>HER2</i> 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. <i>HER2</i> mutations detected by NGS data validated the high sensitivity of the method. Therefore, this new technique can lead to precise molecular-targeted therapies.</p></div

Agarose gel electrophoresis of PCR products.

<p>A: PCR products from real-time PCR experiments were analyzed on a 3% agarose gel. Lanes: 1: TaqMan Probe, 2:, Eprobe 203-10 wt TO 3: C3-blocked 203-10 wt oligonucleotide, 4: Eprobe 205-13 wt TO, 5: Eprobe205-13m TO, 6: C3-blocked 205-13 wt oligonucleotide, 7: Eprobe 215-21 wt TO, 8: Eprobe 215-21m TO, 9: C3-blocked 215-21 wt oligonucleotide, 10: Eprobe 215-21 wt TP, 11: SYBR Green I, M: marker. B: Partial sequence of human EGFR gene. Location of the primers (blue boxes) and the mutation L858R (red box) are indicated.</p

Eprobes in multiplex detection.

<p>Regions of the human EGFR and KRAS genes were amplified from genomic DNA and amplification was monitored by specific Eprobes having different dyes. A: Amplification curves plotting random fluorescent units (RFU) obtained from LightCycler 480 against PCR cycle number (Red: genomic DNA, Green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature (Red: genomic DNA, Green: negative control). Plots in yellow on the left show signals for KRAS using dye D514 (Eprobe KWT14m3 TO); plots in pink on the right show signals for EGFR using dye D570 (Eprobe 215-21 wt TP).</p

Eprobes used for this study.

<p>(T_M values for TO-labeled Eprobes were estimated using the following settings: 0.05 M Na⁺, 0.002 M Mg²⁺, 0.2 µM Eprobe, 60°C Temperature; *T_M values for Eprobe 215-21 wt TP are actual values; in Eprobe name: TO = D514, TP = D570; Z indicating position of modified T).</p

Eprobe derived background.

<p>A. Eprobe 215-21 wt TO labeled with D514 was digested as described in the Materials and Methods and fluorescence excitation spectra from 495 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). B. Eprobe 215-21 wt TP labeled with dye D570 was digested as described in the Materials and Methods and fluorescence excitation spectra from 550 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). C. Plotting random fluorescent units (RFU, mean values from triplicate data for each experiment) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against plasmid DNA template concentration (from 150 to 150,000,000 copies per reaction). Eprobe concentrations are indicated by the different colors (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). D. Plotting random fluorescent units (RFU) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against Eprobe concentration (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). For each data point, mean values plus error bars from triplicate values for each experiment and for all template concentrations are given in the graph.</p

Eprobe mediated mutation detection.

<p>Asymmetric PCR was performed to enrich the reverse strand for mutation detection using different templates and Eprobes. For further details refer to Materials and Methods. A: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 5×10⁴ copies of plasmid DNA using Eprobe 205-13 wt TO. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 1 ng genomic DNA using Eprobe 215-21 wt TP. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control).</p

Sensitivity of Eprobe-PCR for detecting <i>HER2</i> 12-bp duplicated insertion.

<p>MT: <i>HER2</i> 12-bp duplicated insertion mutation type, WT: <i>HER2</i> 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.</p

Primer sets and Eprobe design for <i>HER2</i> mutation detection.

<p>Schematic diagram of primers for the detection of the <i>HER2</i> 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 <i>HER2</i> 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.</p

Comparison of Eprobe-PCR and Sanger methods.

<p>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 (<a href="http://nucleobytes.com/4peaks/" target="_blank">http://nucleobytes.com/4peaks/</a>).</p

Structure and function of Eprimer and Eprobe.

<p>A. Chemical structure of modified deoxythymidine carrying two dye moieties. B. Chemical structure of D514 “Thiazole Orange”. C. Chemical structure of D570 “Thiazole Pink”. D. Signal generation by an arbitrary Eprimer (ECHO), where the two dye moieties are paired in the single-stranded oligonucleotide for signal suppression. Upon hybridization to complementary strand, dye moieties get separated and intercalate into the double-strand leading to emission of strong fluorescence. E. Structure of an arbitrary Eprobe having a blocked 3′ end to prevent primer extension during PCR.</p