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

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

Comparison of three genotyping methods in all clinical samples.

<p>Comparison of three genotyping methods in all clinical samples.</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

Patient characteristics according to <i>HER2</i> mutation status.

<p>Patient characteristics according to <i>HER2</i> mutation status.</p

Evaluation of the nine mutant tumors detected by Eprobe-PCR.

<p>Evaluation of the nine mutant tumors detected by Eprobe-PCR.</p

Preparation of SmartAmp primers to detect the HA segment of the 2009 pdm influenza A(H1N1) virus.

<p>A. Mutation rate and difference score in the consensus sequence of the HA segment. Nucleotide sequences of the HA segment of 2009 pdm influenza A(H1N1) viruses were obtained from the NCBI Influenza Virus Resource database and aligned by using the MUSCLE program to gain the consensus sequence of the HA segment. The mutation rate at each base position was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#s4" target="_blank">Materials and Methods</a>. The difference between 2009 pdm and seasonal A(H1N1) viruses was calculated at each position in the nucleotide sequence of the HA segments to gain the difference score. B: Comparison of data acquired in 2009 and 2011 as to the mutation rates in the HA segment of the 2009 pdm influenza A(H1N1) viruses.</p

Time after the onset of fever and the number of patients who were diagnosed by the RT-SmartAmp assay as positive for infection with the 2009 pdm influenza A(H1N1) virus.

<p>Time after the onset of fever and the number of patients who were diagnosed by the RT-SmartAmp assay as positive for infection with the 2009 pdm influenza A(H1N1) virus.</p

Detection of the 2009 pdm influenza A(H1N1) virus by RT-SmartAmp assay in the fatal case.

<p><b>A:</b> Nasopharyngeal swab samples were collected at 11, 28, and 52 hours after the onset of fever from the patient who was transferred by ambulance to the National Center for Global Health and Medicine. The 2009 pdm influenza A(H1N1) virus was immediately detected by the RT-SmartAmp assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#s4" target="_blank">Materials and Methods</a>. <b>B:</b> Chest radiography of the patient was taken at 11 and 28 hours after the onset of fever. <b>C:</b> Partial sequence of the HA segment of the 2009 pdm influenza A(H1N1) virus was analyzed after extraction of viral genome RNA from the swab samples. An arrow indicates the mutation that caused an amino acid substitution at 185 from aspartate to asparagine (N) in the HA protein.</p