Highly sensitive detection of a HER2 12-base pair duplicated insertion mutation in lung cancer using the Eprobe-PCR method.
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.
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Yoshiaki
Y
Takase
Division of Genomic Technologies, RIKEN Center of Life Science Technologies, Yokohama, Kanagawa, Japan.
Kengo
K
Usui
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Kimihiro
K
Shimizu
Division of Genomic Technologies, RIKEN Center of Life Science Technologies, Yokohama, Kanagawa, Japan.|K.K. DNAFORM, Yokohama, Kanagawa, Japan.
Yasumasa
Y
Kimura
K.K. DNAFORM, Yokohama, Kanagawa, Japan.
Tatsuo
T
Ichihara
Division of Genomic Technologies, RIKEN Center of Life Science Technologies, Yokohama, Kanagawa, Japan.
Takahiro
T
Ohkawa
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Jun
J
Atsumi
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Yasuaki
Y
Enokida
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Seshiru
S
Nakazawa
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Kai
K
Obayashi
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Yoichi
Y
Ohtaki
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Toshiteru
T
Nagashima
K.K. DNAFORM, Yokohama, Kanagawa, Japan.
Yasumasa
Y
Mitani
Department of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Izumi
I
Takeyoshi
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D018734:Genes, erbB-2 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D016254:Mutagenesis, Insertional / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity