Background: EGFR mutation testing is important in the treatment decision for advanced non-small cell lung cancer (NSCLC). Because T790M mutation in exon 20 is associated with approximately 50% of cases resistant to treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), the importance of re-biopsy for detecting T790M mutation during EGFR-TKI therapy is increasing. Circulating tumor DNA (ctDNA) is recognized as a promising target of minimally invasive diagnosis. Digital polymerase chain reaction (dPCR) is a new PCR technology for accurate quantification of the copy number of target molecules from low-input DNA. This study aimed to develop a blood-based EGFR mutation analysis in patients with advanced NSCLC by using a chip-based dPCR system that enables easy handling at a moderate costs.
Methods: From October 2013 to March 2014, 49 patients with NSCLC harboring EGFR-activating mutations detected with the Scorpion ARMS method were enrolled in this study. ctDNA samples were extracted from plasma samples of 21 and 28 patients before treatment and after progression with EGFR-TKI, respectively. dPCR was performed on the QuantStudio 3D (QS3D) Digital PCR System (Life Technologies) by using 20 ng ctDNA, TaqMan probes for three EGFR mutations (exon 19 deletion, L858R in exon 21, and T790M in exon 20), and QS3D Digital PCR 20K Chips (2 chips per assay), according to the manufacturer's instructions. The performance of the dPCR assay was assessed by using HDx Reference Standards (Horizon) containing each mutant sequence serially diluted with that containing wild-type EGFR sequence.
Results: Mutation of 0.1% was successfully detected in the dPCR assay for the three EGFR mutations. The median copy numbers of the EGFR mutation-positive samples were 2.9 copies (range, 0.3-84.2) for exon 19 deletion, 5.1 copies (0.6-742.1) for L858R in exon 21, and 1.4 copies (0.6-36.9) for T790M in exon 20. The sensitivity and specificity of each dPCR assay calculated by comparison with corresponding tumor samples were 61.8% (21/34) and 93.3% (14/15) for exon 19 deletion, and 66.7% (10/15) and 100% (34/34) for L858R, respectively. The T790M mutations were detected in 43% (12/28) of the ctDNA samples after progression with EGFR-TKIs therapy but in none of the samples before treatment with EGFR-TKIs. Among the 12 patients with NSCLC harboring T790M mutation in ctDNA, T790M mutations were detected in 5 of the 11 patients who underwent re-biopsy.
Conclusions: This system showed similar performance to other dPCR assays used in previous studies (sensitivity: 66.6-92.0% and specificity: 95.7-100%). These results indicate the possibility that EGFR mutation testing with ctDNA using the chip-based dPCR for easy handling at a moderate costs is useful as a minimally invasive monitoring method in clinical settings.
Citation Format: Norimitsu Kasahara, Hirotsugu Kenmotsu, Masakuni Serizawa, Rina Umehara, Akira Ono, Kazushige Wakuda, Shota Omori, Kazuhisa Nakashima, Tetsuhiko Taira, Tateaki Naito, Haruyasu Murakami, Noriaki Sunaga, Yasuhiro Koh, Keita Mori, Masahiro Endo, Takashi Nakajima, Masanobu Yamada, Masatoshi Kusuhara, Toshiaki Takahashi. Plasma epidermal growth factor receptor mutation (EGFR) testing in advanced non-small-cell lung cancer patients harboring EGFR mutations by chip-based digital PCR system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2248.