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EBioMedicine. 2017 Jul;21:86-93. doi: 10.1016/j.ebiom.2017.06.003. Epub 2017 Jun 7.

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR.

Author information

1
Third Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan; Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan.
2
Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan; National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan.
3
Clinical Research Center, Department of Thoracic Oncology, National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan.
4
Center for Advanced Medicine and Clinical Research, Nagoya University Hospital, Aichi, Japan.
5
Department of Internal Medicine, National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan.
6
Division of Respiratory Medicine and Allergology, Aichi Medical University School of Medicine, Aichi, Japan; Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan.
7
Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan.
8
Department of Thoracic Surgery, Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan.
9
Department of Thoracic Surgery, National Hospital Organization Hokkaido Cancer Center, Hokkaido, Japan.
10
Department of Thoracic Surgery, National Hospital Organization Nagasaki Medical Center, Nagasaki, Japan.
11
Department of Thoracic Surgery, National Hospital Organization Shibukawa Medical Center, Gunma, Japan.
12
Department of Thoracic Surgery, National Hospital Organization Shikoku Cancer Center, Ehime, Japan.
13
Department of Thoracic Surgery, National Hospital Organization Iwakuni Clinical Center, Yamaguchi, Japan.
14
Clinical Research Institute, National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan.
15
Department of General Thoracic Surgery, National Hospital Organization Toneyama National Hospital, Osaka, Japan.
16
Department of Thoracic Surgery, National Hospital Organization Yokohama Medical Center, Kanagawa, Japan.
17
Department of Surgery, National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan.
18
Third Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan; Department of Respiratory Medicine and Medical Oncology, National Hospital Organization Nagoya Medical Center, Aichi, Japan. Electronic address: ykoh@wakayama-med.ac.jp.

Abstract

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M).

KEYWORDS:

Droplet digital PCR; EGFR mutation; Non–small cell lung cancer

PMID:
28625519
PMCID:
PMC5514407
DOI:
10.1016/j.ebiom.2017.06.003
[Indexed for MEDLINE]
Free PMC Article

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