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非小細胞肺癌患者の末梢血サンプルにおける EGFR 変異の超高感度検出および動的モニタリングのための新しい液滴デジタル PCR アッセイの確立と検証
Establishment and validation of a novel droplet digital PCR assay for ultrasensitive detection and dynamic monitoring of EGFR mutations in peripheral blood samples of non-small-cell lung cancer patients.
PMID: 32645388 DOI: 10.1016/j.cca.2020.07.003.
抄録
背景:
非小細胞肺癌（NSCLC）患者の個別化治療において、液滴デジタルPCR（ddPCR）をベースとしたEGFR変異の血液検出は重要な役割を果たしている。しかし、保健当局に承認された標準的な測定法はまだ存在しない。さらに、この方法を臨床現場で適切に適用するためには、さらなる調査が必要である。
BACKGROUND: Droplet digital PCR (ddPCR)-based blood detection of EGFR mutations plays significant roles in the individualized therapy of non-small-cell lung cancer (NSCLC) patients. However, a standard assay that is approved by health authorities is still lacking. Additionally, the proper application of this method in clinical settings also needs further investigation.
方法:
新たに確立したddPCR法の性能をまず参照試料を用いて評価し、患者の末梢血中の無細胞DNA（cfDNA）を用いた増幅不応性突然変異システム（ARMS）と比較することで検証した。さらに、患者におけるEGFR変異の動的定量とチロシンキナーゼ阻害剤（TKI）治療の臨床成績との相関を検討した。
METHODS: The performance of a newly established ddPCR assay was first evaluated using reference samples and then validated by comparing this method with the amplification refractory mutation system (ARMS) using cell-free DNA (cfDNA) in patients' peripheral blood. Further, the correlation between dynamic quantification of EGFR mutation in the patients and their clinical outcome of tyrosine kinase inhibitors (TKIs) therapy was investigated.
結果:
RESULTS: A total of 77 patients were included, with 50 in the test group and 27 in the validation group. According to the results of the reference samples and the blood samples in the test group, the cut-off value for patient detection was proposed as mutation rate ≥ 0.1% (total copy number of cfDNA ≥1,000) or at least one copy of mutation DNA was detected (total copy number of cfDNA <1,000). With this criterion, superior sensitivity of our assay to that of ARMS was observed (P=0.002 for Ex19Del & L858R and P<0.001 for T790M). The dynamic quantification of EGFR mutations during TKI therapy indicated that an increase in mutation abundance was correlated with resistance, while a decline was associated with response. Notably, a rebound in mutation abundance during chemotherapy may indicate a desirable chance for TKI re-treatment.
結論:
その結果、新規ddPCR法は血中EGFR変異の検出に優れた感度を示した。このアッセイによるEGFR変異の動的定量化は，抵抗性や奏効性のモニタリング，再治療のためのコホートスクリーニングを含めたTKI療法の実施を大いに促進するものと考えられる。
CONCLUSION: The novel ddPCR assay showed superior sensitivity in the detection of EGFR mutation in blood. The dynamic quantification of EGFR mutations by this assay would greatly facilitate the administration of TKI therapy, including the monitoring of resistance and response, as well as cohort screening for retreatment.
Copyright © 2020 Elsevier B.V. All rights reserved.