Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan.
PLoS One. 2018 Nov 16;13(11):e0207001. doi: 10.1371/journal.pone.0207001. eCollection 2018.
Several ultra-sensitive methods for T790M in plasma cell-free DNA (cfDNA) have been developed for lung cancer. The correlation between mutation-allele frequency (MAF) cut-off, drug responsiveness, and outcome prediction is an unmet needs and not fully addressed. An innovative combination of peptide nucleic acid (PNA) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used to proof of concept for monitoring cfDNA T790M in EGFR-mutant patients. Mutant enrichment by PNA was optimized and the detection limit was evaluated through serial dilutions. The cut-off value was identified by receiver-operating-characteristic (ROC) curve analysis utilizing serial sampled plasmas of patients from EGFR-tyrosine kinase inhibitor (TKI) pretreatment to progressive-disease (PD). Results, comparisons, and objective response rate (ORR) were analyzed in 103 patients' tumor and cfDNA T790M, with 20 of them receiving an additional COBAS test. The detection limit was 0.1% MAF. The cut-off for PD and imminent PD was 15% and 5% with an ROC area under the curve (AUC) of 0.96 and 0.82 in 2 ml plasma. Detection sensitivity of cfDNA T790M was 67.4% and overall concordance was 78.6%. ORR was similar in T790M-positive cfDNA (69.6%) and tumor samples (70.6%) treated with osimertinib. Among 65 T790M-positive tumors, 15 were negative in cfDNA (23.1%). Seven of 38 T790M-positive cfDNA samples were negative in the tumors (18.4%). PNA-MALDI-TOF MS had a higher detection rate than COBAS. In conclusion, identification of T790M cut-off value in cfDNA improves cancer managements. We provide a strategy for optimizing testing utility, flexibility, quality, and cost in the clinical practice.
已经开发了几种用于检测血浆无细胞 DNA（cfDNA）中 T790M 的超敏方法，用于肺癌。突变等位基因频率（MAF）截断值、药物反应性和预后预测之间的相关性是一个尚未满足的需求，尚未得到充分解决。肽核酸（PNA）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）的创新组合用于证明监测 EGFR 突变患者 cfDNA T790M 的概念。通过 PNA 优化了突变富集，并通过连续稀释评估了检测下限。通过对 EGFR 酪氨酸激酶抑制剂（TKI）预处理至进展性疾病（PD）的患者连续取样的血浆进行接收者操作特征（ROC）曲线分析，确定了截断值。在 103 名患者的肿瘤和 cfDNA T790M 中分析了结果、比较和客观缓解率（ORR），其中 20 名患者接受了额外的 COBAS 检测。检测下限为 0.1% MAF。PD 和即将发生 PD 的截断值分别为 15%和 5%，在 2 ml 血浆中 ROC 曲线下面积（AUC）为 0.96 和 0.82。cfDNA T790M 的检测灵敏度为 67.4%，总一致性为 78.6%。接受奥希替尼治疗的 T790M 阳性 cfDNA（69.6%）和肿瘤样本（70.6%）的 ORR 相似。在 65 例 T790M 阳性肿瘤中，cfDNA 中 15 例为阴性（23.1%）。在 38 例 T790M 阳性 cfDNA 样本中，肿瘤为阴性的有 7 例（18.4%）。PNA-MALDI-TOF MS 的检测率高于 COBAS。总之，cfDNA 中 T790M 截断值的确定改善了癌症管理。我们为优化检测实用性、灵活性、质量和成本提供了一种策略，以满足临床实践的需要。
