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.
Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan; National Hospital Organization Kinki-Chuo Chest Medical Center, Osaka, Japan.
EBioMedicine. 2017 Jul;21:86-93. doi: 10.1016/j.ebiom.2017.06.003. Epub 2017 Jun 7.
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 (R) 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).
表皮生长因子受体 (EGFR) 突变已被用作评估 EGFR 酪氨酸激酶抑制剂 (TKI) 治疗效果的最强预测指标。已知三种最常见的 EGFR 突变（L858R、外显子 19 缺失和 T790M）是 EGFR-TKI 治疗的主要选择标志物。在这里，我们开发了一种多重皮升级微滴数字 PCR (ddPCR) 检测方法，可在 1 次反应中检测 3 种常见的 EGFR 突变。含有 EGFR 突变的基因组 DNA 的系列稀释实验显示出线性性能，每种突变的分析灵敏度约为 0.01%。通过传统方法在福尔马林固定石蜡包埋 (FFPE) 组织样本中检测到的 33 种 EGFR 激活突变也可通过这种多重检测方法检测到。由于更高的灵敏度，在同一反应中还检测到了另一种突变 (T790M；包括超低水平突变，<0.1%)。双检测和多重检测的回归分析显示，L858R 的相关系数 (R) 为 0.9986，外显子 19 缺失的相关系数为 0.9844，T790M 的相关系数为 0.9959。使用 ddPCR，我们设计了一种用于 3 种常见 EGFR 突变的多重超敏基因分型平台。对临床样本的初步研究结果表明，多重 ddPCR 具有用于同时筛选多种 EGFR 突变和超罕见预处理突变 (T790M) 的临床应用价值。
