Zhu Guanshan, Ye Xin, Dong Zhengwei, Lu Ya Chao, Sun Yun, Liu Yi, McCormack Rose, Gu Yi, Liu Xiaoqing
Translational Science, Asia & Emerging Markets Innovative Medicine, AstraZeneca R&D, Shanghai, China.
Translational Science, Asia & Emerging Markets Innovative Medicine, AstraZeneca R&D, Shanghai, China.
J Mol Diagn. 2015 May;17(3):265-72. doi: 10.1016/j.jmoldx.2015.01.004. Epub 2015 Mar 11.
Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing.
检测肺癌患者血浆游离DNA中的表皮生长因子受体（EGFR）突变是一种新兴的临床工具。然而，与组织检测相比，由于血浆游离DNA高度碎片化、肿瘤DNA比例低以及现有检测技术的局限性，血浆检测的灵敏度仍不尽人意。因此，我们开发了一种用于血浆EGFR突变（外显子19缺失和L858R）检测的高灵敏度和特异性的微滴数字PCR方法。对86例未接受过EGFR酪氨酸激酶抑制剂治疗的肺癌患者的血浆进行检测，并与通过扩增阻滞突变系统检测的匹配肿瘤组织的EGFR突变状态进行比较。通过使用EGFR突变阳性细胞DNA，我们优化了微滴数字PCR检测方法，使其灵敏度达到0.04%。与通过扩增阻滞突变系统检测的匹配肿瘤组织相比，外显子19缺失的血浆检测灵敏度和特异性分别为81.82%（95%CI，59.72%-94.81%）和98.44%（95%CI，91.60%-99.96%），一致性率为94.19%（κ=0.840；95%CI，0.704-0.976；P<0.0001），而L858R的灵敏度和特异性分别为80.00%（95%CI，51.91%-95.67%）和95.77%（95%CI，88.14%-99.12%），一致性率为93.02%（κ=0.758；95%CI，0.571-0.945；P<0.0001）。所报道的用于EGFR突变检测的高灵敏度和特异性微滴数字PCR检测方法在临床血液检测中具有潜力。
