Li Xiaoyan, Liu Yi, Shi Weiwei, Xu Huayan, Hu Haixu, Dong Zhengwei, Zhu Guanshan, Sun Yun, Liu Bing, Gao Hongjun, Tang Chuanhao, Liu Xiaoqing
Department of Lung Cancer, Affiliated Hospital of the Academy of Military Medical Sciences, No. 8 Dongdajie, Beijing, China.
Translational Medicine Center, Laboratory of Oncology, Affiliated Hospital of the Academy of Military Medical Sciences, No. 8 Dongdajie, Beijing, China.
Clin Chim Acta. 2017 Aug;471:177-184. doi: 10.1016/j.cca.2017.06.007. Epub 2017 Jun 8.
Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of nucleic acid. However, its application has not been reported in pleural fluid, which is an ideal sample source for epidermal growth factor receptor (EGFR) mutation analysis in non-small-cell lung cancer (NSCLC) patients.
The extracted DNA from supernatants of pleural fluid was selected from our sample bank and re-analyzed by our previously established ddPCR assay. The results were compared with the former outcomes detected by direct sequencing or the amplification-refractory mutation system (ARMS).
A total of 95 samples were selected, and 64 and 31 of them had been performed with direct sequencing and ARMS tests, respectively. The EGFR mutation detection rate of ddPCR was significantly elevated, compared with both direct sequencing (75.4% vs. 43.8%, P<0.0001) and ARMS (61.3% vs. 38.7%, P=0.016). Compared with ARMS, Fisher's exact test showed that EGFR-positive patients who were redefined by ddPCR had higher objective response rates (ORRs): 57.9% vs. 16.7%, P=0.032. Compared with direct sequencing results, Kaplan-Meier curves demonstrated that EGFR-positive patients who were redefined by ddPCR had longer progression-free survival (PFS): 8.0 vs. 2.0months, P=0.0001.
We have demonstrated the clinical value of ddPCR in pleural fluid samples. The experience obtained from the present study is practical and favorable for the proper application of this new assay.
液滴数字聚合酶链反应（ddPCR）是一种用于分析少量核酸的有前景的方法。然而，其在胸腔积液中的应用尚未见报道，而胸腔积液是分析非小细胞肺癌（NSCLC）患者表皮生长因子受体（EGFR）突变的理想样本来源。
从我们的样本库中选取胸腔积液上清液中提取的DNA，并用我们先前建立的ddPCR检测方法重新进行分析。将结果与先前通过直接测序或扩增阻滞突变系统（ARMS）检测的结果进行比较。
共选取95个样本，其中64个和31个样本分别进行了直接测序和ARMS检测。与直接测序（75.4%对43.8%，P<0.0001）和ARMS（61.3%对38.7%，P=0.016）相比，ddPCR的EGFR突变检测率显著提高。与ARMS相比，Fisher确切概率法显示，经ddPCR重新定义的EGFR阳性患者具有更高的客观缓解率（ORR）：57.9%对16.7%，P=0.032。与直接测序结果相比，Kaplan-Meier曲线表明，经ddPCR重新定义的EGFR阳性患者具有更长的无进展生存期（PFS）：8.0个月对2.0个月，P=0.0001。
我们已经证明了ddPCR在胸腔积液样本中的临床价值。本研究获得的经验切实可行，有利于这种新检测方法的正确应用。
