A rapid liquid biopsy of lung cancer by separation and detection of exfoliated tumor cells from bronchoal-veolar lavage fluid with a dual-layer "PERFECT" filter system
A rapid liquid biopsy of lung cancer by separation and detection of exfoliated tumor cells from bronchoal-veolar lavage fluid with a dual-layer "PERFECT" filter system
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通过使用双层“PERFECT”过滤系统从支气管肺泡灌洗液中分离和检测脱落的肿瘤细胞，对肺癌进行快速液体活检
DOI：
10.7150/thno.44274
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发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Wang, Wei
中科院分区：
文献类型：
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作者：
Li, Tingyu;Liu, Yaoping;Wang, Wei
从支气管肺泡灌洗液（BALF）中分离并检测脱落肿瘤细胞（ETC），即BALF的液体活检，已被证实是诊断肺癌的一种有价值的手段。在此，我们首次基于双层PERFECT（精准、高效、快速、灵活、易操作、可控且轻薄）过滤系统建立了一种快速的BALF液体活检方法。
方法：双层PERFECT过滤系统由上层大微孔滤膜（特征尺寸为49.4±0.5微米）和下层小微孔滤膜（9.1±0.1微米）组成。上层滤膜有助于从BALF样本中分离细胞团并去除黏液，而下层滤膜则用于分离单个ETC。首先，从10毫升临床BALF样本（n = 3）中分离10000个掺入的A549细胞（培养的肺癌细胞），以研究该系统在稀有细胞分离方面的性能。此外，使用该系统从临床BALF样本中分离并检测ETC和ETC团，测试其效果，并与常规细胞离心法进行比较。临床BALF样本采自33例在支气管镜下可见病变的疑似肺癌患者。最终组织病理学结果显示，20个样本来自肺癌阳性患者，另外13例来自肺癌阴性患者。
结果：使用所开发系统从临床BALF样本中回收掺入的A549细胞的回收率（89.8±5.2%）显著高于细胞离心法（13.6±7.8%）。在初步临床试验中，尽管33份体积在6毫升至18毫升之间的临床BALF样本浊度差异很大，但54.6%的样本（18/33）可在3分钟内完成过滤，其余样本最多10分钟完成。事实证明，双层PERFECT过滤系统的灵敏度（80.0%，95%置信区间：55.7% - 93.4%）比常规细胞离心法（45.0%，95%置信区间：23.8% - 68.0%）高得多，p = 0.016（ McNemar检验，双侧）。此外，该平台的灵敏度既不受BALF样本浊度变化的干扰，也与肺癌类型无关。
结论：所建立的双层PERFECT过滤系统能够简便快速地处理不同体积和浊度的BALF样本，具有有竞争力的灵敏度以及对不同肺癌类型的良好通用性，有望成为BALF液体活检的一种有前景的方法。这种高性能的基于BALF的液体活检将改善肺癌的细胞病理学识别与诊断。
Separation and detection of exfoliated tumor cells (ETCs) from bronchoalveolar lavage fluid (BALF), namely the liquid biopsy of BALF, has been proved to be a valuable tool for the diagnosis of lung cancer. Herein, we established a rapid liquid biopsy of BALF based on a dual-layer PERFECT (precise, efficient, rapid, flexible, easy-to-operate, controllable and thin) filter system for the first time. Methods: The dual-layer PERFECT filter system consists of an upper-layer filter with large micropores (feature size of 49.4 ± 0.5 μm) and a lower-layer filter with small micropores (9.1 ± 0.1 μm). The upper-layer filter contributes to the isolation of cell clusters and removal of mucus from BALF samples, meanwhile the lower-layer one targets for the separation of single ETCs. First, separation of 10000 spiked A549s (cultured lung cancer cells) from 10 mL clinical BALF samples (n=3) were performed to investigate the performance of the proposed system in rare cell separation. Furthermore, separation and detection of ETCs and ETC clusters from clinical BALF samples were performed with this system to test its efficacy and compared with the routine cytocentrifuge. The clinical BALF samples were collected from 33 lung cancer-suspected patients with visible lesions under bronchoscope. The final histopathological results showed that 20 samples were from lung cancer positive patients while the other 13 cases were from lung cancer negative patients. Results: The recovery rate of spiked A549 cells from clinical BALF samples with the developed system (89.8 ± 5.2%) is significantly higher than that with the cytocentrifuge (13.6 ± 7.8%). In the preliminary clinical trial, although 33 clinical BALF samples with volume ranging from 6 mL to 18 mL showed greatly varied turbidity, filtrations could be finished within 3 min for 54.6% of samples (18/33), and 10 min at most for the rest. The dual-layer PERFECT filter system is proved to have a much higher sensitivity (80.0%, 95% CI: 55.7%-93.4%) than the routine cytocentrifuge (45.0%, 95% CI: 23.8%-68.0%), p=0.016 (McNemar test, two-tail). Moreover, the sensitivity of this platform is neither interfered by the variations of turbidity of the BALF samples, nor associated with the types of lung cancer. Conclusions: The easy and rapid processing of BALF samples with varying volume and turbidity, competitive sensitivity and good versatility for different lung cancer types will make the established dual-layer PERFECT filter system a promising approach for the liquid biopsy of BALF. The high-performance BALF-based liquid biopsy will improve the cytopathological identification and diagnosis of lung cancer.