Department of Pathology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
Department of Oncology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
Cancer Med. 2024 Sep;13(17):e70197. doi: 10.1002/cam4.70197.
Supernatants from various cytological samples, including body cavity effusion, sputum, bronchoalveolar lavage fluid (BALF), and needle aspiration, have been validated for detecting genetic alterations using cell-free DNA (cfDNA) in patients with non-small cell lung cancer (NSCLC). However, the sensitivity of fusion variations detection remains challenging. The protection of cell-free RNA (cfRNA) is critical for resolving the issue.
A protective solution (PS) was applied for preserving cfRNA in cytological supernatant (CS), and the quality of protected cfRNA was assessed by cycle threshold (CT) values from reverse transcription quantitative polymerase chain reaction (RT-qPCR). Furthermore, we collected an additional set of malignant cytological and matched tumor samples from 84 NSCLC patients, cfDNA & cfRNA extraction and double detection for driver gene mutations was validated using the multi-gene mutations detection by RT-qPCR.
Under the optimal protection system, 91.0% (101/111) of cfRNA were protected effectively. Among the 84 NSCLC patient samples, seven cytological samples failed the tests. In comparison with tumor samples, the overall sensitivity and specificity of detecting driver genes of supernatant cfDNA and cfRNA were 93.8% (74/77) and 100% (77/77), respectively. Notably, when focusing exclusively on patients with fusion gene changes, both sensitivity and specificity reached 100% (11/11) for EML4-ALK, ROS1, RET fusions, and MET ex14 skipping.
These findings suggest that cfDNA & cfRNA extraction and double detection strategy recommended in this study improve the accuracy of driver genes mutations test, especially for RNA-based assay.
已验证从各种细胞学样本（包括体腔积液、痰、支气管肺泡灌洗液（BALF）和针吸）的上清液中提取的无细胞 DNA（cfDNA）可用于检测非小细胞肺癌（NSCLC）患者的遗传改变。然而，融合变异检测的灵敏度仍然具有挑战性。保护无细胞 RNA（cfRNA）对于解决这个问题至关重要。
采用一种保护溶液（PS）来保存细胞学上清液（CS）中的 cfRNA，并通过逆转录定量聚合酶链反应（RT-qPCR）的循环阈值（CT）值来评估保护的 cfRNA 质量。此外，我们从 84 名 NSCLC 患者中收集了另一组恶性细胞学和匹配的肿瘤样本，通过 RT-qPCR 进行了多基因突变检测，验证了 cfDNA 和 cfRNA 提取及对驱动基因突变的双重检测。
在最佳保护系统下，91.0%（101/111）的 cfRNA 得到了有效保护。在 84 名 NSCLC 患者样本中，有 7 个细胞学样本检测失败。与肿瘤样本相比，上清液 cfDNA 和 cfRNA 检测驱动基因的总体灵敏度和特异性分别为 93.8%（74/77）和 100%（77/77）。值得注意的是，当仅关注融合基因改变的患者时，EML4-ALK、ROS1、RET 融合和 MET ex14 跳跃的敏感性和特异性均达到 100%（11/11）。
这些发现表明，本研究推荐的 cfDNA 和 cfRNA 提取及双重检测策略提高了驱动基因突变检测的准确性，特别是对于基于 RNA 的检测。
