Comprehensive Analysis of Best Practices for Clinical Testing of Malignant Pleural Effusion Specimens

项目摘要/摘要 晚期癌症患者通常会出现恶性胸腔积液(MPE)，这是一种积液， 位于肺表面和胸壁之间，胸壁含有恶性肿瘤细胞和良性肿瘤细胞 炎性细胞。在许多情况下，经皮或经支气管组织活检可能是少细胞的或 难以获得，因此MPE可能是唯一可用于病理评估和分子检测的标本。 胸腔穿刺术可以去除这种液体，缓解症状，并提供可以使用的诊断材料。 用于下游分子分析。在目前的临床实践中，胸腔穿刺液通常是离心法和 富含细胞的颗粒用于产生福尔马林固定石蜡包埋(FFPE)细胞颗粒，随后 用于制作苏木精-伊红(H&E)染色的玻片用于诊断，以及附加的未染色的玻片用于 辅助研究。最近的研究强调了这样一个事实，即胸水样本通常含有丰富的细胞- 上清液中的游离DNA(CfDNA)，这可能代表了DNA的替代来源 分子测试。与从血浆中分离的cfDNA类似，从mpes中分离的cfDNA在理论上可以绕过 肿瘤内的异质性和组织可及性问题，同时避免了时间 需要创建细胞块并减少/消除刮除和提取DNA的劳动密集型步骤 未染色的幻灯片。尽管它承诺，但没有关于收集、储存、处理、 以及从MPE中分离的cfDNA的分子检测。我们的提案系统地测试了几个分析前 变量，并直接比较三种不同的cfDNA分离技术，以确定 正在处理MPE中的cfDNA。我们预测，这些分析前步骤的优化和协调 将导致减少假阴性结果，增加重复性，提高效率，并减少周转- 在MPE测试中的周围时间。我们将利用我们在细胞病理学、分子生物学方面的集体专业知识 病理学和测试验证，以开发标准操作程序，这些操作程序可以轻松地适应现有的 临床工作流程。最后，我们将使用CLIA/CAP认证的多基因来验证这些分析前协议 测序分析。

PROJECT SUMMARY/ABSTRACT Patients with advanced cancers often develop malignant pleural effusions (MPEs), a collection of fluid that develops between the surface of the lung and the chest wall that contains malignant tumor cells and benign inflammatory cells. In many cases, percutaneous or transbronchial tissue biopsies may be pauci-cellular or difficult to obtain, thus MPEs may be the only specimen available for pathologic evaluation and molecular testing. Thoracentesis removes this fluid, alleviating symptoms and also providing diagnostic material that can be used for downstream molecular analysis. In current clinical practice, the thoracentesis fluid is typically centrifuged and the cell-rich pellet is used to generate a formalin fixed paraffin embedded (FFPE) cell pellet that is subsequently used to make a hematoxylin & eosin (H&E) stained slide for diagnosis along with additional unstained slides for ancillary studies. Recent studies have highlighted the fact that pleural fluid samples often contain abundant cell- free DNA (cfDNA) within the supernatant fraction and this may represent an alternative source of DNA for molecular testing. Similar to cfDNA isolated from plasma, cfDNA isolated from MPEs could in theory circumvent the problem of intra-tumoral heterogeneity and tissue accessibility while at the same time obviate the time needed to create a cell block and reduce/eliminate the labor-intensive steps of scraping and extracting DNA from unstained slides. Despite its promise, there are no established guidelines for the collection, storage, processing, and molecular testing of cfDNA isolated from MPEs. Our proposal systematically tests several preanalytical variables as well as directly compares three different cfDNA isolation techniques to identify the best practices for processing cfDNA from MPEs. We predict that optimization and harmonization of the these preanalytical steps will lead to reduced false negative results, increased reproducibility, improved efficiency, and reduced turn- around-time in the testing of MPEs. We will leverage our collective expertise in cytopathology, molecular pathology, and test validation to develop standard operating procedures that can be easily adapted into existing clinical workflows. Finally, we will validate these pre-analytical protocols using a CLIA/CAP certified multi-gene sequencing assay.