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

恶性胸腔积液标本临床检测最佳实践综合分析

• 批准号: 10703794
• 负责人: Walter Patrick Devine
• 金额: $ 36.94万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-05 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703794
• 关键词: Advanced Malignant Neoplasm; Affect; Ancillary Study; Area; Benchmarking; Benign; Biological Assay; Biopsy; Blood Tests; Cell Separation; Cells; Centrifugation; Certification; Chemistry; Chest wall structure; Clinical; Collection; Cytology; Cytopathology; DNA; Data; Diagnosis; Diagnostic; Evaluation; Experimental Designs; Formalin; Gene Frequency; Gene Rearrangement; Genes; Goals; Guidelines; Hematoxylin and Eosin Staining Method; Inflammatory; Laboratories; Libraries; Liquid substance; Literature; Lung; Malignant Neoplasms; Malignant Pleural Effusion; Measures; Microsatellite Instability; Molecular; Molecular Analysis; Mutation; Nucleic Acids; Oncogenes; Oncogenic; Paraffin Embedding; Pathologic; Pathologist; Patients; Plasma; Pleural; Pleural effusion disorder; Positioning Attribute; Preparation; Procedures; Process; Protocols documentation; Publishing; Reproducibility; Research; Sampling; Serum; Slide; Source; Specimen; Speed; Stains; Surface; Techniques; Temperature; Testing; Therapeutic; Thoracentesis; Time; Tissues; Tumor Tissue; Urine; Validation; Variant; cancer cell; cell free DNA; clinical practice; detection test; experimental study; gene panel; genetic testing; improved; insertion/deletion mutation; liquid biopsy; molecular pathology; multidisciplinary; mutant; neoplastic cell; next generation sequencing; reduce symptoms; research clinical testing; success; theories; time interval; tumor; tumor heterogeneity

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.

项目摘要/摘要 患有晚期癌症的患者通常会出现恶性胸腔积液（MPE），这是一系列流体，是该液体的集合 在肺表面和包含恶性肿瘤细胞和良性的胸壁表面发展 炎性细胞。在许多情况下，经皮或经支撑组织活检可能是Pauci-ellular或 很难获得，因此MPE可能是唯一用于病理评估和分子测试的标本。 胸腔穿刺消除这种液体，减轻症状，并提供可使用的诊​​断材料 用于下游分子分析。在当前的临床实践中，胸腔穿刺液通常是离心的，并且 富含细胞的颗粒用于生成福尔马林固定的石蜡嵌入（FFPE）细胞颗粒，后来是 用于制作苏木精和曙红（H＆E）染色幻灯片，以诊断以及其他未染色的载玻片 辅助研究。最近的研究强调了一个事实，即胸膜流体样品通常包含丰富的细胞 - 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。与从血浆分离的CfDNA相似，从MPE中分离出的CfDNA可以从理论上讲 肿瘤内异质性和组织可及性的问题同时消除了时间 需要创建一个细胞块，并减少/消除刮擦和从中提取DNA的劳动密集型步骤 未染色的幻灯片。尽管有承诺，但仍未针对收集，存储，处理， 和从MPE分离的CfDNA的分子测试。我们的建议系统地测试了几种精髓 变量以及直接比较三种不同的CFDNA隔离技术，以确定最佳实践 从MPE处理CFDNA。我们预测这些序列步骤的优化和协调 将导致虚假负面结果降低，可重复性提高，提高效率以及降低的转盘 在测试MPE的测试中。我们将利用分子病理学领域的集体专业知识 病理学和测试验证以开发标准操作程序，可以轻松适应现有 临床工作流程。最后，我们将使用CLIA/CAP认证的多基因验证这些分析前协议 测序测定。