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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要晚期癌症患者经常会出现恶性胸腔积液 (MPE)，这是一种积液，发生在肺表面和胸壁之间，含有恶性肿瘤细胞和良性肿瘤细胞炎症细胞。在许多情况下，经皮或经支气管组织活检可能是少细胞或由于难以获得，因此 MPE 可能是唯一可用于病理评估和分子检测的标本。胸腔穿刺术可去除这些液体，缓解症状并提供可用的诊断材料用于下游分子分析。在目前的临床实践中，胸腔穿刺液通常经过离心分离和分离。富含细胞的沉淀物用于生成福尔马林固定石蜡包埋 (FFPE) 细胞沉淀物，随后将其用于制作用于诊断的苏木精和伊红 (H&E) 染色载玻片以及用于诊断的其他未染色载玻片辅助研究。最近的研究强调了这样一个事实：胸水样本通常含有丰富的细胞- 上清液部分中的游离 DNA (cfDNA)，这可能代表 DNA 的替代来源分子测试。与从血浆中分离的 cfDNA 类似，从 MPE 中分离的 cfDNA 理论上可以规避肿瘤内异质性和组织可及性的问题，同时避免了时间创建细胞块并减少/消除从细胞中刮取和提取 DNA 的劳动密集型步骤未染色的载玻片。尽管有这样的承诺，但目前还没有针对收集、储存、处理、以及从 MPE 中分离出的 cfDNA 的分子测试。我们的建议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，以确定最佳实践处理来自 MPE 的 cfDNA。我们预测这些分析前步骤的优化和协调将减少假阴性结果、提高重现性、提高效率并减少周转在 MPE 测试期间。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，以开发可轻松适应现有的标准操作程序临床工作流程。最后，我们将使用 CLIA/CAP 认证的多基因来验证这些预分析方案测序测定。