Technical Development of Molecular Profiling Technologies

分子分析技术的技术发展

• 批准号: 9556822
• 负责人: Daniel Edelman
• 金额: $ 35.84万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556822
• 关键词: Archives; Area; Biological; Biological Assay; Blood; Chemicals; Chemistry; Chloroform; Clinical; Clinical Trials; Clinical Trials Design; Complement; Cytology; DNA; DNA Methylation; DNA copy number; DNA sequencing; Data; Development; Drug Targeting; Ensure; Formalin; Freezing; Future; Genome; Genomic DNA; Genomics; Health Hazards; Human; Laboratories; Libraries; Literature; Malignant Neoplasms; Messenger RNA; Methylation; MicroRNAs; Mission; Molecular Profiling; Mutation; National Cancer Institute; Nucleic Acids; Organic solvent product; Paraffin Embedding; Pathology; Phenols; Procedures; Protocols documentation; Publishing; RNA; Reality Testing; Reproducibility; Research; Research Personnel; Saliva; Sampling; Site; Slide; Somatic Mutation; Specimen; Technology; Time; Tissues; Warm Ischemia; base; comparative genomic hybridization; cost; genomic profiles; improved; innovation; insight; new technology; next generation sequencing; novel; precision medicine; protocol development; research and development; technology development; tumor; tumor growth; wasting

The Clinical Molecular Profiling Core's (CMPC) technology development efforts are directed at expanding the number of clinical samples which can be analyzed and improving next-generation sequencing technologies. Despite the best intentions of clinical researchers, accrual of appropriate biospecimen remains the most challenging aspect of implementing the CMPC's precision medicine mission. For this reason, we have directed efforts to the problem of analyzing formalin fixed paraffin embedded (FFPE) specimens. The ability to use FFPE is extremely attractive since this specimen type fits into routine pathology laboratory practices. An example of a recent project in this area is provided by our study of DNA methylation in cancer. Using a novel microarray based platform, we have established that it is possible to profile sites of DNA methylation in FFPE specimens as accurately as in frozen specimens. This will open up large archives of tissue specimens to this type of research. To complement this tumor specimen type, we regularly use saliva specimens to provide germline DNA to better identify somatic mutations in the cancer specimens. As expected, DNA based assays are relatively robust, however, RNA is a much more labile template. We have explored the possibility of obtaining mRNA signatures from archival material such as FFPE. This is difficult because although the platform technology is not intrinsically limiting, the fragmented RNA found in such compromised samples are subject to many variables in sample processing prior to stabilization (warm ischemia time, processing time, processing chemistry etc.) and after stabilization to varying degrees of time and conditions of storage. In addition to this, we have published in the literature on stabilizing extracted RNA for downstream microarray use in Stevenson et al. (2015), "Long-term stability of total RNA in RNAstable as evaluated by expression microarray"; if implemented in the lab, it could save on -80C storage costs. To help ensure reproducibility and provide for quality results, we have developed refined standard operating procedures for extracting nucleic acid from clinical specimens. We have successfully extracted DNA from FFPE samples for use in methylation assays; significantly, these samples have been very suitable for comparative genomic hybridization and DNA sequencing. We have developed a protocol for extracting DNA from cytology slides and have been able to generate remarkably high quality DNA copy number and mutation profiles from this material. Organic solvents such as phenol and chloroform have been used for decades to purify nucleic acids from blood and tissues. However, the use and waste produced with these chemicals creates health hazard issues and problems of disposal. Therefore, we have investigated and validated new protocols for extraction of DNA, RNA, and microRNA from both research and clinical specimens without the use of organic solvents. These efforts are illustrative of our commitment to extend the utility of genome profiling technologies to realistically obtainable clinical samples. An important new area of technology development is in the application of next-generation sequencing technologies and development of protocols especially in library construction for use with clinical specimens. These new technologies offer the possibility of generating genomic profiling data on tumor specimens in a much deeper and more robust way than has been possible with microarrays. For example, it may become possible to profile large numbers of drug targets for mutations which may promote tumor growth, data which could be incorporated into future clinical trials design. Efforts are ongoing to adapt commercial protocols to the reality of testing human specimens; in one (Chaisaingmongkol et al., 2017) we discovered that changing the blockers had a profound effect on the depth of coverage.

临床分子分析核心（CMPC）技术开发工作旨在扩大可以分析并改善下一代测序技术的临床样本数量。尽管有临床研究人员的最佳意图，但适当的生物循环的应计仍然是实施CMPC精确医学任务的最具挑战性的方面。因此，我们指示努力解决福尔马林固定石蜡嵌入（FFPE）标本的问题。使用FFPE的能力非常有吸引力，因为这种标本类型适合常规病理实验室实践。我们在癌症中对DNA甲基化的研究提供了一个最新项目的例子。使用新型的基于微阵列的平台，我们已经确定可以像冷冻样品中一样准确地介绍FFPE样品中DNA甲基化的位点。这将为此类研究打开大型组织标本的档案。为了补充这种肿瘤标本类型，我们定期使用唾液标本来提供种系DNA，以更好地识别癌症标本中的体细胞突变。如预期的那样，基于DNA的测定相对强大，但是，RNA是一个更不稳定的模板。我们已经探索了从FFPE等档案材料中获得mRNA特征的可能性。这很困难，因为尽管平台技术不是本质上的限制，但是在稳定之前（温暖的缺血时间，加工时间，加工化学等）以及在不同的时间和条件下，在稳定之前（温暖的缺血时间，加工时间，加工化学等）之前，在样本处理（温暖的缺血时间，加工时间，加工化学等）之前，在样本处理中发现了许多变量。除此之外，我们还发表了有关稳定提取的RNA的文献，用于在Stevenson等人的下游微阵列中使用。 （2015年），“通过表达微阵列评估的RNAstable总RNA的长期稳定性”；如果在实验室中实施，则可以节省-80C存储成本。为了帮助确保可重复性并提供质量结果，我们开发了从临床标本中提取核酸的精制标准操作程序。我们已成功从FFPE样品中提取DNA以用于甲基化测定。值得注意的是，这些样品非常适合比较基因组杂交和DNA测序。我们已经开发了一种从细胞学载玻片中提取DNA的方案，并能够从该材料中产生高质量的DNA拷贝数和突变曲线。数十年来，已经使用了有机溶剂（例如苯酚和氯仿）从血液和组织中纯化核酸。但是，这些化学物质产生的使用和废物会造成健康危害问题和处置问题。因此，我们研究并验证了从研究和临床标本中提取DNA，RNA和microRNA的新方案，而无需使用有机溶剂。这些努力说明了我们致力于将基因组分析技术的实用性扩展到现实获得的临床样本。技术开发的一个重要新领域是应用下一代测序技术和协议的开发，尤其是在图书馆构造中用于临床标本。这些新技术提供了与微阵列相比，以更深，更健壮的方式生成有关肿瘤标本的基因组分析数据的可能性。例如，可能有可能为可能促进肿瘤生长的突变概述大量药物靶标，这些数据可以纳入将来的临床试验设计中。正在努力将商业方案调整为测试人类标本的现实；在一个（Chaisaingmongkol等人，2017年）中，我们发现改变阻滞剂对覆盖深度产生了深远的影响。