Technical Development of Molecular Profiling Technologies

分子分析技术的技术发展

• 批准号: 8350130
• 负责人: Daniel Edelman
• 金额: $ 43.69万
• 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/8350130
• 关键词: Anatomy; Archives; Area; Biochemical; Biological; Biological Assay; Biological Markers; Biopsy; Blood; Cell Count; Cells; Chemicals; Chemistry; Chloroform; Clinical; Clinical Investigator; Clinical Trials; Clinical Trials Design; Collaborations; Commerce; Complementary DNA; Custom; Cytology; DNA; DNA Methylation; DNA Sequence; DNA copy number; Data; Development; Disease; Drug Delivery Systems; Ensure; Face; Formalin; Freezing; Future; Gene Expression; Genes; Genome; Genomics; Goals; Government; Health Hazards; Institutes; Laboratories; Leadership; Malignant Neoplasms; Medicine; Messenger RNA; Methylation; MicroRNAs; Microfluidic Microchips; Microfluidics; Mission; Molecular Profiling; Mutation; National Cancer Institute; Nucleic Acids; Organic solvent product; Paraffin Embedding; Pathology; Phenols; Postdoctoral Fellow; Procedures; Process; Protocols documentation; RNA; RNA amplification; Reproducibility; Research; Research Personnel; Sample Size; Sampling; Science; Scientist; Site; Slide; Specimen; Students; Technology; Testing; Time; Tissues; Training; Warm Ischemia; Work; base; comparative genomic hybridization; epigenomics; improved; innovation; insight; nanolitre; new technology; next generation; novel; programs; research and development; technology development; tumor; tumor growth; twelfth grade; volunteer; wasting

The Clinical Molecular Profiling Core's (CMPC) technology development efforts are primarily directed at expanding the number of clinical samples which can be analyzed. Despite the best intentions of clinical researchers, accrual of appropriate biospecimen remains the most challenging aspect of implementing the CMPC's personalized 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. As expected, DNA based assays are relatively robust, however, RNA is much more labile template. We are exploring 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. Despite these challenges, we believe it is worth exploring new technologies and procedures for analyzing short RNA fragments. To help ensure reproducibility and provide for quality results, we have evaluated and are continually develop standard operating procedures for extracting nucleic acid from clinical specimens. For the above mentioned study, we 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. Very recently, 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. A common problem the CMPC faces is that many of the specimens received are biopsies containing relatively few numbers of cells as compared to anatomic specimens. Also, sometimes specimens are in high demand for multiple uses and must be divided into very small amounts. Therefore, the CMPC is working to implement whole genome amplification prior to bringing the sample to testing on our commonly used assays such as DNA sequencing. Data have shown that these amplified sequences are suitable for DNA sequencing, but have generated problems for epigenomic assays such as methylation determination. The work is on going to make the best use of such small sample amounts and when successful will allow the CMPC to either decrease the amount of specimen needed up front or allow us to make use of what would be normally unacceptable starting amounts of very rare and precious specimens. In keeping with our goal to improve the utility of small amounts of sample and yet maximize the amount of information we can derive from such a specimen, an important project of the CMPC is to develop multiplex expression assays. Clinical investigators can utilize these to analyze specimens for particular biomarkers associated with a specific disease they are studying. For example, using the Nanostring platform it is possible to quantitate the gene expression of hundreds of genes using only 100 ng of total RNA. The CMPC is engaged in an inter-government department collaboration developing state of the art microfluidic assays with the National Institute of Standards and Technology (NIST), part of the U.S. Commerce Department. Specifically, in partnership with the NIST Biochemical Science Division we are developing custom fabricated microfluidic devices and procedures for the nanoliter cDNA synthesis and amplification of RNA. Our goal is to reduce sample size down to single cell equivalents for analysis of gene expression. An important new area of technology development is in the application of "next generation" sequencing technologies and development of protocols 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. The leadership of the CMPC understands that training of new students and scientists will be crucial to this new field of personalize cancer medicine and to that end have actively trained in this year one post-baccalaureate, two post-doctoral fellows, multiple summer students, and one high school senior volunteer working with us on a special program.

临床分子分析核心（CMPC）技术开发工作主要是针对可以分析的临床样本数量的。尽管临床研究人员的意图最佳，但适当的生物胶菌的应计仍然是实施CMPC个性化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。如预期的那样，基于DNA的测定相对稳健，但是，RNA更加不稳定。我们正在探索从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。尽管面临这些挑战，我们认为值得探索分析短RNA碎片的新技术和程序。为了帮助确保可重复性并提供质量结果，我们已经评估并不断开发从临床样本中提取核酸的标准操作程序。对于上述研究，我们成功地从FFPE样品中提取了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。最近，我们开发了一种用于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。 CMPC面孔的一个常见问题是，与解剖标本相比，接收到的许多样本是包含相对较少数量的细胞的活检。同样，有时样品对多种用途的需求很高，必须分为很少的。因此，CMPC正在努力实施整个基因组扩增，然后将样品进行测试，例如我们常用的测定法，例如DNA测序。数据表明，这些放大序列适用于DNA测序，但对诸如甲基化测定等表观基因组学测定产生了问题。这项工作是要充分利用这么小的样本量，并且在成功时，CMPC可以提前减少所需的标本量，或者让我们能够利用通常不可接受的非常稀有和珍贵标本的起始数量。为了符合我们改善少量样本的实用性的目标，并最大程度地提高了我们可以从这样的标本中得出的信息量，CMPC的一个重要项目是开发多重表达测定。临床研究人员可以利用它们来分析与他们正在研究的特定疾病相关的特定生物标志物的标本。例如，使用纳米弦平台可以仅使用100 ng的总RNA来定量数百个基因的基因表达。 CMPC与美国商务部一部分的国家标准技术研究所（NIST）进行了政府间的合作，开发了最先进的微流体测定。具体而言，与NIST生化科学部合作，我们正在开发自定义的制作微流体设备和纳米素cDNA合成和RNA扩增的程序。我们的目标是将样本量降低到单细胞当量以分析基因表达。技术开发的一个重要新领域是应用“下一代”测序技术和用于临床标本的协议的开发。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。 CMPC的领导才能理解，对新学生和科学家的培训对于这个新的个性化癌症医学领域至关重要，因此，在今年的一项培训中，一位培训是在今年的一项后核心后的一项培训，两名博士后研究员，两名夏季学生，多个夏季学生和一名高中高中高中的高中志愿者与我们一起从事特殊计划。