Technical Development of Molecular Profiling Technologies

分子分析技术的技术发展

• 批准号: 8158355
• 负责人: Daniel Edelman
• 金额: $ 40.2万
• 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/8158355
• 关键词: Cytology; DNA; Data; Detection; Development; Disease; Microfluidic Microchips; Microfluidics; Molecular Profiling; Technology

The Clinical Molecular Profiling Cores (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 Cores 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 Core 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, we are working on a cell cycle reverse transcriptase PCR assay that incorporates some 25 different genes. The amplicons from the reaction are run on the Beckman Coulter GeXP system, a capillary electrophoresis platform that uses fluorescence for detection and quantification. We are evaluating the results from this prototype assay in comparison to other multiplex platforms such as quantitative real-time PCR and the Fluidigm and Nanostring platforms. The CMPC is actively 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 to 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 alone one post-baccalaureate, one post-doctoral fellow, six summer students, and one high school senior volunteer working with us on a special program.

临床分子特征分析核心（CMPC）技术开发工作主要针对扩大可分析的临床样本数量。尽管临床研究人员的意图是好的，但适当生物标本的积累仍然是实施Cores个性化医学使命的最具挑战性的方面。出于这个原因，我们一直致力于分析福尔马林固定石蜡包埋（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学实验室实践。最近在这一领域的一个项目的例子是我们的研究DNA甲基化在癌症中提供。使用一种新的基于微阵列的平台，我们已经建立了FFPE标本中的DNA甲基化位点的分析与冷冻标本中的分析一样准确。这将为这类研究打开大量的组织标本档案。正如预期的那样，基于DNA的测定是相对稳健的，然而，RNA是更不稳定的模板。我们正在探索从FFPE等档案材料中获得mRNA签名的可能性。这是困难的，因为尽管平台技术本质上不是限制性的，但在这种受损样品中发现的片段化RNA在稳定化之前在样品处理中经受许多变量（热缺血时间、处理时间、处理化学等）。以及在不同程度的时间和储存条件下稳定后。尽管存在这些挑战，但我们认为值得探索用于分析短RNA片段的新技术和程序。为了帮助确保重现性并提供高质量的结果，我们已经评估并不断开发从临床标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DNA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，我们开发了一种从细胞学载玻片中提取DNA的方法，并且能够从这种材料中产生非常高质量的DNA拷贝数和突变谱。有机溶剂如苯酚和氯仿已被用于从血液和组织中纯化核酸数十年。然而，这些化学品的使用和产生的废物造成了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实际可获得的临床样品。 CMPC面临的一个常见问题是，与解剖标本相比，收到的许多标本是含有相对较少数量细胞的活检标本。此外，有时标本是在多种用途的高需求，必须分成非常小的数量。因此，核心正在努力实施全基因组扩增，然后再将样本用于我们常用的检测方法（如DNA测序）。数据表明，这些扩增的序列适合于DNA测序，但对于表观基因组测定如甲基化测定产生了问题。这项工作将充分利用如此小的样本量，当成功时，CMPC将能够减少前期所需的样本量，或者允许我们利用通常不可接受的非常稀有和珍贵的样本的起始量。为了与我们的目标保持一致，以提高少量样品的效用，但最大限度地提高我们可以从这样的标本中获得的信息量，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这些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，我们正在研究一种细胞周期逆转录酶PCR检测方法，其中包含约25种不同的基因。来自反应的扩增子在Beckman Coulter GeXP系统上运行，这是一种使用荧光进行检测和定量的毛细管电泳平台。我们正在评估该原型检测的结果，并与其他多重平台（如定量实时PCR和Fluidigm和Nanostring平台）进行比较。 CMPC积极参与政府部门间合作，与美国商务部下属的国家标准与技术研究所（NIST）合作开发最先进的微流体分析。具体来说，我们与NIST生物化学科学部门合作，正在开发定制的微流体装置和程序，用于纳升cDNA合成和RNA扩增。我们的目标是将样本量减少到单细胞当量，用于基因表达分析。技术发展的一个重要的新领域是将“下一代”测序技术应用于临床标本。这些新技术提供了以比微阵列更深入和更强大的方式生成肿瘤标本基因组分析数据的可能性。例如，它可能成为可能的档案大量的药物靶点的突变，可能会促进肿瘤生长，数据可以纳入未来的临床试验设计。CMPC的领导层了解到，新学生和科学家的培训对于个性化癌症医学这一新领域至关重要，为此，仅在今年就积极培训了一名学士后，一名博士后研究员，六名暑期学生和一名高中高年级志愿者与我们一起工作。