Technical Development of Molecular Profiling Technologies

临床分子分析核心（CMPC）技术的发展主要是为了扩大可分析的临床样本的数量。尽管临床研究人员有最好的意图，但适当的生物标本的积累仍然是实施核心个性化医疗使命的最具挑战性的方面。出于这个原因，我们已经把精力集中在分析福尔马林固定石蜡包埋（FFPE）标本的问题上。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。我们对癌症中DNA甲基化的研究提供了这个领域最近项目的一个例子。使用一种新的基于微阵列的平台，我们已经确定可以像在冷冻标本中一样准确地分析FFPE标本中的DNA甲基化位点。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的分析相对稳定，然而，RNA是更不稳定的模板。我们正在探索从档案材料（如FFPE）中获得mRNA签名的可能性。这是很困难的，因为尽管平台技术在本质上没有限制，但在这些受损样品中发现的片段RNA在稳定之前（热缺血时间，处理时间，处理化学等）和稳定后的不同程度的时间和存储条件下受到许多变量的影响。尽管存在这些挑战，我们认为值得探索分析短RNA片段的新技术和新方法。为了确保可重复性和提供高质量的结果，我们已经评估并正在不断开发从临床标本中提取核酸的标准操作程序。在上述研究中，我们成功地从FFPE样品中提取DNA用于甲基化分析；值得注意的是，这些样品非常适合用于比较基因组杂交和DNA测序。最近，我们开发了一种从细胞学载玻片中提取DNA的方案，并能够从这种材料中产生非常高质量的DNA拷贝数和突变谱。几十年来，苯酚和氯仿等有机溶剂一直用于从血液和组织中纯化核酸。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，我们已经研究并验证了在不使用有机溶剂的情况下从研究和临床标本中提取DNA、RNA和microRNA的新方案。这些努力说明了我们致力于将基因组分析技术的效用扩展到实际可获得的临床样本。CMPC面临的一个共同问题是，与解剖标本相比，许多收到的标本是活组织标本，其中包含的细胞数量相对较少。此外，有时标本有多种用途，需求量很大，必须分成非常小的数量。因此，在将样品用于我们常用的分析（如DNA测序）之前，Core正在努力实现全基因组扩增。数据表明，这些扩增的序列适合DNA测序，但在表观基因组分析（如甲基化测定）中产生了问题。这项工作正在进行中，以充分利用如此少量的样品，如果成功，将允许CMPC减少前期所需的样品数量，或者允许我们利用通常不可接受的非常稀有和珍贵的样品起始数量。为了保持我们的目标，即提高少量样品的效用，并最大限度地从这样的样品中获得信息量，CMPC的一个重要项目是开发多重表达分析。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，我们正在研究一种包含大约25种不同基因的细胞周期逆转录酶PCR测定。反应的扩增子在Beckman Coulter GeXP系统上运行，这是一种利用荧光进行检测和定量的毛细管电泳平台。我们正在评估这个原型分析的结果，并将其与其他多重平台（如实时定量PCR和Panomics QuantiGene）进行比较。CMPC积极参与政府间部门合作，与美国商务部下属的国家标准与技术研究院（NIST）合作开发最先进的微流控分析技术。具体来说，我们正在与NIST生化科学部合作开发定制制造的微流体设备和程序，用于纳米级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，以分析基因表达。技术发展的一个重要新领域是将“下一代”测序技术应用于临床标本。与微阵列相比，这些新技术提供了以更深入、更稳健的方式生成肿瘤标本基因组图谱数据的可能性。例如，有可能描绘出大量的药物靶点的突变，这些突变可能会促进肿瘤的生长，这些数据可以纳入未来的临床试验设计。CMPC的领导明白，培养新的学生和科学家对于个性化癌症医学这一新领域至关重要，为此，仅今年就积极培养了一名博士后、一名博士后和两名暑期学生。

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 Panomics QuantiGene assay. 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 cinical 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, and two summer students.