Technical Development of Molecular Profiling Technologies

临床分子图谱核心(CMPC)技术的开发工作主要是为了扩大可以分析的临床样本的数量。尽管临床研究人员的意图是好的，但获得适当的生物谱学仍然是执行CMPC个性化医学使命的最具挑战性的方面。为此，我们致力于分析福尔马林固定石蜡包埋(FFPE)标本的问题。使用FFPE的能力是非常有吸引力的，因为这种标本类型适合常规的病理学实验室实践。我们对癌症中DNA甲基化的研究提供了这一领域最新项目的一个例子。使用一种新的基于微阵列的平台，我们已经建立了在FFPE样本中像在冰冻样本中一样准确地分析DNA甲基化位点的可能性。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的分析相对稳健，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，因为尽管平台技术在本质上不是限制性的，但在这种受损样本中发现的片断RNA在稳定之前的样本处理中受到许多变量的影响(热缺血时间、处理时间、处理化学等)。并稳定到不同程度的时间和储存条件下。尽管有这些挑战，我们认为值得探索新的技术和程序来分析短RNA片段。为了帮助确保重复性和提供高质量的结果，我们已经评估并正在不断制定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样本中提取了DNA用于甲基化分析；值得注意的是，这些样本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幻灯片中提取DNA的协议，并能够从这种材料中产生非常高质量的DNA拷贝数和突变图谱。几十年来，苯酚和氯仿等有机溶剂一直被用来从血液和组织中提纯核酸。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，而不使用有机溶剂。这些努力说明了我们致力于将基因组图谱技术的用途扩展到实际可获得的临床样本。CMPC面临的一个常见问题是，与解剖标本相比，许多收到的标本是含有相对较少数量的细胞的活组织检查。此外，有时多种用途的标本需求量很大，必须分成非常少量的标本。因此，CMPC正在努力在将样本带到我们常用的分析方法(如DNA测序)上进行测试之前，实施全基因组扩增。数据表明，这些扩增的序列适合DNA测序，但对表观基因组分析(如甲基化测定)产生了问题。这项工作将最大限度地利用如此少量的样品，如果成功，CMPC将能够减少预先需要的样品数量，或者允许我们使用通常不能接受的非常稀有和珍贵的样品的起始量。为了与我们的目标保持一致，提高少量样本的实用性，同时最大化我们可以从这样的样本中获得的信息量，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信息来分析与他们正在研究的特定疾病相关的特定生物标记物的样本。例如，使用纳米串平台，仅使用100 ng总RNA就可以量化数百个基因的基因表达。CMPC参与了一个政府部门间的合作，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控分析。具体地说，我们与NIST生化科学部合作，正在开发定制的微流控设备和程序，用于合成和扩增RNA的纳升DNA。我们的目标是将样本大小降低到用于基因表达分析的单细胞等价物。技术开发的一个重要新领域是“下一代”测序技术的应用和临床标本使用方案的开发。这些新技术提供了在肿瘤标本上以比微阵列更深入和更强大的方式产生基因组图谱数据的可能性。例如，有可能对大量可能促进肿瘤生长的突变的药物靶点进行分析，这些数据可能会被纳入未来的临床试验设计。CMPC的领导层明白，培养新的学生和科学家将是这一个性化癌症医学新领域的关键，为此，我们在今年积极培训了一名学士后、两名博士后研究员、多名暑期学生和一名高中高级志愿者，他们与我们一起开展了一个特殊项目。

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.