Functional genomics of breast cancer

许多技术同时应用于确定给定生物标本的分子特征。这些技术中的大多数目前使用基于微阵列的方法，但它们正在迅速被不断发展的下一代DNA测序技术所补充。多种微阵列用于各种目的，但目前主要的技术方法是使用合成的寡核苷酸结合到固体载体上，并用从感兴趣的生物标本制备的标记核酸进行查询。这种方法的力量在这项技术的当前实施方案中，主要是基于已知基因组序列和完全由计算手段控制的微阵列设计之间的直接联系。这使得研究者能够构建任意设计的阵列，专门针对所需的分析，并将阵列的分辨率调整到非常精细的水平。因此，例如，现在有可能以比单基因分辨率更好的方式确定每个外显子的mrna表达，并观察基因拷贝数（扩增或删除）的变化。从感兴趣的任何细胞或组织源制备的荧光探针然后杂交到这些阵列，提供大规模的高分辨率的基因组视图。我们最近的努力已将这项技术应用于儿童和成人肉瘤。目前，我们的重点是将尽可能多的检测方法过渡到微小样本（例如在常规临床护理过程中通常收集的样本）和福尔马林固定石蜡包埋（FFPE）样本。当一个人考虑到将这项工作过程中的发现转化为临床护理的潜力时，使用FFPE样品的能力尤为重要，在临床实验室中，基于FFPE的方法是稳定生物标本的标准方法。重要的是，我们已经证明，有可能在数百个样品上平行确定超过1500个CpGs的甲基化状态，其结果与从冷冻标本中获得的结果相匹配。这打开了大量现有的FFPE样本档案以供调查。我们现在也经常从FFPE样本中获得出色的拷贝数数据。我们对特定转录因子在确定乳腺癌表型中的作用特别感兴趣，并且已经通过染色质免疫沉淀结合微阵列或测序分析来研究这些。类似的基于阵列的技术已被用于平行识别大量位点的甲基化状态，扩展了表征基因组结构、功能和调控的多个方面的能力。我们最近研究了导管原位癌（DCIS）分级的问题。病理学家能够将DCIS划分为高或低级别，但仍有大量的病例处于中等级别。我们已经证明，可以通过表达分析将这些案例置于高或低等级类别中。这建立了DCIS的二元分类，有可能提高个体诊断，并用于分层临床试验。

A number of technologies are applied in parallel to determine the molecular profile of a given biospecimen. The majority of these technologies currently use microarray based methods, but they are rapidly being supplemented by evolving next generation DNA sequencing technologies. Several varieties of microarray are used for various purposes, but the predominant current technical approaches use synthetic oligonucleotides bound to a solid support and interrogated with labeled nucleic acids prepared from the biospecimen of interest. The power of this approach in the current embodiment of this technology is based largely on the direct connection between known genome sequence and the design of microarrays completely controlled by computational means. This allows the investigator to construct arrays of arbitrary design tailored specifically to the desired analysis and to adjust the resolution of the arrays to a remarkably fine level. Thus, for example, it is now possible to determine the expression of mRNAs exon by exon and to observe changes in gene copy number (amplification or deletion) at better than single gene resolution. Fluorescent probes prepared from any cell or tissue source of interest are then hybridized to these arrays providing a large scale high resolution view of the genome. Our recent efforts have applied this technology to pediatric and adult sarcomas. Currently we are focused on transitioning as many assays as possible to minute samples (such as may typically be collected in the course of routine clinical care) and formalin fixed paraffin embedded (FFPE) specimens. The ability to work with FFPE samples is particularly important when one considers the potential to transition discoveries made in the course of this work to clinical care where FFPE based methods are the standard method of stabilizing biospecimens in the clinical laboratory. Of importance we have demonstrated that it is possible to determine the methylation status of more than 1500 CpGs in parallel on hundreds of samples with results which match those obtained from frozen specimens. This opens vast existing archives of FFPE samples to investigation. We now routinely obtain excellent copy number data from FFPE samples as well. We are particularly interested in the role of specific transcription factors in determining breast cancer phenotypes and have been investigating these through chromatin immunoprecipitation combined with microarray or sequencing analysis. Similar array based technologies have been used to identify the methylation status at a large number of sites in parallel, extending the ability to characterize multiple aspects of genome structure, function and regulation. We have recently investigated the problem of grading ductal carcinoma in situ (DCIS). Pathologists are able to grade DCIS into high or low grade categories, but are left with a large number of cases which are intermediate in grade. We have demonstrated that it is possible to place these cases in either the high or low grade categories by expression profiling. This establishes a binary classification of DCIS which has the potential to improve individual diagnosis and to be used to stratify clinical trials.