Functional Genomics of Sarcoma

同时应用了许多技术来确定特定生物旋转剂的分子轮廓。这些技术中的大多数历史上都使用基于微阵列的方法，但它们正在迅速得到补充，在某些方面，被不断发展的下一代DNA测序技术所取代。这些方法的力量很大程度上是基于已知基因组序列、基因组注释、实验设计和计算分析之间的直接联系。现在有可能以任意精细的细节和多个维度(例如，mRNA、miRNA、lincRNA表达、DNA序列、DNA拷贝数、DNA结构、DNA甲基化、染色质结构、染色质修饰和转录因子结合)来表征癌症基因组。我们最近的努力已经将这项技术应用于儿童和成人肉瘤。目前，我们正致力于将尽可能多的分析方法过渡到微量样本(例如在常规临床护理过程中通常可能收集的样本)和福尔马林固定石蜡包埋(FFPE)样本。当人们考虑到将在这项工作过程中所做的发现转化为临床护理的可能性时，使用FFPE样本的能力尤其重要，在临床实验室中，基于FFPE的方法是稳定生物菌群的标准方法。重要的是，我们已经证明，有可能在数百个样本上并行确定400,000多个CPGS的甲基化状态。这打开了大量现有的FFPE样本档案供调查。我们现在也经常从FFPE样本中获得优秀的拷贝数数据。在过去的一年里，我们越来越多地利用下一代测序的能力来提高这些分析的精确度和吞吐量。我们的实验室拥有长期有趣的肉瘤生物学，最近我们将这些技术应用于儿科骨肿瘤和骨肉瘤。我们已经成功地鉴定了骨肉瘤的高分辨基因表达、miRNA表达、基因拷贝数和SNP谱。这项工作证明了一种反复发生的拷贝数变化的模式，尽管骨肉瘤基因组具有高度混乱的性质，但这种模式仍然很明显。此外，有可能证明拷贝数对骨肉瘤的基因表达有深远的影响。这一模式为进一步研究提供了一些候选基因。为了获得对这种疾病的比较基因组学观点，我们还研究了犬骨肉瘤的基因表达模式，并计划利用人类和犬疾病之间的相似性来加深我们对这种肿瘤的理解。我们还在研究肉瘤中发生的特定突变的分子后果，特别是常见的染色体易位，这种易位产生了几种儿科肉瘤特有的融合基因转录因子。利用染色质免疫沉淀和DNA测序技术，我们正在鉴定致癌转录因子的结合位点，并将这些信息与这些疾病的已知表达谱结合起来。在尤文斯肉瘤中，我们利用RNA干扰技术靶向致癌转录因子EWS-FLI1，以研究该蛋白调控的基因。在肺泡型横纹肌肉瘤中，我们使用染色质免疫沉淀结合下一代测序来确定致癌融合蛋白PAX3-FKHR的靶向基因。这将为构建这些关键致癌事件下游的失调基因网络提供明确的框架。

A number of technologies are applied in parallel to determine the molecular profile of a given biospecimen. The majority of these technologies historically have used microarray based methods, but they are rapidly being supplemented, and in some respects, supplanted by evolving next generation DNA sequencing technologies. The power of these approaches is based largely on the direct connection between known genome sequence, genome annotations, experimental design and computational analysis. It is now possible to characterize cancer genomes in arbitrarily fine detail and in multiple dimensions (e.g. mRNA,miRNA, lincRNA expression, DNA sequence, DNA copy number, DNA structure, DNA methylation, chromatin structure, chromatin modification, and transcription factor binding). 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 400,000 CpGs in parallel on hundreds of samples. This opens vast existing archives of FFPE samples to investigation. We now routinely obtain excellent copy number data from FFPE samples as well. Over the last year, we have increasingly utilized the power of next generation sequencing to improve the precision and throughput of these analyses.Our laboratory has had a long standing interesting sarcoma biology, and we have been most recently applying these technologies to the pediatric bone tumor, osteosarcoma. We have successfully identified the high resolution gene expression, miRNA expression, gene copy number, and SNP profile of osteosarcoma. This work has demonstrated a pattern of recurring copy number changes which are apparent despite the highly chaotic nature of the osteosarcoma genome. In addition, it has been possible to demonstrate that copy number has a profound impact on gene expression in osteosarcoma. This pattern suggests a number of candidate genes for further investigation. To gain a comparative genomics perspective on this disease, we have also investigated the gene expression pattern of canine osteosarcoma, and plan to take advantage of the similarities between human and canine disease to refine our understanding of this tumor.We are also investigating the molecular consequences of specific mutations which occur in sarcoma, particularly the common chromosome translocations which produce the fusion gene transcription factors characteristic of several pediatric sarcomas. Using chromatin immunoprecipitation and DNA sequencing technology, we are identifying the binding sites of oncogenic transcription factors and integrating this information with the known expression profiles of these diseases. In Ewings sarcoma, we have used RNA interference technology to target the oncogenic transcription factor EWS-FLI1 to study the genes which are regulated by this protein. In alveolar rhabdomyosarcoma we have used chromatin immunoprecipitation combined with next generation sequencing to identify the genes which are targeted by the oncogenic fusion protein PAX3-FKHR. This will provide the definitive framework for building the network of dysregulated genes downstream of these critical oncogenic events.