Functional Genomics of Sarcoma

许多技术同时应用于确定给定生物标本的分子特征。历史上，这些技术中的大多数都使用基于微阵列的方法，但它们正在迅速得到补充，并在某些方面被不断发展的下一代DNA测序技术所取代。这些方法的力量主要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。现在可以任意精细地、多维地表征癌症基因组（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）。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400,000个CpGs的甲基化状态。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。在过去的一年里，我们越来越多地利用下一代测序的力量来提高这些分析的精度和通量。我们的实验室长期以来一直在研究有趣的肉瘤生物学，我们最近一直在将这些技术应用于儿童骨肿瘤，骨肉瘤。我们成功地鉴定了骨肉瘤的高分辨率基因表达、miRNA表达、基因拷贝数和SNP谱。这项工作已经证明了一种重复的拷贝数变化的模式，尽管骨肉瘤基因组的高度混乱的性质是明显的。此外，已经有可能证明拷贝数对骨肉瘤中的基因表达有深远的影响。这种模式提示了一些有待进一步研究的候选基因。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，并计划利用人与犬疾病的相似性来完善我们对这种肿瘤的理解。我们也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，它产生了几种儿童肉瘤的融合基因转录因子特征。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相结合。在尤文氏肉瘤中，我们利用RNA干扰技术靶向致癌转录因子EWS-FLI1，研究受该蛋白调控的基因。我们还完成了染色质免疫沉淀研究，该研究确定了EWS-FLI1转录因子在基因组中的位置。生物信息学分析显示EWS-FLI1与细胞周期转录因子E2F3存在显著的共定位。我们的数据表明，EWS-FLI1的转化活性可能部分源于其对细胞周期靶点的影响，这些靶点也受E2F因子的调节。在肺泡横纹肌肉瘤中，我们使用染色质免疫沉淀结合下一代测序来鉴定致癌融合蛋白PAX3-FKHR靶向的基因。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。在胃肠道间质肿瘤中，我们关注的是信号转导激酶缺乏突变的肿瘤组。我们发现这组肿瘤，一致的特征是缺乏琥珀酸脱氢酶，在DNA甲基化中表现出惊人的全球异常。这为疾病机制提供了新的见解，并为这些病例提供了可靠的诊断分类。

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. We have also completed a chromatin immunoprecipitations study which assigns the location of the EWS-FLI1 transcription factor across the genome. The bioinformatics analysis reveals a striking co-location of EWS-FLI1 with the cell cycle transcription factor E2F3. Our data suggests that the transforming activity of EWS-FLI1 may arise in part from its effect on cell cycle targets also regulated by E2F factors. 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. In gastrointestinal stromal tumor, we have focused on the group of tumors which lack mutations in signal transducing kinases. We found that this group of tumors, uniformly characterized by lack of succinate dehydrogenase, demonstrates striking global abnormalities in DNA methylation. This provides a new insight into the mechanism of disease and provides a robust diagnostic classification for these cases.