Functional genomics of cancer

许多技术同时应用于确定给定生物标本的分子特征。这些技术目前大多使用基于微阵列的方法。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癌症、成人肉瘤、淋巴瘤、黑色素瘤、胃肠道肿瘤、乳腺癌和血液病。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。最近，我们与Illumina合作，使用小的FFPE样品获得了基因拷贝数（CGH）、snp和甲基化的优秀数据。也可以研究FFPE中的表达，但主要是候选基因的亚基因组样本。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。我们的原理证明研究比较了滤泡性淋巴瘤和滤泡性增生，并确定了几十个标记物，可以有力地区分这两种实体。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、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。在黑色素瘤中，我们主要集中在分析其祖细胞，即小鼠模型中的黑素细胞。我们与Glenn Merlino （NCI/CCR）和Ed DeFabo （George Washington University）合作，利用一种能够特异标记黑色素细胞的系统，通过流式细胞术从小鼠皮肤中纯化黑色素细胞，研究正常黑色素细胞在小鼠发育过程中的基因表达程序。该系统使我们能够研究黑色素瘤的主要致癌物，紫外线（UV）光对黑素细胞发育的影响。通过使用灵敏的微阵列技术，我们已经能够第一次观察到紫外线辐射对黑色素细胞的体内影响。这些结果为黑素细胞的发育提供了前所未有的见解，并有望促进我们对紫外线致癌作用的理解。在乳腺癌中，我们一直对导管原位癌（DCIS）的问题特别感兴趣。乳腺导管原位癌很容易通过乳房x光检查诊断出来，它可能代表的疾病对患者的长期风险很小，或者是侵袭性癌症的早期表现。然而，病理学家对DCIS分级有很大的困难，因此，临床医生很难对这些患者进行适当的强化治疗。我们与Rosemary Balleine（悉尼Westmead医院）合作，对浸润性癌伴DCIS的标本进行了显微解剖。利用浸润性癌的分级对样本进行分类，我们已经能够建立一个基因表达分类器，可以清晰地区分低分级和高分级的DCIS。我们目前正在开发一种可应用于FFPE DCIS样本的多重基因表达测定方法。经过验证和改进，该方法具有应用于前瞻性样本的潜力，并可能对DCIS的诊断和治疗产生重大影响。同样在乳腺癌领域，我们已经开发了高分辨率的基因表达和基因拷贝数数据，这导致了几个候选基因的出现，正在实验室研究中。我们通过表达谱确定的一个基因是GATA-3，这是一种与雌激素受体阳性乳腺癌相关的转录因子。我们一直怀疑GATA-3在该系统中是一个重要的发育调节因子，最近其他人在小鼠模型中的研究也证实了这一假设。为了确定GATA-3如何调节基因表达，我们在乳腺癌细胞中使用染色质免疫沉淀进行了全基因组转录因子定位研究。这些结果建立了雌激素受体与GATA-3之间的功能相互关系，并为乳腺上皮发育与癌症之间的相互作用提供了新的见解。为了跟进我们的分析研究确定的候选基因，我们正在使用RNA干扰来沉默候选基因面板，这些候选基因与表型终点相结合，这些终点确定了负责肿瘤生长的基因。其中一些研究与我们工作的技术开发方面重叠。例如，我们与安捷伦技术公司合作，利用平铺路径阵列，将染色体8p区域的表达和拷贝数分析推向了可能的最高分辨率，该区域在乳腺癌中经常被扩增。这些研究使我们能够明确地绘制出乳腺癌的这个区域并最终确定在乳腺癌中活跃的8p基因。我们已经使用类似的技术来绘制其他染色体上的基因扩增区域，克隆重排点和鉴定融合基因。我们也在使用类似的技术来研究microrna及其祖细胞的表达模式

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. 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 cancers, adult sarcomas, lymphoma, melanoma, gastrointestinal tumors, breast cancers, and hematologic disease. 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. Very recently in collaboration with Illumina, we have obtained excellent data using small FFPE samples for gene copy number (CGH), SNPs, and methylation. Expression can also be studied in FFPE, but primarily with sub-genomic samples of candidate genes. 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. Our proof-of-principle study compared follicular lymphoma to follicular hyperplasia, and identified dozens of markers which robustly distinguish these two entities. 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, 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. In melanoma, we are primarily focused on profiling its progenitor cell, the melanocyte in a mouse model. In collaboration with Glenn Merlino (NCI/CCR) and Ed DeFabo (George Washington University) we are investigating the gene expression program of normal melanocytes in murine development using and system which specifically tags melanocytes and allows them to be purified from mouse skin by flow cytometry. This system allows us to investigate the effect of the major melanoma carcinogen, ultraviolet (UV) light on melanocyte development. Through the use of sensitive microarray technologies, we have been able for the first time to observe the in vivo effect of UV radiation on melanocytes. These results are providing unprecedented insight into the melanocyte development and promise to advance our understanding of UV carcinogenesis. In breast cancer, we have been particularly interested in the problem of ductal carcinoma in situ (DCIS). DCIS is readily diagnosed by mammography, and may represent disease which carries very little long term risk to the patient or the early presentation of an aggressive cancer. However, pathologists have a great deal of difficulty grading DCIS, and as a result, clinicians have difficulty stratifying these patients to appropriately intense therapy. In collaboration with Rosemary Balleine (Westmead Hospital, Sydney), we have profiled microdissected DCIS lesions from specimens in which invasive cancer was associated with the DCIS. Using the grade of the invasive cancer to index the samples, we have been able to develop a gene expression classifier which can cleanly separate low grad from high grade DCIS. We are currently developing a multiplex gene expression assay which can be applied to FFPE DCIS samples. After validation and refinement, this assay has the potential to be applied to prospective samples and may have a substantial impact on the diagnosis and management of DCIS. Also in the breast cancer field, we have developed high resolution gene expression and gene copy number data which has led to the emergence of several candidate genes under investigation in the laboratory. One gene which we identified by expression profiling is GATA-3, a transcription factor which is associated with estrogen receptor positive breast cancer. We have suspected that GATA-3 is an important developmental regulator in this system, and recent studies by others in mouse models have confirmed this hypothesis. To determine how GATA-3 regulates gene expression, we have carried out whole genome transcription factor localization studies using chromatin immunoprecipitation in breast cancer cells. The results establish a functional inter-relation between estrogen receptor and GATA-3 and provide insight into the interplay between mammary epithelial development and cancer. To follow up on candidate genes identified by our profiling studies, we are using RNA interference to silence panels of candidate genes coupled with phenotypic endpoints which identify genes which are responsible for tumor growth. Some of these studies overlap with technology development aspects of our work. For example, in collaboration with Agilent Technologies, we have pushed the expression and copy number analysis of a region, chromosome 8p, which is frequently amplified in breast cancer to the highest possible resolution using tiling path arrays. These studies have allowed us to map this region in breast cancer definitively and to identify conclusively the 8p genes which are active in breast cancers. We have used similar technology to map regions of gene amplification on other chromosomes, to clone the points of rearrangement and to identify fusion genes. We are also using similar technology to investigate the pattern of expression of microRNAs and their progenitors