Functional genomics of cancer

癌症的功能基因组学

• 批准号: 7592910
• 负责人: PAUL S. MELTZER
• 金额: $ 409.79万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592910
• 关键词: Address; Adult; Affect; Archives; Binding; Biological; Biological Assay; Biological Models; Biology; Bone neoplasms; Breast Cancer Cell; Cancer Biology; Candidate Disease Gene; Canis familiaris; Carcinogens; Caring; Cells; Childhood; Chromatin Structure; Chromosome abnormality; Chromosomes; Clinical; Collaborations; Coupled; DNA; DNA Microarray Chip; DNA Microarray format; DNA Sequence Rearrangement; Data; Deoxyribonuclease I; Deoxyribonucleases; Development; Diagnosis; Dimensions; Disease; Dog Diseases; End Point; Epithelial; Estrogen Receptors; Estrogen receptor positive; Exons; Flow Cytometry; Fluorescent Probes; Follicular Lymphoma; Formalin; Freezing; Gastrointestinal Neoplasms; Gene Amplification; Gene Dosage; Gene Expression; Genes; Genome; Genomics; Goals; Hematological Disease; Hormones; Hospitals; Human; Hyperplasia; In Situ Lesion; Individual; Inherited; Invasive; Investigation; Label; Laboratories; Lead; Lymphoma; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant neoplasm of gastrointestinal tract; Mammary gland; Mammography; Maps; Methods; Methylation; MicroRNAs; Microarray Analysis; Modeling; Molecular Profiling; Mus; Mutation; Nature; Neoplasm Metastasis; Noninfiltrating Intraductal Carcinoma; Nuclear Receptor Coactivator 3; Nucleic Acids; Numbers; Oligonucleotides; Paraffin Embedding; Pathologist; Pathway interactions; Patients; Pattern; Property; Purpose; RNA Interference; Research Personnel; Resolution; Risk; Rosemary; Sampling; Single Nucleotide Polymorphism; Site; Skin; Solid; Somatic Cell; Source; Specimen; Standards of Weights and Measures; Stem cells; Structure; System; Technology; Time; Tissues; Ultraviolet Rays; Universities; Validation; Washington; Work; base; cancer therapy; carcinogenesis; chromatin immunoprecipitation; comparative; design; desire; follow-up; functional genomics; fusion gene; genome sequencing; genome-wide analysis; in vivo; indexing; insight; interest; malignant breast neoplasm; melanocyte; melanoma; mouse model; neoplastic cell; osteosarcoma; outcome forecast; progenitor; programs; prospective; sarcoma; technology development; transcription factor; tumor; tumor growth; tumorigenesis; ultraviolet

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

并行应用许多技术来确定给定的生物测量的分子谱。这些技术中的大多数目前都使用基于微阵列的方法。几种微阵列用于各种目的，但是主要的当前技术方法使用合成寡核苷酸结合到固体支持并用根据感兴趣的生物测量制备的标记的核酸进行审查。该技术在该技术的当前实施方案中的力量主要基于已知基因组序列与完全由计算手段完全控制的微阵列设计之间的直接连接。这使研究人员可以构建专门针对所需分析的任意设计的阵列，并将阵列的分辨率调整到非常良好的水平。因此，例如，现在可以通过外显子确定mRNA外显子的表达，并观察到基因拷贝数（扩增或删除）的变化比单个基因溶液更好。然后将从任何细胞或组织来源制备的荧光探针与这些阵列杂交，从而提供了大规模的基因组高分辨率视图。我们最近的努力将这项技术应用于小儿癌，成人肉瘤，淋巴瘤，黑色素瘤，胃肠道肿瘤，乳腺癌和血液学疾病。目前，我们专注于过渡到尽可能多的分量样品（例如通常在常规临床护理过程中收集）和福尔马林固定石蜡嵌入（FFPE）标本。当人们认为在这项工作中进行过渡到临床护理中的过渡发现的潜力是，基于FFPE的方法是稳定临床实验室中的生物测量的标准方法时，使用FFPE样品的能力尤为重要。最近，与Illumina合作，我们使用了小型FFPE样品获得了出色的数据，用于基因拷贝数（CGH），SNP和甲基化。表达也可以在FFPE中进行研究，但主要使用候选基因的亚基因组样品进行研究。重要的是，我们已经证明，可以在数百个样品上并行确定超过1500个CPG的甲基化状态，并与从冷冻样本获得的结果相匹配。这为FFPE样品的大量档案打开了调查。我们的原理研究将卵泡淋巴瘤与卵泡增生进行了比较，并确定了数十种标记，这些标记可牢固地区分这两个实体。我们的实验室具有很长的有趣的肉瘤生物学，我们最近一直将这些技术应用于小儿骨肿瘤骨肉瘤。我们已经成功识别了骨肉瘤的高分辨率基因表达，基因拷贝数和SNP谱。这项工作表明了一种经常出现的拷贝数变化模式，尽管骨肉瘤基因组具有高度混乱的性质，但显而易见。此外，还可以证明拷贝数对骨肉瘤中的基因表达有深远的影响。这种模式提出了许多候选基因以进行进一步研究。为了获得对这种疾病的比较基因组学的观点，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类疾病之间的相似性来完善我们对这种肿瘤的理解。在黑色素瘤中，我们主要专注于分析其祖细胞（小鼠模型中的黑色素细胞）。与Glenn Merlino（NCI/CCR）和Ed DeFabo（George Washington University）合作，我们正在研究使用和系统在Murine开发中正常的黑素细胞的基因表达程序，该程序专门标记了黑素细胞，并允许通过流式细胞仪从小鼠纯度纯化它们。该系统使我们能够研究主要的黑色素瘤致癌物，紫外线（UV）光对黑素细胞发育的影响。通过使用敏感的微阵列技术，我们首次能够观察紫外线对黑素细胞的体内效应。这些结果为黑素细胞发育提供了前所未有的见解，并有望提高我们对紫外线发生的理解。在乳腺癌中，我们对原位导管癌问题（DCIS）特别感兴趣。 DCI很容易通过乳房X线摄影诊断，并且可能代表疾病，这对患者或早期表现侵袭性癌症带来了很小的长期风险。但是，病理学家对DCIS进行分级有很大困难，因此，临床医生难以对这些患者进行适当的疗法进行分层。与迷迭香Balleine（悉尼Westmead医院）合作，我们从与DCIS相关的标本中介绍了微切除的DCIS病变。使用侵入性癌的等级来索引样品，我们已经能够开发一个基因表达分类器，该分类器可以将低毕业生与高级DCIS清晰分开。我们目前正在开发一个多重基因表达测定法，该测定法可以应用于FFPE DCIS样品。经过验证和完善后，该测定法可能应用于前瞻性样本，并可能对DCI的诊断和管理产生重大影响。同样在乳腺癌领域，我们开发了高分辨率基因表达和基因拷贝数数据，这导致了在实验室研究中正在研究的几个候选基因的出现。我们通过表达分析鉴定的一个基因是GATA-3，这是一种转录因子，与雌激素受体阳性乳腺癌相关。我们怀疑GATA-3是该系统中重要的发展调节剂，而其他人在小鼠模型中的最新研究证实了这一假设。为了确定GATA-3如何调节基因表达，我们使用染色质免疫沉淀在乳腺癌细胞中进行了整个基因组转录因子定位研究。结果建立了雌激素受体和GATA-3之间的功能相互关联，并洞悉了乳腺上皮发育与癌症之间的相互作用。为了跟进通过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的静止板，并与表型终点相结合，这些终点鉴定了识别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，通过与敏捷技术合作，我们推动了一个区域8p染色体的表达和拷贝数分析，该区域经常在乳腺癌中扩增到使用平铺路径阵列的最高分辨率。这些研究使我们能够明确地在乳腺癌中绘制该区域，并最终确定乳腺癌活性的8p基因。我们已经使用类似的技术来绘制其他染色体上基因扩增的区域，以克隆重排点并鉴定融合基因。我们还使用类似的技术研究microRNA及其祖细胞的表达方式