BAC-FISH Assays for Sensitive Karyotyping of Cancer Cells

描述(申请人提供)：对人类肿瘤和癌症组织的详细细胞遗传学分析，特别是揭示了在大多数情况下存在结构染色体异常。潜在的变化通常是特定于肿瘤或癌症的类型和细胞的改变表型的。此外，研究人员可以证明染色体变化的类型或程度、疾病进展和结果之间的相关性。然而，目前用于筛查结构异常的实验室技术在检测微小的“隐蔽”易位方面非常有限。例如，中期染色体的Giemsa(G)显带或基于荧光原位杂交(FISH)的全染色体涂染(WCP)和光谱核型分析(Sky)技术通常会漏掉涉及小于10兆基数(MBP)的片段的易位，即大约一条染色体带的大小。我们推测，在表型正常或患有诸如智力低下、生育能力受损、癌前病变或早期肿瘤等疾病的个体的基因组中，存在着未被检测到的小而隐蔽的易位。有关结构改变和染色体不平衡的知识可能有助于临床医生对疾病的发病和病程做出更准确的预测。这个R21项目将研究快速和廉价地筛查人类基因组中隐蔽易位(OCT)存在的可行性。具体地说，我们将开发和测试FISH分析，使用经过验证的染色体特异性细菌人工染色体(BAC)集合来检测人类癌细胞中的OCTs。由于BAC探针的平均间距为0.9 MBP，并覆盖了人类基因组的整个常染色体部分，我们预计我们的BAC-FISH分析将比WCP或显带试验的灵敏度大大提高。我们针对易位的全基因组敏感筛查的创新检测方法将以结构异常信息有限的癌细胞株为研究对象。在这个项目结束时，我们将有能力对正常人群和肿瘤细胞中OCT的频率进行更大规模的研究，并为研究和临床实验室提供BAC-FISH筛查服务和试剂，以供合作研究。

DESCRIPTION (provided by applicant): The detailed cytogenetic analyses of human tumors and cancer tissues, in particular, have revealed the presence of structural chromosome abnormalities in most of cases. The underlying changes are often specific for the type of tumor or cancer and the cells' altered phenotype. Furthermore, investigators could demonstrate a correlation between the type or extent of chromosome changes, disease progression, and outcome. However, present laboratory techniques for the screening for structural abnormalities are very limited with respect to the detection of small, 'cryptic' translocations. For example, Giemsa (G)-banding of metaphase chromosomes or the fluorescence in situ hybridization (FISH)-based techniques of whole chromosome painting (WCP) and Spectral Karyotyping (SKY) analysis typically miss translocations that involve segments of less than 10 megabasepairs (Mbp), i.e., about the size of a chromosome band. We postulate that small cryptic translocations exist undetected in the genomes of individuals with a normal phenotype or diseases such as mental retardation, impaired fertility, precancerous lesions, or early stage tumors. Knowledge about structural alterations and chromosomal imbalances might help clinicians make more accurate predictions regarding the onset and course of a disease. This R21 project will investigate the feasibility to rapidly and inexpensively screen the human genome for the presence of occult cryptic translocations (OCTs). Specifically, we will develop and test FISH assays using collections of validated chromosome-specific bacterial artificial chromosomes (BACs) for the detection of OCTs in human cancer cells. With BAC probes spaced on average 0.9 Mbp apart and covering the entire euchromatic part of the human genome, we expect our 'BAC-FISH' assay to lead to greatly increased sensitivity compared to WCP or banding tests. Our innovative assay for sensitive genome-wide screening for translocations will be developed with cancer cell lines for which limited information about structural abnormalities is available. At the end of this project, we will be well positioned to conduct a larger study of the frequency of OCTs in the normal population as well as tumor cells and to offer BAC-FISH screening service and reagents to research and clinical laboratories for collaborative studies.