Cancer Study by Flow Cytometric Signal Cell Genotyping

通过流式细胞术信号细胞基因分型进行癌症研究

• 批准号: 7140099
• 负责人: HONG CAI
• 金额: $ 15.13万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-16 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140099
• 关键词: cell line; cell population study; cytodiagnosis; diagnosis design /evaluation; early diagnosis; flow cytometry; fluorescent dye /probe; genetic screening; genotype; hematopoietic stem cells; minimal residual disease; neoplasm /cancer diagnosis; nucleic acid amplification techniques; nucleic acid probes; phenotype

DESCRIPTION (provided by applicant): Sensitive, specific and quantitative measurement of cancer is important in early cancer detection, diagnosis, prognosis and minimal residual disease studies. In hematopoietic malignances, flow cytometry-based immunophenotyping and polymerase chain reaction (PCR or Reverse Transcriptase, RT-PCR)-based sequence identification are the two main methods for detecting submicroscopic levels of leukemia. Flowctyometric detection of MRD based on the identification of immunophenotypic markers expressed on individual leukemia cells is highly sensitive, robust and it allows the detection of 1 target cell in 105 cells. However, surface markers that are highly specific for tumors are hard to find for many cases. PCR-based sequence analysis, on the other hand, relies on the detection of specific genomic abnormality. T cell receptor genes, or breakpoint fusion regions of chromosome aberrations) of a diseased individual. Up to now, researchers have discovered > 20 subtypes of leukemia. Although the PCR-based nucleic acid assay is highly specific, it does not provide a quantitative analysis desired for MRD studies. In this proposal, we will develop a novel fluorescent labeling strategy that is capable of in-situ labeling a subtype of leukemia target (with a single base resolution) for the subsequent flow cytometric single cell analysis. Such a labeling strategy enables us to develop a quantitative assay for specific cancer population detection and isolation. The hematopoietic cells will be fixed, in-situ labeled with a subtype-specific fluorescent oligomer probe using a modified in-situ rolling circle amplification scheme, and analyzed individually with a high-speed flow cytometer. The tumor cell population labeled with specific fluorescent oligomers will be detected and quantified. The feasibility of the assay will be initially demonstrated on well-characterized acute leukemia cell lines, and the sensitivity will be determined on the mixture of normal and tumor cells. Once the assay is optimized, we plan to do future larger scale validation on clinical samples. In summary, we propose to develop a general in-situ nucleic acid-based genotyping strategy that enables the quantitative, specific and sensitive detection/isolation of rare tumor cell populations. This new approach expands the conventional flow cytometric immunophenotyping to the next generation gene-based flow cytometric diagnostics by combining the strength of quantitative analysis of flow cytometric immunophenotyping and specificity of sequence-based genotype analysis ultimately providing sensitive and quantitative characterization/isolation of a subpopulation of cancer cells at molecular genetic level with a single base resolution.

描述（由申请人提供）：癌症的灵敏、特异和定量测量在早期癌症检测、诊断、预后和微小残留病研究中非常重要。在造血系统恶性肿瘤中，基于流式细胞术的免疫分型和基于聚合酶链反应（PCR或逆转录酶，RT-PCR）的序列鉴定是检测亚显微水平白血病的两种主要方法。基于单个白血病细胞上表达的免疫表型标志物的鉴定的MRD的流式检测是高度灵敏的、稳健的，并且其允许在105个细胞中检测1个靶细胞。然而，在许多情况下，很难找到对肿瘤高度特异性的表面标志物。另一方面，基于PCR的序列分析依赖于特定基因组异常的检测。T细胞受体基因或染色体畸变的断点融合区）。到目前为止，研究人员已经发现了超过20种白血病亚型。尽管基于PCR的核酸测定具有高度特异性，但它不能提供MRD研究所需的定量分析。在这个提议中，我们将开发一种新的荧光标记策略，能够原位标记白血病靶点的亚型（具有单碱基分辨率），用于随后的流式细胞术单细胞分析。这种标记策略使我们能够开发用于特定癌症群体检测和分离的定量测定。将造血细胞固定，使用改良的原位滚环扩增方案用亚型特异性荧光寡聚体探针原位标记，并使用高速流式细胞仪单独分析。将检测和定量用特异性荧光寡聚体标记的肿瘤细胞群。该试验的可行性将首先在充分表征的急性白血病细胞系上证明，并将在正常细胞和肿瘤细胞的混合物上确定灵敏度。一旦检测方法得到优化，我们计划在未来对临床样本进行更大规模的验证。总之，我们建议开发一种通用的基于原位核酸的基因分型策略，该策略能够定量、特异性和灵敏地检测/分离罕见肿瘤细胞群。这种新方法通过结合流式细胞免疫分型的定量分析的强度和基于序列的基因型分析的特异性，将传统的流式细胞免疫分型扩展到下一代基于基因的流式细胞诊断，最终在分子遗传水平上以单碱基分辨率提供癌细胞亚群的灵敏和定量表征/分离。