Cancer Study by Flow Cytometric Signal Cell Genotyping

描述（由申请人提供）：肿瘤的敏感、特异和定量测量对于早期癌症检测、诊断、预后和微小残留病研究具有重要意义。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。基于对单个白血病细胞上表达的免疫表型标记物进行鉴定的流式细胞法MRD检测具有高度敏感性和鲁棒性，并且可以在105个细胞中检测1个靶细胞。然而，在许多情况下，对肿瘤具有高度特异性的表面标记物很难找到。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。一个患病个体的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。在本提案中，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标亚型（具有单一碱基分辨率），用于随后的流式细胞术单细胞分析。这样的标签策略使我们能够开发特定癌症人群检测和分离的定量分析。将造血细胞固定，用亚型特异性荧光低聚物探针原位标记，使用改进的原位滚动圈扩增方案，并使用高速流式细胞仪单独分析。用特定荧光低聚物标记的肿瘤细胞群将被检测和定量。该试验的可行性将首先在具有良好特征的急性白血病细胞系上进行验证，敏感性将在正常细胞和肿瘤细胞的混合物上进行测定。一旦检测得到优化，我们计划在临床样本上进行未来更大规模的验证。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，使罕见肿瘤细胞群的定量、特异性和敏感性检测/分离成为可能。这种新方法通过结合流式细胞免疫表型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表型分析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一碱基分辨率提供癌细胞亚群的敏感和定量表征/分离。

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.