Cancer Study by Flow Cytometric Signal Cell Genotyping

描述(由申请人提供)：灵敏、特异和定量的癌症测量在癌症早期检测、诊断、预后和最小残留疾病研究中非常重要。在血液系统恶性肿瘤中，基于流式细胞术的免疫表型分析和基于聚合酶链式反应的序列鉴定是检测亚微观水平白血病的两种主要方法。基于单个白血病细胞表达的免疫表型标志物的流式细胞术检测MRD是一种高度敏感、可靠的方法，它允许在105个细胞中检测到1个靶细胞。然而，对于许多病例来说，对肿瘤具有高度特异性的表面标志物很难找到。另一方面，基于聚合酶链式反应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，或染色体异常的断点融合区)。到目前为止，研究人员已经发现了20种白血病亚型。尽管基于聚合酶链式反应的核酸分析具有很高的特异性，但它并不能提供MRD研究所需的定量分析。在这项提案中，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的一个亚型(具有单一碱基分辨率)，用于后续的流式细胞仪单细胞分析。这种标记策略使我们能够开发一种用于特定癌症群体检测和分离的定量检测方法。将造血细胞固定，使用改进的原位滚动圆扩增方案，用亚型特异性荧光寡聚物探针进行原位标记，并用高速流式细胞仪进行单独分析。用特定的荧光寡聚体标记的肿瘤细胞群将被检测和定量。该分析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，并将在正常细胞和肿瘤细胞的混合物上确定其敏感性。一旦测试得到优化，我们计划未来在临床样本上进行更大规模的验证。综上所述，我们建议开发一种通用的基于原位核酸的基因分型策略，从而能够对罕见的肿瘤细胞群进行定量、特异和灵敏的检测/分离。这一新的方法将传统的流式细胞仪免疫表型分析扩展到基于基因的新一代流式细胞仪诊断，结合了流式细胞仪免疫表型定量分析的优势和基于序列的基因分析的特异性，最终以单一碱基分辨率在分子遗传水平上提供了对癌细胞亚群的敏感和定量的表征/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.