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. ? ?
