The goal of this proposal is to continue to develop and evaluate fluorescence in situ hybridization (FISH) and associated analytical techniques for detection and characterization of chromosomal abnormalities relevant to research and clinical questions of human malignancy. We will focus on detection of low frequency structural and numerical abnormalities in residual disease and disease progression in chronic myelogenous leukemia (CML), and detection and characterization of structural abnormalities in breast cancer. In both diseases, we will determine genetic status and immunophenotype for individual interphase cells. We will: A) Develop multi-color FISH to permit analysis of simultaneous hybridizations using probes for at least 7 different targets. B) Develop hardware and software for computer assisted microscopy to allow convenient analysis of 7 probe hybridizations, automatic slide scanning and recognition of cancer-specific genotypes, and double pass scanning and analysis for sequentially determining cell immunophenotype and genotype. C) Acquire or develop improved probes spanning the BCR and ABL breakpoint regions of chromosomes 9 and 22 involved in CML; and a set of probes distributed along chromosome 17 at about 4 Mb intervals, with additional elements targeted at specific genes implicated in breast cancer. The utility of these probe sets for genetic and phenotypic analysis of CML and breast cancer will be evaluated: A) CML. We will determine the lowest frequency of residual leukemic cells that can be detected by hybridization to interphase cells, determine the frequency, phenotype and proliferation status of residual leukemic cells in patients undergoing interferon therapy, and determine how early genetic progression characteristic of CML blast crisis can be detected. B) Breast Cancer. We will test the ability of the chromosome 17 probe set to detect structural abnormalities in cell lines, breast tumors, potential precursor lesions, and metastases. We will study the spatial distribution of heterogeneity and clonality in breast cancer, and correlate genotype with phenotype on a cell by cell basis.
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