BREAST CANCER MOLECULAR CYTOGENETICS

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The aim of this project is to characterize cytogenetic changes present in interphase breast tumor cells. Cytogenetic analysis leads to a better understanding of molecular defects in tumor DNA, is an independent prognostic marker for tumor behavior, and will help to pinpoint chromosome regions of particular interest for further molecular analysis. The utility of classical cytogenetics by analysis of metaphase chromosomes has been limited in solid tumors due to the difficulty in preparing adequate tumor metaphases from biopsy tissue. Analysis of tumor cells after short term culture is selective for diploid cells capable of proliferating in restricted culture conditions. Techniques are now available for cytogenetic analysis of interphase tumor cells on a cell by cell basis. Chromosome copy number and major structural aberrations will be detected using fluorescence in situ hybridization with chromosome-specific DNA probes. Copy number of each chromosome will be detected using peri-centromeric repetitive probes specific for chromosomes 1, 7, 11, 133, 16, and 17. These chromosomes have been chosen based on their likely involvement in DNA aberrations. Probe libraries specific for low copy number sequences lining the entire chromosome will be used to "paint" these chromosomes in order to detect translocations or duplications of major chromosome regions. As further probes specific for chromosomal subregions of interest are developed by our collaborators, they will be used to detect amplifications, deletions, and translocations of smaller regions. Similar techniques will be used to characterize to what extent cytogenetically defined subpopulations of tumor cells have proliferative advantage over other subpopulations. Cell proliferative status will be determined by immunofluorescent characterization of bromodeoxyuridine incorporation simultaneously with cytogenetic characterization by fluorescence in situ hybridization. Two and three color analysis will allow comparison of proliferation between populations defined by chromosome copy number or the presence of translocations. The degree of heterogeneity of chromosome abnormalities in tumors can be defined using fluorescence in situ hybridization with chromosome specific probes. Cytogenetic heterogeneity is related to genetic instability and thus to tumor development and progression. The distribution of chromosome abnormalities will be determined using in situ hybridization in tissue sections, and will be compared to tumor architecture and to the distribution of DNA content and antigenic markers. Within the Program Project, cytogenetic information will be correlated with tumor pathology, clinical followup, and related molecular studies.