The purpose of this proposal is to pursue a comprehensive analysis of the chromosomal and molecular genetic abnormalities that have been identified in human neuroblastoma. The most common cytogenetic abnormalities in human neuroblastoma cells are double minute chromatin bodies (DMs), homogeneously staining regions (HSRs) and partial monosomy for the distal short arm of chromosome 1. DMs and HSRs are cytogenetic manifestations of gene amplification. We have demonstrated that a sequence called N-myc, with homology to the cellular oncogene c-myc, is amplified in virtually all neuroblastoma cell lines and in 38% of primary untreated tumors. Indeed, we have shown that amplification of N-myc is highly correlated with advanced stages of disease, and hence with a poor survival. Very little is known about the mRNA expression of N-myc in neuroblastomas, and little is known about genomic amplification or mRNA expression of another neuroblastoma-derived oncogene, N-ras. I propose to analyze genomic emplification (by Southern hybridization) and mRNA expression (by Northern hybridization) of both N-myc and N-ras in about 10 neuroblastoma cell lines and in at least 100 primary tumors. These data will be correlated with clinical and laboratory features of these patients with neuroblastomas. We have identified deletion of the short arm of chromosome 1 in about 70% of primary neuroblastomas and tumor-derived cell lines. I plan to do fine-structure mapping of this deletion with both cytogenetic and molecular genetic techniques to focus on the smallest region of common involvement in these tumors. In addition to high-resolution cytogenetic analysis, I will obtain single-copy molecular probes for the distal short arm of chromosome 1 from a chromosome-specific recombinant DNA library. These probes will be used to analyze DNA from the somatic cells and the tumors of patients with neuroblastoma. I will look for abnormalities of restriction fragment length polymorphisms or gene copy number between the two DNA sources from each patient. Analysis of these data may identify probes that can be used to narrow the deleted segment to a region that is more amenable to molecular analysis of the presumptive neuroblastoma gene. In summary, the proposed studies should provide a considerable amount of information about the possible role of these genes in malignant transformation or progression of human neuroblastoma. These data may well permit us to better determine prognosis, and they may provide tools that could be used to detect persons with genetic predisposition.
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