Sandra H. Bigner, MD - US grants

The long term goal of this laboratory is to determine the molecular basis of the cytogenetic abnormalities which characterize human gliomas and to define the role of these alterations in the genesis and progression of these tumors. During the previous funding period, we have determined that loss of heterozygosity (LOH) for 17p is associated with the TP53 gene mutations, that double minute chromosomes (DMs) are associated with amplification, often with rearrangement, of the epidermal growth factor receptor (EGFR) gene, that loss of 9p is associated with homozygous deletion of the CDKN2 gene, and that loss of all or part of chromosome 10 often reflects deletion or mutation of the PTEN/MMAC1 gene. In the present application we will use these genetic alterations, along with histologic parameters and immunohistochemical markers, to precisely and reproducibly separate glioma patients into prognostic groups and to identify patients who are likely to respond to specific forms of treatment. In Specific Aim 1 we will test the ability of age of the patient, histologic features, proliferation index, p53 gene mutations, gene amplification, and LOH for 1p, 9p, chromosome 10, and 19q to distinguish between astrocytic and oligodendroglial tumors and to grade tumors within these cytologic groups. We will also use immunohistochemical and biochemical markers to identify patients whose tumors contain antigens or molecular defects which render them susceptible or resistant to specific therapeutic modalities. Since loss of chromosome 10 tumor suppressor genes which are altered in these tumors, including PTEN, DMBT1, and new suppressor genes, particularly on 10p. We believe that these studies will provide accurate classification of brain tumor patients, for the prospective analysis of specific therapeutic regimens. Our goal is to select therapy for individual patients based on immunohistochemical, biochemical, or molecular genetic characteristics of their tumors, and by doing so have the best chance of avoiding resistance and achieving response or even cure in a subset of malignant glioma patients.

The long-term goal of these studies is to identify tumor suppressor genes which are lost on inactivated in medulloblastomas. Cytogenetic and restriction fragment length polymorphism (RFLP) analyses have demonstrated loss of chromosomal region 17p in a subset of medulloblastomas which suggests that a suppressor gene normally located in this region may be important in these tumors. Although the TP53 gene is located within this deleted region point mutations of this gene are uncommon in medulloblastomas which suggests that either a different type of TP53 alteration is involved in these tumors or that the loss of 17p is targeting a previously undescribed tumor suppressor gene in this location. The following aims have been generated to answer this question. AIM 1). Specific chromosomes and chromosomal regions that are deleted in medulloblastoma will be identified by allelotyping, using both minisatellite probes and analysis of microsatellite sequences by polymerase chain reaction (PCR), and karyotyping. These studies will establish the incidence and specificity of 17p loss as compared to other chromosomes and will identify additional chromosomal regions, the loss of which is important in medulloblastomas. AIM 2). The TP53 will be analyzed in medulloblastomas to exclude the possibility that it is the target of 17p loss in these tumors. Specifically, gross rearrangements and deletions will be analyzed by Southern blotting, expression of the gene will be determined by Northern analysis, immunohistochemical studies will be performed to evaluate expression of the TP53 protein, and mutations will be sought by sequencing. AIM 3). This aim will be focused on the isolation of the region harboring the 17p suppressor gene in medulloblastoma. Polymorphic probes will be isolated from a human chromosome 17 genomic library, and dinucleotide repeats on 17p will be defined. These probes will be regionally mapped on chromosome 17p, and used to define a small region of consistent loss in medulloblastomas by RFLP analysis. The definition of the regional 17p loss will provide the basis for eventual identification of the suppressor gene in that location. These comprehensive studies will determine whether the loss, mutation, or inactivation of known tumor suppressor gene TP53 is critical to the pathogenesis of medulloblastomas. In additionl regions of chromosomal loss will be narrowly defined on 17p and other chromosomes which are lost in these tumors will be determined forming the basis for the isolation and characterization of tumor suppressor genes. Characterization of such genes and their products will help to elucidate the mechanisms of growth control which are operative in normal and neoplastic cells and which are important in development, differentiation,a nd in regulation of cells in repair and regeneration. Gene probes emerging from these studies can be used to detect specific genetic alterations in medulloblastomas as well as in the tumors and the products of these genes can be used for the preparation of specific antibodies which are potentially useful in clinical diagnosis and therapy.

tissue resource /registry; cancer registry /resource; biomedical facility; cytogenetics; tissue /cell preparation; karyotype; in situ hybridization; fluorescent dye /probe;

DESCRIPTION: (Applicant's Abstract) Medulloblastoma, the most common malignant central nervous system neoplasm in children, has a dismal prognosis despite multi-institutional clinical trials using increasingly sophisticated neurosurgical and therapeutic interventions, with the majority of children with advanced medulloblastomas eventually dying of progressive disease. Progress has been hindered by technical inabilities to discriminate prospectively between patients with aggressive and indolent neoplasms. Recent advances in molecular biology, cytogenetics, and immunohistochemistry now convincingly demonstrate the phenotypic and genotypic heterogeneity of these neoplasms. Cytogenetic analysis and restriction fragment length polymorphism analysis using Microsatellite probes have shown that approximately 1/3 of these tumors have loss of the 17p chromosomal arm. Approximately 5% of medulloblastomas have mutations of the p53 gene, which is located on 17p and approximately 5% of these tumors have amplification of myc family genes. Preliminary studies from the applicant's laboratory and others suggest that loss of heterozygosity (LOH) for 17p, may identify patients with aggressive neoplasms and significantly shorter survivals. Furthermore, recent immunohistochemical phenotypic studies suggest that evidence of glial or neuroblastic differentiation in the desmoplastic pattern may predict a long-term survival. The applicant's hypothesis is that patients with medulloblastomas exhibiting LOH for 17p, have a significantly worse prognosis than patients whose tumors lack these alterations and that the identification of neuroblastic or glial differentiation by immunohistochemistry can be used to define these prognostic groups more fully. In Specific Aim 1 she will analyze paraffin-embedded human medulloblastoma specimens for LOH of chromosome 17p using microsatellite markers, fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). In Aim 2, she will reclassify all medulloblastomas as "classic," "desmoplastic", "mixed", or "other" and will define, by immunohistochemistry, the distribution of synaptophysin, neuron-specific enolase, neurofilament proteins, and glial fibrillary acidic protein, in these specimens. Using these data, in Specific Aim 3, the applicant will define the biological relevance of LOH for 17p, and phenotypic profile, with regard to initial tumor staging, tumor recurrence, and clinical outcome. She believes that these studies will provide a rational, molecular-based classification scheme for these neoplasms which has prognostic significance. In addition, these observations will establish the scientific basis for further investigations into the basic mechanisms of medulloblastoma etiology and progression.

tissue resource /registry; cancer registry /resource; neoplasm /cancer; cytogenetics; biomedical facility;