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Biopsies of 33 malignant human gliomas were karyotyped and evaluated for amplification (more than eight gene copies per cell) of the epidermal growth factor receptor (EGFR), N-myc, c-myc, and gli genes by Southern blot analysis. Fifteen of 33 tumors showed amplification of EGFR, none had amplified c-myc, one tumor had amplified N-myc, and one had amplification of gli. Thirteen of the 16 (81%) evaluable tumors with gene amplification contained double minutes (DM), and only four of 16 (25%) tumors without demonstrable amplification contained these structures. Polysomy for chromosome #7, in contrast, occurred in 58% of tumors with EGFR amplification and 53% of tumors without amplification of the gene. Structural abnormalities of 7p occurred in two tumors with EGFR amplification and in one tumor without amplification of this gene. These studies suggest that DM are the usual locus for amplified genes (usually EGFR) in human glioma biopsies, but that structural abnormalities of 7p may be associated with EGFR amplification in a small proportion of these tumors. The presence of polysomy 7, however, probably is unrelated to amplification of the EGFR gene.

Gene amplification occurs in 45-50% of malignant human gliomas (MHG). In the present study, 64 genetically characterized gliomas were evaluated to determine if tumors with amplification of the epidermal growth factor receptor (EGFR), N-myc, c-myc, or gli genes had distinctive histopathologic features. There was no significant difference in age (p = 0.10) or gender (p = 0.78) between patients whose tumors contained amplified genes and those whose tumors did not exhibit this characteristic. Although the patients with amplified genes in their tumors survived slightly longer than patients whose tumors had no detectable gene amplification, these differences were not statistically significant (p = 0.21). The 28 tumors with amplification included 24/48 (50%) glioblastoma multiforme, 2/6 (33%) anaplastic astrocytomas and 2/5 (40%) gliosarcomas. No amplification was seen in one oligodendroglioma, three anaplastic mixed gliomas or one giant cell glioblastoma multiforme. Necrosis and endothelial proliferation were equally prevalent among tumors with and without amplification. Comparison of tumors with gene amplification and tumors without this characteristic revealed similar distributions of most morphologic cells types. Although prominent perivascular lymphocytic infiltrates were more frequent in tumors without amplification, this association was of borderline significance statistically. In situ hybridization of tumors with amplification using an EGFR mRNA probe showed intense labeling of different neoplastic cell types including fibrillary and protoplasmic astrocytes, gemistocytes, anaplastic cells, and multinucleated giant cells. Non-neoplastic cells such as hyperplastic endothelium within the tumors did not express detectable EGFR mRNA. These studies demonstrate that (a) cells with quite different morphology within the same tumor can contain the same genetic alteration; (b) tumors of identical histological appearance may have arisen and evolved by different molecular mechanisms; and (c) molecular analyses are necessary to evaluate gene amplification in MHG since this characteristic cannot be accurately predicted by the morphologic or clinical criteria used in this study.

Monoclonal antibody 81C6, which is directed against a human gliomamesenchymal extracellular matrix antigen, was used to evaluate the potential advantage of intracarotid (i.c.) administration versus i.v. delivery to D-54 MG human glioma intracranial xenografts in immunosuppressed rats. Two approaches were taken. In paired-label analysis, 125I-labeled 81C6 and 131I-labeled isotype control antibody were given to separate groups of animals by either the i.v. or i.c. route. Biodistribution measurements as a function of time were analyzed in terms of the percentage of injected dose/g of tissue and localization indices. No significant difference (P greater than 0.19 to P greater than 0.56) was demonstrated between the i.v. and i.c. routes. To control for the large localization variation inherent in the animal model used, an alternative experimental design, paired-injection analysis, was performed in which 125I- and 131I-labeled 81C6 were simultaneously administered by the i.c. and i.v. routes to the same animal. Significantly higher levels of percentage of dose/g of tissue and localization ratios (P less than 0.05 to P less than 0.005) were shown from Day 1 to Day 3 for 81C6 given i.c. Approximately 20% more antibody was delivered to the D-54 MG intracranial tumor by the i.c. route during the experimental period of 5 days. No difference in the levels of normal tissue exposure between the two routes of administration was seen. These data suggest an advantage exists for whole monoclonal antibody given i.c. and that, theoretically, a greater advantage may be present for smaller molecules such as Fab and F(ab')2 fragments.

Karyotypic analysis of 54 malignant human gliomas (5 anaplastic astrocytomas, 43 glioblastoma multiformes, 3 gliosarcomas, 2 giant cell glioblastomas, 1 anaplastic mixed glioma) has demonstrated that 12 tumors contained normal stemlines or only lacked one sex chromosome. The 42 tumors with abnormal karyotypes included 38 tumors which could be completely analyzed. Six of these 38 cases had near-triploid or near-tetraploid stemlines and 32 had near-diploid stemlines. Statistically significant numerical deviations in the near-diploid group were gains of chromosome 7 (26 of 32; P less than 0.001), and losses of chromosome 10 (19 of 32; P less than 0.001). Double minutes occurred in 18 of 32 near diploid tumors. The distribution of structural abnormalities was analyzed statistically by comparing the incidence of breakpoint in each chromosomal arm to the expected value based on chromosomal arm length. This analysis demonstrated that structural abnormalities of 9p and 19q were significant statistically (P less than 0.005 and P = 0.02, respectively). Although chromosome 1, 6p, the centromeric region of chromosome 11, 13q, and 15q were also frequently involved in structural abnormalities, the incidence of these breaks did not reach statistical significance. This demonstration of specific chromosomal abnormalities in near-diploid gliomas provides the basis for the investigation of genes which may be quantitatively or qualitatively altered in these neoplasms.

DNA content, as determined by flow cytometry (FCM), and ploidy, as determined planimetrically from chromosomes, were measured for 17 established human glioma-derived cell lines. Values obtained by these methods corresponded well for two of two near-diploid lines, two of two hyperdiploid-hypotriploid lines, four of four hypertriploid-hypotetraploid lines, two of six hypertetraploid-hypopentaploid lines, the one hyperpentaploid line and two of two multiclonal lines. For the remaining four lines, ploidy values obtained by karyotyping were more than 10% lower than those obtained by FCM. Since karyotypic ploidy was determined by planimetric measurements of the chromosomes, which corrects for deviations in chromosome size, the lower values could not be explained by large marker chromosomes. Technical problems, such as random chromosomal loss in karyotypic preparations, adherent bits of cytoplasm in nuclear preparations for FCM or differences in nuclear stainability, are possible reasons for the discrepancies. Alternatively, chromosomes in some glioma cell lines may actually contain an increased amount of DNA. FCM and karyotyping provide complementary information in the initial evaluation of cultured cell lines. For sequential studies, such as are used for monitoring cultured lines, the rapidity of FCM makes it the more practical method.