Oncogene Amplification in Pediatrie Brain Tumors'

Despite a considerable amount of information concerning chromosomal and molecular abnormalities found in gliomas in adults, relatively little is known regarding these abnormalities in pediatrie brain tumors. We have analyzed DNA from 37 primary brain tumors and 4 tumor-derived cell lines for oncogene amplification. Probes utilized represent 11 known oncogenes (erbBl, gli, neu, myc, L-myc, N-myc, H-ras, K-ras, N-ras, sis, and src). Of 20 primary medulloblastomas studied, only one tumor was found to have erbBl amplification. In contrast, of the 4 medulloblastoma cell lines studied, 1 had c-myc amplification, 1 had erbBl amplification, and 1 had amplification of N-myc. Twelve glial brain tumors were analyzed, and only 1 case with amplification of the erbBl oncogene was found. Other tumors studied include 1 meningioma, 2 epcndymomas, 1 anaplastic ependymoma, and 1 cerebral primitive neuroectodermal tumor, none of which had oncogene amplification. These results suggest that oncogene amplification is relatively uncommon in primary medulloblas tomas, but the frequency and diversity of oncogene amplification is greater in tumors that can be established as cell lines. The lower frequency of erbBl amplification in glial brain tumors in children compared to adults is consistent with the generally lower grade of glial tumor histology seen in pediatrie patients. However, the case with amplification of the erbBl oncogene represented 1 of 2 cases of glioblastoma multiforme we studied, which suggests that pediatrie glioblastoma multiforme may have a similar frequency of erbBl oncogene amplification to glioblastomas seen in adults. Our results suggest that oncogene amplification is a relatively uncommon mechanism of oncogene activation in pediatrie brain tumors, and they provide molecular evidence for heterogeneity in tumors classified as medulloblastomas. and quantitated by fluorometric assay as previously described (13). Samples (5 Â¿ig)of DNA from each tumor or cell line were digested to completion with EcoRi, separated by electrophoresis on 0.8% agarose gels, and transferred to nitrocellulose or nylon filters by a modification of the method of Southern (13). Positive controls utilized include the neuroblastoma cell line NGP, which has 150-fold amplification of N-myc (14, 15), the epidermoid carcinoma cell line A431, which has 40- fold amplification of erbBl (16), the breast carcinoma cell line MCF-7, which has 10-fold amplii imi imi of N-ras (17), and the colon carci noma cell line COLO-320, which has 50-fold amplification and re arrangement of c-myc (18). Filters were baked and hybridized with "P-labeled probes representing 10 known oncogenes (c-erbBl, neu, c-myc, L-myc, N-myc, H-ras, K.-ras,N-ras, sis, and src) and were washed under high stringency conditions. The rationale for choosing these 10 onco genes was the existence, at the time the study was initiated, of published data suggesting the involvement of these oncogenes in tumors of either the central or peripheral nervous systems. The following individuals generously provided DNA probes for these studies: J. M. Bishop, human N-mj'c pNb-1 J. D. Minna, human L-myc pLmyc 10(19); R. A. Weinberg, human neu J. M. Trent, human erbBi probe pE7 Other probes were obtained from the American Type Culture Collection. Filters were exposed for 18-24 h at â€”70Â°C with an intensifying screen. The gene copy number was determined by serial dilution and laser densitometry. Filters were also stripped and rehybridized with other oncogene probes so that the densitometric confirmation of the single copy nature of these other oncogenes would represent an internal control to confirm the loading of equal amounts of DNA in each lane (data not shown). A probe for human gli was also tested. The gli probe pKK380 was generously provided by B. Vogelstein and is derived from an amplified, expressed gene in an adult glioma that is a zinc finger protein with homology to the KrÃ¼ppelfamily of genes in Drosophila (21-23). Al though there is no direct evidence that gli actually functions as an oncogene in transfection assays, it is also amplified and overexpressed in a subset of childhood sarcomas (24). and in some glial tumors (21).