Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification.

Abstract

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.In vitro experiments have shown that greatly increased expression of some protooncogenes can lead to neoplastic transformation (1-3). In naturally occurring tumors, increases in gene expression have been postulated to occur through two kinds of mechanism (4-6). One class of mechanism involves structural changes within or surrounding the expressed gene, either through DNA amplification (7,8) or rearrangement (9). The other class of mechanism includes changes in DNA-binding proteins (10) or DNA methylation (11) in the absence of structural alterations of the expressed gene. In several human tumors, increased expression of protooncogenes apparently takes place in the absence of genetic changes at the protooncogene locus (see, e.g., refs. 12-15) and these increases have been suggested to play an active role in tumor formation. The presence of genomic alterations of a protooncogene in a tumor provides strong evidence for involvement of the protooncogene in formation of the tumor. However, in the absence of such structural alterations, it is difficult to know whether increased expression of a protooncogene is causally related to the tumorigenic process or simply reflects the abnormal growth status or unusual microenvironment present in tumors.The epidermal growth factor receptor (EGFR) is a protooncogene that has been extensively studied (reviewed in ref. purified from frozen blocks of tissue, 1.5-to 4-ptg samples were cleaved with EcoRI, separated by electrophoresis through a 1% agarose gel, and blotted on nitrocellulose. Prehybridization, hybridization, and washing conditions were as described (19). The EGFR probe used was the 1.6-kilobase EcoRI fragment of pE7 (21), a cDNA clone of EGFR mRNA generously provided by G. Merlino and I. Pastan (National Institutes of Health). Filters were rehybridized sequentially with three other probes: a 1.0-kb EcoRI/ BamHI fragment of pNB-1 (22), containing part of the second exon of the N-myc (human, NMYC) gene; a 1.6-kb Sst I fragment of pHSR-1, containing the second exon of c-myc (human, MYC) (23); a 1.55-kb Pst I insert of pKK36P1, containing gli sequences from chromosome 12 (24); and a 5.0-kb EcoRI insert of pAW101, containing sequences from chromosome 14 (25).In Situ Hybridization. Tissue sections of 6 ,um thickness were cut from paraffin...

Tandem duplication of the epidermal growth factor receptor tyrosine kinase and calcium internalization domains in A-172 glioma cells

“…As a result of multistep tumor progression, malignant gliomas are genetically heterogeneous tumors (Louis and Gusella, 1995). The most consistent genetic alterations identi®ed to date include p53 abnormalities (Bogler et al, 1995;Rasheed et al, 1994), alterations in the p16/CDKN2/pRb pathway (Ueki et al, 1996) and EGFR gene ampli®cation and rearrangement (Libermann et al, 1985;Wong et al, 1987;Humphrey et al, 1988;Bigner et al, 1990). Levels of EGFR in gliomas are proportional to tumor invasiveness (Lund-Johansen et al, 1990) and overexpression of wild type EGFR has been shown to induce ligand-dependent cellular transformation of astrocytes, indicating a direct role for the receptor in oncogenesis (Frisa et al, 1996;O'Rourke et al, 1997).…”

“…EGFR ampli®cation occurs in up to 50% of malignant gliomas with gene rearrangement in approximately one-half of those tumors leading to frequent co-expression of both wild type and mutant receptors (Libermann et al, 1985;Wong et al, 1987;Humphrey et al, 1988;Bigner et al, 1990;Ekstrand et al, 1991). Expression of these EGFR mutants has been reported to be associated with a poor prognosis (Schlegel et al, 1994).…”

Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor

“…Epidermal growth factor receptor (EGFR) gene (protooncogene of v-erbB) is amplified and overexpressed in about 40% of cases of glioblastoma, the major malignant tumour of human brain Wong et al, 1987;Humphrey et al, 1988). This amplification is frequently correlated with structural rearrangement of EGFR, resulting in in-frame deletion mutations in the extracellular domains (Humphrey et al, 1988(Humphrey et al, , 1991Yamazaki et al, 1988Yamazaki et al, , 1990Bigner et al, 1990;Wong et al, 1992).…”

Epidermal growth factor receptors in intracranial and breast tumours: their clinical significance

“…In malignant gliomata, over-expression of the EGFR protein appears to be associated with gene amplification (Wong et al, 1987). In culture, the growth of glial tumour cells can be stimulated by EGF (Frappaz et al, 1988) and antibodies against the EGFR can inhibit such stimulation (Werner et al, 1988).…”