Mapping of homozygous deletions on human chromosome 10q23 has led to the isolation of a candidate tumor suppressor gene, PTEN, that appears to be mutated at considerable frequency in human cancers. In preliminary screens, mutations of PTEN were detected in 31% (13/42) of glioblastoma cell lines and xenografts, 100% (4/4) of prostate cancer cell lines, 6% (4/65) of breast cancer cell lines and xenografts, and 17% (3/18) of primary glioblastomas. The predicted PTEN product has a protein tyrosine phosphatase domain and extensive homology to tensin, a protein that interacts with actin filaments at focal adhesions. These homologies suggest that PTEN may suppress tumor cell growth by antagonizing protein tyrosine kinases and may regulate tumor cell invasion and metastasis through interactions at focal adhesions.

The epidermal growth factor receptor (EGFR) gene is amplified in 40% of lignant gliomas, and the amplified genes are frequently rearranged. We have characterized the genetic alterations asoated with these rearrangements in five malignant gliomas. In one tumor the rearrangement resulted in the deletion of most of the extracytoplasmic domain of the receptor, resulting in a hybrid mRNA between new sequences and the truncated EGFR sequence. The predicted amino acid sequence of the protein from this tumor was remarkably similar to that described for several viral erbB oncogenes. Four other tumors were noted to have internal deletions of the EGFR gene. These rearrangements brought about in-frame deletions affecting either of two cysteine-rich domains in the extracytoplasmic portion of the molecule. The clonal nature of these alterations, and the fact that Identical alterations were seen in more than one tumor, suggests a role for these mutant receptor proteins in tumorigenesis. Further, these studies document the existence of tumor-specific cell surface molecules resulting from somatic mutation.

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...