EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.Glioblastoma multiforme is an incurable disease with a median survival time that is typically less than 1 year. Surgery and radiation have been shown to increase survival times, but the disease responds poorly to chemotherapy. One of the most common genetic abnormalities found in glioblastoma is amplification of the gene for EGFR.1 Along with amplification, the EGFR gene is frequently mutated. The most common mutation is a deletion of exons 2-7. This deletion is in-frame and results in the expression of a truncated EGFR, known as EGFRvIII, in which amino acids 6 -273 of the extracellular domain are replaced by a single glycine residue (1-4). Multiple studies have demonstrated that EGFRvIII is expressed in approximately half of all glioblastomas (5-8), and there is evidence that its expression is associated with a poorer prognosis (9). Consistent with this, EGFRvIII has been shown to greatly enhance the tumorigenicity of human glioblastoma cells grown as xenografts in nude mice (10).Studies in mouse models of glioblastoma show that EGFRvIII cooperates with mutations at the INK4a/ARF locus to promote the formation of glioblastoma (11). Thus, whereas EGFRvIII expression in normal mice does not give rise to tumors, expression of EGFRvIII in INK4a/ARF(Ϫ/Ϫ) mice does give rise to tumors with a high frequency. The INK4a/ARF locus contains genes coding for two different proteins (12). One of these is Ink4a, a repressor of cyclin-dependent kinase 4 (cdk4); the other is Arf, which has a role in regulating p53 levels. EGFRvIII will also form tumors in mice if cdk4 is overexpressed at the same time, indicating that these two pathways cooperate in the formation of glioblastoma. It is likely that these two ...

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Background Sarcoma mortality remains high despite adjuvant chemotherapy. Biomarker predictors of treatment response and outcome could improve treatment selection. Methods Tissue microarrays (TMAs) were created using pre- and posttreatment tumor from two prospective trials (MGH pilot and RTOG 9514) of neoadjuvant/adjuvant MAID chemotherapy and preoperative radiation. Biomarkers were measured using automated computerized imaging (AQUA or ACIS). Expression was correlated with disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS). Results Specimens from 60 patients included 23 pretreatment (PRE), 40 posttreatment (POST), and 12 matched pairs (MPs). In the MP set, CAIX, GLUT1, and PARP1 expression significantly decreased following neoadjuvant therapy, but p53 nuclear/cytoplasmic (N/C) ratio increased. In the PRE set, no biomarker expression was associated with DFS, DDFS, or OS. In the POST set, increased p53 N/C ratio was associated with a significantly decreased DFS and DDFS (HR 4.13, p=0.017; HR 4.16, p=0.016), while increased ERCC1 and XPF expression were associated with an improved DFS and DDFS. No POST biomarkers were associated with OS. Conclusions PRE biomarker expression did not predict survival outcomes. Expression pattern changes after neoadjuvant chemoradiation supports the concepts of tumor reoxygenation, altered HIF-1α signaling, and a p53 nuclear accumulation DNA damage response. Clinical Trial Registration NRG Oncology RTOG 9514 is registered with ClinicalTrials.gov. The ClinicalTrials.gov Identifier is NCT00002791.

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