Expression of activated epidermal growth factor receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens.

OBJECTIVE Amplification of the epidermal growth factor receptor (EGFR) is a common event in the molecular pathogenesis of high-grade astrocytic tumors, occurring in 50% of glioblastoma multiforme (GBM) cases. A subset of GBMs also express a constitutively phosphorylated truncated receptor (EGFRvIII). Expression of transfected EGFRvIII in cells has been reported to activate the Ras-mitogen-activated protein kinase pathway and to provide a growth advantage. Novel therapeutic agents targeting signal transduction pathways are entering early clinical trials; determination of which GBMs express EGFRvIII might help identify patients who might benefit from these biological agents. METHODS A cohort of 15 flash-frozen surgical specimens (12 GBMs, 2 gliosarcomas, and 1 adult low-grade glioma) were evaluated for EGFR and EGFRvIII expression and for EGFR activation status using immunohistochemical (IHC) analysis, Western blotting, and reverse transcription-polymerase chain reaction assays. Levels of activated Ras-guanosine triphosphate were measured using a nonradioactive luciferase-based technique. Mitogen-activated protein kinase activation was determined using a myelin basic protein assay. IHC analysis was performed on paraffin-embedded, formalin-fixed, pathological specimens. Normal control samples included white matter specimens distal to tumors (n = 5), a sample obtained during a lobectomy for treatment of epilepsy (n = 1), and cultured fetal human astrocytes (n = 1). RESULTS We demonstrated higher levels of activated Ras and mitogen-activated protein kinase in GBM specimens, compared with normal brain tissue or the low-grade glioma. There was a very good correlation between results obtained using specialized molecular techniques and those obtained using routine IHC techniques. Screening for EGFRvIII expression may be of prognostic importance, because patients with EGFRvIII-positive tumors exhibited shorter life expectancies (mean survival time for patients with EGFRvIII-positive tumors, 4.5 +/- 0.6 mo; mean survival time for patients with EGFRvIII-negative tumors, 11.2 +/- 0.9 mo). CONCLUSION We demonstrated that routine IHC techniques using commercially available antibodies are capable of identifying which GBM specimens express EGFRvIII and whether the EGFRs are activated. Such a molecular classification of GBMs might allow us to determine which patients might benefit from biologically targeted therapies. In addition, characterization of specimens with respect to their EGFRvIII status seems to be of prognostic value.

[1]  T. Libermann,et al.  Expression of epidermal growth factor receptors in human brain tumors. , 1984, Cancer research.

[2]  W. McGuire,et al.  Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. , 1987, Science.

[3]  B. Vogelstein,et al.  Gene Amplification in Malignant Human Gliomas: Clinical and Histopathologic Aspects , 1988, Journal of neuropathology and experimental neurology.

[4]  M. Shibuya,et al.  Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors , 1988, Molecular and cellular biology.

[5]  W. Yung,et al.  Expression of an altered epidermal growth factor receptor by human glioblastoma cells. , 1988, Cancer research.

[6]  J. L. Bos,et al.  ras oncogenes in human cancer: a review. , 1989, Cancer research.

[7]  P. Humphrey,et al.  Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[8]  P. Humphrey,et al.  Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. , 1990, Cancer research.

[9]  C. James,et al.  Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[10]  N. Lemoine,et al.  Avoiding Punishment Is Its Own Reward , 1991, British Journal of Cancer.

[11]  M. Noble,et al.  Retinoblastoma gene deletions in human glioblastomas. , 1991, Oncogene.

[12]  Georg Breier,et al.  Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo , 1992, Nature.

[13]  P. Humphrey,et al.  Structural alterations of the epidermal growth factor receptor gene in human gliomas. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Tykocinski,et al.  Loss of tumorigenicity of rat glioblastoma directed by episome-based antisense cDNA transcription of insulin-like growth factor I. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R Akita,et al.  Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. , 1993, Cancer research.

[16]  G. Reifenberger,et al.  Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. , 1993, Cancer research.

[17]  D. Bigner,et al.  Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas. , 1993, Cancer research.

[18]  S. Shamah,et al.  Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells , 1993, Molecular and cellular biology.

[19]  Michael J. Fry,et al.  Phosphatidylinositol-3-OH kinase direct target of Ras , 1994, Nature.

[20]  W. Cavenee,et al.  A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. Louis.  The p53 Gene and Protein in Human Brain Tumors , 1994, Journal of neuropathology and experimental neurology.

[22]  V. P. Collins,et al.  Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplification. , 1994, Oncogene.

[23]  M. Karin,et al.  JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain , 1994, Cell.

[24]  W Arap,et al.  Loss of P16INK4 expression is frequent in high grade gliomas. , 1995, Cancer research.

[25]  D. Louis,et al.  A tiger behind many doors: multiple genetic pathways to malignant glioma. , 1995, Trends in genetics : TIG.

[26]  G. Boss,et al.  Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: comparison of parental and Ras-overproducing NIH 3T3 fibroblasts. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[27]  C. Marshall,et al.  Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation , 1995, Cell.

[28]  R. McLendon,et al.  Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. , 1995, Cancer research.

[29]  Jonathan A. Cooper,et al.  Differential Modulation of Mitogen-activated Protein (MAP) Kinase/Extracellular Signal-related Kinase Kinase and MAP Kinase Activities by a Mutant Epidermal Growth Factor Receptor (*) , 1995, The Journal of Biological Chemistry.

[30]  J. Biegel,et al.  Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. , 1995, Cancer research.

[31]  W. Gullick,et al.  Specific targeting of a mutant, activated egf receptor found in glioblastoma using a monoclonal antibody , 1995, International journal of cancer.

[32]  A. Guha,et al.  Expression of PDGF and PDGF receptors in human astrocytoma operation specimens supports the existence of an autocrine loop , 1995, International journal of cancer.

[33]  P. Black,et al.  Expression of platelet derived growth factor and platelet derived growth factor receptor mRNA in a glioblastoma from a patient with Li-Fraumeni syndrome. , 1995, Journal of neurology, neurosurgery, and psychiatry.

[34]  W. Cavenee,et al.  Tyrphostin AG 1478 preferentially inhibits human glioma cells expressing truncated rather than wild-type epidermal growth factor receptors. , 1996, Cancer research.

[35]  D Tripathy,et al.  Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. , 1996, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[36]  O. Bogler,et al.  A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. , 1996, Cancer research.

[37]  M. Shibuya,et al.  Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor. , 1996, British Journal of Cancer.

[38]  J R Feramisco,et al.  Enhanced Tumorigenic Behavior of Glioblastoma Cells Expressing a Truncated Epidermal Growth Factor Receptor Is Mediated through the Ras-Shc-Grb2 Pathway* , 1996, The Journal of Biological Chemistry.

[39]  R. B. Montgomery,et al.  Transformational and altered signal transduction by a naturally occurring mutant EGF receptor. , 1996, Oncogene.

[40]  A. Wong,et al.  A naturally occurring mutant human epidermal growth factor receptor as a target for peptide vaccine immunotherapy of tumors. , 1997, Cancer research.

[41]  A. Pawson,et al.  Proliferation of human malignant astrocytomas is dependent on Ras activation , 1997, Oncogene.

[42]  D. Bigner,et al.  Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate. , 1997, Cancer research.

[43]  H. Wiley,et al.  The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling* , 1997, The Journal of Biological Chemistry.

[44]  W. K. Alfred Yung,et al.  Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers , 1997, Nature Genetics.

[45]  Jan Mollenhauer,et al.  DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3–26.1 is deleted in malignant brain tumours , 1997, Nature Genetics.

[46]  D. Bigner,et al.  In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent. , 1997, Nuclear medicine and biology.

[47]  D. Gutmann,et al.  O-20-295 RAS-GTP levels are elevated in human NF1 peripheral nerve tumors , 1996, Clinical Neurology and Neurosurgery.

[48]  M. Wigler,et al.  PTEN, a Putative Protein Tyrosine Phosphatase Gene Mutated in Human Brain, Breast, and Prostate Cancer , 1997, Science.

[49]  J. Olson,et al.  Gene amplification as a prognostic factor in primary brain tumors. , 1998, Clinical cancer research : an official journal of the American Association for Cancer Research.

[50]  D Tripathy,et al.  Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. , 1998, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[51]  Alexander Levitzki,et al.  Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation , 1998, Molecular and Cellular Biology.

[52]  D. O’Rourke,et al.  Inhibition of a naturally occurring EGFR oncoprotein by the p185neu ectodomain: implications for subdomain contributions to receptor assembly , 1998, Oncogene.

[53]  M. Ciesielski,et al.  Tandem duplication of the epidermal growth factor receptor tyrosine kinase and calcium internalization domains in A-172 glioma cells , 1998, Oncogene.

[54]  W. Wels,et al.  Expression of an oncogenic mutant EGF receptor markedly increases the sensitivity of cells to an EGF‐receptor‐specific antibody‐toxin , 1998 .

[55]  L. Norton,et al.  Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. , 1998, Cancer research.