Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

Background: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/ 8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma

Somatic mutations affect key pathways in lung adenocarcinoma

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well- classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers - including NF1, APC, RB1 and ATM - and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.National Human Genome Research InstituteWe thank A. Lash, M.F. Zakowski, M.G. Kris and V. Rusch for intellectual contributions, and many members of the Baylor Human Genome Sequencing Center, the Broad Institute of Harvard and MIT, and the Genome Center at Washington University for support. This work was funded by grants from the National Human Genome Research Institute to E.S.L., R.A.G. and R.K.W.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62885/1/nature07423.pd

Drug-sensitive FGFR2 mutations in endometrial carcinoma

Oncogenic activation of tyrosine kinases is a common mechanism of carcinogenesis and, given the druggable nature of these enzymes, an attractive target for anticancer therapy. Here, we show that somatic mutations of the fibroblast growth factor receptor 2 (FGFR2) tyrosine kinase gene, FGFR2, are present in 12% of endometrial carcinomas, with additional instances found in lung squamous cell carcinoma and cervical carcinoma. These FGFR2 mutations, many of which are identical to mutations associated with congenital craniofacial developmental disorders, are constitutively activated and oncogenic when ectopically expressed in NIH 3T3 cells. Inhibition of FGFR2 kinase activity in endometrial carcinoma cell lines bearing such FGFR2 mutations inhibits transformation and survival, implicating FGFR2 as a novel therapeutic target in endometrial carcinoma

EGFR Missense Mutations Are Transforming and Tumorigenic

<div><p>(A) Anchorage-independent growth of NIH-3T3 cells expressing various <i>EGFR</i> alleles as mean number of colonies ± standard deviation (bar graph, above). The lanes (below) show EGFR and actin immunoblots of whole cell lysates from NIH-3T3 subclones plated in soft agar. EGF (10 ng/ml) was added to the top agar where indicated.</p> <p>(B) Tumorigenicity of NIH-3T3 cells stably expressing the indicated <i>EGFR</i> alleles in nude mice. Mean tumor size ± standard deviation was determined 3–4 wk after subcutaneous inoculation into nude mice (<i>n</i> = 6 per cell line).</p></div

<i>EGFR</i> Missense Mutations Sensitize Cells to EGFR Kinase Inhibitors

<div><p>(A) Effect of increasing concentrations of the EGFR inhibitor erlotinib (0–10 μM) on the viability of IL-3 independent Ba/F3 subclones expressing EGFR ectodomain mutants (R108K, T263P, A289V, G598V, and EGFRvIII), the EGFR kinase domain mutants (L858R and L861Q), or the erlotinib-resistant EGFR double mutant L858R-T790M (LTM). Parental Ba/F3 cells and Ba/F3 cells expressing wild-type EGFR are not IL-3 independent and were included as controls. Viability (a mean percent of control ± standard deviation) was determined after exposure to erlotinib for 48 h.</p> <p>(B) Oncogenic EGFR ectodomain mutations map to interdomain interfaces. Shown are ribbon and surface diagrams of the EGFR [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#pmed-0030485-b046" target="_blank">46</a>] with sites of amino acid substitutions highlighted. Blue, domain I; green, domain II; red, domain III; and yellow, domain IV. Sites of the most prevalent amino acid substitutions are shown in red. Images were created with PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>). P596 is not visible in this view.</p></div

EGFR Missense Mutations in Glioblastoma Cluster in the Extracellular Domain and Are Associated with Increased <i>EGFR</i> Gene Dose

<div><p>(A) Location of missense mutations within the EGFR protein in a panel of 151 gliomas (132 glioblastomas, 11 WHO grade III gliomas, and eight glioblastoma cell lines). Each diamond represents one sample harboring the indicated mutation. Amino acid (AA) numbers are based on the new convention for EGFR numbering, which starts at the initiator methionine of pro-EGFR. Ligand-binding domains (I and III), cysteine-rich domains (II and IV), kinase domain (kinase), and the extracellular deletion mutant EGFRvIII [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#pmed-0030485-b045" target="_blank">45</a>] are indicated as reference.</p> <p>(B) Increased <i>EGFR</i> gene dose in tumors harboring <i>EGFR</i> missense mutations. The array (left) shows a high-resolution view of Affymetrix 100K SNP array at the <i>EGFR</i> gene locus for ten glioblastoma tumors and three normal controls (sample numbers are indicated above each column). <i>EGFR</i> mutation and log₂ ratio (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#st2" target="_blank">Methods</a>) are indicated below each column. The plot (left) shows a comparison of <i>EGFR</i> gene copy number determination by SNP array (y-axis, EGFR log₂ ratios) and FISH (x-axis). AMP, amplified; NON-AMP, non amplified.</p> <p>(C) RT-PCR for <i>EGFRvIII</i> and full-length <i>EGFR</i> in 14 fresh-frozen glioblastoma tumors (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030485#st2" target="_blank">Methods</a>). The upper band represents full-length <i>EGFR</i> (1,044 bp), the lower band <i>EGFRvIII</i> (243 bp), and the inset shows glyceraldehyde-3-phosphate dehydrogenase <i>(GAPDH)</i> RT-PCR results.</p></div