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

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