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.


A B S T R A C T 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.

Introduction
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that regulates fundamental processes of cell growth and differentiation. Deletion of the EGFR gene is embryonically lethal in mice, and increased EGFR signaling has been linked to a variety of human malignancies. Mechanisms for oncogenic conversion of EGFR in cancer include EGFR gene amplification, structural rearrangements of the receptor, overexpression of epidermal growth factor (EGF)-family ligands by tumor cells and/or surrounding stroma, and-as was recently shown in lung cancer-activating mutations in the EGFR kinase domain [1].
The evidence for a role of EGFR in oncogenesis is particularly compelling in glioblastoma, the most aggressive human brain tumor with a two year survival of less than 5% despite surgery, radiation, and chemotherapy [2,3]. About 40% of glioblastomas show amplification of the EGFR gene locus [4], and about half of these tumors express a mutant receptor (EGFRvIII) that is constitutively active due to an inframe truncation within the extracellular ligand-binding domain [5][6][7]. Perhaps the strongest evidence for a role of EGFR in the biology of glioblastoma stems from clinical trials in which 15%-20% of glioblastoma patients experienced significant tumor regression in response to small-molecule EGFR kinase inhibitors [8,9]. Our recent data indicate that expression of EGFRvIII in the context of an intact PTEN (phosphatase and tensin homolog) pathway is associated with these clinical responses [9].
To explore the possibility that EGFR might be the target of oncogenic mutations outside the kinase domain, we sequenced the entire EGFR coding region in a panel of 151 glioma tumors and cell lines.

DNA Samples
Genomic DNA was extracted from eight glioblastoma cell lines (A172, SF268, SF295, SF539, T98G, U87, U118, and U251) and 143 fresh frozen glioma samples. The clinical glioma samples comprised glioblastomas (n ¼ 132), World Health Organization grade III anaplastic astrocytomas (n ¼ 3), grade III mixed gliomas (n ¼ 4), and grade III oligodendrogliomas (n ¼ 4). Germline genomic DNA was extracted from peripheral blood samples. To confirm the match between germline and tumor DNA for each patient, we performed mass spectrometric genotyping of 24 single-nucleotide polymorphism (SNP) loci. These loci included 23 SNP loci represented on both 50K Xba and Hind arrays (Affymetrix, http://www.affymetrix.com) and one AmelXY locus for sex determination (Table S1). Collection and analysis of all clinical samples was approved by the University of California Los Angeles Institutional Review Board.

Sequencing and Mass Spectrometric Genotyping
PCR reactions for each exon and flanking intronic sequences contained 5 ng of genomic DNA, 13 HotStar Buffer, 0.8 mM dNTPs, 1 mM MgCl 2 , 0.2 U HotStar Enzyme (Qiagen, http://www.qiagen.com), and 0.2 lM forward and reverse primers in a 6 or 10 ll reaction volume. PCR cycling parameters were: one cycle of 95 8C for 15 min; 35 cycles of 95 8C for 20 s, 60 8C for 30 s, and 72 8C for 1 min; followed by one cycle of 72 8C for 3 min.
The resulting PCR products were sequenced using bidirectional dye-terminator fluorescent sequencing with universal M13 primers. Sequencing fragments were detected via capillary electrophoresis using ABI Prism 3730 DNA Analyzer (Applied Biosystems, http://www.appliedbiosystems.com). PCR and sequencing were performed at Agencourt Bioscience Corporation (http://www.agencourt.com) or at the Broad Institute of Harvard and MIT (http://www.broad.mit.edu). Forward (F) and reverse (R) chromatograms were analyzed in batch with Mutation Surveyor 2.51 (SoftGenetics, http:// www.softgenetics.com), followed by manual review.
A minimum of 21 of 28 (75%) EGFR exon sequence coverage was accomplished for 151 samples. An exon for each individual sample was considered covered if 90% of the sequence trace within the exon had a phred quality score of 30 or greater, a signal-to-background noise ration of 15% or less, and signal intensity greater than 25% of the signal intensity of the sequencing plate. High-quality sequence variations found in one or both directions were scored as candidate mutations. Exons harboring candidate mutations were reamplified from the original DNA sample and resequenced.

Affymetrix 100K SNP Arrays
Genomic DNA was processed and hybridized following the guidelines of the manufacturer (Affymetrix) and arrays were scanned with a GeneChip Scanner 3000. Genotyping calls and signal quantification were obtained using GeneChip Operating System 1.1.1 and Affymetrix Genotyping Tools 2.0 software. Data were normalized at the probe level to a baseline array with median signal intensity using invariant set normalization. After normalization, the signal values for each SNP in each array were obtained with a model-based (perfectmatch/mismatch) method [11]. Signal intensities at each probe locus were compared with a set of normal reference samples representing 36 ethnically matched individuals to generate log 2 ratios. Log 2 ratios were smoothed using the breakpoint analysis method in the R package GLAD (Gain and Loss Analysis of DNA) [12]. Regions were considered amplified if their smoothed log 2 ratio exceeded 0.3 (half the variation seen with a single-copy gain).

Fluorescence In Situ Hybridization
Dual-probe fluorescence in situ hybridization (FISH) was performed on paraffin-embedded sections with locus-specific probes for EGFR and the centromere of Chromosome 7 as previously described [9].

Quantification of Mutant EGFR Alleles
The abundance of missense and wild-type EGFR alleles in tumor DNA samples was determined by PCR-cloning and sequencing of respective EGFR exons. PCR products were ligated into PCR2.1-Topo vectors (Invitrogen) and transformed into E. coli. After transformation, bacteria were plated onto selection plates and grown overnight. For each DNA sample, 65-94 colonies were isolated using a colony picking robot (QPix2, Genetix Limited, http://www.genetix.com), grown overnight, and bidirectionally sequenced at the Broad Institute. Sequence traces were analyzed using Mutation Surveyor software (SoftGenetics).

Expression of EGFR Alleles in NIH-3T3 Cells
Cells cultured in DMEM supplemented with 10% calf serum were infected with pBabe-Puro-based viral stock in the presence of polybrene. Beginning 2 d after infection, cells were selected in puromycin (2 lg/ml) for 3 d. Pooled NIH-3T3 cells stably expressing respective EGFR alleles at comparable EGFR protein levels were examined for their ability to induce colony formation in soft agar and tumor growth in nude mice. For soft agar assays, 1 3 10 5 NIH-3T3 cells were suspended in a top layer of DMEM supplemented with 10% calf serum and 0.4% Select Agar (Gibco/Invitrogen) and plated on a bottom layer of DMEM supplemented with 10% calf serum and 0.5% Select Agar. EGF (10 ng/ml) was added to the top agar where indicated. Pictures of colonies were taken 2-3 wk after plating. Colonies were counted from ten random images (403 magnification) taken from each well. Colonies were counted from three replicate wells with the average number represented. In vivo tumorigenicity assays were performed in three mice (two injections/mouse) for each cell line. For each injection, 2 3 10 6 cells were injected subcutaneously into each nude mouse (Taconic, http://www. taconic.com) and three-dimensional tumor volumes calculated 3-4 wk following injection.

Expression of EGFR Alleles in Human Astrocytes
Viral supernatants (pLXSN-neo-EGFR) were used to infect immortalized human astrocytes expressing the catalytic subunit of the telomerase holoenzyme and human papillomavirus 16 E6/E7 [16]. Astrocytes were then selected in G418 (Invitrogen) for approximately 10 d.

Results
Missense Mutations in Glioblastoma Cluster in the Extracellular Domain of EGFR Encouraged by the recent success in identifying oncogenic kinase mutations through resequencing of kinase-encoding genes [17][18][19], we sequenced the entire coding sequence of EGFR in 143 human glioma samples and eight glioblastoma cell lines. Analysis of the initial Sanger sequencing results in these 151 samples revealed several novel sequence variations in the coding region of the EGFR. To validate these candidate mutations via a complementary method, all DNA samples were reexamined using allele-specific genotyping by MALDI-TOF mass spectrometry.
In all, we identified EGFR missense mutations in 14.4% (19/ 132) of glioblastomas, 12.5% (1/8) of glioblastoma cell lines, and none (0/11) in lower-grade gliomas. Only one tumor sample harbored a missense mutation in the EGFR kinase domain (L861Q), the location of EGFR mutations in lung cancer, supporting the recent conclusion from other groups that EGFR kinase domain mutations appear to be a rare event in this disease [20][21][22]. The remainder of the EGFR mutations (18/132 glioblastomas) were located in the extracellular ligand-binding (I, III) or cysteine-rich (II, IV) domains of the receptor ( Figure 1A). Two evolutionarily highly conserved amino acid residues ( Figure S1) were affected by mutations in five samples each (R108 and A289). Examination of peripheral blood DNA, matched to the tumor DNA by genotyping of 24 SNP loci, showed that eight of the 12 distinct missense mutations were unambiguously somatic, and one mutation (E330K) was germline. Three additional missense mutations (A289D, A289T, and R324L) were found in tumors for which no normal tissue was available ( Table 1). None of the missense mutations were detected in germline DNA from 270 normal control individuals.
To define which fraction of the EGFR pool represented the mutant allele in gliomas with EGFR missense mutations, we employed a PCR-cloning strategy previously used by our laboratories for mutation detection in clinical samples [23]. The mutant EGFR allele represented 30%-98% of the receptor pool in two-thirds (10/16) of all examined cases and over 50% in at least one tumor representing the most common amino acid changes: R108K, T263P, A289V, and G598V (Table 1). Lower abundance of the mutant EGFR allele in other samples might be due to contaminating stromal tissue, because genomic DNA was extracted from frozen tumor aliquots without prior microdissection.
We also genotyped genomic DNA from 119 primary lung tumors to detect EGFR ectodomain mutations. While 13.4% (16/119) of these lung tumor samples harbored mutations in the EGFR kinase domain, we did not detect any of the gliomarelated EGFR ectodomain mutations in this sample set.

EGFR Ectodomain Mutations Are Associated with Increased EGFR Gene Dose
Since EGFR is amplified in about 40% of human glioblastomas [4], we determined the relationship between EGFR missense mutation and EGFR gene dose in our tumor samples. Of 17 tumors with EGFR missense mutations, 58.8% (10) showed evidence for EGFR amplification by FISH and/or Affymetrix 100K SNP genotyping arrays ( Figure 1B; Table 1). This distribution suggests that EGFR missense mutations are associated with EGFR amplification and raises the question of whether EGFR missense mutations in glioblastoma co-occur with or are mutually exclusive of the EGFRvIII mutation, which is found almost exclusively in glioblastomas with increased gene dosage [24]. Using at least two independent assays for the determination of EGFRvIII status, we identified the EGFRvIII allele in 28.3% (13/46) of gliomas without EGFR missense mutation and 6.3% (1/16) tumors with EGFR missense mutation ( Figure 1C; Table 1); note that this tumor showed vastly lower levels of EGFRvIII ( Figure 1C, lane 12). These findings suggest that EGFR ectodomain mutations occur independently of EGFRvIII in glioblastoma and provide an alternative mechanism for EGFR activation in this disease.

EGFR Ectodomain Mutants Are Oncogenic
To test the oncogenicity of the glioma-related EGFR missense mutations, we transduced NIH-3T3 fibroblasts with retroviruses encoding either wild-type EGFR or selected EGFR missense mutants (encoding R108K, T263P, A289V, G598V, and L861Q). Ectopic expression of all EGFR mutants examined in NIH-3T3 cells conferred anchorage-independent colony formation in soft agar (Figure 2A). In contrast, expression of wild-type EGFR induced a transformed phenotype only in the presence of exogenous EGF, as previously reported [25,26].
To further analyze the oncogenic potential of the EGFR mutants, NIH-3T3 subclones stably expressing the same missense mutant receptors (encoding R108K, T263P, A289V, G598V, and L861Q) were inoculated subcutaneously into nude mice. NIH-3T3 cells infected with empty vector or wildtype EGFR-expressing virus did not yield any measurable tumors within the four-week observation period. In contrast, NIH-3T3 cells expressing each of the tested EGFR missense mutants produced large tumors at the inoculation site in all mice within three to four weeks ( Figure 2B).

EGFR Ectodomain Mutants Are Basally Phosphorylated and Are Responsive to Ligand
Signal transduction through EGFR is determined by its basal catalytic activity, receptor activation by ligand, and signal termination through intracellular compartmentalization of the receptor-ligand complex, receptor dephosphorylation, and degradation [27]. To explore the biochemical basis for the gain of function observed with EGFR ectodo-main mutants, we first examined the basal catalytic activity of A289V-EGFR in transiently transfected 293T cells using EGFR autophosphorylation as a readout for receptor activation. EGFR autophosphorylation was determined by measuring the total phosphotyrosine content of the immunoprecipitated receptor ( Figure 3A, left blot) and by immunoblotting of whole cell lysates with several phosphosite-specific anti-EGFR antibodies ( Figure 3A, right blot). Compared to wild-type EGFR, the ectodomain mutant A289V-EGFR showed a marked increase in receptor autophosphorylation in the absence of ligand or serum. We subsequently examined a more extensive panel of EGFR (B) Increased basal activity of EGFR missense mutants in human astrocytes. Immortalized human astrocytes were stably infected with wild-type EGFR or the indicated EGFR missense mutants. Shown are total phosphotyrosine (PY), Y1068-EGFR, total EGFR, and PI3K p85 (loading control) immunoblots of whole cell lysates from cells following 12 h of serum starvation. The solid arrow at the PY position represents tyrosine-phosphorylated EGFR, and the interrupted arrows indicated other differentially tyrosine-phosphorylated proteins. The inset shows an anti-EGFR immunoblot of parental astrocytes (far-left lane) and stable astrocyte subclones (designated in remaining five lanes) growing in full serum. (C) Basal receptor phosphorylation and EGF-responsiveness of wild-type EGFR and four different EGFR ectodomain mutants stably expressed in Ba/F3 murine hematopoietic cells. Shown are immunoblots of stable Ba/F3 subclones after 12 h of serum starvation (À EGF) and 15 min following EGFinduction (0.5 or 5 ng/ml EGF). doi:10.1371/journal.pmed.0030485.g003 missense mutants (T263P, A289V, G598V, L861Q) in immortalized human astrocytes stably transduced with these receptors. Compared to astrocytes overexpressing wild-type EGFR, sublines expressing EGFR missense mutants showed an increased phosphotyrosine content of EGFR and several other unidentified proteins under serum-free conditions ( Figure 3B).
We also expressed selected EGFR mutants (R108K, T263P, A289V, G598V, L861Q) in murine hematopoietic cells (Ba/F3 cells) which do not express any EGFR family members [14] but otherwise retain functional properties of the EGF-signaling pathway [28][29][30]. Consistent with our findings in 293T cells and astrocytes, all examined EGFR ectodomain mutants showed increased tyrosine phosphorylation under serum-starved conditions and were responsive to exogenous EGF ( Figure 3C). We also noted that EGF stimulation led to a more pronounced drop of EGFR levels in Ba/F3 cells expressing wild-type EGFR than in subclones expressing EGFR ectodomain mutants ( Figure 3C), reminiscent of the impaired ligand-induced receptor downregulation reported for selected EGFR kinase domain mutants [31].

Sensitivity of EGFR Ectodomain Mutants to EGFR Kinase Inhibitors
The presence of identical missense mutations in multiple patient samples and their oncogenicity in standard transformation assays suggest that these mutants play a role in gliomagenesis. It also raises the question whether these mutations might sensitize transformed cells to EGFR kinase inhibitors. Ba/F3 cells provide a unique model system to examine kinase inhibitor sensitivity [32][33][34][35] because stable expression of oncogenic kinases in these cells can relieve them from their intrinsic dependence on IL-3 for survival [15,36]. As expected from our results in NIH-3T3 cells, expression of the tested EGFR missense mutants but not wildtype EGFR was able to relieve Ba/F3 cells from IL-3 dependence. Addition of the EGFR kinase inhibitor erlotinib to the media had little or no effect on the viability of parental Ba/F3 cells growing in the presence of IL-3 or on Ba/F3 cells expressing the drug-resistant EGFR double-mutant L858R/ T790M-EGFR. However, erlotinib did induce dose-dependent cell death in Ba/F3 subclones expressing the EGFR ectodomain mutants (missense and vIII truncation) or EGFR kinase domain mutants (L858R and L861Q) ( Figure 4A). Of note, erlotinib-induced cell death of Ba/F3 cells expressing EGFR ectodomain mutants occurred at IC-50 values of 50-150 nM, drug concentrations that are well below the concentrations achieved in human plasma [37]. These data suggest that EGFR missense mutants sensitize transformed cells to EGFR kinase inhibitors similar to EGFRvIII or lung cancer-related kinase domain mutants, both of which have been associated with clinical responses to EGFR kinase inhibitor therapy [9,19,38,39].
We recently reported the results of a glioblastoma clinical trial with EGFR kinase inhibitors which associated clinical responses to the coexpression of EGFRvIII and PTEN [9]. To investigate whether clinical responses might also be linked to the presence of EGFR ectodomain mutations, we reexamined all available tumor DNA samples from this clinical trial. We identified the ectodomain mutant R108K-EGFR in 14% (1/7) gliomas that responded to erlotinib. This tumor, however, also expressed EGFRvIII, raising the possibility of independent clones arising from a common progenitor with EGFR amplification. We also identified the R108K EGFR mutation in 7% (1/5) gliomas that failed EGFR kinase inhibitor therapy, but loss of PTEN in this tumor provides a potential explanation for treatment failure (Table S3). Larger clinical trials are required to ascertain the contribution of EGFR missense mutants to EGFR kinase inhibitor response in glioblastoma.

Discussion
We have identified novel oncogenic missense mutations in the ectodomain of EGFR in glioma. The association of these mutations with increased EGFR gene dosage raises the  question of whether similar ectodomain missense mutations might exist in other malignancies with EGFR amplification or polysomy of Chromosome 7. More broadly, our results suggest that ectodomain missense mutations in other tyrosine kinase genes may be transforming events in multiple cancers, arguing for an extension of current kinase gene resequencing efforts beyond the kinase domains [40,41].
The ligand-independent basal phosphorylation of the EGFR missense mutants in our study is consistent with their ability to confer NIH-3T3 cells with the ability to grow in soft agar in the absence of exogenous EGF. Whether all EGFR ectodomain mutants share a common mechanism of oncogenic receptor conversion warrants further study. A common mechanism is suggested by the structural observation fact that many of the resulting amino acid substitutions map to interdomain interfaces. R108K and A289V/D/T occur at the domain I/II interface, P569L and G598V occur at the domain II/IV contact, and T263P occurs in domain II just before the extended loop that contacts domain IV ( Figure  4B). Differences in constitutive receptor activity (G598V.A289V.T263P), on the other hand, point toward alternative mechanisms of oncogenic receptor conversion.
Three of the EGFR missense mutations (encoding P596L, G598V, and A289V) were previously observed in smaller cohorts of glioblastoma tumors [24,42]. The identification of additional ectodomain mutations in our study might have been facilitated by the large number of tumors, nearcomplete coverage of the EGFR coding sequence, and use of MALDI-TOF mass spectrometry genotyping in addition to Sanger sequencing. Since most of the patients in our study were of Northern European descent, we were unable to establish whether the prevalence of EGFR ectodomain mutations in glioblastoma might be affected by ethnicity as has been shown for EGFR kinase domain mutations. The distribution of EGFR missense mutations in glioblastoma (largely extracellular) and lung cancer (exclusively kinase domain) suggests fundamental differences in oncogenic EGFR signaling between these two tumor types. Importantly, however, both classes of mutants-as well as EGFRvIIIappear to sensitize transformed cells to EGFR kinase inhibitors in a preclinical model system that has been predictive of clinical responses [33,43]. Based on the experience with kinase inhibitors for chronic myeloid leukemia [44], the development of sensitive methodologies to monitor the EGFR pool before and during therapy will constitute an important step in advancing the current use of EGFR kinase inhibitors for cancer. Figure S1. Protein Sequence Alignment for EGFR Missense Mutations Alignment (ClustalW) of the human EGFR protein sequences ERBB2, ERBB3, ERBB4, and the EGFR protein sequence of Mus musculus, Rattus norvegicus, Sus scrofa, Danio rerio, and Drosophila melanogaster and Xiphophorus for the residues affected by missense mutations. Found at doi:10.1371/journal.pmed.0030485.sg001 (29 KB PDF).  Table S3. Ectodomain Mutations in Glioblastomas and Response to EGFR Kinase Inhibitor Therapy Patients were classified as responders (patients 1-7) or nonresponders (patients 8-26) based on radiographic criteria [9]. Found at doi:10.1371/journal.pmed.0030485.st003 (16 KB XLS).