Epidermal Growth Factor Receptor Extracellular Domain Mutations in Glioblastoma Present Opportunities for Clinical Imaging and Therapeutic Development

Abstract

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFR^A289D/T/V). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFR^A289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFR^A289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFR^A289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFR^A289V mutation in glioblastoma, postulating EGFR^A289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.

Keywords: A289D/T/V; EGFR; EGFR oncogenes; EGFR targeted therapy; GBM; glioblastoma; glioma; radiogenomics; radiomics; survival.

Figure 1.. EGFR^A289D/T/V Missense Mutations Are Associated with Inferior Survival in GBM

(A and B) 2D representation of EGFR protein with functional domains indicated by colored segments and summary of missense mutations identified in the UPenn cohort (n = 260) (A) and the TCGA cohort (n = 591) (B). The location of mutated amino acids is indicated by a bar with a green circle. The height of the bar shows the number of patients in each cohort with the specific mutation. (C–F) Kaplan-Meier (KM) survival curves for the UPenn cohort, comparing EGFR^A289D/T/V with EGFR^A289 (C), EGFR^R108G/K to EGFR^R108 (D), EGFR^G598V to EGFR^G598 (E), and EGFR amplified to non-amplified (F). See also Figure S1 and Table S1.

Figure 2.. MRI Signatures of EGFR Missense Mutants Suggest an Invasive and Proliferative Phenotype

(A) Examples of the four basic/structural MRI modalities used to segment all brain scans into healthy and tumor labels, along with the major axis, minor axis, and the color legend for each label. WM, white matter; GM, gray matter; CSF, cerebrospinal fluid. (B) Selected quantitative imaging phenotypes (QIP) features found in the ET region presenting a picture of increased rCE due to decreased T1 signal in EGFR^A289D/T/V mutant tumors. (C) QIP features found in advanced imaging in the ET region. (D) Morphological features found in the CTE region. (E)Basic and advanced imaging features found in the ED region. See also Figures S2 and S3, Tables S2 and S3.

Figure 3.. Mice Bearing Intracranial EGFR^A289V Tumors Have Attenuated Survival and an Invasive Phenotype

(A and B) KM survival curves comparing mice implanted with U87 (A) or HK281 (B) tumors expressing either WT EGFR or EGFR^A289V, n = 6 per group. (C and D) Representative H&E and Ki67 stained sections of U87 (C) and HK281 (D) tumors expressing WT EGFR or EGFR^A289V harvested at time of sacrifice from mice in (A and B) Scale bars, 100 μm. (E and F) T2 weighted MRI (left) and 3D volume segmentation map (right) of mouse brains and tumors for WT EGFR (E) and EGFR^A289V U87 (F) tumors at 20 days after orthotopically implantation. Whole brain is in red and tumor is in green. See also Figure S4.

Figure 4.. EGFR^A289V Missense Mutation Induces ERK Activation and Increased MMP1 Expression

(A) RT-PCR analysis of MMP1 expression in U87 glioma cells expressing WT EGFR, EGFRvIII (V3), EGFR^R108K, EGFR^A289V, or EGFR^G598V. (B) RT-PCR analysis of MMP1 in HK281 GBM-spheres expressing WT EGFR or EGFR^A289V. (C) Western blotting analysis of the indicated proteins in serum starved U87 glioma cells expressing WT EGFR, EGFRvIII, EGFR^R108K, EGFR^A289V, or EGFR^G598V in the presence or absence of 100 ng/mL EGF for 10 min at 37°C. (D) Densitometric quantification of (C). (E) Western blot analysis of the indicated proteins in U87 glioma cells expressing either WT EGFR or EGFR^A289V following treatment with gefitinib (Gef) or lapatinib (Lap) (4 μM, 24 hr). (F) Densitometric quantification of (E). (G) Western blot analysis of the indicated proteins in U87 glioma cells expressing either WT EGFR or EGFR^A289V following treatment with U0126 or PD98059 (10 μM, 24 hr). (H) Densitometric quantification of (G). (I) RT-PCR analysis of MMP1 expression in U87 glioma cells expressing EGFR^A289V following treatment with gefitinib or lapatinib (4 μM, 24 hr). (J) RT-PCR analysis of MMP1 expression in U87 glioma cells expressing EGFR^A289V following treatment with U0126 or PD98059 (10 μM, 24 hr). (K) Western blot analysis of the indicated proteins in HK281 GBM-spheres expressing either WT EGFR or EGFR^A289V following treatment with gefitinib (4 μM), lapatinib (4 μM), or U0126 (10 μM) for 24 hr. (L) Densitometric quantification of (K). (M) RT-PCR analysis of MMP1 expression in HK281 GBM-spheres expressing EGFR^A289V following treatment with gefitinib (4 μM), lapatinib (4 μM), or U0126 (10 μM) for 24 hr. RT-PCR data shown are fold-change gene expression relative to GAPDH. Error bars are SEM of at least three replicates and represent at least three independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Figure 5.. Constitutive EGFR^A289V/ERK/MMP1 Signaling Results in Increased Invasion and Proliferation In Vitro

(A and B) Quantification of U87 glioma cells (A) and HK281 GBM-spheres (B) expressing WT EGFR or EGFR^A289V cell invasion following treatment with U0126 (10 μM) or control for 24 hr. (C and D) Quantification of invaded EGFR^A289V U87 glioma cells (C) and HK281 GBM-spheres (D) expressing an shMMP1 vector compared with control shRNA. (E and F) Quantification of % bromodeoxyuridine (BrdU)-positive U87 (E) or HK281 (F) cells treated with DMSO control or U0126 (10 μM) 24 hr prior to BrdU incorporation. (G and H) H&E staining of intracranial U87 (G) and HK281 (H) tumors without (shControl) or with MMP1 knockdown (shMMP1). Scale bars, 100 μm. Error bars are SEM of at least three replicates and represent at least three independent experiments. ns, not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See also Figure S6.

Figure 6.. mAb806 as a Therapeutic Option for Patients Expressing EGFR^A289V

(A and B) Fluorescent-activated cell sorter analysis of U87 glioma cells (A) and HK281 GBM-spheres (B) expressing WT EGFR, EGFRvIII, or EGFR^A289V. Serum-starved cells were incubated with either mAb806 or mAb528 (1 μg/1 × 10⁶ cells) followed by secondary staining with a fluorescein isothiocyanate-conjugated antibody. Results are shown as mAb806 staining normalized to mAb528 (total EGFR). (C) Mice bearing subcutaneous U87 tumors expressing WT EGFR, EGFRvIII (V3), or EGFR^A289V were treated with PBS (100 μL/mouse) or mAb806 (0.1 mg/100 μL/mouse) intraperitoneally (i.p.) 3 times per week for 2 weeks once tumors reached an average of 100 mm³. Mean tumor growth after treatment is shown as a function of time. (D) Representative images by fluorescence molecular topography (FMT) at day 23 of mice implanted intracranially with iRFP720 expressing U87 glioma cells expressing WT EGFR, EGFRvIII, or EGFR^A289V and treated with PBS (100 μL) or mAb806 (1 mg/100 μL/mouse) i.p. every other day from days 0 to 14. (E) Quantification of FMT signal intensity on day 23 post-implantation for each region of interest of mice in (D). (F) KM survival curve of mice in (D and E). (G) KM survival curve of mice bearing HK281 intracranial tumors as described in (D). n = 5 for each animal group. Error bars are SEM. ns, not significant; *p < 0.05, **p < 0.01. See also Figure S7.