Glioblastoma multiforme (GBM) frequently involves amplification and alteration of the epidermal growth factor receptor (EGFR) gene, resulting in overexpression of varied mutations, including the most common mutation, EGFRvIII, as well as wild-type EGFR (EGFRwt). To test the prognostic value of EGFR, we retrospectively analyzed the relationship between treatment outcomes and the EGFR gene in 87 newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. The EGFR gene status was assessed by Southern blots and EGFR expression by immunohistochemistry using three monoclonal antibodies (EGFR.25 for EGFR, EGFR.113 for EGFRwt, and DH8.3 for EGFRvIII). EGFR amplification was detected in 40 (46%) of the 87 GBM patients; in 39 (97.5%) of these, EGFR was overexpressed. On the other hand, in 46 of 47 patients without EGFR amplification (97.9%), no EGFR overexpression was present. There was a close correlation between EGFR amplification and EGFR overexpression (P < 0.0001). EGFRwt was overexpressed in 27 of the 40 (67.5%) patients with, and in none without, EGFR amplification (P < 0.0001). Similarly, EGFRvIII was overexpressed in 18 (45.0%) of 40 patients with and in 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). The finding that 8 (20%) of the patients with EGFR amplification/EGFR overexpression manifested overexpression of neither EGFRwt nor EGFRvIII indicates that they overexpressed other types of EGFR. Multivariate analysis demonstrated that EGFR amplification was an independent, significant, unfavorable predictor for overall survival (OS) in all patients (P = 0.038, HR = 1.67). With respect to the relationship of age to EGFR prognostication, the EGFR gene status was a more significant prognosticator in younger patients, particularly in those <60 years (P = 0.0003, HR = 3.15), whereas not so in older patients. EGFRvIII overexpression, on the other hand, was not predictive for OS. However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71). This finding indicates that EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis. In GBM patients, EGFR is of significant prognostic value for predicting survival, and the overexpression of EGFRvIII with amplification plays an important role in enhanced tumorigenicity.

GBM3 is the most common primary malignant neoplasm of the central nervous system in adults. Treatment outcomes even after multimodal therapies, including surgical resection, radiotherapy, and chemotherapy, remain poor; the median survival is ∼1 year. Nevertheless, there is considerable variation among GBM patients with respect to survival. Many studies, undertaken to improve the clinical management of this lethal tumor by identifying prognostic factors, confirmed that the patient age at diagnosis plays a significant role (1, 2, 3, 4, 5, 6, 7). Advances in molecular biology disclosed the presence of molecular genetic alterations in GBM (8, 9, 10, 11). The most frequent alteration of GBM oncogenes consists of amplification of the EGFR gene that results in overexpression of EGFR, a transmembrane tyrosine kinase receptor (12, 13, 14, 15, 16, 17, 18). EGFR amplification is present in 30–50% of all GBM; it occurs more frequently in primary (de novo) GBM (12, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26) and confers advantages of growth and invasiveness and radio- and chemo-resistance on tumor cells (27, 28, 29, 30, 31, 32). Furthermore, the majority of GBM with EGFR amplification exhibit a considerable variety of qualitative EGFR alterations, resulting in different EGFR mutations (16, 17, 21, 33, 34, 35). The most common EGFR mutation is EGFRvIII (also known as ΔEGFR and de2–7EGFR), which is characterized by the deletion of exons 2–7 in EGFR mRNA that correspond to cDNA nucleotides 275-1075 encoding amino acids 6–273. This mutation presumably occurs through alternative splicing or gene rearrangements (33, 34, 36, 37). The extracellular ligand-binding domain of EGFRvIII is truncated, and unlike EGFRwt, EGFRvIII displays ligand-independent constitutive activity (38, 39, 40) and enhances tumorigenicity in vivo(32, 41, 42). There is experimental evidence that EGFR amplification may result in a less favorable prognosis; however, clinical studies are inconclusive (6, 13, 18, 43, 44, 45, 46, 47, 48, 49). Simmons et al.(43) suggested that differences in patient populations may explain the divergent results. To evaluate the prognosis of GBM patients, we examined the relationship between outcome and EGFR gene status/EGFR expression in a uniform Japanese population selected from newly diagnosed adult patients with supratentorial GBM enrolled in clinical trials. Feldkamp et al.(50) suggested that GBM patients with EGFRvIII may have a shorter life expectancy; however, they were unable to produce statistical evidence for this supposition. Therefore, we also assessed whether EGFRvIII expression plays a determining role in the prognosis of GBM patients. We subjected our data to multivariate analysis and now present clinical evidence that EGFR amplification and EGFR overexpression, including EGFRvIII, play a significant role in the prognosis of GBM patients. We also document that routine immunohistochemical studies that use combinations of antibodies are useful for the assessment of EGFR expression.

Patient Population.

Our patient population consisted of 87 newly diagnosed adults with histologically verified supratentorial GBM. Histopathological examination was conducted by two neuropathologists (J-i. K. and Y. I.) according to criteria published by WHO (51, 52); tumors exhibiting prominent microvascular proliferation and/or necrosis, in addition to high cellularity, marked nuclear atypia, and remarkable mitotic activity were diagnosed as GBM. The presence of necrosis was a requisite for a diagnosis of GBM; cases without necrosis were excluded. After surgery, patients enrolled in two prospective randomized Phase III trials conducted at Kumamoto University Hospital, Kagoshima University Hospital, and affiliated hospitals received combined radiotherapy and nitrosourea (ACNU)-based chemotherapy. Protocol 8701 (active from December 1987 to June 1995) compared the effectiveness of intra-arterial ACNU administration versus intravenous ACNU administration (53). Protocol 9501 (active from July 1995 to April 2003) compared the effectiveness of PAV versus PAV plus IFN-β. Patients enrolled in protocol 8701 were ≥15 years; 8 patients were ≥70 years. Patients treated according to protocol 9501 were from 15 to 69 years of age; none were ≥70 years. Some of the patients enrolled in these trials were excluded from our study because they did not receive combined radiotherapy and chemotherapy, because they died of other diseases, or because their follow-up was <6 months on the day of analysis or their last known day of life. After histopathological reexamination, also excluded were enrolled patients whose tumors, because they had a significant oligodendroglial component, were reclassified as anaplastic oligoastrocytomas (54, 55). On the day of analysis, 166 patients were eligible to participate in this study (75 of 84 from protocol 8701 and 91 of 99 from protocol 9501). Adequate tumor samples for gene analysis by Southern blots were obtained from 87 patients (23 enrolled in protocol 8701 and 64 in protocol 9501).

In a comparison of 23 analyzable versus 52 unanalyzable patients enrolled in protocol 8701, there was no statistically significant difference in the median age (53 versus 55 years, respectively, P = 0.36, Mann-Whitney U test), the median KPS (70 versus 60, respectively, P = 0.07, Mann-Whitney U test), the gender distribution (analyzable group: 12 males, 11 females; unanalyzable group: 30 males, 22 females; P = 0.66, χ2 test), and the distribution of surgery (GTR, PR, and biopsy in the analyzable and unanalyzable groups: 43.5, 52.2, and 4.3% versus 32.7, 50, and 17.3%; P = 0.28, χ2 test). In a comparison of 64 analyzable versus 27 unanalyzable patients enrolled in protocol 9501, there was no statistically significant difference in the median age (55 versus 58 years, P = 0.23), the median KPS (70 versus 80, P = 0.5), and the gender distribution (analyzable group: 42 males, 22 females; unanalyzable group: 13 males, 14 females; P = 0.12). The only characteristic that was significantly different among patients in protocol 9501 was the extent of surgery; of the analyzable group, 32.8, 56.3, and 10.9% underwent GTR, PR, and biopsy, respectively, compared with 11.1, 48.2, and 40.7%, respectively, of unanalyzable patients (P < 0.01). Among analyzable patients, thus, in many cases, the biopsy procedure yielded an insufficient amount of tumor tissue for gene analysis. In addition, the number of analyzable patients in the earlier protocol (#8701) was smaller than in protocol 9501 (30.7 versus 70.3%, P < 0.01; χ2 test), because in many cases, the amount of frozen, stored samples from all of the procedures was insufficient for further analysis. With respect to OS, there was no difference between patients who were assayed and those who were not; the median OS was 1.232 and 1.103 years, respectively (P = 0.67; Log-rank test), among patients in protocol 8701 and 1.366 and 1.114 years (P = 0.09) among those in 9501; it was 1.262 and 1.114 years, respectively (P = 0.18), for all patients in both protocols.

Written informed consent to participate in the clinical trials and in gene analysis was obtained from all patients and/or their family members.

Samples for Gene Analysis.

Tumor tissue samples were immediately frozen and stored at −80°C until the extraction of genomic DNA. The mean quantity of tumor sample judged sufficient for subsequent Southern blot analysis was 0.22 ± 0.118 gram. Control specimens from 4 patients operated for diseases other than brain tumors consisted of histologically normal brain tissues; these were handled in the identical manner. We verified electrophoretically that there was no oligonucleosomal DNA fragmentation in the tumor samples, confirming that the tumor samples contained no significant necrotic components.

Analysis of the EGFR Gene Status.

For quantitative detection of the EGFR gene status, we performed Southern blot analysis using full-length human EGFR cDNA (56) as the probes. The probes hybridized to all EGFR exons and for them to be considered adequate, they had to permit the detection of EGFRwt and in-frame deletion mutations, including EGFRvIII. To prepare non-RI, digoxigenin-11-dUTP-labeled random primed DNA probes, EGFR cDNA, and full-length human α-tubulin cDNA (pEGFP-Tub Vector; BD Biosciences, Clontech, Palo Alto, CA) were labeled using a DIG DNA-labeling kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Genomic DNA from frozen tissue samples was extracted by methods described previously (57, 58, 59). Genomic DNA (10 μg) digested with EcoRI was electrophoretically separated on 0.8% agarose gels, and DNA fragments were then transferred to nylon membranes (Roche). The blots were hybridized with EGFR cDNA probes in a hybridization buffer [5× standard saline citrate, 0.02% (w/v) SDS, 0.1% (w/v) N-laurosylsarcosine NaCl, and 1% (w/v) blocking reagent (Roche)]. After overnight incubation at 65°C, the membranes were washed and probed with Anti-Digoxigenin-AP (Roche). The blots were subjected to luminescence reaction using CSPD (Roche). Autoradiographs were taken using X-ray film (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom) and intensifying screens. The blots were then stripped and rehybridized with α-tubulin reference probes. Using the Scion Imaging software program (Scion Corp., Frederick, MD), the signal intensities emitted by EGFR fragments from tumor- and normal human brain samples were compared, and the normalized ratio was determined using a reference gene. A cutoff value was set after determining the EGFR gene status of normal human brain tissue samples, and a value of 2 was chosen as the threshold. Normalized ratios ≥2 were considered indicative of amplification; values <2 threshold were recorded as no amplification.

Monoclonal Antibodies.

Mouse monoclonal antibodies were used as primary antibodies: (a) clone EGFR.25 (Novocastra Laboratories Ltd., Newcastle, United Kingdom), which recognizes 200 amino acids of the intracellular domain of the EGFR molecule, excluding the conserved tyrosine kinase domain; (b) clone EGFR.113 (Novocastra), which recognizes the extracellular domain of EGFR molecule (25); and (c) clone DH8.3 (Novocastra), which recognizes only the junctional truncated extracellular domain of EGFRvIII. It has been confirmed that DH8.3 does not cross-react with full-length EGFR (60, 61, 62).

Immunofluorescence Microscopy.

To confirm the specificity of monoclonal antibodies used in this study for each EGFR, fluorescence immunocytochemistry was performed on stable cell lines: (a) U87 MG parental cells; (b) U87 MG.wtEGFR cells (EGFRwt overexpressed); and (c) U87 MG.ΔEGFR cells (EGFRvIII overexpressed) kindly gifted by Cavenee et al.(41, 63). The cells were grown on a 35-mm Petri dish, fixed with 4% paraformaldehyde in phosphate buffer (pH. 7.4) for 15 min at room temperature, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. The cells were blocked with 5% goat serum/PBS for 60 min at room temperature and then incubated with each primary antibody (1:300 dilution in 0.2% BSA/PBS) at room temperature for 60 min. The primary antibodies were visualized with fluorescein-conjugated goat anti-mouse IgG (1:300 dilution; Biosource, Camarillo, CA) for 45 min. The stained cells were mounted with 2.5% 1,4-diazabicyclo [2.2.2] octane/glycerol and analyzed under a fluorescent microscope (BX 52; Olympus Optical Co., Ltd., Japan).

Immunohistochemical Technique to Determine EGFR Expression.

Paraffin-embedded tissue sections (3 μm) were placed on glass slides and dried. After routine deparaffinization, rehydration, and blocking of endogenous peroxidase activity, microwave-enhanced antigen retrieval was performed (64). Slide-mounted sections immersed in 0.01 m sodium citrate buffer (pH 6.0) were placed for 15 min into a 700-W microwave oven at maximum power. After blocking nonspecific protein binding with 3% BSA/PBS, the sections were incubated with primary antibodies: EGFR.25 (dilution 1:100), EGFR.113 (dilution 1:100) at room temperature for 1 h, and DH8.3 (dilution 1:20) at 4°C overnight. In subsequent steps, we used the Vectastain ABC kit and 3,3′-diaminobenzidine as the chromogen (Vector, Burlingame, CA). The sections were lightly counterstained with hematoxylin. Positive and negative controls were included with each batch of sections to confirm the consistency of the analysis. Sections were examined for immunoreactivity of each EGFR by at least one independent neuropathologist who was unaware of the patients’ outcomes or clinical features. The membrane and/or cytoplasm of cells were typically stained for EGFR. EGFR expression was scored according to the intensity of staining and number of stained tumor cells as 0 (no staining), 1 (light or focal), 2 (moderate), and 3 (strong). For statistical analysis, scores of 0 or 1 were defined as no overexpression; scores of 2 and 3 as overexpression.

Clinical Details.

Clinical details, including the patient’s age at entry into the trial, gender, preoperative KPS score, extent of surgical resection, protocol number, and the recorded date of disease progression or death, were notated. The goal of the operation was to remove as much tumor as possible. Except for the deep-seated lesions, such as thalamus and basal ganglia, craniotomy and surgical resection were carried out. To identify the extent of resection, contrast-enhanced neuroimaging data, i.e., computed tomograms or magnetic resonance images, were obtained within 1–2 weeks; starting in 1994, these were obtained within 72 h of surgery to easily exclude the effect of time-lapse changes attributable to the surgical procedure (53, 65). GTR was recorded when there were no contrast-enhanced lesions, subtotal resection when <10% of the preoperatively contrast-enhanced lesion remained, and PR when ≥10% of the contrast-enhanced lesion was noted. Subtotal resection and PR were subsumed into the PR classification. When the lesion was deep seated and considered inaccessible for direct removal, biopsy was performed by stereotactic surgery techniques using the Leksell apparatus. To harvest diagnostic tissue specimens, we selected one or two targets in the enhanced lesions on 3-mm-thick contrast-enhanced magnetic resonance image.

Statistical Analysis.

For outcome analysis, patients were classified according to the presence or absence of EGFR amplification or EGFR overexpression. OS was calculated as the interval between trial entry and day of death attributable to tumor recurrence. Patients whose day of death was uncertain were censored on the last known day of life; patients alive on the day of analysis were censored on April 30, 2003. Other potential prognostic variables were age (≥55 versus <55 years), gender, surgery (GTR versus PR), preoperative KPS score (40–60 versus 70–100), and enrollment protocol (8701 versus 9501); there was no significant difference in survival time between the two treatment arms of the protocols (data not shown). The Log-rank test was used for univariate analysis to estimate differences in survival times for these variables. To plot survival curves, we used the Kaplan-Meier method. Using the Cox proportional hazards regression model, multivariate analysis was performed in a backward manner. Possible correlations between patient age and EGFR gene status/EGFR expression were based on the unpaired t test and the correlation between the EGFR gene status and EGFR expression score on the Mann-Whitney U test. All calculations were performed with commercially available software (Statview, Version 5.0; Abacus Concepts, Inc., Berkeley, CA). A probability value of <0.05 was considered statistically significant. This study was approved by The Committee for the Development of Advanced Medicine at Kumamoto University Hospital.

Assessment of Clinical Characteristics.

Table 1 shows the clinical characteristics of the 87 GBM study subjects. All 87 patients (54 males and 33 females; ratio 1.64:1) were Japanese. Their median age was 54 years (range 17–78 years); 45 patients (51.7%) were <55, 39 (44.8%) were from 55 to 69 years, and 3 (3.5%) were ≥70 years. The number of patients in protocol 8701, which enrolled subjects ≥70 years, was small (23 of 87; 26.4%). The median preoperative KPS score was 70 (range 40–100); 31 (35.6%) patients underwent GTR, 48 (55.2%) underwent PR. Because the number of biopsied patients (n = 8; 9.2%) was small, biopsy was subsumed into the PR classification in statistical analyses. The median OS was 1.262 (range 0.142–7.422) years. On the day of analysis, 74 patients (85.1%) were dead; 13 (14.9%) were alive and censored.

EGFR Gene Status.

To determine the cutoff value for EGFR amplification, the EGFR gene status of normal human brain tissue samples was examined. The normalized ratio of EGFR varied between >0.5 and <2 (data not shown); 2 was chosen as the threshold, and normalized ratios ≥2 were considered indicative of gene amplification. When we examined the EGFR gene status of tumor samples (Fig. 1), 40 (46%) of 87 patients manifested EGFR amplification with normalized ratios ranging from 2.1 to 75.5.

Specificities of Primary Antibodies.

To confirm the specificity of the primary antibodies for each EGFR, fluorescent immunocytochemistry was performed using the following cell lines: (a) U87 MG.parental cells; (b) U87 MG.wtEGFR cells whose cell surface overexpressed EGFRwt; and (c) U87 MG.ΔEGFR cells whose cell surface overexpressed EGFRvIII. Because U87 MG.parental cells express little endogenous EGFR, it is impossible to detect EGFR. However, it is possible to detect EGFR if cells with exogenous overexpression of EGFR are used and cells with EGFR overexpression are suitable for evaluation of the specificity of anti-EGFR antibodies. As shown in Fig. 2, EGFR.25 was reactive to the cell surface of both U87 MG.wtEGFR− and U87 MG.ΔEGFR cells (Fig. 2, D and G) but not U87 MG.parental cells (Fig. 2,A). On the other hand, EGFR.113 was reactive to the cell surface of only U87 MG.wtEGFR cells (Fig. 2,E); there was no immunoreactivity with either U87 MG.ΔEGFR− or U87 MG.parental cells (Fig. 2, B and H). As in other reports (60, 61, 62), the specificity of DH8.3 for U87 MG.ΔEGFR cells expressing EGFRvIII was confirmed (Fig. 2, C, F, and I). Therefore, we used EGFR.25 for the evaluation of EGFR, including EGFRwt and EGFRvIII, EGFR.113 for the evaluation of EGFRwt, and DH8.3 for the evaluation of EGFRvIII.

EGFR Expression.

Table 2 shows the correlation between the EGFR gene status and the EGFR expression level in 87 GBM patients. Of 40 patients with EGFR amplification, 39 (97.5%) manifested EGFR overexpression, i.e., scores ≥2, whereas 46 (97.9%) of 47 patients without EGFR amplification had no EGFR overexpression; there was a close correlation between the presence of the EGFR gene and EGFR expression (P < 0.0001). In one case (GB164) with EGFR amplification but no EGFR overexpression (expression score 1), DH8.3 revealed overexpression of EGFRvIII (Fig. 3, D–F). Of 40 patients with EGFR amplification, 27 (67.5%) manifested EGFRwt overexpression; no tumors without EGFR amplification overexpressed EGFRwt (P < 0.0001). EGFR.25 showed that all tumors with EGFRwt overexpression overexpressed EGFR (Fig. 3, A and B). Irrespective of the presence or absence of EGFR amplification, 70 of the 87 (80.5%) patients manifested EGFRvIII expression; overexpressed EGFRvIII was found in 18 (45%) of 40 patients with and 4 (8.5%) of 47 patients without EGFR amplification (P < 0.0001). There were 18 amplification-positive, EGFRvIII-overexpressing tumors; EGFR.25 revealed that all but one (GB164) manifested EGFR overexpression (Fig. 3, A and C); 4 amplification-negative tumors with EGFRvIII overexpression had EGFR expression scores of 1, i.e., no overexpression, by EGFR.25 (data not shown). As shown in Table 3, of 40 EGFR amplification-positive patients, 8 (20%) had neither EGFRwt nor EGFRvIII overexpression (Fig. 3, H and I); however, EGFR.25 showed that all 8 manifested overexpression of EGFR (Fig. 3 G).

Statistical Analysis.

We next examined the possibility of a correlation between patient age and the EGFR gene status/EGFR expression (Table 4). Although the mean age of patients with EGFR amplification or EGFR overexpression tended to be higher than that of patients without, no statistically significant difference was found (P = 0.085 versus 0.088).

The results of univariate analysis for OS are shown in Table 5. The median OS of patients with EGFR amplification was significantly shorter than in those without (1.199 versus 1.684 years, P = 0.007; Fig. 4 A). Similarly, compared with patients without overexpression, OS was significantly shorter in patients with overexpressed EGFRwt (P = 0.014). However, EGFRvIII overexpression did not have a significant negative impact on OS (P = 0.081). Higher age and a worse preoperative KPS also had a significant negative impact on OS (P = 0.0001, 0.041, respectively). Gender, the extent of surgery, and the protocol did not have a significant negative impact on OS (P = 0.42, P = 0.8, and P = 0.79). Gender and protocol were excluded as covariates in subsequent multivariate analysis.

To test the prognostic value of the EGFR gene status and of EGFR expression, we performed multivariate analysis for OS on the 87 GBM patients (Table 6). We found that EGFR amplification was an independent, significant, poor prognostic factor for OS (P = 0.038, HR = 1.67). The prognostic value of EGFRwt and EGFRvIII expression was not sufficient to reach statistical significance. Age was the only other independent significant predictor for OS (P = 0.001, HR = 2.26). KPS was eliminated as a significant variable, although it was an independent significant factor, unless EGFR was also included in this model as a covariate (data not shown).

We next tested the relationship of age to EGFR prognostication in GBM patients. As shown in Table 7, we chose the typical median age of 55 years and 60 years as the thresholds and divided the patients into two groups, i.e., an older versus a younger age group (patients ≥ 60 versus <60 years and patients ≥ 55 versus <55 years, respectively). Then, the prognostic significance of the EGFR gene status in individual groups was tested by univariate and multivariate analysis. Multivariate analysis by the Cox regression model in a backward manner included adjustments for KPS, surgery, EGFRwt, and EGFRvIII. In each younger age group, especially in patients < 60 years, EGFR gene amplification played a stronger role in survival than in all 87 patients (1.133 versus 2.324 years, P = 0.0002; Fig. 4 B). Similarly, by multivariate analysis, the prognostic significance of the EGFR gene status was more pronounced in each younger age group (P = 0.0054, HR = 2.72; P = 0.0003, HR = 3.15). On the other hand, in neither of the two older age groups was the EGFR gene status/EGFR expression of prognostic significance. With respect to other significant variables, KPS was the only independent significant variable in each older age group; there was no significant variable except for the EGFR gene status in each of the younger groups (data not shown). The prognostic value of EGFRvIII expression was not sufficient to reach statistical significance in any of the groups categorized by age.

To determine the clinical significance of EGFRvIII expression, further analysis was carried out. As shown in Fig. 4,C, of 40 patients with EGFR amplification, those with EGFRvIII overexpression manifested significantly shorter OS than did patients without (median OS 0.893 versus 1.374 years, P = 0.0031). In addition, multivariate analysis on the 40 amplification-positive patients revealed that EGFRvIII overexpression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71; Table 8). Age was eliminated as a significant variable.

There is experimental evidence that in GBM, EGFR amplification resulting in EGFR overexpression may signal an unfavorable prognosis. However, the results of clinical studies are currently inconclusive or inconsistent (6, 13, 18, 43, 44, 45, 46, 47, 48, 49, 50). Using the Log-rank test, Hurtt et al.(47) demonstrated that in supratentorial GBM, EGFR amplification was significantly associated with shorter survival. However, their study lacked confirmation by multivariate analysis of the prognostic value of the EGFR alteration. On the other hand, Waha et al.(13) reported that multivariate analysis attributed no statistical value to EGFR amplification in terms of the survival of patients with grade 2–4 astrocytic gliomas. It is not clear from their report whether patient populations with the different tumor grades were clinically uniform. In fact, Simmons et al.(43) suggested that differences in the studied patient populations may explain the divergence in reported results. For our multivariate analysis of the prognostic value of the EGFR gene/EGFR protein status, we selected a uniform population among Japanese GBM patients enrolled in clinical trials carried out at our institutions.

We found that the frequency of EGFR amplification/EGFR overexpression was consistent with data reported by others (12, 13, 14, 20, 21, 22, 23). As noted previously (12, 19), there was a close correlation between the EGFR gene status determined by Southern blots and the EGFR protein expression levels assessed by immunohistochemical analysis, especially when EGFR.25, which recognizes the intracellular domain of the EGFR molecule, was used (Table 2). Because full-length EGFR cDNA and EGFR.25 were able to widely detect EGFR, including EGFRwt and EGFRvIII, the gene status and expression level of EGFR was thought to be closely correlated. Interestingly, 8 (20%) of 40 GBM with EGFR amplification manifested neither EGFRwt nor EGFRvIII overexpression; however, EGFR.25 detected EGFR overexpression (Fig. 3, G–I; Table 3). The incidence of 20% was too high for attribution to differences in the affinity of the different antibodies we used. This observation led us to suspect that these eight tumors expressed EGFR type(s) other than EGFRwt and EGFRvIII. EGFR.25 recognizes 200 amino acids of the intracellular domain of the EGFR molecule excluding the conserved tyrosine kinase domain. This antibody is thought to recognize the receptor internalization domain and the kinase inhibitory domain that are located nearer to the COOH-terminal tails than the tyrosine kinase domain. Our finding suggests the existence of other types of mutations that conserve the cytoplasmic domain near the COOH-terminal tails reported by others (34, 35, 66). EGFR.25 revealed that in 1 of 47 (2.1%) amplification-negative tumors, there was a distributed pattern of EGFR-overexpressed regions with an expression score of 2. This minimal inconsistency between the gene status and protein expression level may be explicable by sampling errors attributable to regional heterogeneity in these tumors.

It has been documented that EGFR amplification/EGFR overexpression is significantly more frequent in older patients (26, 43, 46, 48). In our series, the mean age of patients with EGFR amplification and EGFR overexpression was 55.1 ± 10.1 years; however, their age was not significantly different from amplification- and overexpression-negative patients (Table 4). This may be attributable to the fact that only 3 of our 87 patients (3.5%) were ≥70 years (Table 1).

Simmons et al.(43), who also studied GBM patients enrolled in clinical trials, demonstrated that immunohistochemically confirmed EGFR overexpression was an independent, unfavorable prognostic factor in only a limited subgroup, i.e., a cohort whose age was less than the median age of 55 years of their study population and whose TP53 status was normal. On the other hand, our multivariate analysis confirmed that EGFR amplification was an independent, unfavorable predictor for survival in our study population (Table 6). However, we cannot rule out the possibility that our study carried an age bias because 96.5% of our patients (84 of 87) were <70 years. In fact, the median survival time for our 87 patients was 1.262 years, similar to that of patients enrolled in protocol 9501, which excluded patients ≥70 years (1.194 years, data not shown) and longer than that reported in other GBM series that included patients ≥70 years. We recognize that our study carries a bias for the younger group of GBM patients, and our results support the suggestion of Simmons et al.(43) that EGFR is most negatively prognostic in younger patients with GBM. In fact, in our patients younger than the typical median age of 55–60 years, the prognostic significance of the EGFR gene status was more pronounced than in all 87 patients; this was not true for the older age group (Fig. 4,B; Table 7).

In the small cohort of GBM patients studied by Feldkamp et al.(50), those with EGFRvIII-positive tumors appeared to have shorter survival periods than did those with EGFRvIII-negative tumors. However, no statistical documentation was presented. We carried out multivariate analysis and found that the predictive value of EGFRvIII overexpression for survival was not sufficient to reach statistical significance (Table 6). However, among our GBM patients with EGFR amplification, those manifesting EGFRvIII overexpression had significantly shorter survival periods than those who did not (Fig. 4,C). Multivariate analysis confirmed that EGFRvIII overexpression was an independent, unfavorable predictor for survival (Table 8). On the basis of the results presented here, we suggest that the overexpression of EGFRvIII in the presence of EGFR amplification is the strongest indicator of a poor survival prognosis.

It is controversial whether EGFRvIII occurs through alternative splicing or by gene rearrangements after amplification (33, 34, 35, 36, 37). In our series of 87 cases, 32 (36.8%) manifested EGFRvIII expression (scores ≥1) in the absence of EGFR amplification detected by Southern blots (Table 2). Although EGFR and TP53 are reportedly mutually exclusive in GBM (25), Okada et al.(67), who used fluorescence in situ hybridization, recently demonstrated that GBM with mutated TP53 frequently manifested EGFR gene amplification at the cellular level. It is possible that EGFRvIII occurs through gene rearrangements after low-level amplification of the EGFR gene in scattered cells and that lysate-based approaches, such as Southern blot analysis, fail to detect this phenomenon.

Our studies included only Japanese patients, i.e., a unique and racially homogeneous population. To develop targeted therapies against tumors expressing EGFR, we must have clinical evidence of the importance of the EGFR gene status/EGFR expression in racially diverse GBM patients. Our results are a step toward the development of therapies to treat GBM patients with EGFR amplification and/or EGFR overexpression. They also indicate that routine immunohistochemical studies that use combinations of antibodies are useful for assessing the EGFR expression status in GBM patients.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by Grant-in-Aid 11307025 for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by Grant 1961SC from the Pacific Rim Research Program of the University of California (San Francisco).

3

The abbreviations used are: GBM, glioblastoma multiforme; EGFR, epidermal growth factor receptor; EGFRwt, wild-type epidermal growth factor receptor; OS, overall survival; KPS, Karnofsky performance score; HR, hazards ratio; GTR, gross total resection; PR, partial resection; ACNU, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride; PAV, procarbazine, ACNU, vincristine.

Fig. 1.

EGFR gene amplification detected by Southern blot analysis in GBM tumor samples. Comparison of the signal intensity of a 6.1-kb EcoRI fragment of an EGFR gene and a 3-kb fragment of an α-tubulin gene in tumor samples (GB99, GB249, AA36, and GB37) and normal brain tissue samples (NB). The normalized ratio of the signal intensity of the EGFR fragment was determined by that of α-tubulin. The numbers at the bottom of the figure are the normalized ratios. EGFR amplification was found in GB99, GB249, and GB37.

Fig. 1.

EGFR gene amplification detected by Southern blot analysis in GBM tumor samples. Comparison of the signal intensity of a 6.1-kb EcoRI fragment of an EGFR gene and a 3-kb fragment of an α-tubulin gene in tumor samples (GB99, GB249, AA36, and GB37) and normal brain tissue samples (NB). The normalized ratio of the signal intensity of the EGFR fragment was determined by that of α-tubulin. The numbers at the bottom of the figure are the normalized ratios. EGFR amplification was found in GB99, GB249, and GB37.

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Fig. 2.

Immunoreactivity with EGFRwt and EGFRvIII of monoclonal antibodies EGFR.25, EGFR.113, and DH8.3 by fluorescent immunocytochemistry. Shown are cell lines U 87MG. parental cells (A–C), U87 MG.wtEGFR cells (D–F), and U87 MG.ΔEGFR cells (G–I). The cells were treated with the following monoclonal antibodies: EGFR.25 (left panels), EGFR.113 (center panels), and DH8.3 (right panels). EGFR.25 was reactive to both, U87 MG.wtEGFR cells whose cell surface overexpressed EGFRwt, and U87 MG.ΔEGFR cells whose cell surface overexpressed EGFRvIII. EGFR.113 was reactive to only U87 MG.wtEGFR cells, and DH8.3 was reactive to only U87 MG.ΔEGFR cells.

Fig. 2.

Immunoreactivity with EGFRwt and EGFRvIII of monoclonal antibodies EGFR.25, EGFR.113, and DH8.3 by fluorescent immunocytochemistry. Shown are cell lines U 87MG. parental cells (A–C), U87 MG.wtEGFR cells (D–F), and U87 MG.ΔEGFR cells (G–I). The cells were treated with the following monoclonal antibodies: EGFR.25 (left panels), EGFR.113 (center panels), and DH8.3 (right panels). EGFR.25 was reactive to both, U87 MG.wtEGFR cells whose cell surface overexpressed EGFRwt, and U87 MG.ΔEGFR cells whose cell surface overexpressed EGFRvIII. EGFR.113 was reactive to only U87 MG.wtEGFR cells, and DH8.3 was reactive to only U87 MG.ΔEGFR cells.

Close modal
Fig. 3.

Expression patterns of each EGFR in three cases with EGFR amplification. Shown are three illustrative cases with EGFR amplification. The expression of EGFR, EGFRwt, and EGFRvIII is shown in the left, center, and right panels, respectively. GB249 (A–C), the same case as shown in Fig. 1 manifested overexpression of EGFR (A, expression score 3), EGFRwt (B, expression score 3), and EGFRvIII (C, expression score 2) in the same area. GB164 (D–F) manifested neither overexpression of EGFR (D, expression score 1) nor EGFRwt (E, expression score 0). However, there was overexpression of EGFRvIII (F) in the same area (expression score 2). GB254 (G–I) manifested overexpression of neither EGFRwt (H, expression score 1) nor EGFRvIII (I, expression score 1). However, there was overexpression of EGFR (G, expression score 3) in the same area. Each original magnification: ×100.

Fig. 3.

Expression patterns of each EGFR in three cases with EGFR amplification. Shown are three illustrative cases with EGFR amplification. The expression of EGFR, EGFRwt, and EGFRvIII is shown in the left, center, and right panels, respectively. GB249 (A–C), the same case as shown in Fig. 1 manifested overexpression of EGFR (A, expression score 3), EGFRwt (B, expression score 3), and EGFRvIII (C, expression score 2) in the same area. GB164 (D–F) manifested neither overexpression of EGFR (D, expression score 1) nor EGFRwt (E, expression score 0). However, there was overexpression of EGFRvIII (F) in the same area (expression score 2). GB254 (G–I) manifested overexpression of neither EGFRwt (H, expression score 1) nor EGFRvIII (I, expression score 1). However, there was overexpression of EGFR (G, expression score 3) in the same area. Each original magnification: ×100.

Close modal
Fig. 4.

OS according to EGFR gene status and EGFRvIII expression in 87 patients with GBM. Shown are Kaplan-Meier survival curves of OS for all 87 patients, irrespective of the presence or absence of amplification (A), 60 patients < 60 years, irrespective of the presence or absence of amplification (B), and 40 amplification-positive patients with or without EGFRvIII overexpression (C). Among all 87 patients, those with EGFR amplification (n = 40, solid line) had significantly shorter survival periods than did patients without (n = 47, dotted line; A, P = 0.007). Among 60 patients < 60 years, those with EGFR amplification (n = 30, solid line) had significantly shorter survival periods than did patients without (n = 30, dotted line; B, P = 0.0002). Among 40 patients with EGFR amplification, those with EGFRvIII overexpression (n = 18, solid line) had significantly shorter survival periods than did patients without (n = 22, dotted line; C, P = 0.0031).

Fig. 4.

OS according to EGFR gene status and EGFRvIII expression in 87 patients with GBM. Shown are Kaplan-Meier survival curves of OS for all 87 patients, irrespective of the presence or absence of amplification (A), 60 patients < 60 years, irrespective of the presence or absence of amplification (B), and 40 amplification-positive patients with or without EGFRvIII overexpression (C). Among all 87 patients, those with EGFR amplification (n = 40, solid line) had significantly shorter survival periods than did patients without (n = 47, dotted line; A, P = 0.007). Among 60 patients < 60 years, those with EGFR amplification (n = 30, solid line) had significantly shorter survival periods than did patients without (n = 30, dotted line; B, P = 0.0002). Among 40 patients with EGFR amplification, those with EGFRvIII overexpression (n = 18, solid line) had significantly shorter survival periods than did patients without (n = 22, dotted line; C, P = 0.0031).

Close modal
Table 1

Clinical characteristics of 87 GBM patients

Median age yrs (range) 54 (17–78) 
No. (%)  
<55 45 (51.7) 
55–69 39 (44.8) 
≥70 3 (3.5) 
Median KPS  
Score (range) 70 (40–100) 
Gender  
Male/female ratio 1.64/1 
Surgery no. (%)  
GTR 31 (35.6) 
PR 48 (55.2) 
Biopsy 8 (9.2) 
Protocol no. (%)  
8701 (n = 23)  
IA-ACNUa 10 (11.5) 
IV-ACNUb 13 (14.9) 
9501 (n = 64)  
PAV 34 (39.1) 
PAV plus IFN-β 30 (34.5) 
OS  
No. of censored (%) 13 (14.9) 
Median, years (range) 1.262 (0.142–7.422) 
Median age yrs (range) 54 (17–78) 
No. (%)  
<55 45 (51.7) 
55–69 39 (44.8) 
≥70 3 (3.5) 
Median KPS  
Score (range) 70 (40–100) 
Gender  
Male/female ratio 1.64/1 
Surgery no. (%)  
GTR 31 (35.6) 
PR 48 (55.2) 
Biopsy 8 (9.2) 
Protocol no. (%)  
8701 (n = 23)  
IA-ACNUa 10 (11.5) 
IV-ACNUb 13 (14.9) 
9501 (n = 64)  
PAV 34 (39.1) 
PAV plus IFN-β 30 (34.5) 
OS  
No. of censored (%) 13 (14.9) 
Median, years (range) 1.262 (0.142–7.422) 
a

intra-arterial ACNU.

b

intra-venous ACNU.

Table 2

Correlation between EGFR gene status and EGFR expression score in 87 patients with GBM

EGFR scoreEGFR gene statusP
Amplification (n = 40)No amplification (n = 47)
EGFR   <0.0001 
0 (0)a 34.1 (16)  
2.5 (1) 63.8 (30)  
20.0 (8) 2.1 (1)  
77.5 (31) 0 (0)  
EGFRwt   <0.0001 
20.0 (8) 63.8 (30)  
12.5 (5) 36.2 (17)  
27.5 (11) 0 (0)  
40.0 (16) 0 (0)  
EGFRvIII   <0.0001 
5.0 (2) 31.9 (15)  
50.0 (20) 59.6 (28)  
32.5 (13) 8.5 (4)  
12.5 (5) 0 (0)  
EGFR scoreEGFR gene statusP
Amplification (n = 40)No amplification (n = 47)
EGFR   <0.0001 
0 (0)a 34.1 (16)  
2.5 (1) 63.8 (30)  
20.0 (8) 2.1 (1)  
77.5 (31) 0 (0)  
EGFRwt   <0.0001 
20.0 (8) 63.8 (30)  
12.5 (5) 36.2 (17)  
27.5 (11) 0 (0)  
40.0 (16) 0 (0)  
EGFRvIII   <0.0001 
5.0 (2) 31.9 (15)  
50.0 (20) 59.6 (28)  
32.5 (13) 8.5 (4)  
12.5 (5) 0 (0)  
a

Percentage with raw numbers in parenthesis.

Table 3

Correlation between EGFRwt and EGFRvIII expression in 40 GBM patients with EGFR amplification

EGFRwtEGFRvIII
+ab
32.5 (13)c 35.0 (14) 
− 12.5 (5) 20.0 (8) 
EGFRwtEGFRvIII
+ab
32.5 (13)c 35.0 (14) 
− 12.5 (5) 20.0 (8) 
a

Overexpression.

b

No overexpression.

c

Percentage with raw numbers in parenthesis.

Table 4

Correlation between age and EGFR gene status/EGFR expression in 87 GBM patients

Mean age (yrs ± SD)P
EGFR gene status  0.085 
Amplification 55.1 ± 10.1  
No amplification 49.9 ± 16.2  
EGFR  0.088 
Overexpression 55.1 ± 10.0  
No overexpression 50.0 ± 16.3  
EGFRwt  0.13 
Overexpression 55.7 ± 8.37  
No overexpression 50.8 ± 15.6  
EGFRvIII  0.14 
Overexpression 56.1 ± 13.8  
No overexpression 51.0 ± 13.8  
Mean age (yrs ± SD)P
EGFR gene status  0.085 
Amplification 55.1 ± 10.1  
No amplification 49.9 ± 16.2  
EGFR  0.088 
Overexpression 55.1 ± 10.0  
No overexpression 50.0 ± 16.3  
EGFRwt  0.13 
Overexpression 55.7 ± 8.37  
No overexpression 50.8 ± 15.6  
EGFRvIII  0.14 
Overexpression 56.1 ± 13.8  
No overexpression 51.0 ± 13.8  
Table 5

Univariate analysis for OS in 87 GBM patients

VariableNo. (%)Median OS (yrs)P
Age (yrs)   0.0001 
≥55 42 (48.3) 0.920  
<55 45 (51.7) 1.621  
Gender   0.42 
Male 54 (62.1) 1.336  
Female 33 (37.9) 1.361  
KPS   0.041 
70–100 62 (71.3) 1.418  
40–60 25 (28.7) 0.958  
Surgery   0.80 
GTR 31 (35.6) 1.366  
PR 56 (64.4) 1.194  
Protocol   0.79 
8701 23 (26.4) 1.232  
9501 64 (73.6) 1.366  
EGFR gene status   0.0070 
Amplification 40 (46.0) 1.199  
No amplification 47 (54.0) 1.684  
EGFRwt   0.014 
Overexpression 27 (31.0) 1.342  
No overexpression 60 (69.0) 1.336  
EGFRvIII   0.081 
Overexpression 22 (25.3) 0.966  
No overexpression 65 (74.7) 1.394  
VariableNo. (%)Median OS (yrs)P
Age (yrs)   0.0001 
≥55 42 (48.3) 0.920  
<55 45 (51.7) 1.621  
Gender   0.42 
Male 54 (62.1) 1.336  
Female 33 (37.9) 1.361  
KPS   0.041 
70–100 62 (71.3) 1.418  
40–60 25 (28.7) 0.958  
Surgery   0.80 
GTR 31 (35.6) 1.366  
PR 56 (64.4) 1.194  
Protocol   0.79 
8701 23 (26.4) 1.232  
9501 64 (73.6) 1.366  
EGFR gene status   0.0070 
Amplification 40 (46.0) 1.199  
No amplification 47 (54.0) 1.684  
EGFRwt   0.014 
Overexpression 27 (31.0) 1.342  
No overexpression 60 (69.0) 1.336  
EGFRvIII   0.081 
Overexpression 22 (25.3) 0.966  
No overexpression 65 (74.7) 1.394  
Table 6

Multivariate analysis by the Cox proportional hazard regression model in a backward manner in 87 GBM patients

VariableHR (95% CI)aP
Age (yrs)   
≥55 vs. <55 2.26 (1.40–3.67) 0.0010 
KPS   
40–60 vs. 70–100  nsb 
Surgery   
GTR vs. PR  ns 
EGFR gene status   
Amplification vs. no amplification 1.67 (1.03–2.72) 0.038 
EGFRwt   
Overexpression vs. no overexpression  ns 
EGFRvIII   
Overexpression vs. no overexpression  ns 
VariableHR (95% CI)aP
Age (yrs)   
≥55 vs. <55 2.26 (1.40–3.67) 0.0010 
KPS   
40–60 vs. 70–100  nsb 
Surgery   
GTR vs. PR  ns 
EGFR gene status   
Amplification vs. no amplification 1.67 (1.03–2.72) 0.038 
EGFRwt   
Overexpression vs. no overexpression  ns 
EGFRvIII   
Overexpression vs. no overexpression  ns 
a

CI, confidence interval.

b

ns, not significant.

Table 7

Prognostic value of EGFR gene status by univariate and multivariate analysis in older vs. younger groups according to age thresholds

Age groupMedian OS, yrs (no. of cases)Univariate PMultivariate (Amp. vs. no amp.)
Amp.aNo amp.bHR (95% CI)dP
55 yrsc      
Older (n = 42) 0.931 (21) 0.901 (21) 0.71  nse 
Younger (n = 45) 1.374 (19) 2.324 (26) 0.0040 2.72 (1.34–5.50) 0.0054 
60 yrsc      
Older (n = 27) 1.262 (10) 0.865 (17) 0.53  ns 
Younger (n = 60) 1.133 (30) 2.324 (30) 0.0002 3.15 (1.69–5.89) 0.0003 
Age groupMedian OS, yrs (no. of cases)Univariate PMultivariate (Amp. vs. no amp.)
Amp.aNo amp.bHR (95% CI)dP
55 yrsc      
Older (n = 42) 0.931 (21) 0.901 (21) 0.71  nse 
Younger (n = 45) 1.374 (19) 2.324 (26) 0.0040 2.72 (1.34–5.50) 0.0054 
60 yrsc      
Older (n = 27) 1.262 (10) 0.865 (17) 0.53  ns 
Younger (n = 60) 1.133 (30) 2.324 (30) 0.0002 3.15 (1.69–5.89) 0.0003 
a

Amplification.

b

No amplification.

c

Age threshold.

d

CI, confidence interval.

e

ns, not significant.

Table 8

Multivariate analysis by the Cox proportional hazard regression model in a backward manner in 40 GBM patients with EGFR amplification

VariableHR (95% CI)aP
Age (yrs)   
≥55 vs. <55  nsb 
KPS   
40–60 vs. 70–100  ns 
Surgery   
GTR vs. PR  ns 
EGFRwt   
Overexpression vs. no overexpression  ns 
EGFRvIII   
Overexpression vs. no overexpression 2.71 (1.36–5.39) 0.0044 
VariableHR (95% CI)aP
Age (yrs)   
≥55 vs. <55  nsb 
KPS   
40–60 vs. 70–100  ns 
Surgery   
GTR vs. PR  ns 
EGFRwt   
Overexpression vs. no overexpression  ns 
EGFRvIII   
Overexpression vs. no overexpression 2.71 (1.36–5.39) 0.0044 
a

CI, confidence interval.

b

ns, not significant.

We thank Dr. M. Shibuya, Institute of Medical Science, University of Tokyo, Dr. K. Ueki, Department of Neurosurgery, Dokkyo University School of Medicine, and Dr. R. Nishikawa, Department of Neurosurgery, Saitama Medical School, for the gift of full-length human EGFR cDNA; Dr. M. Nagane, Department of Neurosurgery, Kyorin University School of Medicine, Dr. K. Mishima, Department of Neurosurgery, Saitama Medical School, and Dr. Webster K. Cavenee, Ludwig Institute for Cancer Research, University of California at San Diego, for the gift of the cell lines U87 MG, U87 MG.wtEGFR, and U87 MG.ΔEGFR. We also thank Dr. K. Semba, Division of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, for technical assistance in gene analysis; Dr. D. Murakami, Tumor Genetics and Biology, Kumamoto University School of Medicine, for technical assistance in fluorescence immunocytochemistry; and Masayo Obata for technical assistance in immunohistochemistry.

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