| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Departments of Neurosurgery [N. S., K. T., S. S., T. K., M. K., H. N., K. M., Y. U.] and Tumor Genetics and Biology [H. S.], Kumamoto University School of Medicine, Kumamoto 860-8556 and 860-0811; Neurosurgery, Kagoshima University School of Medicine, Kagoshima 890-8520 [H. H., J-i. K.]; Neurosurgery, Nakamura Memorial Hospital, Sapporo 060-8570 [K. O.]; and Division of Pathology, Kumamoto University College of Medical Science, Kumamoto 862-0976 [Y. I.], Japan
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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 
EGFR 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. |
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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, 
2 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, 2 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; 2 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 
-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 [5x 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 -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.EGFR 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.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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.
|
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.
|
EGFR 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.EGFR cells (Fig. 2, D and G)
but not U87 MG.parental cells (Fig. 2A)
. On the other hand, EGFR.113 was reactive to the cell surface of only U87 MG.wtEGFR cells (Fig. 2E)
; there was no immunoreactivity with either U87 MG.EGFR- 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.EGFR 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.
|
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. 3G)
.
|
|
|
. 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 median OS of patients with EGFR amplification was significantly shorter than in those without (1.199 versus 1.684 years, P = 0.007; Fig. 4A
). 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.
|
|
. 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 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. 4B
). 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.
|
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.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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. 4B
; 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. 4C)
. 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.
|
ACKNOWLEDGMENTS |
|---|
EGFR. 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. |
FOOTNOTES |
|---|
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). 
2 To whom requests for reprints should be addressed, at Department of Neurosurgery, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Phone: 81-96-373-5219; Fax: 81-96-371-8064; E-mail: 008m9020{at}med.stud.kumamoto-u.ac.jp
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.
Received 1/15/03. Revised 7/14/03. Accepted 7/24/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, and epidermal growth factor and their expression in human gliomas in vivo.. Cancer Res., 51: 2164-2172, 1991.This article has been cited by other articles:
|
|
 |
|
  U. Rajcevic, K. Petersen, J. C. Knol, M. Loos, S. Bougnaud, O. Klychnikov, K. W. Li, T. V. Pham, J. Wang, H. Miletic, et al. iTRAQ-based Proteomics Profiling Reveals Increased Metabolic Activity and Cellular Cross-talk in Angiogenic Compared with Invasive Glioblastoma Phenotype Mol. Cell. Proteomics, November 1, 2009; 8(11): 2595 - 2612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. Jane, D. R. Premkumar, S. O. Addo-Yobo, and I. F. Pollack Abrogation of Mitogen-Activated Protein Kinase and Akt Signaling by Vandetanib Synergistically Potentiates Histone Deacetylase Inhibitor-Induced Apoptosis in Human Glioma Cells J. Pharmacol. Exp. Ther., October 1, 2009; 331(1): 327 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Neyns, J. Sadones, E. Joosens, F. Bouttens, L. Verbeke, J.-F. Baurain, L. D'Hondt, T. Strauven, C. Chaskis, P. In't Veld, et al. Stratified phase II trial of cetuximab in patients with recurrent high-grade glioma Ann. Onc., September 1, 2009; 20(9): 1596 - 1603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mahajan and R. A. Prayson Repeated Molecular Testing in Gliomas: A Retrospective Study of 53 Cases Am J Clin Pathol, July 1, 2009; 132(1): 118 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Karpel-Massler, U. Schmidt, A. Unterberg, and M.-E. Halatsch Therapeutic Inhibition of the Epidermal Growth Factor Receptor in High-Grade Gliomas: Where Do We Stand? Mol. Cancer Res., July 1, 2009; 7(7): 1000 - 1012. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Okamura, S. Singh, J. Buolamwini, T. Haystead, H. Friedman, D. Bigner, and F. Ali-Osman Tyrosine Phosphorylation of the Human Glutathione S-Transferase P1 by Epidermal Growth Factor Receptor J. Biol. Chem., June 19, 2009; 284(25): 16979 - 16989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-S. Guillamo, S. de Bouard, S. Valable, L. Marteau, P. Leuraud, Y. Marie, M.-F. Poupon, J.-J. Parienti, E. Raymond, and M. Peschanski Molecular Mechanisms Underlying Effects of Epidermal Growth Factor Receptor Inhibition on Invasion, Proliferation, and Angiogenesis in Experimental Glioma Clin. Cancer Res., June 1, 2009; 15(11): 3697 - 3704. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. A. Kohutek, C. G. diPierro, G. T. Redpath, and I. M. Hussaini ADAM-10-Mediated N-Cadherin Cleavage Is Protein Kinase C-{alpha} Dependent and Promotes Glioblastoma Cell Migration J. Neurosci., April 8, 2009; 29(14): 4605 - 4615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M.-E. Sauvageot, J. L. Weatherbee, S. Kesari, S. E. Winters, J. Barnes, J. Dellagatta, N. R. Ramakrishna, C. D. Stiles, A. L.-J. Kung, M. W. Kieran, et al. Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells Neuro-oncol, January 1, 2009; 11(2): 109 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. J. Becher, K. M. Peterson, S. Khatua, M. R. Santi, and T. J. MacDonald IGFBP2 Is Overexpressed by Pediatric Malignant Astrocytomas and Induces the Repair Enzyme DNA-PK J Child Neurol, October 1, 2008; 23(10): 1205 - 1213. [Abstract] [PDF]
|
|
|
|
 |
|
 H.-D. Kim, T. W. Guo, A. P. Wu, A. Wells, F. B. Gertler, and D. A. Lauffenburger Epidermal Growth Factor-induced Enhancement of Glioblastoma Cell Migration in 3D Arises from an Intrinsic Increase in Speed But an Extrinsic Matrix- and Proteolysis-dependent Increase in Persistence Mol. Biol. Cell, October 1, 2008; 19(10): 4249 - 4259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C M West, L Joseph, and S Bhana Epidermal growth factor receptor-targeted therapy Br. J. Radiol., October 1, 2008; 81(Special_Issue_1): S36 - S44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. P. Mason and J. G. Cairncross Invited Article: The expanding impact of molecular biology on the diagnosis and treatment of gliomas Neurology, July 29, 2008; 71(5): 365 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Brandes, E. Franceschi, A. Tosoni, M. E. Hegi, and R. Stupp Epidermal Growth Factor Receptor Inhibitors in Neuro-oncology: Hopes and Disappointments Clin. Cancer Res., February 15, 2008; 14(4): 957 - 960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Schlomm, P. Kirstein, L. Iwers, B. Daniel, T. Steuber, J. Walz, F. H.K. Chun, A. Haese, J. Kollermann, M. Graefen, et al. Clinical Significance of Epidermal Growth Factor Receptor Protein Overexpression and Gene Copy Number Gains in Prostate Cancer Clin. Cancer Res., November 15, 2007; 13(22): 6579 - 6584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M.P. Omuro, S. Faivre, and E. Raymond Lessons learned in the development of targeted therapy for malignant gliomas Mol. Cancer Ther., July 1, 2007; 6(7): 1909 - 1919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Barnett, D. L. Urbauer, G. I. Murray, G. N. Fuller, and A. B. Heimberger Cytochrome P450 1B1 Expression in Glial Cell Tumors: An Immunotherapeutic Target Clin. Cancer Res., June 15, 2007; 13(12): 3559 - 3567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Pelloski, K. V. Ballman, A. F. Furth, L. Zhang, E. Lin, E. P. Sulman, K. Bhat, J. M. McDonald, W.K. A. Yung, H. Colman, et al. Epidermal Growth Factor Receptor Variant III Status Defines Clinically Distinct Subtypes of Glioblastoma J. Clin. Oncol., June 1, 2007; 25(16): 2288 - 2294. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Sok, F. M. Coppelli, S. M. Thomas, M. N. Lango, S. Xi, J. L. Hunt, M. L. Freilino, M. W. Graner, C. J. Wikstrand, D. D. Bigner, et al. Mutant Epidermal Growth Factor Receptor (EGFRvIII) Contributes to Head and Neck Cancer Growth and Resistance to EGFR Targeting Clin. Cancer Res., September 1, 2006; 12(17): 5064 - 5073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Wei, L. Clarke, D. K. Scheidenhelm, B. Qian, A. Tong, N. Sabha, Z. Karim, N. A. Bock, R. Reti, R. Swoboda, et al. High-grade glioma formation results from postnatal pten loss or mutant epidermal growth factor receptor expression in a transgenic mouse glioma model. Cancer Res., August 1, 2006; 66(15): 7429 - 7437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ji, X. Zhao, Y. Yuza, T. Shimamura, D. Li, A. Protopopov, B. L. Jung, K. McNamara, H. Xia, K. A. Glatt, et al. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors PNAS, May 16, 2006; 103(20): 7817 - 7822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Ramnarain, S. Park, D. Y. Lee, K. J. Hatanpaa, S. O. Scoggin, H. Otu, T. A. Libermann, J. M. Raisanen, R. Ashfaq, E. T. Wong, et al. Differential Gene Expression Analysis Reveals Generation of an Autocrine Loop by a Mutant Epidermal Growth Factor Receptor in Glioma Cells Cancer Res., January 15, 2006; 66(2): 867 - 874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Aghi, P. Gaviani, J. W. Henson, T. T. Batchelor, D. N. Louis, and F. G. Barker II Magnetic Resonance Imaging Characteristics Predict Epidermal Growth Factor Receptor Amplification Status in Glioblastoma Clin. Cancer Res., December 15, 2005; 11(24): 8600 - 8605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Van Schaeybroeck, A. Karaiskou-McCaul, D. Kelly, D. Longley, L. Galligan, E. Van Cutsem, and P. Johnston Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy Clin. Cancer Res., October 15, 2005; 11(20): 7480 - 7489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-H. Zhou, K. R. Hess, L. Liu, Mark. E. Linskey, and W.K. A. Yung Modeling prognosis for patients with malignant astrocytic gliomas: Quantifying the expression of multiple genetic markers and clinical variables Neuro-oncol, October 1, 2005; 7(4): 485 - 494. [Abstract] [PDF]
|
|
|
|
|
|
V. Damiano, D. Melisi, C. Bianco, D. Raben, R. Caputo, G. Fontanini, R. Bianco, A. Ryan, A. R. Bianco, S. De Placido, et al. Cooperative Antitumor Effect of Multitargeted Kinase Inhibitor ZD6474 and Ionizing Radiation in Glioblastoma Clin. Cancer Res., August 1, 2005; 11(15): 5639 - 5644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Heimberger, R. Hlatky, D. Suki, D. Yang, J. Weinberg, M. Gilbert, R. Sawaya, and K. Aldape Prognostic Effect of Epidermal Growth Factor Receptor and EGFRvIII in Glioblastoma Multiforme Patients Clin. Cancer Res., February 15, 2005; 11(4): 1462 - 1466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Blum, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, and Y. Kloog Ras Inhibition in Glioblastoma Down-regulates Hypoxia-Inducible Factor-1{alpha}, Causing Glycolysis Shutdown and Cell Death Cancer Res., February 1, 2005; 65(3): 999 - 1006. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Heimberger, E. C. McGary, D. Suki, M. Ruiz, H. Wang, G. N. Fuller, and M. Bar-Eli Loss of the AP-2{alpha} Transcription Factor Is Associated with the Grade of Human Gliomas Clin. Cancer Res., January 1, 2005; 11(1): 267 - 272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nakahara, T. Shiraishi, H. Okamoto, T. Mineta, T. Oishi, K. Sasaki, and K. Tabuchi Detrended fluctuation analysis of genome-wide copy number profiles of glioblastomas using array-based comparative genomic hybridization Neuro-oncol, October 1, 2004; 6(4): 281 - 289. [Abstract] [PDF]
|
|
|
|
|
|
H. Ohgaki, P. Dessen, B. Jourde, S. Horstmann, T. Nishikawa, P.-L. Di Patre, C. Burkhard, D. Schuler, N. M. Probst-Hensch, P. C. Maiorka, et al. Genetic Pathways to Glioblastoma: A Population-Based Study Cancer Res., October 1, 2004; 64(19): 6892 - 6899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Steinbach, A. Klumpp, H. Wolburg, and M. Weller Inhibition of Epidermal Growth Factor Receptor Signaling Protects Human Malignant Glioma Cells from Hypoxia-Induced Cell Death Cancer Res., March 1, 2004; 64(5): 1575 - 1578. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
