Expression of EGFRvIII in glioblastoma: prognostic significance revisited

Abstract

The epidermal growth factor receptor variant III (EGFRvIII) is associated with increased proliferation of glioma cells. However, the impact of EGFRvIII on survival of patients with glioblastoma (GBM) has not been definitively established. In the present study, we prospectively evaluated 73 patients with primary GBM treated with surgical resection and standard radio/chemotherapy. The EGFRvIII was assessed by reverse transcription-polymerase chain reaction (PCR), O(6)-methylguanine methyltransferase (MGMT) promoter methylation was assessed by methylation-specific PCR, and phosphatase and tension homolog (PTEN) expression was assessed by immunohistochemistry. In 14 patients of this series, who presented with tumor recurrence, EGFRvIII was determined by real-time PCR. Sensitivity to temozolomide (TMZ) was assessed in vitro on GBM neurosphere cell cultures with different patterns of EGFRvIII expression. Age 60 years or younger, preoperative Karnofsky Performance Status score of 70 or higher, recursive partitioning analysis score III and IV, methylated MGMT, and Ki67 index of 20% or less were significantly associated with longer overall survival (OS; P = .0069, P = .0035, P = .0007, P = .0437, and P = .0286, respectively). EGFRvIII identified patients with significantly longer OS (P = .0023) and the association of EGFRvIII/Ki67 of 20% or less, EGFRvIII/normal PTEN, EGFRvIII/methylated MGMT, and EGFRvIII/normal PTEN/methylated MGMT identified subgroups of GBM patients with better prognosis. In recurred GBMs, EGFRvIII expression was approximately two-fold lower than in primary tumors. In vitro, the EGFRvIII-negative GBM neurosphere cells were more resistant to TMZ than the positive ones. In conclusion, in contrast with previous studies, we found that EGFRvIII is associated with prolonged survival of GBM patients treated with surgery and radio/chemotherapy. Depletion of EGFRvIII in recurrent GBMs as well as differential sensitivity to TMZ in vitro indicates that the EGFRvIII-negative cell fraction is involved in resistance to radio/chemotherapy and tumor repopulation.

Figure 1

Kaplan-Meier survival curves of 73 GBM patients stratified by EGFRvIII, MGMT, and Ki67. The presence of EGFRvIII in tumors (A, blue line), methylated MGMT (B, blue line), and Ki67 index of 20%or less (C, blue line), conferred a favorable survival advantage (P =.0023, P = .0437, and P = .0286, respectively).

Figure 2

Kaplan-Meier survival curves of 73 GBM patients stratified by EGFRvIII/Ki67, EGFRvIII/PTEN, EGFRvIII/MGMT methylation, and MGMT methylation/PTEN/EGFRvIII. (A) Patients stratified by EGFRvIII/Ki67. EGFRvIII-positive GBMs and Ki67 index of 20% or less (blue line) was related with longer OS (P = .0275). There were no differences in survival times between the following three unfavorable groups: EGFRvIII+/Ki67 greater than 20% (gray line), EGFRvIII-/Ki67 20% or less (yellow line), and EGFRvIII-/Ki67 greater than 20% (red line). (B) Patients stratified by EGFRvIII/MGMT methylation. EGFRvIII-positive GBMs, and methylated MGMT (blue line) was related with longer OS (P = .0108). There were no differences in survival times between the following three unfavorable groups: EGFRvIII+/unmethylated MGMT (gray line), EGFRvIII-/methylated MGMT (yellow line), and EGFRvIII-/unmethylated MGMT (red line). (C) Patients stratified by EGFRvIII/PTEN. The presence of EGFRvIII and normal expression of PTEN (blue line) was associated with longer OS (P = .0223). There were no differences in survival times between the following three unfavorable groups: EGFRvIII+/hypoexpression of PTEN (-) (gray line), EGFRvIII-/PTEN- (yellow line), and EGFRvIII-/PTEN+ (red line). (D) Patients stratified by MGMT methylation/PTEN/EGFRvIII. The association of methylated MGMT, normal expression of PTEN, and presence of EGFRvIII (blue line) was associated with longer OS (P = .004).

Figure 3

Expression of EGFRvIII in recurrent GBM. (A) Box-and-whisker plots showing significant reduction of EGFRvIIImRNA levels in recurrent GBM in comparison to the primary tumors (Wilcoxon signed rank test). (B)Graph showing the relationship between survival after tumor recurrence and level of EGFRvIII mRNA expression in the recurrent tumor (Spearman correlation coefficient).

Figure 4

Cell viability percentages of GBM neurosphere cells with different patterns of EGFRvIII expression, MGMT methylation status, and PTEN expression (Table W4) treated for 24, 48, 72, and 96 hours with TMZ at 125, 250, and 500 µM. Each experiment was repeated three times, and mean values were plotted. (A) Cell survival curves comparing sensitivity to TMZ of cell line 1A (blue; EGFRvIII-/MGMT unmethylated/PTEN+) versus cell line 1B (orange; EGFRvIII+/MGMT unmethylated/PTEN+). (B) Semiquantitative RT-PCR analysis of Bcl-XL mRNA expression by the EGFRvIII-positive cell line 1B and by EGFRvIII-negative cell line 1A at 0, 48, and 72 hours exposure to 500 µM TMZ in culture. Upper panel, representative experiment (MW, molecular weight; -, negative control). Lower panel, semi-quantitative analysis of Bcl-xL mRNA expression from three different experiments in EGFR-positive (orange) and in EGFR-negative (blue) neurosphere cell line. (C) Cell survival curves comparing sensitivity to TMZ of cell line 2A (orange; EGFRvIII+/MGMT methylated/PTEN+) versus cell line 2B (blue; EGFRvIII-/MGMT methylated/PTEN+). (D) Cell survival curves comparing sensitivity to TMZ of cell line 3A (light blue; EGFRvIII-/MGMT methylated/PTEN-) versus cell line 3B (dark blue; EGFRvIII-/MGMT unmethylated/PTEN-).