Abstract
Aim: To analyze the correlation between epidermal growth factor receptor (EGFR) expression and clinicoradiological features in patients with glioblastoma. Patients and Methods: Select clinical (age, Karnofsky performance status, neurological performance status) and neuroradiological (multiple tumor incidence, peritumoral edema, response to treatment) prognostic variables were correlated with EGFR expression levels estimated on pathological samples in 144 patients who had undergone postoperative radiochemotherapy treatment. Results: Clinical and radiological prognostic parameters, including synchronous multiple tumor, extent of edema, and radiological response after therapy, showed a statistically significant correlation with the EGFR expression score. Patients with a high EGFR expression seemed to present worse cliniconeurological status and radiological features of tumor aggressiveness (higher incidence of multiple tumor, major extent of peritumoral edema and poorer radiological response to treatment). Conclusion: A potential correlation between high EGFR expression and aggressive clinical and radiological features of GB seems to exist, leading to worse prognosis in these patients.
Glioblastoma (GB) is the most frequent brain malignancy and remains lethal in almost all patients, despite the improvement in knowledge of biological and molecular features (1, 2), and in treatment modalities (3).
Active proliferation, invasiveness, and angiogenesis are the main characteristics of GB. The amplification of epidermal growth factor receptor (EGFR), resulting in mRNA and protein overexpression of this gene, is found in 50-60% of all GBs. (4, 5). An abnormal activity of EGFR seems to play a key role in the development of the neoplastic phenotype, influencing cell survival, proliferation, migration and invasion processes (6, 7); the extreme aggressiveness of GB appears to be related to these features. Pre-clinical research in cellular (8, 9) and animal models (10, 11) deals mostly with tumor development, whereas clinical studies primarily address the role of EGFR as a prognostic marker (12-14), although correlations between EGFR expression and clinical parameters are less satisfactorily evaluated in patient cohorts. However, evidence exists for a causal role of aberrant EGFR signaling in establishing resistance to radiotherapy (RT) and chemotherapy (CHT) in patients with GB (7). Therefore, further data are required concerning the role of EGFR in determining the clinical features and response to therapy of GB, and of the possible underlying molecular mechanisms. Previously, we analyzed the potential correlation of combined expression of EGFR and autophagic protein beclin-1 as prognostic markers (15).
The aim of the present study was to investigate, more extensively, through a retrospective analysis of a consecutive series of patients, the potential correlations between EGFR expression levels alone and well-known clinical and radiological features known to be related to GB aggressiveness.
Patients and Methods
Data collection. One hundred and forty-four patients affected by GB [grade IV by WHO 2007 classification (16)] were submitted to a conventional postoperative RT-CHT treatment from February 2008 to December 2013. During this period, all patients eligible for full-course RT-CHT management were included in this study; patients enrolled in experimental RT or CHT trials were excluded. After surgery, on referral at the Radiation Oncology Unit of the University Hospital of Siena, general and neurological examinations were performed in all cases. The following data, concerning this study were collected: age, Karnofsky performance status (KPS), Neurological performance scale (NPS), results of preoperative and postoperative magnetic resonance imaging (MRI), and extent of surgical procedure. Patients with no available data were excluded from the present study. This study was authorized by the Institutional Review Board and local Ethics Committee and signed informed consent was obtained for any treatment and for the anonymous use of clinical data.
Postoperative treatment. RT consisted of 54-60 Gy total dose administered with three-dimensional conformal (3D-RT) or intensity-modulated RT in all cases with a unifocal presentation. The planning target volume was obtained by computed tomographic-MRI image fusion and included tumor mass (T1-weighted scans after gadolinium contrast) or postoperative cavity with a 2-2.5 cm margin. Patients with multifocal lesions underwent whole brain irradiation, up to 50 Gy. Five weekly 1.8-2 Gy sessions were given during the entire RT course, in all cases. All patients received temozolomide (75 mg/m2/day) concurrent with RT for a maximum of 7 weeks. The majority of them received also sequential temozolomide (150-200 mg/m2 for 5 days, every 28 days), unless tumor progression or relevant toxicity occurred. The patients included in this evaluation completed at least the 80% of the planned treatment.
Follow-up. All patients were included in a follow-up program after completion of RT and concurrent temozolomide administration. General and neurological examinations, including blood counts and chemistry, were performed every 3 months. Brain MRI was performed after 2-3 weeks, 3-4 months and then every 6 months, or in any case showing clinical signs suggesting tumor progression.
Clinical and radiological variables. Age was taken into account as a prognostic variable in GB, with a cut-off value of 50 years (17). Two categories were considered for KPS (100-80; ≤70), and four for NPS (0-1: no or some neurological impairment, respectively; 2: moderate impairment; 3: major functional impairment) according to the Medical Research Council Brain Tumor Working Party (18). All patients were referred after a surgical procedure and classified according to the surgical records into two categories: biopsy-subtotal resection (B-STR) and gross total resection.
Pre- and postoperative brain MRI scans included unenhanced sagittal, coronal and axial T1-weighted, T2-weighted images, and gadolinium-enhanced axial, coronal and sagittal T1-weighted images, and these were reviewed by a neuroradiologist skilled in brain tumor assessment. Pre-operative brain MRI assessed the single or multiple (including both multicentricity and multifocality) presentation of the tumor and maximal extent of peritumoral edema measured from the outer ring of the tumor margin to its maximum extent (19) in patients naïve with respect to antitumor and antiedema therapy.
Treatment response was assessed using the Response Assessment in Neuro-Oncology (RANO) criteria (20); patients accrued before 2010 were reviewed and re-classified according to these criteria. The results were grouped into complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). Radiological response to treatment was assessed on brain MRIs at least 5-6 months after treatment. Patients with clinical deterioration not attributable to other non-tumor causes were considered to have PD in cases without MRI assessment.
Evaluation of EGFR expression. EGFR protein expression was evaluated by immunohistochemical staining. In each case, 3-μm-thick sections were cut from paraffin blocks of 10% formalin fixed tumor fragments and processed for immunohistochemistry. Briefly, after de-paraffinization and rehydratation, before applying the mouse monoclonal antibody to EGFR (clone 31G7, diluted 1:200; Zymed, Milan, Italy), sections were pre-treated either with Pronase XIV of Streptomyces griseus (Bio-Optica, Milan, Italy) at 37°C for 10 min, or with WCAP citrate buffer pH 6.0 (Bio-Optica) at 98.5°C for 40 min. The evaluation of the signal was performed by UltraVision LP Large Volume Detection System HRP Polymer (Bio-Optica), with diaminobenzidine chromogen (Dako, Milan, Italy) for 8 min. Sections were then counterstained with Meyer's hematoxylin. In all cases, negative controls were performed by repeating the procedure and omitting the primary antibody.
The samples were evaluated by an experienced neuropathologist, at medium resolution (×20 objective, ×1.25 eyepiece), throughout all tumor sections. Membranous and cytoplasmic EGFR immunoreactivity were scored 0 if negative, and from 2 to 5, if positive, on the basis of both the staining intensity (1=weak, 2=moderate, 3=strong) and the percentage of positively stained cells (1: ≤50%, 2: >50%). As previously reported (15), we considered the total score 0–2 as a low (L), and score 3-5 as a high (H) protein expression, respectively.
Molecular determination of the methylation status of the O6-methylguanin-DNA-methyltransferase (MGMT) gene promoter. MGMT gene promoter methylation was assessed by methylation-specific polymerase chain reaction (PCR). Briefly, genomic DNA was extracted from paraffin-embedded tumor sections and treated with sodium bisulfite using the EZ DNA Methylation-Gold kit (HISS Diagnostics, GmbH, Freiburg, Germany). Primer sequences were used to detect methylated and un-methylated MGMT promoter sequences. PCR products were separated on 2% agarose gel. A glioma cell line with a completely methylated MGMT promoter and peripheral blood mononucleated cells served as positive and negative control samples, respectively. A methylation percentage of 5% was used as a cut-off value, samples with methylation <5% were classified as unmethylated, and thosewith a value >5% as methylated.
Statistical analysis. A Fisher's exact test between EGFR expression and categorical clinical and radiological data was performed. A correlation analysis using Spearman's rho two-tailed was used to explore correlation between EGFR expression and both continuous and discrete clinical (NPS) and radiological (peritumoral edema and response to treatment) variables.
Overall survival was calculated with the Kaplan–Meier method. A univariate survival analysis of validated known prognostic parameters was used in this patients series. We used statistical tests (log-rank) to assess the significance of survival differences for the studied parameters. A multivariate analysis (Cox regression method) was performed to individualized independent parameters correlated to survival. All reported p-values are two-sided test results values of 0.05 or less were considered statistically significant. All statistical analysis were performed with SPSS 15.0 software (IBM Corporation, New York, USA) package for Windows.
Results
The median age was 63 (range=35-84) years: 18 patients (12.5%) were ≤50 years old; 126 patients (87.5%) were older than 50 years. The KPS score was 80-110 in 116 patients (80.7%), and ≤70 in 28 (19.3%). The NPS score was 0 in 30 patients (20.8%), 1 in 52 (36.1%), 2 in 38 (26.4%), and 3 in 24 (16-7%). A total of 116 patients were in the B-STR category (80.6%), and 28/144 in the GTR (19.4%). Fifty-six patients (38.9%) received a RT dose of <54 Gy (due to a multifocal presentation or to constraints, e.g. critical structures very close to the tumor), and 88 (61.1%) a dose of ≥54 Gy. All patients received temozolomide concurrently with RT; sequential temozolomide was administered in 100 out of 144 patients (69.4%) until tumor progression or severe toxicity occurred; 44 (30.6%) patients did not follow the treatment scheduled due to early tumor progression or toxicity at the end of the concurrent RT-CHT administration.
Preoperative MRI showed a single tumor in 112 patients (77.1%) and multiple tumors in 32 (22.9%). The mean maximum extent of the measureable (in 122 out of 144 patients) peritumoral edema was 26.33 mm (range=1-71 mm). Response after RT-CHT (concurrent temozolomide administration) was PR in 22 (16.2%), CR in 23, SD in 29 and PD in 70 patients at the post-RT MRI evaluation. Metachronous multiple tumor occurred in 18 out of 100 evaluable patients (18%) with an initially unifocal tumor.
EGFR expression and MGMT methylation status. The EGFR expression was low in 51 out of 144 patients (35.4%), and high in the other 93 (64.6%). The determination of MGMT status was performed in 116 patients; 62 cases (53.3%) had MGMT expression and 54 (47.7%) a methylated MGMT promoter.
Relationship of EGFR score and clinical variables (Table I). No significant correlation was demonstrated between EGFR score and MGMT methylation status, or age (p=0.12 and p=0.057, respectively). The relationship between EGFR expression and extent of surgical resection was not significant (p=0.67). An elevated EGFR score was significantly correlated with low KPS (p=0.003) and a worse NPS (p=0.013). Interestingly, EGFR score was inversely related to administration of sequential temozolomide (p=0.001).
EGFR expression and radiological features (Table I). A significant correlation was found between EGFR expression and synchronous tumor multifocality (p=0.001), tumor in 29 out of 32 patients were high EGFR-expressing cases.
Furthermore, EGFR expression score strongly correlated with the preoperative extent of edema on initial MRI scan (p=0.0001). The mean extent was 18.8 mm (±11.66 mm) in the L-EGFR group and 31.38 mm (±12.65mm) in the group with high EGFR expression, with a maximum extent ≤20 mm in more than a half of patients for L-EGFR group (Figure 1). According to the RANO criteria, patients with high EGFR expression had a stronger tendency for worse radiological response to treatment (p=0.001), with a higher incidence of PD at post-treatment MRI.
Patients with initial unifocal presentation who developed other new lesions after treatment were in almost all cases those with high EGFR levels (16/18) (p=0.02).
Survival results. After a median follow-up time of 14 months (range=6-61 months), the median overall survival of the 144 patients included in this study was 11 months; 1 and 2 year-overall survival rate values were 42.2% and 26.6%, respectively.
Specific well-known prognostic factors (3, 21-23) confirmed their significance in overall survival in this series. (Table II). In particular, despite age (p=0.01), MGMT methylation status (p=0.001), extent of resection (p=0.001) and radiation dose (p=0.001), clinical prognostic factors such as KPS (p=0.001), NPS (p=0.001) and sequential temozolomide (p=0.001) and radiological findings being correlated with EGFR expression, others such as synchronous single/multiple tumor (p=0.002), peritumoral extent (p=0.03) and radiological response to treatment (p=0, 001) were related to prognosis of patients in this series. In fact, univariate analysis of EGFR expression showed it to significantly inversely correlate with overall survival (p=0.001) (Figure 2). Patients with high vs. low EGFR expression levels had, in fact, a worse median overall survival (8 months vs. 20 months). Furthermore, the multivariate analysis of survival factors showed that age >50 years (hazard ratio(HR)=2.45, 95% confidence interval (CI)=1.055-5.698; p=0.037), unmethylated MGMT promoter (HR=2.19, 95% CI=1.315-3.663; p=0.002), sub-total resection or biopsy of tumor (HR=2.02, 95% CI=1.806-3.759; p=0.026), but in particular EGFR-related prognostic factors such as no sequential temozolomide administration (HR=1.78, 95% CI=1.105-2.854; p=0.018), high NPS (HR=1.41, 95% CI=1.130-1.781; p=0.003) and no response to treatment (HR=2.03, 95% CI=1.578-2.613; p=0.0001) were independently associated with a shorter survival.
Discussion
Glioblastoma generally determines a poor survival but multiple prognostic predictors have been recognized in analyses of both clinical trials and retrospective case series. Natural history and treatment-related parameters were identified, associated with a relatively better survival (i.e. young age, macroscopic surgical ablation and high RT dose, i.e. 60 Gy) (17, 21, 23). Beyond these factors, only a better knowledge of the biological factors underlying resistance to traditional therapeutic approaches can improve therapeutic results. This is probably related to specific to biomolecular features such as the MGMT methylation status (22). Extensive multi-platform genomic characterization is improving our knowledge over the molecular basis underlying the resistance of GB to therapy and EGFR alterations (mutations, amplification and overexpression) are highlighted as the most common biological alterations in GB (2). However, results of clinical studies (12-14) and issues over EGFR alterations (overexpression, amplification, mutated form) alone and prognosis in patients affected by GB are very controversial, and shed very little light on possible biological and clinical correlations.
EGFR is a 170-kDa receptor, consisting of a single chain polypeptide of 1186 amino acids (24), that is expressed on the surface of the majority of normal cells. Its binding with growth factors leads to receptor dimerization and allosteric modification, with consequent activation of the intracellular tyrosine kinase domain (25). Several studies showed hyperactivity of EGFR is linked to cell acquisition of neoplastic phenotype (26, 27) and EGFR activates several intracellular signaling cascades linked to proliferation, differentiation and cell migration (28).
A characteristic feature of GB is its intrinsic resistance to RT-CHT. Several studies have demonstrated that EGFR could also play a role as a cell survival factor in response to ionizing radiation and CHT damage (10, 29, 30). GB invasiveness and inherent resistance to RT-CHT treatment are somehow related to EGFR overexpression, and both can lead to unsuccessful therapy.
We previously reported the clinical implication of EGFR expression analysis combined with expression of autophagic protein beclin-1 (15). The present report might add some significant elements to the knowledge of the role of EGFR in the clinical context, given the amount of recorded clinical data and the uniformity of recruitment criteria and treatment modalities, despite the partial retrospective approach of the present study.
Some studies show that expression of matrix metalloproteases (31), as well as many biophysical processes that drive cell motility (32), are directly related to the activity of EGFR (33, 34). Furthermore, Sommers et al. suggested that phosphorylation by EGFR tyrosine residues of β-catenin (a protein associated with E-cadherin) may lead to loss of ability to promote cell-cell adhesion and therefore to an increased capacity for invasion (35). It is possible, for these reasons, that increased receptor activity might be linked to an accentuation of invasiveness and cell migration, leading to increased local invasiveness and multiple tumor in vivo. In our series, one of these aspects was clinically confirmed in that higher EGFR expression level was significantly correlated with a multiple tumor presentation (both synchronous and metachronous) in patients with GB when compared to those with a low score and this seemed to be related to a shorter survival. An unfavorable clinical course of GB was also reported to be related to the amount of peri-tumoral edema (19). The extent of peritumoral edema at preoperative MRI scans, in fact, was significantly related to EGFR expression in our experience, and the extent of edema was found to have prognostic significance at a cut-off level of a maximum extent of 2 cm.
The interaction of EGFR with other growth factors and hypoxia was demonstrated in regulation of vascular endothelial growth factor (VEGF) activity via a phosphatidylinositide 3-kinases (PI3K) pathway (36). Other experience indicated that Rat sarcoma (RAS)/PI3K is involved in transcriptional regulation of the VEGF promoter by EGFR, distinctly from signals induced by hypoxia (37). VEGF secretion can, in turn, induce some typical pathological and clinical features of GB, such as an increased angiogenesis and edema, respectively. Furthermore, peritumoral edema can contribute to the development of disabling symptoms, determining low KPS and high NPS scores in patients with high EGFR expression as found in the present study.
A characteristic feature of GB is its intrinsic resistance to RT-CHT. Several studies demonstrated that EGFR may also play a role as survival factor in response to RT and CHT damage (10, 29, 30). This function was linked mainly to the ability of suppression of apoptosis (38). Acting on PI3K/AKT and MAPK pathways (39), EGFR leads to the inactivation of regulatory molecules of the apoptotic process such as Bcl-2-associated death promoter, procaspase-9 and the forkhead transcription factor Foxo-1. Furthermore, EGFR appears to play a key role in the repair of double-strand-breaks (DSBs) induced by ionizing radiation. This receptor promotes non-homologous end-joining that is determinant in the repair of DSBs, either indirectly via PI3K/AKT, or directly, moving to the nucleus and stimulating the activity the catalytic subunit of protein kinase 5-dependent DNA, key enzyme in non-homologous end-joining (40). Interestingly, a worse response to postoperative therapy was more frequently seen in patients with GB with high EGFR expression according to our results. This represented one of the most important prognosticators in our series.
All these clinical and radiological findings related to EGFR expression may explain the difference in overall survival observed in patients according to the different EGFR expression scores, an inherent effect of the more aggressive course of the disease in patients with high EGFR expression.
Conclusion
Our results provide a preliminary assessment on the role of EGFR expression, extrapolated from a uniformly treated series of patients with GB regarding clinical features at referral, imaging data, response to RT-CHT, tumor evolution and survival. EGFR expression alone appears to be significantly related to some parameters of aggressiveness in GB, such as multiple tumor incidence (probably related to invasiveness), extent of peritumoral edema, and reduced response to postoperative treatment. These aspects are discussed herein, also taking into account the remarkable laboratory investigations by other authors, which deserve further study in order to drive research for a targeted-therapy, effective against EGFR that adversely influence s the clinical course of patients with GB.
Footnotes
↵* These Authors contributed equally to the present study.
Conflicts of Interest
The Authors declare that they have no conflict of interest in regard to this study.
- Received April 8, 2015.
- Revision received May 5, 2015.
- Accepted May 7, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved