|Year : 2014 | Volume
| Issue : 3 | Page : 390-395
|Revisiting epidermal growth factor receptor in glioblastoma multiforme: Does it play a role in response to therapy?
Priyanka Soni1, Nuzhat Husain1, Rishi Awasthi2, Anil Chandra3, Bal K Ojha3, Rakesh K Gupta2
1 Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Radiodiagnosis, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Neurosurgery, King George Medical University, Lucknow, Uttar Pradesh, India
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|Date of Web Publication||14-Aug-2014|
| Abstract|| |
Background and Aim: Glioblastoma multiforme (GBM) are the most aggressive class of cancer of central nervous system with hallmark characteristics that include rampant proliferation, necrosis, and endothelial proliferation. Epidermal growth factor receptor (EGFR) has been implicated as the primary contributor to glioblastoma initiation and succession. The present study was designed to evaluate EGFR protein expression in GBM as predictor of response to therapy and survival. Materials and Methods: Epidermal growth factor receptor was assessed by immunohistochemistry as a percentage of positive tumor cells in hot spots (10 high-power fields). The study group comprised of 35 cases of GBM. All cases underwent surgical resection and subsequently underwent radiotherapy (n = 17) or radiotherapy with adjuvant temozolomide chemotherapy (n = 18). Immediate response to therapy was assessed at 3 months using World Health Organization response evaluation criteria in solid tumors criteria and cases followed up for survival. Results: Twenty-four cases (68.6%) expressed EGFR while 11/35 (31.4%) cases were negative. Response to therapy was evident in 21/35 cases (60.0%) and 14/35 were (40.0%) nonresponders. Mean EGFR protein expression in responders was 37.23 ± 33.70 and in nonresponders was 59.5 ± 39.46 (P = 0.542). The percentage of responders which were EGFR negative was 72.7% and while response in EGFR positive cases was observed in 54.2%. Mean survival in EGFR positive and negative GBM was 394.37 ± 189.11 and 420.54 ± 191.23 days, respectively. Conclusion: The EGFR negative cases appear to respond better to therapy, however, the difference is not statistically significant (P = 0.298). Further, EGFR protein expression does not play a definitive role in predicting survival. This is an original study evaluating EGFR in terms of therapeutic response.
Keywords: Epidermal growth factor receptor, glioblastoma multiforme, immunohistochemistry, nonresponders, responders
|How to cite this article:|
Soni P, Husain N, Awasthi R, Chandra A, Ojha BK, Gupta RK. Revisiting epidermal growth factor receptor in glioblastoma multiforme: Does it play a role in response to therapy?. Indian J Pathol Microbiol 2014;57:390-5
|How to cite this URL:|
Soni P, Husain N, Awasthi R, Chandra A, Ojha BK, Gupta RK. Revisiting epidermal growth factor receptor in glioblastoma multiforme: Does it play a role in response to therapy?. Indian J Pathol Microbiol [serial online] 2014 [cited 2023 Feb 8];57:390-5. Available from: https://www.ijpmonline.org/text.asp?2014/57/3/390/138725
| Introduction|| |
Glioblastoma multiforme (GBM) is a World Health Organization (WHO) grade IV neoplasm with a very poor prognosis and a mean survival of a year after diagnosis. ,, The current standard treatment of GBM involves surgical resection, radiation, and chemotherapy. ,, Molecular profiling of GBM reveals significant genetic heterogeneity which specifically involves amplification of epidermal growth factor receptor (EGFR) gene, resulting in overexpression of the EGFR. ,,, In the present study, we have evaluated EGFR protein expression in GBM as a predictor of marker of response to therapy and survival and to perceive whether chemotherapy in addition to postsurgical irradiation influences survival.
| Materials and methods|| |
Biopsy of 35 patients was fixed in 4% buffered formaldehyde at room temperature and embedded in paraffin. Paraffin-embedded tissues of GBM were sectioned, 3-4 μm, using a microtome (Leica, Germany), mounted on tissue bond-coated slides (Biocare, USA) and stained with hematoxylin and eosin for histopathological examination by neuropathologist. All the cases were classified morphologically and graded according to WHO criteria.  Immunohistochemistry (IHC) was done by the standard protocol. Briefly, section on coated slides was fixed overnight at 60°C in a dry oven, deparaffinized in xylene and rehydrated through graded ethanol series. Sections were blocked with 3% hydrogen peroxide in methanol for 30 min to quench any endogenous peroxidase activity, if present, and were then processed for antigen retrieval in Pascal (DAKO Cytomation, California) by placing in sodium citrate buffer (pH-6.0). Sections were incubated for an hour with anti-EGFR-pan, rabbit monoclonal (Biogenex, Hyderabad), followed by treatment with polymer-based secondary antibody kit (Dakopatts, Envision kit, Denmark). Bound antibody was visualized using diaminobenzidine, according to the manufacturer's instructions. Sections were counter-stained with hematoxylin and mounted. Positive and negative (by omitting primary antibody) controls were run with all batches. Evident cytoplasmic-membrane staining in tumor cells was scored as positive/overexpressed. EGFR was assessed by IHC as a percentage of positive tumor cells in hot spots (10 high power fields). Patients were divided into EGFR positive and EGFR negative. Cases with tumors demonstrating >20% EGFR immunohistochemical expression were considered positive [Figure 1]f, while below 20% were assigned negative [Figure 1]c. ,
|Figure 1: (a) Magnetic resonance imaging of 60-year-old male with glioblastomas multiforme (GBM) in right frontoparietal region before surgery (b). Three months after treatment (responder) (c). Microphotograph showing negative epidermal growth factor receptor (EGFR) expression by immunohistochemistry (d). 40-year-old female with GBM in right temporal region before surgery (e). Nonresponder (f) positive EGFR expression (diaminobenzidine ×125 × digital magnification)|
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The study group was homogenous in terms of surgical therapy and extent of surgical resection was near total in all 35 cases. After the surgery, patients with GBM were stratified into two treatment regime obtaining (i) radiation (ii) adjuvant chemo-radiation. Randomization of cases was not possible and stratification was dependent on financial capacity of patient to afford added chemotherapy. Seventeen patients received postoperative radiation alone of at least 60 Gy for 6 weeks given and eighteen underwent chemo-radiation temozolomide (TMZ).The TMZ schedules was 100 mg/m 2 daily during radiotherapy then up to 6 adjuvant cycles of 150-200 mg/m 2 for 5 out of 28 days.
Response to radiotherapy and/or chemo-radiotherapy was assessed by magnetic resonance imaging (MRI) and computed tomography (CT) scans done at the time of patient's admission with neurologic symptoms to neurosurgery department. These scans were compared with the postoperative MRI/CT scans, done after 3 months of therapy completion, for the assessment of tumor response. The tumor volume was calculated for all the cases to assess treatment response. The patient was graded as complete responder (CR), partial responder (PR) [Figure 1]a,b, progressive disease (PD), and stable disease (SD) [Figure 1]d,e.
Response evaluation criteria in solid tumors (RECIST) guidelines  were applied to assess response: Complete response: 100% reduction, that is disappearance of all target lesions, partial response: >30% reduction in the sum of longest diameter (LD) of target lesions; PD: >20% increase in the sum of LD of target lesions; SD: Treatment result lies between partial response and PDs.
All the cases were followed-up at regular intervals till death. At the time of this analysis, all 35 patients had died. Information on patient morbidity and mortality was collected by telephonic/personal means. Morbidity was assessed in terms of Karnofsky performance score (KPS). Overall survival time was defined as the time interval between initial craniotomy and the day of the patient's death.
This study has been approved by the Institutional Ethics Committee. Consent was taken. Clinical evaluation of all the patients was done including age, gender, site of tumor, extent of surgical resection, preoperative KPS score, date of surgery, history of any other disease, treatment given, and cases followed up for disease progression till death. All cases included underwent near total surgical excision (to avoid confounding due to variation in surgical procedure).
Statistical analysis was performed using the IBM-Statistical Package for Social Sciences (SPSS, International Business Machines Corporation, New York, USA) analysis software, version 16. Kaplan-Meier curves were used to assess survival. Chi-square test was used for the categorical variables (sex, age, KPS, treatment and response). All P were calculated with two-sided tests and P ≤ 0.05 was considered significant and highly significant when P ≤ 0.01.
Using the Log-rank test the mean survival of the patients was calculated with respect to their basic characteristics. Univariate and multivariate Cox-regression analysis was performed to identify predictors of good survival. All effects were quantified by odds-ratio (OR) estimates with 95% confidence intervals (CIs).
| Results|| |
The study group comprised of 35 cases of GBM. The mean age of patients was 44.31 years (range, 10-76); 16 patients (45.7%) were ≤40 years and 19 (54.3%) were >40 years. There were 77.14% males and 22.85% females. The mean preoperative KPS score was 62 (ranged 30-90). The intracranial location of the tumor was 51.4%, 28.6%, 14.3%, and 5.7%, respectively, in frontal, parietal, temporal, and occipital sites. All 35 cases underwent surgical resection initially and subsequently 48.6% underwent radiotherapy (n = 17) or 51.4% radiotherapy with adjuvant chemotherapy (n = 18). Immediate response to therapy was assessed at 3 months using WHO RECIST criteria and cases followed-up for survival. Patient characteristics and demographics are scheduled in [Table 1].
Response to treatment was categorized in four response groups (CR = 11 [31.4%], PR = 10 [28.6%], SD = 6 [17.1%] and PD = 8 [22.9%]. For statistical evaluation, CR and PR were categorized as responders (21/35 cases; 60.0%) and group SD and PD were categorized as nonresponders (14/35 cases; 40.0%), [Table 1]. The percentage of responders in the radiotherapy with adjuvant TMZ group was 13.8% higher and clinically better [Table 1], compared to radiation group alone but the difference was statistically insignificant (P = 0.407).
The mean EGFR protein expression in responders was 37.23 ± 33.70 and was less than nonresponders at 59.5 ± 39.46.
These two values are significantly different (P = 0.542). The percentage of responders in EGFR negative versus positive cases was 72.7% and 54.2% respectively. On the other hand, the percentage of nonresponders in EGFR negative versus positive cases was 27.3% and 45.8%, respectively. This difference was not significant (P = 0.298). EGFR protein expression in relation to clinical characteristics of cases is detailed in [Table 2].
|Table 2: Association of EGFR protein expression with basic characteristics and treatment response of GBM patients|
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Mean survival time was 402.6 days after diagnosis (57.5 weeks). Mean survival in EGFR positive and negative GBM was 394.37 ± 189.11 and 420.54 ± 191.23, respectively, [Table 1] and this difference was not statistically significant.
Univariate and multivariate analysis was done to analyze the impact of all the parameters on overall survival (OS) by using the Cox-regression analysis for identifying predicators of good survival. Kaplan-Meier method survival curves and differences in survival were assessed with the Log-rank test [Table 3].
|Table 3: Mean survival (days) of GBM patients in relation to basic characteristics, treatment response, and EGFR protein expression|
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The preoperative KPS and the response to therapy reached significance in OS [P = 0.010 and 0.002 respectively, [Table 4] by univariate analysis. However, multivariate analysis revealed that age (P = 0.041) and treatment response (P = <0.001) were independent significant prognostic predictors of OS. Insignificant association of EGFR with OS was observed by univariate and multivariate analysis with P = 0.789 and P = 0.479, respectively. Results were reported using the P and the estimated OR with their 95% CIs.
|Table 4: Univariate and multivariate Cox-regression analysis for identifying predictors of good survival|
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| Discussion|| |
In the current study, we have analyzed EGFR protein expression in GBM with the primary intention to study its role, if any, in predicting response to treatment and as well as OS. All cases underwent near total surgical resection. It was not definitely possible in all our cases to histologically distinguish primary and secondary glioblastomas, since representative biopsies were sent and remaining tumor was aspirated. Hence, it is not possible that existing low grade areas in some tumor may have been aspirated. Primary and secondary GBMs are indistinguishable to the neurosurgeon as well as neuropathologist, and the clinical management of these two GBM subtypes is identical.
Seventeen cases took subsequent radiation therapy alone eighteen cases underwent chemo-radiation therapy with addition of TMZ. The proportion of cases on radiation and chemo-radiation were approximately the same in both response groups. Hence, this does not appear to confound EGFR results. We analyzed other known prognostic predictive confounders and hence other secondary data were also generated. Patient age, which is an established independent predictive factor for survival in glioblastoma, , and a potential confounding factor showed a significant effect on survival by multivariate analysis with cases ≤40 years survived longer days than cases with >40 years (P = 0.041). The EGFR overexpression was not significantly related to survival and treatment response in terms of the sex of the cases. Cases were divided for the purpose of analysis into cases with a KPS score of ≤50 and cases than with KPS >50. High KPS score was significantly associated with better survival by univariate analysis [P = 0.010, [Figure 2]a. Previous studies have shown an association between young age at diagnosis, high KPS, and longer survival.  The results of multivariate analysis in our study showed age (P = 0.041) and treatment response (P = <0.001) were independent significant prognostic predictors of OS.
|Figure 2: Kaplan-Meier showing survival in days of patients according to (a). Karnofsky performance score (b). Treatment protocols: Radiation and radiation + temozolamide (c). Complete responders (CRs),|
Partial responders (PRs), Progressive disease, Stable disease (SD) (d). Responders (CR + PR) versus nonresponders (PR + SD) (e) epidermal growth factor receptor negative and positive cases
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Review of studies on EGFR protein overexpression in glioblastoma have shown ambiguous results regarding its prognostic significance and several studies have found no association of these alterations with survival. ,,,, In the present study, we have not found significant association of EGFR protein overexpression and OS. The mean OS was 394.38 days for patients where tumors overexpressed EGFR as compared to a mean survival of 420.55 days in patients where tumors were negative for EGFR protein expression (P = 0.789), [Figure 2]e. Hurtt et al. and Shinojima et al. have demonstrated that in supratentorial GBM, high EGFR expression was significantly associated with shorter survival. , Study performed by Heimberger et al. showed lack of prognostic effect of the overexpressed EGFR or the EGFR VIII on OS compared with other studies.  Other studies done on astrocytic neoplasms of multiple grades, EGFR protein overexpression has been shown to be associated with a worse prognosis, , while other reports have found no prognostic relationship. ,
Within our cohort of GBM patients treated with radiotherapy and chemo-radiotherapy using TMZ a survival benefit of 15.4 months was observed [P = 0.055, [Table 3]. The Kaplan-Meier [Figure 2]b showed good prognosis in the group of patients treated with TMZ (R + T) compared to the radiotherapy (R) treatment alone.  Complete response was observed in 31.4% of cases and these cases showed a maximum overall mean survival OS of 563.64 days. OS of PRs forming 28.6% cases was 381.80 days. SD was present in 17.1% cases with an OS of 295 days and PD was observed in 22.9% cases with an OS of 287 days. There was a decreasing trend in OS in all four treatment response groups starting from CR to PD [Figure 2]c.
Response to therapy was evident in 60% cases in this study. Responders had better survival of 477.05 days [Figure 2]d compared to the nonresponders with OS of 290.93 days (P = 0.001). Mutations involving the EGFR gene could lead to its constant activation resulting in uncontrolled cell division, predisposing to GBM.  Mutations of EGFR have been identified in several types of cancer, and it is the target of an expanding class of anticancer therapies. 
Radiotherapy is the most used traditional method in almost all cancer types and is now considered the standard of care, but due to its short time relief along with multiple side effects there appears the necessity of some drugs that may act at molecular level for better treatment with prolong effect. TMZ has been shown to confer better result when used as an adjuvant to radiation therapy. TMZ is known for its excellent penetration into all body tissues, including the brain. Evidence from the other studies has shown better survival in high grade gliomas as well as GBM patients with low concentrations of O6-alkylguanine-DNA alkyltransferase AGT. The adverse short term events associated with TMZ are low but can be severe, while the long term effects are unknown. TMZ have also been shown to induce a G2-M arrest in glioma cells, thus, synchronizing the cell cycle in a radiosensitive phase.  In our study, the percent responder with addition of TMZ was 66% while on radiation alone was 52.9%. This difference was not statistically significant (P = 0.40).
| Conclusion|| |
Epidermal growth factor receptor protein overexpression is a significant and the frequent mutation in glioblastomas, EGFR negative cases appear to respond better to therapy, however, the difference is not statistically significant. Further, EGFR does not play a definitive role in predicting survival. The study is limited by a small number of cases. This is the first study evaluating EGFR in terms of therapeutic response. The article may be considered as a pilot for further studies and evaluation for significance. An insignificant small difference survival benefit and better treatment response was observed in cases on concomitant TMZ therapy. However, MGMT gene methylation was not studied in our cases. This may account for the insignificant difference in response with addition of TMZ therapy which is dependent on the MGMT gene methylation status.
| Acknowledgment|| |
Indian Council of Medical Research-Senior Research Fellow (ICMR-SRF) Grant.
| References|| |
Shinojima N, Tada K, Shiraishi S, Kamiryo T, Kochi M, Nakamura H, et al.
Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme. Cancer Res 2003;63:6962-70.
Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. Am J Pathol 2007;170:1445-53.
Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, et al.
Malignant astrocytic glioma: Genetics, biology, and paths to treatment. Genes Dev 2007;21:2683-710.
Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008;359:492-507.
Van Meir EG, Hadjipanayis CG, Norden AD, Shu HK, Wen PY, Olson JJ. Exciting new advances in neuro-oncology: The avenue to a cure for malignant glioma. CA Cancer J Clin 2010;60:166-93.
Etcheverry A, Aubry M, de Tayrac M, Vauleon E, Boniface R, Guenot F, et al.
DNA methylation in glioblastoma: Impact on gene expression and clinical outcome. BMC Genomics 2010;11:701.
Burton EC, Lamborn KR, Forsyth P, Scott J, O'Campo J, Uyehara-Lock J, et al.
Aberrant p53, mdm2, and proliferation differ in glioblastomas from long-term compared with typical survivors. Clin Cancer Res 2002;8:180-7.
Stark AM, Witzel P, Strege RJ, Hugo HH, Mehdorn HM. p53, mdm2, EGFR, and msh2 expression in paired initial and recurrent glioblastoma multiforme. J Neurol Neurosurg Psychiatry 2003;74:779-83.
Hill C, Hunter SB, Brat DJ. Genetic markers in glioblastoma: Prognostic significance and future therapeutic implications. Adv Anat Pathol 2003;10:212-7.
Wemmert S, Ketter R, Rahnenführer J, Beerenwinkel N, Strowitzki M, Feiden W, et al.
Patients with high-grade gliomas harboring deletions of chromosomes 9p and 10q benefit from temozolomide treatment. Neoplasia 2005;7:883-93.
Kleihues P, Burger PC, Scheithauer BW. The new WHO classification of brain tumours. Brain Pathol 1993;3:255-68.
Cox G, Jones JL, O'Byrne KJ. Matrix metalloproteinase 9 and the epidermal growth factor signal pathway in operable non-small cell lung cancer. Clin Cancer Res 2000;6:2349-55.
Choe G, Park JK, Jouben-Steele L, Kremen TJ, Liau LM, Vinters HV, et al.
Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype. Clin Cancer Res 2002;8:2894-901.
Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al.
New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:228-47.
Smith JS, Tachibana I, Passe SM, Huntley BK, Borell TJ, Iturria N, et al.
PTEN mutation, EGFR amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J Natl Cancer Inst 2001;93:1246-56.
Simmons ML, Lamborn KR, Takahashi M, Chen P, Israel MA, Berger MS, et al.
Analysis of complex relationships between age, p53, epidermal growth factor receptor, and survival in glioblastoma patients. Cancer Res 2001;61:1122-8.
Curran WJ Jr, Scott CB, Horton J, Nelson JS, Weinstein AS, Fischbach AJ, et al.
Recursive partitioning analysis of prognostic factors in three radiation therapy oncology group malignant glioma trials. J Natl Cancer Inst 1993;85:704-10.
Olson JJ, Barnett D, Yang J, Assietti R, Cotsonis G, James CD. Gene amplification as a prognostic factor in primary brain tumors. Clin Cancer Res 1998;4:215-22.
Smith JS, Jenkins RB. Genetic alterations in adult diffuse glioma: Occurrence, significance, and prognostic implications. Front Biosci 2000;5:D213-31.
Prados MD, Gutin PH, Phillips TL, Wara WM, Larson DA, Sneed PK, et al
. Highly anaplastic astrocytoma: A review of 357 patients treated between 1977 and 1989. Int J Radiat Oncol Biol Phys 1992;23:3-8.
Hurtt MR, Moossy J, Donovan-Peluso M, Locker J. Amplification of epidermal growth factor receptor gene in gliomas: Histopathology and prognosis. J Neuropathol Exp Neurol 1992;51:84-90.
Heimberger AB, Hlatky R, Suki D, Yang D, Weinberg J, Gilbert M, et al.
Prognostic effect of epidermal growth factor receptor and EGFRvIII in glioblastoma multiforme patients. Clin Cancer Res 2005;11:1462-6.
Korkolopoulou P, Christodoulou P, Kouzelis K, Hadjiyannakis M, Priftis A, Stamoulis G, et al
. MDM2 and p53 expression in gliomas: A multivariate survival analysis including proliferation markers and epidermal growth factor receptor. Br J Cancer 1997;75:1269-78.
Etienne MC, Formento JL, Lebrun-Frenay C, Gioanni J, Chatel M, Paquis P, et al.
Epidermal growth factor receptor and labeling index are independent prognostic factors in glial tumor outcome. Clin Cancer Res 1998;4:2383-90.
Rainov NG, Dobberstein KU, Bahn H, Holzhausen HJ, Lautenschläger C, Heidecke V, et al.
Prognostic factors in malignant glioma: Influence of the overexpression of oncogene and tumor-suppressor gene products on survival. J Neurooncol 1997;35:13-28.
Waha A, Baumann A, Wolf HK, Fimmers R, Neumann J, Kindermann D, et al.
Lack of prognostic relevance of alterations in the epidermal growth factor receptor-transforming growth factor-alpha pathway in human astrocytic gliomas. J Neurosurg 1996;85:634-41.
Krex D, Klink B, Hartmann C, von Deimling A, Pietsch T, Simon M, et al.
Long-term survival with glioblastoma multiforme. Brain 2007;130:2596-606.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al.
Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-39.
Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R, et al.
ErbB receptors: From oncogenes to targeted cancer therapies. J Clin Invest 2007;117:2051-8.
Hirose Y, Berger MS, Pieper RO. p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 2001;61:1957-63.
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow - 226 003, Uttar Pradesh
Source of Support: Indian Council of Medical Research-Senior Research
Fellow (ICMR-SRF) Grant., Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
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