Original article
Epidermal growth factor receptor gene expression in high grade gliomas

Introduction

Anaplastic astrocytoma (AA) and glioblastoma (GBL) are the most frequent primary brain malignancies in adults. In the general population the frequency of these two high grade gliomas ranges from 0.2 to 0.5 per 100 000 children aged below 14 years and from 1.7 to 4.5 per 100 000 adults aged more than 45 years. The most often occurring genetic disturbances in gliomas are mutations in the p53 gene [10,25,50], amplification and rearrangements of the MDM2 proto-oncogene [36] and amplification of such oncoge­nes as N-myc, C-myc, N-ras, K-ras and PDGFR- [5,7,8]. Several underlying cytogenetic abnormalities are observed, such as loss of heterozygosity (LOH) in chromosomes 17p [10-12], 10 [46,47], 9p [19] and 19q [40,47]. Additionally, in AA several deletions in chromosomes 13q, 17 p and 22p are observed. However, the strongest stimulus for the in vitro-observed tu­mour growth in malignant gliomas is the gene-level activation of receptors for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) [35]. It is suggested that the degree of astrocytoma malignancy is positively correlated with both the concentration of EGFR protein and with EGFR gene amplifications [26]. In primary glioblastomas, which typically affect older patients, amplifications of the epidermal growth factor receptor gene (EGFR) occur along with deletions/mutations of phosphatase and tensin ho­mologue tumour suppressor gene (PTEN), a negative regulator of the phosphatidyl inositol 3 kinase/Akt signalling pathway [43].

Amplifications and overexpression of the EGFR gene are seen in 30-50% of cases of adult high grade gliomas. However, some differences are observed between primary and secondary GBL. Watanabe et al. observed EGFR overexpression in 63% of primary GBL, but only in 10% of secondary GBL. In the same study, p53 mutations were found in 11% of primary GBL and 67% of secondary GBL, but in 37% of primary GBL accompanied by the accumulation of p53 protein.

The human EGFR gene is located on chromosome 7. Its high expression is observed in many ty­pes of cancer, such as in lung [33,42], breast [37], head and neck [15], bladder carcinoma [31], as well as in gliomas [26]. EGFR overexpression can induce abnormal proliferation of cells, and neoplastic phenotype. Pathway stimulation may be a result of gene amplification, but also ligand-independent activation can induce protein phosphorylation and binding with adaptor proteins [19,53].

Technically, the analysis of EGFR over-expression should take into account the frequent mutations of this gene. Deletions can appear both in mRNA encoding intracellular domains of the receptor, as well as in sequences of its intracellular part. Loss of the tyrosine kinase domain inhibits signal transduction even in the presence of a ligand [18,39,55]. EGFRvIII, the large deletion located in the extracellular domain of EGFR, is the most frequent mutation of the EGF receptor, appearing in over 50% of low and high grade astrocytomas [19,23,34,39,44,45,55].

Some differences in the EGFR-related pathways leading to the formation of de novo primary

GBL are suggested between children and adults [6,13,39,55]. Although EGFR protein overexpression is noted in nearly 1/4 of paediatric cases of GBL, corresponding amplification of the EGFR gene in this population is relatively rare. This fact may be of clinical importance, as younger patients with primary GBL exhibit relatively long overall survival compared to older patients [23,34,44].

Aim of the study

The aim of the study was to assess the expression level of EGFR gene in high grade brain gliomas, for both wild type and mutated form. The particular aim was to relate the differences in EGFR expression to patients’ age.

Material and methods

EGFR gene expression was analysed in 75 tumour samples obtained from patients treated in the Department of Neurosurgery of the Medical University of Silesia in Katowice. The study was approved by the local Bioethics Committee. Median age was 58 years (28-75 years). 49.3% were women. All pa­tients were operated on for supratentorial intrahaemispheric tumour. A primary tumour was resected in 61.2% of patients, while 38.8% were reoperated due to recurrent tumour. All of them had postoperative radiation therapy. Expression of EGFR in 26 samples of breast cancer was also analysed and used as a control group. Detailed histopathological findings are presented in Table I.

Total RNA was isolated from tumour tissue, using the TRIzol method and followed by purification with the RNeasy Mini Kit method (QIAGEN). Quantification of cDNA for the EGFR gene and 18S RNA (internal reference gene) was performed using real-time quantitative PCR (ABI PRISM 7700 Sequence Detection System – Taqman; Applied Biosystems).

EGFR expression was studied using three amplicons, complementary to different fragments of the mRNA molecule for EGFR (three exon junctions: 2-3, extracellular domain of EGFR molecule, 13-14, extracellular domain of EGFR molecule, 18-19, intracellular domain of EGFR molecule).

Exons 2 and 3 (in the region of EGFRvIII mutation): sense starter, F: 5'-GCAGAG GAATTA TGATCT TTCCTT CT-3', antisense starter, R: 5'-TGTTGA GGGCAA TGAGGA CA-3', probe: FAM-5'-AACCAG CCACCT CCTGGA TGGTCT TT-3'-TAMRA.

Exons 13 and 14: sense starter, F: 5'-TGCGTC TCTTGCC GGAAT-3', antisense starter, R: 5'-GGCTCA CCCTCC AGAAGC TT-3', probe: FAM-5'-ACGCAT TCCCTG CCTCGGC TG-3'-TAMRA.

Exons 18 and 19: sense starter, F: 5'-GCGTTC GGCACG GTGTAT-3', antisense starter, R: 5'-TTGATA GCGACG GGAATT TTAAC-3', probe: FAM-5'-TTCTCA CCTTCT GGGATC CAGAGT CCCT-3'-TAMRA.

As the reference gene, 18S rRNA was used: sense starter, F: 5'-CGGCTA CCACAT CCAAGG AA-3', antisense starter, R: 5'-GCTGGA ATTACC GCGGCT-3', probe: FAM-5'-TGCTGG CACCAG ACTTGC CCTC-3'-TAMRA.

A standard curve was made from cDNA serial dilutions of a sample mixture of high grade gliomas. The results after normalization by 18S RNA expression are given in arbitrary units (au, one arbitrary unit is the EGFR gene expression in a reference RNA sample, after normalization with 18S RNA) (Table II).

Immunohistochemical analysis of EGFR expression was performed on paraffin slides with Dako­ Cytomation anti-EGFR antibody, Clone 11 H, diluted 1 : 200. Labelling was performed by the streptavidin-biotin procedure (LSAB K 0690), staining with DAB Dako 3468 reagent.

Results

In the first step we performed immunohistoche­mical (IHC) analysis of EGFR expression in brain tu­mour samples available for IHC analysis. In 52.8% of high grade gliomas EGFR expression was strongly positive (Table III, Fig. 1).

Gene expression level was measured by quantitative real-time PCR. At the first step the non-normalized data were assessed. We found the highest levels of mean expression in the set containing linking of exons 13 and 14 (mean 0.165 au). Similar results were obtained for the set containing linking of exons 18 and 19 (mean 0.155 au). The results for the set containing linking of exons 2 and 3 (extracellular domain) revealed relatively low expression (mean 0.119 au). Detailed results are presented in Table IV. For non-normalized data, we did not find any statistically significant differences in expression levels of EGFR between subgroups of primary and secondary GBL.

Further analysis was carried out on EGFR gene ex­pression normalized to 18S RNA. Correlation analysis of gene expression of the three amplicons studied was performed. It revealed a strong correlation amongst analysed sets. Detailed results are presented in Fig. 2.

The highest values of expression were observed for exons 18-19 of the EGFR gene (median 0.99 au), slightly lower in exons 13-14 (median 0.85 au) and exons 2-3 (median 0.81 au). The difference in the ex­pression levels was statistically significant (Friedman’s test, p = 0.021).

As a reference, we also analysed the expression of EGFR in the set of epithelial cancers (breast cancer) (Table VB). When the EGFR expression levels in brain gliomas were compared with breast cancers, signi­ficantly higher EGFR expression was found in the glioma set (Table VC).

In the next step we tried to find cases with EGFRvIII mutation, which is a deletion of the extracellular domain containing exons 2-3. For this purpose mean values of expression of exons 13-14 and exons 18-19 were calculated. In samples with lower expression of exons 2-3 compared to mean expression of the other two domains probably EGFRvIII mutation is present. The mutation in selected samples was confirmed in RT-PCR (Fig. 3).

In the whole group of brain tumours EGFRvIII mutation was found in 26 samples (34.67% of all tumours). In the GBL group mutation was present in 34.21% of cases and in 40% of the AA group. In the control group of breast cancer EGFRvIII mutation was present in 24% of samples.

Comparison of subgroups with present EGFRvIII mutation with expression levels of exons 13-14 and 18-19 revealed that tumours without mutation have higher expression levels (Mann-Whitney U test p < 0.05). The differences were statistically significant for the whole group of tumours, but we did not obtain significant results within histological subgroups of GBL and AA.

When EGFR gene expression, obtained both at protein (IH) and gene (QPCR) level, was compared, we observed significant differences in gene expression between samples with different protein staining levels (Fig. 4), although there were four outlier samples with low protein level, highly expressing EGFR mRNA.

In the next step, we attempted to estimate the frequency of EGFR gene over-expression. We approached this by relating gene expression in gliomas to epithelial cancer (breast cancer), and we have taken the me­dian value of gene expression in breast cancer as the non-overexpressed level of gene expression. The me­dian EGFR expression in breast cancer is approximately 0.5 au. We found that EGFR expression > 0.5 au is significantly more frequent in brain gliomas (47%) than in breast cancers (17%) (Fig. 5).

We also divided the whole group of patients with brain tumours according to age (< 50 and > 50 years). We did not find, however, any statistically significant difference in the expression level of EGFR according to age group. Nevertheless, the younger patients (below 50 yrs) exhibited a trend towards lower expression of the EGFR gene in both mean expression and in two intracellular domains. The same results were seen for subgroups with GBL or AA tumours (Table VI).

Analysis of EGFRvIII mutation in age subgroups revealed that patients older than 50 years of age more often exhibited a mutated EGFRvIII gene (48.6%) than younger patients (23.5%). This difference was also seen for the GBL subgroup, where none of the patients younger than 50 years of age had the EGFRvIII mutation, while in the subgroup of older patients 36.67% exhibited this mutation (Fisher exact test, p < 0.05). For AA tumours we did not find a statistically significant difference according to age (older than 50 years old 66.67%, younger 50%).

In comparison of EGFR gene overexpression frequency in the context of age, we did not find significantly higher EGFR expression in the older patient subgroup.

Discussion

In our study we analysed the expression of wild-type EGFR: it was found to be expressed in 61.8% of brain gliomas, with strongly positive expression in 12.2% of them. The results are in the range observed by others: Yoon et al. (RT-PCR, 36 patients with GBL) found EGFR expression in 36% of cases [54]; Schlegel et al. (RT-PCR and Southern blot, 42 GBL) observed in­creased EGFR expression in 64%, but gene amplification in 45% [38]; Arjona et al. (86 brain gliomas) ob­served overexpression in 40% of cases, but no EGFR expression in 3.5% of analysed tumours [2]. Aldape and colleagues analysed EGFR expression by both RT-PCR and immunohistochemistry; they found gene expression in 66% of cases, and presence of mutated EGFRvIII form in 44%. Deletion was observed in parallel to gene over-expression.

In our study, for a detailed analysis of EGFR transcript expression we used three different amplicons located within the EGFR gene: within the exon 2-3 junction, extracellular domain, including the most frequent EGFRvIII deletion site; exon 13-14 junction, anchoring domain of receptor within the cellular membrane (Voldborg et al. observed that a deletion in the transmembrane domain makes the protein unable to anchor in the membrane [45]), with relatively low mutation rate in gliomas; and the third amplicon was located at the exon 18-19 junction, corresponding to the intracellular gene domain. Simultaneous analysis of three different amplicons allowed us to obtain a reliable estimate of gene expression. We simultaneously analysed by RT-PCR the EGFRvIII status and found the deletion in 21.3% of tumours, in the range observed by other authors (20 to 67% of cases [16,49]). It was found that gene amplifications can be found in both wild and mutated form of the molecule [27,32,40,51,52], and this seems to be confirmed by our data.

One of the consequences of mutation of tyrosine kinase genes such as EGFR or PDGFR is stimulation of cell proliferation and their transformation under stimulation of activated Ras proteins [48]. Malignant phenotype of glial tumours may also be a result of high expression of factors promoting invasion and cell migration (such as protein kinase C – PKC [29], or mitogen activated protein kinase – MAPK [9]), as well as autocrine loops activating receptors propagating invasion (e.g. EGFRvIII [17]). Production of proteolytic factors such as MMP is also essential for infiltration of nerve tissue [24].

EGFR has been a target for successful anti-cancer therapeutic strategies in different malignancies, including colon, head and neck and lung cancer. Studies concerning EGFR biology in glioma biology play a significant role, especially to understand the inherent differences underlying the growth and therapy resistance of these tumours [15]. EGFR activation promotes dedifferentiation of cells, disturbs natural processes of proliferation and simultaneously has anti-apoptotic capabilities. Moreover, stimulation of EGFR also causes an increase of neoplastic angiogenesis and promotes mechanisms of cell migration and invasion of tumours into surrounding nervous tissue. Additionally, frequent mutations of the gene (including EGFRvIII), especially in combination with amplification, lead to hyperactivity of the receptor indepen­dently of ligands, and are important factors in ma­­lignant biology of gliomas [4]. EGFRvIII has also been shown to confer resistance to both radiotherapy and chemotherapy [24]. Very interesting in a clinical context is the fact of different EGFR expression and its mutation according to age of patients. Malignant gliomas in children seem to be clinically and biologically distinct from their adult counterparts [3,22]. As mentioned above, GBLs in the adult population are classified as primary or secondary based on progression from pre-existing low-grade tumours and on distinct patterns of molecular abnormalities. The primary (de novo) GBLs which typically affect older patients are characterized by amplification of EGFR along with deletion or mutation of the phosphatase and tensin homologue tumour suppressor gene (PTEN), a negative regulator of the phosphatidyl inositol 3 kinase/Akt signalling pathway. Secondary GBLs, which occur in younger individuals, often have mutations of the tumour suppressor gene TP53 but only infrequently have amplification of EGFR or alterations of PTEN [3,30,43]. It seems (considering molecular biology) that paediatric cases are similar to secondary adult GBLs [3,30,43]. But on the other hand, these neoplasms rarely originate from pre-existing low-grade lesions. What is important, also other malignant primary brain tumours (e.g. anaplastic ependymomas) show similar differences when comparing paediatric, young adult and older populations [28]. The aforementioned molecular differences concern EGFR expression and its mutation. The EGFR gene is also overexpressed in the most common malignant primary brain tumours in children – medulloblastomas [21]. EGFRvIII mutation was described in 4 of 6 cases of astrocytoma in children, and in 6 of 7 cases of medulloblastomas [45].

In our group EGFRvIII mutation was significantly more frequent in patients older than 50 years of age (48.6%) than in younger patients (23.5%). There was also a statistically significant difference in subgroups of patients with GBL, where no tumours derived from patients younger than 50 years of age had EGFRvIII mutation whereas in the older subgroup of patients the frequency of this mutation reached 36.67%. On the other hand, we did not find any statistically significant difference in the frequency of EGFR overexpression between age groups. Nevertheless, younger patients (below 50 years of age) had lower expression levels in comparison to older patients in both mean expression of all three analysed domains as well as in mean expression level for two intracellular domains. The same results were obtained for subgroups with GBL or AA tumours.

Certainly, the role of EGFR should be discussed widely in the context of MGMT epigenetic regulation, confirmed as an important prognostic factor in glioma and clearly related to cell sensitivity to genotoxic factors. Similarly, its relation to mutations in the isocitrate dehydrogenase 1 gene (IDH1), possibly correlated with overall survival [34], needs to be discus­sed, especially due to its potential importance in young­er patients. To shed more light on the age-dependent differences in EGFR expression we intend to compare the expression of the gene in three age groups, i.e. paediatric, young adults and older patients with high-grade gliomas, in a subsequent study, stratified ac­cor­d­ing to MGMT and IDH1 status.

To conclude, in our analysis we observed EGFRvIII mutation to be more frequent in patients older than 50 years, with a non-significant trend toward higher gene expression in older subjects. As nearly 1/3 of high grade gliomas do not demonstrate abnormal gene expression levels, EGFR status should be taken into account in any targeted therapy attempt. The sig­nificance of EGFR axis-related differences between young and old glioma patients and their impact on the prognosis warrant further study.

Acknowledgments

This study was supported by the Polish Ministry of Scientific Research and Information Technology (PBZ/MNiSW/07/2006/21).

References

 1. Aldape KD, Ballman K, Furth A, Buckner JC, Giannini C, Burger PC, Scheithauer BW, Jenkins RB, James CD. Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance. J Neuropathol Exp Neurol 2004; 63: 700-707.  

2. Arjona D, Bello MJ, Alonso ME, Aminoso C, Isla A, De Campos JM, Sarasa JL, Gutierrez M, Villalobo A, Rey JA. Molecular analysis of the EGFR gene in astrocytic gliomas: mRNA expression, quantitative-PCR analysis of non-homogeneous gene amplification and DNA sequence alterations. Neuropathol Appl Neurobiol 2005; 31: 384-394.  

3. Bax DA, Gaspar N, Little SE, Marshall L, Perryman L, Regairaz M, Viana-Pereira M, Vuononvirta R, Sharp SY, Reis-Filho JS, Stáva­le JN, Al-Sarraj S, Reis RM,Vassal G, Pearson AD, Hargrave D, Ellison DW, Workman P, Jones C. EGFRvIII deletion mutations in pediatric high-grade glioma and response to targeted the­rapy in pediatric glioma cell lines. Clin Cancer Res 2009; 15: 5753-5761.  

4. Bianco R, Troiani T, Tortora G, Ciardiello F. Intrinsic and acquired resistance to EGFR inhibitors in human cancer therapy. Endocr Relat Cancer 2005; 12 Suppl 1: 159-171.  

5. Bigner SH, Vogelstein B. Cytogenetics and molecular genetics of malignant gliomas and medulloblastoma. Brain Pathol 1990; 1: 12-18.  

6. Brat DJ, Shehata BM, Castellano-Sanchez AA, Hawkins C, Yost RB, Greco C, Mazewski C, Janss A, Ohgaki H, Perry A. Congenital glio­blastoma: a clinicopathologic and genetic analysis. Brain Pathol 2007; 17: 276-281.  

7. Collins VP, James CD. Gene and chromosomal alterations associated with the development of human gliomas. FASEB J 1993; 7: 926-930.  

8. Collins VP. Oncogenesis and Progression in Human Gliomas. In: Brain tumor invasion. Mikkelsen T, Bjerkvig R, Laerum OD, Rosenblum ML (eds.). Wiley-Liss, New York 1998; pp. 71-85.  

9. Feldkamp MM, Lau N, Guha A. Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects. Oncogene 1999; 18: 7514-7526.

10. Frankel RH, Bayona W, Koslow M, Newcomb EW. p53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency. Cancer Res 1992; 52: 1427-1433.

11. Fults D, Brockmeyer D, Tullous MW, Pedone CA, Cawthon RM. p53 mutation and loss of heterozygosity on chromosomes 17 and 10 during human astrocytoma progression. Cancer Res 1992; 52: 674-679.

12. Fults D, Tippets RH, Thomas GA, Nakamura Y, White R. Loss of heterozygosity for loci on chromosome 17p in human malignant astrocytoma. Cancer Res 1989; 49: 6572-6577.

13. Ganigi PM, Santosh V, Anandh B, Chandramouli BA, Sastry Kolluri VR. Expression of p53, EGFR, pRb and bcl-2 proteins in pediatric glioblastoma multiforme: a study of 54 patients. Pediatr Neurosurg 2005; 41: 292-299.

14. Grandis JR, Tweardy DJ. Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res 1993; 53: 3579-3584.

15. Harari PM. Epidermal growth factor receptor inhibition strategies in oncology. Endocr Relat Cancer 2004; 11: 689-708.

16. Heimberger A, Suki D, Yang D, Shi W, Aldape K. The natural history of EGFR and EGFRvIII in glioblastoma patients. J Transl Med 2005; 3: 38. doi: 10.1186/1479-5876-3-38.

17. Holbro T, Hynes NE. ErbB receptors: directing key signaling networks throughout life. Annu Rev Pharmacol Toxicol 2004; 44: 195-217.

18. Holland EC, Hively WP, DePinho RA, Varmus HE. A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma- like lesions in mice. Genes Dev 1998; 12: 3675-3685.

19. Izycka-Swieszewska E, Brzeskwiniewicz M, Wozniak A, Dro­zyn­ska E, Grajkowska W, Perek D, Balcerska A, Klepacka T, Limon J. EGFR, PIK3CA and PTEN gene status and their protein product expression in neuroblastic tumors. Folia Neuropathol 2010; 48: 238-245.

20. James CD, He J, Carlbom E, Nordenskjold M, Cavenee WK, Collins VP. Chromosome 9 deletion mapping reveals interferon alpha and interferon beta-1 gene deletions in human glial tumors. Cancer Res 1991; 51: 1684-1688.

21. Kagawa N, Maruno M, Suzuki T, Hashiba T, Hashimoto N, Izumo­to S, Yoshimine T. Detection of genetic and chromosomal aberrations in medulloblastomas and primitive neuroectodermal tu­mors with DNA microarrays. Brain Tumor Pathol 2006; 23: 41-47.

22. Kleihues P, Ohgaki H. Phenotype vs genotype in the evolution of Astrocytic brain tumors. Toxicol Pathol 2000; 28: 164-170.

23. Krakstad C, Chekenya M. Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics. Mol Cancer 2010; 9: 135.

24. Lammerng D, Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, McLendon RE, Moscatello D, Peg­ram CN, Reist CJ. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 1995; 55: 3140-3148.

25. Lang FF, Miller DC, Pisharody S, Koslow M, Newcomb EW. High frequency of p53 protein accumulation without p53 gene mutation in human juvenile pilocytic, low grade and anaplastic astrocytomas. Oncogene 1994; 9: 949-954.

26. Libermann TA, Razon N, Bartal AD, Yarden Y, Schlessinger J, So­req H. Expression of epidermal growth factor receptors in human brain tumors. Cancer Res 1984; 44: 753-760.

27. Liu W, James CD, Frederick L, Alderete BE, Jenkins RB. PTEN/ MMAC1 mutations and EGFR amplification in glioblastomas. Cancer Res 1997; 57: 5254-5257.

28. Massimino M, Buttarelli FR, Antonelli M, Gandola L, Modena P, Giangaspero F. Intracranial ependymoma: factors affecting outcome. Future Oncol 2009; 5: 207-216.

29. Meng QH, Zhou LX, Luo JL, Cao JP, Tong J, Fan SJ. Effect of 7-hydroxystaurosporine on glioblastoma cell invasion and mi­gration. Acta Pharmacol Sin 2005; 26: 492-499.

30. Nakamura M, Shimada K, Ishida E, Higuchi T, Nakase H, Sakaki T, Konishi N. Molecular pathogenesis of pediatric astrocytic tumors. Neuro Oncol 2007; 9: 113-123.

31. Neal DE, Marsh C, Bennett MK, Abel PD, Hall RR, Sainsbury JR, Harris AL. Epidermal-growth-factor receptors in human bladder cancer: comparison of invasive and superficial tumours. Lancet 1985; 1: 366-368.

32. 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: 215-222.

33. Ozanne B, Richards CS, Hendler F, Burns D, Gusterson B. Over-expression of the EGF receptor is a hallmark of squamous cell carcinomas. J Pathol 1986; 149: 9-14.

34. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, Olivi A, McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y, Busam DA, Tekleab H, Diaz LA Jr, Hartigan J, Smith DR, Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ, Bigner DD, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW. An integrated genomic analysis of human glioblastoma multiforme. Science 2008; 321: 1807-1812.

35. Pollack IF, Randall MS, Kristofik MP, Kelly RH, Selker RG, Vertosick FT. Response of malignant glioma cell lines to epidermal growth factor and platelet-derived growth factor in a serum-free medium. J Neurosurg 1990; 73: 106-112.

36. Riemenschneider MJ, Buschges R, Wolter M, Reifenberger J, Bostrom J, Kraus JA, Schlegel U, Reifenberger G. Amplification and overexpression of the MDM4 (MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation or MDM2 amplification. Cancer Res 1999; 59: 6091-6096.

37. Sainsbury JR, Malcolm AJ, Appleton DR, Farndon JR, Harris AL. Presence of epidermal growth factor receptor as an indicator of poor prognosis in patients with breast cancer. J Clin Pathol 1985; 38: 1225-1228.

38. Schlegel J, Merdes A, Stumm G, Albert FK, Forsting M, Hynes N, Kiessling M. Amplification of the epidermal-growth-factor-receptor gene correlates with different growth behaviour in human glioblastoma. Int J Cancer 1994; 56: 72-77.

39. Shih HA, Betensky RA, Dorfman MV, Louis DN, Loeffler JS, Batchelor TT. Genetic analyses for predictors of radiation response in glioblastoma. Int J Radiat Oncol Biol Phys 2005; 63: 704-710.

40. Smith JS, Tachibana I, Passe SM, Huntley BK, Borell TJ, Iturria N, O’Fallon JR, Schaefer PL, Scheithauer BW, James CD, Buckner JC, Jenkins RB. PTEN mutation, EGFR amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J Natl Cancer Inst 2001; 93: 1246-1256.

41. Smits A. Low-grade gliomas: clinical and pathobiological aspects. Histol Histopathol 2002; 17: 253-260.

42. Sobol RE, Astarita RW, Hofeditz C, Masui H, Fairshter R, Royston I, Mendelsohn J. Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody. J Natl Cancer Inst 1987; 79: 403-407.

43. Suri V, Das P, Pathak P, Jain A, Sharma MC, Borkar SA, Suri A, Gupta D, Sarkar C. Pediatric glioblastomas: a histopathological and molecular genetic study. Neuro Oncol 2009; 11: 274-280.

44. Ulutin C, Fayda M, Aksu G, Cetinayak O, Kuzhan O, Ors F, Bey­zadeoglu M. Primary glioblastoma multiforme in younger pa­tients: a single-institution experience. Tumori 2006; 92: 407-411.

45. Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 1997; 8: 1197-1206.

46. von Deimling A, Louis DN, von Ammon K, Petersen I, Hoell T, Chung RY, Martuza RL, Schoenfeld DA, Yasargil MG, Wiest­ler OD. Association of epidermal growth factor receptor gene amplification with loss of chromosome 10 in human glioblastoma multiforme. J Neurosurg 1993; 77: 295-301.

47. von Deimling A, Louis DN, von Ammon K, Petersen I, Wiestler OD, Seizinger BR. Evidence for a tumor suppressor gene on chromosome 19q associated with human astrocytomas, oligodendrogliomas, and mixed gliomas. Cancer Res 1992; 52: 4277-4279.

48. Watanabe K, Tachibana O, Sata K, Yonekawa Y, Kleihues P, Ohgaki H. Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas. Brain Pathol 1996; 6: 217-223; discussion 23-24.

49. Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, McLendon RE,

Moscatello D, Pegram CN, Reist CJ, et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 1995; 55: 3140-3148.

50. Wolańczyk M, Hułas-Bigoszewska K, Witusik-Perkowska M, Papierz W, Jaskólski D, Liberski PP, Rieske P. Imperfect oligodendrocytic and neuronal differentiation of glioblastoma cells. Folia Neuropathol 2010; 48: 27-34.

51. Wygoda Z, Kula D, Bierzyńska-Macyszyn G, Larysz D, Jarzab M, Właszczuk P, Bazowski P, Wojtacha M, Rudnik A, Stepień T, Kaspera W, Etmańska A, Składowski K,Tarnawski R, Kokocińska D, Jarzab B. Use of monoclonal anti-EGFR antibody in the radioimmunotherapy of malignant gliomas in the context of EGFR expression in grade III and IV tumors. Hybridoma (Larchmt) 2006; 25: 125-132.

52. Wygoda Z, Tarnawski R, Brady L, Steplewski Z, Bazowski P, Wojtacha M, Stepien T, Kula D, Skladowski K, Kokocinska D, Wygoda A, Pawlaczek A, Etmanska A, Larysz D, Jarzab B. Simultaneous radiotherapy and radioimmunotherapy of malignant gliomas with anti-EGFR antibody labelled with iodine 125. Preliminary results. Nucl Med Rev Cent East Eur 2002; 5: 29-33.

53. Yamauchi T, Ueki K, Tobe K, Tamemoto H, Sekine N, Wada M, Honjo M, Takahashi M, Takahashi T, Hirai H, Tushima T, Akanuma Y, Fujita T, Komuro I, Yazaki Y, Kadowaki T. Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone. Nature 1997; 390: 91-96.

54. Yoon KS, Lee MC, Kang SS, Kim JH, Jung S, Kim YJ, Lee JH, Ahn KY, Lee JS, Cheon JY. p53 mutation and epidermal growth factor receptor overexpression in glioblastoma. J Korean Med Sci 2001; 16: 481-488.

55. Zawrocki A, Biernat W. Epidermal growth factor receptor in glioblastoma. Folia Neuropathol 2005; 43: 123-132.