Screening for EGFR Amplifications with a Novel Method and Their Significance for the Outcome of Glioblastoma Patients

Abstract

Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (i.e. TP53 and IDH1 mutations, CDKN2A deletion, EGFR amplification, chromosome 7 polysomy and EGFRvIII expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for EGFR amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient’s age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, EGFR amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, EGFR amplification was related to a better prognosis in combination with the homozygous CDKN2A deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased EGFR gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient’s tumour molecular characteristics in the selection of the therapy.

Identification of Postoperative Margins of Glioblastoma Multiforme Using Gold Nanoparticles Conjugated to Epidermal Growth Factor Antibodies

“…To determine EGFR expression in the GBM samples, we measured the expression of the GPER and RNaseP genes, as recently described [6]. The primer sequence was kindly provided by Dr. Bieńkowski and his group.…”

“…A ratio of GPER expression to RNaseP expression above 1.5 is considered indicative of chromosome 7 polysomy. A ratio of EGFR expression to RNase P expression above 1.5 is considered indicative of EGFR amplification [6]. Cut off was calculated according to a previous publication [6].…”

“…The following materials were added to the reaction mixture: Sybr Green (5 μl), 10 μM reverse primer (0.5 μl), 10 μM forward primer (0.5 μl), DNA (10 μl) and ddH 2 0 (3 Melting curves were generated to analyze the specificity of gene amplification. The expression levels of each gene of interest were calculated from the generated data [6,9,16].…”

“…Despite current therapies, it has a rapidly progressive and fatal course [5]. A recent study of the molecular genetics of GBM reported overexpression of the EGFR gene in the majority of tumors evaluated, either by amplification, which results in multiple copies of the gene in chromosomal regions (amplicons) [2] or by polysomy of chromosome 7 which contains the EGFR gene [6]. In very rare cases, EGFR overexpression may be due to a de novo mutation [4] .…”

Biomarkers in Glioblastoma multiforme. Evidences from a literature review

“…Studies reporting the prognostic or predictive value of EGFRvIII are listed in Table 1 [8,15,16,[21][22][23][24][25][26][27][28][29][30][31][32][33][34]. We found 17 studies considering 1656 GBM patients overall.…”