Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor—EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor—EGFRvIII

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

<div><p>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>i.e. TP53</i> and <i>IDH1</i> mutations, <i>CDKN2A</i> deletion, <i>EGFR</i> amplification, chromosome 7 polysomy and <i>EGFRvIII</i> 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 <i>EGFR</i> 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, <i>EGFRvIII</i> expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, <i>EGFR</i> 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, <i>EGFR</i> amplification was related to a better prognosis in combination with the homozygous <i>CDKN2A</i> 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 <i>EGFR</i> 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.</p></div

Selected results of the statistical analyses.

<p>Cox’s Proprtional Hazard values pertain to the univariate analysis for age and to the multivariate analysis (adjusted for age) for other analyses. HR values refer to the presence of given feature.</p><p>For example:</p><p>In the group of patients younger than 60 years old, the risk of death over given time is 3.745 times higher in those with <i>EGFR</i> amplification than in those without the amplification.</p><p>Abbreviations:</p><p><i>TP53</i>– <i>TP53</i> mutation;</p><p><i>EGFR</i> – <i>EGFR</i> amplification;</p><p>Poly 7– chromosome 7 polysomy;</p><p><i>EGFRvIII</i> – <i>EGFRvIII</i> expression;</p><p><i>CDKN2A</i> – <i>CDKN2A</i> deletion;</p><p>y. o. – years old.</p

Kaplan-Meier diagrams depicting differences in survival times related to the molecular aspects.

<p>The attached table presents statistical data for each diagram. Cox’s proportional hazard refers to multivariate analysis. The calculated HR values pertain to the second subgroup listed, while the HR values of the first subgroup equal to 1. ♦ - complete responses; Δ - censored responses. A. <i>EGFRvIII</i> expression; B. <i>CDKN2A</i> deletion; C. The combination of <i>CDKN2A</i> deletion with <i>EGFR</i> amplification; D. the combination of chromosome 7 polysomy with <i>EGFR</i> amplification.</p

Kaplan-Meier diagrams depicting differences in survival times related to the clinical aspects.

<p>The attached table presents statistical data for each diagram. Cox's proportional hazard refers to univariate analysis for diagram A and to multivariate analysis for diagrams B, C, D. The calculated HR values pertain to the second subgroup listed (“total” subgroup for diagram B), while the HR values of the first subgroup (cumulatively of “partial” and “subtotal” subgroups for diagram B) equal to 1. ♦ - complete responses; Δ - censored responses. A. age of the patient, the threshold of 60 years included in the “younger” subgroup; B. extent of resection; C. radiotherapy; D. radio-chemotherapy.</p

Kaplan-Meier diagrams depicting differences in survival times related to the <i>EGFR</i> amplification and clinical aspects.

<p>The attached table presents statistical data for each diagram. Cox’s proportional hazard refers to multivariate analysis. The calculated HR values pertain to the second subgroup listed, while the HR values of the first subgroup equal to 1. ♦ - complete responses; Δ - censored responses. A. <i>EGFR</i> amplification in patients aged 60 years and less; B. <i>EGFR</i> amplification in patients treated with radiotherapy; C. comparison of patients not treated with radiotherapy with those with the <i>EGFR</i> amplification treated with radiotherapy; D. <i>EGFR</i> amplification in a cumulative group of younger patients and those treated with radiotherapy.</p

A diagram depicting the premises upon which the <i>EGFR</i> gene copy number analysis is based.

<p>In normal cells both the ratio of <i>EGFR</i> to <i>GPER</i> and the ratio of <i>GPER</i> to <i>RNase</i> is equal to 1. In cells with chromosome 7 polysomy the ratio of <i>GPER</i> to <i>RNase</i> increases, while in cells with <i>EGFR</i> amplification the ratio of <i>EGFR</i> to <i>GPER</i> increases. In cells with both the polysomy and the amplification both ratios are increased and the ratio of <i>EGFR</i> to <i>RNase</i> is equal to their product. A. normal cell; B. cell with chromosome 7 polysomy; C. cell with extrachromosomal <i>EGFR</i> amplification.</p