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Screening for EGFR amplifications with a novel method and their significance for the outcome of glioblastoma patients.

Bieńkowski M, Piaskowski S, Stoczyńska-Fidelus E, Szybka M, Banaszczyk M, Witusik-Perkowska M, Jesień-Lewandowicz E, Jaskólski DJ, Radomiak-Załuska A, Jesionek-Kupnicka D, Sikorska B, Papierz W, Rieske P, Liberski PP - PLoS ONE (2013)

Bottom Line: At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04).We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University of Lodz, Lodz, Poland. michal.bienkowski@gmail.com

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.

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A diagram depicting the premises upon which the EGFR gene copy number analysis is based.In normal cells both the ratio of EGFR to GPER and the ratio of GPER to RNase is equal to 1. In cells with chromosome 7 polysomy the ratio of GPER to RNase increases, while in cells with EGFR amplification the ratio of EGFR to GPER increases. In cells with both the polysomy and the amplification both ratios are increased and the ratio of EGFR to RNase is equal to their product. A. normal cell; B. cell with chromosome 7 polysomy; C. cell with extrachromosomal EGFR amplification.
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pone-0065444-g001: A diagram depicting the premises upon which the EGFR gene copy number analysis is based.In normal cells both the ratio of EGFR to GPER and the ratio of GPER to RNase is equal to 1. In cells with chromosome 7 polysomy the ratio of GPER to RNase increases, while in cells with EGFR amplification the ratio of EGFR to GPER increases. In cells with both the polysomy and the amplification both ratios are increased and the ratio of EGFR to RNase is equal to their product. A. normal cell; B. cell with chromosome 7 polysomy; C. cell with extrachromosomal EGFR amplification.

Mentions: To determine the EGFR gene dosage level in each sample quantitative Real-Time PCR was performed using Rotor-Gene 6000 system (Qiagen, Germany). Each sample was amplified in triplicate in a 10 µl reaction volume containing 10 ng of DNA, a 1x reaction mixture containing Syto9 (Life Technologies, US) and 35 ng each of the forward and reverse primers. The cycling conditions for the Real-Time PCR reactions were as follows: 3 min at 95°C (polymerase activation) followed by 40 cycles of 20 s at 95°C (denaturation), 30 s at 60°C (annealing) and 20 s at 72°C (extension). All primer sequences are listed in Table S1. The gene dissociation curve was analysed for each sample to confirm the specificity of the amplification signal. The normalised relative gene dosage level of the tested samples compared to the control sample was calculated using the method previously described by Pfaffl et al. based on each sample’s average CT value and each gene’s average PCR efficiency [9]. DNA derived from non-tumorous tissue (peripheral blood leukocytes) was used as a control and the gene dosage in normal tissue was assumed to be 1. The technique presented is based on the assumption that the ratio of EGFR to the other marker, located within chromosome 7, would be equal to 1 if there are no amplicons (assuming no LOH within either of the analysed loci). The selection of the chromosome 7 marker was based on two criteria. Firstly, the locus of the marker had to be located within the same chromosomal arm at an appropriate distance from the EGFR gene locus (7p12) in order to minimise the probability of its inclusion into the amplicons [10], [11]. Secondly, the locus of the marker had to be located within a region which is retained in gliomas. These criteria were fulfilled by the GPER gene (7p22) [12], [13]. The following method of result interpretation was applied: the cumulative EGFR gene dosage was assessed by the ratio of EGFR to RNaseP (the RPP25 gene located within chromosomal region 15q24.2) [14], [15]; chromosome 7 polysomy was identified when the ratio of GPER to RNaseP was higher than 1.5; while EGFR amplification was identified when the ratio of EGFR to GPER was higher than 1.5 (Fig. 1).


Screening for EGFR amplifications with a novel method and their significance for the outcome of glioblastoma patients.

Bieńkowski M, Piaskowski S, Stoczyńska-Fidelus E, Szybka M, Banaszczyk M, Witusik-Perkowska M, Jesień-Lewandowicz E, Jaskólski DJ, Radomiak-Załuska A, Jesionek-Kupnicka D, Sikorska B, Papierz W, Rieske P, Liberski PP - PLoS ONE (2013)

A diagram depicting the premises upon which the EGFR gene copy number analysis is based.In normal cells both the ratio of EGFR to GPER and the ratio of GPER to RNase is equal to 1. In cells with chromosome 7 polysomy the ratio of GPER to RNase increases, while in cells with EGFR amplification the ratio of EGFR to GPER increases. In cells with both the polysomy and the amplification both ratios are increased and the ratio of EGFR to RNase is equal to their product. A. normal cell; B. cell with chromosome 7 polysomy; C. cell with extrachromosomal EGFR amplification.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675194&req=5

pone-0065444-g001: A diagram depicting the premises upon which the EGFR gene copy number analysis is based.In normal cells both the ratio of EGFR to GPER and the ratio of GPER to RNase is equal to 1. In cells with chromosome 7 polysomy the ratio of GPER to RNase increases, while in cells with EGFR amplification the ratio of EGFR to GPER increases. In cells with both the polysomy and the amplification both ratios are increased and the ratio of EGFR to RNase is equal to their product. A. normal cell; B. cell with chromosome 7 polysomy; C. cell with extrachromosomal EGFR amplification.
Mentions: To determine the EGFR gene dosage level in each sample quantitative Real-Time PCR was performed using Rotor-Gene 6000 system (Qiagen, Germany). Each sample was amplified in triplicate in a 10 µl reaction volume containing 10 ng of DNA, a 1x reaction mixture containing Syto9 (Life Technologies, US) and 35 ng each of the forward and reverse primers. The cycling conditions for the Real-Time PCR reactions were as follows: 3 min at 95°C (polymerase activation) followed by 40 cycles of 20 s at 95°C (denaturation), 30 s at 60°C (annealing) and 20 s at 72°C (extension). All primer sequences are listed in Table S1. The gene dissociation curve was analysed for each sample to confirm the specificity of the amplification signal. The normalised relative gene dosage level of the tested samples compared to the control sample was calculated using the method previously described by Pfaffl et al. based on each sample’s average CT value and each gene’s average PCR efficiency [9]. DNA derived from non-tumorous tissue (peripheral blood leukocytes) was used as a control and the gene dosage in normal tissue was assumed to be 1. The technique presented is based on the assumption that the ratio of EGFR to the other marker, located within chromosome 7, would be equal to 1 if there are no amplicons (assuming no LOH within either of the analysed loci). The selection of the chromosome 7 marker was based on two criteria. Firstly, the locus of the marker had to be located within the same chromosomal arm at an appropriate distance from the EGFR gene locus (7p12) in order to minimise the probability of its inclusion into the amplicons [10], [11]. Secondly, the locus of the marker had to be located within a region which is retained in gliomas. These criteria were fulfilled by the GPER gene (7p22) [12], [13]. The following method of result interpretation was applied: the cumulative EGFR gene dosage was assessed by the ratio of EGFR to RNaseP (the RPP25 gene located within chromosomal region 15q24.2) [14], [15]; chromosome 7 polysomy was identified when the ratio of GPER to RNaseP was higher than 1.5; while EGFR amplification was identified when the ratio of EGFR to GPER was higher than 1.5 (Fig. 1).

Bottom Line: At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04).We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University of Lodz, Lodz, Poland. michal.bienkowski@gmail.com

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.

Show MeSH
Related in: MedlinePlus