Molecular analysis of the EGFR gene in astrocytic gliomas: mRNA expression, quantitative‐PCR analysis of non‐homogeneous gene amplification and DNA sequence alterations

Abstract

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with tyrosine kinase activity. This report investigates the presence of mutations, amplification and/or over‐expression of the EGFR gene in 86 glial tumours including 44 glioblastomas, 21 anaplastic astrocytomas, and 21 WHO grade II astrocytomas, using polymerase chain reaction/single‐strand conformation polymorphism, semiquantitative reverse‐transcription‐polymerase chain reaction (RT‐PCR) and Southern Blot techniques. Gene amplification values were found in 34 tumours. Amplification levels were not uniform, as the transmembrane region presented lower amplification rates than extra‐ and intracellular domains. For the 19 samples with sufficient available tumour tissue we found over‐expression in 11, and no EGFR mRNA expression in three. Ten cases showed deletion transcripts, and EGFR VIII was identified in all of these cases. One of the cases with EGFR vIII also presented a truncated form, C‐958, while another showed an in frame tandem duplication of exons 18–25. We found 14 cases with sequence/structure gene alterations, including seven on which genomic novel DNA changes were identified: a missense mutation (1052C > T/Ala265Val), an insertion (InsCCC2498/Ins Pro748), three intronic changes (E6 + 72delG, E22–14C > G and E18–109T > C), a new polymorphic variant E12 + 22A > T, and one case that presented a 190 bp insertion, that was produced by the intron‐7–exon‐8 duplication and generated a truncated EGFR with intact exons 1–8 followed by an additional amino acidic sequence: Val‐Ile‐Met‐Trp. These findings corroborate that EGFR is non‐randomly involved in malignant glioma development and that different mutant forms participate in aberrant activation of tyrosine kinase pathways.

EGFR and PTEN Gene Mutation Status in Glioblastoma Patients and their Prognostic Impact on Patient's Survival

“…In that way, the present study contributes to a better understanding of glioblastoma genetics, confirming the relatively high frequency of EGFR extracellular domain mutations. Indeed, novel missense mutations of the EGFR extracellular domain have been recently reported in glioblastomas [23][24][25] and few of our mutations that were detected in GBM patients are consistent to these studies. The 13.5% rate of missense mutations of the EGFR extracellular domain in our series is in agreement with the findings of Lee et al [22] but the frequency observed was very less as compared with Lori et al [24].…”

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

“…This receptor plays a prominent role in carcinogenesis and the growth of many solid tumours due to the occurrence in its encoding gene of point mutations, truncations, copy number gene amplification, overexpression and/or deregulated transactivation with distinct G protein-coupled receptors [4][5][6][7]. Therefore, the EGFR has been widely targeted for cancer therapy [8].…”

Gene dosage and mutational analyses of EGFR in oligodendrogliomas

“…All the samples were analyzed in duplicate. Genomic amplification of exons was performed with an initial amount of DNA of 50 ng/μl and using the Light Cycler FarstStart DNA Master SYRB Green kit (Roche Molecular Biochemical) (12,13).…”

“…PCR was carried out as described (12,13) briefly: after an initial (pre-PCR) 10-min pre-incubation step at 95˚C, 45 amplification cycles were run, each consisting of 95˚C for 10 sec, 63-55˚C for 10 sec, 72-82˚C for 10 sec and (exon 11, exon 25) an additional step for exon 11 and 25, at 80˚C or 85˚C for 2 sec, respectively, was also performed. The relative amounts of exon products were compared with the reference gene (18S) and calculated with Light Cycler Relative Quantification Software (Roche Molecular Biochemicals).…”