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Genetic-molecular study of the EGFR/PI3K/AKT pathway in high and low grade gliomas

      Gliomas account for 70% of all central nervous system (CNS) tumors and are classified according to the WHO in low (I and II) or high (III and IV or glioblastoma multiforme, GBM) grade malignancies. The survival of patients with a high grade is less than 2 years. The EGFR/PI3K/AKT pathway promotes cellular survival, inhibits apoptosis and differentiation. This pathway is regulated by phosphatase homologous of tensin (PTEN). In gliomas, amplification of EGFR and PI3K has been reported, as well as mutations in PI3KCA and PTEN, which are associated with permanent activation of this signaling pathway and with decreased expression of pro-apoptotic genes such as Bax and increased expression of anti-apoptotic genes such as Bcl2. We obtained 30 samples of gliomas with high and low grade. Amplification was assessed by real-time PCR with TaqMan probes for the EGFR, PI3K and AKT genes. Mutations in PI3KCA (9 and 20 exons) and PTEN (5 and 6 exons) were determined by direct sequencing from DNA using specific primers for each exon. The results of each sample were analyzed in 5′–3′ and 3′–5′ orientations using the program FINCH TV. Subsequent alignment was performed for each sample with the human reference sequence (obtained from Gene Bank) using the GENE RUNNER program. To assess the expression levels of the PI3K, AKT, Bax, and Bcl2 genes, cDNA obtained by reverse transcription was amplified by real-time PCR with SYBR Green using specific primers for each gene. The results were analyzed by calculating the CT. We did not find any high level EGFR amplifications. Only one grade IV sample showed an increase in gene dosage (2.8 times). PI3K was found amplified in 50% of grade I and II (0.4 times) and in 45% of grade III and IV (between 0.5 and 0.9 times). However, in 18% of the grade IV samples, the increase was greater than 1. AKT was not found amplified in the samples analyzed. Also, no mutations were found in PI3K and PTEN. In low-grade gliomas we found an increase (>1 time) in PI3K (62%), AKT (12.5%), Bcl2 (50%), and Bax (25%) gene expression. In high-grade tumors over-expression of PI3K (50%), AKT, Bcl2, and Bax (all 32%) was found.
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