Characterization of the amplicon on chromosomal segment 4q12 in glioblastoma multiforme

Abstract

A subset of glioblastomas (GBMs) carry gene amplifications on chromosomal segment 4q12. To characterize this amplicon in detail, we analyzed a set of 100 samples consisting of 65 GBMs, 10 WHO grade III astrocytomas, 12 oligodendrogliomas, and 13 glioma cell cultures. We applied multiplex ligation-dependent probe amplification to determine the gene dosage of PDGFRA, KIT, and KDR and the flanking genes USP46, RASL11B, LNX1, CHIC2, SEC3L1, and IGFBP7. The amplicon was highly variable in size and copy number and extended over a region of up to 5 Mb. Amplifications on 4q12 were observed in 15% of GBMs and 23% of GBM cell cultures but not in 22 other gliomas. We analyzed transcription and translation of some genes within this amplicon. Gene amplification generally correlated with high transcript levels but did not necessarily result in increased protein levels. However, we detected frequent expression of proteins encoded by PDGFRA, KIT, and KDR in GBMs and GBM cell cultures independent of the amplification status. Future treatment of GBM patients may include drugs targeting multiple kinases that are encoded by genes on chromosomal segment 4q12.

Fig. 1

Electropherogram and gene amplification pattern of glioblastomas (GBMs). (A) Electropherogram of multiplex ligation-dependent probe amplification (MLPA) products of GBM 21830 and lymphocyte DNA. Product size is shown on the x-axis and ranges from 86 to 168 base pairs. Peak areas corresponding to amplified genes are enhanced relative to unaffected control genes. (B) The diagram shows gene amplification pattern of GBMs 468 and 21830 with high copy numbers.

Fig. 2

Quantification of CHIC2 and PDGFRA transcript levels in GBMs, GBM cell cultures, and temporal lobe (brain) by real-time PCR. Bars correspond to the mean value of triplicates. *Samples with low-grade amplification (<10); **samples with high-grade amplification (>10).

Fig. 3

Protein levels of PDGFRA, KIT, and KDR in GBMs and GBM cell cultures determined by Western blot. Amplification status of the genes is depicted under the lanes. Samples that were also analyzed by real-time PCR are gray. Note that Ponceau S staining showed that in the U-373 MG sample total protein was comparable to the other samples despite the weaker β-actin band. *Samples with low-grade amplification (<10); **samples with high-grade amplification (>10).