Distinct phenotypic differences associated with differential amplification of receptor tyrosine kinase genes at 4q12 in glioblastoma

Abstract

Gene amplification at chromosome 4q12 is a common alteration in human high grade gliomas including glioblastoma, a CNS tumour with consistently poor prognosis. This locus harbours the known oncogenes encoding the receptor tyrosine kinases PDGFRA, KIT, and VEGFR2. These receptors are potential targets for novel therapeutic intervention in these diseases, with expression noted in tumour cells and/or associated vasculature. Despite this, a detailed assessment of their relative contributions to different high grade glioma histologies and the underlying heterogeneity within glioblastoma has been lacking. We studied 342 primary high grade gliomas for individual gene amplification using specific FISH probes, as well as receptor expression in the tumour and endothelial cells by immunohistochemistry, and correlated our findings with specific tumour cell morphological types and patterns of vasculature. We identified amplicons which encompassed PDGFRA only, PDGFRA/KIT, and PDGFRA/KIT/VEGFR2, with distinct phenotypic correlates. Within glioblastoma specimens, PDGFRA amplification alone was linked to oligodendroglial, small cell and sarcomatous tumour cell morphologies, and rare MGMT promoter methylation. A younger age at diagnosis and better clinical outcome in glioblastoma patients is only seen when PDGFRA and KIT are co-amplified. IDH1 mutation was only found when all three genes are amplified; this is a subgroup which also harbours extensive MGMT promoter methylation. Whilst PDGFRA amplification was tightly linked to tumour expression of the receptor, this was not the case for KIT or VEGFR2. Thus we have identified differential patterns of gene amplification and expression of RTKs at the 4q12 locus to be associated with specific phenotypes which may reflect their distinct underlying mechanisms.

Figure 1. Gene-specific BAC probes used for FISH.

Probes (green bars) were selected according the hg19 (February 2009) build of the human genome to selectively overlap with the coding sequence for either PDGFRA (RP11-819D11, RP11-58C6), KIT (RP11-452H23, RP11-586A2) or VEGFR2 (CTD-2169F23, RP11-1125E19, RP11-643K20) alone.

Figure 2. Amplification of 4q12 genes in glioblastoma.

(A) Representative FISH images showing individual amplification of PDGFRA, KIT, and VEGFR2 in glioblastoma samples RMH6862, RMH6427 and RMH6881, respectively. Original magnification ×1000. (B) Pie charts showing the relative proportions of the predominant cellular morphology in glioblastoma samples for cases with PDGFRA, KIT, and VEGFR2 amplification. Fibrillary, purple; gemistocytic, blue; giant cell, green; small cell, light green; oligodendroglial, yellow; and sarcomatous, orange. (C) Kaplan-Meier curves for the effect of amplification of PDGFRA, KIT, and VEGFR2 on overall survival in glioblastoma patients. P values are given using the log-rank test for overall survival.

Figure 3. Differential patterns of 4q12 gene amplification.

(A) Pie chart showing the relative proportions of distinct gene amplification subtypes in glioblastoma. All three genes, black; PDGFRA and KIT, purple; PDGFRA-only, red; normal copy number of all three genes, grey. (B) Boxplot showing the age distribution of the gene amplification subtypes. * p<0.05, t test. (C) Kaplan-Meier curves for the effect of gene amplification subtypes on clinical outcome.

Figure 4. Expression of PDGFRA, KIT and VEGFR2 receptors in glioblastoma tumour cells.

(A) Representative images showing tumour cell positivity for PDGFRA, KIT, and VEGFR2 in glioblastoma samples RMH6374, RMH6357 and RMH6691, respectively. Original magnification ×400. (B) Pie charts showing the relative proportions of the predominant cellular morphology in glioblastoma samples for cases overexpressing PDGFRA, KIT, and VEGFR2. Fibrillary, purple; gemistocytic, blue; giant cell, green; small cell, light green; oligodendroglial, yellow; and sarcomatous, orange. (C) Kaplan-Meier curves for the effect of overexpression of PDGFRA, KIT, and VEGFR2 on overall survival in glioblastoma patients. P values are given using the log-rank test for overall survival.

Figure 5. Vascular endothelial cell expression of PDGFRA, KIT and VEGFR2 receptors in glioblastoma.

(A) Representative images showing endothelial cell expression of PDGFRA, KIT, and VEGFR2 in glioblastoma samples RMH6401, RMH6657 and RMH6713, respectively. Original magnification ×400. (B) Pie charts showing the relative proportions of the predominant cellular morphology in glioblastoma samples for cases with vessels expressing PDGFRA, KIT, and VEGFR2. Fibrillary, purple; gemistocytic, blue; giant cell, green; small cell, light green; oligodendroglial, yellow; and sarcomatous, orange. (C) Kaplan-Meier curves for the effect of vascular expression of PDGFRA, KIT, and VEGFR2 on overall survival in glioblastoma patients. P values are given using the log-rank test for overall survival.