Characterization of novel and complex genomic aberrations in glioblastoma using a 32K BAC array

Abstract

Glioblastomas (GBs) are malignant CNS tumors often associated with devastating symptoms. Patients with GB have a very poor prognosis, and despite treatment, most of them die within 12 months from diagnosis. Several pathways, such as the RAS, tumor protein 53 (TP53), and phosphoinositide kinase 3 (PIK3) pathways, as well as the cell cycle control pathway, have been identified to be disrupted in this tumor. However, emerging data suggest that these aberrations represent only a fraction of the genetic changes involved in gliomagenesis. In this study, we have applied a 32K clone-based genomic array, covering 99% of the current assembly of the human genome, to the detailed genetic profiling of a set of 78 GBs. Complex patterns of aberrations, including high and narrow copy number amplicons, as well as a number of homozygously deleted loci, were identified. Amplicons that varied both in number (three on average) and in size (1.4 Mb on average) were frequently detected (81% of the samples). The loci encompassed not only previously reported oncogenes (EGFR, PDGFRA, MDM2, and CDK4) but also numerous novel oncogenes as GRB10, MKLN1, PPARGC1A, HGF, NAV3, CNTN1, SYT1, and ADAMTSL3. BNC2, PTPLAD2, and PTPRE, on the other hand, represent novel candidate tumor suppressor genes encompassed within homozygously deleted loci. Many of these genes are already linked to several forms of cancer; others represent new candidate genes that may serve as prognostic markers or even as therapeutic targets in the future. The large individual variation observed between the samples demonstrates the underlying complexity of the disease and strengthens the demand for an individualized therapy based on the genetic profile of the patient.

Fig. 1

Frequency of copy number changes in glioblastoma and blood samples calculated for all autosomal clones and plotted relative to the position along the chromosome for each clone, for the 78 tumor samples (B) and for 117 hybridizations run on peripheral blood DNA (D), including 71 samples from healthy subjects and 46 blood samples derived from the glioblastoma (GB) patients included in this study. Red bars above the horizontal line indicate the incidence in percent copy number gains (CNC = 1, 2, 3), and yellow bars represent higher copy number gains (CNC = 2, 3). Green bars below the horizontal line indicate the frequency of copy number losses (CNC = −1, −2), and blue bars indicate the incidence of homozygous deletions (CNC = −2). The 32K array was supplemented with 22_B, a different set of clones representing 22q used in our previously reported chromosome 22-specific array, indicated on the figure as 22b. The chromosome 22_B clone set was printed on the same slide as the 32K clones, and both sets identified the same frequency of copy number aberrations. To identify tumor-specific aberrations, the array-based comparative genomic hybridization results from the series of 78 GB tumor samples were compared to the data obtained from 117 hybridizations run on peripheral blood DNA. Fisher’s exact test was used to evaluate statistically significant differences between gained and nongained clones as well as between deleted and nondeleted clones in tumor versus blood groups. (A and C) The 3,000 top ranked gained (red) and deleted (green) clones, including clones representing chromosomes 7, 19, 20, and 21 for gains and chromosomes 10, 9, 22, 13, and 1 for deletions.

Fig. 2

Identification of multiple, narrow, and high copy number amplicons on chromosomes 7 and 12 in glioblastoma using 32K array. (A) Whole-genome profile of tumor G24064 identifies several narrow amplicons on chromosome 7, in addition to 9p deletion and monosomy 10. (Inset) Chromosome 7 profile reveals several highly amplified regions encompassing candidate oncogene loci. (B) Whole-genome profile of tumor G22576 identifies several independent amplicons on chromosome 12; a narrow amplicon on 9p; two amplified regions on chromosome 4; trisomy 1, 7, and 18; monosomy 2, 10, 11, 13; and partial deletion of 12. (Inset) Chromosome 12 profile reveals several different amplicons encompassing amplified gene loci, including CDK4, RASSF3, and MDM2. Amplicons are indicated in gray bars. The x-axis shows the clone positions for each chromosome, and the y-axis depicts fluorescence ratios. The samples were hybridized against a healthy female reference (F1). Clone mapping information was obtained from Ensembl, and coordinates from human genome assembly of March 2006 (National Center for Biotechnology Information Build 36, hg18) were used.