Genome-wide analyses identify recurrent amplifications of receptor tyrosine kinases and cell-cycle regulatory genes in diffuse intrinsic pontine glioma

Abstract

Purpose: Long-term survival for children with diffuse intrinsic pontine glioma (DIPG) is less than 10%, and new therapeutic targets are urgently required. We evaluated a large cohort of DIPGs to identify recurrent genomic abnormalities and gene expression signatures underlying DIPG.

Patients and methods: Single-nucleotide polymorphism arrays were used to compare the frequencies of genomic copy number abnormalities in 43 DIPGs and eight low-grade brainstem gliomas with data from adult and pediatric (non-DIPG) glioblastomas, and expression profiles were evaluated using gene expression arrays for 27 DIPGs, six low-grade brainstem gliomas, and 66 nonbrainstem low-grade gliomas.

Results: Frequencies of specific large-scale and focal imbalances varied significantly between DIPGs and nonbrainstem pediatric glioblastomas. Focal amplifications of genes within the receptor tyrosine kinase-Ras-phosphoinositide 3-kinase signaling pathway were found in 47% of DIPGs, the most common of which involved PDGFRA and MET. Thirty percent of DIPGs contained focal amplifications of cell-cycle regulatory genes controlling retinoblastoma protein (RB) phosphorylation, and 21% had concurrent amplification of genes from both pathways. Some tumors showed heterogeneity in amplification patterns. DIPGs showed distinct gene expression signatures related to developmental processes compared with nonbrainstem pediatric high-grade gliomas, whereas expression signatures of low-grade brainstem and nonbrainstem gliomas were similar.

Conclusion: DIPGs comprise a molecularly related but distinct subgroup of pediatric gliomas. Genomic studies suggest that targeted inhibition of receptor tyrosine kinases and RB regulatory proteins may be useful therapies for DIPG.

Fig 1.

Copy-number abnormalities in diffuse intrinsic pontine glioma (DIPG). (A) Heat map showing segmentation analysis of normalized data from Affymetrix SNP 6.0 arrays to identify copy-number gains (red) and losses (blue) in 43 DIPGs and eight brainstem low-grade gliomas (LGGs). Chromosome positions are indicated along y-axis and separated by dashed line. Histologic subtypes are indicated across top. Scale bar shows color gradient to indicate copy number. Comparison of frequencies of most common large-scale genomic (B) gains or (C) losses in DIPG, compared with adult or pediatric nonbrainstem glioblastoma (GBM). Large-scale gains or losses were scored when more than half of markers on chromosome arm had copy-number gains or losses, respectively. All frequencies and P values listed in Data Supplement. (*) Indicates tumors obtained before adjuvant therapy.

Fig 2.

Heterogeneity of focal amplification in diffuse intrinsic pontine glioma. (A) Fluorescent in situ hybridization showed high-level amplification of PDGFRA in focal area of tumor (PDGFRA, red; control chromosome 4, green). Focus of tumor with PDGFRA amplification and remainder of tumor lacking high-level PDGFRA amplification (data not shown) demonstrated similar histopathologic features; both consisted of densely packed tumor cells with minimal normal tissue. PDGFRA amplification was found in both solid groups of tumor cells (B: hematoxylin and eosin [HE] staining) and scattered infiltrating tumor cells (C: HE). Coamplification of PDGFRA and MET in BSG009T (D to F) and BSG037T (G to I). Cells with amplified PDGFRA (D, G: PDGFRA, red; control, green) and MET (E, H: MET, red; control, green) showed coamplification (F, I: PDGFRA, red; MET, green) of (F) both genes in same tumor cells (arrows) or (I) independent amplification in different tumor subclones (arrows).

Fig 3.

Unsupervised hierarchical clustering (UHC) of diffuse intrinsic pontine glioma (DIPG) showed subgroups similar to those previously identified in adult and pediatric high-grade gliomas. (A) Dendrogram of UHC using top 1,000 most variable probe sets selected using median absolute deviation scores, and heat map featuring top 150 signature probe sets of each subgroup. Three main subgroups were identified. Tumors with focal gains of components in receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) or retinoblastoma protein (RB) pathway are indicated in red at top of heat map. (B, C, D) Gene set enrichment analysis to evaluate coordinate expression in DIPG of gene sets defining subclasses of adult and pediatric high-grade gliomas^, showed that DIPG subgroups identified by UHC are highly similar to subgroups previously identified. Plots of running enrichment scores showed highly significant enrichment of mesenchymal markers in HC1, proliferative markers in HC2, and pediatric proneural markers in HC3; 33,928 genes were analyzed. (B) 132 genes in gene set; P = 0, false discovery rate (FDR) = .00162. (C) 176 genes in gene set; P = .0457, FDR = .216. (D) 616 genes in gene set; P = 0, FDR = .0445.

Fig A1.

Principal component analysis (PCA) showed differences in expression signature based on tumor location for diffuse intrinsic pontine glioma (DIPG) but not for brainstem low-grade gliomas (LGGs). (A) PCA generated using 1,000 most variable probes, comparing 29 DIPGs (red), including two patient cases of DIPG obtained before treatment, as previously described (Paugh BS, Qu C, Jones C, et al: J Clin Oncol 28:3061-3068, 2010), and 51 nonbrainstem pediatric high-grade gliomas (HGGs; blue). Arrowheads indicate autopsy samples clustering with tumors outside brainstem. Arrows indicate matched diagnostic (with asterisk) and autopsy sample from same patient. (B) PCA shows DIPG (red), nonbrainstem pediatric HGGs (blue), brainstem LGGs (yellow), and nonbrainstem LGGs (green). (*) Indicates samples obtained before treatment.