Multimodal molecular analysis of astroblastoma enables reclassification of most cases into more specific molecular entities

Abstract

Astroblastoma is a rare and controversial glioma with variable clinical behavior. The diagnosis currently rests on histologic findings of a circumscribed glioma with astroblastomatous pseudorosettes and vascular hyalinization. Immunohistochemical studies have suggested different oncogenic drivers, such as BRAF p.V600E, but very few cases have been studied using genome-wide methodologies. Recent genomic profiling identified a subset of CNS embryonal tumors with astroblastoma-like morphology that harbored MN1 gene fusions, termed "CNS high-grade neuroepithelial tumors with MN1 alteration" (CNS-HGNET-MN1). To further characterize the genetic alterations that drive astroblastomas, we performed targeted next-generation sequencing (NGS) of 500 cancer-associated genes in a series of eight cases. We correlated these findings with break-apart fluorescence in situ hybridization (FISH) analysis of the MN1 locus and genome-wide DNA methylation profiling. Four cases showed MN1 alteration by FISH, including two pediatric cases that lacked other pathogenic alterations, and two adult cases that harbored other cancer-associated gene mutations or copy number alterations (eg, CDKN2A/B homozygous deletion, TP53, ATM and TERT promoter mutations). Three of these cases grouped with the CNS-HGNET-MN1 entity by methylation profiling. Two of four MN1 intact cases by FISH showed genetic features of either anaplastic pleomorphic xanthoastrocytoma (BRAF p.V600E mutation, CDKN2A/B homozygous deletion and TERT promoter mutation) or IDH-wildtype glioblastoma (trisomy 7, monosomy 10, CDK4 amplification and TP53, NRAS and TERT promoter mutations) and these cases had an aggressive clinical course. Two clinically indolent cases remained unclassifiable despite multimodal molecular analysis. We conclude that astroblastoma histology is not specific for any entity including CNS-HGNET-MN1, and that additional genetic characterization should be considered for astroblastomas, as a number of these tumors likely contain a methylation profile or genetic alterations that suggest classification as other tumor entities. Our heterogeneous molecular findings help to explain the clinical unpredictability of astroblastoma.

Keywords: CNS-HGNET-MN1; DNA methylation profiling; astroblastoma; next-generation sequencing.

Figure 1

Histologic features of astroblastoma cases. Case numbers are designated in the upper left of each panel. Scale bar = 50 μm and applies to all images.

Figure 2

Examples of immunohistochemical variability in tumors diagnosed as astroblastoma. Scale bars = 50 μm. A. GFAP and OLIG2 show a reciprocal pattern of staining in some cases. B. The EMA pattern varies from focal paranuclear dot‐like staining, to a combination of membranous, cytoplasmic and paranuclear dot‐like staining.

Figure 3

Molecular features of cases classified as MN1 breakapart tumors. A. Representative FISH results using an MN1 breakapart probe, and quantitation of abnormal signal frequency in 100 examined nuclei. B. Variability of EMA immunohistochemical staining in this category of cases. The finding in Case 1 was isolated to a single focus in the tumor. Scale bars = 50 μm. C. Copy number profiling for selected chromosomes from Case 1 demonstrate homozygous deletion of CDKN2A/B on chromosome 9p21, and focal copy number loss on chromosome 22. D. Complex structural rearrangement of chromosome 22 involving the NF2 locus in Case 8. Gray bars represent sequencing reads with homology to the displayed region, and colored bars represent transition to an area of mismatch, which corresponded to a distal region of chromosome 22q.

Figure 4

Key findings in astroblastoma tumors where molecular studies are not able to definitively reclassify the tumors. Case 2 demonstrated focal extensively hyalinized vessels (A) and numerous whole chromosome losses (C). Case 6 demonstrated extensive vascular hyalinization (B) and numerous whole chromosome losses (D). E. FISH studies were negative for MN1 breakapart in both cases, but showed the indicated 22q copy number changes in a subset of cells.

Figure 5

Summary of molecular findings (top) and grouping of astroblastoma cases (bottom) by next‐generation molecular profiling.