Targeted next-generation sequencing panel (GlioSeq) provides comprehensive genetic profiling of central nervous system tumors

Abstract

Background: Identification of genetic changes in CNS tumors is important for the appropriate clinical management of patients. Our objective was to develop a next-generation sequencing (NGS) assay for simultaneously detecting the various types of genetic alterations characteristic for adult and pediatric CNS tumors that can be applied to small brain biopsies.

Methods: We report an amplification-based targeted NGS assay (GlioSeq) that analyzes 30 genes for single nucleotide variants (SNVs) and indels, 24 genes for copy number variations (CNVs), and 14 types of structural alterations in BRAF, EGFR, and FGFR3 genes in a single workflow. GlioSeq performance was evaluated in 54 adult and pediatric CNS tumors, and the results were compared with fluorescence in-situ hybridization, Sanger sequencing, and reverse transcription PCR.

Results: GlioSeq correctly identified 71/71 (100%) genetic alterations known to be present by conventional techniques, including 56 SNVs/indels, 9 CNVs, 3 EGFRvIII, and 3 KIAA1549-BRAF fusions. Only 20 ng of DNA and 10 ng of RNA were required for successful sequencing of 100% frozen and 96% formalin-fixed, paraffin-embedded tissue specimens. The assay sensitivity was 3%-5% of mutant alleles for SNVs and 1%-5% for gene fusions. The most commonly detected alterations were IDH1, TP53, TERT, ATRX. CDKN2A, and PTEN in high-grade gliomas, followed by BRAF fusions in low-grade gliomas and H3F3A mutations in pediatric gliomas.

Conclusions: GlioSeq NGS assay offers accurate and sensitive detection of a wide range of genetic alterations in a single workflow. It allows rapid and cost-effective profiling of brain tumor specimens and thus provides valuable information for patient management.

Keywords: CNS tumors; gene fusions; mutations; next generation sequencing; paraffin.

Fig. 1.

GlioSeq next-generation sequencing (NGS) panel design. It includes 30 genes (>1360 CNS tumor- related hot spots) analyzed for point mutations and small insertions and deletions, 24 genes (in bold) for copy number changes, 16 subtypes of BRAF and FGFR3 gene fusions, and EGFRvIII structural alterations. In addition, it tests for the expression of 3 housekeeping genes (GUSB, HPRT1, PGK) for evaluation of RNA integrity.

Fig. 2.

Boxplot demonstrating the range of coverage depth values for known (hot spot) variants detected in 30 genes in GlioSeq next-generation sequencing panel. Horizontal line indicates the threshold for minimum coverage (300x) for reporting sequence variants.

Fig. 3.

Boxplot comparing the normalized depth of coverage for sequenced amplicons between formalin-fixed paraffin embedded (FFPE) and frozen tissue specimens.

Fig. 4.

Genomic landscape of 54 CNS tumors profiled using the GlioSeq panel. Left pane indicates the mutational rate for each target across all the samples. Each vertical bar represents an individual case. A, astrocytoma (WHO grade II); AA, anaplastic astrocytoma (WHO grade III); AO, anaplastic oligodendroglioma (WHO grade III); GBM, glioblastoma multiforme (WHO grade IV); MB, medulloblastoma (WHO grade IV); MNG, meningioma; O, oligodendroglioma (WHO grade II); PA, pilocytic astrocytoma (WHO grade I); PMA, pilomyxoid astrocytoma (WHO grade II; *, gliosarcoma.

Fig. 5.

Application of GlioSeq panel for detection of genetic alterations relevant to different subtypes and grades of both adult and pediatric gliomas, medulloblastomas, and meningiomas. GBM, glioblastoma; GG; PA, pilocytic astrocytoma; PMA, pilomyxoid astrocytoma; SHH, sonic hedgehog; WNT, wingless.