gms | German Medical Science

Background: Molecular genetic approaches are becoming increasingly important in brain tumor diagnostics to assess for diagnostic, prognostic and predictive markers. Frequent genetic alterations detectable in adult glioma patients include mutations in the IDH1/IDH2, TP53 and ATRX genes in diffuse astrocytic gliomas, mutations in the IDH1/IDH2, CIC, FUBP1 and TERT genes as well as 1p/19q co-deletion in oligodendrogliomas, and mutations in the TERT, PTEN, TP53 and NF1 genes as well as copy number alterations of EGFR and CDKN2A/B in glioblastomas. Examples of common alterations in pediatric patients include H3F3A mutations in pediatric high-grade gliomas as well as BRAF mutations in subsets of low-grade gliomas.

Methods: We developed a customized glioma panel covering commonly altered genes in adult and pediatric gliomas for next-generation sequencing (NGS) on the Ion Torrent PGM or Proton platforms. Specificity and sensitivity of the NGS-based detection of sequence variants and copy number changes were validated by conventional sequencing, pyrosequencing, qRT-PCR data and microsatellite-based analysis. Application of gene panel NGS was optimized for use on formalin-fixed paraffin embedded tissue including stereotactic biopsy samples.

Results: Sequencing runs with read output of one to two million reads per sample were performed to ensure high sensitivity. Known glioma-associated sequence variations and copy number variations of e.g. EGFR and CDKN2A as well as co-deletion of 1p/19q were successfully detected and validated. C>T artefacts were detected in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material and effectively eliminated by using Uracil-DNA Glycosylase treatment during DNA extraction. To assess the relationship of mutation profiles with histological classification, we screened 98 gliomas of different histologies for alterations in selected candidate genes. Cluster analysis of mutation data revealed mutation profiles characteristic for the distinct glioma entities.

Conclusions: Our results demonstrate that glioma-tailored gene panel NGS is a sensitive, specific and reliable method for the diagnostic detection of gene mutations in gliomas. The method can be successfully applied to routinely processed tissue samples and requires low amounts of DNA, so that even small tissue samples like stereotactic biopsies can be analyzed.