Can next‑generation sequencing be used to test for molecular markers in glioblastoma multiforme?

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Last updated: October 13, 2026 • View editorial policy

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NGS Testing for Glioblastoma Multiforme Molecular Markers

Yes, next-generation sequencing (NGS) should be used to test for molecular markers in glioblastoma multiforme, as it enables comprehensive assessment of multiple diagnostic, prognostic, and potentially actionable alterations in a single assay.

Why NGS is Recommended

The 2025 EANO guidelines explicitly endorse NGS panel diagnostics for glioma classification and treatment target identification 1. NGS offers the critical advantage of simultaneously detecting mutations, gene fusions, and copy number alterations in one run, which is essential for both WHO 2021 classification requirements and identifying therapeutic targets 1.

The 2021 EANO diagnostic guidelines further support this approach, stating that "next-generation sequencing-based gene panels could enable the assessment of all or most genetic and chromosomal aberrations relevant for diagnosis using a single assay" 2.

Essential Markers NGS Should Detect in GBM

For glioblastoma multiforme specifically, NGS panels must capture:

Diagnostic Markers:

  • IDH1/IDH2 mutations (codon 132 and 172) - defines IDH-wildtype vs IDH-mutant status 2
  • TERT promoter mutations - characteristic of IDH-wildtype GBM 2
  • EGFR amplification - common in IDH-wildtype GBM 2
  • +7/-10 cytogenetic signature (chromosome 7 gain/chromosome 10 loss) 2
  • CDKN2A/B homozygous deletions 2

Predictive/Treatment-Guiding Markers:

  • MGMT promoter methylation - predicts benefit from temozolomide 2
  • Actionable fusions (FGFR, MET, NTRK) - found in 11% of glioblastomas 1
  • PTEN/PI3K pathway alterations 1

Clinical Evidence Supporting NGS

Recent validation studies demonstrate NGS feasibility:

  • A 2026 study using the Oncomine Comprehensive Assay showed 100% concordance with FISH for critical biomarkers (1p/19q, +7/-10, EGFR amplification, CDKN2A/B deletion) while reducing required FISH studies by 90% and shortening turnaround times 3

  • Analysis of 55 GBM patients identified 166 actionable mutations across 36 genes, with most patients harboring 3+ actionable alterations affecting PI3K/AKT/MTOR and RAS/RAF/MEK/MAPK pathways 4

  • Comprehensive panels identified 24 additional variants compared to glioma-specific panels, revealing potential therapeutic targets 5

Practical Implementation Considerations

Panel selection matters: The 2025 EANO guidelines note that 50-80% of patients may have identified targets, but common GBM alterations (EGFR amplification, PTEN loss, PIK3CA mutations, CDK4/6 amplifications) have not been successfully targeted therapeutically 1. This highlights a critical caveat - identifying alterations doesn't guarantee treatment benefit.

Tumor cellularity threshold: NGS requires ≥30% tumor cellularity for reliable results 3. Samples below this threshold should be excluded or enriched.

Fusion detection: RNA sequencing-based approaches are particularly valuable for detecting oncogenic gene fusions with diagnostic/predictive value in rare GBM subsets 2. The 2025 guidelines found potentially targetable fusions in 11% of glioblastomas, most commonly in FGFR, MET, and NTRK genes 1.

Common Pitfalls to Avoid

  1. Don't delay treatment for NGS results: Molecular diagnostic algorithms should not delay radiotherapy or pharmacotherapy 2

  2. Database variability: Different pathogenicity databases may yield different interpretations of the same variant 1. Ensure your laboratory uses validated, standardized calling algorithms.

  3. Distinguish diagnostic from actionable: While NGS may identify many alterations, the ESCAT scoring system (used by EANO) stratifies which targets have actual clinical evidence for treatment benefit 1

  4. Age-appropriate testing: For patients >55 years with histologically typical GBM, retained nuclear ATRX, and non-midline location, immunohistochemical negativity for IDH1 R132H suffices without full sequencing 2

Integration with Standard Testing

NGS should complement, not replace, essential immunohistochemistry:

  • IDH1 R132H immunostaining remains first-line 2
  • ATRX nuclear expression assessment 2
  • H3 K27M immunostaining for midline tumors 2

The optimal approach uses NGS as a comprehensive platform that captures multiple biomarkers simultaneously, reducing the need for sequential single-gene testing while maintaining diagnostic accuracy 3.

References

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This information is intended for healthcare professionals. Any medical decision-making should rely on clinical judgment and independently verified information. The content provided herein does not replace professional discretion and should be considered supplementary to established clinical guidelines. Healthcare providers should verify all information against primary literature and current practice standards before application in patient care. Dr.Oracle assumes no liability for clinical decisions based on this content.