Next-generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets

Abstract

With the number of prognostic and predictive genetic markers in neuro-oncology steadily growing, the need for comprehensive molecular analysis of neuropathology samples has vastly increased. We therefore developed a customized enrichment/hybrid-capture-based next-generation sequencing (NGS) gene panel comprising the entire coding and selected intronic and promoter regions of 130 genes recurrently altered in brain tumors, allowing for the detection of single nucleotide variations, fusions, and copy number aberrations. Optimization of probe design, library generation and sequencing conditions on 150 samples resulted in a 5-workday routine workflow from the formalin-fixed paraffin-embedded sample to neuropathological report. This protocol was applied to 79 retrospective cases with established molecular aberrations for validation and 71 prospective cases for discovery of potential therapeutic targets. Concordance of NGS compared to established, single biomarker methods was 98.0 %, with discrepancies resulting from one case where a TERT promoter mutation was not called by NGS and three ATRX mutations not being detected by Sanger sequencing. Importantly, in samples with low tumor cell content, NGS was able to identify mutant alleles that were not detectable by traditional methods. Information derived from NGS data identified potential targets for experimental therapy in 37/47 (79 %) glioblastomas, 9/10 (90 %) pilocytic astrocytomas, and 5/14 (36 %) medulloblastomas in the prospective target discovery cohort. In conclusion, we present the settings for high-throughput, adaptive next-generation sequencing in routine neuropathology diagnostics. Such an approach will likely become highly valuable in the near future for treatment decision making, as more therapeutic targets emerge and genetic information enters the classification of brain tumors.

Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy

“…2 High-throughput sequencing has made possible the ability to molecularly profile patients and tumors at time of diagnosis, thus leading to cancer diagnoses and treatment beyond histopathology and traditional, standard-ofcare therapies. 3,4 Genomic profiling has been carried out across a multitude of pediatric CNS tumors, revealing targetable driver mutations and molecularly distinct tumor subtypes. 5 Certainly, medulloblastoma is a prime example of how molecular profiling has improved risk stratification, prognostic prediction, and focused treatment strategies.…”

“…8 With these advancements, institutions are working to incorporate genomic profiling into routine patient care to improve diagnostic accuracy, better predict outcome, and personalize therapy. 3,4 Our medical center is implementing these techniques in the pediatric neuro-oncology population and now has a year of practice in doing so. Herein we describe our experience using next-generation sequencing to diagnose and treat children with a variety of brain tumors and document how genomic profiling has significantly augmented the comprehensive nature of our treatment strategy.…”

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

“…Numerous studies using nextgeneration sequencers have revealed clinically relevant genetic alterations in various tumors, and some researchers have recently advocated the clinical use of next-generation sequencers. [15][16][17][18][19][20] This is the first study to establish a clinical sequencing system for meningioma, which is the most common brain tumor.…”

Next Generation Sequencing and Pediatric Brain Tumors: Detection of Cancer Predisposition Syndromes in Patients and Their Families

“…DNA extraction from tumor tissue, sequencing, and bioinformatic analysis at NP has been described previously [6]. In parallel, DNA extraction from blood lymphocytes and NGS are performed to detect germline mutations.…”

“…DNA is extracted from both tumor and blood. Targeted NGS is next performed for both sample types using a gene panel of 129 genes known to be involved in brain tumorigenesis or frequently mutated in brain tumor tissue [6].…”