Pan-Mutation Test for Brain Tumors

Researchers at the University of Pittsburgh have developed a sequencing test to analyze a broad spectrum of genetic alterations in central nervous system (CNS) tumors. The test can be used to study the genetic alterations of adult and pediatric brain tumors (high-grade gliomas, meningiomas, and medulloblastomas) for which a small brain biopsy is available. Results from a validation analysis of the test were published in Neuro-Oncology.

The test, an amplification-based next generation sequencing assay called GlioSeq, analyzes 30 genes previously identified to be important in brain tumors. The test detects single nucleotide variants (SNVs) and indels among 30 genes, analyzes copy number variations (CNVs) among 24 genes, and can detect 14 types of structural alterations in BRAF, EGFR, and FGFR3 genes in a single test.

Marina Nikiforova, MD, of the department of pathology at the University of Pittsburgh and her colleagues, compared the performance of GlioSeq to three other assays: fluorescence in-situ hybridization (FISH), Sanger sequencing, and reverse transcription PCR, each of which detect different types of genomic alterations using 54 adult and pediatric CNS tumor samples.

The new test was able to identify 100% (71 of 71) genetic alterations that were detected using the conventional mutation-detecting methods. The alterations included 56 SNVs/indels, three EGFR structural variants, and three BRAF fusion genes.

"The diagnosis of brain tumors has been based primarily on cellular features seen under the microscope," said Nikiforova in a news release. "However, patients with tumors that look identical may experience different clinical outcomes and responses to treatment because the underlying genetic characteristics of their tumors differ. We designed this panel to quickly identify those traits from very small biopsies of the brain lesion."

Only a minute amount of tumor DNA and RNA was needed for the detection-20 nanograms of DNA and 10 nanograms of RNA, for sequencing of 100% of frozen and 96% of formalin-fixed, paraffin-embedded tissue specimens.

Sensitivity of the assay was 3% to 5% of mutant alleles for SNVs and 1% to 5% for gene fusions.

The most commonly detected genetic alterations the researchers detected in high-grade gliomas were IDH1, TP53, TERT, ATRX, CDKN2A, and PTEN. In low-grade gliomas, the most frequent alteration detected was BRAF fusions and H3F3A mutations in pediatric gliomas.

“GlioSeq next-generation sequencing assay offers accurate and sensitive detection of a wide range of genetic alterations in a single workflow,” concluded the study authors. “It allows rapid and cost-effective profiling of brain tumor specimens and thus provides valuable information for patient management.”

In identifying genetic variations in brain tumors, the test may help guide treatment choices for patients as well as to guide patient eligibility for clinical trials with molecularly targeted agents.

According to Dr. Nikiforova, the team is now working to improve the test, including the addition of recently uncovered genetic alterations found in brain tumors.