GlioSeq® - NGS Panel for Brain Tumors
Test DetailsGlioSeq® is a targeted NGS panel designed to assist in diagnosis, prognostication and treatment of adult and pediatric CNS tumors including low and high grade gliomas, meningiomas, and medulloblastomas. It allows for the simultaneous analysis of a broad spectrum of genetic alterations, including point mutations in 30 CNS tumor-related genes (>1360 hot spots), copy number alterations in 24 genes, and 14 fusion types involving BRAF, FGFR3 and EGFRvIII in a single workflow. It can be performed on small brain tumor biopsies and resected tumor specimens.
|Mutations and Copy Number Variations|
|Structural Alterations / Fusions|
Genes and genetic alterations included in GlioSeq® NGS panel. Genes tested for both mutations (SNVs and indels) and copy number variations (CNVs) are in bold. From Nikiforova et al. Targeted Next Generation Sequencing Panel (GlioSeq®) Provides Comprehensive Genetic Profiling of CNS Tumors. 2015 Neuro-Oncology
Among primary adult and pediatric CNS tumors, diffuse gliomas are the largest and most diverse group. They usually arise in the cerebral hemispheres and are defined by their widely infiltrative properties and tendency for biological progression. According to the World Health Organization (WHO) criteria, gliomas are classified as astrocytomas, oligodendrogliomas or oligo-astrocytomas grades II to III and glioblastoma multiforme (GBM) grade IV, which is the most aggressive astrocytic tumor with a dismal prognosis (Fig. 1 below). Less infiltrative gliomas of children and young adults include WHO grade I pilocytic astrocytomas and gangliogliomas. Other major classes of CNS tumors include cerebellar medulloblastomas and extra-axial meningiomas. The diagnosis of CNS tumors has historically been based primarily on histopathologic features. However, patients with morphologically identical tumors may experience different clinical outcomes and responses to treatment because the underlying genetic characteristics of the tumors differ.
Therefore, many molecular markers became deeply integrated into CNS tumors diagnosis and are now used to guide patient prognostication and treatment.
Circumscribed low grade gliomas such as pilocytic astrocytoma, pilomyxoid astrocytoma, ganglioglioma, and pleomorphic xanthoastrocytoma are often harbor BRAF V600E mutation or gene fusions involving BRAF and KIAA1549 or FAM131B. A number of genetic alterations have been discovered in lower grade gliomas (WHO Grades II-III), including IDH1/2, TP53, and ATRX mutations in infiltrating astrocytomas of adults and secondary GBMs and IDH1/2 with 1p/19q co-deletion, CIC, FUBP1, and TERT mutations in oligodendrogliomas (Fig. 1). Primary GBMs demonstrate dysregulation of several critical signaling pathways. i.e. receptor tyrosine kinase (RTK)/RAS/PI(3)K pathways via amplification and mutations in EGFR/ (EGFRvIII), PIK3CA, RAS, NF1, and MET and TP53 and RB1 suppressor pathways via mutations/ loss of TP53, CDKN2A, and RB1 genes (Fig. 1). Pediatric gliomas are unique, featuring mutations in H3F3A, ATRX, and BRAF, but IDH mutations are rare unless the patient is an adolescent (Fig. 1). Therefore, analysis of gliomas for multiple molecular markers helps not only the establishing of correct morphological diagnosis but also highlights the biological differences between morphologically similar tumors and can help with the clinical management of patients. For example, patients with IDH1/TERT/CIC/FUBP1 positive lower grade gliomas (WHO grades II-III) have a significantly longer median overall survival than those with IDH1/TP53/ATRX mutations. In addition, several molecular biomarkers showed promise in predicting of response to targeted therapies in gliomas. For example, clinical trials are now open for EGFRvIII mutated GBMs, vemurafenib is being evaluated in BRAF V600E mutant gliomas, and a clinical response has already been observed in FGFR3-TACC3-positive patients treated with an FGFR inhibitor.
Medulloblastomas have been recently divided into several subtypes based on specific driver mutations including WNT (wingless), SHH (sonic hedgehog), Group 3, and Group 4 (Fig. 1). Mutations in CTNNB1 or DDX3X identify Wnt pathway of medulloblastomas that tend to have a much better prognosis. In contrast, MYC or MYCN/CDK6 amplification is characteristic of group 3 and 4 medulloblastomas, respectively, which are far more likely to metastasize and have a poor prognosis even with therapy. Tumors with PTCH and SMO belong in the Shh class, and have a relatively intermediate prognosis between Wnt and group 3/4 tumors. Therefore, molecular characterization of medulloblastomas has lasting clinical value.
Meningiomas often have mutations in the NF2, AKT1, SMO, and KLF4 genes (Fig. 2) NF2-driven meningiomas are far more likely to exhibit atypical grade II features than the other subtypes. Recurrent mutations in KLF4, AKT1, and SMO genes are often present in NF2-negative sporadic meningiomas. Clinical trial of SMO/AKT/NF2 Inhibitors is open for patients with progressive meningiomas with SMO/AKT/NF2 mutations.
Figure 1. Application of GlioSeq® panel for detection of genetic alterations relevant to different subtypes and grades of both adult and pediatric gliomas, medulloblastomas, and meningiomas. From Nikiforova et al. Targeted Next Generation Sequencing Panel (GlioSeq®) Provides Comprehensive Genetic Profiling of CNS Tumors. 2015 Neuro-Oncology
- Nikiforova MN. et al. Targeted next-generation sequencing panel (GlioSeq®) provides comprehensive genetic profiling of central nervous system tumors. Neuro-Oncology 2015 Ahead of print.
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- Cin H, Meyer C, Herr R, et al. Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma. Acta neuropathologica. 2011; 121(6):763-774.
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- Wu G, Broniscer A, McEachron TA, et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet. 2012; 44(3):251-253.
- Zhang J, Wu G, Miller CP, et al. Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nat Genet. 2013; 45(6):602-612.
- Koelsche C, Sahm F, Capper D, et al. Distribution of TERT promoter mutations in pediatric and adult tumors of the nervous system. Acta Neuropathol. 2013; 126(6):907-915.
- Yip S, Butterfield YS, Morozova O, et al. Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers. J Pathol. 2012; 226(1):7-16.
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GlioSeq® NGS Panel is performed on DNA and RNA isolated from formalin-fixed, paraffin-embedded (FFPE) or fresh/frozen tumor tissue. For enrichment of tumor cell population, all surgically removed tumor specimens are microdissected from unstained slides under the microscope with H&E guidance. Specimens with a minimum of 20-50% of tumor cells or at least 300 tumor cells in a microdissection target are accepted for analysis.
Using Ion AmpliSeq technology (Life Technologies) genomic DNA is used for multiplex PCR amplification of 396 amplicons, which target >1200 hotspots in 30 key brain cancer-related genes and total RNA is used for cDNA synthesis and multiplex PCR amplification to target 14 gene fusions known to occur in brain cancers.
Amplicons are barcoded, purified and ligated with specific adapters. A final check of library preparation is performed using the 2200 Tapestation. The Ion One Touch2 and One Touch ES are used to prepare and enrich templates and enable testing via Ion Sphere Particles on a semi-conductor chip. Next generation bidirectional sequencing was performed on the Ion Torrent Personal Genome Machine (PGM) or Ion Proton and analyzed with the Torrent Suite Software. Variant annotation and reporting was performed with Variant Explorer and SeqRepoter software (UPMC). The analytical sensitivity is 3-5% of mutant alleles for detection of mutations and 1% for detection of gene fusions. The minimal required sequencing depth is 300x.
- Input DNA: 20ng
- Input RNA: 10ng
Specimen Requirements and Shipping Instructions
Paraffin embedded tissure sections
- Tissue should be fixed in formalin and not exposed to decalcification solution. The paraffin block should contain no less than 3 mm area of tumor.
- Slides should prepared by histology using a specific protocol for cutting molecular sections to avoid contamination of the tissue sections (available upon request).
- One H&E and 6 unstained sections are required for most of the tests. Ten unstained sections or more are required for some tests (indicated by * in the test menu) or if the tissue is small. Please call the lab if you have questions.
- Slides should be properly labeled with a block label that matches the surgical pathology specimen number on the surgical pathology report.
- Slides should be sent ambient temperature in proper storage containers (plastic slide boxes) to protect them during shipment.
- A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.
- A minimum of 2 x 2 x 2 mm of frozen tissue is required; however, 5 x 5 x 5 mm is optimal.
- Collection date and time should be stated.
- Tissue specimen containing at least 50% of tumor cells can be either placed into cryogenic tube and snap frozen in liquid nitrogen, or placed into a tube with preservative solution provided by the Molecular & Genomic Pathology laboratory (request solution from the lab) and frozen at -20° C.
- Ship overnight on dry ice. A surgical pathology and/or cytology report and completed requisition form must accompany all specimens.
7 - 14 days
* For insurance or Institutional Price, please call.