Molecular Diagnostics of Gliomas Using Next Generation Sequencing of a Glioma‐Tailored Gene Panel

Abstract

Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma-tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS-based mutation detection was optimized for application on formalin-fixed paraffin-embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α-thalassemia/mental retardation syndrome X-linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.

Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas: Prognostic Role and Comparison between Primary and Recurrent Tumors

“…sanger.ac.uk/cosmic) and have been previously reported in other studies as summarized in Supplementary Table S3. NGS analysis also detected EGFRvIII, however, at somewhat lower sensitivity when compared with RT-PCR analysis (8/10 investigated tumors with EGFRvIII positivity), in line with published data (31). EGFR SNVs were found in 8 of 13 EGFR-amplified primary tumors (including 7 of 11 EGFRvIII-positive tumors and one of two EGFRvIII-negative tumors), but only in one of 14 EGFR-nonamplified primary tumors (P < 0.01, Fisher exact test).…”

“…Detection of EGFR gene amplification by real-time PCR was performed as reported (29)(30)(31). The following primers were used for EGFR: EGFR-F (5 0 -cactgcctcatctctcaccatc-3 0 ) and EGFR-R…”

“…In a subset of 27 primary and recurrent paired glioblastoma samples, including 13 pairs with and 14 pairs without EGFR amplification, we performed targeted next-generation sequencing (NGS) of the EGFR coding sequence employing an ampliconbased approach for a predefined glioma gene panel and the Ion Proton sequencing platform as reported (31). In total, 59 amplicons covering the entire EGFR coding region from exon 1 to exon 28 were amplified from tumor DNA and sequenced.…”

“…In total, 59 amplicons covering the entire EGFR coding region from exon 1 to exon 28 were amplified from tumor DNA and sequenced. Evaluation of the NGS data for sequence variations and copy number alterations was performed as described in detail elsewhere (31).…”

Expression of HLA-DR genes in gliomas: correlation with clinicopathological features and prognosis

“…in both the suppression and promotion of cancer [7,8]. Gliomas is a disease carries profound genomic alterations, and a variety of tumor-specific genetic changes have been identified as prognostic indicators and/or therapeutic targets in glioma [9][10][11]. However, there are few papers regarding the roles of immune-related genetic changes in gliomas [12].…”