A Custom DNA-Based NGS Panel for the Molecular Characterization of Patients With Diffuse Gliomas: Diagnostic and Therapeutic Applications

Abstract

The management of patients with Central Nervous System (CNS) malignancies relies on the appropriate classification of these tumors. Recently, the World Health Organization (WHO) has published new criteria underlining the importance of an accurate molecular characterization of CNS malignancies, in order to integrate the information generated by histology. Next generation sequencing (NGS) allows single step sequencing of multiple genes, generating a comprehensive and specific mutational profile of the tumor tissue. We developed a custom NGS-based multi-gene panel (Glio-DNA panel) for the identification of the correct glioma oncotype and the detection of its essential molecular aberrations. Specifically, the Glio-DNA panel targets specific genetic and chromosomal alterations involving ATRX chromatin remodeler (ATRX), cyclin dependent kinase inhibitor 2A (CDKN2A), isocitrate dehydrogenase (NADP+) 1 (IDH1) and the telomerase reverse transcriptase (TERT) promoter while also recognizing the co-deletion of 1p/19q, loss of chromosome 10 and gain of chromosome 7. Furthermore, the Glio-DNA panel also evaluates the methylation level of the O-6-methylguanine-DNA methyltransferase (MGMT) gene promoter that predicts temozolomide efficacy. As knowledge of the mutational landscape of each glioma is mandatory to define a personalized therapeutic strategy, the Glio-DNA panel also identifies alterations involving “druggable” or “actionable” genes. To test the specificity of our panel, we used two reference mutated DNAs verifying that NGS allele frequency measurement was highly accurate and sensitive. Subsequently, we performed a comparative analysis between conventional techniques - such as immunohistochemistry or fluorescence in situ hybridization - and NGS on 60 diffuse glioma samples that had been previously characterized. The comparison between conventional testing and NGS showed high concordance, suggesting that the Glio-DNA panel may replace multiple time-consuming tests. Finally, the identification of alterations involving different actionable genes matches glioma patients with potential targeted therapies available through clinical trials. In conclusion, our analysis demonstrates NGS efficacy in simultaneously detecting different genetic alterations useful for the diagnosis, prognosis and treatment of adult patients with diffuse glioma.

The Sesquiterpene Lactone Cynaropicrin Manifests Strong Cytotoxicity in Glioblastoma Cells U-87 MG by Induction of Oxidative Stress

“…Considering the high heterogeneity of glioblastoma as emerging from the deep molecular characterization of brain tumors by next-generation sequencing technology to integrate the histological analysis [ 44 ], the cytotoxicity of Cyn has been tested against different cell lines. In particular, cell lines here used were chosen according to the common predictive markers IDH 1/2 and MGMT gene promoter methylation [ 45 , 46 ].…”

Molecular profile and clinical features of patients with gliomas using a broad targeted next generation‑sequencing panel

“…Therefore, the implementation of a pan-cancer panel enables the identification of a larger number of mutations as well as rare genomic events, thus providing more therapeutic choices for patients with gliomas. Consequently, these findings may help with glioma patient diagnosis, prognosis, eligibility for clinical trial enrollment, and treatment as previously described (12,28,43). The application of broad panel sequencing also provided insightful information about clinicopathological traits and patient outcomes.…”

Next-Generation Sequencing Comparative Analysis of DNA Mutations between Blood-Derived Extracellular Vesicles and Matched Cancer Tissue in Patients with Grade 4 Glioblastoma

“…To date, additional techniques such as FISH and immunohistochemistry are currently employed beyond the NGS, where often they are reported as discordant. Despite this, the high sensitivity and specificity of NGS still offers a versatile strategy to obtain an accurate landscape of useful markers for diagnostic purposes [ 50 ]. In fact, more genes can be simultaneously tested by NGS, and several panels of gene can be designed according to different clinical and research needs [ 51 ].…”