4.6 Article

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

Journal

FRONTIERS IN ONCOLOGY
Volume 12, Issue -, Pages -

Publisher

FRONTIERS MEDIA SA
DOI: 10.3389/fonc.2022.861078

Keywords

glioma; next generation sequencing; biomarkers; molecular biology; diagnosis; targeted therapy; immunohistochemistry; fluorescence in situ hybridization

Categories

Funding

  1. Research Plan of the University of Catania PIACERI - linea di intervento 2 - entitled (MultiDisciplinar RESEarch and Targeted Therapy for malignant GLIOmas (MD_RESETT_GLIO)
  2. Piano Sanitario Nazionale 2015, Linea Progettuale 6 - Azione 6.3

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In this study, a custom NGS-based multi-gene panel was developed to accurately identify the tumor type and detect molecular abnormalities in gliomas. The panel targets specific genetic and chromosomal alterations, as well as methylation levels of gene promoters. NGS showed high concordance with conventional techniques and has the potential to replace multiple time-consuming tests. Furthermore, NGS can identify actionable genetic alterations, matching glioma patients with potential targeted therapies.
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.

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