Exploring glioblastoma heterogeneity: the impact of ecDNA on tumor evolution and progression

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults, characterized by rapid progression, invasion and high genomic instability. The presence of subpopulations named glioma stem cells (GSCs), expressing stemness-related markers, contribute to molecular heterogeneity, resistance to therapies and relapses. Extrachromosomal DNA (ecDNA) appears to be one of the major causes of complexity in GBM pathogenesis since it drives high oncogene copy number, correlating with poor prognosis. In this study we investigated the presence of ecDNA in four patient-derived GSCs, isolated in our laboratory, in order to understand its role in the onset and progression of the disease. For this purpose, conventional karyotyping, array-CGH, sequencing and FISH (Fluorescent in situ hybridization) analysis were performed. Array-CGH showed high amplification of several oncogenes, such as EGFR, in both GSC cultures and the matched bulk tumors. Moreover, targeting sequencing confirmed EGFR amplification even up to 190 copies. Karyotyping data revealed an heterogeneous presence of double minutes (DMs) in GSC cultures, reinforcing the idea that oncogene amplification can be maintained as small ecDNAs during cell proliferation. In order to confirm this, we performed a dual colour FISH using probes specific for the HER1-EGFR locus and the centromere of chromosome 7. As expected, all the DMs were positive to the EGFR probe; conversely, centromeric signals were evidenced only on the canonical chromosome sites. Thus, we concluded that DMs in GSCs are small ecDNAs carrying at least the amplification of EGFR locus, and are preserved even without centromere during mitosis. EGFR amplification was also corroborated by FISH on nuclei of formalin-fixed paraffin-embedded (FFPE) tumors from which the patient-derived GSCs were isolated. This result excluded the possibility of an in vitro acquisition of the amplification during GSCs isolation, and confirmed the driver significance of this mutation in the tumor initiation.The exact content of ecDNA, by whole genome sequencing (WGS), could clarify its role in GBM evolution and progression. Finally, EcDNA could represent a new target for therapy in GBM and a potential biomarker for the early diagnosis of relapse.