*2.1. Heterogeneity, Stem Cells and Therapy Resistance*

Common molecular abnormalities involved in the evolution of glioblastomas include aberrations in the oncogenes (EGFR, PDGF and its receptors) and tumor suppressor genes (p16INK4a, p14ARF, PTEN, RB1, and TP53), which are often observed in other human cancers as well [23]. GBM is also characterized by inter-tumoral heterogeneity, which is highlighted by the classification of GBMs into three subgroups: proneural, classical and mesenchymal [24,25]. Each subtype is characterized by specific gene expression patterns and molecular abnormalities, resulting in different clinical treatment outcomes [25,26]. Proneural subtype has the most favorable prognosis among the three subtypes; aberrations in the isocitrate dehydrogenase 1 (IDH1) gene and the platelet-derived growth factor receptor A (PDGFRA) define this subgroup. The classical subgroup is characterized by the amplification of EGFR, lack of TP53 mutations and often with homozygous CDKN2A deletions [26]. Lastly, the mesenchymal subtype is the most aggressive and it is characterized by aberrations in the neurofibromin 1 (NF1) and PTEN genes [23]. It is also characterized by a pro-inflammatory environment compared with the other subtypes [27]. It was hypothesized that one underlying cause for this was the higher incidence of tumorassociated antigens (TAAs), however this could not be proven, as specific tumor antigens are expressed in each subtype [27]. Nevertheless, this classification has not led to altered or adapted treatment approaches [28,29].

Apart from intertumoral heterogeneity, intra-tumoral heterogeneity poses another therapeutic obstacle in treatment of GBM, allowing escape of subclones from (targeted) therapies and driving treatment resistance. This heterogeneity was captured by genomewide and single cell RNA studies, which showed tumor cells with different transcriptional profiles within the same tumor [30,31]. In addition, it was shown that within the same tumor, different subtypes can coexist, highlighting the heterogeneity that characterizes GBM [31]. In another study, paired primary and recurrent tumor tissue samples were analyzed to determine the persistence of possible drug targets. The results showed that the molecular targets between primary and recurrent tumors changed by 90% [32]. This may explain the failure of drugs that target specific molecular mutations in GBM, such as the EGFR [33].

Eventually, most of the patients experience tumor relapse due to therapeutic resistance [29]. This therapeutic resistance is mainly attributed to glioblastoma stem cells (GSCs), which activate DNA repair mechanisms to promote survival after chemo- and radiotherapy [34]. Additionally, outgrowth of resistant subclones and downregulation of targeted molecules contribute to drug resistance. Furthermore, the highly infiltrative nature of GSCs makes total surgical resection of the tumor impossible [35]. The remaining and/or treatment-resistant clones will eventually generate functional vessels for the nutrient transport and develop tumor recurrence [34].
