The mechanisms of action of effective systemic standard of care treatments for GBM are summarized in Fig. 1. The most frequently used drug in the first-line setting is TMZ. TMZ is a prodrug that works by being converted to the monomethyl triazene 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (23). The biological actions of TMZ appear to be mediated by methylation at the O⁶ position of guanine (24), leading to mutations that ultimately escape the mismatch repair system (MMR). The MMR promotes a signaling cascade that activates cell cycle checkpoints and causes G2-M cell cycle arrest and apoptosis through single-and double-strand breaks in DNA (25). Tumor cells with methylated MGMT are more susceptible to the cytotoxic effects of TMZ than cells with functioning MGMT (26).

Carmustine (also known as BCNU) is a small alkylating agent and nitrogen mustard compound. It causes guanine and cytosine bases in DNA to form interstrand crosslinks. Lomustine is an oral alkylating anti-tumor therapy, which has anti-GBM efficacy due to its high lipophilicity and small size, which facilitates BBB crossover (27).

A high degree of tumor vascularization as a result of increased production of proangiogenic growth factors, including VEGF, is observed in GBM, which has led to the development of treatment methods aimed at targeting proangiogenic signaling pathways. The randomized, multicenter, open-label phase II BRAIN trial comparing the efficacy of bevacizumab in combination with irinotecan vs. bevacizumab alone contributed to the accelerated Food and Drug Administration (FDA) approval of bevacizumab for the treatment of recurrent glioblastoma in 2009 (28). The primary endpoints in this trial were 6-month progression-free survival (PFS) and objective response rate (ORR). The rate of PFS at 6 months was 42.6 and 50.3% for the bevacizumab-alone and the bevacizumab-plus-irinotecan groups, respectively. The ORR was 28.2 and 37.8%, respectively. The median OS was 9.2 and 8.7 months, respectively. Grade 3 adverse events occurred in 46.4% of individuals treated with bevacizumab alone, with hypertension (8.3%) and convulsions (6.0%) being the most common. In comparison, 65.8% of patients in the bevacizumab-plus-irinotecan group experienced grade 3 adverse events, including convulsions (13.9%), neutropenia (8.9%) and fatigue (8.9%) (29).

In patients treated with bevacizumab and standard therapy, PFS was better than with standard therapy in two studies at 4.4 months (P<0.0001) in the AVAglio and 3.4 months (P=0.004) in the RTOG0825 trial (30,31). However, the median OS was not different between treatment groups in both trials. In recurrent GBM, studies failed to achieve an improvement in OS and PFS in patients treated with combination therapy with bevacizumab compared to the use of bevacizumab alone (32). The combination of bevacizumab and lomustine was also studied. OS at 9 months was chosen as the primary endpoint. This combination increased the median OS to 12 months compared to bevacizumab or lomustine alone with 8 months each. The combination also increased 6-month PFS to 42%. Several patients in the study had grade 4 or grade 3 thrombocy-topenia (33). However, the increase in PFS may be related to the phenomenon of 'pseudo-response'. This phenomenon consists of improved contrast enhancement due to the normalization of vascular permeability (34). However, the usage of FLAIR or T2-weighted images revealed an increase in the nonenhacing part of the tumor. The Response Assessment in Neuro-Oncology criteria consider FLAIR/T2 hyperintensity as a surrogate for the nonenhancing component of the tumor (35).

Cediranib, a multi-kinase inhibitor targeting VEGFR, failed to achieve a significant improvement in PFS in a phase III randomized study of either cediranib alone or cediranib in combination with lomustine vs. lomustine based on independent or local review of postcontrast T1-weighted MRI (36).

Aflibercept is a recombinantly generated fusion protein that scavenges both VEGF and placental growth factor. In a phase II study, it had minimal evidence of single-agent activity in unselected patients with recurrent malignant glioma (37).

Finally, regorafenib is a recently approved oral multikinase agent that is now endorsed by the NCCN for the treatment of relapsed GBM post-radiation and TMZ (12). This drug demonstrated significant clinical activity and superiority to lomustine in a recent phase 2 trial (13).

TTF is a non-invasive antimitotic therapy, delivered by an alternating electric field by the Optune^® system. Preclinical studies have indicated that TTF is able to alter microtubule formation, causing mitotic arrest and death. During cyto-kinesis, it also induces the dielectrophoretic migration of polar molecules. The phase III EF-14 study demonstrated a significant improvement in PFS and OS of patients with newly diagnosed GBM when Optune^® was used with TMZ compared to chemotherapy alone. Compliance was associated with a better clinical outcome. The use of TTF with TMZ significantly improved median OS compared to chemotherapy alone (20.9 vs. 16.0 months, respectively; P<0.001) (38). The clinical efficacy and tolerability of TTF in glioblastoma have been established in 2 large phase III studies and have been validated in real-world settings. TTF is associated with minimal adverse events (local or systemic). A limitation of TTF is that it must be worn continuously with minimal interruption. This inevitably leads to major lifestyle modifications. Furthermore, the total monthly therapy cost is ~$21,000 (39). TTF has proven its clinical efficacy, but its usage is associated with certain disadvantages and side effects in patients with GBM, including significantly higher rates of localized skin toxicity (38). Following regulatory approval, NCCN guide-lines included TTF in conjunction with TMZ for the treatment of patients with both newly diagnosed (Category 1) and recurrent glioblastoma (Category 2B) (12). Treatment strategies in conjunction with TTF provide important clinical benefits in limiting additional toxicity in patients with brain cancer and may be extended to patients with other types of solid tumor.

Despite the absence of randomized trials, surgery appears effective and the main principle of glioblas-toma surgery is currently gross-total resection (40). Maximal resection improves survival irrespective of the age of the patient or the molecular status of the tumor (41). When resection is contraindicated, stereotactic biopsy is the method of choice for both histological verification and molecular evaluation of the tumor (42). The use of fluorescence-guided surgery with 5-aminolevulinic acid assessed in a prospective study provided an improvement (46 vs. 28.3%) of 6-month PFS (43). To perform safer interventions and reduce postoperative complications, functional MRI and assessment of diffusion-tensor fibers should be routinely used. As a mandatory requirement, postoperative contrast-enhanced MRI should be performed within 48 h of resection to determine the extent of the intervention (44). In case of recurrence, the most radical resection of the focus is performed, particularly if >6 months have passed since the intervention or in patients with a good functional status (high Karnofsky performance score) or a young age (45). Currently, there are no data from ongoing randomized clinical trials regarding the surgical treatment of recurrent glioblastomas.

Aberrant constitutive activation of the NF-κB signaling pathway is common in GBM. The constitutive NF-κB hyperactivation is oncogenic due to the stimulation of tumor growth and invasion, apoptosis suppression and development of resistance to therapy (53). The most common form of NF-κB protein dimer is the heterodimer of p65-p50. This dimer is able to bind to a specific sequence (i.e., NF-κB sites) of the target gene to regulate gene transcription. NF-κB regulates cell activity through slight differences in the binding of these NF-κB dimers to target sequences (52). In non-stimulated cells, NF-κB dimers are inactive due to binding to three inhibitory factors (IκBα, IκBβ and IκBε) of NF-κB in the cytoplasm, which blocks the nuclear localization sequence and prevents the transfer of NF-κB into the nucleus. In the nucleus, NF-κB dimers bind to κB-sites in the regulatory regions of genes participating in a wide range of cellular processes (86). NF-κB in neurons maintains neuronal health, synapse growth and plasticity-related functions (87).

Activation of the NF-κB signaling pathway is common in various cancer types. Numerous mechanisms have been proposed that may lead to the disruption of NF-κB signal regulation in gliomas. For instance, RTK, primarily EGFR and PDGFR that are frequently activated in glioblastomas, may be triggered secondary to the activation of NF-κB through several mechanisms such as AKT-related and -unrelated signaling pathways. In murine models, it has been demonstrated that inhibition of NF-κB by depletion of IκB kinase 2, expression of an IκBaM super repressor or using an NF-κB essential modifier-binding domain attenuated tumor proliferation and prolonged survival (88). In one of the studies, NF-κB p65 subunit was indicated to be overexpressed in 81% of cases of GBM (89).

Oncogenic mechanisms of EGFR and PDGFR signal transduction make a major contribution to the growth and invasion of GBM. The NF-κB pathway interplays with these receptors, which may lead to the development of GBM (90). Loss of the tumor suppressors, such as neurofibromin 1 is also associated with the disruption of activation of NF-κB in GBM due to the upregulated activity of PI3K (91). Disruption of the tumor suppressor Krüppel-like factor 6, which serves as a negative regulator of NF-κB, also contributes to the activation of NF-κB (92).

Exposure of GBM cell lines to NF-κB-p65 small interfering (si)RNA and NF-κB inhibitors resulted in a significant decrease in GBM-cell viability. Treatment of GBM with NF-κB inhibitors overcame cisplatin resistance and led to an increase in the effects of cisplatin and doxorubicin. Importantly, normal astrocytes were less sensitive to NF-κB inhibition, implying tumor cell selectivity (93).

It has been proposed that numerous other mechanisms have an important role in the disruption of signal transduction of NF-κB, including facilitation of NF-κB by peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, mixed lineage kinase 4 and heterozygous deletion of the NFKBIA gene, which encodes IκBα (94). These observations emphasize the potential role of abnormal NF-κB signaling pathways in various mechanisms of GBM pathogenesis.

Amentofavone is a flavonoid that is able to cross the BBB and inhibit the NF-κB pathway by inhibiting IκB kinase degradation. Treatment with this compound reduces the viability and proliferation of GBM cells, resulting in the emergence of sub-G1 population, which indicates apoptosis. To date, amen-tofavone has not been evaluated in any randomized clinical trial (95).

The Wnt signaling pathway is a primordial instructive genetic program (96). Depending on the type of interaction between Wnt and Frizzled protein and consecutive involvement of β-catenin, there are one β-catenin-dependent canonical pathway and two β-catenin-independent noncanonical pathways (planar cell polarity and Wnt/Ca²⁺).

Wnt/β-catenin pathway activity has been linked to neural progenitor cell proliferation in the early stages of brain development, while it reduces the self-renewal capacity and promotes neuronal differentiation in later stages (97). Recent reports from a small cohort have reported mutations of adenomatous polyposis coli (which is responsible for Wnt activation) in ~13% of GBM cases with a mutation frequency of close to 14.5% (98). It has been reported that the increased activity of the canonical Wnt pathway is responsible for GBM resistance to chemotherapy and RT (99), contributing to the growth, aggressiveness and invasive potential of GBM (100). It also generates CSCs from differentiated cells (97).

It should be noted that the hypoxic environment influences Wnt-induced differentiation. Furthermore, hypoxia-inducible factor 1α is required to sustain the expression of transcriptional associates of β-catenin the transcription factor T cell factor and lymphoid enhancer binding factor) (101). In addition, Wnt-induced differentiation inhibits Notch signal transduction and thus, enhancement of Wnt and Notch suppression lead to the activation of pre-neuronal differentiation (102).

Although the Wnt pathway has been thoroughly studied and numerous molecular targeted drugs have entered the clinical trial stage, insight into the efficacy of these medications is lacking (40). In the canonical Wnt signaling pathway, the Wnt protein interacts with the Frizzled and low-density lipoprotein receptor-related protein 5/6 receptor. This binding was inhibited by monoclonal antibodies, including vantic-tumab (OMP18R5) and ipafricept (OMP54F28), thus blocking the Wnt signaling pathway (103). Vantictumab has been indicated to be well tolerated in a phase I trial with nearly no gastrointestinal adverse effects at the effective dose (104). While clinical trials are ongoing, it remains elusive at this time whether these drugs have brain efficacy or whether any GBM trials are planned with these agents.

TERT encodes the catalytic subunit of the telomerase complex. Since telomerase activity is a function of this catalytic subunit, mutations of the TERT gene promoter are frequently associated with cancer. These mutations are most often represented by nucleotide substitutions in the two most common 'hot spots': At position-124 up from the transcription initiation site (nucleotide polymorphism chr5: 1,295,228 G>A, also called C228T) and at region-146 (nucleotide polymor-phism chr5: 1,295,250 G>A, known as C250T (105).

The TERT promoter (TERTp) mutation (pTERTmut) was originally detected in melanoma. Follow-up studies also revealed a high frequency of pTERTmut in IDH-wt GBM, as well as in IDHmut oligodendroglioma and oligodendroglioma with 1p/19q co-deletion, and demonstrated its potential use for glioma classification (106).

Most glioblastomas may be divided into molecular subgroups based on mutations in TERTp and IDH 1/2. These molecular subgroups use different genetic mechanisms to maintain telomeres: This is either a TERTp mutation leading to telomerase activation or an a α-thalassemia/mental retardation, X-linked mutation leading to an alternative extension of the telomere phenotype. TERTp-mutant GBM demonstrates telomerase activation due to the de novo generation of transcription factor binding sites, leading to increased TERT expression. These tumors, designated glioblastomas TERT pWT-IDH WT, do not have any well-established genetic biomarkers or specific mechanisms for maintaining telo-meres (107).

In one of the studies, overexpression of the C-terminal fragment of human TERT (hTERTC27) was demonstrated to inhibit the growth and oncogenicity of HeLa cells (108). The therapeutic effect and molecular mechanisms of gene therapy for hTERTC27-mediated malignant tumors were further studied in vivo on human glioblastoma xenografts in thymus-free mice. Intra-tumor injection of the hTERTC27-carrying adeno-associated virus (rAAV-hTERTC27) has been demonstrated to be effective in slowing the growth of subcutaneously transplanted glioblastoma tumors. Histological analysis suggested that treatment with rAAV-hTERTC27 led to deep necrosis, apoptosis, infiltration of polymorphonuclear neutro-phils and a decrease in the density of microvessels in tumor samples (109). Another pre-clinical study reported increased expression of the hTERT gene in patients with high-grade glioma that may be associated with the aggressiveness of the tumor (patients with low hTERT mRNA levels in the tumors had a median PFS of 24 months and patients with low hTERT levels had a PFS of 11 months). It was indicated that when hTERT mRNA expression was reduced by siRNA, this led to a decrease in the cell viability. Therefore, targeting TERT using small molecules or other approaches may lead to the development of novel therapeutic agents in the future (110).

Liu et al (103) demonstrated the feasibility of gene editing as a pre-clinical therapeutic approach, utilizing CRISPR-associated protein 9 from Streptococcus pyogenes or Campylobacter jejuni, together with chimeric guide RNA (sgRNA), which is a programmable endonuclease that may be used to modify, regulate or label genomic loci in a variety of cells. Local injection of adeno-associated viruses expressing sgRNA-controlled Campylobacter jejuni CRISPR-associated protein 9-fused adenine base editor suppressed the growth of gliomas carrying mutations of the TERT promoter (111).

The PI3K/AKT/mTOR intracellular signaling pathway is responsible for growth, cell proliferation and metabolism (54). There are three classes (I, II and III) of PI3K, which differ in substrate specificity and products. Class I kinases are the most well-studied; they are heterodimers of regulatory and catalytic subunits. These enzymes may be activated by G-protein-associated receptors and RTK. After ligand binding, RTK autophosphorylate tyrosine residues in their cytoplasmic domains. The regulatory subunits of PI3K contain an SH2 domain that allows them to recognize and bind phosphotyrosine residues of RTK. As a result, kinases are in close proximity to the membrane, and hence their substrates, so the synthesis of PI3K begins (112). PI3K inhibitors may be clas-sified into pan-PI3K, isoform-selective and dual PI3K/mTOR types (113). The frequency of mutations of PIK3CA in GBM (encodes p110α, which is part of a catalytic subunit of class IA PI3K) ranges from 4 to 27% (113). Through decreased AKT and FAK activation, PIK3CA knockdown significantly decreases cell survival, migration and invasion of GBM cells (114). The p110α isoform-selective inhibitors A66 or PIK-75 effectively suppressed GBM cell growth, survival and migration in vitro (115). In the absence of PTEN, p110β has a critical role in GBM cell proliferation, survival and migration. In vitro and in vivo, knockdown of PIK3CB (encodes p110β) suppresses cell proliferation and triggers caspase-dependent apoptosis in GBM, and it works in tandem with PTEN restoration (116). The selective inhibitor of p110β TGX-221 significantly reduces cell migration in GBM cells while having a minimal effect on survival and invasion (115). Thus, AKT-phosphorylated forkhead box O proteins may have tumor suppressor functions unless they are degraded by E3 ubiquitin ligases (117).

mTOR also belongs to the PI3K-related kinases (118). mTOR is a core component of two functionally different multi-subunit protein complexes named mTORC1 and mTORC2 (119). Various extracellular stimuli, such as growth factors, nutrients or amino acids, cause a strong association of mTOR with various protein molecules. PI3K/AKT/mTOR regulates various growth signals by directly phosphorylating the immediate substrates (120). In a normal cell, various RTKs, such as EGFR, insulin receptor and G-protein coupled receptor avails extracellular stimuli from various growth factors. RTKs stimulate the recruitment of a family of lipid kinases known as class 1 PI3Ks to the plasma membrane, where they phosphorylate the glycerophospholipid phosphati-dylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the D-3 position of the inositol ring, converting it to PtdIns(3,4,5)P3. PTEN, the tumor suppressor which reverses phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 4,5-bisphosphate, counteracts this activity (121). Hyperactivation of the mTOR signaling pathway occurs in ~90% of glioblastomas. The mTOR inhibitor rapamycin has failed in clinical studies to demonstrate efficacy in patients with GBM, partially as a consequence of persistent mTORC2 signaling (122). Expression of activated mTORC2 was indicated to be nearly undetectable in normal brain tissue but was high in tumor cell lines. These same investigators discovered that 86% of tumor samples had rapamycin-insensitive companion of mammalian target of rapamycin overexpression and 70% of them had strong mTORC2 activity, which matched the in vitro observations (123).

The deregulation of the mTOR pathway was also correlated with radioresistance and in pre-clinical studies, it has also been proposed that PI3K/mTOR inhibition rendered GBM tumors radiosensitive (124).

mTORC1 inhibitors include rapamycin (sirolimus) and its analogues, such as RAD001 (everolimus), CCL-779 (temsiro-limus) and AP23573 (ridaforolimus) (55). These medications are first-generation mTOR inhibitors. A total of 171 patients with newly diagnosed GBM took part in the everolimus phase II study. RT with concurrent and adjuvant TMZ, with or without daily everolimus (10 mg), was provided to patients. When comparing patients in the everolimus group to those in the control group, there was no significant difference in PFS. Patients who received everolimus, on the other hand, had a considerable increase in toxicities (125). The only known peri-surgical phase 1 study of ridaforolimus in grade IV malignant glioma was suspended due to slower than expected patient accrual and postsurgical drug administration challenges (126). In the phase II study of RT with temsirolimus vs. radiochemotherapy with TMZ, a total of 257 patients were enrolled. In the temsirolimus arm, the median OS was 14.8 months, while in the control arm, it was 16.0 months. The temsirolimus arm had a median PFS of 5.4 months, while the control arm had a median PFS of 6.0 months (127). The combination of RT and temsirolimus is currently being further studied in a phase I/IIa trial that seeks to selectively match patients with targeted therapies based on known alterations (N2M2 trial) (128). Over the past several years, great effort has been put into developing second-generation ATP-competitive mTOR kinase inhibitors (TORKi), including INK128, Torin 1 and AZD8055, and third-generation bivalent mTOR inhibitors that specifically target mTOR resistance mutations (129). However, TORKi have not yet demonstrated clinical effectiveness in GBM, likely due to the limited capacity of mTOR inhibitors to cross the BBB and compensatory AKT activation (130).

c-Met is an RTK, expressed on the surfaces of various cells. Hepatocyte growth factor (HGF) is the ligand for this receptor. HGF binding leads to a sequence of intracellular signals that mediate embryogenesis and wound healing in normal cells. In cancer cells, aberrant HGF/c-Met axis activation, which is closely related to c-Met gene mutations, overexpression and amplification, promotes tumor development and progression by stimulating several signaling pathways (131). Approximately 37% of patients with GBM have c-Met overexpression (132). c-Met also has a role in the resistance mechanism that drives GBM invasion in xenografts. Resistance to anti-angiogenic drugs may be mediated by upregulation of c-Met gene expression (acquired resistance) or due to selective survival (intrinsic resistance) of tumor cell subpopulations overexpressing c-Met (133). It is worth noting that the shortest time to progression and OS was observed in GBM accompanied by overexpression of c-Met and VEGFR2, which indicates the primary/innate activation of the two pathways (134).

Several drugs targeting c-Met have been studied in clinical trials. Onartuzumab, which is an anti-c-met monoclonal anti-body, is highly specific for binding c-Met. This antibody is able to block the binding of c-Met-HGF by blocking the HGF α-chain and forming a complex with the Sema-PSI domain of c-Met (135). This process does not lead to any agonistic activity or trigger c-Met dimerization. Recent clinical trials did not indicate any clinical benefit with onartuzumab in GBM (136). Other types of drugs that may affect c-Met are small molecule inhibitors. Crizotinib is an effective small molecule inhibitor of c-Met, derived from the first-generation series c-Met inhibitor, PHA-66752. Crizotinib targets the TK domain of c-Met and is approved by the FDA for the treatment of advanced non-small-cell lung carcinoma (131). In the context of GBM, a recent phase Ib dose-escalation study followed by an extension phase with crizotinib was added to standard RT and TMZ. This study indicated highly promising efficacy for newly diagnosed GBM, warranting further investigation (137). Other small molecule inhibitors of c-Met include cabozantinib, foretinib, LY280163, MK2461, capmatinib, tivantinib, which may be tested in future GBM trials (131).

FGFRs control numerous biological functions, including cell proliferation, survival and cytoskeletal regulation. The FGFR signal is important in the embryonic development of the CNS and serves as the survival mechanism of adult neurons and astrocytes (138). In addition, it was indicated that FGFR signaling may promote the self-renewal and fate specification of neural stem cells (139). FGFR expression changes in astrocytes may prompt malignant transformation and GBM progression due to the activation of mitogenic, migratory and antiapoptotic reactions (140). Whole-genome analyses of patient samples have uncovered that the rate of FGFR mutations and amplifications are exceptionally low in GBM (<2%) (141). Several FGFR inhibitors have been developed over the past years. Fisogatinib is an inhibitor of the FGFR4 gene. Clinical trials suggested that fisogatinib has high activity and selectivity, resulting in considerable anti-tumor efficacy (142). Fisogatinib is able to covalently bind to a specific cysteine residue identified in FGFR4 (Cys 552), giving it a high degree of selectivity over other FGFR family members (133). Currently, this drug has only been studied in phase I clinical trials for the treatment of hepatocellular carcinoma (143). However, the study in mice indicated that brain accumulation of fisogatinib is significantly limited by ATP binding cassette subfamily B member 1 P-glycoprotein in the BBB, while oral availability of fisogatinib is markedly constrained by CYP3A activity (144). Futibatinib is also an irreversible inhibitor of FGFR. Several tumor cell lines with distinct genetic abnormalities of FGFR exhibited effective and specific growth suppression with futibatinib (145). However, to date, it has not been studied in the settings of GBM or CNS tumors. Another drug is AZD4547, which is a selective FGFR1–3 inhibitor. In an FGFR3-transforming acidic coiled-coil containing protein 3 (TACC3) glioma xenograft model, oral administration of AZD4547 resulted in longer survival compared with that of mice that were given the vehicle control (146). AZD4547 has been studied in patients with recurrent IDH-wt gliomas with FGFR1-TACC1 or FGFR3-TACC3 fusions (147).

The BRAF proto-oncogene serine/threonine kinase (BRAF) is a member of the Raf kinase family (148), which consists of three kinases: ARAF, CRAF (RAF-1) and BRAF. BRAF has an important role in regulating of the MAPK/ERK pathway. Hyperactivation of this pathway may cause cell cycle arrest, while aberrant regulation of the pathway may lead to carcinogenesis (149). BRAF activation in human neural stem and progenitor cells not only triggers tumor growth, but also subsequently leads to oncogene-induced senescence in certain low-grade brain tumors (150). This may explain the relatively high frequency of BRAF mutant brain tumors associated with good prognosis. BRAF gene alterations, on the other hand, are also seen in diffusely developing malignancies in adults, which are associated with poor prognosis (151).

In total, >40 mutations have been discovered in the BRAF gene, with a single thymine-to-adenine nucleotide base change at position 1,799 accounting for 90% of them. This missense mutation causes a substitution of glutamine for valine at position 600 (V600E). BRAF^V600E leads to a ~500-fold increase in gene activity. It allows signaling cascade activation in the absence of external stimuli such as growth signals, allowing cells to become self-sufficient in this pathway (152). The BRAF^V600E mutation is rare in primary and metastatic CNS neoplasms, found in 4% of cases (151).

BRAF^V600E mutation has a higher frequency in certain brain tumors, such as pleomorphic xanthoastrocytoma, ganglioglioma and pilocytic astrocytoma, and in epithelioid and giant cell glioblastoma (148). Genetic analyses have indicated a particularly high percentage of BRAF^V600E (50–93%) in epithelioid glioblastoma (153). This tumor also possesses TERT promoter mutations (70%) and homozygous deletions of CDKN2A/2B (79%) (153).

BRAF inhibitors targeting the BRAF^V600E mutation, such as dabrafenib and vemurafenib, provided a big step forward in the treatment of patients with malignant melanoma. Currently, BRAF inhibition is a treatment option for a small subset of patients with recurrent GBM if the V600E mutation is present (154).

Another study reported a marked radiological response and a stable clinical outcome in patients with malignant BRAF V600E-mutated glioma with leptomeningeal tumor appearance who received dabrafenib alone for up to 27 months (155). In one of the three instances in this case series, histology revealed glioblastoma, whereas the other diagnoses were compatible with anaplastic pleomorphic xanthoastrocytomas. Primary treatment with BRAF and MEK inhibitors has been indicated to lead to tumor regression in patients with BRAF V600E mutant glioblastoma. As a result, it was proposed that all young patients with GBM, particularly those with an unusually aggressive tumor behaviour, should be tested for BRAF^V600E mutation (156).

Preclinical and human tumor studies support a potentially important role for Src in human glioblastoma. In transgenic mice expressing v-Src, glioblastomas may potentially develop due to this alteration (157). Dasatinib is a TKI that reduces Src autophosphorylation and downstream signaling to AKT and phosphor-S6 in GBM cell lines, also reducing the growth and invasion of glioblastoma cells. Inhibition of src-family kinases by dasatinib also causes death of autophagic glioblastoma cells in vitro (158). Src signaling is also markedly enhanced in patients with invasive glioblastoma after administration of bevacizumab. The Src family kinase inhibitor dasatinib effectively blocked bevacizumab-induced invasion of glioma in preclinical models, leading to the hypothesis that combining bevacizumab with dasatinib may increase the efficacy of bevacizumab in patients with recurrent GBM (159).

Despite encouraging pre-clinical data, in the clinical study of Galanis et al (159), the combination of bevacizumab with dasatinib did not significantly improve outcomes in patients with recurrent GBM compared to treatment with bevacizumab alone. Although the combination had an acceptable tolerance profile in this sample of 121 patients and an improvement in PFS at 6 months was observed in the bevacizumab-dasatinib group, the efficacy threshold was not reached with a PFS-6 of 28.9% for bevacizumab with dasatinib vs. 18.4% for bevaci-zumab alone (P=0.22).