Signs and symptoms arising from GBM vary depending on its location within the brain. Focal neurological/cognitive deficits, headaches, seizures and increased intracranial pressure are common and often progress within days to weeks (11). Less commonly, ~25% of patients present with seizures and benefit from anticonvulsant medication administration (12). Corticosteroids may also be prescribed for symptom alleviation related to peritumoral edema (PTE) (13). Brain MRI typically reveals enhanced tumor regions with central mass necrosis and increased T2/FLAIR signal intensity in PTE (14-16). Pathological confirmation after biopsy or maximal safe surgical resection leads to diagnosis of GBM per WHO 2021 classification.

Following surgical resection, the current standard of care for high-grade gliomas includes adjuvant RT with concomitant and maintenance systemic TMZ. TMZ is an FDA-approved DNA alkylating agent, and when combined with RT, it has shown to increase OS in patients with GBM; with a median OS time of 14.6 months compared with 12.1 months with radiation alone (17-19). Treating patients with tumor-treating fields (TTF), or alternating electric fields, in combination with maintenance TMZ, has been shown to increase both PFS and OS time. The former increases to 6.7 months compared with 4 months with TMZ alone, while the latter increases to 20.9 months compared with 16 months with TMZ alone (20).

Upon recurrence of GBM, there are two main pathways that begin to differentiate the treatment: i) local tumor recurrence and ii) diffuse/multiple tumors outside of the initial resection cavity. With local tumor recurrence, the patient must be assessed with brain MRI for the feasibility of additional surgical resection. After resection, non-invasive treatment should be considered. If the tumor is unable to be resected, not recommended, or not elected by the patient, then the patient is directly diverted to non-invasive treatments. Such treatments include clinical trials, systemic chemotherapy, reirradiation, alternating electric field therapy and palliative care. For diffuse/multiple tumors outside of the initial resection cavity, the treatments are the same as the non-invasive therapy with the addition of surgical resection of symptomatic or large lesions. Resection of the recurrent tumor also allows for genetic and histologic analysis to understand the progression of the tumor from the genetics identified at initial diagnosis.

Preferred regimens of systemic chemotherapy in recurrent high-grade gliomas include re-challenging with TMZ, lomustine or carmustine, PCV (procarbazine, carmustine and vincristine), regorafenib, or bevacizumab. If there has been a long interval between initial treatment with TMZ and recurrence of the glioma, then it is reasonable to consider rechallenging with TMZ therapy at variable proposed dosages, which was shown to have a response rate of 64% in the study reported by Perry et al (21). Another therapeutic option is the use of a nitrosourea such as lomustine or carmustine (22-25). This therapy should be particularly considered in those with methylguanine-DNA methyltransferase (MGMT) unmethylated promoter status, whose response to TMZ is suboptimal (26,27). In PCV therapy, carmustine may be substituted for lomustine. In a randomized, placebo-controlled clinical trial, traditional carmustine administration was compared with a carmustine-biodegradable polymer placed surgically at the tumor site in order to understand its impact on OS and effects of systemic toxicities in recurrent gliomas. The median survival time of the treatment group was 31 weeks compared with 23 weeks in the placebo group and 6-month survivability was 50% higher in the carmustine-polymer treatment group (28,29). Regorafenib was compared with lomustine in a randomized, open-label phase II clinical trial studying its effectiveness in treating recurrent gliomas and increased average survival time by 1.8 months compared with lomustine (30).

Bevacizumab, an FDA-approved, anti-VEGF monoclonal antibody, has failed to show a survival advantage against recurrent GBMs while it has led to significantly improved PFS (31-33). The increase in PFS may be due to its mechanism of action, which alters the blood-brain barrier (BBB), and it may also play a role in the prevention or improvement of rapid neurologic deterioration (34,35).

RT should be considered if there is a sufficient time period since the last RT in order to prevent additional RT-related complications, or if there was a favorable response to RT in the past. RT may be used in combination with bevacizumab even when bevacizumab monotherapy fails, if retaining the steroid-sparing effects of bevacizumab are desired. An FDA-approved biosimilar agent may be used in place of bevacizumab. A meta-analysis reviewed 50 eligible non-comparative studies including 2,095 patients to determine the efficacy and toxicity of reirradiation of recurrent GBM (36). The meta-analysis demonstrated an OS at 6 and 12 months of 73 and 36%, respectively. A PFS 6-month rate of 43% and PFS 12-month rate of 17% was also observed.

Alternating electric field therapy has been FDA-approved for safety in recurrent glioma therapy based on the results of the EF-11 clinical trial (37). This treatment is a non-invasive, low-intensity and intermediate frequency electric field therapy which interferes with cell division by interrupting microtubule formation during mitosis (38). This trial compared chemotherapy-free treatment using alternating electric field therapy vs. traditional chemotherapy in recurrent GBM. There was no increase of survival between the two groups. Median survival time was 6.6 vs. 6.0 months, respectively [hazard ratio, 0.86; 95% confidence interval (CI), 0.66-1.12; P=0.27]. The analysis of the Patient Registry Dataset on all patients with recurrent GBM receiving TTF from 2011-2013 showed 1/3 of patients received treatment at first recurrence as opposed to 9% in the EF-11 clinical trial. The overall median survival in the study was 9.6 months, displaying a significant improvement as compared with the results of the EF-11 trial.

EGFR is the most common amplification/mutation in GBMs, observed in ~58% of cases (43). Tumors with EGFR amplification have also been identified to harbor the EGFRvIII mutation. This mutation is caused by the deletion of exons 2-7, producing a constitutively active form of EGFR (44). This form of EGFR has direct consequences on cell proliferation, tumor initiation and resistance to apoptosis due to continuous autophosphorylation of downstream signaling proteins (45-47).

Inda et al (48) reported heterogeneity of EGFR expression in GBM cells, showing co-expression of both wt-EGFR and EGFRvIII. The expression of both forms of EGFR plays critical roles in GBM proliferation and creates a feedback loop between them. It was reported that cells overexpressing wt-EGFR had a higher proliferative ability, and that the cells expressing EGFRvIII produced cytokines, such as IL-6, which activate signaling pathways responsible for expressing wt-EGFR, indicating a harmonious relationship between the two populations of cells. This correlation was shown in cell lines, immortalized murine astrocytes and clinical samples (48).

Rb is a tumor suppressor protein encoded by the gene RB1. In healthy cells, Rb protein functions to control the cell cycle, only allowing cycle progression when appropriate. When the cell cycle progresses, Rb is inactivated via phosphorylation by the cyclin D and CDK4/6 complex, releasing E2F and allowing the cell cycle to enter the S phase. This protein is mutated in numerous different cancers, allowing cells to proliferate uncontrollably (49). In 2001, a study was performed on 56 samples of GBMs to investigate the loss of RB1 expression. Results demonstrated that 85% of the samples without RB1 expression were hypermethylated at the promoter region of RB1 (50). Another study in 2010 evaluated genetic networks in known dysregulated pathways in GBM. Cyclins CDK4, CDK6, and CCDN2 were commonly mutated and negatively affected the Rb pathway (51). GBM samples analyzed by EGFR expression levels revealed a significant correlation between Rb dysregulation and tumor size. When the pathway was unaltered, tumor size averaged 2.5 cm³. GBM samples harboring mutations in the Rb pathway had an average tumor size of 4.1 cm³ (52). This tumor size discrepancy indicates that dysregulation of Rb leads to increased tumor growth, ultimately leading to more aggressive disease.

The p53 protein isoforms are referred to as the 'guardians of the genome' and are encoded by the TP53 gene. P53 proteins protect DNA by promoting cell cycle arrest and DNA repair mechanisms when damaged. They also initiate apoptosis if DNA damage is beyond repair. It is one of the most frequently mutated genes in all cancers (53). In GBM, the pathway is altered in 85% of patients. The p53 protein alone is modified in 28% of GBM samples, but its frequency varies depending on the molecular subtype: Proneural, 54%; mesenchymal, 32%; neural, 21%; and classical, 0% (54). In GBMs with an amplification of PDGFRA, which is a marker of the proneural classification, a loss of wt-p53 has been revealed to constitute a more invasive tumor (55). Most p53 mutations are gain-of-function (GOF) missense mutations in the DNA binding domain (55). This gene signature has also been associated with the increased inflammation and decreased OS in patients (56). Some studies have reported that reactivating p53 in GBM cell lines can increase drug sensitivity and inhibit growth in vitro (57,58). These studies have shown that the loss of wt-p53 and/or GOF of p53 aids in the progression of GBM and may be an effective clinical target.

PI3K is a family of kinases upstream of the PI3K/Akt/mTOR pathway. This pathway coordinates pro-survival signaling throughout cells. PI3K is regulated by both PTEN and PIK3R1 (59). The function of PTEN is to prevent uncontrolled proliferation in healthy cells (60). In GBM, PI3K frequently has GOF mutations in its catalytic domain, promoting Akt overactivation and cell growth (61). The loss of heterozygosity mutation in PTEN is found in ~60% of GBMs. Mutations in PTEN tend to occur more frequently at later stages of cancer development and have a similar effect as the PI3K GOF mutations (62). The repressive subunit of PI3K, PIK3R1, has also been demonstrated to be mutated/altered in GBM samples at a higher frequency than PI3K alterations (59,63,64). Numerous studies have demonstrated that inhibition of the PI3K/Akt pathway in GBM cell lines inhibits growth in vitro and in vivo (65,66). However, clinical trials have shown that inhibitors of this pathway are ineffective in improving the long-term survival of patients (67).

There are several other genetic and epigenetic alterations that lead to propagation of growth, viability and invasion of GBM and carry prognostic and therapeutic implications. For the sake of discussion in this paper, we will be focusing on these aforementioned pathways as they are most significantly upregulated or downregulated in GBMs. Altogether, the complex heterogeneity of GBM leads to the survival and proliferation of malignant cells. These alterations/mutations not only affect the tumorigenicity of cells, but allow them to evade chemotherapeutics and RT. The intricate web of interactions these pathways have on one another and have independent of one another indicates the dire need for novel therapeutic targets. It also strengthens the need for multimodal treatment strategies to combat the heterogenous nature of GBM. The literature shows that not all cells harbor the same mutations within the same tumor. Consequently, by targeting a specific protein, this treatment strategy may only be targeting a subpopulation of tumor cells present and lead to inevitable relapse and treatment-resistant tumors.

TMZ, which was granted FDA approval in 2005, is the standard chemotherapeutic drug used concurrently with RT for patients with GBM. At physiological pH, TMZ is converted into methyl-diazonium ions. Methyl-diazonium ions cause formation of DNA adducts by transferring a methyl group to DNA, which ultimately causes cytotoxicity. Methylation of the O³ position of adenine and the N⁷ and O⁶ position of guanine causes DNA breaks, leading to G2/M cell cycle arrest and apoptosis (68).

Although TMZ is an important part of GBM therapy, resistance to this drug is common. After exposure, almost 50% patients exposed to TMZ stop responding positively. MGMT is the primary contributor to resistance to TMZ (Fig. 2). MGMT is involved in direct repair of DNA in the presence of methylated residues. If MGMT is present, it directly removes O⁶-methylguanine residues, essentially rendering the drug ineffective. Hypermethylation of MGMT's promoter region leads to decreased expression of MGMT, preventing the removal of O⁶-methylguanine residues and resulting in cells being sensitive to TMZ treatment. Stupp et al (19) showed the clinical implications of MGMT promoter methylation status while diagnosing and treating patients. They demonstrated that when comparing methylation status alone, patients who had the MGMT promoter region methylated had an increase of OS by 6 months compared with patients with an unmethylated MGMT status. The effects of methylation status in response to treatment were also reported and an improved response was observed to both TMZ and radiation therapy in patients who harbored a methylated MGMT promoter status (69). Several published clinical trials have stratified treatment plans according to MGMT methylation status, signifying its importance in drug efficacy and patient response (69-71).

Besides DNA repair proteins, cancer stem cells also contribute to TMZ resistance. Ligands of the Wnt/β-catenin pathway such as Wnt3a, Wnt7a and Wnt1 are known to induce stemness and have been reported to be highly expressed in GBM cells. As compared with healthy cells, Wnt signaling is upregulated in glioma stem cells (GSCs). This upregulation results in higher self-renewal capabilities, motility and altered epithelial-to-mesenchymal transition (EMT) activator expression (72). A previous study showed that EMT in GBM promotes resistance to chemotherapy, including TMZ, which can be reversed by knocking out β-catenin. Moreover, β-catenin has been linked to genes involved in EMT such as ZEB1, Snail, Slug and Twist (73).

Bevacizumab, a humanized monoclonal antibody against VEGF-A, has received FDA approval for the treatment of a variety of cancers (74). The FDA approved bevacizumab in 2009 to treat recurrent GBMs (75,76). While the use of anti-angiogenetic therapies provides some benefit to PFS, patients ultimately become resistant to these anti-angiogenic therapies (77).

The use of bevacizumab leads to the creation of intratumoral hypoxia due to a decrease in blood vessel formation (78). This creation of a hypoxic microenvironment results in an increased expression of hypoxia-inducible factor 1-alpha (HIF1α), which increases the expression of VEGF (79). Alongside an increase in HIF1α and VEGF, hypoxia also causes upregulation of c-Met and phospho-c-Met (p-Met). Increases in expression of these proteins have been reported to have direct consequences on downstream signaling pathways that are involved in resistance to anti-angiogenic treatments, leading to more invasive tumors. The hepatocyte growth factor (HGF) and c-Met pathway has been extensively examined in the context of anti-angiogenic-resistant tumors (78). Multiple studies have shown the effects of the HGF/c-Met pathway on invasiveness and tumor growth. HGF interacts with c-Met, causing activation of several pathways such as the pathways mentioned previously as well as the MAPK/ERK and NF-κB pathways (78,80,81).

Interestingly, VEGF has been reported by Lu et al (82) to inhibit c-Met in GBM murine models and human GBM samples. It was reported that the inhibitory effect of VEGF on c-Met actually prevented tumor invasion. The group reported that when cells were stimulated with 10 ng/ml of HGF and increasing concentrations of VEGF, there was a decrease in p-Met. However, p-Met levels were elevated when cells were stimulated with increasing concentrations of HGF and 100 ng/ml of VEGF; indicating that there is an antagonistic relationship between HGF and VEGF. Analysis of samples of patients with GBM that had relapsed with bevacizumab treatment revealed a 70% increase in p-Met staining. Tumor samples also revealed an increase in proteins associated with mesenchymal GBM (82). This finding indicated that anti-VEGF bevacizumab may actually increase the functionality of the HGF/c-Met pathway in GBM of recurrent patients, leading to bevacizumab failure.

Etoposide, also known as VP-16, was approved by the FDA in 1983 (83) and has been used to treat a wide variety of cancers. The mechanism of action is through the inhibition of topoisomerase type II (TopoII). By inhibiting the TopoII cleavage complexes (TopoIIcc), replication fork stalling occurs, leading to double-strand breaks (DSBs) and the failure of TopoII to re-ligate the DNA, ultimately resulting in cell death (84,85). It has been reported that cancer cells have used autophagy as a way around etoposide treatment conferring to decreased sensitivity to etoposide. Biasoli et al (85) reported that Rb may play a role in GBM's resistance to etoposide by regulating autophagy and apoptosis. They reported that a knockdown of Rb produced an increase in apoptosis with etoposide. It was also reported that p62, which is degraded in auto-phagolysosomes, was decreased in their negative control groups treated with etoposide. In their Rb knockdown cells treated with etoposide, p62 levels were increased, suggesting that Rb may play a role in blocking etoposide-induced autophagy (85).

Mouse Double Minute 2 (MDM2) has been shown to be amplified in ~14% of GBMs (86). Senturk et al (87) showed that osteosarcoma cell lines that had amplification of MDM2 were responsive to DNA-damaging agents, but not as sensitive to TopoII inhibitors such as etoposide. Conradt et al (88) reported that MDM2 may be involved in repairing DNA damage caused by etoposide in murine models of pancreatic ductal adenocarcinoma. It was shown that MDM2 interacts with Nijmegen breakage syndrome 1, which is part of a DNS DBS repair complex. They identified that using an MDM2 inhibitor (PXN822) resulted in a decrease of DNA repair capabilities and an increase in sensitivity to etoposide (88). MDM2 has also been reported to be responsible for resistance to etoposide in GBM by Kondo et al (89); it was demonstrated that MDM2 causes an increase in expression of p-glycoprotein (P-gp) by transfecting human MDM2 into U87-MG GBM cells that do not express MDM2. In addition to the expression of P-gp, cells that expressed MDM2 were less sensitive to etoposide compared with U87-MG parental cells (89). These findings indicated that MDM2 may be responsible not only for an increase in tumor proliferation, but also in the resistance to certain drugs such as etoposide.

Approved by the FDA in the 1980s, carboplatin has been used for different cancers over the years. The drug creates DNA lesions, thus disrupting replication and transcription which ultimately leads to cell death. Although carboplatin is an established drug, resistance after persistent use is common. In total, three primary mechanisms are involved: Decreased drug availability, DNA repair mechanism alteration and changes in microenvironmental responses. CTR1 downregulation, ATP7A/7B and MRP2 upregulation help cancer cells in keeping the intracellular concentration of the drug low by reduced uptake and increased efflux. Any drug that reaches the tumor site and gets inside of the cell is neutralized by high levels of glutathione (GSH). Carboplatin-resistant cells have high levels of GSH and GSH-supporting proteins such as γ-glutamyl-cysteine synthetase and glutathione s-transferases (90,91).

Similar to other DNA-damaging chemotherapeutics, a key resistance mechanism involves DNA repair. Most carboplatin-induced lesions are excised by nucleotide excision repair (NER). Proteins involved in NER such as ERCC1, ERCC4 and XPF are observed to be upregulated in resistant tumors. High levels of ERCC1 are a marker of poor prognosis in numerous cancers, rendering drugs such as carboplatin and cisplatin ineffective. Other enzymes including MMR proteins MSH2 and MLH 1, and specific DNA polymerases such as REV1 and REV3 also play a part, albeit an indirect one, in chemoresistance (92). High expression levels of these proteins cause an increase in DNA replication, which can prevent tumor recurrence. However, in the event of relapse, they make DNA-damaging chemotherapies obsolete.

The initial therapy for GBM consists of maximal surgical resection followed by adjuvant RT to a dose of 60 Gy with concurrent TMZ, per the so-called Stupp protocol, followed by maintenance TMZ and the use of TTF. This regimen became the standard of care following a series of landmark trials which established the role for the respective adjuvant therapies. The benefit of adjuvant RT was initially demonstrated in trials conducted by the Brain Tumor Study Group, which found that the addition of RT roughly doubled survival time compared with post-operative observation (93). This benefit was also found to be dose-dependent, with patients receiving higher RT doses living longer (94). After preclinical data demonstrated reduced tumor cell survival with the combination of TMZ and RT, this combination was investigated clinically (95).

This work ultimately culminated in the landmark 2005 EORTC-NCIC trial by Stupp et al (19), which demonstrated significant improvements in 2-year OS with combination of adjuvant TMZ and RT without significant toxicity. Unfortunately, disease progression is virtually inevitable despite the current standard of care, with a median time to recurrence/progression of <12 months for patients receiving maximal safe resection and definitive adjuvant therapies (96). When recurrence or progression occur, effective salvage options are limited, with median OS after recurrence of 6 months (97). This poor survival is due in part to the development of resistance to systemic therapies as previously outlined and also radioresistance. While radioresistance has been empirically observed, the mechanisms underlying this phenomenon are not entirely understood. Thus far, several relevant pathways have been identified.

Solid tumors have abnormal vasculature, and consequently varying degrees of oxygenation. GBM is a rapidly growing, hypoxic tumor, and the degree of hypoxia is further associated with increased neoangiogenesis and accelerated endothelial proliferation. In turn, this neoangiogenesis causes remodeling of the extracellular matrix, and increased overall invasiveness of tumor cells (98). Furthermore, hypoxia also reduces the lethal effect of irradiation by reducing the generation of the reactive oxygen species that mediate RT-induced DNA damage (99,100). Thus, tumor hypoxia is a crucial driver of aggressiveness of GBM and reduces RT effectiveness (Fig. 3). Hypoxia also results in downstream signaling via HIFs, with multiple targets including vascular endothelial growth factor A (VEGF) (101).

Given these findings, agents targeting this pathway have been developed, most prominently bevacizumab. This humanized monoclonal antibody binds the circulating VEGF-A ligand, reducing its ability to bind to receptors, altering the kinetics of ligand binding to endothelial cells and downregulating angiogenesis. Despite compelling preclinical data of the benefits of RT with bevacizumab, real-world outcomes have been less than optimal. A total of two phase III clinical trials both failed to demonstrate significant improvements in OS, though improvements in PFS were noted (102,75). Interestingly, the use of bevacizumab actually reduced survival time in the most favorable subgroup (MGMT-methylated with favorable gene signatures from the 9-gene molecular profile) from 25 months to 16.7 months. The 9-gene molecular profile used for stratification in the trial came from the work of Colman et al (103), who reported the development of a 9-gene array that can be used as a predictor of survival in patients with GBM. The 9 genes that were selected as prognostic indicators were: Aquaporin 1 (AQP1), YKL-40 (CHI3L1), epithelial membrane protein 3 (EMP3), glycoprotein (GPNMB), insulin-like growth factor binding protein 2 (IGFBP2), galectin 3 (LGALS3), oligodendrocyte lineage transcription factor 2 (OLIG2), podoplanin (PDPN) and reticulon 1 (RTN1). These genes were shown to give survival time predictions independently of MGMT status in patients and were observed to provide similar predictions of survival as MGMT methylation status (103). Further investigation is necessary to successfully exploit hypoxia therapeutically.

Moreover, hypoxia has been linked to increased stemness characteristics of GBM (104). Cancer stem cells (CSCs) are considered to exist as a subpopulation of tumor cells with the ability to potently repopulate and have been demonstrated as a common feature of several tumor histologies (105). These stem cells contribute significantly to therapy resistance, and thus represent a potential target for intervention (106). Attempts to distinctly identify and preferentially eradicate CSCs have been largely unsuccessful, due to genotypic and phenotypic heterogeneity and plasticity in response to therapy and environmental cues (107). CSCs are particularly refractory to cancer therapies because cancer therapies are largely mediated by DNA damage and subsequent mitotic catastrophe (108). In response to genotoxic insults, CSCs adopt a quiescent, dormant phenotype, which may subsequently repopulate tumors long after initial treatment (109).

CD133 (prominin-1) is identified as a putative hallmark of stem cells, both in tumors and neural progenitor cells (106,110,111). CD133-positive (CD133⁺) GSCs have more robust DNA repair mechanisms and greater growth checkpoint activation following DNA damage vs. CD133-negative (CD133^-) GBM tumor cells (112). Consequently, irradiation exerts a potent evolutionary pressure that inadvertently selects for the survival of CD133⁺ cells. This ultimately contributes to eventual repopulation of the surviving niche of tumor cells, which likely underlies the observation of the distinct genetic profile of recurrent GBM (113). More specifically, exome sequencing has demonstrated that some recurrent tumors appear to originate from clonal expansion of specific subpopulations of the original tumor (113).

Specific targeting of the DNA repair mechanisms upregulated in CD133⁺ GSCs is a compelling proposition that may prove useful. This is perhaps especially advantageous in the recurrent setting, in which tumors are CD133⁺ enriched (114,112). In response to irradiation, CD133⁺ glioma cells have more robust activation of ATM, Rad17, Chk1 and Chk2 compared with CD133^-glioma cells, preferentially inducing cell cycle arrest and repair (112). Irradiation of CD133⁺ glioma cells pretreated with the Chk 1/2 inhibitor debromohymenialdisine has been shown to significantly increase efficacy of irradiation in vitro (112).

Other molecular signaling pathways have also been shown to contribute to radioresistance, both in the de novo and recurrent setting. Alterations of the EGFR are among the most common mutations in GBM, present in >50% of tumors (115). Specifically, mutations of EGFR-wt to a specific, constitutively active variant, EGFRvIII are highly oncogenic (116). EGFRvIII has been demonstrated to confer radioresistance compared with EGFR-wt by multifold activation of pro-proliferative signaling via mitogen-activated protein kinase (MAPK). It has also been shown to cause robust stimulation of anti-apoptotic pathways via the Akt/phosphatidylinositol-3-kinase pathways (PI3K-Akt) in response to irradiation (117). This hyperactivation of the PI3K/Akt signaling pathway by EGFRvIII reduces radiosensitivity via enhanced repair of DNA DSBs (118). Unfortunately, response to EGFR inhibition has generally been modest (119). This has been attributed to poor tumor penetrance as well as due to redundant mutations of these downstream signaling cascades. However, inhibition of downstream PI3K-Akt has been demonstrated to radio-sensitize glioma cells in vitro (120). Ongoing investigations of the Akt pathways and other molecular cascades downstream of EGFR may prove productive.

While targeting the mechanisms underlying the radioresistance of GBM remains a largely preclinical endeavor, the inevitability of recurrence has prompted clinical efforts to improve the efficacy of RT. In the upfront setting, this was primarily investigated from the perspective of RT target delineation. It has been demonstrated that GBM exists as distinct subpopulations of cells with unique roles in the growth, signaling and invasiveness of the tumor (121). Thus, one area of inquiry is that of incorporating novel imaging modalities to localize and characterize GBM more granularly. For example, multiparametric MRI sequences to assess hyper-cellularity and hyper-perfusion has been shown to be predictive of subsequent sites of failure (122). Similarly, hypoxia imaging is an ongoing area of investigation, given its association with increased invasiveness and radioresistance (123). Imaging that more robustly correlates the known intratumoral heterogeneity with location and may allow for more optimal, biologically-driven, RT dose distribution, often referred to as 'dose-painting'. Novel PET agents such as [¹¹C] methionine-PET (MET-PET); [¹⁸F] fluoro-ethyl-L-tyrosine (FET-PET), and [¹⁸F]-FDOPA-PET are being investigated in dose-escalation trials (124).

In the recurrent setting, several reirradiation approaches have been attempted as salvage options. These are complicated by considerations of the location of the recurrent lesion relative to the initial course (in-field, marginal, out-of-field), prior RT dose to adjacent organs-at-risk, volume of recurrent/progressive disease and changes in tumor biology. GBMs fail overwhelmingly in-field, and thus additional radiation doses often overlap significantly with the initial course (125). Consequently, this may increase the risk of radio-necrosis or other toxicity if full-dose, conventionally fractionated reirradiation, namely 60 Gy/30 fractions, was attempted (125). The use of more conformal, stereotactic approaches, whether single-fraction radiosurgery or fractionated stereotactic RT (SRS/SRT), may limit the toxicity of reirradiation (126). For larger volume recurrences, more conservative hypo-fractionated approaches should be utilized to reduce toxicity (127). Additionally, the changes in biology at the time of recurrence may dictate response to RT. Specifically, recurrent GBM has been observed to shift to a more aggressive, mesenchymal phenotype (126). Intriguingly, it has been observed that RT itself (as well as other therapies) may play a crucial role in the mesenchymal transition of recurrent GBM, which in turn is more treatment-resistant (128).