1. Molecular Classification

Glial tumors can be divided into two categories: diffuse and circumscribed [

Glial tumors can be divided into two categories: diffuse and circumscribed ^[1]. Diffuse tumors are highly likely to recur due to their nature of malignancy by infiltrating surrounding brain tissue, as opposed to the benign growth pattern of circumscribed tumors. Diffuse gliomas can further be categorized as WHO grades II, III, or IV tumors. Glioblastoma multiforme (GBM) is synonymous with a WHO grade IV malignancy and accounts for more than half of all adult primary brain tumors ^[1][2]. In adult populations, primary tumors are typically more likely to affect elderly patients, whereas secondary tumors typically affect patients 45 years of age or younger ^[2][3]. GBMs can be primary tumors, signifying they are grade IV at baseline or secondary tumors that have evolved from lower grade tumors. Low grade histology divisions include astrocytoma, oligodendroglioma, oligoastrocytoma, and the three aforementioned anaplastic forms ^[1][3]. The four major genetic and epigenetic irregularities noted in GBM are derived from mutations in the metabolic enzyme isocitrate dehydrogenase 1 and 2 genes (IDH1/2), amplification in the epidermal growth factor receptor (EGFR), amplification of platelet derived growth factor alpha (PDGFRA), and the loss or mutation of neurofibromatosis type 1 gene (NF1) ^[1][3][4][5]. Primary tumors often show a high level of gene expression or mutation in oncoproteins such as EGFR or NF1 loss or mutation, while secondary GBMs typically express mutations in IDH1/2 ^[1][3][4][5]. IDH wild type is most consistent in GBM primary tumors, whereas IDH mutant is consistent with low-grade gliomas and secondary GBM ^[4]. GBMs can be further divided into four subtypes based on genomic abnormalities. These four subtypes are proneural, neural, classical, and mesenchymal. Previous studies have shown that mesenchymal subtypes have lower NF1 expression, but more specifically, focal hemizygous deletions of a region at 17q11.2 which contains the gene NF1 ^[5]. Proneural subtypes are often associated with younger age patients ^[3]. They express alterations in the PDGFRA gene with either higher amplification of the locus at 4q12 or multiple point mutations, and they also express point mutations in IDH1 ^[5]. Higher levels of PDGFRA amplifications are most often seen in pediatric GBMs, although childhood GBM is less common [

1]. Diffuse tumors are highly likely to recur due to their nature of malignancy by infiltrating surrounding brain tissue, as opposed to the benign growth pattern of circumscribed tumors. Diffuse gliomas can further be categorized as WHO grades II, III, or IV tumors. Glioblastoma multiforme (GBM) is synonymous with a WHO grade IV malignancy and accounts for more than half of all adult primary brain tumors [1,2]. In adult populations, primary tumors are typically more likely to affect elderly patients, whereas secondary tumors typically affect patients 45 years of age or younger [2,3]. GBMs can be primary tumors, signifying they are grade IV at baseline or secondary tumors that have evolved from lower grade tumors. Low grade histology divisions include astrocytoma, oligodendroglioma, oligoastrocytoma, and the three aforementioned anaplastic forms [1,3]. The four major genetic and epigenetic irregularities noted in GBM are derived from mutations in the metabolic enzyme isocitrate dehydrogenase 1 and 2 genes (IDH1/2), amplification in the epidermal growth factor receptor (EGFR), amplification of platelet derived growth factor alpha (PDGFRA), and the loss or mutation of neurofibromatosis type 1 gene (NF1) [1,3,4,5]. Primary tumors often show a high level of gene expression or mutation in oncoproteins such as EGFR or NF1 loss or mutation, while secondary GBMs typically express mutations in IDH1/2 [1,3,4,5]. IDH wild type is most consistent in GBM primary tumors, whereas IDH mutant is consistent with low-grade gliomas and secondary GBM [4]. GBMs can be further divided into four subtypes based on genomic abnormalities. These four subtypes are proneural, neural, classical, and mesenchymal. Previous studies have shown that mesenchymal subtypes have lower NF1 expression, but more specifically, focal hemizygous deletions of a region at 17q11.2 which contains the gene NF1 [5]. Proneural subtypes are often associated with younger age patients [3]. They express alterations in the PDGFRA gene with either higher amplification of the locus at 4q12 or multiple point mutations, and they also express point mutations in IDH1 [5]. Higher levels of PDGFRA amplifications are most often seen in pediatric GBMs, although childhood GBM is less common [1]. The neural subtype is classified by expression of neuron markers including NEFL, GABRA1, SYT1, and SLC12A5 [5]. Neuron projection and axon and synaptic transmission are gene ontologies associated with this subtype [5]. The classical subtype is commonly characterized by EGFR amplification or mutation [5]. Knowledge of the genetic discrepancies, tumor origination, histology, and DNA methylation patterns allow for more precise identification of tumors which predicts patient prognosis and guides possible treatment options.

]. The neural subtype is classified by expression of neuron markers including NEFL, GABRA1, SYT1, and SLC12A5 ^[5]. Neuron projection and axon and synaptic transmission are gene ontologies associated with this subtype ^[5]. The classical subtype is commonly characterized by EGFR amplification or mutation ^[5]. Knowledge of the genetic discrepancies, tumor origination, histology, and DNA methylation patterns allow for more precise identification of tumors which predicts patient prognosis and guides possible treatment options.

2. Cellular Pathways in GBMs

GBMs rely heavily on different cellular pathways for growth, signaling, proliferation, and migration, among other things. The receptor tyrosine kinase (RTK) pathway is a major pathway in which GBM malignancies capitalize. Receptors include EGFR, vascular endothelial growth factor receptor (VEGFR), PDGFR, hepatocyte growth factor receptor (HGFR/c-MET), fibroblast growth factor receptor (FGFR), and insulin-like growth factor 1 receptor (IGF-1R) ^[6]. When these receptors are bound with a ligand, they trigger two RTK pathways: Ras/MAPK/ERK and PI3K/ATK/mTORC ^[6]. In the Ras/MAPK/ERK pathway, the Ras protein is activated through phosphorylation of GDP to GTP ^[6]. Ras activation leads to MAP kinase activation which then activates ERK through phosphorylation ^[6]. Activation of this pathway promotes tumorigenesis, cell proliferation, cell migration, and angiogenesis through increased VEGF expression ^[6]. The PI3K/ATK/mTORC pathway is activated by transmembrane tyrosine kinase growth factor receptors and integrins, and G-protein-coupled receptors ^[6]. A series of events occur to activate ATK, mTORC, and S6K1 ^[6]. PTEN works to counteract the activation of PI3K signaling by dephosphorylating PIP

₁

and PIP

₂

, which are directly responsible for activating ATK ^[6]. This pathway is also responsible for inhibiting p53 and I

_K

B, which are known for anti-tumor progression ^[6]. The PI3K/ATK/mTORC pathway leads to GBM cell survival, growth, proliferation, and even angiogenesis due to increased VEGF expression ^[6]. This pathway is found to be altered in nearly 86–90% of GBM cases studied in a recent review ^[6].

3. Current Treatment Options

Despite advances in molecular studies and multimodal treatment approaches, the prognosis of GBM patients remains dismal [

Despite advances in molecular studies and multimodal treatment approaches, the prognosis of GBM patients remains dismal ^{[7][8][9][10][11][12]}[

7], with a median survival of ~14 months [8]. Therefore, there is a critical demand for new, life-extending approaches. Upon diagnosis, GBM patients typically follow the current standard of care, known as the Stupp protocol, undergoing maximal safe tumor resection. This is most often followed by adjuvant radiation and chemotherapy. Temozolomide, a DNA alkylating agent approved more than two decades ago, remains the primary chemotherapeutic for newly diagnosed GBMs [9]. Unfortunately, recurrence is observed in almost all patients, with limited therapeutic options available thereafter [7,10]. Most often recurrent GBM patients receive bevacizumab (brand name: Avastin

], with a median survival of ~14 months ^[8]. Therefore, there is a critical demand for new, life-extending approaches. Upon diagnosis, GBM patients typically follow the current standard of care, known as the Stupp protocol, undergoing maximal safe tumor resection. This is most often followed by adjuvant radiation and chemotherapy. Temozolomide, a DNA alkylating agent approved more than two decades ago, remains the primary chemotherapeutic for newly diagnosed GBMs ^[9]. Unfortunately, recurrence is observed in almost all patients, with limited therapeutic options available thereafter ^[7][10]. Most often recurrent GBM patients receive bevacizumab (brand name: Avastin

^®

), a monoclonal antibody, for palliative support. Other options for the newly diagnosed and recurrent treatment include application of an FDA approved physical device, non-invasive alternating electric field therapy or ‘tumor treating fields’ (TTFs), including its concomitant use with standard of care. TTFs, administered through use of the Optune

^®

device, are most commonly applied to supplement treatment therapies to halt tumor growth ^[11]. Vaccines and immunotherapy have shown a degree of effectiveness for prostate cancer and melanoma, albeit responses are not durable ^[12]. Trials are ongoing with both approaches for a subset of qualifying GBM patients. Vaccines offer a boost to a patient’s immune system, which may prompt a response to tumor antigens ^[12]. The intent is that vaccinations, following the completion of the standard of care, will initiate an immune response for tumor antigens in the event of recurrence.

4. Barriers to Identifying Effective Treatment

Barriers to the development of new therapeutic agents for GBMs include: (1) lack of selective, novel “druggable” targets; (2) inability of most drugs to cross the blood-brain barrier (BBB), penetrate the brain-tumor barrier (BTB), and selectively accumulate in tumor cells ^[13]; (3) molecular heterogeneity of GBMs ^[14]. Regarding the BBB/BTB, dysfunctional BBB/BTB as well as abnormal blood vessels, stem from hypoxic environments caused by metabolic demands of gliomas which increase angiogenesis and VEGF expression ^[11]. Abnormal blood vessels allow oxygen and nutrient delivery to the tumor and enable cell migration ^[15]. It is also important to note that the majority of patients undergoing treatment for GBMs develop resistance to standard of care therapy ^[13].