Isocitrate Dehydrogenase 1 and 2

Isocitrate dehydrogenases (IDHs) are enzymes that catalyse the oxidative decarboxylation of isocitrate to 2-oxoglutarate resulting in isocitrate dehydrogenases 1 (IDH1) and isocitrate dehydrogenases 2 (IDH2) reaction products which are involved with cellular metabolism, epigenetic regulation, redox states and deoxyribonucleic acid (DNA) repair. ^{Reference Molenaar, Maciejewski and Wilmink9} The IDH1 gene is located on the long arm of chromosome 2 at position 34 (2q34) and encodes the IDH1 enzyme which resides in the cytosol and peroxisomes, whereas the IDH2 gene is located on the long arm of chromosome 15 at position 26.1 (15q26.1) and encodes the IDH2 enzyme which localises to mitochondria. IDH1/2 are metabolic genes whose mutations have been found in multiple forms of cancers, including glioblastoma multiforme (GBM). ^{Reference Yang, Ye and Guan12–Reference Suchorska, Kraus and Biczok14} The contributions of IDH1/2 mutations to tumourigenesis are reported to be linked to cell fate determination at a specific stage of stem or progenitor cell differentiation and are predominantly somatic, heterozygous, occur at early stages of tumourigenesis and are most commonly present in grade II–III gliomas and secondary glioblastomas but not in primary GBM. ^{Reference Yang, Ye and Guan12,Reference Yan, Parsons and Jin15} Cohen et al. ^{Reference Cohen, Holmen and Colman16} have reported that most low-grade diffuse gliomas (astrocytomas and oligodendrogliomas) and a small percentage of glioblastomas are IDH-mutant. According to Louis et al., ^{Reference Louis, Perry and Reifenberger17} IDH gene mutations are increasingly being recognised as a potential genetic prognostic biomarker for diffuse gliomas and have been included in the 2016 update of diffuse astrocytomas in the World Health Organisation (WHO) classification of brain tumours. Yang et al. ^{Reference Yang, Ye and Guan12} reported that over 75% of IDH1/2 mutations occur in grade 2–3 gliomas and secondary glioblastomas but are much less frequent in primary GBM and other brain tumours. Therefore, suggesting that IDH1/2 mutations can be used to distinguish between primary and secondary GBM which are pathologically indistinguishable but clinically distinct entities with different prognoses. ^{Reference Yang, Ye and Guan12} Currently, the standard diagnostic assays for the detection of IDH1/2 mutations include polymerase chain reaction (PCR), immunohistochemistry (IHC) and magnetic resonance spectroscopy.

Several studies ^{Reference Yang, Ye and Guan12–Reference Yan, Parsons and Jin15,Reference Juratli, Kirsch and Geiger18,Reference Chen, Yao and Xu19} have investigated the prognostic potential of IDH1/2 for brain tumours. Yan et al. ^{Reference Yan, Parsons and Jin15} investigated IDH1/2 genes mutations in 445 brain tumours samples and the enzymatic activity of the proteins that are produced from normal and mutant IDH1/2 genes were also determined in cultured glioma cells that were transfected with the genes. They identified IDH1 mutations in more than 70% of WHO grade II and III astrocytomas, oligodendrogliomas and glioblastomas. IDH1/2 mutant tumours had distinctive genetic and clinical characteristics, and patients with those tumours had a better outcome than those with wild-type IDH genes. ^{Reference Yan, Parsons and Jin15} Mukasa et al. ^{Reference Mukasa, Takayanagi and Saito13} analysed 250 glioma cases to investigate the prevalence of IDH mutations and the impact on prognosis for glioma patients. They observed IDH1/2 mutations in 29 and 1% of the tissue samples, respectively, and reported that the detected mutations were heterozygous. They found a relatively high frequency of IDH mutations in 71% (37/52) of oligodendroglial and 58·6% (17/29) of diffuse astrocytomas tumours. However, they observed relatively less frequent mutations in 27·6% (8/29) of anaplastic astrocytomas and 10·4% (13/125) of glioblastomas tumours. Furthermore, they reported that IDH mutation was strongly associated with increased progression-free survival and overall survival for patients with grade 3 gliomas without 1p/19q co-deletion. Juratli et al. ^{Reference Juratli, Kirsch and Geiger18} investigated the prognostic value of IDH mutations in 99 patients with secondary high-grade gliomas (48 secondary anaplastic gliomas and 51 secondary glioblastomas). The median survival time for all patients with IDH mutation was 4 years compared to 1·2 years for patients with wild-type IDH as well as patients with secondary anaplastic glioma and IDH mutation had a significantly improved outcome. This suggests that IDH mutations are potentially powerful prognostic biomarker with regard to both progression-free survival and mean survival. Chen et al. ^{Reference Chen, Yao and Xu19} performed a meta-analysis of several trails to examine the association of IDH1/2 mutations with overall survival and progression-free survival in patients with glioblastomas and reported that IDH1/2 mutations were associated with better overall survival and progression-free survival in these patients.

Phosphatase and Tensin Homolog Deletion on Chromosome 10 (PTEN)

PTEN is a tumour suppressor gene which is located on the long arm of chromosome 10 at position 23.31 (10q23.31). It encodes for a 403-amino acid multifunctional protein which possesses lipid and protein phosphatase activities. ^{Reference Milella, Falcone and Conciatori20,Reference Li, Yen and Liaw21} The PTEN gene was identified in 1997 as a mutation that was common in human cancers ^{Reference Li, Yen and Liaw21} and was reported to regulate several critical cell functions such as proliferation, survival, genomic stability, cell motility through both enzymatic and non-enzymatic activities, and phosphatidylinositol 3-kinase (PI3K)-dependent and PI3K-independent mechanisms which are aberrant at multiple levels in glioblastoma. ^{Reference Bazzichetto, Conciatori and Pallocca22,Reference Chen, Chen and He23} Furthermore, loss of PTEN function can occur through a mix of genetic or epigenetic mechanisms including point mutations, chromosomal deletions, promoter hypermethylation and post-translational modifications which can lead to a wide spectrum of human diseases. ^{Reference Bazzichetto, Conciatori and Pallocca22,Reference Chen, Chen and He23} Koul ^{Reference Koul24} reported that cell cycle arrest in the G1 (Gap 1) phase is a mechanism for the proliferation defect induced by PTEN in most glioblastomas and that PTEN mutations and deletions are frequently observed in primary glioblastomas, with higher rates of inactivation in advanced stages, whereas its occurrence is very rare in low-grade stages. This suggests that PTEN inactivation is not required for tumour initiation, but its loss is an indication for progression to highly malignant cancer. Furthermore, PTEN functions as a tumour suppressor by negatively regulating protein kinase signalling cascades and may regulate tumour cell invasion and metastasis through interactions at focal adhesions. ^{Reference Koul24}

Han et al. ^{Reference Han, Hu and Yang25} conducted a meta-analysis study based on nine cohort studies which involved 1,173 patients to investigate the relationship between PTEN genetic mutation and the prognosis of patients with glioma. They reported that PTEN genetic mutation is associated with poor prognosis and shorter overall survival time. Sasaki et al. ^{Reference Sasaki, Zlatescu and Betensky26} investigated PTEN status in 72 anaplastic oligodendrogliomas tumours and reported that PTEN mutations are predictive of poor prognosis. In addition, Parsa et al. ^{Reference Parsa, Waldron and Panner27} reported a link between PTEN mutation and immunoresistance in glioma cells. Yang et al. ^{Reference Yang, Shao and Luo28} investigated PTEN gene mutations in high-grade gliomas patients and concluded that PTEN mutations are late events in the malignant progression of glioma, and their occurrences are significantly correlated with patients’ short-term survival. Yang et al. ^{Reference Yang, Schiapparelli and Nguyen29} investigated PTEN mutations, expressions, activities and membrane localisations in different cancer-related PTEN defects from 16 glioblastoma patients’ primary tumour cells. They concluded that their results provided a molecular mechanism for cancer-associated PTEN defects and may lead to brain cancer treatments that target PTEN mono-ubiquitination. According to Bazzichetto et al., ^{Reference Bazzichetto, Conciatori and Pallocca22} the assessment of PTEN status in human tumours is yet to provide clinically prognostic, predictive or therapeutic information, possibly due to the exceptionally complex regulation of PTEN function. Several studies have, however, highlighted that PTEN status has a high but yet unfulfilled potential as a prognostic and predictive biomarker to prospectively select groups of patients that are most likely to benefit from specific therapeutic interventions. ^{Reference Bazzichetto, Conciatori and Pallocca22}

Tumour Protein P53 Gene

The tumour protein P53 (TP53) gene is located on the short arm of human chromosome 17 at position 13.1 (17p13.1) and encodes the TP53 protein that regulates the cell cycle and functions as a tumour suppressor. TP53 plays a major role in several cellular processes including cell cycle, response of cells to DNA damage, cell death, cell differentiation, neovascularisation, cell proliferation, maintenance of genomic stability, inhibition of angiogenesis and regulation of cell metabolism and tumour microenvironment. ^{Reference Ohgaki and Kleihues30–Reference Liu, Bhatt and Oltvai34} Furthermore, it also functions as a transcriptional regulator to integrate stress signals and promote cell cycle arrest, senescence and damaged cell apoptosis to prevent damaged cells from propagating. ^{Reference Ohgaki and Kleihues30–Reference Liu, Bhatt and Oltvai34} According to Zhang et al., ^{Reference Zhang, Dube and Gibert31} TP53 gene employs tumour suppressor activity primarily by altering the expression of several genes involved in cell cycle arrest, apoptosis, stem cell differentiation and cellular senescence. Furthermore, it is frequently activated in response to DNA damage, genotoxicity, oncogene activation, aberrant growth signals and hypoxia. ^{Reference Zhang, Dube and Gibert31} TP53 is reported as one of the most frequently deregulated genes in cancer and its pathway plays a crucial role in the evolution of secondary glioblastomas. ^{Reference Zhang, Dube and Gibert31} The TP53–Alternate Reading Frame–Mouse Double Minute 2 homolog pathway is reported to be deregulated in 84% of glioblastoma patients and 94% of glioblastoma cell lines, whereas TP53 gene mutations are reported to occur in 30% of primary glioblastomas cases. ^{Reference Zhang, Dube and Gibert31,Reference Fulci, Ishii and Van Meir33} Deregulated TP53 pathway components have been implicated in glioblastoma cell invasion, migration, proliferation, evasion of apoptosis and cancer cell stemness. ^{Reference Zhang, Dube and Gibert31,Reference Bogler, Huang and Kleihues32} Furthermore, the mutational status of TP53 has been associated with GBM progression and its inactivation is correlated with a more invasive, lower apoptotic, increased proliferative and stem-like phenotype. ^{Reference Zhang, Dube and Gibert31,Reference Fulci, Ishii and Van Meir33} Zhang et al. ^{Reference Zhang, Dube and Gibert31} reported that TP53 mutations in GBM are usually point mutations that can result in a high expression of gain of function, oncogenic variants of the TP53 protein and can aid GBM malignancy by acting as transcription factors on a set of genes other than those regulated by wild-type TP53. Mutant TP53 expression is reported to correlate with a worse prognosis, suggesting its potential significance as a biomarker and target for GBM therapy. ^{Reference Zhang, Dube and Gibert31,Reference Fulci, Ishii and Van Meir33} Therefore, understanding the functions of mutant TP53 can lead to the development of novel methods to restore its activity or promote its degradation for future GBM therapies. ^{Reference Zhang, Dube and Gibert31,Reference Fulci, Ishii and Van Meir33} Wild-type TP53 gene is reported as a potential candidate for treatment against astrocytic gliomas due to its cell autonomous role in cell cycle arrest after DNA damage, its activation of apoptosis or senescence and its cell extrinsic potential to induce secretion of anti-angiogenic molecules. ^{Reference Fulci, Ishii and Van Meir33}

Badie et al. ^{Reference Badie, Kramar and Lau35} investigated adenovectors bearing human wild-type TP53 gene and Escherichia coli beta-galactosidase gene on glioma cells in rat brain and observed that TP53 gene transduction inhibited tumour growth by about 40%. Iwadati et al. ^{Reference Iwadati, Fujimoto and Tagawa36} studied TP53 gene mutation and chemo-sensitivities in malignant gliomas from 34 patients and demonstrated that mutation of TP53 inactivates the TP53-dependent apoptotic pathway and induces resistance to chemotherapy. However, the replacement of normal TP53 function or stimulation of the apoptotic pathway can result in re-establishment of the chemo-sensitivity. ^{Reference Fulci, Ishii and Van Meir33} Hsiao et al. ^{Reference Hsiao, Tse and Carmel37} investigated a liposome wild-type TP53 complex injected into a cavitary glioblastoma model which was developed by injecting glioma cells into the peritoneal cavity of nude mice to simulate postoperative surgical cavity after glioblastoma excision in patients. They reported a 54% growth reduction in the in-vitro growth assay and the mice bearing TP53-treated tumours survived significantly longer compared to those treated with vector controls. Fulci et al. ^{Reference Fulci, Ishii and Van Meir33} reviewed the molecular and physiological functions of TP53 and examined how its loss favours brain tumour development. They reported that TP53 gene is important in astrocytic tumour formation and allows the development of therapies for tumour cells with disrupted TP53 tumour suppressor pathways. Quan et al. ^{Reference Quan, Li and Jin38} studied the expression of TP53-inducible gene 3 (a downstream molecule of TP53, which is involved in apoptosis and oxidative stress response) in a cohort of 104 glioma [27 low-grade gliomas (grade I and II) and 77 high-grade gliomas (grade III and IV)] specimens and investigated the correlations of the protein expression with patient characteristics, clinical and pathological variables as well as patient survival. They reported that suppression of TP53-inducible gene 3 promoted glioblastoma progression, high expression of TP53-inducible gene 3 is significant for glioblastoma inhibition and it independently shows good prognosis in patients, which suggest that it is a novel prognostic biomarker or potential therapeutic target in glioblastoma. Furthermore, patients with high TP53-inducible gene 3 expression had significantly longer median survival time (15 months) than those with low TP53-inducible gene 3 expression (8 months). Although the study supports an effect of TP53 status on improving prognostic value, while others show a limited impact on clinical outcome. Karsy et al. ^{Reference Karsy, Neil and Guan39} reported that the lack of a clear correlation of TP53 with prognosis may be due to the complexity of the TP53 signalling pathway, the importance of other regulators in the TP53 pathway that may be altered in GBM and the heterogeneity of TP53 mutations and effects.

Alpha Thalassemia Mental Retardation X-Linked Protein

The alpha thalassemia mental retardation X-linked (ATRX) gene is located on the long arm of the X chromosome at position 21.1 (Xq21.1) and encodes for the ATRX protein which plays an essential role in normal development of several systems, including the central nervous, genital, skeletal systems and blood cells. ^{Reference Haase, Garcia-Fabiani and Carney40–Reference Nandakumar, Mansouri and Das42} The ATRX protein is a member of the family of chromatin remodelling proteins whose main function is the deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. ^{Reference Nandakumar, Mansouri and Das42} The ATRX status has been shown to correlate with patient age, tumour histopathology and prognosis, and ATRX mutations are reported to be widely distributed in gliomas which correlate with alternative lengthening of telomeres development and confer a better progression-free survival and overall survival in low-grade glioma harbouring IDH mutations without 1p/19q co-deletion. ^{Reference Haase, Garcia-Fabiani and Carney40–Reference Nandakumar, Mansouri and Das42} Haase et al. ^{Reference Haase, Garcia-Fabiani and Carney40} reported that ATRX absence is linked to DNA damage and replicative stress, and ATRX loss may occur by mutations, deletions or gene fusions and correlates with molecular changes, such as the alternative lengthening of telomeres phenotype, platelet-derived growth factor receptor alpha amplification and TP53 mutation. Mutation in ATRX is associated with gliomas, and it is very rare in adult primary gliomas (i.e., glioblastoma) but commonly found in younger adults with lower grade (WHO grade II–III) glioma. In a genome-wide sequencing of human gliomas, it has been observed that approximately 30% of younger glioblastoma patients had ATRX mutation. ^{Reference Koschmann, Lowenstein and Castro41}

Koschmann et al. ^{Reference Koschmann, Lowenstein and Castro41} used a mouse model to investigate ATRX deficiency in GBM and observed that the loss of ATRX accelerated GBM growth rate, reduced median survival and promoted both alternative lengthening of telomeres and genetic instability (e.g., impairment of non-homologous end joining activity and decrease in the expression of enzymes involved in the non-homologous end joining DNA repair pathway). Furthermore, they investigated whether the association between ATRX loss and genetic instability observed in a mouse model is applicable in human GBM, using an integrated multiple genome-wide dataset of human GBM. They reported that ATRX mutation in paediatric GBM is associated with increased mutation rate at the single nucleotide variant level but not with alterations at the chromosomal or copy number level and that treatment of ATRX-deficient GBM would be to select agents that most effectively promote double-strand breaks, such as topoisomerase inhibitors. ^{Reference Koschmann, Lowenstein and Castro41} Jiao et al. ^{Reference Jiao, Killela and Reitman43} investigated ATRX mutational data of 363 brain tumours and reported that ATRX was frequently mutated in 71% grade II–III astrocytomas, 68% oligoastrocytomas and 57% secondary glioblastomas tumours. Furthermore, they observed that ATRX mutations were associated with IDH1 mutations with an alternative lengthening of telomeres phenotype. Schwartzentruber et al. ^{Reference Schwartzentruber, Korshunov and Liu44} undertook a comprehensive mutation analysis in protein-coding genes by performing whole-exome sequencing on 48 well-characterised paediatric GBMs. They reported identifying 44% of somatic mutations in the H3.3–ATRX–DAXX (death-domain associated protein) chromatin remodelling pathway and 31% of mutations in ATRX and DAXX of the tumours.

Telomerase Reverse Transcriptase

The telomerase reverse transcriptase (TERT) gene is located on the short arm of chromosome 5 at position 15.33 (5p15.33) and encodes for the catalytic component of the ribonucleoprotein enzyme complex telomerase. According to Spiegl-Kreinecker et al., ^{Reference Spiegl-Kreinecker, Lotsch and Ghanim45} the telomerase together with a ribonucleic acid (RNA) template is required to elongate telomeres by adding hexameric 5′-TTAGGG-3′ tandem repeats at the ends of chromosomes. In non-cancerous somatic cells, telomere lengths are shortened after each cell replication and when a critical telomere length is reached, the cell undergoes senescence and apoptosis. However, mutations in TERT can lead to telomerase reactivation (i.e., are able to escape from telomere shortening) and the cells can proliferate indefinitely since the telomeres are being lengthened. ^{Reference Spiegl-Kreinecker, Lotsch and Ghanim45} According to Vinagre et al., ^{Reference Vinagre, Almeida and Populo46} germline and somatic mutations of the human TERT have only been observed in the promoter region of the gene (TERTp) and Spiegl-Kreinecker et al. ^{Reference Spiegl-Kreinecker, Lotsch and Ghanim45} reported observing two point mutations (C228T and C250T) in the promoter region after whole genome sequencing. The two point mutations are both cytosine-to-thymine single nucleotide polymorphisms that occur at the positions 124 and 146 base pairs upstream from the start codon and they both generate a novel-binding site for E-twenty-six/ternary complex transcription factors (Ets/TCFs) that may regulate TERT gene expression. ^{Reference Spiegl-Kreinecker, Lotsch and Ghanim45}

Kim et al. ^{Reference Kim, Kwon and Song47} investigated the clinical implications of TERT promoter mutation on IDH mutation and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in 67 diffuse gliomas patients and observed that patient age at diagnosis (over 60 years), frontal location and grade IV gliomas could be predictive factors of TERTp mutation. They identified TERTp mutations in 56·7% (38/67) patients which comprise of 7·9% (3/38) in grade II, 21% (8/38) in grade III and 71·1% (27/38) in grade IV tumours. Furthermore, they reported that TERTp mutation resulted in poor prognosis in overall diffuse gliomas and showed statistically significant survival differences between MGMT-unmethylated/TERTp-mutated and MGMT-unmethylated/TERTp-wild-type subgroups in grade II gliomas. In addition, TERTp mutation also showed statistically significant survival differences of overall survival between IDH-wild-type/TERTp-mutated and IDH-mutated/TERTp-mutated subgroups in grade III gliomas. They concluded that the prognostic and predictive properties of TERTp mutation are correlated with the presence of MGMT methylation, IDH mutation and 1p19q co-deletion. ^{Reference Kim, Kwon and Song47} Lee et al. ^{Reference Lee, Koh and Kim48} studied the frequency and prognostic effect of TERTp mutation in diffuse gliomas carrying various genetic mutations in 168 patients. They reported TERTp mutations at a frequency of 96·9% (63/65) in oligodendroglioma IDH-mutant and 1p/19q co-deletion; 4·4% (1/23) in anaplastic astrocytoma IDH-mutant; 76·9% (10/13) in glioblastoma IDH-mutant; 20% (3/15) in anaplastic astrocytoma IDH-wild-type and 84·6% (44/52) in glioblastoma IDH-wild-type patients. They further reported that TERTp mutant carriers had worse overall survival than TERTp-wild-type carriers and that TERTp mutation was strongly correlated with poor outcome in patients with IDH-wild-type glioblastoma, suggesting that it may be of prognostic value in this subgroup of patients. ^{Reference Lee, Koh and Kim48}

Avian Myelocytomatosis Viral Oncogene Homolog (MYC)

The proto-oncogenes that make up the MYC family of transcription factors consist of three human-related genes, namely MYCC, MYCN and MYCL, and while their activity is tightly regulated in non-malignant cells, their constitutive expression is directly linked to the pathogenesis of a wide variety of human cancers. ^{Reference Westermann, Muth and Benner49,Reference Poole and van Riggelen50}

Vascular Endothelial Growth Factor

The vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a highly conserved receptor-binding cystine-knot structure and comprises of five related growth factors including VEGF-A, VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PGF). ^{Reference Neufeld, Cohen and Gengrinovitch62} The VEGFs function by binding to VEGF tyrosine kinase receptors (VEGF-Rs) which initiate multiple signalling pathways affecting cell proliferation, survival, promoting cell migration and inhibiting apoptosis and tissue permeability. ^{Reference Neufeld, Cohen and Gengrinovitch62,Reference Holmes and Zachary63} The VEGF-A gene is located on the short arm of chromosome 6 at position 21.1 (6p21.1) and encodes the VEGF-A protein; VEGF-B gene encodes the VEGF-B protein and is located on the long arm of chromosome 11 at position 13.1 (11q13.1); VEGF-C gene encodes the VEGF-C protein and is located on the long arm of chromosome 4 at position 34.3 (4q34.3); VEGF-D gene encodes the VEGF-D protein and is located on the short arm of the X chromosome at position 22.2 (Xp22.2) and the PGF gene encodes the PGF protein and is located on the long arm of chromosome 14 at position 24.3 (14q24.3). ^{Reference Holmes and Zachary63} According to Neufeld et al., ^{Reference Neufeld, Cohen and Gengrinovitch62} VEGF is a highly specific mitogen for vascular endothelial cells and plays a central role in the regulation of vasculogenesis. Deregulation of VEGF expression has been reported to contribute to the development of solid tumours by promoting tumour angiogenesis; however, it has also been indicated that the inhibition of VEGF signalling pathways could inhibit the development of a wide variety of cancers. ^{Reference Neufeld, Cohen and Gengrinovitch62} According to Montano et al. ^{Reference Montano, D’Alessandris and Izzo64} and D’Alessandris et al., ^{Reference D’Alessandris, Martini and Cenci65} VEGF is the main regulator of angiogenesis in GBM, exists in several isoforms with different molecular weights and biological properties and its production is triggered by tumour hypoxia.

Several studies ^{Reference Montano, D’Alessandris and Izzo64–Reference Zhao, Hou and Hou68} have investigated both the predictive and prognostic value of VEGF expression in brain tumours, whereas some have observed that VEGF overexpression has a positive prognostic value, others have reported a negative prognostic value. Furthermore, VEGF overexpression has been reported to be predictive of response to anti-angiogenic therapy in some studies. ^{Reference Montano, D’Alessandris and Izzo64} D’Alessandris et al. ^{Reference D’Alessandris, Martini and Cenci65} examined the VEGF status of 25 adult patients with recurrent GBM who received bevacizumab as an alternative treatment. They reported that quantitative analysis of VEGF isoforms is a promising biomarker for response to bevacizumab in patients with GBM. They observed similar results in an earlier study ^{Reference D’Alessandris, Montano and Cenci66} where they prospectively enrolled 10 adult patients suffering from recurrent glioblastoma who had undergone surgical resection and standard chemo-radiotherapy. Reardon et al. ^{Reference Reardon, Desjardins and Vredenburgh67} evaluated the VEGF status and bevacizumab treatment with metronomic etoposide among 59 recurrent malignant glioma patients in a phase 2, open-label trial. They reported encouraging clinical and survival benefit of treatment with bevacizumab and VEGF status among recurrent GBM and grade 3 malignant glioma patients. Although, they indicated that increased understanding of the mechanisms of VEGF inhibitor resistance is needed to further improve the outcome with anti-angiogenic therapy for malignant glioma patients. Zhao et al. ^{Reference Zhao, Hou and Hou68} investigated the clinical significance of VEGF-C in 86 glioblastoma cases and observed high expression of VEGF-C was associated with poorer survival rate and can be identified as an independent prognostic factor in glioblastoma. Furthermore, high expression of VEGF-C was observed to be predictive of unfavourable prognosis, suggesting that VEGF-C signalling pathway is a potential and promising drug target in glioblastoma therapy. ^{Reference Zhao, Hou and Hou68}

Sex-Determining Region Y-Box 2 (SOX2)

The SOX2 is a single-exon, intronless gene belonging to the SOX family of transcription factors and it is associated with the control of different developmental processes related to the maintenance of undifferentiated state of cancer stem cells in several tissues including the brain. ^{Reference Schmitz, Temme and Senner69–Reference Annovazzi, Mellai and Caldera71} The SOX2 gene is located on the long arm of chromosome 3 at position 26.33 (3q26.33) and encodes a 317 amino acid protein. Furthermore, it exhibits highly dynamic expression patterns during the development of different tissues and cell types and also performs important functions during embryogenesis. ^{Reference Schmitz, Temme and Senner69–Reference Annovazzi, Mellai and Caldera71} According to Garros-Regulez et al., ^{Reference Garros-Regulez, Garcia and Carrasco-Garcia70} SOX2 has a role in cell fate and maintenance of the progenitors’ identity during embryogenesis, and it also plays an important role in tissue homeostasis and regeneration by maintaining stem cell activity in several compartments particularly in the CNS in adults. Furthermore, SOX2 has been identified to control the expression of several important developmental genes and plays an important role in both neurogenesis and gliogenesis in gliomas. ^{Reference Annovazzi, Mellai and Caldera71} Garros-Regulez et al. ^{Reference Garros-Regulez, Garcia and Carrasco-Garcia70} reported that elevated levels of SOX2 increase the capacity of cell invasion and migration, cell proliferation and self-renewal activity in glioma cell lines. Khan et al. ^{Reference Khan, Ullah and Hussein72} also reported that knockdown of SOX2 in glioblastoma cancer stem cells resulted in slowing down cell proliferation and in the loss of tumourigenic ability. According to Annovazzi et al., ^{Reference Annovazzi, Mellai and Caldera71} the expression of SOX2 in tissues can be measured using real-time PCR, IHC and Western blot, and its overexpression has been observed in most gliomas whereas no expression has been found in normal CNS. ^{Reference Schmitz, Temme and Senner69,Reference Annovazzi, Mellai and Caldera71} Furthermore, Garros-Regulez et al. ^{Reference Garros-Regulez, Garcia and Carrasco-Garcia70} reported increased levels of SOX2 in biopsies of glioblastoma patients and high levels of SOX2 have been associated with tumour aggressiveness and poor prognosis, suggesting that silencing SOX2 in brain tumours might be a novel therapeutic approach to combat brain tumours.

The prognostic and predictive value of SOX2 has been investigated in several studies. ^{Reference Schmitz, Temme and Senner69–Reference Gangemi, Griffero and Marubbi74} Annovazzi et al. ^{Reference Annovazzi, Mellai and Caldera71} investigated SOX2 expression status in primary glioblastomas and oligodendrogliomas cell lines and observed a positive correlation between SOX2 expression and grade of malignancy in gliomas and observed the highest SOX2 expression levels in the hypercellular and hyperproliferative areas of glioblastomas. They concluded that SOX2 is a potential marker for undifferentiated and proliferating cells and that its expression is up-regulated in the most anaplastic areas of glioblastomas and oligodendrogliomas. Schmitz et al. ^{Reference Schmitz, Temme and Senner69} screened a large DNA chip-based expression database and found SOX2 overexpression in the majority of GBM samples, whereas expression in normal brain and other non-malignant tissues was almost negligible. Garros-Regulez et al. ^{Reference Garros-Regulez, Garcia and Carrasco-Garcia70} reviewed current studies targeting SOX2 activity in glioblastoma treatment and reported that temozolomide used in combination with rapamycin inhibited tumourigenesis in glioblastoma patients with high SOX2 levels. Although cells with elevated SOX2 expression are more resistant to temozolomide, its inhibition sensitises glioma cells to this agent, suggesting that targeting SOX2 activity may offer an attractive therapeutic approach to treat glioblastoma patients. ^{Reference Garros-Regulez, Garcia and Carrasco-Garcia70} Alonso et al. ^{Reference Alonso, Diez-Valle and Manterola73} investigated SOX2 status in 42 glioblastoma samples at the molecular level and also explored its role to malignant phenotype of GBM. They observed SOX2 amplification in 11·5% and overexpression in all samples and observed that high SOX2 expression was sufficient to induce invasion and migration of glioma cells. Furthermore, the treatment of SOX2-negative glioma cell lines with 5-azacitidine resulted in re-expression of SOX2 and in a change in the methylation status of the SOX2 promoter. They also analysed data from GBM cases generated by the Cancer Genome Atlas (CGA) project and observed 86% SOX2 overexpression (n = 414), 8·5% SOX2 gene amplification (n = 492) and 100% SOX2 promoter hypomethylation (n = 258), signifying the relevance of this biomarker in the malignant phenotype of GBMs and suggesting that SOX status could be used as a therapeutic target in GBM. Gangemi et al. ^{Reference Gangemi, Griffero and Marubbi74} investigated the role of SOX2 in the atypical growth of glioblastoma by silencing SOX2 expression in glioblastoma tumour-initiating cells derived from mice. They reported that SOX2 silenced glioblastoma tumour-initiating cells prevented proliferation and inhibited tumourigenicity in immune-deficient mice, suggesting that SOX2 or its immediate downstream effectors could be an ideal target for glioblastoma therapy.

Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) or p16INK4a

The CDKN2A (also known as p16INK4a) gene is a tumour suppressor gene which regulates gene expression at different levels by modifying functional equilibrium of transcription factors involved in cell cycle regulation. It has been implicated in some processes, such as apoptosis, cell invasion and angiogenesis, and these activities may be related to its overexpression in cancer. ^{Reference Agarwal, Kabir and DeInnocentes75,Reference Romagosa, Simonetti and Lopez-Vicente76} The CDKN2A gene which encodes for the CDKN2A protein is located on the short arm of chromosome 9 at position 21.3 (9p21.3) and its alterations can lead to aberrant cell cycle pathway. ^{Reference Serrano77–Reference Sibin, Bhat and Narasingarao79} It has been reported that the CDKN2A gene is either mutated or deleted in various human tumours, including over 70% of human glioblastoma and glioma cell lines. ^{Reference Romagosa, Simonetti and Lopez-Vicente76,Reference Sibin, Bhat and Narasingarao79,Reference Liu, Lv and Li80} Furthermore, Sibin et al. ^{Reference Sibin, Bhat and Narasingarao79} reported that CDKN2A status is associated with poor prognosis and its expression can be used in various cancers as a prognostic biomarker along with methylation and deletion status of the gene. According to Park et al. ^{Reference Park, Won and Kim78} the loss or inactivation of CDKN2A protein as a result of a homozygous deletion or promoter methylation of CDKN2A/2B is observed in about 60% of GBMs and 11% of low-grade gliomas including oligodendroglioma, pleomorphic xanthoastrocytoma and pilocytic astrocytoma. A genome-wide association study observed that CDKN2A is a susceptibility locus for gliomas and the CDKN2A-CDK4/6-retinoblastoma protein pathway plays a crucial role in malignant glioma pathogenesis. ^{Reference Liu, Lv and Li80}

Romagosa et al. ^{Reference Romagosa, Simonetti and Lopez-Vicente76} reviewed the role of CDKN2A in cancer diagnosis and prognosis in some tumours and hypothesised that CDKN2A could be a diagnostic tool that could differentiate benign from malignant lesions in all tumours in which the malignant transformation is associated with CDKN2A loss. In addition, CDKN2A could also be used as a prognostic tool in those tumours in which malignant transformation is associated with CDKN2A overexpression. ^{Reference Romagosa, Simonetti and Lopez-Vicente76} Sibin et al. ^{Reference Sibin, Bhat and Narasingarao79} performed messenger RNA (mRNA) quantification analysis on 48 high-grade glioma tissues and investigated its potential prognostic role. They demonstrated that CDKN2A mRNA expression could potentially independently predict patient survival in overall survival and progression-free survival. Reis et al. ^{Reference Reis, Pekmezci and Hansen81} conducted a study to investigate whether CDKN2A gene loss is associated with overall survival across pathologically and genetically defined glioma subtypes (grade II or grade III astrocytoma, oligodendroglioma or oligoastrocytoma) in patients over 18 years. They observed that CDKN2A deletion was strongly associated with poorer overall survival among patients with astrocytomas but not in patients with oligodendrogliomas or oligoastrocytomas after adjusting for IDH mutation, sex and age. ^{Reference Reis, Pekmezci and Hansen81} Liu et al. ^{Reference Liu, Lv and Li80} investigated the overexpression and knockdown of CDKN2A in human glioma cell lines from 61 patients for potential correlations with tumour grade and the role that CDKN2A plays in malignant glioma pathogenesis. They observed that CDKN2A expression was associated with grade of glioma (patients with high-grade glioma had lower expression level) and lower level of CDKN2A was correlated with a worse prognosis. Moreover, CDKN2A overexpression inhibited growth of glioma cell lines by suppression of cyclin D1 gene expression; however, knockdown of CDKN2A promoted low-grade gliomas to high-grade gliomas. They further reported that decreased CDKN2A levels in high-grade glioma are indicative that CDKN2A may be involved in malignant glioma carcinogenesis and suggested that CDKN2A could be useful for predicting behaviour of high-grade malignant gliomas. Furthermore, the therapeutic targeting of the CDKN2A-cyclin-Rb pathway (which plays a key role in malignant gliomas formation) can potentially be useful in malignant gliomas treatment. ^{Reference Liu, Lv and Li80}

Sonic Hedgehog

The Hedgehog (HH) pathway is a signalling cascade that directs patterning in most animals as well as being essential for proper development at the molecular level and its ligands drive cell proliferation in some cell types while causing others to undergo differentiation. ^{Reference Evangelista, Tian and de Sauvage82} The three homologs of HH found in mammals include the SHH, Desert Sonic HH and Indian Sonic HH, and they act as secreted, intercellular morphogens affecting cell fate, differentiation, survival and proliferation in the developing embryo and in most organs. ^{Reference Ruiz i Altaba, Stecca and Sánchez83,Reference Shahi, Rey and Castresana84} The SHH gene is the most investigated and it is located on the long arm of chromosome 7 at position 36.3 (7q36.3). It encodes the SHH protein which plays an important role in cell embryonic development, cell specialisation and the normal shaping of the body. ^{Reference Shahi, Rey and Castresana84} According to Sasai et al., ^{Reference Sasai, Romer and Lee85} gene expression profiling shows that the SHH pathway is active in about 30% of human medulloblastomas, suggesting that it could provide a useful therapeutic target. Evangelista et al. ^{Reference Evangelista, Tian and de Sauvage82} reported that SHH signalling is required for the normal growth and regeneration of organs from stem cells, and aberrant activation of SHH pathway during tissue repair and regeneration could promote tumourigenesis, survival and/or metastasis. The aberrant activation of the SHH pathway in cancers is caused either by mutations in the pathway (ligand independent) or through HH overexpression (ligand dependent). Therefore, inhibition of the SHH pathway may provide a way to directly target cell populations that ultimately cause cancer. ^{Reference Evangelista, Tian and de Sauvage82} Evangelista et al. ^{Reference Evangelista, Tian and de Sauvage82} further indicated that cyclopamine can inhibit the growth of medulloblastoma tumours in vivo and in vitro, and most human medulloblastoma tumours tested seemed to be responsive, suggesting a potential broad application of SHH antagonist in treating this particular brain tumour, of which about 25% harbour mutations in the SHH pathway. ^{Reference Evangelista, Tian and de Sauvage82}

Dahmane et al. ^{Reference Dahmane, Sánchez and Gitton86} investigated the role of SHH-zinc finger protein (SHH-Gli) pathway in normal and abnormal precursor proliferation in the dorsal brain. They demonstrated that the SHH-Gli pathway is deregulated in brain tumours and has a general role in controlling progenitor cell number in the developing dorsal brain. Furthermore, they reported that SHH is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, and in-vitro and in-vivo assays demonstrated that SHH is a mitogen for neocortical and tectal precursors and modulates cell proliferation in the dorsal brain. ^{Reference Dahmane, Sánchez and Gitton86} They also reported that SHH signalling controls the development of the three major dorsal brain structures and that cyclopamine (an SHH signalling inhibitor) can inhibit the proliferation of tumour cells. ^{Reference Dahmane, Sánchez and Gitton86} Sasai et al. ^{Reference Sasai, Romer and Lee85} investigated the response of medulloblastoma cells in a mouse model propagated in flank allografts to HhAntag-691 (HH pathway inhibitor) and demonstrated that HhAntag-691 suppressed the SHH pathway in vivo leading to the elimination of spontaneous mouse medulloblastoma. Robinson et al. ^{Reference Robinson, Orr and Wu87} conducted two prospective phase II studies in 9 paediatric and 20 adult patients with recurrent medulloblastoma and also assessed the efficacy of vismodegib treatment. Furthermore, they evaluated the genomic correlation of clinical responses to vismodegib. They observed that treatment with vismodegib resulted in longer progression-free survival in patients with SHH-medulloblastoma than in those with non-SHH-medulloblastoma.

BRAF Proto-Oncogene

The BRAF proto-oncogene is a serine–threonine protein kinase and it is located on the long arm of chromosome 7 at position 34 (7q34). It encodes the BRAF protein and belongs to the RAS-MAPK (RAt sarcoma-mitogen-activated protein kinase) signalling pathway which affects cell proliferation, differentiation, migration and apoptosis. ^{Reference Maraka and Janku88} Cheng et al. ^{Reference Cheng, Lopez-Beltran and Massari89} reported that over 97% of BRAF mutations are located in codon 600 of the BRAF gene and the dominant mutation detected in BRAF-mutated cancers is the V600E mutation which represents about 70–90% of all BRAF mutations. There also exist other less frequent BRAF mutations including V600K (8–20%), V600D (0·1%), V600M (0·3%) and V600R (1%). ^{Reference Maraka and Janku88–Reference Schreck, Guajardo and Lin90} The BRAF-V600 mutation is a thymine to adenine transversion at nucleotide 1799 (T1799A), resulting in a substitution of valine for glutamic acid at position 600 (V600E) or for the less common mutations, substitutions of valine for lysine (V600K), arginine (V600R), leucine (V600) and aspartic acid (V600D). ^{Reference Maraka and Janku88,Reference Schreck, Guajardo and Lin90} BRAF-V600 mutations are reported to be found in different types of adult and paediatric brain cancers including pleomorphic xanthoastrocytoma (42–66%), ganglioglioma (18–70%), astroblastoma (38%), pilocytic astrocytoma (5–16%), anaplastic astrocytoma (0–3%), low-grade glioma (5–15%), glioblastoma (1–8%), gliosarcoma (0–22%), rhabdoid meningioma (0–3%) and oligodendroglioma (0–3%). ^{Reference Maraka and Janku88,Reference Schreck, Guajardo and Lin90}

Several BRAF inhibitors such as vemurafenib, dabrafenib and trametinib are either approved or currently in clinical development or are being used in clinical trial studies for targeting BRAF-mutant cancers. ^{Reference Schreck, Guajardo and Lin90–Reference Hawkins, Walker and Mohamed92} Hawkins et al. ^{Reference Hawkins, Walker and Mohamed92} investigated BRAF alterations in 146 paediatric low-grade astrocytoma patients’ tumour samples using reverse transcriptase PCR, FISH and single nucleotide polymorphism array analysis and correlated with progression-free survival. They observed that 60% of the tumours were BRAF-KIAA1549 fusion positive and 5-year progression-free survival was 61 and 18% for fusion positive and negative patients, respectively. Furthermore, they reported that BRAF-KIAA1549 fusion was the most significant favourable prognostic factor in incompletely resected paediatric low-grade astrocytomas, independent of location, pathology and age and confers a less aggressive clinical phenotype on the tumour. ^{Reference Hawkins, Walker and Mohamed92} Del Bufalo et al. ^{Reference Del Bufalo, Ceglie and Cacchione91} investigated the safety and efficacy of vemurafenib (a BRAF-V600 inhibitor) in paediatric patients with low-grade gliomas who are BRAF-V600E positive. They found vemurafenib to be a potential treatment option in paediatric patients who are affected by low-grade gliomas and harbouring BRAF-V600E mutation. Schreck et al. ^{Reference Schreck, Guajardo and Lin90} reported on two cases of malignant brain tumour patients with BRAF-V600 mutations that were resistant to radiation and temozolomide and treated with concurrent BRAF and MEK inhibitors. They demonstrated that targeting BRAF-V600 mutation with dual BRAF–MEK inhibition in recurrent primary brain tumours has the potential to produce a durable response. Hence, further studies are needed to determine response rates, response duration and impact of the therapy on survival. ^{Reference Schreck, Guajardo and Lin90}

Glioma-Associated Oncogene Homolog 1 (GLI1)

The glioma-associated oncogenes consist of a family of three genes: GLI1, GLI2, GLI3 which are located on the long arm of chromosome 12 at position 13.3 (12q13.3), on the long arm of chromosome 2 at position 14.2 (2q14.2) and on the short arm of chromosome 7 at position 14.1 (7p14.1), respectively. These genes encode the GLI1, GLI2 and GLI3 proteins which are zinc finger transcription factors, SHH-dependant and are expressed in vertebrates. ^{Reference Basile Carballo, Ribeiro Honorato and Farias de Lopes93} The transcription factors play a major role in the HH signalling pathway, where GLI1 and GLI2 are activators and GLI3 is a repressor to regulate the expression of downstream targets such as PTCH1, cyclin D and MYC which are involved in cell survival, proliferation and differentiation. ^{Reference Huang and Yang94} According to Basile et al., ^{Reference Basile Carballo, Ribeiro Honorato and Farias de Lopes93} the role of the HH–GLI signalling pathway in cancer initiating stem cells suggests that it regulates self-renewal and has tumourigenic potential. According to Gruber et al., ^{Reference Gruber, Hutzinger and Elmer95} the GLI proteins act as potent oncogenic drivers by promoting a variety of malignant traits including proliferation, survival, invasion and metastasis, and GLI1 is reported as a critical determinant of tumour-initiating cancer stem cells in several cancers including glioblastoma. Furthermore, the oncogenic properties and the capability of GLI1 to integrate and relay common Smoothened (SMO)-independent cancer-promoting signals such as receptor-tyrosine kinase pathways, PI3K and MAP kinase signalling make GLI1 an attractive molecular target for cancer therapy. ^{Reference Gruber, Hutzinger and Elmer95}

Malatesta et al. ^{Reference Malatesta, Steinhauer and Mohammad96} reported that the inhibition of aberrant HH–GLI signalling pathway has the potential as an attractive approach for anticancer therapy. Basile et al. ^{Reference Basile Carballo, Ribeiro Honorato and Farias de Lopes93} have reported the development of several SHH antagonists that directly target GLI (i.e., GLI inhibitors) in brain tumours which are either biologically based (e.g., curcumin, resveratrol, epigallocatechin-3-gallate, physalin B, physalin F, zerumbone and staurosporinone) or chemically based (e.g., arsenic trioxide, sodium arsenite, GANT 58, and GANT 61). Furthermore, some of these inhibitors such as the FDA-approved arsenic trioxide drug (which has been shown to inhibit GLI-dependent growth in medulloblastoma mouse model), itraconazole and genistein are already being tested in brain tumours in clinical trials. ^{Reference Basile Carballo, Ribeiro Honorato and Farias de Lopes93} Shih et al. ^{Reference Shih, Northcott and Remke97} retrospectively collected 673 medulloblastoma samples from 43 cities worldwide to investigate the presence of molecular biomarkers. They reported that GLI2 amplification can potentially identify patients with poor prognosis and that GLI2 and patient’s metastatic status can practically and reliably predict prognosis for patients with SHH-positive medulloblastoma. Gruber et al. ^{Reference Gruber, Hutzinger and Elmer95} used human medulloblastoma cells culture to investigate the dual-specificity-tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) as a potent therapeutic target in HH–GLI-dependent cancer cells with SMO inhibitor resistance. They reported that DYRK1B is a positive regulator of GLI signalling downstream of SMO and that the inhibition of DYRK1B with harmine resulted in marked repression of GLI1 expression in both human and mouse cancer cell models, suggesting that DYRK1B inhibitors could potentially be used to treat medulloblastoma cancers with GLI amplification. ^{Reference Gruber, Hutzinger and Elmer95} Malatesta et al. ^{Reference Malatesta, Steinhauer and Mohammad96} investigated the potential role of histone acetyltransferase PCAF in the HH–GLI signalling pathway in human medulloblastoma and glioblastoma cell models. They observed that PCAF interacts with GLI1 (which is a downstream effector in the HH–GLI signalling pathway) and that GLI1 loss reduces the levels of H3K9 acetylation on HH target gene promoters. Furthermore, they reported that acetyltransferase PCAF is a positive cofactor of the HH–GLI signalling pathway and therefore proposed PCAF as a therapeutic target for the treatment of patients with medulloblastoma and glioblastoma. ^{Reference Malatesta, Steinhauer and Mohammad96} Zhang et al. ^{Reference Zhang, Zheng and Luan98} investigated the potential link between metabotropic glutamate receptor subtype 4 (mGluR4) and regulation of GLI1 levels in human glioblastoma cell lines. They reported that GLI1 expression was significantly downregulated by mGluR4 activation, and downregulation of GLI1 expression by gene-targeted siRNA resulted in both inhibition of cell proliferation and promotion of apoptosis. Thus, the activation of mGluR4 inhibits GLI1 expression and may block the SHH pathway activation, suggesting that mGluR4 can be employed as a potential drug target for therapy of glioblastomas. ^{Reference Zhang, Zheng and Luan98}

Neuroblastoma Rat Sarcoma Viral Oncogene Homolog (NRAS)

NRAS is a member of the RAS family [Kirsten-RAS (KRAS), Harvey-RAS (HRAS), neuroblastoma-RAS (NRAS)] of membrane-associated G proteins and encodes a protein which belongs to the intrinsic guanosine triphosphatase (GTPase) superfamily involved in cell proliferation, differentiation, cytoskeletal reorganisation and survival. ^{Reference Yapijakis, Adamopoulou and Tasiouka99,Reference Kodaz, Kostek and Hacioglu100} The NRAS gene is located on the short arm of chromosome 1 at position 13.2 (1p13.2) and due to its regulatory nature, a mutation in the gene can potentially lead to carcinogenesis. ^{Reference Yapijakis, Adamopoulou and Tasiouka99,Reference Kodaz, Kostek and Hacioglu100} Yapijakis et al. ^{Reference Yapijakis, Adamopoulou and Tasiouka99} have reported evidence suggesting the involvement of NRAS in some types of brain tumours including glioblastomas, gliomas and neuroblastomas. According to Shih et al., ^{Reference Shih, Yip and McDonald101} activating mutations in the RAS gene (K, N or H) are present in about 20% of all human cancers and mutations in the NRAS gene accounts for about 15% of all RAS mutations. Furthermore, the mutations occur primarily in codons 12, 13 or 61 and lead to constitutive activation of RAS GTPase in the absence of growth factor signalling and ultimately neoplastic growth. ^{Reference Shih, Yip and McDonald101}

Yapijakis et al. ^{Reference Yapijakis, Adamopoulou and Tasiouka99} investigated the genotypes of specimens from various types of brain tumours (neuroglial tumours, astrocytomas, oligodendrogliomas, glioblastomas multiforme, meningiomas) from 35 patients who underwent craniotomy for brain tumour surgery and were also screened for mutations in the HER2, NRAS and NFL oncogenes. They reported the detection of NRAS mutations in 54% (19/35) of biopsies comprising 69% (9/13) in neuroglial tumours, 43% (6/14) in meningiomas, 33% (1/3) in other nervous system tumours and 60% (3/5) in metastatic tumours. Furthermore, they reported that activated NRAS is a major oncogene in CNS malignancies and plays a major role in the activation of the HER2–NRAS–NFL pathway. ^{Reference Yapijakis, Adamopoulou and Tasiouka99} Fang et al. ^{Reference Fang, Boehling and Koay102} investigated the outcomes (local and distant brain failure and overall survival) of 235 melanoma brain metastases in molecularly characterised patients who received conventional treatment for BRAF and NRAS mutations. They studied associations between outcomes and clinical–pathological features of the patients with known mutation status following initial treatment. They reported NRAS mutation prevalence rate of 18% (43/235) and that the NRAS mutation status predicted increased local failure following treatments. Jacob et al. ^{Reference Jakob, Bassett and Ng103} reviewed the clinical and pathologic features of 296 melanoma patients with brain metastasis and known BRAF and NRAS mutation status and observed NRAS mutation prevalence rate of 17%. They reported that the presence of NRAS mutations predicted worse local control, however combining radiation therapy improved local tumour control. Therefore, suggested a combination of radiotherapy with targeted therapies for the treatment of brain metastasis of melanoma patients who may harbor activating NRAS-mutation.

Programmed Cell Death (PD-1)

The PD-1 gene is located on the long arm of chromosome 2 at position 37.3 (2q37.3) and encodes for the PD-1 protein which belongs to the immunoglobulin superfamily. It is a transmembrane immune checkpoint protein which is expressed on lymphocytes and binds the two programmed cell death ligands 1 and 2 (PD-L1 and PD-L2). ^{Reference Zeng, See and Phallen104,Reference Wilmotte, Burkhardt and Kindler105} PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is mostly expressed on the surface of tumour cells and antigen-presenting cells in various solid malignancies including carcinomas of the brain. ^{Reference Zeng, See and Phallen104,Reference Wilmotte, Burkhardt and Kindler105} Furthermore, it plays an important role in the inhibition of T cell proliferation, cytokine production, enhancement of T cell activation and also serves as a receptor that transmits anti-apoptotic signals to tumour cells to protect them from apoptosis. ^{Reference Wilmotte, Burkhardt and Kindler105,Reference Wang, Teng and Kong106} Zeng et al. ^{Reference Zeng, See and Phallen104} reported that PD-1 is implicated as a mediator of immune suppression in a variety of tumours, including GBM, and its binding to the PD-L1 or PD-L2 ligands induces apoptosis or exhaustion of activated immune cells; however, blocking this interaction has been shown to enhance antitumour activity. According to Wang et al., ^{Reference Wang, Teng and Kong106} the PD-1-PD-L1 pathway plays an important role in immune regulation by delivering inhibitory signals to maintain the balance in T cell activation, tolerance and immune-mediated tissue damage. Furthermore, PD-1 signalling in T cells in normal tissues regulates immune responses to decrease damage to adjacent tissues and also counteracts the development of autoimmunity by promoting tolerance to self-antigens. Wang et al. ^{Reference Wang, Teng and Kong106} reported that PD-L1 expression on normal tissues is very rare but is inducibly expressed on tumour cells, suggesting that the selective expression of PD-L1 could have some relationship with the clinical outcomes of cancer patients and could potentially be a selective target for anticancer therapy. Xue et al. ^{Reference Xue, Hu and Iyer107} reviewed the role of PD-L1 expression, PD-L1-mediated immunosuppressive mechanisms and the clinical applications of PD-1-PD-L1 inhibitors in gliomas. They reported that PD-L1 is expressed in glioma cells, correlates with tumour grade and contributes to immune-resistance and that the PD-1-PD-L1 pathway plays an important role in glioma biology.

Jacobs et al. ^{Reference Jacobs, Idema and Bol108} investigated the immune suppressive mechanisms in 83 human brain tumours using ultrasonic tumour aspirates as a biosource. They reported PD-L1 expression in 61% of the malignant tumours while the inhibitory PD-1 receptor was expressed in 26% of tumours. Furthermore, they identified regulatory T cells and the PD-L1-PD-1 pathway as immune suppressive mechanisms in malignant but not benign human brain tumours. Wilmotte et al. ^{Reference Wilmotte, Burkhardt and Kindler105} investigated the expression level of PD-L1 in biopsies collected from 55 patients with brain tumours (low to high-grade astrocytoma) aged 18 years and above. They reported PD-L1 expression in human glioma both in vitro and in vivo and that PD-L1 expressed on glioma cells impaired T cell responses in vitro. Furthermore, considering that PD-L1 exerts negative regulatory effects on T cell proliferation and activation, they suggested that PD-L1 may contribute to the immunosuppressed environment of human glioma and that the targeting of this pathway may be useful to consider for brain tumour immunotherapies. Yao et al. ^{Reference Yao, Tao and Wang109} investigated the expression levels of PD-L1 and the malignancy grade of human astrocyte tumours specimens from 48 patients. They demonstrated a correlation between the expressions of PD-L1 and the malignancy grade of human astrocytomas, suggesting that PD-L1 may be a novel tumour marker and target for therapy. Zeng et al. ^{Reference Zeng, See and Phallen104} examined how a combination treatment of anti-PD-1 immunotherapy with stereotactic radiosurgery could improve local tumour control and overall survival in a mouse model. They reported improved survival benefit for a combination of anti-PD-1 therapy plus radiation compared with either modality alone. The median survival was 25, 27, 28 and 53 days in the control, anti-PD-1 antibody, radiation and radiation plus anti-PD-1 therapy arms, respectively. Liu et al. ^{Reference Liu, Carlsson and Ambjørn110} investigated PD-L1 in neurons and gliomas in tumours from 17 GBM patients and associated the findings with clinical outcome. They observed that upregulation of PD-L1 by neurons in tumour-adjacent brain tissue is associated positively with GBM patient survival, whereas lack of neuronal PD-L1 expression was associated with high PD-L1 in tumours and an unfavourable prognosis.

Wingless-Related Integration Site (WNT) Pathway Mutations

The WNT gene family consists of structurally related genes which encode secreted signalling glycoproteins and their signalling pathway is implicated in the regulation of important biological functions such as embryonic development, proliferation, self-renewal, maintenance, differentiation and migration of stem cells in embryonic and adult tissues. ^{Reference Zuccarini, Giuliani and Ziberi111} The WNT signalling pathway is usually divided into canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) WNT pathways which differ in the way signals are transmitted. ^{Reference Zuccarini, Giuliani and Ziberi111–Reference Denysenko, Annovazzi and Cassoni113} McCord et al. ^{Reference McCord, Mukouyama and Gilbert112} and Denysenko et al. ^{Reference Denysenko, Annovazzi and Cassoni113} reported that the central feature of the canonical WNT pathway is the stabilisation of cytosolic β-catenin followed by translocation to the nucleus, and the WNT-β-catenin signalling pathway is involved in the maintenance of tumour stem cells by inhibiting differentiation, conditioning radio- and chemoresistance and inducing an invasive phenotype in gliomas. However, the aberrant activation of this signalling pathway contributes to glioma development, malignant progression and invasion with prognostic implication. ^{Reference Denysenko, Annovazzi and Cassoni113–Reference Pu, Zhang and Kang115} Zuccarini et al. ^{Reference Zuccarini, Giuliani and Ziberi111} reported that the critical role the WNT pathway plays in brain development and function is well established and the dysregulation of the WNT signal has been implicated in the development, progression and invasiveness of GBM tumours. Denysenko et al. ^{Reference Denysenko, Annovazzi and Cassoni113} also reported that the aberrant activation of the canonical WNT β-catenin signalling pathway contributes to glioma development and malignant progression. Therefore, pharmacological modulation of the WNT pathway could be a potential therapeutic modality for gliomas ^{Reference Zuccarini, Giuliani and Ziberi111} and hence has generated growing investigational interest in the potential role of WNT signalling in malignant glioma pathogenesis, and how it could potentially be targeted therapeutically. ^{Reference McCord, Mukouyama and Gilbert112} Furthermore, WNT plays an essential role in the development of CNS vasculature and the establishment of key structural and functional blood–brain barrier features; therefore, the WNT can be targeted to potentially improve blood–brain barrier permeability and drug delivery. ^{Reference McCord, Mukouyama and Gilbert112}

Götze et al. ^{Reference Götze, Wolter and Reifenberger114} investigated 70 human glioma tissues of different malignancy grades for promoter hypermethylation in 8 genes encoding members of the secreted frizzled-related families of WNT pathway inhibitors. They demonstrated that promoter hypermethylation leading to transcriptional downregulation of certain WNT pathway inhibitor genes is a common event in astrocytic gliomas and is associated with malignancy. Furthermore, they reported that primary and secondary glioblastomas show a distinct pattern of hypermethylated WNT pathway genes, further suggesting that these glioblastoma subtypes differ in their genetic alterations and in their DNA methylation profiles. ^{Reference Götze, Wolter and Reifenberger114} They implicated the role of epigenetic inactivation of WNT pathway inhibitor genes in the malignant progression of astrocytic gliomas to potentially provide novel methods for targeted therapeutic approaches in the future. Pu et al. ^{Reference Pu, Zhang and Kang115} examined the expression of WNTs in 45 resected primary astrocytic glioma specimens (with different tumour grades) to determine the role of WNT signalling in gliomagenesis. They reported overexpression of WNT2, WNT5a, frizzled 2 and β-catenin in gliomas and knockdown of WNT2 and β-catenin by siRNA in human glioma cells, effectively inhibited malignant cell proliferation and invasive ability, and induced apoptotic cell death. Furthermore, treating nude mice carrying established subcutaneous gliomas with siRNA targeting WNT2 and β-catenin intratumourally also delayed tumour growth. Suggesting that the canonical WNT signalling pathway may be a new potential therapeutic target for human glioma. Denysenko et al. ^{Reference Denysenko, Annovazzi and Cassoni113} examined protein expression of WNT (WNT3a, β-catenin) and transcription factor 4 (TCF4) in 74 adult human brain tumours (31 glioblastomas and 43 grade I–III gliomas). They reported WNT3a was overexpressed at the protein and mRNA levels in malignant astrocytic tumours and cell lines, and cytoplasmic expression of β-catenin was detected in high-grade gliomas and cell lines. Furthermore, expression of WNT3a, cytoplasmic β-catenin and TCF4 was significantly associated with the histological malignancy grade and with a worse prognosis for patients with malignant glioma. Thus, WNT3a and β-catenin may be regarded as potential prognostic factors in the entire group of gliomas.

Insulin-like Growth Factor-IR

The insulin-like growth factor (IGF) signalling pathway is implicated in regulating growth and development in normal tissues by promoting cellular proliferation, differentiation and prevention of apoptosis, thereby playing a major role in oncogenesis. ^{Reference Maris, D’Haene and Trépant116,Reference Simpson, Petnga and Macaulay117} The IGF axis comprises insulin and two related ligands: IGF-1 and IGF-2, that regulate cellular processes by interacting with specific cell-surface receptors, namely IGF-IR and IGF-2 R, which are members of the receptor tyrosine kinase class 2 family of receptors. ^{Reference Simpson, Petnga and Macaulay117} The IGF-1 gene is located on the long arm of chromosome 12 at position 23.2 (12q23.2) whereas the IGF-2 gene is located on the short arm of chromosome 11 at position 15.5 (11p15.5) and encodes proteins which are similar to insulin in function and structure. ^{Reference Simpson, Petnga and Macaulay117} Sachdev and Yee ^{Reference Sachdev and Yee118} reported that IGF-IR is involved in maintaining malignant phenotype and disruption of its activation impedes growth and motility of cancer cells in vitro and in mouse models, thereby making it a potential target for cancer therapy. Circulating levels of IGF ligands and/or IGF-IR overexpression have been observed in various cancers including glioblastoma, and this overexpression is linked to faster disease progression and poor prognosis. ^{Reference Simpson, Petnga and Macaulay117,Reference Sachdev and Yee118} Maris et al. ^{Reference Maris, D’Haene and Trépant116} and Yin et al. ^{Reference Yin, Girnita and Strömberg119} reported that IGF-1 R and IGF-2 R are involved in tumours of the cerebellum, neuroblastomas and glioblastomas, and in-vitro studies have shown that IGFs promote proliferation, survival and migration of glioblastomas cell lines. Yin et al. ^{Reference Yin, Girnita and Strömberg119} also reported that IGF-1 R is a transmembrane heterotetramer whose cytoplasmic tyrosine kinase domain activates the PI3K–AKT and RAS–RAF–MAPK signalling pathways. It has been reported that since IGF signalling in tumour cells is driven by the presence of the ligands rather than receptor aberrations, a low circulating IGF-1 concentration can protect against tumourigenesis. ^{Reference Simpson, Petnga and Macaulay117,Reference Sachdev and Yee118}

Maris et al. ^{Reference Maris, D’Haene and Trépant116} studied the expression levels of IGF ligands and receptors in 218 human glioblastoma specimens to determine their prognostic value and observed overexpression of IGF-1 R and IGF-2 R in glioblastoma compared with normal brain. They identified the overexpression as an independent prognostic factor which was associated with shorter survival and a less favourable response to temozolomide, thus suggesting that IGF-1 R could be a potential target for glioblastoma therapy. However, they reported that further studies are needed to investigate the role of IGF-1R in the chemo-resistance of glioblastomas and to determine which patients could benefit from a combination therapy of temozolomide and an IGF-IR inhibitor. Zamykal et al. ^{Reference Zamykal, Martens and Matschke120} examined the effect of IMC-A12 (an IGF-1 R blocking antibody) on glioblastoma growth in two different in-vivo animal models (a U87 cells line used to establish rapidly growing, angiogenesis-dependent tumours in the brains of nude mice, and an GS-12 cell line used to generate highly invasive tumours). They observed that the IMC-A12 treatment inhibited growth of U87 tumour by 75%, moderately decreased cell proliferation and increased intratumoural vascularisation in the U87 tumours, whereas the treatment inhibited the growth of GS-12 tumours by 50%, significantly reduced invasive tumour extension and proliferation rate, increased apoptosis and blocked the activation of IGF-1 R by IGF-1 and IGF-2 in the GS-12 tumour cells. Thus, blocking IGF-1 R may be useful to target highly proliferative, angiogenesis-dependent glioblastoma tumours. Yin et al. ^{Reference Yin, Girnita and Strömberg119} also investigated the effect of cyclolignan picropodophyllin (an IGF-IR inhibitor) on human glioblastomas. They observed that cyclolignan picropodophyllin inhibits the growth of human glioblastoma cell and reduces the phosphorylation of IGF-1 R. Furthermore, in-vivo cyclolignan picropodophyllin treatment resulted in significant tumour regression in both subcutaneous xenografts and intracerebral xenografts, indicating its passage across the blood–brain barrier. Thus, suggesting that cyclolignan picropodophyllin targeting IGF-1 R is a promising treatment option for patients with glioblastoma.

Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) also referred to as ErbB1/HER1 belongs to the ErbB family consisting of 4 receptors (ErbB1-4/HER1-4) with tyrosine kinase activity and 13 polypeptide extracellular ligands with a conserved EGF domain. ^{Reference Paul, Bhattacharya and Chatterjee121–Reference Andersson, Schwartzbaum and Wiklund123} The EGFR gene is located on the short arm of chromosome 7 at position 11.2 (7p11.2) and encodes for the EGFR receptor protein ^{Reference Jaros, Perry and Adam124} , and its amplification, overexpression and mutations have been implicated in various cancers including glioma development. ^{Reference Paul, Bhattacharya and Chatterjee121,Reference Xu, Zong and Ma122,Reference Golding, Morgan and Adams125} Activation of EGFR by ligand binding or in a transient, ligand-independent manner by radiation will trigger a cascade of cellular signaling events associated with increased cell proliferation, angiogenesis, invasion and metastasis and thus, are crucial for tumorigenesis. ^{Reference Paul, Bhattacharya and Chatterjee121,Reference Andersson, Schwartzbaum and Wiklund123,Reference Golding, Morgan and Adams125} Xu et al. ^{Reference Xu, Zong and Ma122} and Andersson et al. ^{Reference Andersson, Schwartzbaum and Wiklund123} have reported that EGFR is overexpressed in about 50–60% of primary glioblastomas and in about 10% of secondary glioblastomas, and it is characteristic of more aggressive glioblastoma phenotypes. According to Golding et al., ^{Reference Golding, Morgan and Adams125} the upregulation of wild type or expression of mutant EGFR is associated with high tumour radio-resistance and poor clinical outcome.

EGFR signalling pathway has been the central target for GBM therapy and hence there have been investigations into developing EGFR-directed antitumour therapeutic strategies, some of which have already shown preliminary antitumour effects in GBM. These include blocking of the tyrosine kinase activity of EGFR, using competitive and noncompetitive kinase inhibitors (e.g., erlotinib, gefitinib) and other second generation inhibitors (e.g., afatinib, dacomitinib) often in combination with radiation and temozolomide and using monoclonal antibodies (mAbs) (e.g., cetuximab, nimotuzumab). ^{Reference Paul, Bhattacharya and Chatterjee121,Reference Xu, Zong and Ma122} Golding et al. ^{Reference Golding, Morgan and Adams125} have shown that increased EGFR signalling has a global and multi-faceted effect on the DNA damage response to influence double-strand break repair. Thus, targeting double-strand break repair in conjunction with radiotherapy either alone or in combination with a TKI may be a potential interventional therapeutic approach for GBM. Andersson et al. ^{Reference Andersson, Schwartzbaum and Wiklund123} investigated the genetic variations of EGF, EGFR, ERBB2, LRIG2, LRIG3, VEGF and VEGFR2 genes on risk of glioma and glioblastoma in 725 glioma patients. They reported that EGFR genotypes are important determinants of glioma risk and that certain haplotypes in EGFR and ERBB2 may be involved in glioblastoma development and therefore may be used to identify patients who would benefit from therapies targeting EGFR receptor pathways. Wu et al. ^{Reference Wu, Yang and Barth126} evaluated boronated mAb (cetuximab) as a delivery agent for boron neutron capture therapy of a human EGFR gene-transfected rat glioma model. They demonstrated the therapeutic efficacy of molecular targeting of EGFR with boronated mAb either alone or in combination with boronophenylalanine. Jaros et al. ^{Reference Jaros, Perry and Adam124} investigated the expression levels of EGFR with tumour proliferative activity and patients’ progress in biopsies of 16 types of human brain tumours and 43 astrocytomas. They reported that the proportion of tumours expressing EGFR increased with grade of malignancy, and the expression of EGFR could occur at early stages of tumourigenesis, was associated with malignant progression and poor prognosis and was also associated with a significantly reduced survival in astrocytomas.

Co-Deletion of 1p and 19q (1p19q)

1p19q co-deletion is the complete deletion of the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q) and is commonly found in oligodendrogliomas. ^{Reference Jansen, Yip and Louis127} According to the National Center for Biotechnology Information, ¹²⁸ chromosome 1 is the largest human chromosome containing over 3,000 genes, whereas chromosome 19 is one of the shortest human chromosomes and contains over 1,700 genes and both chromosomes are geometrically metacentric. Co-deletion of 1p and 19q has been reported in roughly 80–90% of grade II oligodendroglioma cases and about 50–70% of grade III oligodendroglioma cases with around 70% overall. Furthermore, co-deletion of 1p/19q is also found in mixed glial tumours, oligoastrocytomas, in a lower proportion to regular oligodendrogliomas (20–30%) and has demonstrated prolonged progression-free survival. ^{Reference Jansen, Yip and Louis127} Jansen et al. ^{Reference Jansen, Yip and Louis127} have reported that although the responsible oncogenic genes on chromosomes 1p and 19q remain unknown, many correlations have been made regarding 1p and/or 19q co-deletion such as increased IDH1 and IDH2 mutations, tumours frequent appearance of classic histology, an inverse correlation with TP53 mutations, 10q deletions and EGFR amplification. Furthermore, the location of brain tumour is associated with 1p19q co-deletion; thus, low-grade oligodendroglioma in the frontal, parietal and occipital lobes is more likely to show 1p19q loss than tumours involving the temporal lobe, insula and diencephalon. ^{Reference Jansen, Yip and Louis127}

1p19q co-deletion is reported to be a pathognomonic clinical biomarker that defines a distinct group of gliomas and is characteristic of oligodendrogliomas, ^{Reference Chaturbedi, Yu and Linskey129} a strong independent prognostic biomarker associated with improved survival in both diffuse low-grade and anaplastic tumours ^{Reference Zhao, Ma and Zhao130} and has predictive value for response to chemotherapy in anaplastic oligodendrogliomas. ^{Reference Cairncross, Wang and Shaw131} Chaturbedi et al. ^{Reference Chaturbedi, Yu and Linskey129} investigated the detection of 1p/19q deletions in low-grade oligodendroglioma (n = 21), anaplastic oligodendroglioma (n = 15) and mixed oligoastrocytoma tumours (n = 8) using quantitative PCR. They observed 1p/19q deletions in 81% of low-grade oligodendroglioma tumours, 47% of anaplastic oligodendroglioma tumours and 13% of mixed oligoastrocytoma tumours. They reported that 1p/19q deletion is a potential prognostic factor in oligodendroglial tumours and may predict better survival after both chemotherapy and radiotherapy. Zhao et al. ^{Reference Zhao, Ma and Zhao130} conducted a meta-analysis of 28 studies involving 3,408 glioma cases to evaluate the association between loss of heterozygosity of 1p/19q and progression-free survival and overall survival. They reported that co-deletion of 1p and 19q was associated with better progression-free survival and overall survival rates in gliomas and the association is independent of detection method, the grade and subtypes of gliomas. Cairncross et al. ^{Reference Cairncross, Wang and Shaw131} investigated the efficacy of procarbazine, lomustine and vincristine (PCV) chemotherapy plus radiotherapy (n = 148) versus radiotherapy alone (n = 143) on 291 patients with anaplastic oligodendroglioma and anaplastic oligoastrocytoma and the potential prognostic and predictive value of 1p and 19q allelic loss in the tumours. They reported that tumours lacking 1p and 19q alleles were less aggressive or more responsive or both to the treatments and demonstrated that for patients with 1p/19q co-deleted anaplastic oligodendroglioma and anaplastic oligoastrocytoma, PCV chemotherapy plus radiotherapy has survival advantage.

MGMT Promoter Methylation

The MGMT gene is located on the long arm of chromosome 10 at position 26.3 (10q26.3) and in most glioblastomas, one allele of this chromosome is usually lost, leaving the remaining gene copy to drive function. ^{Reference Mansouri, Hachem and Mansouri132} The MGMT gene encodes a highly conserved DNA repair enzyme that provides resistance to alkylating chemotherapies such as carmustine, lomustine and temozolomide ^{Reference Mansouri, Hachem and Mansouri132–Reference Brandes, Franceschi and Paccapelo134} and the methylation of MGMT promoter has been observed in about 50% of grade IV gliomas. ^{Reference Esteller, Garcia-Foncillas and Andion135} MGMT protein expression is observed in a wide variety of tumours including glioblastomas and the loss of expression of the protein results in decreased DNA repair and retention of alkyl groups, thereby allowing alkylating agents to have greater efficacy in patients whose tumours exhibit hypermethylation of the MGMT promoter and reduce the MGMT protein concentration. ^{Reference Esteller, Garcia-Foncillas and Andion135,Reference Rivera, Pelloski and Gilbert136} Esteller et al. ^{Reference Esteller, Garcia-Foncillas and Andion135} and Revera et al. ^{Reference Rivera, Pelloski and Gilbert136} have reported that proper functioning of the MGMT gene is important for maintaining cell integrity; however, epigenetic silencing of the gene by methylation of the cytidine phosphate guanosine islands of the promoter region has been shown to correlate with loss of gene transcription and protein expression.

Hypermethylation of the MGMT gene promoter has been observed as a potential predictive biomarker of sensitivity to alkylating agents (e.g., carmustine, lomustine and temozolomide) chemotherapy ^{Reference Mansouri, Hachem and Mansouri132,Reference Rivera, Pelloski and Gilbert136–Reference Gilbert, Wang and Aldape139} and as a strong prognostic factor for newly diagnosed glioblastoma. ^{Reference Brandes, Franceschi and Paccapelo134} Hegi et al. ^{Reference Hegi, Diserens and Gorlia137} investigated the relationship between MGMT silencing in glioblastomas and the survival of patients who participated in a randomised trial comparing radiotherapy alone and radiotherapy with temozolomide chemotherapy. They reported that 45% (93 of 206) of cases had methylated MGMT promoters and observed that irrespective of the treatment arm, MGMT promoter methylation was an independent favourable prognostic factor. Furthermore, they reported a longer survival in patients whose tumour harboured methylated MGMT promoter and were treated with temozolomide and radiotherapy (median survival was 21·7 months) compared with patients who were assigned to only radiotherapy (median survival was 15·3 months). Esteller et al. ^{Reference Esteller, Garcia-Foncillas and Andion135} analysed 47 newly diagnosed grade III or IV glioma (18 anaplastic astrocytoma and 29 GBM) samples obtained from patients who were treated with carmustine to determine whether methylation of the MGMT promoter is related to the tumour responsiveness to the alkylating agent. They reported that MGMT promoter methylation was associated with a response to carmustine, an increase in overall survival and the time to progression of disease. Moreover, the methylation status of the promoter was more predictive of the outcome of carmustine treatment than the grade of the tumour, Karnofsky performance score or the age of the patient. Revera et al. ^{Reference Rivera, Pelloski and Gilbert136} assessed the MGMT methylation status in 225 patients with newly diagnosed glioblastoma who were treated with radiation therapy (n = 172) and adjuvant chemotherapy (n = 52). They observed an approximately 50% reduction in the rate of tumour progression during radiation therapy in MGMT methylated tumours compared to unmethylated tumours. They also demonstrated that methylation of the MGMT promoter resulted in a nearly 2-fold delay in tumour progression as opposed to tumours with unmethylated MGMT promoter, even in the absence of alkylating agent chemotherapy. Stupp et al. ^{Reference Stupp, Mason and Van Den Bent138} conducted a similar study on 573 patients from 85 centres with newly diagnosed, histologically confirmed glioblastoma and MGMT status, who were randomly assigned to receive either radiotherapy alone or radiotherapy plus temozolomide. They reported a median survival of 14·6 months for patients treated with radiotherapy and temozolomide and 12·1 months for patients treated with radiotherapy alone at a median follow-up of 28 months. Furthermore, they indicated a 2-year survival rate of 26·5% with radiotherapy and temozolomide and 10·4% with radiotherapy alone and concluded that the addition of temozolomide to radiotherapy for newly diagnosed glioblastoma resulted in a clinically meaningful and statistically significant survival benefit with minimal toxicity. Gilbert et al. ^{Reference Gilbert, Wang and Aldape139} conducted a phase III trial to investigate whether temozolomide improves overall survival or progression-free survival in 833 patients with newly diagnosed GBM and MGMT status who were randomly assigned to either radiation alone or radiation and temozolomide. They reported that MGMT methylation was associated with improved overall survival or progression-free survival and confirmed the prognostic significance of MGMT methylation.

Copy Number Alterations

Copy number alterations (CNAs) are somatic changes to chromosome structure that result in either a gain or loss of copies in sections of the DNA and are found to correlate with the development and progression of cancer. ^{Reference Hughesman, Lu and Liu140,Reference Lombardi and Assem141} According to Nawaz et al., ^{Reference Nawaz, Patil and Thinagararjan142} CNAs include amplifications and duplications (gain) or deletions (loss) and are responsible for changing the diploid status of a specific locus, thereby disrupting the balanced genome. These modifications continually shape the structure of the cancer genome as they enable its adaptation, evolution and distortion. Furthermore, CNAs influence cancer phenotype through the amplification of oncogenes and deletions of tumour suppressor genes, form the sources of genomic instability and structural dynamism (which are the important classifiers of cancer cells) and alter the dosage of a gene product independent of the transcription factors. ^{Reference Nawaz, Patil and Thinagararjan142} Hughesman et al. ^{Reference Hughesman, Lu and Liu140} reported that copy number gains at chromosome 8q24.1, which include within the MYC oncogene family, occur in 45·9% of all cancers. According to Nawaz et al., ^{Reference Nawaz, Patil and Thinagararjan142} glioblastomas have a unique landscape of CNAs, with the associated appearance of numerous driver amplifications and deletions and are observed to be severely affected by the CNAs, as there are numerous important genes that get derailed due to CNAs leaving a significant portion of the genome heavily distorted. Lombardi. ^{Reference Lombardi and Assem141} reported that the most common CNAs in glioblastoma include the complete or partial loss of chromosome 9 and 10, polysomy of chromosome 7, 19, 20, the focal deletion of CDKN2A/B locus located at 9p21.3 and focal high level amplification of EGFR locus located at 7p11.2.

Cimino et al. ^{Reference Cimino, McFerrin and Wirsching143} investigated the frequency of copy number subtype in glioblastoma patients who were selected from the CGA, the German Glioma Network, the phase II trial ARTE in elderly patients with newly diagnosed glioblastoma and a multi-institutional cohort which was focused on paired resected initial/recurrent glioblastoma. They reported that it is important to include molecular profiling, such as CNAs, when enrolling patients for clinical trials in order to ensure more appropriate cohort distributions and therapeutic strategies that are applicable to the general population. Seifert et al. ^{Reference Seifert, Friedrich and Beyer144} used a regulatory network inferred from gene expression and gene copy number data of 768 human cancer cell lines to quantify the impact of patient-specific CNAs on survival signature genes. They observed that rare patient-specific gene CNAs have stronger effects on signature genes than frequent gene CNAs and that the integration of indirectly acting gene CNAs significantly improves survival analysis. Cimino et al. ^{Reference Cimino, Zager and McFerrin145} used Oncoscape (a web-based platform) and applied multidimensional scaling-derived molecular groups to the 2D visualisation of the 2016 WHO classification of diffuse gliomas. They observed that diffuse glioma survival is associated with whole chromosome and gene level CNAs and that CNAs predict patient survival and form prognostic molecular subtypes in diffuse gliomas, and subtypes with clear differences in median survival.