Astrocytes Oligodendrocytes And Ependymal Cells Biology Essay

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Gliomas are sub-categorized as low- and high- grade tumours, with low and high degree of malignancy respectively. Low grade gliomas include oligodendroglioma, ependymoma, mixed gliomas and diffuse astrocytoma (grade II), all without an anaplastic feature. But then, high grade gliomas are the most malignant tumours with element of anaplastic features and these include: anaplastic astrocytoma (grade III) and Glioblastoma multiforme (grade IV) [x]

Current study conducted across Europe, declared that patients with astrocytic tumour have the worst prognosis, with a low survival rate. High grade astrocytic tumours exhibits 5% survival rate whilst low grade astrocytic tumours present a more moderate rate: oligodendroglia (54.5%), ependymal (74.2 %) (Crocetti.E et al., 2012). Low survival rate reported in Europe may be due to the fact, that EU regions have less accessibility to Magnetic resonance imaging (MRI). Furthermore, lack of surgical, radiotherapy and chemotherapeutical facilities across Europe may also contribute to the low survival rate observed across EU regions. (Crocetti.E et al., 2012). Life-threatening gliomas continues to plague across varying geographical regions, where countries like Australia, Canada, Denmark, Finland, New Zealand and the US presents fourfold difference in incidence of primary malignant brain tumours when compared to other countries such as Mumbai in India and Rizal in the Philippines.(Schwartzbaum Judith. A et al., 2006) Recent researches have statistically proven that gliomas are more common in men than in women. However, it is quite evident that gender does not factor too much in the tumour development, i.e oligodendroglioma (Schwartzbaum Judith. A et al., 2006).

Prognosis of patients diagnosed with gliomas is extremely low, with "approximately 2% of patients aged 65 years or older, and only 30% of those under the age of 45 years at GM diagnosis, survive for 2 years or more" (Schwartzbaum Judith. A et al., 2006). With the advent of the cure for gliomas yet to arise, strategies to limit the pathogenesis surrounding the brain tumour and ultimately lead to a cure are being looked into and these include the genetic and molecular factors behind the tumour.

The objective behind this study is to explicitly provide a detailed account of the pathogenesis responsible for the invasiveness and aggressiveness of gliomas type tumour in the brain. The focus of this study will be centred on the hallmarks of cancer including the pre-metastatic niche formation, vascular proliferation and the cellular mechano-signal behind the tumour development.

Consequently, the molecular findings presented here may help shed light on promising areas for future research.

Main section

Origin of gliomas

Our current knowledge on the cell of origin for glioma is still highly vague and inconclusive as this task have proven difficult due to acquired plasticity feature which allows the true cellular origin of the cancerous cells to be concealed . Complete understanding of the cellular origin of glioma poses as the missing element needed to fully master the biology of glioma. Thanks to the collective information obtained from cancer patients, developmental biologist and experimental glioma models, three targets cells have been acknowledged and suggested to have the capability to be the origin of glioma, which includes astrocytes, neural stem cells and oligodendrocytes precursor cells (Jiang & Uhrbom, 2012).

There are substantial evidences to support the hypothesis which states astrocytes as the purported cell of origin for glioma. The fact that astrocytic molecular marker known as GFAP is often expressed in human glioma tissue makes astrocytes a plausible target (Jiang & Uhrbom, 2012). On the other hand, the possibility that gliomas can also originate from other cell types have not be eliminated as the expression of GFAP also take place in NSCs in the adult sub-ventricular zone and radial glial cells (Jiang & Uhrbom, 2012). [7] (Fig.17)

Furthermore, the hypothesis supporting neural stem cells (NSC) situated in the subventricular zone as a valid cell candidate of origin of gliomas cannot be ignored. Findings obtained from the histopathological analysis of glioma tissue indicated that presymptomatic lesions were located within the SVZ, thus suggesting SVZ stem cells as a cell of origin for glioma (Jiang & Uhrbom, 2012). Furthermore, another strong experimental support for NSC as a valid candidate is that the signalling pathways responsible for regulating self-renewal, differentiation and proliferation of NSCs frequently appear to be altered in gliomas (Jiang & Uhrbom, 2012). Oligodendrocyte precursor cells (OPC) are also regarded as a potential cell of origin for glioma. This hypothesis is supported by the fact that PDGFRα signalling pathway required for controlling the proliferation and migrations of OPCs appear to be altered in gliomas. Additionally, OPC molecular markers such as NG2 and PDGFR, are expressed readily in glioblastoma and oligodendroglia (Jiang & Uhrbom, 2012).

Characteristics of low grade and high grade gliomas

Low grade astrocytoma (LGA) such as oligoastrocytoma, oligodendroglioma and diffuse astrocytoma all display very little if any propensity to metastasize to neighbouring environment. However, low grade astrocytoma present pathological and morphological features such as elevated rate of transformation, invasive ability, micro-vascular network proliferation, some indication of pleomorphism, elevated cellularity in neoplastic astrocytes and indication of pseudopalisading necrosis. Furthermore, deposition of calcium particles was also observed in LGA. {{526 BURGER,PC 1985;}} (BURGER et al.,1987)

Anaplastic astrocytoma (AA) is a type of infiltrative brain tumour with apparent lesions. AA is characterized by anaplastic features such as vascular proliferation, pleomorphic cells with hyperchromatic nuclei and high cellular mitotic rate (BURGER et al.,1987).

Glioblastoma multiforme (GBM) is a type of astrocytic tumour which can arise anew or from the malignant progression of a low grade astrocytoma. GBM presents significant increase in tumour cell density, necrosis tissue, cellular proliferation, establishment of large vascular network and high malignant tendency. Furthermore, this tumour also display activity of glomeruloid body deposition, a vascular like- structure associated with the tumour which accounts to the pathological features of GBM. (BURGER et al.,1987)

analysis of the PATHOPHYSIOLOGY FEATUREs in gliomas using neuroimaging data

Magnetic resonance imaging (MRI) is the best type of diagnostic neuroimaging technique employed to visualize pathological & anatomical features in gliomas such as hypoxia & angiogenesis (MENDICHOVSZKY and JACKSON 2011) (Bansal et al.,2011). This imaging technique uses magnetic fields combined with radio waves to visualize the internal structure of the brain tissue via manipulation of protonic atoms in brain tissue.

The low grade astrocytoma and anaplastic astrocytoma is evidently apparent in these T1 weighted MRI scans unlike the GBM T1 weighted scan. LGA and AA tumour is situated in the occipital region of the brain, although LGA is localised to the bottom of the occipital region whilst AA appears to demonstrate malignancy towards the centre of the brain (Keith A.Johnson et al.,2013) The T1 weighted scan demonstrates area of mixed signals as the size and actual location of the GBM tumour is difficult to elucidate at this stage (Keith A.Johnson et al.,2013). Nonetheless, the AA tumour appears to be considerably bigger than LGA, at least three times the size of the LGA tumour. The size of AA and GBM compared to LGA explains the morbidity and mortality rate presented by these tumours. Brain tumours of this size often present pathophysiological features such as raised intracranial pressure, elements of intracranial neoplasm, tumour growth in bilateral nature and aberrant hyperplastic blood vessels. The invasive and aggressive nature of AA and GBM tumour is illuminated and potentiated by the aggregation of these pathophysiological features.

Furthermore, in the T1 weighted scan for the AA tumour, the strong signal originating from water is due to the fact that the gadolinium molecules might have shortened its relaxation time (Bansal et al., 2011)(Keith A.Johnson et al., 2013). It is possible that such signal might be an indication of the blood brain barrier breakdown which corresponds to the pathological feature common in patients diagnosed with malignant AA or GBM. This phenomenon is possibly due to a newly acquired ability by the malignant tumour to actively attacking the structural component of the BBB termed endothelial tight junctions using soluble factors (Castejón, 2012). The disruption of the blood-brain barrier (BBB) by these secreted soluble factors (VEGF?) also results in cerebral oedema (Castejón, 2012).

T2-weighted scans are more suitable for reflecting vasogenic oedema and clinical diagnosis of brain tumours. (Bansal et al.,2011) Upon observation, LGA demonstrate minimal degree of invasive ability whilst AA showed a tumour mass surrounded by mix of high and low signal. (Keith A.Johnson et al., 2013)GBM appeared as a spheroid mass with necrotic centre with evidence of cerebral oedema. Similarly, the AA scan conferred similar pathological features as a lower signal and appears to be situated in the left hemisphere of the brain. These underlying features correspond to the pathological criterion for AA and GBM. (Keith A.Johnson et al., 2013)Cerebral oedema arises from the convergence of BBB disruption and the accumulation of water in the brain which in turn shifts the intracranial pressure in favour of a pathological criterion. (Castejón, 2012) Tissue necrosis observed in AA and GBM is caused by a hypoxic environment, a pathological condition where the oxygen level is below normal. These multiple hypoxic regions are stimulated by the activation and stabilization of hypoxia-inducible factor (HIF) (Kaur et al.,2005) HIF activation is amongst the governing force behind the pathological angiogenesis observed in gliomas.

Proton density weighted MRI scan primarily depend on the quantitative density of proton within a given tissue. High density of proton correlates with signal intensification, thus a more contrast PD weighted MRI image is generated. PD weighted MRI scan of AA and GBM tumour reflects high concentration of protons in the tumour mass and neighbouring tissues, which in turn allows greater net magnetization, hence, a brighter contrasted PD weighted MRI image. The PD weighted MRI images of AA and GBM reflected on the pathological features such as the invasive nature of gliomas, tissue necrosis and lesions. In addition, AA and GBM are surrounded by the mix of hyper- and hypo- intensity which possibly correlates with the active stages of gliomagenesis. (Keith A.Johnson et al., 2013) These active stages are possibly driven by the altered vascular biology of the gliomas tumour elicited by the upregulation of HIF.

Thallium (201TI ) SPECT imaging is centred around the active Ionic movement of thallium molecules via ATP pump which correlates with tumour growth rate, hence thallium uptake interrelates with the tumour metabolism (Zaidi et al.,2006) . 201TI SPECT scan of LGA revealed a small area of thallium uptake which in turn correlates with minimal increase in tumour metabolism, which indicates a low risk of tumour recurrence. On the other hand, the 201TI SPECT scan of AA and GBM featured high uptake of thallium, which interrelate with high risk of the glioma recurrence and poor prognosis. In addition, significant thallium uptake in different regions is observed in the GBM 201TI SPECT which indicate possible area of tumour metastasis . Increase in the tumour metabolism can be explained by the switch to cytoplasmic glycolysis from mitochondrial oxidative phosphorylation by the highly tumorigenic GBM.* Metabolic modulation observed in these tumorigenic tumours is complemented by mitochondrial hyperpolarization and it is thought that the change in the mitochondrial membrane potential may allow the proapoptotic mediators to efflux via the mitochondrial transition pore. (Michelakis et al., 2012) Consequently, glioma becomes less susceptible to cell-program death (apoptosis) which in turn further potentiate the risk of tumour metastasis. The metabolic shift exhibited by GBM is required to fuel the accelerated mitosis. Furthermore, lactic acid, a by-product of cytoplasmic glycolysis confers some proliferative advantages as this by -products are able to facilitate the interstic matrix breakdown, alter mitochondrial function and promote angiogenesis (Michelakis et al., 2012). Consequently, GBM and AA tumour occurrence is often observed.

Technetium based SPECT imaging technique is centred around the radioactive tracer known as 99mTc which emits low gamma rays which allows the vascular nature of the brain to be mapped based on the uptake of the radio-ligand (Zaidi et al.,2006) High uptake of technetium correlates with the blood flow within the brain which is coupled to the organ's energy and metabolic requirement. 99mTc based SPECT scan for LGA reflect a small uptake of technetium compared to AA and GBM which corresponds to its pathological feature. Alternatively, AA and GBM 99mTc based SPECT scan indicated high uptake of technetium which corresponds to increased neo-vascularization exhibited by both tumours. The increase in 99mTc uptake correlates with the increased blood flow due to the presence of aggressive vascular proliferation established by the tumour. This again can be explained by the alternation in AA and GBM vascular biology. Angiogenesis initiated by hypoxia allows the upregulation of factors required for the formation of blood vessel. As a result, gliomas vascular proliferation drives the malignant tumour growth.


During tumorigenesis, the development of vasculature is essential for sustaining tumour proliferation. Tumour associated neovasculature is developed by a process known as angiogenesis. This biological process is marked by the appearance of branching, enlarged vessels with unsettled blood flow, microheamorrhaging, and by aberrant endothelial cell proliferation (fig.1). {{519 Douglas 2011;}}

Normal vascular network and gliomas vascularization

Normal vascular network originate from blood vessels created during early stages of embryogenesis via a biological process known as vasculogenesis. Blood vessels are formed from endothelial cell precursors called angioblasts which proliferates to form a network of vessels term primary capillary plexus(PAPETTI & HERMAN, 2012). This network is used as a scaffold to construct new vessels from pre-existing ones via branching and sprouting remodelling, both which are angiogenic processes. (PAPETTI & HERMAN, 2012) Mechanisms involved in normal angiogenesis include vessel destabilization, vessel hyperpermeability, endothelial proliferation & migration, tube formation, mesenchymal proliferation, pericytes differentiation and lastly vessel stabilization (PAPETTI & HERMAN, 2012). Angiopoietin -2 is responsible vessel destabilization while vascular endothelial growth factor (VEGF) induces vessel hyperpermeability and endothelial proliferation & migration. Fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and epidermal growth factors (EGF) are responsible for the proliferation of endothelial and mesenchymal cells as well as tube formation. . (PAPETTI & HERMAN, 2012) Furthermore, transforming growth factor-β (TGF- β) and tumour necrosis factor-α (TNF- α) governs pericytes proliferation and mediates vessel stabilization. (PAPETTI & HERMAN, 2012)

Fig.2 Diagrammatic representation of normal angiogenesis. (PAPETTI & HERMAN, 2012)

Highly tumorigenic tumours such as AA or GBM are capable of formulating their own blood vessels from pre-existing vascular network via a process similar to that of normal angiogenesis. (PAPETTI & HERMAN, 2012) Fig.3 describes 2 similar models for tumour associated neovascularization. The first model describes how an avascular tumour establishes its blood supply by growing until hypoxic regions are established inwardly (PAPETTI & HERMAN, 2012). Consequently, this tumour favoured- pathological condition enables upregulated secretion of angiogenic factors such as interleukin-8(IL-8), FGF, TNF- α, PDGF & VEGF which all play key roles in tumour-induced angiogenesis (PAPETTI & HERMAN, 2012) .The latter model explains how a vascularized tumour induces the secretion of angiopoietin-2 (ang-2) in peripheral vessels which then becomes structurally regressed due to apoptotic death induced in the endothelial cells. (PAPETTI & HERMAN, 2012)

Consequently, the tumour becomes avascular and becomes able to utilize the aforementioned mechanism to establish its own vascular network. Angiogenic factors involved in malignant gliomas are summarized in table 2




Fig.3 (A) shows a schematic diagram of the models formulated in tumour neovascularization by Michael et al.

.(PAPETTI & HERMAN, 2012 ) (B) Mechanisms of hypoxia initiated angiogenesis in gliomas

Hypoxia-induced angiogenesis in gliomas

Tumour hypoxia-induced angiogenesis aforementioned is mediated by hypoxia-inducible factor 1 (HIF-1). The function of this factor is affected by both hypoxic conditions and by molecular mechanisms which are capable of regulating its level of degradation, synthesis & transcriptional activity. (Kaur et al., 2005) HIF-1 is synthesised as a heterodimeric protein, made up of α and β subunits. (Kaur et al., 2005) HIF-1 activation takes place under a hypoxic condition, a low oxygenated pathological condition commonly featured by tumorigenic gliomas. Blood vessels have an integrated domain used to detect oxygen level around them term as prolyl hydroxylase domain proteins (PHD1-3) "[18]. Under normoxia condition, a feature not frequently seen in glioma, proline and asparagine residues in HIF-1/2α are hydroxylated. (Kaur et al., 2005) This reaction is mediated by prolyl hydroxylase domain and asparaginyl hydroxylase respectively. Hydroxylation of the asparagine residues (FIH-1) renders the HIF unable to binds to its co-activator CBP/p300 which is required to activate HIF complex. (Kaur et al., 2005) Unstable HIF-1 is targeted for degradation via pVHL-mediated ubiquitination & proteasomal pathway, resulting in low HIF transcriptional activity. (Kaur et al., 2005) However, under low oxygen tension condition, PHDs and FIH-1 are less active. Therefore, HIF-1 is able to interact with SUMO-1 which mediates the sumoylation of the factor, resulting in its increased transcriptional activity and molecular stabilization. (Kaur et al., 2005) Stabilized HIF-1α- HIF-1β complex translocate to the nucleus, where the complex interacts with CBP/p300. This recruitment process is mediated by Ref-1 resulting in the transcriptional activation of angiogenic target genes with HREs. (Kaur et al., 2005) HIF-1 interaction with hypoxia-responsive elements (HREs) initiates the transcription of various angiogenic genes responsible for the aggravated vascular hyperplasia observed in gliomas as well as stressing favourable features like cell survival and increase tumour cell metabolism. (Kaur et al., 2005)

Activation of the HIF-1 pathway induces increased rate of VEGF gene expression as well as stabilizing the VEGF mRNA. (Gerald,2000) Consequently, expression of the VEGF protein is upregulated, a prominent phenomenon responsible for the tumour vascularization and aggressiveness during gliomagenesis. (Gerald,2000) {{519 Douglas 2011;}} (Kaur et al., 2005) . The HIF-1 pathway can also be activated by the interaction between the transmembrane receptor tyrosine kinases (RTKs) and growth factors. Furthermore, this interaction further stabilizes HIF-1. Active form of RTKs interacts with p85, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), resulting in the activation of PI3K. PI3K, a lipid kinase, function as generator of a signalling molecule known as phosphatidylinositol 3,4,5-triphosphate. This molecule is generated by phosphorylating its precursor phosphatidylinositol 4,5-biphosphate. Activated PI3K is able to induce the phosphorylation and activation of AKT as a result. AKT is a serine /threonine kinase and its activation has been associated with antiapoptotic and prosurvival activity in a cell. More importantly, the activation of AKT has also been linked to the intensification of HIF-1 protein translation via the AKT/FRAP/mTOR pathway. (Kaur et al., 2005)

Ultimately, activation of the HIF-1 pathway allows a series of cascade reaction to occur which leads to the activation of factors such as VEGF. The VEGF family consist of six structural superfamily, including VEGF, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. (Gerald,2000). These proteins are secreted as dimeric glycoproteins with cysteine knot motif, where the 8 cysteine residues are spaced regularly. (Gerald,2000). VEGF is known to elicit various effects which further potentiate gliomas neovascularization and this may include the enhancement of vesicular -vacuolar and clustered vesicles activity which is responsible for metabolic transport in order to increase endothelial cell permeability (PAPETTI & HERMAN, 2012 ). Conversely, changes in endothelial permeability may arise from structural weakening of adheren junctions which is facilitated by rearranging cadherin complexes between endothelial cells (PAPETTI & HERMAN, 2012 ).

Figure 4 schematic diagram of the HIF signalling pathway under normoxia and hypoxia condition (Kaur et al., 2005).

Other aberrant genetic alteration which affects HIF signalling in gliomas

Furthermore, the PI3K/AKT pathway can also be activated by the adhesion of extracellular matrix (ECM) , which is mediated by integrins. Subsequently, this results in the activation of integrin-linked kinases (ILK), which in turn causes an increase in HIF-1 α and VEGF production via PI3K/AKT/FRAP/mTOR pathway. Activating the PI3K/AKT pathway also result in increased heat shock proteins 90 and 70, which interact with one another to help stabilize HIF-1 α. The presence of helix-loop-helix and Per/ARNT/Sim (PAS) domains in the HIF-1 subunits (α & β) are responsible for the induction of hypoxic condition. (Kaur et al., 2005)

Figure 5 : Genetic signalling modulating HIF signalling via RKT activation, a component of the PI3K/AKT/mTOR pathway. Additionally, TP53 signalling can negatively regulate the HIF signalling via proteasome pathway. (Kaur et al., 2005)Upregulated ILK activity has been observed in gliomas, which help pronounce the aggressiveness in the glioma tumour biology.

Evaluation of morphological analysis of the tumour blood vessels in gliomas

GBM revealed significant differences in the structural features of the blood vessels compared to AA and GBM. The micrographic image of blood vessels in LGA informed that the vessels in diffuse astrocytoma (DA) appears similar to vessels in normal brain as these vessels were observed through digital examination to be thin and straight with very little branching, although DA featured more branching than normal vessels. This cytological findings are evidently presented by the graphical representation of Kruskal-Wallis &Dunn's statistical test result (Sato et al.,2011) . On the other hand, the vascular parameters presented by AA and GBM are significantly different than the ones in normal vessels and DA . These tumours had larger network of vessels and these were thicker and more branched than normal. However, GBM featured the largest network of vessel with abberant thickness.GBM exhibited vessel density 3-fold more than the normal vessel specimen. Furthermore, the specimen observed was also infused with a glomeruloid vessel (Sato et al.,2011).

The results obtainted from this study correlate to pathophiosiology features presented by both high grade and low grade gliomas. Intensified angiogenic stimuli exhibited by GBM and AA explains the distorted vascular network and glomeruloid formation. Weak structural integrity and abnormal vessel branching feature by both high grade-and low grade gliomas is caused by the amplified growth factor which causes the discordant vessel wall strengthing. Furthermore, this coalescence vasculature generates further facilitate more hypoxic regions, resulting in the upregulation of HIF-1 expression. Consequently, accumlation of hypoxic micro-regions cause tissue necrosis observed in higly tumorigenic gliomas, which further potentiate angiogenesis and tumour growth via increased HIF expression. Additionally, these factors into therapy resistance featured by high grade gliomas.





Figure 6 Micrograph image of tumour vessel in DA (A) , AA(B) and GBM (C & D). Present results obtained from the morphological analysis of the structural features of blood vessels in gliomas by Sato et al. Probability value used to describe statistical significance between vascular parameter: **P < 0.01, *P < 0.05 & áµ» P < 0.001. (Sato et al.,2011).

Aberrant cellular signalling in gliomas

Summary of genetic alteration in gliomas

Over the years, genetic analysts have managed to identify the major mutational events associated with the neoplastic transformation of normal cells to abnormal cells such as gliomas. Fig. 1 & 5 summarises the aberrant signalling and gene expression linked to gliomagenesis.

Low grade astrocytomas feature slow growth, propensity to diffuse to surrounding brain structure and also the ability to eventually undergo malignant transformation, all of which accounts for the mortality rate presented in cancerous patients (Watanabe et al., 2003). Frequent genetic abnormalities often observed are the mutation of the IDH1 gene which codes for isocitrate dehydrogenase and the mutation of p53 tumour suppressor gene (Knobbe et al 2002, Dang, et al 2009) Furthermore, PDGF/R overexpressions which further potentiate the ontogenesis of LGA have also been discovered. However, evidence of RB proteins mutation, CDK4 amplification, PTEN loss, chromosomal deletion of 11q and 19q, INKa/ARF loss and DMBT1/mxi loss have been observed in anaplastic astrocytoma. *

New scientific advances which came from the efforts from both TCGA consortium and genetic analysts have led to the discovery of new genetic alterations in GBM along with the hypothesis formulated, which states that GBM can be sub-divided into several subtypes (Meir et al.,2010) These subtypes are termed as follow: classical, mesenchymal, proneural and neural GBM (Meir et al.,2010), (Fig.15) .

Evidently, these GBM subtypes commonly shared genetic abnormalities such as p53 & Rb inactivation and upregulated tyrosine kinase pathway. Based on these scientific findings, it is possible that previous hypotheses may need to be further elucidated to accommodate for these new discoveries in order to fully understand the biology behind gliomas ontogenesis. As these discoveries indicate different cells of origin for GBM (Meir et al.,2010)

IDH mutation in gliomagenesis

Isocitrate dehydrogenase 1 (IDH1) gene mutations have been identified in 70%-85% of grade II-III gliomas. (Meir et al.,2010) (Dang et al ,2009) IDH1 mutations mostly occur at a single amino acid residue, arginine 132, which becomes mutated to histidine (R132H) 1, 3, 4. (Dang et al ,2009) The R132H mutation allows the residues in the enzyme active site to shift, allowing the structural changes which enable the conversion of α-ketoglutarate to 2-hydroxyglutarate (2HG) (Dang et al ,2009). Although the exact role of IDH1 mutation in gliomagenesis is still yet to be fully elucidated, however recent studies proposed that excess accumulation of 2HG is associated with higher risk gliomas development in adults. Furthermore, elevated 2HG has been linked to increased reactive oxygen species (ROS) levels, which explains the elevated risk of cancer due to the potential genetic damage possessed by ROS. (Dang et al ,2009) Additionally, heterozygous mutation in IDH gene also reduces the cellular level of α-ketoglutarate and NADPH metabolism which further potentiate the risk of tumorigenesis imposed by increased susceptibility of oxidative stress. In addition, IDH mutation is also associated with upregulated expression level of hypoxia-inducible factor-1α which is known to potentiate survival and angiogenesis. [6] Common method employed for detecting all IDH mutagenic variants in gliomas is the Sanger sequencing technique.

PTEN / RTK/PI3K/AKT signalling transduction pathway in gliomas

PTEN (phosphatase and tensin homolog) is a tumour suppressor gene protein which translates to form phosphatase type enzymic protein known as phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (Ptdlns(3, 4,5)3-P) (Knobbe et al. 2002, UniProt 2013) . The PTEN protein catalytic subunit termed p110α catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidylinositol 3,4,5-bisphosphate (PiP2). PIP3 function to recruit pleckstrin homologue (PH)- domain type protein such as ATK1 to activate the RTKs/PI3K/Akt pathway involved in cellular processes such as proliferation, cell growth, motility & survival. (UniProt 2013) However, the generated PiP2, mediated by the catalytic subunit p110α antagonizes this signalling pathway, thus resulting in its inhibition. The catalytic p110α protein is encoded by PIK3CA gene and oncogenic mutation resulting in the amplification of the PIK3CA gene is often observed in brain tumours, primarily GBM (Reifenberger et al. 1993) PIK3CA mutations are observed in patients with GBM between 5% -13% of cases (Reifenberger et al. 1993). Consequently, this increases the expression of PIK3CA protein which in turn elevates the level of PIP2. This potentiates uncontrollable cell growth, resulting in aberrant neoplastic transformation of normal cells to become cancerous cells.

Although, PTEN associated mutations are hardly seen in astrocytomas, hypermethylation of the PTEN promoter which leads to the overactivity of the downstream Akt/PI3K signalling is observed in astrocytoma (Smith et al., 2001). Overactivity of the RTKs/PI3K/Akt signal transduction pathway potentiate angiogenesis, cell survival and cellular proliferation, which in turn explains the aggressiveness of gliomas.

The RTK/PI3K/Akt pathway controls several cellular processes which includes cellular proliferation, cytoskeletal rearrangement, apoptosis and growth. This pathway comprises of molecular components such as RKTS, PI3K, mTOR and Akt. Receptor tyrosine kinases (RTK)s function as cell surface receptor with high affinity for growth factors, cytokines and hormones and this may include EGFR and PDGFR and VEGFR. When the surface of RTK (EGFR or PDGFR) is occupied by growth factors, RTK interacts with the p85 subunit of PI3k, which in turn activations its catalytic subunit, p110. This subunit catalyses the phosphorylation of PI 3,4-bisphosphate (PiP2) into 3,4,5-triphosphate (PiP3). PiP3 activates phosphoinositide-dependent kinase-1 (PDK1), which in turn phosphorylate Thr308 of Akt. Akt is subjected to further phosphorylation at Ser473 before it's activated. Activated Akt interacts with TSC1/TSC2 suppressor complex to inactivate the complex. This results in the activation of mTORC1, which affects cellular processes such as motility, proliferation, angiogenesis and cell survival (Nakada et al 2011).

Results obtained from recently conducted studies help cement the hypothesis I formulated which states that malignant gliomas feature such unusual activation of this signalling pathway, due to the genetic changes.

Alterations in RTKs are often observed in gliomas. Firstly, amplification of the EGFR gene is mostly featured by GBM, accounting for 40% of genetic alteration. However, structural alteration have also be linked with EGFR gene amplification and the most common mutant is known as EGFRvIII, which is capable of sending growth signal without the need for ligand binding. Amassed evidences indicate a strong correlation between alterations in EGFR with increased aggressiveness of GMB. Increased expression of the EGFR results in increased expression of tissue factors which in turn promotes proliferation, angiogenesis and cell survival. This potentiates the aggressiveness in GBM (Nakada et al.,2011) . Similarly, overexpressions of platelet-derived growth factor receptor (PDGFR) are also featured by all gliomas grade. This receptor is able to potentiate the aggressiveness of GBM using similar mechanism aforementioned. Additionally, amplified PDGFRA is also presented by proneural subtype of GBM. (Nakada et al.,2011) Yet, despite the significant increase in the depth of our knowledge of gliomas ontogenesis, anti-PDGFR therapy and EGFR inhibitors have not provided clinical response in clinical trials with GBM. This may be due to the possibility that these genetic alterations in RTKs are not the governing factor behind ontogenesis of gliomas but contributes only to increase its risk and further potentiate its effects, which supports my aforementioned hypothesis.

Furthermore, activation of EGFR/EGFRvIII via ligand binding leads to the PI3K pathway activation which in turn results in the amplification of HIF-1 α via PI3K/AKT/FRAP/mTOR pathway.

P53 /ATM/CHK2, p14ARF/MDM2/p53 & RB pathway in gliomas

The CDKN2B and CDKN2A genes located to chromosome 9p21 are responsible for coding for the p15INK4b and p16INK4a tumour suppressor protein respectively whilst p14ARF protein is encoded by exon 1β & exon 2-3 of p16INK4a. The p14ARF protein functions as a tumour suppressor by inducing cell cycle arrest in G1 and G2 phases. This protein is capable of inhibiting the oncogenic action of MDM2 by directly binding to the MDM2 and blocks its nucleocytoplasmic shuttling in the nucleolus (UniProt 2013). Consequently this prevents the oncogenic action of MDM2, thus, the p53 degradation and p53 dependent transactivation and apoptosis mediated by MDM2 is inhibited (UniProt 2013). p15INK4b and p16INK4a are critical components in the RB1 pathway, whilst p14ARF is involved in the TP53 pathway, acting as an upstream regulator (Watanabe et al., 2003) p15INK4b and p16INK4a are also capable of interacting strongly with CDK6 and CDK4 respectively, thus inhibiting the cyclin D-CDK4 and cyclin D-CDK6 kinase activity. As a result, the CDK4-and CDK6-mediated phosphorylation of retinoblastoma protein (RB) is averted. Therefore, cell cycle progression is regulated at the G1 checkpoint via negative control (Watanabe et al., 2003) The Murine double minute 2 (MDM2) genes encode a cellular protein which is able to form an oligomeric complexes with the p53 gene products and inhibit its function (Reifenberger et al. 1993). Scientists have identified the MDM2 gene to be amplified and overexpressed in gliomas such as AA & GBM (Reifenberger et al. 1993). The MDM2 gene is the second most frequently gene amplified in gliomas behind epidermal growth factor receptor gene. (Reifenberger et al. 1993) MDM2 gene is located at chromosome 12q14.3-q15 and it encodes an E3 ubiquitin-protein ligase which binds tightly to the N-terminal transactivation domain, mediating the ubiquitination and proteasomal degradation of p53 (Nakada et al.,2011, UniProt 2013). AS a result, it is possible that the tumour with MDM2 amplification and overexpression is then able to escape from growth control by p53 pathway (Reifenberger et al. 1993). As the full function of the MDM2 gene mutation is not fully understood yet, it is also possible that the overexpression of the MDM2 gene is able to promote neoplastic growth via other mechanisms. Furthermore, inactivation of the tumour suppressor pathway has also been implicated to be caused by frequent hypermethylation of p14ARF, p15INK4b and p16INK4a tumour suppressor in low grade gliomas. Scientist have also hypothesize that p53 is able to inhibit HIF activity by promoting the degradation of HIF-1 α and MDM2-mediated ubiquitination. Activating the HIF pathway results in the promotion of tumour vascularization in gliomas. The resulting vasculature lead to further hypoxia and the upregulation of HIF which further potentiate gliomas angiogenesis. Furthermore, wild type CDKN2, p53 & pRB are involved in cell cycle transition: Go or G1 to the S phase, G1-S and the G2-M respectively. Inability to correctly regulate cell cycle transition explains the elevated mitotic activity featured in AA and GBM.

P53 gene located to chromosome 17q13.1 locus, encodes a protein with a diverse function which includes regulating target genes responsible for inducing cell cycle arrest, cell differentiation, senescence, DNA repair, and neovascularization & cell death when subjected to cellular stresses (Nakada et al., 2011)

It's also been reported that the loss of components in the ATM/Chk2/p53 pathway promotes glioma development and contributes to radiation resistance (UniProt 2013, Reifenberger et al., 1993;}} Ataxia telangiectasia mutated (ATM), a serine protein kinase responsible for activating checkpoint signalling when exposed to genotoxic stresses becomes activated (UniProt 2013). Activation of ATM leads to the activation of checkpoint kinase known as CHK2 and p53 (Nakada et al.,2011) Consequently, genes such as p21Waf1/Cip1 which function as cell cycle progression regulator are transcribed and expressed.


Defects in the RAS signalling have also been implicated in the ontogenesis of gliomas. RAS signalling is responsible for cellular transformation and the induction of anti-apoptotic signalling. The RAS signalling transduction cascade involves Ras proteins, a 21 kDa GTPase which acts as a transducer to relay information to other signalling pathways. Ras acts as a switch and it is switched on by various Ras guanine nucleotide exchange factors (RasGEFs) such as Ras guanine nucleotide release-inducing factors (RasGRFs), Ras guanine nucleotide releasing proteins (RasGRPs) and Son-of-sevenless (SoS). On the other hand, the OFF switch is induced by Ras GTPase-activating proteins (RASGAPS) such as Ca2+ -promoted Ras inactivator (CAPRI), neurofibromin, Ras GTPase-activating-like (RASAL) and SynGAP by accelerating the hydrolysis of GTP to GDP. The addition of GTP to Ras proteins, mediated by RasGEFs activates the protein whilst the conversion of Ras-GTP to RAS-GDP, mediated by RasGRPs inactivates the protein [19]. RTKs and NF-1 stimulate the GTPase activity of Ras, thus controlling the binary switch. Activated Ras interacts with RAF, serine/theorine kinase which goes on to phosphorylate and activate MEK (aka mitogen-activated protein kinase kinase (MAPKK)) (Nakada et al., 2011). The activation of MAPKK allow transcriptor factors like PPARÏ’, Ets, E1K1, c-myc and STAT 1/3 to become activated, which are able to induce cellular transformation and anti-apoptotic activity. NF-1 is a tumour suppressor gene which encodes a protein known as neurofibromin, which acts as a negative regulator of Ras signalling. There are mounting evidences from TCGA studies which implies that periodic homozygous deletions and mutation of NF-1 gene are observed in gliomas. The studies revealed that the mesenchymal sub-type of GBM exhibit 37% of NF-1 inactivation. (Nakada et al., 2011) However, Ras and Raf mutation are rare in gliomas, with only 2% of all cases. (Nakada et al., 2011) Inactivation of the neurofibromin tumour suppressor gene results in the inability of the cell to prevent uncontrollable cell cycle division. Consequently, uncontrollable cellular division occur and the eventuality of gliomas ontogenesis is promoted.

Glioma stem cell pathway in gliomas

Glioma stem cells (GSCs) are characterized by their self-renewal ability, the expression of neural stem cell markers and the ability to differentiate into variety of different neural cell such as astrocytes and oligodendrocytes. Sonic hedgehog (SHH) is activated and utilized in this pathway. The dimerization of SHH ligands with their receptors results in the activation of the transducer known as Gli (Nakada et al., 2011). This result in the activation of Gli transcription factors which up-regulates the expression of Gli gene and the translation of Gli protein. Gli protein mediate the cell survival by inducing anti-apoptotic signal, activation of GSCs and the promotion of G1/S phase (Nakada et al., 2011) In addition, the activation of notch signalling within GSC signalling pathway has been postulated to be present in gliomas. This tumorigenic effected is thought to be induced to due to the activation of the p53 pathway and the promotion of neural stem cell growth mediated by upregulated notch signalling (Nakada et al., 2011). The involvement of STAT3 activation mediated by Janus kinase tyrosine (JAK) has also been indicated in gliomas ontogenesis. (Nakada et al., 2011).


Table of summary for gliomas:

Tumour type

Low-grade astrocytoma (LGA)

Anaplastic type gliomas (AA)

Glioblastoma multiforme (GBM)

Evidence from experimental studies

WHO grade classification





Tumour types/ subtypes

-Diffuse astrocytoma (DA)

- Oligodendroglioma (OD)

- Oligoastrocytoma (OA)

- Subependymal giant cell astrocytomas (SGCAs)*

- Optic nerve gliomas *

- Brain stem gliomas *

- Anaplastic astrocytoma (AA)

- Anaplastic -oligodendroglioma (AO)







Headaches, Elevated intracranial pressure, seizure, numbness/weakness in limbs, nausea , vomiting & vision loss [2]


Prognostic survival time

5-10 years

2-3 years

9- 12 months

Patient prognostic survival time was obtained from the analysis of several large randomized trial

Genetic markers

Mutations in IDH1 gene, PDGF/R and p53 tumour suppressor

RB proteins mutation, CDK4 amplification, PTEN loss, loss of heterozygosity on 11q and 19q, INKa/ARF loss and DMBT1/mxi loss are observed in AA

PTEN loss, ,amplification of EGFR & PDGFR gene and mutations in CDKN2A, TP53 & NF-1 are often featured by GBM

Loss of chromosomal heterozygosity is often featured by both astrocytoma and anaplastic astrocytoma as 50%-70% of both tumours present loss at chromosomes 1p and 19p. [33, 34]. Furthermore, Loss of heterozygosity predicts survival and chemosensitivity in patients with LGA. Possible genetic testing that can be employed are : fluorescence in situ hybridization (FISH), LOH (traditional gel-based assays

or capillary electrophoresis) or comparative genomic hybridization (CGH)

Significant genetic alternations in gliomas have been identified in the meta-analysis study conducted by Jonathan et al. Results obtained from this experimental

Identified aberrant HIF pathway as the most statistically significant aberrant signalling in GBM. FISH based dual probe screen was conducted with specificity for PTEN and EGFR gene in high grade gliomas. Both oncogenic genes were found to be amplified in high grade gliomas. [5]

Immunohistochemical markers

Cytokeratin (AE1/3)

and GFAP expression.

Ki67 nuclear antigen

MIB-1 prognostic marker

Cytokeratin (AE1/3)

and GFAP expression

Cytokeratin (AE1/3)

and GFAP expression

Ki-67 /MIB-1 Immunohistochemical predictive marker for proliferating nucleus is used to predict prognostic factors associated with the patient with LGA. Reaction

with KI-67 nuclear antigen yielded a labelling index of 1-2% in LGA and 15-20% in high-grade gliomas. Furthermore, elevated level of olig expression, which encodes factors which control neural cell differentiation and proliferation have also been associated with glial neoplasm. As a result, Immunohistochemical study of olig factors with glial fibrillary acidic protein (GFAP) (Olig+/GFAP) can employed to identify LGA such as oligodendroglioma

as these proteins are primarily expressed by oligodendroglioma. Additionally, all GBM tumours tested positive for GFAP & β-tubulin III immuno-staining [1] [6]

Treatment plan

Currently, there is no available cure for LGA however the tumour related symptoms can be managed to make the patient's life more comfortable. Generally, a "watch & wait" approach is often employed initially due to the unpredictability nature of the tumour. However, decision to perform resection surgery or receive chemotherapy or radiotherapy is consider when symptoms worsen. Additionally, factors such as tumour location, patient age & the patient's preference are also considered. Tumour resection is often employed to reduce intracranial pressure and also correct neurological deficits such as uncontrollable seizures caused by the tumour mass. Radiotherapy is often undertaken when the tumour confer signs tumour recurrence due to irregular mitotic activity.

In poor prognosis patient with highly tumorigenic tumour such as AA and GBM, hypo-fractionated radiotherapy treatment is undertaken. This is a treatment plan where in which the duration of the therapy is shortened. The patient's age also factors into considering this treatment. Treatment such as resection surgery & chemotherapy are also employed to minimize the damage conferred by the aggressiveness of high grade gliomas. [4]

Treatment related complications

Neurotoxicity from radio-/chemo-therapy can cause deterioration of cognitive function

Secondary malignancy conferred by alkylating agents from chromosomal damage

Treatment-related effects includes

Surgery-related damage

Neurotoxicity with both radiotherapy

& chemotherapy, anticonvulsant therapy and

Endocrine dysfunction

Overall, the aim and objective of this study was achieved. GBM emerged as the most significant pathological and most harmful tumour as previously hypothesized followed by AA and LGA. Furthermore mutations in both VEGFR and FDGFR appear to drive the LGA tumorigenesis whilst the HIF pathway emerged to herald the active phases of high grade ontogenesis as previously stated.