Glioblastoma Multiforme

GLIOBLASTOMA: Glioblastoma (aka Glioblastoma Multiforme (GBM)) is a primary brain tumor which arises from glial cells (the supporting cells of the brain). The tumor is most often located in the cerebral hemispheres and a small percentage occurs in the spinal cord, brainstem and cerebellum (1). GBM has heterogeneous features, characterized by cystic and gelatinous areas, multifocal hemorrhage, and necrosis (2,3). The tumor is usually manifested by a single, irregularly shaped, relatively large lesion, usually arising within the white matter (4). GBM can be subdivided into primary and secondary categories based on clinical characteristics and markers (3).

Primary:

·       Arise without previous clinical or radiological evidence.

·       Hallmark alterations: EGFR gene amplification and mutation, MDM2 overexpression, p16 deletion, chromosome 10q loss of heterozygosity (LOH) holding phosphatase and tensin homologue (PTEN) as well as TERT promoter mutation. In primary glioblastomas, IDH gene status is wild type (i.e. not mutated).

Secondary:

·       Arise slowly from a preexisting low-grade astrocytoma.

·       Hallmark alterations: overexpression of PDGFA, and PDGFA/PDGFRa, RB, 19q LOH, mutations of ATRX, TP53 and IDH1/2.

Secondary and primary GBMs can be divided into transcriptional subclasses (3 & 23).

The tumor is most often initially detected using computer tomography (CT) and/or magnetic resonance imaging (MRI). However, to classify a patient’s subtype, a diagnostically important move, the diagnosis is based on invasive approaches, such as invasive biopsy or surgical resection which then may be used for genomic analysis. Non-invasive techniques, such as proton magnetic resonance spectroscopy (MRS) (15), are a field of study which the community hopes to develop in the future to avoid invasive probing.

GBM is the most frequently occurring malignant type of primary astrocytoma, and it accounts for over 60% of tumors arising from brain in adults (7). Usually, patients have a median survival between 14 to 15 months from the diagnosis (8,9). The global incidence is relatively rare - less than 10 per 100,000 people. However, the poor prognosis make it an important public health issue. GBM accounts for 50% of all gliomas across all age groups and can occur at any age. Peak age, however, is 55 to 60 years (10). The tumor is more prevalent in men than women, and in the western world, with Afro-Caribbeans being less prone to it compared to Caucasians, Latinos and Asians (11).

The etiology of GBM is largely unknown. Thus far, there is no conclusive correlation between GBM incidence and potential carcinogens such as smoking, electromagnetic fields or dietary risk factors (3,5). The only confirmed risk factor is high dosage of ionizing radiation. GBM treatment is difficult largely due to location of the tumor, and its biological heterogeneity. Effective care of high-grade gliomas involves not just therapeutic management, but also effective supportive care. This entails management of signs and symptoms of GBM, including seizures, cerebral edema, GI disturbances, cognitive impairment, venous thromboembolism and mood disorders (6). Therapeutic management sequence involves surgical resection, radiation and adjuvant temozolomide (TMZ) therapy (16,17). TMZ mechanism of action involves methylation of DNA at the O6 and N7 position on guanine, leading to the DNA mismatch repair system failing to find a complementary base, generating long live nicks in DNA. As a result, the cell cycle is halted, triggering apoptosis. Optune® device treatment is a recent innovation, using tumor-treating fields (alternating electrical fields) to cause cell death. The clinical benefit of Optune combined with current standard of care (surgery, radiotherapy and TMZ is 20.5 months OS compared with 15.6 months with TMZ alone (18).

CLINICAL TRIALS: Historically, most GBM clinical trials failed before even reaching Phase III. Presently there are many Phase III and/or Phase II clinical trials that broadly can be categorized into ones testing novel compounds and ones that are based on prior/known agents. A number of facilities are concentrated on investigating novel compounds against GBM, and many are actively conducting or recruiting for clinical trials (12).

Started in August 2015, GLOBE clinical trial is a pivotal, randomized multicentre phase 3 study aiming to compare VB-111 (an antiangiogenic inducing apoptosis in tumor endothelial cells (19) with bevacizumab against bevacizumab in adults with recurrent GBM. It involves 252 participants, and is sponsored by Vascular Biogenics Ltd.

CheckMate143 is a clinical trial started in January 2014, set to compare the safety and efficacy of nivolumab (a human immunoglobulin G4 monoclonal antibody; binds the PD-1 receptor. Potentiating the immune response against tumour cells20) and bevacizumab in patients with recurrent GBM. It also aims to evaluate the tolerability and safety of administering nivolumab alone or combined with ipilimumab, a T-cell stimulating monoclonal antibody (21) in patients using different GBM therapy lines. Sponsored by Bristol Myers, Squibb has enrolled 626 patients.

CheckMate498 is a clinical trial involving 550 patients aiming to evaluate the action of Nivolumab in combination with radiotherapy in GBM patients with unmethylated MGMT. The study started in February 2016 and is estimated to be completed by October 2019

COMMERCIAL POTENTIAL AND FUTURE PROSPECTS: GBM is an unmet medical need (22) and improving the situation is an important goal for the cancer research community. In the 2014-2024 period, the GBM treatment market is expected to grow fivefold, rising from $659 million to $3.3 billion, expecting a robust Compound Annual Growth Rate (CAGR) of about 17.4% (13). TMZ is the established chemotherapeutic against GBM, however, carmustine and lomustine are also used in some settings. According to GlobalData, Bristol-Myers Squibb’s immunotherapy Opdivo (nivolumab) could replace TMZ as the standard of care by 2024, with improving therapeutic profile and patents going off. GlobalData’s report covers seven nations, including USA, Spain, UK, German, France, Japan and Italy, and claims that these will be most prominent in the GBM market, with Japan expected to grow from $47 million in 2014 to $268 million in 2024, due to higher pricing of nivolumab (14).

USEFUL LABORATORY RESOURCES: When experimenting with GBM cells, they need to be kept in an environment that will support their growth and maintenance. Cell culture media, such as DMEM or F-12, are some of the media that can be used to do that.

https://www.thermofisher.com/uk/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media.html

https://www.biowest.net/products/cell-culture-media/

https://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts.html

PCR (i.e. qPCR, RT-PCR) is a useful process in glioblastoma research, helping with many essential operations, such as amplification, DNA and RNA quantification, DNA sequencing, probe generation and many more. There are many providers of PCR thermocyclers on the market, whose tools allow conduction of all these processes.

http://www.bio-rad.com/featured/en/thermocycler.html

https://www.thermofisher.com/uk/en/home/life-science/pcr/thermal-cyclers-realtime-instruments.html

https://online-shop.eppendorf.com/OC-en/PCR-44553.html#goto-Cyclers-WebPSub-44554

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) is an important step in conducting western blotting. The operation allows separation of proteins by size, which can then be further processed to deduce important genetic information. SDS-PAGE kits can be purchased from:

https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels.html

http://www.bio-rad.com/en-uk/category/protein-electrophoresis-blotting?ID=da4d07ba-052c-4156-89ef-16ab7b17f243

https://www.thomassci.com/scientific-supplies/Sds-Page-Apparatus

 

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