Definition Gliomas and Glioblastomas
Isocitratdehydrogenase- Gene & the Enzyme IDH
Epidemiology and Prognosis
Cell of origin, Epigenetics & Genetic Pathways of Tumorigenesis
Treatment Implications of IDH
Indications for a Systematic Review
Study Analysis/ Quality Assessment
Study and participant characteristics
Analysis of Data & Discussion
Appendix 1- the Search Strategy
Appendix 2- PRISMA flowchart
Appendix 3- Quality Assessment
A) Newcastle-Ottawa Scale
Appendix 4- Results-Table
Figure 1 — Diffuse Gliomas: from histology, IDH status and other genetic parameters
Figure 2 - Potential mechanism implicated in tumorigenesis induced by IDH MUTATION
Therapy response and survival of patients with glioma or glioblastoma and IDH mutation - A Systematic Review
1 University of Southampton, Faculty of Medicine/Kassel School of Medicine, Gesundheit Nordhessen Holding AG, Monchebergstrafte 41-43, 34125 Kassel, Germany
Introduction: Gliomas are the most common type of human brain cancers and make up 1/3 of all tumours of the central nervous system. Mutations in the IDH-gene are frequent and regarded as an early event in gliomagenesis. IDH mutations are considered to be associated with better outcome. If this accounts for all histological subtypes and for low- as well as for high grade gliomas, has yet to be clarified.
Aim: A systematic review was conducted to consolidate recent evidence whether mutations of the IDH-gene affect outcome of patients with glioma/glioblastoma.
Methods: Electronic databases were searched for studies published after 2008 combining terms like “glioma”, “isocitrate-dehydrogenase”, “mutation” and “outcome”. This yielded 1.160 articles. Reference lists of included studies were hand-searched. Quality of included studies was critically evaluated. Overall Survival, Progression Free Survival and their p- values were analysed to assess the relation between IDH mutation and outcome. Results were extracted, analysed and compared to gain a thorough overview of the current evidence base.
Results: 23 studies (4356 patients) were included in the analysis. Results on gliomas of WHO grade lll/IV are consistent, whereas evidence on WHO grade II gliomas are heterogeneous. Current evidence shows a strong trend towards improved outcome in patients with gliomas ll-lll and secondary GBM. Low grade gliomas show diverse outcomes depending on the tumour histology. Oligodendrogliomas seem to be associated with 1p/19q co-deletion (marker for good clinical outcome), whereas astrocytomas were linked to TP53 mutations (markerfor worse clinical outcome).
Conclusion: Current evidence indicates a strong positive influence of IDH mutations on outcome for high grade gliomas (WHO grade lll/IV). Positive impact on complete resection in high grade gliomas was shown. Results on low grade gliomas (WHO grade II) depend on the histological subtype and other genetic parameters - Oligodendrogliomas seem to have a better prognosis than astrocytomas.
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I would like to express my special thanks to my supervisor, Professor Irina Berger, who entrusted me with the project in the first place and met up with me very regularly to discuss my progress and who was always there for guidance, feedback and support. She challenged me to think critically about the results. The way she guided and supervised me throughout the whole time made me grow personally and academically and I am very thankful to be her mentee.
Also, I would like to thank Ms Paula Sands and Mr Ric Paul at the University of Southampton Health Sciences Library for their support and feedback regarding my initial search strategy and for answering my questions along the way.
As my project was changed due to organisational issues with the Landesarztekammer Hessen, I would like to reflect on my learning over that time.
The Neurology department was an opportunity to reflect on how I face and master problems. My former supervisor and I did not know how difficult it would be to get German ethics- approval for a project like mine. It demanded more paperwork than we could foresee (see LaboratoryNotebook). Due to misunderstandings with the LAKH about timeline and scope of the project, we were unable to get full German ethics-approval until end of October. We mutually agreed to change my project enabling me to conduct a well-structured and well- supervised Systematic Review. This process demanded mental strength and communication skills. I became more confident and improved my negotiation and communication skills. Search strategy and study selection
Before creating a search-strategy, I read the relevant chapters of the WHO classification of tumours of the CNS and a paper my supervisor advised me to. I went through the references of that paper for background information and did a scoping search with MeSH-terms in Ovid MEDLINE to get an overall idea of the existing literature on the topic and to familiarise myself with the subject background. I then started to create a search-strategy in MEDLINE including the relevant terms.
To get feedback on my search, I contacted Paula Sands, an academic librarian from the University of Southampton Health Library. She agreed with me on the terms I chose and advised me to add another MeSH-term. Later I skyped with Ric Paul, validated my search strategy and clarified questions regarding different databases.
I then began database searching. I searched MEDLINE, Embase and the Cochrane library and had a look into the Trip Database and PROSPERO to look for ongoing research on my topic and to prevent publication bias. Searching PubMed found over 100.000 records. After discussion with Ric Paul, I decided to not include a PubMed search -among other reasons due to the time constraints mentioned above. I extracted the results to the referencing software Endnote Web. I filtered the results through extraction of duplicates and application of the in-and exclusion criteria that we formulated in the beginning. I screened the full papers again and refined my results until the final studies for inclusion and evaluation had been identified. Having decided which studies to include, I produced a PRISMA flow-chart for incorporation into my report.
I critically appraised the quality of the papers using the CASP framework1 and Newcastle Ottawa Scale2 and summarised my findings in a results-table. I evaluated and analysed the results. In regular meetings with my supervisor we discussed my findings. During that time, I wrote the discussion and the conclusion.
While searching and selecting the papers, I consecutively wrote the introduction to my project report. With guidance from my supervisor, I selected most relevant evidence from reputable sources. Simultaneously, I produced the rest of the report, the tables and figures for the appendix.
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DEFINITION GLIOMAS AND GLIOBLASTOMAS
About 30% of all tumours of the central nervous system are gliomas and 80% of all malignant brain cancers are classified as being a form of glioma3. Gliomas arise from glia cells which support and protect the neurons of the central- and peripheral nervous system (CNS or PNS respectively). Glia cells do not belong to the neuronal cells and rather sustain homeostasis or form the myelin sheath around neurons of the CNS/PNS. Glia cells of the CNS comprise astrocytes, oligodendrocytes, ependymal cells and microglia4. Most commonly, cancers of the CNS resemble the morphology of astrocytes and oligodendrocytes.
Classification of gliomas and glioblastomas is an ongoing and dynamic process and due to recent findings in the genetics of gliomas, the WHO Classification for tumours of the CNS was updated in 2016. This allows the summary but also clear distinction of gliomas regarding their genotype. Therewith, the WHO provides a guidance for pathogenic classification based on phenotype and genotype of diffuse gliomas. Growth pattern and behaviour is considered as well. From a clinical point of view, this classification groups tumours sharing similar prognostic markers to guide therapeutic decisions.
Diffuse Glioma is an umbrella term, comprising tumours of the CNS arising from the glia cells. The WHO grade I (Pilocytic) astrocytic tumour is not discussed in the review at hand as it is regarded to be an exception. Pilocytic tumours are commonly seen in children and young adults and are not associated with IDH mutations. Furthermore, this review will focus on cerebral tumours and will not discuss spinal neoplasms. Since 2016, the following tumours are grouped together:
- Astrocytic tumours of WHO grade II and III (diffuse- and anaplastic astrocytomas)5
- Oligodendrogliomas of WHO grade II and III (diffuse- and anaplastic oligodendrogliomas)5
- Oligoastrocytomas of WHO grade II and III (diffuse- and anaplastic oligoastrocytomas)5
- Glioblastomas - WHO grade IV 5
Histologically, a WHO grade II tumour shows cytological atypia alone -simply meaning a deviation from the normal morphology of the cell. To be classified grade III, the tumour has to demonstrate anaplasia, loss of differentiation and orientation, as well as more than one mitotic spindle. A WHO grade IV brain tumour is termed glioblastoma (GBM) and has to show microvascular proliferation and/or necrosis as additional feature5.
The process of progression through the WHO grades can be described as follows:
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adapted from Louis DN, Ohgaki H, Wiestler OD, et al., WHO Classification of Tumours ofthe Central Nervous System; 20165
The role of tumour genetics is known to be of importance and mutations in TP53, EGFR or ATRX have been discussed elsewhere5,6. Relative recent research discovered the crucial role of Isocitrat-Dehydrogenase 1 and 2 mutations in the development, progression and behaviour of diffuse gliomas. Underlining the importance of this discovery, the WHO classification of CNS tumours updated the distinction of diffuse gliomas accordingly - classifying diffuse gliomas as being either a) IDH-mutant or b) IDH-wildtype5 (Figure 1).
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Figure 1 — Diffuse Gliomas: from histology, IDH status and other genetic parameters adapted from Louis DN, Ohgaki H, Wiestler OD, et al., WHO Classification of Tumours ofthe Central Nervous System; 2016 5
ISOCITRATDEHYDROGENASE- GENE & THE ENZYME IDH
The wildtype IDH gene codes for the enzyme IDH, responsible for catalysing the oxidative decarboxylation of isocitrate into alpha-ketoglutarate (a-KG) under concomitant production of NADPH. IDH1 is located within the cytoplasm and peroxisomes whereas IDH2 is found in mitochondria5,7. The activity of IDH makes up about two-thirds of the NADPH production in the brain. Among other functions, NADPH is critically important in reducing glutathione and thioredoxin to prevent oxidative stress in the tissues. It is the main antioxidant of the body. Alpha-KG is an important substrate in the Kreb’s cycle, and thus in the body’s energy metabolism8. Alpha-KG can be transaminated to glutamate and stands in a delicate equilibrium with this physiologically important amino acid. Glutamate plays a crucial role in the physiology of glial cells. Hence, a disruption of this balance is thought to contribute to formation of malignant gliomas9.
If needed, IDH catalyses the reverse reaction as well: conversion of alpha-ketoglutarate into isocitrate which can be further converted to acetyl-CoA -a major substrate for multiple biochemical reactions all over the body10.
NADPH scavenges oxygen radicals and is especially important in the brain as it is the organ being least capable of tolerating oxidative stress (Figure 2)5,10. IDH mutations reduce the brain’s capacity to produce NADPH by about 40%8 and hence the brains capacity to tolerate oxidative stress.
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Figure 2 - Potential mechanism implicated in tumorigenesis induced by IDH mutation adapted from Liu A, Hou C, Chen H, et al. Genetics and Epigenetics of Glioblastoma:
Applications and Overall Incidence of IDH1 Mutation. Front Oncol. 201610
The mutant IDH gene is associated with a loss of function. Due to the mutation at the active site of the enzyme IDH, it has less enzymatic activity and the production of NADPH is reduced9. Therefore, the susceptibility to oxidative stress and DNA damage is increased and leads to accumulation of further mutations, enhancing progression of the tumour11. A majority of IDH mutations seems to be associated with a mutation on TP53 in astrocytic WHO grade II, III gliomas and secondary GBMs. Both mutations together are regarded as the earliest and most frequently detected mutation in WHO grade II astrocytomas5,11. In an IDH-mutant glioma with co-occurring TP53 mutation, reactive apoptosis, induced by oxidative damage, is avoided, enhancing the damaging effect of reduced amount of NADPH11.
It was found that a single amino acid missense mutation in exon 4 at arginine at codon 132 (R132) is the most commonly found mutation in IDH1 (about 90%)1°. The analogous mutation site in IDH2 is at codon 172 (R172) and much less frequently found10. IDH1 and 2 mutations seem to be mutually exclusive and are found in about 70% of gliomas of WHO grade II to III and in secondaryglioblastomas12.
In addition to the loss of function described above, mutations in the IDH gene result in an increased NADPH-dependent reduction of alpha-KG to 2-Hydroxyglutarate (2HG) with concomitant NADP+ production. This is a gain of function (neomorphic activity) of IDH9,13. While reducing the affinity of IDH for isocitrate, its affinity for alpha-ketoglutarate and NADPH is increased10 thus favouring the NADPH-dependent reduction of alpha-ketoglutarate to 2HG which has direct toxic- and oncogenic effects.
The overproduction of the oncometabolite 2HG was found to have profound physiological consequences on cellular epigenetic states and on gene regulation (Figure 2)5,6,9,13. 2HG is structurally similar to alpha-KG. It is now thought that 2HG is the major driver of gliomagenesis. Among others, by competitively inhibiting alpha-KG-dependent dioxygenases (and additionally glutamate-dependent enzymes). Over 60 enzymes are thought to be alpha- KG-dependent. Furthermore, degradation of hypoxia-inducible factor 1-alpha (HIF1-alpha) is blocked which, under normal conditions, plays a crucial role in the cell’s adaptation to low oxygen levels and angiogenesis8. Under IDH-mutant conditions, HIF1-alpha is upregulated and drives tumour progression14. Another important mechanism in gliomagenesis of IDH- mutant gliomas, is DNA and histone tail hypermethylation and resulting silencing of several gene promotor regions.
Mutations in the IDH gene are relatively specific for gliomas and it is assumed that they play a crucial role in early gliomagenesis5. IDH mutations are common in WHO grades II and III as well as in secondary glioblastomas. They are less frequent in primary GBMs and almost absent in grade I (Pilocytic) gliomas5,15. In about 70-80% of low grade gliomas, 50% of anaplastic gliomas (WHO grade II and III respectively) and in more than 5% of (mostly secondary) glioblastomas (WHO grade IV), mutations in IDH can be demonstrated9,16. Screening for IDH mutations can be used to distinguish secondary from primary GBMs as the gene shows differential expression between these10. A further finding is the association of IDH mutations with other mutations. Mutations of the IDH gene seem to be closely linked to 1p/19q co-deletion, MGMT promotor hypermethylation, TP53 mutations and mutually exclusive with EGFR amplified tumours15.
Another clinical use of screening for IDH mutations might be the implication on prognosis and response to therapy concepts. This still is subject of intense research since the discovery in 2008.
EPIDEMIOLOGY AND PROGNOSIS
Both, epidemiology and prognosis have to be considered in the light of IDH mutations, at the latest since the incorporation into the WHO classification in 2016. IDH-mutant and -wildtype gliomas show distinct characteristics - in terms of patients as well as in terms of tumour behaviour. For example, patients with an IDH-mutant glioma tend to be significantly younger and the tumour is believed to be generally less aggressive5.
Astrocytomas are IDH-mutant in about 80% of cases. Most are of WHO grade III anaplastic astrocytoma and have no less malignant precursor lesion. The median age at presentation is 36 years versus 53 years if IDH-wildtype gliomas are considered. The annual incidence is estimated at 0.37/100.00 cases with a male to female ratio of 1.4:15.
Oligodendrogliomas mostly develop between 35 and 44 years of age with a male predominance (male to female ratio: 1.3:1). IDH-mutant oligodendrogliomas, like the other IDH-mutant diffuse gliomas, preferentially grow in the frontal lobe and account for 5.9% of all gliomas. The estimated annual incidence is 0.26/100.000 cases. About one third of all oligodendrogliomas are anaplastic (WHO grade III) at diagnosis. Mixed oligoastrocytomas (OA) account for 3.3% of all gliomas with an estimated annual incidence of 0.21/100.0005.
Glioblastomas (GBMs) are the most common malignant brain tumours and have a frequency of about 45-50% of all primary malignant brain neoplasms. The peak incidence lies between 45 and 70 years of age and is about 3 to 4/100.000 annually in most parts of the world. There is a clear distinction between primary and secondary GBMs. Whereas primary GBMs develop de novo, secondary GBMs progress from a less malignant precursor cell.
Primary GBMs are almost 100% IDH-wildtype and the median patient age is about 62 years at diagnosis. The length of clinical history is about 3.9 months. There is a male predominance (male to female ratio of 1.35:1).
Secondary GBMs are much less frequent with a prevalence of 9% of all GBMs. They are almost always IDH-mutant and have progressed from diffuse or anaplastic astrocytomas in most cases. The median age at diagnosis is 43 years with a median length of clinical history of 15.2 months. No male predominance has been found (male to female ratio of 1).
Generally, GBMs tend to be highly resistant to chemotherapy and due to their aggressiveness the median survival is about 12-18 months5.
CELL OF ORIGIN, EPIGENETICS & GENETIC PATHWAYS OF TUMORIGENESIS
Even though the classification as glioma implies histogenesis from glial cells, the evidence is only based on common morphological features of the neoplastic, compared to normal glia cells. From a histological point of view, gliomas are classified as being Oligodendrogliomas (O), Astrocytomas (A) or mixed Oligoastrocytomas (OA). However, it is assumed that the IDH-mutant- as well as the IDH-wildtype diffuse glioma each develop from a distinct cell of origin which has yet to be demonstrated5. Despite being in the same pathological class, IDH- mutant and IDH-wildtype gliomas seem to have a different etiological make-up. They show different degrees of aggressiveness, develop in distinct regions of the brain and respond differently to therapy. Clear diagnosis based solely on histological grounds is often difficult and, for example, primary and secondary GBMs are histologically indistinguishable5.
A concrete cell of origin of diffuse gliomas is yet unknown. The fact that gliomas resemble the morphology of astrocytes or oligodendrocytes does not mean they develop from these cell types. As said above, one hypothesis is that they arise from a distinct cell of origin. Brain tumour stem cells were proposed5 which, after some kind of carcinogenic event, begin selfrenewal and growth, thus leading to tumour progression. It was proposed further that IDH- mutant gliomas may share a cell of origin distinct from IDH-wildtype gliomas (e.g. Pilocytic- or juvenile gliomas). As oligodendroglial precursor cells might develop either into astrocytic gliomas or oligodendrogliomas, it was hypothesised that oncogenic signals are able to override the cell of origin determining the phenotype of the neoplasm5.
The term epigenetics describes changes in gene expression due to mitotically heritable mechanisms and not by changes in the underlying DNA sequence. Methylation of the DNA and modifications of histones are known to affect gene expression and are considered a hallmark of human cancers10,17.
Diverse genetic alterations have been studied elsewhere and discuss the role of mutations in EGFR, TP53, PTEN, ATRX, loss of heterozygosity or total allele deletion5,6,10. IDH mutation is frequently found to be the first event in the development of astrocytomas (WHO grade ll/lll) and in secondary GBMs resulting from progression of IDH-mutant grade II or III tumours6,9,11. In comparison to WHO grade II and III Oligodendrogliomas/Astrocytomas, the frequency of IDH mutations is less in secondary GBMs. This might be due to the aggressiveness ofthe tumour resulting in death ofthe patient before progression6.
Mutations in the IDH gene induce the glioma-CpG island methylator phenotype (G-CIMP) which leads to hypermethylation of gene promotor regions and modifications of histone tails resulting in silencing of differentiation factors18,19. Considered an important mechanism in gliomagenesis, is the silencing of O-6-methyltransferase (MGMT), responsible for DNA repair8. Silencing of MGMT is thought to make the glioma more sensitive to chemotherapy. Exact mechanisms are still unclear. However, several studies have shown that MGMT silencing is an independent positive prognostic factor8,20,21. Another hypothesis of gliomagenesis is that G-CIMP induction keeps glioma cells in a stem-cell-like state physiologically and thus being capable of self-renewal; thereby promoting tumorigenesis. It can be said that mutations in IDH promote formation of gliomas by influencing homeostasis and epigenetics leading to oncogene expression5.
TREATMENT IMPLICATIONS OF IDH
A clinical use of screening for IDH mutations is the implication on prognosis and response to therapy. This has been subject of intense research since the discovery in 2008. Mutations on IDH can be detected via diverse procedures. Most commonly, Polymerase Chain Reaction (PCR), Sanger Sequencing and Immunohistochemistry is used10. The body’s production of 2HG may be utilized to identify patients with IDH-mutant gliomas or glioblastomas13. Elevated 2HG levels can be detected by MR spectroscopy, a non-invasive method to determine the IDH status, or in body fluids5.
Surgery is usually the very first step to remove as much of the cancerous cells as possible. After this, the patient will be given a combination of radiotherapy (RT) and/ or chemotherapy (CT)22.
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