Genetics and epigenetics of gliomas

Gliomas are the most common primary intrinsic brain tumours. Their classification is based on phenotypic resemblance to normal glial cells (astrocytomas, oligodendrogliomas, mixed oligoastrocytomas) and pathological grading. Whereas this system is clinically relevant and has been the basis for our understanding of gliomas, systematic use of next-generation sequencing has transformed our knowledge of their pathogenesis and has uncovered genetic changes in an unanticipated number of genes and regulatory elements. In the past few years, in-depth analysis of low-grade astrocytomas and glioblastomas in both paediatric and adult populations has clarified our molecular understanding of these diseases, with distinct molecular events occurring in different age groups. In oligodendrogliomas, recent studies have highlighted mutations in candidate tumour suppressor genes located on 1p/19q, chromosome arms frequently deleted in this tumour. In this review, we discuss recent discoveries in the genetics of adult and paediatric gliomas, and highlight how some of the founding genetic mutations reshape the cancer epigenome. These studies provide an in-depth view of the molecular routes leading to brain tumour development and will be key for refining classification systems and improving clinical care.


Introduction
Despite more than a century of biomedical research, only little progress has been made in the clinical management of diffuse gliomas.Low grade tumour progression to high grade malignancies remains the rule in adult patients, while the most aggressive form, grade IV astrocytoma (glioblastoma), is incurable with the current standard of care therapy [1].Equally unsuccessful have been molecular therapies targeting angiogenesis [2,3].These frustrating results demand a better understanding of the underlying biology, with at least two roads warranting in-depth exploration.First, the emergence of molecular therapies targeting cancer-specific genetic events, as evidenced by recent successes in targeting BRAF mutant melanoma [4], EGFR mutant lung cancer [5], HER2 amplified breast cancer [6], BCR-ABL translocation in chronic myeloid leukaemias [7] and EML4-ALK translocation in lung cancer [8], provide a proof-of principle that histopathologic diagnosis must be complemented with genetic information to exploit tumour dependencies.To that end, recent international efforts in cancer genome sequencing, reviewed here, transformed our understanding of genetic changes in gliomas and should provide a basis for meaningful clinical exploitation.Second, the importance of epigenetic modifications in cancer fitness and the rapid emergence of epigenetic drugs offer unprecedented opportunities for novel targeted approaches.In this review, we focus on epigenetic alterations shaped by genetic events relevant to gliomas, such as mutations affecting IDH1/2, the histone methyltransferase SETD2, as well as histone H3.3 and its chaperones ATRX/DAXX.

Recent advances in the genetics of primary glioblastoma, WHO grade IV
Our modelling of the molecular routes leading to primary glioblastoma has been refined by systematic usage of whole-exome sequencing technologies.Adult glioblastoma is characterised by a complex genetic landscape [9].One of the critical components of these genetic aberrations is amplification and intragenic re-arrangement of receptor tyrosine kinases (RTK), affecting EGFR (~50%), PDGFRA (~15%) or MET (~5%).The most prevalent re-arrangements affect the extracellular domains of EGFR (EGFRvIII with in frame deletion of exons 2-7) or PDGFRA (PDGFRA8-9 with in frame deletion of exons 8-9).While these genetic events have been known for many years and transduce aberrant signalling, their inhibition has yet to show significant clinical efficacy.This is most likely rooted in the inter-and intra-tumoural heterogeneity observed in glioblastoma.Indeed, recent studies demonstrated a mosaic amplification of multiple RTK in the same tumour sample [10].This is further supported by recent single cell RNA sequencing efforts that highlighted heterogeneity in expression of different transcriptional programmes and surface receptors in primary glioblastoma cells [11].In addition, alternate routes to transduce aberrant RTK signalling have recently been discovered: two studies showed that ~3% of glioblastoma, without EGFR, PDGFRA or MET amplification, actually harbour an oncogenic chromosomal translocation fusing the tyrosine kinase coding domains of FGFR1/3 (fibroblast growth factor receptor 1 or 3) to, respectively, the coding domains of TACC1/3 (transforming acidic coiled-coil 1 or 3) [12,13].A study by Frattini et al. also identified, in rare cases, the novel fusion proteins EGFR-SEPT14 and EGFR-PSPH as mediators of RTK signalling in glioblastoma [14,15].Clearly, the complexity of RTK aberrations combined to intra-tumoural heterogenity poses significant challenges for clinical therapies.Recent advances also highlighted how genetic events affect glioma stem cells self-renewal.In the same study, Frattini et al. identified mutations in LZTR1, a gene normally expressed in brain and functioning as adaptor of the cullin3 ubiquitin ligase complex.Through advanced bioinformatics and functional experiments, the study showed that LZTR1 inactivation promotes glioma stem cells self-renewal and growth.Similarly Morris et al. uncovered the genetic basis for aberrant Wnt signalling in a subset of glioblastoma.The study found that in addition to previously described cases driven by PLAGL2 amplification/ gain, ~20% of glioblastoma harboured recurrent somatic mutations in FAT1 [16].The normal function of FAT1 is to bind beta-catenin and antagonise its nuclear localisation.Its mutational inactivation thus promotes Wnt signalling and tumourigenesis.These studies shed light on genetic bases for the reactivation of neurodevelopmental pathways that play a key role in glioblastoma cell properties [17,18].Glioblastoma cells self-renewal also requires telomere maintenance and Killela et al. have identified TERT gene promoter mutations in ~80% of primary glioblastoma, leading to increased telomerase expression.Of note, these mutations are mutually exclusive with ATRX mutations observed in adult secondary glioblastoma or in paediatric populations, and that are correlated with the ALT (alternative lengthening of telomeres) phenotype (see below).Thus it seems that either increased telomerase expression or ALT mechanisms ensure telomeres maintenance in primary or secondary glioblastoma respectively.

Recent advances in the genetics of adult astrocytic tumours, WHO grade II and III, and secondary glioblastoma, WHO grade IV
Frequent involvement of the tumour suppressor TP53 in astrocytomas has been known for decades while mutations in IDH1/2 have been more recently identified [9,19].Detection of these mutations, combined with assessment of chromosome 7 copy number, thus represents a sensitive diagnostic panel for low-grade glioma in challenging biopsies [20,21].Closely correlated with IDH1/2 and TP53 mutations, three studies published in 2012 identified missense mutations in the chromatin regulator ATRX (thalassemia/ mental retardation syndrome X-linked) in the majority (~70%) of grade II and III astrocytoma [22][23][24].ATRX mutations were mutually exclusive with 1p/19q co-deletion, the molecular hallmark of oligodendroglioma, and were identified at a lower frequency in mixed oligo-astrocytic tumours.ATRX mutations thus could represent a novel candidate marker of astrocytic lineage in diffuse gliomas [22][23][24].As the frequency of ATRX mutations is similar in grade II and III astrocytoma, they likely represent early events in the development of the disease, and could provide a genetic mechanism responsible for telomere maintenance.Indeed, ATRX is a SWI/SNF helicase playing a key role in assisting in the deposition of histone variant H3.3 in chromatin, in particular in telomeric and sub-telomeric regions and its mutations are correlated with the ALT phenotype [25].Involvement of chromatin regulators in gliomagenesis is also illustrated by the identification of missense and truncating mutations in SETD2, a histone H3 lysine 36 (H3K36) tri-methyltransferase, observed in 8% of adult high-grade gliomas (WHO grades III and IV).The absence of detectable mutations in SETD2 in low-grade gliomas suggests a role in glioma progression (see also below) [26].The role of BRAF genetic alterations in diffuse infiltrative gliomas is limited, although the rare diffuse grade II gliomas with BRAF V600E mutation may have a better prognosis [27].BRAF V600E hotspot mutations are more commonly found in pleomorphic xanthoastrocytoma, gangliogliomas and extra-cerebellar pilocytic astrocytoma.They can be readily detected by immunohistochemistry or molecular testing, providing an ancillary approach to the diagnosis of such tumours in difficult biopsies.

Recent advances in the genetics of oligodendroglial tumours, WHO grade II and III
Oligodendrogliomas have attracted substantial interest because of their remarkable sensitivity to treatment with alkylating agents.Sensitivity to DNA damaging agents cor- relates with signature genetic aberrations, in particular with co-deletion of the whole arms of chromosomes 1p and 19q.Although somatic mutation of the IDH1 or IDH2 genes may be the earliest and candidate founding event in this cancer, Killela et al. identified mutation in TERT promoter in ~80% of oligodendrogliomas, as observed in primary glioblastoma.These mutations lead to increased telomerase expression, as has previously been described in melanoma, thus possibly contributing to early disease pathogenesis.In addition, the frequent co-deletion of 1p/19q suggests that these chromosomal arms may contain tumour suppressor genes adding to disease pathogenesis.In the recent past, three studies described CIC (homologue to Drosophila gene capicua) on chromosome 19q as a candidate tumour suppressor gene, somatically mutated in 70% of oligodendrogliomas with 1p/19q co-deletion [22,28,29].In the same studies, mutations of FUBP1 (far-upstream element (FUSE) binding protein) on chromosome 1p have been observed in some tumours, although at a lower frequency than CIC mutations (fig.1).Finally, rare cases of BRAF gain, by duplication or fusion to KIAA1549, classically observed in pilocytic astrocytoma, have been observed in diffuse low grade gliomas, more commonly in oligodendrogliomas [30,31].

Recent advances in the genetics of paediatric diffuse gliomas, WHO grade II
Adult and paediatric diffuse gliomas have very different underlying genetic events and clinical behaviour, despite similar histopathological aspects.For example, IDH1 or IDH2 mutations, found in the majority of grade II and III diffuse gliomas in adults are very rare in children.Paediatric gliomas also less frequently progress toward highergrade than adult tumours.While these differences have been known for many years, it was only in 2013, using whole-genome sequencing, that signature genetic events were identified in paediatric populations [32].One of the major findings was that most paediatric tumours only harbour a single somatic event affecting protein coding sequences, suggesting that few oncogenic hits are required for transformation.The most common genetic event identified is a duplication of the tyrosine kinase domain of FGFR1, present in approximately one quarter of cases (fig.1).This duplication results in FGFR1 autophosphorylation and activation of the downstream MAPK/ERK and PI3K pathways.FGFR was also subject to other rearrangements, albeit at much lower frequency.These include FGFR1-TACC1 and FGFR3-TACC3 translocations, as reported in rare cases of adult glioblastoma (see above).A total of 25% of cerebral gliomas, including a few cases of angiocentric gliomas, harboured recurrent genetic rearrangements and amplifications affecting MYB or MYBL1.These cases also showed higher MYB protein expression levels detectable by immunohistochemistry. Several abnormalities affecting RAF were also identified, including different fusions (FXFR1-BRAF, QKI-RAF1, BRAF-MACF1, FAM131B-BRAF).Finally recurrent genetic events affecting histones and chromatin regulators were present in a subset of tumours, affecting ATRX, EP300, H3F3A, CHD2 or WHSC1.The functionality of some of these genes is detailed below.

Recent advances in the genetics of paediatric high-grade gliomas, WHO grade III-IV
Paediatric high-grade gliomas also have genetic underpinnings distinct from low-grade lesions and from their adult counterparts.Using whole-genome or whole-exome sequencing, two independent studies [33,34] have discovered recurrent mutations in the H3F3A gene, encoding histone H3.3, in approximately 30% of cases of paediatric glioblastoma.These mutations affect either lysine 27 (K27M), or less commonly glycine 34 (G34R/V) of histone H3.3.Notably, in a minority (~20%) of cases of diffuse intrinsic pontine gliomas (DIPGs), an anatomical variant of high-grade glioma, K27M mutations were also identified in the canonical H3.1 (HIST1H3B).All histone alterations identified are heterozygous, suggesting a gain-of-function mutation.Genetic mutations affecting many different residues were also discovered in ATRX and DAXX (death domain associated protein), chaperones which mediate deposition of H3.3 in telomeric regions.Overall, in cohorts of hundreds of cases, approximately 40% of paediatric highgrade gliomas had mutations either in H3F3A, ATRX or DAXX.Most mutations in ATRX correlate with loss of its expression and with an ALT phenotype.Subsequent studies highlighted that distinct H3F3A mutations define epigenetic and clinicopathological entities and are mutually exclusive with IDH1 mutations [35].In particular, K27M mutations exclusively affect the brain stem or thalamus [36], while G34R/V mutations are found in hemispheric cases.The mutations in H3F3A and HIST1H3B discovered in these studies are located in the histone tail that is subject to extensive post-translational modification (see below).

Genetics events that reshape the epigenome of gliomas
Links between chromatin and cancer were originally evidenced by oncogenic fusions containing chromatin modifying proteins and have been recently strengthened by wholeexome and whole-genome sequencing studies that have identified mutations in histone modifying enzymes and chromatin remodelers.In 2012, these links were further reinforced by the discovery of somatic mutations directly affecting modifiable residues in histone H3 proteins themselves in paediatric glioblastoma.A number of studies have together provided growing insight into the interplay between histone modifications and tumourigenesis.The human histone H3 family consists of H3.1 and H3.2 that both represent "canonical" H3 as well as the H3.3 variant, the testes-specific variants H3t and H3.5 and finally the centromeric variant CenH3 (reviewed in [37]).In humans, there are two genes, H3F3A and H3F3B that produce identical H3.3 proteins, differing only by their mRNA untranslated regions.H3.3 is preferentially deposited into genomic regions displaying active or poised transcriptional status as well as in telomeric and pericentromeric regions.Unlike H3.1 and H3.2, H3.3 deposition is replication in- dependent.It is incorporated into chromatin by two major histone chaperones complexes: ATRX/DAXX (see above) that mediates deposition in pericentromeric and telomeric regions and HIRA (histone regulator A) that incorporates H3.3 in active regions of the genome.The reported mutations in ATRX/DAXX in paediatric glioblastoma, adult low grade gliomas [22] and in neuroblastoma [38], suggests that impaired deposition of H3.3 in pericentromeric and telomeric regions would represent a critical axis leading to gliomas.H3F3A alterations are also likely to interfere with transcriptional regulation.Indeed, Lewis et al. identified that introduction of H3K27M leads to global decrease in H3K27 methylation and global increase in H3K27 acetylation, a mark associated with activated regulatory elements [39].Introduction of K27M peptide allosterically inhibits the methytransferase activity of EZH2 (enhancer of zeste homologue), a sub-unit of PRC2 (polycomb repressive complex 2) complex, thus representing a dominant-negative mechanism to alter gene expression [39].This loss of H3K27 trimethylation in cancer cells may be visualised by immunohistochemistry and may find use in diagnostic applications [40].Therefore H3.3 K27M mutation alters gene regulation by affecting global levels of H3K27me3 through inhibition of PRC2 in regions where the mutant histone is deposited.Deregulation of the PRC2 complex has been involved as an underlying mechanism in many malignancies, including adult gliomas [41,42].Both gain and lossof-function mutations of the polycomb complex have been shown to contribute to transformation in a variety of cancers.However, additional genetic events are likely to be required in paediatric gliomas, as the introduction of H3.3K27M into p53-null, nestin-expressing progenitors in the neonatal mouse brainstem was insufficient to generate gliomas [39].The role of mutations affecting G34 is less well established.Introduction of the H3G34R/V mutant results in decrease H3K36me3 in the same and nearby nucleosomes, through inhibition of the H3K36 tri-methyltransferase SETD2.Notably, Fontebasso et al. identified mutations in SETD2 in up to 15% of paediatric high-grade gliomas, supporting the notion that a disruption of H3K36 through either SETD2 or H3F3A mutations plays a key role in gliomagenesis [26].A suggested mechanism could be that H3G34 mutations cause profound upregulation of MYCN, potentially driving glioblastoma when expressed in the correct developmental context [43].Importantly, nearly all G34 mutant tumours also bear alterations in ATRX/DAXX, and display the ALT phenotype.The association between TP53/H3F3A mutations and TP53/IDH1 mutations in paediatric and adult gliomas, respectively, suggests at least a partial functional overlap between IDH1 and H3F3A genetic alterations.Up to 80%-90% of adult grade II/III gliomas and secondary glioblastomas harbour a genetic mutation in IDH1 or IDH2 affecting Arg residues 132 or 172 respectively [19,44].Glioblastomas bearing the mutations have significantly better prognosis than IDH wild-type tumours [45].These IDH mutations are sufficient to establish the CpG island methylator phenotype (CIMP), a distinct subclass of tumours in a number of human malignancies, including in glioblastoma [46].CIMP is associated with extensive and coordinated hypermethylation at specific genomic loci, one of which is the O 6-methylguanine-DNA-methyltransferase (MGMT) promoter (see below).The underlying mechanism relies on the fact that mutant IDH enzymes are not catalytically inactive, but rather have altered activity and produce the oncometabolite (R)-2-hydroxyglutarate [(R)-2HG], normally only present in a very low quantity of cells [44,47].Several mechanisms of (R)-2HG mediated transformation have been suggested, mostly relying on the structural similarity between (R)-2HG and 2OG.The dominant model is that (R)-2HG transforms cells by competitively inhibiting tumour suppressor enzymes that depend on 2OG.While there are many candidate enzymes that could be the target of (R)-2HG, a lot of the attention has been drawn towards TET2, in the context of myeloid tumours [44].TET enzymes are thought to be epigenetic regulators of gene expression by mediating demethylation of DNA.Specifically, TET family members hydroxylate 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC).It would thus be expected that loss or inhibition of TET2 activity would result in DNA hypermethylayion.However, the studies in AML have been variable and contradictory, reporting both hyperand hypo-methylation in TET2 mutant cases [48,49].A unifying hypothesis could be that TET2 mutations alter the methylation status of DNA in specific genomic sites while having variable effects on the global level of DNA methylation [44].Very strong evidence for a link between IDH and TET2 mutations derives from the observation that they appear to be mutually exclusive in acute myeloid leukaemia, suggesting they act on the same leukomogenic pathway [48].While TET family member mutations have been identified in myeloid tumours, they are not present in gliomas.However, rare cases of TET2 promoter methylation have been reported in IDH1 WT grade II and III gliomas [50].This would suggest that TET2 inhibition by IDH1 mutation or its down-regulation by promoter methylation play similar roles in gliomagenesis.Other enzyme candidate targets of (R)-2HG include JmJC histone demethylases that regulate gene expression by modulating methylation of histone tail residues and appear to function as tumour suppressors [44].Affecting their activity through IDH mutations would suggest a functional overlap with the mutually exclusive H3F3A mutations observed in paediatric populations.As mentioned above, MGMT promoter methylation is part of the CIMP phenotype associated with IDH1/2 mutations.It is also observed in ~40% of primary (IDH1/2 wild-type) glioblastoma, but the underlying mechanisms of its silencing are less clear [51].In clinical settings, MGMT promoter methylation is predictive of clinical response to alkylating agents, most notably in the elderly (65-70 years), MGMT promoter methylated patients benefitting the most from temozolomide therapy [52].Importantly, MGMT silencing is also thought to increase genetic instability, by facilitating G>A transition mutations.This hypothesis is supported by the more frequent mutation in TP53 and PTEN observed in MGMT-methylated glioblastoma than in MGMT-nonmethylated cases [53].It is also supported by the frequent induction of a "hypermutator phenotype" due to mutational inactivation of mismatch repair genes in temozolomide treated MGMT-methylated cases [53].While MGMT somatic mutations have been observed in certain malignancies, they are exceptional in gliomas [51].

Conclusion
The studies reviewed here have certainly furthered our understanding of brain tumour development.In addition to the wealth of genomic alterations previously identified in gliomas (fig.1), these recent genetic and epigenetic studies further underscore that gliomas are very complex ecosystems with significant inter-and intra-tumoural genetic diversity.The prominent involvement of histone genes and chromatin regulators mutations, and their impact on the epigenome, emphasises the critical roles of epigenetic mechanisms in cancer development [54].While a comprehensive catalogue of genetic and epigenetic events in cancer will require further efforts, we have already gained significant information about major molecular events and their significance.These discoveries challenge our grading systems, with certain mutations such as IDH1/2 being powerful predictors of clinical outcome, so that we have begun to integrate them into our classifications schemes [55].More systematic genomics and epigenomics approaches will likely become part of future clinical assessment.The novel findings provide a rational basis for renewed attempts at improving clinical care of brain tumour patients.

Figure 1
Figure 1 Genomic alterations underlying the development of gliomas.(A) Gliomagenesis in adults.Secondary glioblastomas (10% of cases) affect younger individuals and develop over a long period of time from lower-grade astrocytomas.Almost all cases are characterised by a mutation in IDH1/2, and a majority has mutations in ATRX.Primary glioblastomas (90% of cases) occur in older individuals and do not have a known precursor lesion.Almost all cases are wildtype for IDH1/2 and ATRX.Oligodendrogliomas are characterised by mutations in IDH1/2, wild-type ATRX, and loss of 1p/19q.(B) Paediatric gliomagenesis is molecularly distinct.Low-grade tumours are characterised by a single genetic event affecting FGFR, MYB/MYBL1 or RAF.Paediatric glioblastoma hallmark mutations target H3F3A at different residues, depending on tumour location.See text for gene abbreviations.In bold are highlighted proteins routinely tested to guide diagnosis.In addition to this, MGMT promoter methylation is frequently tested for therapeutic decisions (see text).Larger panels of genetic mutations are screened in clinical trials.

Funding / potential competing
Figures (large format) Figures (large format)

Figure 1
Figure 1 Genomic alterations underlying the development of gliomas.(A) Gliomagenesis in adults.Secondary glioblastomas (10% of cases) affect younger individuals and develop over a long period of time from lower-grade astrocytomas.Almost all cases are characterised by a mutation in IDH1/2, and a majority has mutations in ATRX.Primary glioblastomas (90% of cases) occur in older individuals and do not have a known precursor lesion.Almost all cases are wild-type for IDH1/2 and ATRX.Oligodendrogliomas are characterised by mutations in IDH1/2, wild-type ATRX, and loss of 1p/19q.(B) Paediatric gliomagenesis is molecularly distinct.Low-grade tumours are characterised by a single genetic event affecting FGFR, MYB/MYBL1 or RAF.Paediatric glioblastoma hallmark mutations target H3F3A at different residues, depending on tumour location.See text for gene abbreviations.In bold are highlighted proteins routinely tested to guide diagnosis.In addition to this, MGMT promoter methylation is frequently tested for therapeutic decisions (see text).Larger panels of genetic mutations are screened in clinical trials.