Background
Oligodendrogliomas belong to the class of diffuse gliomas that represent the most frequent primary brain tumors in adults [
1]. About 4 to 8% of all diagnosed tumors of the central nervous system are oligodendrogliomas [
2]. Diffuse gliomas are generally characterized by infiltration of the surrounding brain tissue, and fast progression and relapse are common [
3]. Traditionally, histological similarities to normal glial cells (astrocytes and oligodendrocytes) were used to distinguish between different types of diffuse gliomas according to the World Health Organization (WHO) 2007 grading system [
4]. Known downsides of this histological classification include a considerable variability of diagnoses between neuropathologists and difficulties in discriminating oligodendrogliomas from other types of diffuse gliomas like astrocytomas and “mixed-type” oligoastrocytomas, which complicates diagnostics and treatment decisions for individual patients [
5,
6]. These challenges led to the exploration of molecular markers for glioma diagnostics [
7]. The majority of oligodendrogliomas shows a characteristic allelic loss of chromosomal arms 1p and 19q (1p/19q) that contributes to better chemotherapy sensitivity and longer recurrence-free survival [
8,
9]. Three different gene expression subtypes of 1p/19q co-deleted oligodendrogliomas have recently been revealed, but the analysis of the clinical relevance of these subtypes requires additional studies [
10]. Further, specific heterozygous somatic point mutations of the isocitrate dehydrogenase gene (IDH1/2) were found in more than three-fourths of all oligodendrogliomas and nearly three-fourths of all astrocytomas of WHO grades II and III [
11‐
13] and in all 1p/19q codeleted gliomas [
14]. These mutations are associated with the glioma-CpG island methylator phenotype (G-CIMP) [
15,
16] and with a better prognosis compared to IDH wild-type tumors [
11,
17].
These molecular markers were integrated into a recent update of the classification of tumors of the central nervous system by the WHO [
18]. As a consequence, some diffuse glioma classes became obsolete, like the “mixed-type” oligoastrocytomas that should now be classified as either oligodendrogliomas or astrocytomas. According to this new classification, oligodendrogliomas are characterized by the co-occurrence of the mutation of
IDH1/2 and the 1p/19q co-deletion. Notably, this class does not accommodate
IDH-mutated tumors with 1p/19q wild-type that were classified as oligodendrogliomas based on histology before. Such discrepancies between histological and molecular tumor classification still remain a great challenge for further improvements of glioma diagnostics, but in terms of prognosis molecular markers can outweigh histological characteristics. Recently, it has been shown that glioma subgroups can be defined based on
IDH mutation and 1p/19q co-deletion status deriving genetic subgroups that are more reflective of disease subtypes than glioma classes defined by histology [
19]. These results were further refined through the analysis of DNA methylation profiles revealing clinically relevant molecular subtypes [
20]. In addition, single cell transcriptome data has allowed to gain novel insights into the molecular architecture of oligodendrogliomas showing that the majority of tumor cells express either a specialized astrocyte-like or oligodendrocyte-like program, whereas a subpopulation of cells remains undifferentiated and is associated with a neural stem cell expression program that most likely drives tumor development [
21]. This has been further extended by analyzing single cell transcriptomes of oligodendrogliomas and astrocytomas suggesting a common stemness program for both tumor types that drives tumor growth, whereas differences between both types are mainly driven by the tumor microenvironemt and specific genetic signatures [
22]. This has important consequences for the clinical management of oligodendrogliomas and may also explain in part differences between molecular and histological classifications. All these and many other studies have greatly contributed to a better understanding of molecular characteristics of oligodendrogliomas. Still, also in the light of differences between histological and molecular classifications, our knowledge about specific molecular characteristics of oligodendrogliomas is incomplete.
Here, we present an in-depth computational analysis of histologically classified oligodendrogliomas from The Cancer Genome Atlas (TCGA) revealing novel differences between molecular subgroups at the level of individual genes, pathways, and gene regulatory networks. We first stratified these tumors based on their gene copy number profiles into three subgroups utilizing unsupervised clustering. Additional screening for the presence of known glioma markers showed that these subgroups largely resembled already known molecular glioma subtypes. To further characterize molecular differences, we derived a signature of differentially expressed genes distinguishing tumors with 1p/19 co-deletion and IDH mutation from tumors that predominantly showed an IDH mutation. We further learned a gene regulatory network that is capable to explain this observed expression signature. This enabled us to identify novel putative major regulators that are potentially involved in the manifestation of differences between both subgroups. Interestingly, this network also contained a characteristic expression signature of HOX and SOX genes that distinguishes both subgroups indicating the activity of different glioma stemness programs.
Discussion
First, we analyzed gene copy number data of histologically classified oligodendrogliomas from TCGA and revealed three molecular subgroups by hierarchical clustering of gene copy number data alone (Fig.
1). We used additional information about the presence of a 1p/19q co-deletion [
8] and an
IDH mutation [
11] to further characterize these subgroups. In accordance with previous findings for histologically classified oligodendrogliomas [
10,
71] and gliomas in general [
19], we observed a large 1p/19q subgroup characterized by concurrent 1p/19q co-deletion and
IDH mutation, an intermediate IDHme subgroup of tumors that mainly show an
IDH mutation but no commonly overrepresented gene copy number alterations, and a small 7a10d subgroup showing a concurrent duplication of chromosome 7 and a deletion of chromosome 10 where most tumors lacked
IDH mutations. In addition, considering Verhaak [
26] and G-CIMP [
15] classes, the 1p/19q and the IDHme subgroup resembled each other, whereas the 7a10d subgroup strongly deviated from these two subgroups also in terms of significantly lower overall patient survival (Fig.
2). This, in combination with the molecular characteristics of the 7a10d subgroup, suggests that these tumors might rather represent glioblastoma-like tumors [
3]. This is also supported by a refined molecular classification of gliomas in [
20]. Thus, tumors of our small 7a10d subgroup may have been falsely classified as oligodendrogliomas based on histology alone, which is not unlikely considering difficulties of pure histological classifications [
6]. We therefore decided to focus our further analyses on the comparison of the 1p/19q and the IDHme subgroups.
Second, we performed an in-depth analysis of the 1p/19q and IDHme subgroups deriving a characteristic gene expression signature that distinguished tumors of both groups (Fig.
3). Interestingly, many of these signature genes were part of signaling pathways involved in the regulation of cell proliferation, differentiation, migration, and cell-cell contacts (Fig.
4). Several of these pathways have already been reported to be involved in glioma development (e.g. PI3K-AKT, MAPK, VEGF signaling) [
27,
33,
72,
73]. The strong downregulation of these pathways in the 1p/19q subgroup compared to the IDHme subgroup might be associated with a better sensitivity to treatment and prognosis of (1p/19q) oligodendrogliomas compared to other low-grade gliomas [
74,
75].
Third, to better understand differences between the 1p/19q and the IDHme subgroup, we reconstructed a gene regulatory network capable to explain gene expression differences between both subgroups (Figs.
5 and
6). Interestingly, we revealed that several potential hub transcription factors involved in remodeling of the cytoskeleton (e.g.
APBB1IP,
VAV1,
ARPC1B), apoptosis (
CCNL2,
CREB3L1), and neural development (e.g.
MYTIL,
SCRT1,
MEF2C) were differentially expressed between both subgroups. Since all or the vast majority of tumors of these two subgroups show
IDH mutations, the globally observed expression differences are likely to be strongly influenced by the 1p/19q co-deletion. Moreover, we observed characteristic expression differences between
HOX and
SOX transcription factors (Fig.
7). All
HOX genes included in our network were downregulated and all
SOX genes were upregulated in 1p/19q compared to IDHme. This indicates that the 1p/19q subgroup and the IDHme subgroup express different stemness programs. Recent findings of specific
HOX and
SOX gene expression patterns for different types of gliomas indicate an important role of both gene families in brain tumors [
20‐
22]. This is also supported by the recent finding that
SOX2 repression is an early driver of gliomagenesis that blocks the differentiation of neural stem cells in an
in-vitro model of low-grade astrocytomas [
76]. Further experimental studies are required to analyze our revealed stemness signatures.
Finally, it is important to discuss the revealed molecular subtypes in the light of the new WHO 2016 brain tumor classification scheme [
18]. All oligodendrogliomas that we analyzed have been classified by the TCGA according to the WHO 2007 brain tumor classification scheme [
4], which was state-of-the-art when the tumors were obtained. This older classification is purely based on histology, whereas the new WHO 2016 classification additionally considers the 1p/19q-co-deletion and the
IDH mutation status. There would be differences in the grouping of tumors, but a reclassification of the analyzed tumors is not straightforward and would require expert knowledge of neuropathologists that have to consider histological and molecular data. Therefore, we cannot realize this reclassification for the considered TCGA data set, but we can interpret our subgroups with respect to the new WHO 2016 classification. Considering our 7a10d subgroup, information about the gain of chromosome 7 and the deletion of chromosome 10 are not considered at all in the new WHO 2016 classification system [
18]. Thus, tumors of these subgroup would still not be classified as glioblastomas if no clear signs of high malignancy (necrosis, pathological vascular proliferation) are observed in histology. It is likely that such signs were not present in nearly half of the 7a10d tumors (6 of 15), otherwise these tumors would have been assigned the WHO grade IV instead of grade II according to the WHO 2007 brain tumor classification system. Therefore, these tumors of our 7a10d subgroup might rather be classified as astrocytoma
IDH-wildtype or
IDH-mutant (if histological and molecular data are conclusive) or even as oligodendroglioma, NOS (if histological and molecular data are inconclusive) according to the WHO 2016 brain tumor classification system. This may change in future [
77]. Such low-grade gliomas without any signs of high malignancy and without
IDH mutation still represent an area of ongoing research [
78]. Further, like for the WHO 2007 brain tumor classification, all tumors of our 1p/19q subgroup would also be classified as oligodendrogliomas (
IDH-mutant and 1p/19q-codeleted) according to the WHO 2016 brain tumor classification system. This is also supported by the characteristic overexpression of
SOX genes. In contrast, tumors of our IDHme subgroup would now be classified as astrocytoma
IDH-mutant or
IDH-wildtype also when oligodendroglia-like features are present in histology. This is further supported by the presence of characteristic
ATRX (30 of 45 tumors) or
TP53 (35 of 45 tumors) mutations in
IDH-mutated tumors [
18]. It is important to note that the new WHO 2016 brain tumor classification system does not change the results of our study. The observed molecular differences between subgroups exist independent of the underlying classification system. Still, one should always be aware of the underlying classification system. In the light of the new WHO 2016 brain tumor classification system, we performed an in-depth comparison of oligodendrogliomas (
IDH-mutant and 1p/19q co-deleted) represented by our 1p/19q subgroup to astrocytomas (vast majority
IDH-mutant) represented by our IDHme subgroup. This is supported by our finding that the 1p/19q subgroup expressed an oligodendrocyte-like program and that the IDHme subgroup expressed an astrocyte-like program [
22].
Conclusions
Our study confirms prior findings about the molecular subtyping of histologically classified oligodendrogliomas and further provides novel insights into gene expression differences between subtypes. It is important to note that we were able to derive these subtypes purely based on gene copy number data alone. Additional information about the presence of a 1p/19q co-deletion and an IDH mutation were only considered subsequently to further characterize these subgroups. The in-depth comparison of the 1p/19q and IDHme subgroups provides novel insights into differences at the level of single genes, pathways, and regulatory networks that have not been reported so far. We identified a characteristic gene expression signature that distinguishes both subgroups including several known signaling pathways that impact on cell proliferation, migration, and angiogenesis. We derived a gene regulatory network that can explain expression differences between both subgroups. Our network-based analysis enabled us to predict novel putative major regulators that contribute to the manifestation of differences between both subgroups. Several of these major regulators are known to be involved in the regulation of cytoskeleton remodeling, apoptosis, and neural development. Moreover, we also revealed a characteristic HOX and SOX gene expression signature that distinguishes both subgroups suggesting the activity of different glioma stemness programs.
Further, the analyzed oligodendroglioma data set represents an important resource for future research, but researchers have to be aware that these tumors were classified by TCGA according to the WHO 2007 brain tumor classification system. We hope that the discussion of our findings in the context of the new WHO 2016 classification will raise awareness for the fact that brain tumor classification systems can vary considerably. This is important for the interpretation of the results of our retrospective study and for future studies based on the considered TCGA data set.
In summary, our in-depth study focused on the analysis of molecular data of histologically classified oligodendrogliomas. Especially with respect to an oligodendroglial phenotype, characteristic expression differences associated with histological classification may also exist for other types of gliomas. Future studies with already existing molecular data of histologically classified oligodendrogliomas, oligoastrocytomas, and astrocytomas could search for such patterns and evaluate their value for molecular tumor classification.