Keywords

Histological Classification of Low-Grade Diffuse Gliomas

The 2007 WHO classification recognizes three histological types of grade II low-grade diffuse glioma: diffuse astrocytoma, oligoastrocytoma, and oligodendroglioma. Diffuse astrocytoma is a well-differentiated and slow-growing tumor but shows a consistent tendency to diffusely infiltrate surrounding brain structures. Therefore, these tumors tend to recur after surgical resection, and this is often associated with progression to more malignant histological types, i.e., anaplastic astrocytoma (WHO grade III) and eventually secondary glioblastoma (WHO grade IV) [1].

Oligodendroglioma is a well-differentiated, slow-growing, diffusely infiltrating tumor of adults, typically located in the cerebral hemispheres and composed predominantly of cells morphologically resembling oligodendroglia [1]. Progression from oligodendroglioma to anaplastic oligodendroglioma (WHO grade III) is not consistent [1].

Oligoastrocytoma is composed of a conspicuous mixture of two distinct neoplastic cell types morphologically resembling the neoplastic cells in oligodendroglioma and low-grade ­astrocytoma [1]. However, cases with discrete tumor areas exhibiting either oligodendroglial or astrocytic differentiation are rare. More commonly, ­oligoastrocytomas show an intimate mixture of oligodendroglial and astrocytic tumor cells. Oligoastrocytoma also includes cells with phenotypic characteristics that are intermediate to those of the two cell types, i.e., with oligodendroglial and astrocytic differentiation [1].

Histological criteria for the diagnosis of low-grade diffuse glioma, in particular oligoastrocytoma, may be subjective owing to the pronounced phenotypic heterogeneity of the astroglial and oligodendroglial cell lineages and the lack of reliable immunohistochemical markers to define oligodendroglioma cells [1]. Therefore, there is considerable interobserver variability in the histological diagnosis of low-grade diffuse glioma, particularly oligoastrocytoma [2–5].

Primary and Secondary Glioblastoma

Most glioblastomas (WHO grade IV) develop very rapidly after a short clinical history without evidence of a less malignant precursor lesion. These “primary” or “de novo” glioblastomas typically affect elderly patients [1, 6, 7]. Much less frequently, glioblastoma develops through progression from diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III). These “secondary” glioblastomas typically develop in younger patients [1, 6, 7]. In the past, the distinction between primary and secondary glioblastomas was made on the basis of clinical data: tumors were considered to be primary if the diagnosis of glioblastoma was made at the first biopsy, without clinical or histological evidence for the presence of less malignant precursor lesion, whereas the diagnosis of secondary glioblastoma required histological and/or clinical evidence of a preceding low-grade or anaplastic astrocytoma [6, 8].

Accumulating evidence suggests that primary and secondary glioblastomas are characterized by distinct genetic and epigenetic alterations as well as different expression profiles at RNA and protein levels [6]. Genetic alterations that are significantly more frequent in primary glioblastomas than secondary glioblastomas include loss of heterozygosity (LOH) 10p (47 % vs. 8 %), EGFR amplification (36 % vs. 8 %), and PTEN mutations (25 % vs. 4 %) [6, 9]. Alterations that are significantly more frequent in secondary than primary glioblastomas are TP53 mutations (65 % vs. 28 %), LOH 19q (54 % vs. 6 %), and LOH 22q (82 % vs. 41 %) [6, 10, 11]. However, until the identification of IDH1 mutations, none of these alterations could reliably distinguish between glioblastoma subtypes. Since primary and secondary glioblastomas are histologically largely indistinguishable [12], these subtypes remained conceptual and were not used for diagnosis or treatment decisions [6, 8, 12].

IDH1/2 Mutations Are Frequent and Early Genetic Alterations Shared by Astrocytic and Oligodendroglial Diffuse Gliomas

The presence of IDH1 mutations (at codon 132) in glioblastomas was first reported in an analysis of 20,661 protein-coding genes [13]. IDH2 mutations (at codon 172) were also found in gliomas lacking IDH1 mutations, although much less frequently [14]. Subsequent studies in several laboratories demonstrated that IDH1/2 mutations are not only very frequent (>80 %) in secondary glioblastomas and their precursor lesions ­(diffuse astrocytoma and anaplastic astrocytoma) but also similarly frequent (>80 %) in oligodendrogliomas (oligodendroglioma WHO grade II and anaplastic oligodendroglioma WHO grade III) and oligoastrocytomas (oligoastrocytoma WHO grade II and anaplastic oligoastrocytoma WHO grade III) [14–16]. In contrast, IDH1/2 mutations are very rare (<5 %) or absent in primary glioblastomas and pilocytic astrocytomas, as well as other neoplasms of the nervous system, including ependymomas, medulloblastomas, and meningiomas [14–17]. IDH1/2 mutations are also largely absent or very rare in tumors at other organ sites, including the bladder, breast, stomach, colorectum, lung, liver, ovary, and prostate [14, 17]. The exceptions so far reported include chondrosarcoma (∼55 %) [18], cholangiocarcinomas of intrahepatic origin (23 %) [19], acute myeloid leukemia (AML, up to 20 %) [20–25], angioimmunoblastic T-cell lymphoma (AITL, 20 %) [26], melanomas (∼10 %) [27], and anaplastic thyroid cancer (approx. 10 %) [28] (Fig. 5.1).

Fig. 5.1
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Frequency of IDH1/2 mutations in human neoplasms

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In low-grade diffuse gliomas, in addition to frequent IDH1/2 mutations, about 60 % of diffuse astrocytomas carry a TP53 mutation, while oligodendrogliomas show frequent 1p/19q loss (∼70 %) [1, 15, 29–31]. Most low-grade diffuse gliomas have either IDH1/2 mutations plus TP53 mutations or IDH1/2 mutations plus 1p/19q loss (Fig. 5.2) [5, 15]. IDH1/2 mutations are likely to occur before TP53 mutations or 1p/19q loss. Of 40 patients with diffuse glioma who had both IDH1 mutations and other genetic alterations (TP53 mutation or 1p/19q loss) at the last biopsy, 33 (83 %) had both IDH1 mutations and other genetic alterations from the first biopsy [15]. In four patients, the first biopsy had an IDH1 mutation alone, while the second biopsy showed both IDH1 and TP53 mutations. In three patients, the first biopsy had an IDH1 mutation alone, while the second biopsy showed both IDH1 mutations and 1p/19q loss [15]. There was no case in which an IDH1 mutation occurred after the acquisition of a TP53 mutation or loss of 1p/19q [5, 15]. Furthermore, low-grade diffuse gliomas carrying only IDH1/2 mutations are more frequent (17 %) than those carrying only a TP53 mutation (2 %) or those showing only 1p/19q loss (3 %) [5].

Acquisition of 1p/19q loss in cells with IDH1/2 mutations may be the driving force toward oligodendroglial differentiation in low-grade diffuse gliomas [5, 15, 30]. It has been shown that oligodendrogliomas with the typical histological signature of oligodendroglioma (e.g., honeycomb appearance of most neoplastic cells) showed loss at 1p/19q in the vast majority of cases (>90 %) [30]. The astrocytic phenotype/astrocytic differentiation may be associated with IDH1/2 mutations, since the majority (66 %) of low-grade diffuse gliomas containing only an IDH1/2 mutation were histologically diagnosed as diffuse astrocytoma; alternatively the astrocytic phenotype may develop in cells with IDH1/2 mutations that subsequently acquire TP53 mutations [5].

Molecular Bases of Common Genetic Alterations in Low-Grade Diffuse Gliomas

IDH1/2 Mutations

IDH1 and IDH2 are enzymes that catalyze the interconversion of isocitrate and α-ketoglutarate (α-KG), resulting in the production of NADPH in the citric acid (Krebs) cycle [21, 32–36].

Heterozygous IDH1 mutations impair the enzyme’s affinity for its substrate and dominantly inhibit wild-type IDH1 activity through the formation of catalytically inactive heterodimers [37]. IDH1 mutations decreased the enzyme activities in oligodendroglioma cells [14], and downregulation of either IDH1 or IDH2 significantly reduced the proliferative capacity of cancer cells [38]. Forced expression of mutant IDH1 in cultured cells reduced formation of α-KG and increased the levels of hypoxia-­inducible factor-1α (HIF-1α), a transcription factor, and its targets such as GLUT1, VEGF, and PGK1 [37], which are involved in angiogenesis, survival, and invasion in malignant glioma cells. However, immunohistochemistry revealed HIF-1α upregulation in tumor cells adjacent to areas of necrosis in gliomas irrespective of IDH1 mutations [39].

IDH1/2 mutation results in a gain of enzymic function in the form of the NADPH-dependent reduction of α-KG to produce 2-­hydroxyglutarate (2-HG) in gliomas [40] as well as in AMLs [38]. 2-HG production, but not the dominant-negative effect, is a shared function of the several different IDH1/2 mutants analyzed (IDH1-R132H, IDH1-R132C, IDH1-R132G, IDH1-R132L, IDH1-R132S; IDH2-R172K, IDH2-172 G, IDH2-R172M) [38, 40, 41]. It has been shown that malignant gliomas carrying an IDH1 mutation contain an increased (up to 100-fold) concentration of 2-HG [40]. It is of interest to note that children with excessive accumulation of 2-HG due to inborn errors of 2-HG metabolism have an elevated risk of brain tumor [42], while IDH2 heterozygous germline mutations were detected in 15 unrelated patients with 2-HG aciduria [43].

IDH1/2 mutations are associated with a hypermethylation phenotype in gliomas and AMLs. Noushmehr et al. [44] reported a distinct subset of glioblastomas with concerted CpG island methylation at a large number of loci frequently carry IDH1/2 mutations. Similarly, in a study of 131 brain tumors, hypermethylation of CpG loci was strongly associated with IDH1/2 mutation [45]. Similar hypermethylation signatures were detected in AMLs carrying IDH1/2 mutations [35]. In AMLs, IDH1/2 mutations were mutually exclusive with mutations in 5-methlycytosine hydroxylase 2 (TET2, an α-KG-dependent enzyme), and TET2 mutations were associated with epigenetic defects similar to those seen in IDH1/2 mutants [35]. TET2 promoter methylation, but not TET2 mutation, was detected in a small fraction of gliomas lacking IDH1/2 mutations [46]. Recent evidence suggests that 2-HG plays a critical role in abnormal methylation, since it is a competitive inhibitor of multiple α-KG-dependent dioxygenases, such as histone demethylases and the TET family of 5-methlycytosine hydroxylases [47].

In summary, IDH1 and IDH2 mutations lead to simultaneous downregulation of α-KG and upregulation of 2-HG [35, 40], resulting in HIF-1α upregulation, and a hypermethylation phenotype due to genome-wide histone and DNA-methylation alterations.

TP53 Mutations

TP53 plays an important role in many cellular processes, such as the cell cycle, response to DNA damage, apoptosis, and cell differentiation [48]. After DNA damage, TP53 is activated and induces transcription of genes such as p21Waf1/Cip1 [49, 50]. MDM2 is induced by wild-type TP53 [51, 52] and binds to mutant and wild-type TP53, thereby inhibiting the ability of TP53 to activate transcription [53, 54]. p14ARF binds to MDM2 and inhibits MDM2-mediated TP53 degradation and transactivational silencing [50, 55]. p14ARF is negatively regulated by TP53 [50]. Thus, loss of normal TP53 function may result from alterations in TP53, MDM2, or p14ARF. Promoter methylation of the p14 ARF gene was observed in 20–30 % of diffuse astrocytomas [56, 57] and oligodendrogliomas [58], whereas p14 ARF homozygous deletion and MDM2 amplification are largely absent in low-grade diffuse gliomas.

The type and distribution of TP53 mutations in diffuse astrocytomas and secondary glioblastomas are similar, being characterized by frequent G:C → A:T mutations at CpG sites, particularly at codons 248 and 273 [8, 29]. This contrasts with findings for primary glioblastomas, in which TP53 mutations are more evenly distributed throughout the exons, and G:C → A:T mutations are less frequent than in secondary glioblastomas [7, 8]. These results suggest that the acquisition of TP53 mutations in primary and secondary glioblastomas may occur through different molecular mechanisms [8].

1p/19q Loss

Oligodendrogliomas are characterized by frequent co-deletion of 1p and 19q; in most cases, the entire 1p/19q arms are involved [59–62]. Jenkins et al. [63] showed that this is due to unbalanced translocation between chromosomes 1 and 19 [t(1;19)(q10;p10)]. A balanced whole-arm translocation between chromosomes 1 and 19 forming two derivative chromosomes, one composed of 1q and 19p, the other of 1p and 19q, and subsequent loss of der(1;19)(p10;q10) then results in the simultaneous 1p and 19q loss observed in oligodendroglioma with retention of the der(1;19)(q10;p10) seen in these cases [64]. Isolated deletions of 19q are also common in astrocytic and oligodendroglial tumors [62, 65, 66], but isolated deletions of 1p are rare in gliomas and are associated with a poorer prognosis [61, 67].

Molecular cytogenetic deletion mapping studies have suggested that the minimal regions of deletion and, by implication, the putative candidate genes reside within 1p36 and 19q13.3 [61, 62, 68]. Recent exomic sequencing in seven oligodendrogliomas showed somatic mutations in the CIC gene (homologue of the Drosophila gene capicua) at 19q13.2 in six cases and in the FUBP1 gene that encodes far-upstream element (FUSE) binding protein on chromosome 1p in two tumors [69]. Similarly, another study of exome sequencing showed somatic mutations and insertions/deletions in the CIC gene in 13/16 (81 %) oligodendrogliomas with 1p/19q co-deletion [70]. This finding was validated by deep sequencing of 13 additional tumors, which revealed 7 others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69 %) [70]. Astrocytomas and oligoastrocytomas lacking 1p/19q loss revealed that CIC alterations were very rare (2 %) [70]. In contrast, Bralten et al. [71] reported the absence of common somatic alterations in genes on 1p and 19q in seven ­oligodendrogliomas analyzed.

Prognostic Value of Common Genetic Alterations in Low-Grade Diffuse Gliomas

IDH1/2 Mutations

It is well established that IDH1 mutations are a significant prognostic marker of favorable outcome in patients with glioblastoma [14, 72]. This is, however, likely to be due to distinct biological behaviors of primary glioblastomas typically lacking IDH1 mutations and secondary glioblastomas typically carrying IDH1 mutations.

In low-grade diffuse gliomas, interpretation of survival data may be more complex, because IDH1 mutations are frequently copresent with either TP53 mutations or 1p/19q loss [5, 15]. In one study (n  =  49), there was a significant association between the presence of IDH1 mutations and longer overall survival in patients with diffuse astrocytoma [73]. In another study (404 gliomas of grades II–IV; 100 being classified as WHO grade II), univariate and multivariate analyses showed that IDH1 mutations were prognostic for a more favorable outcome [74]. Houillier et al. [75] showed that 1p/19q loss, but not IDH mutation, was associated with prolonged progression-free survival of low-grade diffuse glioma patients, although IDH1 mutation and 1p/19q co-deletion were associated with prolonged overall survival. In our recent study, when low-grade diffuse gliomas (diffuse astrocytoma, oligoastrocytoma, oligodendroglioma; n  =  360) were combined in univariate or multivariate analyses, the presence of IDH1/2 mutations was not prognostic for patient survival [5].

TP53 Mutations

The prognostic value of TP53 mutations in low-grade gliomas has been controversial. In a study of diffuse astrocytomas and oligoastrocytomas (159 cases), TP53 mutation was significantly associated with progression-free survival, but not with overall survival [76]. Ishii et al. [77] reported a tendency for shorter survival in patients (34 diffuse astrocytomas/oligoastrocytomas) with TP53 mutations, but the results were not statistically significant. Watanabe et al. [57] found that TP53 mutations were not significantly prognostic of survival of patients with diffuse astrocytomas (n  =  46). We have previously reported in a population-based study that TP53 mutations are predictive of shorter survival in patients with low-grade diffuse gliomas (n  =  122) [29]. In our recent study (n  =  360), when low-grade diffuse gliomas were combined, TP53 mutations were prognostic for shorter survival [5].

1p/19q Loss

Concurrent deletion of chromosomes 1p and 19q, a typical genetic alteration in oligodendroglioma, is a well-established predictive marker in oligodendrogliomas [63, 78, 79], i.e., associated with increased chemosensitivity and a more favorable clinical outcome [61, 63, 80, 81]. In our recent study (n  =  360), when results for low-grade diffuse gliomas were combined, 1p/19q loss remained to be prognostic for longer survival [5].

Molecular Classification of Low-Grade Diffuse Gliomas

Since the vast majority (>90 %) of WHO grade II diffuse gliomas carry at least one of these alterations (IDH1 mutation, TP53 mutation, and/or 1p/19q loss) (Fig. 5.2) [5], it may be possible to develop a molecular classification that complements and eventually replaces histological typing. In our recent study (n  =  360), patients with low-grade diffuse glioma with IDH1/2 mutations plus 1p/19q loss survived significantly longer than those with IDH1/2 mutation plus TP53 mutation [5]. Patients with diffuse astrocytoma showed a similar survival curve to that of patients with low-grade diffuse glioma with IDH1/2 mutation plus TP53 mutation; survival of patients with oligodendroglioma was similar to that of patients with low-grade diffuse glioma with IDH1/2 mutation plus 1p/19q loss [5]. Thus, with respect to clinical outcome of patients with low-grade diffuse gliomas, the power of molecular classification on the basis of IDH1/2 mutations, TP53 mutations, and 1p/19q loss is similar to that of histological classification [5].

A molecular classification of low-grade diffuse gliomas would be valuable, since histo­logical diagnosis of these tumors may be difficult in a substantial fraction of cases, with marked ­interobserver variability, particularly for ­oligoastrocytomas. Oligoastrocytomas carry either IDH1 mutation plus TP53 mutation (approx. 40 %) or IDH1 mutation plus 1p/19q loss (approx. 45 %) [1, 29]. However, TP53 mutations and 1p/19q loss are mutually exclusive [29, 30, 82, 83], indicating that, despite their histologic heterogeneity, oligoastrocytomas are genetically monoclonal and carry genetic alterations similar to either ­diffuse astrocytomas or oligodendrogliomas. This was also supported by our recent analyses (n  =  360), showing that the frequency and combination of genetic alterations in oligoastrocytomas are similar to those when all diffuse gliomas combined [5, 11]. Thus, oligoastrocytoma is not a distinct tumor entity, but one subset appears to be genetically related to diffuse astrocytomas, while another is genetically related to oligodendrogliomas.

Fig. 5.2
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Frequency and combinations of genetic alterations in diffuse astrocytomas and oligodendrogliomas

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A small fraction (7 %) of low-grade diffuse gliomas lack common alterations, i.e., are triple negative for IDH1/2 mutations, TP53 mutations, and 1p/19q loss [5]. This may suggest the presence of not yet identified additional genetic pathway(s) in the development of low-grade diffuse gliomas. Array CGH analysis in triple-­negative low-grade diffuse gliomas showed loss at 9p21 (p14 ARF, p15 INK4b, p16 INK4a loci) and 13q14–13q32 (containing the RB1 locus) in several cases. Further analyses revealed that alterations in the RB1 pathway (­homozygous deletion and promoter methylation of the p15 INK4b, p16 INK4a, and RB1 genes) were significantly more frequent in triple-negative than in non-­triple-negative cases, and they were significantly associated with unfavorable patient outcome [84]. These results suggest that a fraction of low-grade diffuse gliomas lacking common genetic alterations may develop through a distinct genetic pathway, which may include loss of cell-cycle control regulated by the RB1 pathway.

In summary, the molecular profile of low-grade diffuse gliomas based on IDH1/2 mutations, TP53 mutations, and 1p/19q loss provides a more objective classification and correlates well with clinical outcome. Despite their histological heterogeneity, oligoastrocytomas are genetically clonal neoplasms, one subset being genetically related to diffuse astrocytomas, while the other type is genetically related to oligodendroglial tumors, indicating that oligoastrocytoma is not a distinct entity. We recommend that the working group of the next WHO classification (5th edition) reevaluate this issue.

Genetic Alterations Useful for Diagnosis of Low-Grade Diffuse Gliomas

Much progress has been made in establishing the genetic profile of gliomas, in particular through next-generation sequencing [13, 85]. Some genetic alterations are helpful in confirming the histopathological diagnosis (Table 5.1). Screening for IDH1/2 mutations and/or immunohistochemistry using antibodies to specific IDH1 mutants [86] is useful for reliably distinguishing between primary and secondary glioblastomas, between diffuse gliomas and pilocytic astrocytomas or other CNS neoplasms, and between the infiltrating zone of low-grade diffuse gliomas and non-­tumorous tissues [14–16, 87]. Since pilocytic astrocytomas are characterized by frequent BRAF-KIAA1549 fusion (70 %) [88], combined molecular analysis of BRAF and IDH1 reliably distinguishes pilocytic astrocytoma from diffuse astrocytoma [87] (Table 5.1).

Table 5.1 Genetic alterations diagnostic for astrocytic and oligodendroglial gliomas
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Genetic Pathways in the Development of Astrocytic and Oligodendroglial Diffuse Gliomas

The identification of IDH1/2 mutations was a breakthrough, since it significantly changed our understanding of genetic pathways in the development of gliomas. Primary and secondary glioblastomas are now reliably defined by the absence or presence of IDH1/2 mutations, respectively [72]. Taking IDH1 mutations as a genetic marker of secondary, but not primary, glioblastomas corresponds to the respective clinical diagnosis in 385/407 (95 %) glioblastomas at the population level [72]. IDH1/2 mutations are very early and frequent genetic alterations common to diffuse astrocytic and oligodendroglial tumors [11, 14–16], suggesting that they may originate from the common precursor cells. The additional loss of 1p/19q in cells with IDH1/2 mutations may lead to the acquisition of the oligodendroglial phenotype. Among glioblastomas, only secondary glioblastomas share a common cellular origin with ­oligodendrogliomas, whereas primary glioblastomas may derive from different precursor cells lacking IDH1/2 mutations. Our current concept of genetic pathways to astrocytic and oligodendroglial diffuse gliomas is summarized in Fig. 5.3.

Fig. 5.3
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Current concept of genetic pathways to astrocytic and oligodendroglial gliomas

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