Genetic profile of astrocytic and oligodendroglial gliomas
Low-grade diffuse gliomas WHO grade II (diffuse astrocytoma, oligoastrocytoma, oligodendroglioma) are characterized by frequent IDH1/2 mutations (>80%) that occur at a very early stage. In addition, the majority of diffuse astrocytomas (about 60%) carry TP53 mutations, which constitute a prognostic marker for shorter survival. Oligodendrogliomas show frequent loss at 1p/19q (about 70% of cases), which is associated with longer survival. With respect to clinical outcome, molecular classification on the basis of IDH1/2 mutations, TP53 mutations, and 1p/19q loss showed a predictive power similar to histological classification. IDH1/2 mutations are frequent (>80%) in secondary glioblastomas that have progressed from low-grade or anaplastic astrocytomas. Primary (de novo) glioblastomas with IDH1/2 mutations are very rare (<5%); they show an age distribution and genetic profile similar to secondary glioblastomas and are probably misclassified. Using the presence of IDH1/2 mutations as a diagnostic criterion, secondary glioblastomas account for approximately 10% of all glioblastomas. IDH1/2 mutations are the most significant predictor of favorable outcome of glioblastoma patients. The high frequency of IDH1/2 mutations in oligodendrogliomas, astrocytomas, and secondary glioblastomas derived thereof suggests these tumors share a common progenitor cell population. The absence of this molecular marker in primary glioblastomas suggests a different cell of origin; both glioblastoma subtypes acquire a similar histological phenotype as a result of common genetic alterations, including the loss of tumor suppressor genes on chromosome 10q.
KeywordsAstrocytoma Primary glioblastoma Secondary glioblastoma Oligodendroglioma IDH1 mutation TP53 mutation 1p/19q loss
Low-grade diffuse gliomas
The 2007 World Health Organization (WHO) classification recognizes three main histological types of low-grade diffuse glioma grade II: diffuse astrocytoma, oligoastrocytoma, and oligodendroglioma . Diffuse astrocytomas tend to progress to more malignant histological types, i.e., anaplastic astrocytoma (WHO grade III), and eventually secondary glioblastoma (WHO grade IV), whereas progression of oligodendrogliomas to anaplastic oligodendrogliomas is less predictable .
Frequency and combination of genetic alterations in low-grade diffuse gliomas
IDH1/2 mutations are the only genetic alteration with a high prevalence (>80% of cases) in all WHO grade II diffuse gliomas, and their frequency does not change during the progression from diffuse astrocytoma (WHO grade II) to anaplastic astrocytoma (WHO grade III) and secondary glioblastoma (WHO grade IV). Similarly, oligodendroglioma (WHO grade II) shows a frequency of IDH1 mutation similar to that in anaplastic oligodendroglioma (WHO grade III) .
IDH1/2 mutations are likely to occur before TP53 mutations or 1p/19q loss because low-grade diffuse gliomas carrying only IDH1/2 mutations are more frequent (17% of all low-grade diffuse gliomas) than those carrying only a TP53 mutation (2%) or those showing 1p/19q loss (3%) . Furthermore, analysis of multiple biopsies from the same patients revealed that there was no case in which an IDH1/2 mutation occurred after the acquisition of a TP53 mutation or loss of 1p/19q [2, 9].
We hypothesize that loss of 1p/19q in cells with IDH1/2 mutations favors an acquisition of the oligodendroglial phenotype [2, 5, 9], whereas cells with IDH1/2 mutations that subsequently acquire TP53 mutations tend to develop an astrocytic phenotype. The majority of tumors containing only an IDH1/2 mutation are histologically diagnosed as astrocytoma (66%), and less frequently as oligoastrocytoma (20%) and oligodendroglioma (15%) .
Molecular classification of low-grade diffuse gliomas
Because most (>90%) of WHO grade II gliomas carry at least one of these alterations (IDH1/2 mutation, TP53 mutation, or 1p/19q loss)  (Fig. 2), it is possible to develop a molecular classification that may complement and eventually replace 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 . Survival of patients with diffuse astrocytoma was similar to that of patients with low-grade diffuse glioma with IDH1/2 mutation plus TP53 mutation, and survival of patients with oligodendroglioma was similar to that of patients with low-grade diffuse glioma with IDH1/2 mutation plus 1p/19q loss . 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 . When all low-grade diffuse gliomas are combined in a multivariate analysis, TP53 mutation is significantly associated with shorter survival and 1p/19q loss with longer survival, but IDH1/2 mutations are not prognostic for patient outcome .
A molecular classification of low-grade diffuse gliomas would be valuable, because the histological diagnosis of low-grade diffuse gliomas may be difficult in a substantial fraction of cases, with marked interobserver variability. This problem is particularly apparent for oligoastrocytomas, which are composed of a conspicuous mixture of two distinct neoplastic cell types morphologically resembling oligodendroglioma or diffuse astrocytoma . Genetic profiling suggests that oligoastrocytoma is not a distinct tumor entity, but that these are monoclonal neoplasms which differ in their biology and clinical behavior. This possibility is supported by the fact that the frequency and combination of genetic alterations in oligoastrocytomas are similar to those in all diffuse gliomas combined (Figs. 1 and 2).
Primary and secondary glioblastomas
The majority of glioblastomas (WHO grade IV) affect elderly patients and manifest rapidly after a short clinical history and without evidence of a less malignant precursor lesion: these are designated primary or de novo glioblastomas. In contrast, secondary glioblastomas develop in younger patients through progression from diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III) . In the past, the distinction between primary and secondary glioblastomas was based on clinical data: tumors were considered to be primary if the diagnosis of glioblastoma was made at the first biopsy, without clinical or histological evidence of a preexisting, less malignant precursor lesion; the diagnosis of secondary glioblastoma required histological or clinical evidence of a preceding low-grade or anaplastic astrocytoma [10, 11].
Evidence has accumulated that primary and secondary glioblastomas develop through different genetic pathways . Genetic alterations that are significantly more frequent in primary glioblastomas than secondary glioblastomas are loss of heterozygosity (LOH) 10p (47% vs. 8%), epidermal growth factor (EGFR) amplification (36% vs. 8%), and PTEN mutations (25% vs. 4%) [10, 12]. Alterations significantly more frequent in secondary glioblastomas include TP53 mutations (28% vs. 65%), LOH 19q (6% vs. 54%), and LOH 22q (41% vs. 82%) [10, 13]. However, none of these alterations reliably separates these glioblastoma subtypes. Because primary and secondary glioblastomas are histologically largely indistinguishable , these subtypes have remained conceptual without being used for diagnostic or treatment decisions [10, 11, 14].
IDH1/2 mutation as reliable molecular marker of secondary glioblastomas
Comparison of age and IDH mutations in glioblastomas
Mean age of patients (years)
With IDH mutation
Without IDH mutation
All glioblastoma (n = 123)
Yan et al. 
All glioblastoma (n = 407)
Nobusawa et al. 
All glioblastoma (n = 183)
Ichimura et al. 
All glioblastoma (n = 98)
Bleeker et al. 
Primary glioblastoma (n = 99)
Balss et al. 
Primary glioblastoma (n = 123)
Yan et al. 
Primary glioblastoma (n = 377)
Nobusawa et al. 
Primary glioblastoma (n = 86)
Toedt et al. 
Although rare, there are also secondary glioblastomas that lack IDH1/2 mutations (Fig. 3). Such secondary glioblastomas have infrequent TP53 mutations, and the patients have a short clinical history . Furthermore, most of these secondary glioblastomas (seven of eight) had developed through progression from an anaplastic glioma (WHO grade III), whereas the majority of secondary glioblastomas with IDH1/2 mutations had progressed from a WHO grade II glioma . The possibility exists, therefore, that some neoplasms diagnosed as anaplastic glioma were actually primary glioblastomas that were misdiagnosed as the result of a sampling error, e.g., because of the absence of necrosis or microvascular proliferation in the available histological sections.
IDH1/2 mutations, as a genetic marker of secondary but not primary glioblastomas, correspond to the respective clinical diagnosis in 385 of 407 (95%) of glioblastomas (Fig. 3) . Using the presence of IDH1/2 mutation as a molecular criterion, secondary glioblastomas account for 6–13% of all glioblastomas in hospital-based studies [15, 16, 19] and for 9% of all glioblastomas at the population level .
In conclusion, the IDH1/2 mutation is reliable genetic marker of secondary glioblastomas and their precursor lesions and should be assessed in translational investigations and clinical trials.
IDH1/2 mutation correlates with favorable outcome for patients with glioblastoma
Genetic alterations that are prognostic for glioblastoma patients at the population level (multivariate analysis)
No. of cases
Nobusawa et al. 
Ohgaki et al. 
Ohgaki et al. 
Ohgaki et al. 
Ohgaki et al. 
Homma et al. 
Homma et al. 
Ohgaki et al. 
IDH1/2 mutations are associated with typical expression and methylation phenotypes in glioblastomas
Glioblastomas have also been classified on the basis of cDNA expression profiles, with proneural, neural, classic, mesenchymal, and proliferative patterns being described [22, 23]. Most glioblastomas with IDH1/2 mutations (11/12, 92%) showed a proneural expression signature; conversely, approximately 30% of glioblastomas with a proneural signature had IDH1 mutations . Glioblastomas lacking IDH1/2 mutations were classified as classical, mesenchymal, neural, or proneural . Toedt et al.  clustered data on the basis of the expression signature in glioblastomas reported by Phillips et al. . One cluster corresponded to IDH1 mutated tumors with a proneural signature, and another cluster broadly corresponded to IDH1 wild-type tumors with proliferative or mesenchymal signatures .
Promoter methylation of MGMT, RB1, p14ARF, p16INK4a, and TIMP3 is significantly more frequent in secondary than in primary glioblastomas . In a study by Noushmehr et al. , a distinct subset of glioblastomas displayed concerted CpG island methylation at a large number of loci; these tumors belonged to the proneural subgroup and were tightly associated with IDH1/2 mutations. Similarly, in a study of 131 brain tumors, hypermethylation of CpG loci was strongly associated with IDH1/2 mutations . It is of interest to note that similar hypermethylation signatures were detected in acute myeloid leukemia (AML) cases carrying IDH1/2 mutations . This finding suggests that genome-wide epigenetic abnormalities associated with IDH1/2 mutations may cause the distinct expression profiles and favorable biological behavior of glioblastomas with IDH1/2 mutations.
Genetic pathways to astrocytic and oligodendroglial diffuse gliomas
Origin of primary and secondary glioblastomas
Primary and secondary glioblastomas may develop from different cells of origin. The indirect evidence for this hypothesis includes the observations that (1) only secondary glioblastomas, but not primary glioblastomas, share common IDH1/2 mutations with oligodendrogliomas; (2) primary and secondary glioblastomas develop in patients of different age groups; and (3) these glioblastoma subtypes have significantly different biological behavior.
If this is the case, cancer stem cells in primary and secondary glioblastomas may also be different. One study showed that the relative content of CD133+ cells was significantly higher in primary than in secondary glioblastomas, and that CD133+ expression was associated with neurosphere formation only in primary but not in secondary glioblastomas . Lottaz et al.  compared gene expression profiles in 17 glioblastoma cancer stem cell (CSC) lines and in their different putative founder cells. Two subgroups of glioblastoma were distinguished: type I CSC lines displaying “proneural” signature genes and resembling fetal neural stem cell (NSC) lines, and type II CSC lines showing “mesenchymal” transcriptional profiles and resembling adult NSC lines . Since glioblastomas with IDH1 mutations typically have a proneural signature , secondary glioblastomas may derive from cells that have preserved or acquired the properties of fetal NSC lines, distinct from those of adult NSC lines .
Another possibility would be that primary and secondary glioblastomas develop from the same precursor cells, but show distinct biological behavior because of different genetic alterations, most importantly, the absence or presence of IDH1/2 mutations. If this is the case, specific glial progenitor cells present in younger patients could be more susceptible to the acquisition of IDH1/2 mutations and the consequent growth advantage than those cells present in older patients.
Genetic pathways to primary and secondary glioblastoma
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