The incidence of diffuse astrocytomas differs somewhat regionally, but recent estimates suggest an incidence rate of 0.4 per 100,000 people for WHO grade II astrocytomas and an incidence rate of 3.2 per 100,000 people for glioblastomas. The histological grade shows a direct correlation with the age at presentation, as WHO grade II tumors tend to present in younger adults in their 4th or 5th decades, while glioblastomas (WHO grade IV) peak in the elderly (mean age at diagnosis 61 years). Males appear to be more affected than females with a male:female ratio of 1.5:1.0 for all astrocytic tumors [15].

WHO grade II diffuse astrocytomas are morphologically heterogeneous and characterized by a higher degree of cellular differentiation, relatively slow growth, low mitotic activity, diffuse infiltration, and spread into adjacent brain structures (Fig. 2.2a). Tumor cells express GFAP (glial fibrillary acidic protein), a protein typically found in astrocytomas. WHO grade II diffuse astrocytomas can be found at any site within the CNS, but preferentially within the cerebral hemispheres, particularly within the subcortical and deep white matter of frontotemporal lobes. Although these lesions are rare in children, the main site in pediatric patients is the brain stem (so-called brain stem glioma). The 2016 WHO classification removed two variants, fibrillary and protoplasmic astrocytoma, due to lack of reproducible definition. The gemistocytic variant, which shows a very distinct appearance with eccentrically placed nuclei and dense cytoplasm, remains. Further, gliomatosis cerebri, previously defined by the diffuse involvement of several cerebral lobes, was also removed as separate entity, and it is simply viewed as an extreme example of widespread dissemination of tumor cells [3, 16].

Anaplastic astrocytomas (WHO grade III) are defined as diffuse astrocytomas with focal or dispersed anaplasia. These tumors are grossly more discernible since they are more cellular and form a more readily identifiable tumor mass. The infiltrative nature tends to create an overall increase in tissue volume without inducing a destructive effect. They are seen to arise from low-grade astrocytomas, but are also frequently diagnosed at first biopsy, without indication of a less malignant precursor lesion. In comparison with low-grade tumors, these neoplasms are microscopically remarkable for increased cellularity and enlarged, irregular hyperchromatic nuclei (Fig. 2.2b). Capillaries are lined by a single layer of endothelium, and frank vascular proliferation and necrosis are not present. Immunoreactivity for GFAP is less consistent than that for grade II lesions. In contrast to low-grade astrocytomas, these lesions display increased mitotic activity with a proliferative index (Ki-67/MIB-1 labeling) of 5–10% [3].

Glioblastomas (WHO grade IV) are the most malignant tumors within the spectrum of diffuse astrocytomas and account for up to 60% of all astrocytic tumors [15]. They affect mainly adults with a peak incidence between 40 and 70 years. Less than 10% of glioblastomas arise from a lesion of lower malignancy grade (secondary glioblastoma) and manifest in younger patients (mean age of 45 years). Most are found de novo (primary glioblastoma) after a short clinical history and are seen in older individuals (mean age 62 years). The majority of tumors are located within the cerebral hemispheres and show a tendency to infiltrate deep nuclei and spread along white matter tracts to the contralateral hemisphere. Brain stem involvement is rare and mainly present in children. Sites such as spinal cord or cerebellum are infrequently involved. Microscopically, glioblastomas are extremely heterogeneous and show a higher degree of cellularity, nuclear atypia, cellular pleomorphism, and mitotic activity, in addition to microvascular proliferation and (palisading) necrosis (Fig. 2.2c). The latter two features are the cardinal diagnostic features of glioblastomas and help distinguish them from grade III astrocytomas. Three distinct glioblastoma variants are part of the 2016 classification, which are giant cell glioblastoma, gliosarcoma, and the recently added variant of epithelioid glioblastoma. [3].

Epithelioid glioblastoma is a newly accepted and rare variant, which is characterized by the presence of predominantly epithelioid or focally rhabdoid morphology. This entity poses a diagnostic challenge due to its resemblance to poorly differentiated carcinomas [3, 17].

Oligodendrogliomas form a group of diffusely infiltrative glial tumors with features reminiscent of oligodendrocytes. Oligodendrogliomas account for approximately 5–6% of all glial neoplasms, and overall for 2–3% of all primary brain tumors. The annual estimated incidence rate lies within a range of 0.27–0.35 per 100,000 individuals. Although oligodendrogliomas can develop at any age, the majority of tumors arise within the 4th–5th decade, and less than 2% of oligodendrogliomas are found in children younger than 14 years. Males are more affected than females [15]. Oligodendrogliomas can arise anywhere within the central nervous system, but the majority of tumors are found within the frontal and temporal lobes of the cerebral hemispheres. Other cortical regions are less involved and oligodendrogliomas are rare within deep nuclei or spinal cord. Microscopically, these tumors are composed of a relatively monomorphous population of cells with round-to-oval nuclei with delicate chromatin pattern, and surrounded by perinuclear ‘halos’ (cytoplasmic clearing), an artifact seen in formalin-fixed and paraffin-embedded sections. The vasculature is typically thin-walled and described by some authors as ‘chicken-wire’ vasculature (Fig. 2.2e).

The WHO classification assigns two grades to oligodendrogliomas: well-differentiated relatively slow-growing tumors correspond to WHO grade II, whereas oligodendrogliomas with anaplastic features are assigned WHO grade III. Anaplastic oligodendrogliomas are characterized by an increase in cellularity and nuclear atypia, increased cellular pleomorphism, in addition to increased mitotic activity, endothelial proliferation, and necrosis. In contrast to other gliomas, such as astrocytomas and ependymomas, oligodendrogliomas show a more slowly progressive clinical course [3].

Although oligoastrocytomas appear to have a mixed phenotypic appearance, they seem to demonstrate either an astrocytic or oligodendroglial genotype. This indicates that these tumors do not form a separate entity. The new WHO classification recommends molecular testing to assign these tumors a definitive lineage. Therefore, the new WHO classification discourages the diagnoses of oligoastrocytoma and anaplastic oligoastrocytoma. The term ‘oligoastrocytoma, NOS’ and ‘anaplastic oligoastrocytoma, NOS’ should be used in cases when gliomas are morphologically mixed or ambiguous and cannot be resolved using molecular testing [2, 18].

The hallmark genetic alterations in diffuse gliomas are somatic mutations in the gene encoding human cytosolic NADPH-dependent isocitrate dehydrogenase 1 (IDH1), a citric acid cycle component. Less frequently involved are mutations of IDH2. The IDH1 enzyme normally catalyzes the oxidative carboxylation of isocitrate to alpha-ketoglutarate (α-KG), resulting in the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. Numerous studies uncovered several mechanisms to explain the tumorigenic potential of IDH proteins. There is convincing evidence that mutant IDH, such as IDH1 (R132H), acquires neomorphic activity that converts alpha-ketoglutarate (α-KG) to 2-hydroxygluturate (2-HG) [19]. 2-HG in turn inhibits α-KG-dependent dioxygenases, including the members of the TET family of 5-methylcytosine hydroxylases and Jumonji-C domain-containing histone lysine demethylases. It has been shown that inhibition of these enzymes increases DNA and histone methylation, which eventually triggers the aberrant methylation of multiple cytosine–phosphate–guanine (CpG) dinucleotide-rich islands across the genome [20]. This ‘glioma CpG-island methylator phenotype (G-CIMP)’ is a characteristic profile seen in diffuse gliomas [21, 22] and likely contributes to the neoplastic transformation of neural stem or progenitor cells. Additional studies have shown that the production of 2-HG stimulates the activity of prolyl-hydroxylase domain isoform 3 (PHD3/EGLN) and prolyl 4-hydroxylases, which leads to reduced levels of hypoxia-inducible factor (HIF) and consequently to the enhanced proliferation of human astrocytes [23]. Increased oxidative stress due to decreased intracellular NADPH levels as a result of IDH mutations additionally promotes tumorigenesis [24].

It has been postulated that IDH mutations likely represent an initiating event, but that they are probably not sufficient to induce tumor growth on their own, instead they have to be accompanied by additional genetic mutations. Mutations involving tumor protein 53 (TP53) and ATRX genes play a role in diffuse and anaplastic astrocytomas. Co-deletions of 1p/19q and mutations involving the promoter region of telomerase reverse transcriptase (TERT) have been described for oligodendrogliomas [25].

These mutational profiles appear to be mutually exclusive and define tumor lineages: The combination of IDH1/p53/ATRX and IDH1/1p19q/TERT are mutational signatures for astrocytic tumors and oligodendrogliomas, respectively [26] (see Fig. 2.1). The new WHO classification requires the demonstration of both IDH1 mutation and 1p19q co-deletion for the diagnosis of oligodendroglioma and anaplastic oligodendroglioma. Similarly, the diagnosis of diffuse or anaplastic astrocytoma requires molecular testing for IDH mutations, with additional demonstration of ATRX mutation or loss of nuclear ATRX expression confirming an astrocytic lineage [27, 28].

Numerous studies established that IDH1 mutations are present at high frequency in secondary glioblastomas that originate from prior low-grade gliomas (~85%), which is contrasted by the fact that these mutations rarely occur in primary or de novo glioblastomas (<1%), which are found in the absence of low-grade precursor lesion. IDH1 mutations are further identified in the vast majority of diffuse low-grade (WHO grade II) and anaplastic (WHO grade III) astrocytomas (~70–80%), oligodendrogliomas (80%), anaplastic oligodendrogliomas (85%), and mixed oligoastrocytomas (100%). The IDH1 mutation frequency appears to be similar for WHO grade II and WHO grade III tumors [29]. Interestingly, the mutation rate in pilocytic astrocytomas (WHO grade I), ependymal tumors, or other less common glial tumors is extremely low or absent [13]. It was further demonstrated that IDH1 mutations do not exist in reactive conditions related to cerebral ischemia or infarctions, viral infections, or radiation change [30]. These findings are of particular diagnostic value as they enable the distinction of reactive gliosis from low-grade diffuse astrocytoma, a diagnostically challenging task, especially in the context of small biopsy samples.

It is of clinical importance that IDH 1 and IDH 2 mutations are found to be associated with a favorable prognosis and overall prolonged survival time independent of treatment. The survival of patients with the mutant form of IDH1 in astrocytomas or oligodendrogliomas (WHO grade II-III) and glioblastoma is longer than that of their IDH1 wild-type counterparts. Interestingly, patients with IDH1-mutated glioblastomas (WHO grade IV) show better survival than patients with wild-type anaplastic astrocytomas (WHO grade III). The IDH status, however, does not predict treatment-specific responses of patients with glioma [31].

In 2010, an antibody (Fig. 2.3a) was developed which is able to specifically recognize the mutant IDH1-R132H protein, which represents the majority (90%) of glioma-associated hotspot mutations [32, 33]. In the case of IDH1-R132H-negative immunostaining, testing for other IDH1 or IDH2 mutations is required for WHO grade II and III gliomas as well as glioblastomas from young patients and secondary glioblastomas [34]. This is usually accomplished by direct DNA sequencing or pyrosequencing using DNA extracted either from frozen tissue or more commonly formalin-fixed tissue [35, 36].

The combined deletion of the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q) together with IDH1 mutations defines oligodendrogliomas and anaplastic oligodendrogliomas [37]. Mechanistically, this co-deletion results from of an unbalanced centromeric translocation and leads to loss of entire chromosomal arms t(1;19) (q10;p10). The frequency of 1p/19q co-deletions has been estimated to be 80–90% in WHO grade II oligodendrogliomas and 50–70% in WHO grade III oligodendrogliomas. In spite of a strong association between 1p/19q loss and classic oligodendroglioma morphology, morphology alone cannot predict the 1p/19q status. Interestingly, the chromosomal regions of 1p and 19q have been mapped in great detail; however, no definitive candidate genes have been identified which could explain the tumorigenic effect. Although the genes on 1p/19q remain enigmatic, numerous correlations have been established demonstrating that many tumors with 1p/19q co-deletions also show IDH1/IDH2 mutations; however, 1p/19q loss appears to be absent in cases with tumor protein p53 (TP53) mutations or EGFR amplifications. Notably, the combined loss of 1p/19q is also found in mixed glial tumors (oligoastrocytomas), but extremely rare in non-glial malignancies.