Opinion & Special Article: Glioma Classification

Histology

Histologic tissue evaluation is central and the first step in the diagnostic process (Figure 2). Much of the basis for classification and grading of diffuse glioma can be obtained by assessment of differentiation status, cell density, mitotic activity, the presence or absence of necrosis, and/or microvascular proliferation. In addition to classical histology, several immunohistochemical markers provide direct information on essential molecular parameters such as IDH1-R132H, H3, and ATRX status. However, classification and grading of many diffuse gliomas demand additional molecular analyses as a second step (Figure 2).

Figure 2 Stepwise Approach and Timeline of the Diagnostic Process

In clinical practice, information regarding the studied material becomes available in a stepwise manner. First, the material is studied using microscopic techniques. This includes the analysis of morphological features (e.g., cell atypia, mitotic activity, necrosis, and/or vascular proliferations) supplemented in most cases by immunohistochemical stains to investigate the presence of specific (mutated) proteins (e.g., ATRX, p53, and/or IDH-R132H). This first step of the investigation takes up to 1–3 working days to come up with a histologic diagnosis that is then shared with the clinicians. In most cases, a second step featuring molecular investigations is needed to arrive at the integrated diagnosis. The method picked is determined by the histologic diagnosis. In morphological glioblastoma cases in patients older than 55 years with a negative IDH1-R132H immunohistochemical analysis, it requires only testing for MGMT promotor status. For other cases, NGS and/or methylation profiling using the brain tumor classifier will also be required to investigate specific molecular markers. Depending on local protocols and the technique used, information regarding the methylation class, chromosomal gains, losses, amplifications, and/or specific mutations can be obtained. With the required information on the presence or absence of an IDH mutation, TERT promotor mutation, 1p/19q codeletion, EGFR amplification, +7/−10, and a CDKN2A/B homozygous loss, the final integrated diagnosis based on the histologic diagnosis and molecular alterations can be rendered. Sometimes, the required techniques are initiated in parallel, but when inconclusive results are obtained or when limited material is available, the techniques can also be initiated subsequently, delaying the rendering of an integrated diagnosis. Note: the times listed are only an estimation and can differ based on local protocols. In addition, because the analyses are performed in batches, further delays can be introduced in the case of low sample counts and/or infrequent analysis runs. ATRX = ATP-dependent X-linked helicase; CDKN2A/B = cyclin-dependent kinase inhibitor 2A/B; EGFR = epidermal growth factor receptor; IDH = isocitrate dehydrogenase; MGMT = O6-methylguanine-DNA methyl-transferase; NGS = next-generation sequencing; TERT = telomerase reverse transcriptase.

IDH

Hotspot mutations in isocitrate dehydrogenase (IDH) enzymes facilitate the production of 2-hydroxyglutarate, which leads to competitive inhibition of alpha-ketoglutarate–dependent enzyme activity, resulting in distinct epigenetic histone and DNA modification, followed by metabolic reprogramming.⁷ This effect can be mediated by mutations in both the IDH1 and IDH2 genes. IDH mutations are considered early driver mutations and are now the basis of molecular glioma classification. The IDH status constitutes a fundamental divide with IDH-mutant astrocytoma and oligodendroglioma on one side and glioblastoma and some rare diffuse gliomas on the other side (Figure 1). A consequence of this central divide among diffuse gliomas has been renaming the formerly IDH-mutant glioblastoma to the current astrocytoma, IDH-mutant, WHO grade 4. The term glioblastoma, a glioma with the worst clinical prognosis, is reserved exclusively for IDH-wildtype grade 4 glioma.

Approximately 90% of the supratentorial IDH mutations are of the IDH1-R132H type, and this can be detected with a mutation-specific antibody (immunohistochemistry). Other IDH1 (R132C, R132L, R132S) or IDH2 (R172G, R172K, R172M) mutations require DNA sequencing for detection. In patients older than 55 years with a supratentorial tumor, IDH mutations are rare. Therefore, when no IDH1-R132H mutation is found, additional sequencing is required only for patients aged 55 years or younger.⁷ Non-IDH1-R132H mutations constitute roughly 80% of IDH alterations in infratentorial IDH-mutant astrocytoma.⁸

1p/19q

Oligodendrogliomas have, by definition, an IDH mutation and a second molecular alteration that defines the entity: the combined full losses of the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q), better known as a 1p/19q codeletion. The exact role of the 1p/19q loss in the tumor biology is currently unclear. Of special importance here is that the 1p/19q status is decisive for the separation of astrocytoma from oligodendroglioma (both IDH-mutant) and that morphological aspects are second in relevance.

ATRX

The ATP-dependent X-linked helicase (ATRX) gene encodes for a protein involved with telomerase activity. Mutations in ATRX, immunohistochemically identified as loss of nuclear expression of this protein, are next to mutually exclusive with 1p/19p loss (oligodendrogliomas). Therefore, loss of nuclear ATRX expression can be used as surrogate marker for IDH-mutated astrocytoma. When an IDH mutation is present, it eliminates the need for further 1p/19q testing.³

TP53

TP53, a tumor suppressor gene, is mutated in the majority of grade 2, 3, and 4 IDH-mutated astrocytomas. Strong staining of p53 protein suggests a TP53 mutation; however, this has a lower specificity than loss of ATRX expression for the diagnosis of an astrocytoma.

CDKN2A/B

In IDH-mutated astrocytoma, the cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) status is an important prognostic factor because homozygous deletion of CDKN2A/B is associated with increased aggressiveness. Homozygous CDKN2A/B loss overrules the histologic grade, so even without microvascular proliferation and/or necrosis, the histologic criteria for a grade 4 tumor, homozygous CDKN2A/B loss also warrants a grade 4 diagnosis in IDH-mutated astrocytomas.⁹ Owing to the prognostic significant implications of CDKN2A/B, IDH-mutant astrocytoma without 1p/19q codeletion should therefore always be tested for homozygous CDKN2A/B loss.

EGFR Amplification, +7/−10, and/or TERT Promotor Mutation

Three different molecular markers independently warrant the diagnosis of a grade 4 glioblastoma in IDH-wildtype diffuse gliomas because they are associated with very poor clinical outcome:

1. Epidermal growth factor receptor (EGFR) amplification.

2. The complete gain of chromosome 7 in combination with a complete loss of chromosome 10, often referred to as +7/−10. It is unclear whether tumors harboring variants of +7/−10 (e.g., +7p/−10 or +7/−10q) are as aggressive as tumors with the full +7/−10 loss.¹⁰

3. Mutations in the promotor of the telomerase reverse transcriptase (TERT) gene. TERT promotor mutations lead to the reactivation of telomerase, resulting in maintained telomere length and thus limitless cellular lifespan.

Thus, in an IDH-wildtype astrocytoma without microvascular proliferation and/or necrosis, the histologic criteria for a grade 4 tumor, testing for these alterations is important because of the prognostic and possible therapeutic consequences. Overall, TERT mutations in this triad of markers are most sensitive but least specific, while both EGFR mutation and +7/−10 signature have high specificity with lower sensitivity. Very frequently, these alterations occur together, and the presence of any 2 of the 3 features is highly specific for glioblastoma. TERT promotor mutations do also occur in tumors within the differential diagnosis of glioblastoma such as pleomorphic xanthoastrocytoma. Furthermore, these mutations occur in most oligodendrogliomas.

H3 K27M and H3 G34

Two diffuse glioma entities have histone 3 (H3) alterations. Both are rare, IDH-wildtype, and have an aggressive course (grade 4). These tumors occur most often in children and young adults. Diffuse midline gliomas, H3 K27–altered, are gliomas with H3 K27-trimethylation alterations (H3F3A/B K27M mutation being most common), are located in midline CNS structures (i.e., brainstem, thalamus, or spinal cord), and have a diffuse infiltrative growth pattern.³ The presence of all criteria is important because H3 K27M can also be found in other entities that may have different pathogenesis and prognosis.

Diffuse gliomas that feature H3 G34 mutations are located most often in a supratentorial hemisphere and can have a nonenhancing appearance on MRI. These tumors are termed diffuse hemispheric glioma, H3 G34–mutant. H3-mutated gliomas have a prognosis similar to that of glioblastoma, IDH-wildtype.^11,12

MGMT

Hypermethylation of the O6-methylguanine-DNA methyl-transferase (MGMT) gene promotor leads to reduced expression of this DNA repair protein. MGMT repairs the damage by alkylating chemotherapeutic agents, rendering the tumor more resistant to alkylating chemotherapy.¹³ MGMT promotor methylation status is a biomarker of both prognostic and therapeutic significance in glioblastoma, IDH-wildtype. Glioblastoma with MGMT promotor hypermethylation have a better prognosis and more favorable response to chemotherapy (eTable 1, links.lww.com/WNL/C324).¹⁴

FGFR1/NF1/BRAF

The RAS/mitogen-activated protein kinase (MAPK) pathway plays an important role in cell regulation, including proliferation and differentiation. Although MAPK-pathway alterations occur most often in pediatric low-grade gliomas, they are also observed in adult diffuse gliomas. Several gene alterations can lead to an upregulation of this pathway. Genes involved with this pathway include fibroblast growth factor receptor 1 (FGFR1), neurofibromin 1 (NF1, affected in neurofibromatosis type 1), and BRAF.¹⁵ The most common MAPK pathway alteration is a BRAF p.V600E mutation that occurs in approximately 2% of adult glioblastoma cases. The exact incidence of BRAF fusions, FGFR, and other MAPK alterations remains to be determined as novel techniques to detect them become more widely available.⁴ For some MAPK-activating alterations, antagonistic therapeutic options are available (eTable 1, links.lww.com/WNL/C324).

Methylation Brain Tumor Classifier

In recent years, methylation patterns generated by genome-wide DNA methylation profiling have been increasingly used to assist tumor diagnosis for their potential to predict specific tumor types. Methylation profiling requires a classification tool for tumor class prediction, a calibrated score for the validity of the prediction, and a copy number profile. The methylation-based approach has been used for the identification of many novel brain tumor entities (e.g., high-grade astrocytoma with piloid features). Methylation analysis is a powerful emerging diagnostic tool, and its use is encouraged for all diagnostically unresolved brain tumor samples.

NOS/NEC

In some cases, molecular testing is not performed or interpretable because of sparse resources or for intentional reasons (e.g., no therapeutic consequences). In those cases, the term “not otherwise specified (NOS)” should be added to the histologic diagnosis: for example, astrocytoma, WHO grade 2, NOS (note: no IDH status listed).

In the event that molecular testing was performed and valid results were obtained, but the results did not lead to a CNS5-compatible diagnosis, the term “not elsewhere classified (NEC)” should be added to the diagnosis. For example, a nondiffuse hemispheric glioma with a K27M mutation results in nondiffuse nonmidline K27-altered glioma, NEC.³

Relevance to Neurologists and Trainees

Molecular alterations have become increasingly important in the diagnosis and classification of glioma. For many entities, specific molecular alterations are required to render a WHO CNS5–compatible diagnosis.

In clinical practice, information regarding the studied material becomes available in a step-wise manner (Figure 2). This has resulted in a layered integrated diagnosis starting with a histopathologic classification days after surgery. The histopathologic findings are used to select subsequent molecular testing. For example, in histologic low-grade diffuse glioma, it is important to determine the IDH status and the presence of specific markers that determine the diagnosis (e.g., 1p/19q codeletion for oligodendroglioma) or grade (homozygous CDKN2A/B loss for astrocytoma) (Figure 1 and eTable1, links.lww.com/WNL/C324). This additional information is added to the report, after which the integrated diagnosis is rendered. Alterations that can have potential therapeutic consequences (e.g., MGMT promotor methylation) will be listed under the “molecular alterations” layer.

For example:

• Integrated diagnosis: astrocytoma, IDH-mutant, WHO CNS5 grade 2

• Histopathologic classification: low-grade diffuse glioma

• CNS5 WHO grade: 2

• Molecular information: IDH1-R132H–mutated, ATRX mutation, TP53 mutation, no CDKN2A/B loss, no 1p/19q codeletion