Diffuse glioma overview based on the 2021 WHO classification part 1 – adult type

Czech version

Authors: M. Hendrych ¹; M. Barák ²; H. Valeková ²; T. Kazda ³; P. Pospíšil ³ ; R. Lakomý ⁴; J. Šána ^4,5; R. Jančálek ²; M. Hermanová ¹
Authors‘ workplace: I. ústav patologie, LF MU a FN u sv. Anny v Brně ¹; Neurochirurgická klinika LF MU a FN U sv. Anny v Brně ²; Klinika radiační onkologie LF MU a Masarykův onkologický ústav, Brno ³; Klinika komplexní onkologické péče LF MU a Masarykův onkologický ústav, Brno ⁴; CEITECH – Středoevropský technologický institut, MU, Brno ⁵
Published in: Cesk Slov Neurol N 2023; 86(6): 359-368
Category: Review Article
doi: https://doi.org/10.48095/cccsnn2023359

Overview

Recent findings in the field of molecular-genetic alterations in diffuse gliomas, encompassing both adult and pediatric types, have initiated significant changes in their classification and diagnostics. These changes are presented in the fifth edition of the WHO Classification of Tumors of the Central Nervous System emphasizing a pivotal shift from the conventional categorization of tumors based on morphology to an integrated diagnostic approach, which incorporates characteristic molecular-genetic and epigenetic alterations alongside traditional morphological features. This review presents a summary of the units that are included in the group of diffuse gliomas of the adult type, with an emphasis on diagnostic criteria and grading according to the fifth edition of the WHO Classification of Tumors of the Central Nervous System, published in 2021.

Keywords:

astrocytoma – glioblastoma – oligodendroglioma – diffuse glioma – WHO CNS 2021 – integrated diagnostics

This is an unauthorised machine translation into English made using the DeepL Translate Pro translator. The editors do not guarantee that the content of the article corresponds fully to the original language version.

Introduction

Gliomas represent a highly heterogeneous group of the most frequently occurring primary brain tumours in adults and children [1]. Their current classification according to the 5th edition of the 2021 WHO Classification of Central Nervous System Tumours [2,3] incorporates the rules of integrated diagnosis already established in the previous revised 4th edition of the 2016 WHO Classification of Central Nervous System Tumours (summarised in the review paper [4]) and supplemented by the latest findings published in the form of the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy (cIMPACT-NOW) recommendations [5-10].

The traditional division of gliomas into diffuse and circumscribed gliomas has been newly extended by the division of diffuse gliomas into two groups - paediatric and adult-type diffuse gliomas. Paediatric-type diffuse gliomas are further divided into low-grade and high-grade tumours (Table 1). This new division of diffuse gliomas into adult and paediatric types is due to their markedly different biological behaviour and molecular genetic alterations, although morphologically they are often virtually indistinguishable. At the same time, the division into adult and paediatric type of diffuse gliomas corresponds to the dominant occurrence of tumour units but does not exclude the occurrence of paediatric type of diffuse glioma in adult patients or, on the contrary, the rare occurrence of adult type of diffuse glioma in paediatric patients [2]. The first part of this review will be devoted to adult-type diffuse gliomas, while the second part will deal with high-grade and low-grade paediatric-type diffuse gliomas.

It must be stated that the diagnosis and classification of gliomas is currently far ahead of therapeutic options. For a long time, updates of treatment recommendations were rather limited to various modifications of the known postoperative radiotherapy and chemotherapy [11] (mainly with the alkylating cytostatic temozolomide). Only recently, studies pointing to the possible effect of other therapies, such as tumour treating fields in glioblastomas [12] or vorasidenib in IDH-mutated low-grade gliomas [13], have been reported.

Types of glioma growth

The differentiation of the type of growth of glial differentiated tumours is the initial and essential step of diagnosis. Diffusely growing gliomas are characterized by unrestricted infiltrative growth of CNS tissues predominantly through preformed spaces around blood vessels, under the pia mater or around/along axons of white matter pathways. The characteristic morphological signs of diffuse growth are a gradual decrease in tumour cell density from the centre to the periphery of the tumour, where the so-called secondary features of diffuse glioma are evident - perineuronal satellitosis and perivascular or subpial spread. Immunohistochemically (IHC), diffuse growth can be visualized by examination of neurofilaments showing fragmented residual axons persisting in the diffusely growing glioma tumour mass (Figure 1A). In contrast, circumscribed gliomas grow expansively and tend to push away the surrounding CNS tissue, which can be verified by IHC examination of neurofilaments (Figure 1B) [4]. The subsequent diagnostic process involves a sequence of molecular genetic examinations, which, together with morphology, are necessary for the correct classification of the tumour according to the integrated diagnosis. The diagnostic process is simplified in Figure 2.

Adult-type of diffuse gliomas

Astrocytoma, IDH-mutant (WHO G2-4)

Astrocytomas are diffusely growing gliomas genetically defined by mutations in the IDH1/2 gene with concomitant IHC evidence of loss of ATRX expression and/or mutations in the TP53 gene in the absence of the 1p/19q codeletion (Table 2). Astrocytomas arise throughout the craniospinal axis, but typically occur supratentorially, especially in the frontal and temporal lobes. The highest incidence of astrocytomas is between 30-34 years of age [14].

Morphologically, astrocytomas typically consist of diffusely growing atypical fibrillary astrocytes or, less frequently, gemistocytic elements (Figure 3). However, genetically defined astrocytomas also include tumours with oligoastrocytic or purely oligodendroglial differentiation [3,4]. Only rarely have cases of composite IDH-mutated diffuse gliomas with a dual oligoastrocytic genotype - loss of ATRX expression, TP53 mutation, no 1p/19q codeletion in the astrocytic component, and 1p/19q codeletion without alterations in both ATRX and TP53 genes in the oligodendroglial component - been described [15]. The status of ATRX and TP53 must be investigated if the 1p/19q codeletion has not been examined (Figure 4). However, only 70-80% of astrocytomas have the inactivating ATRX mutation, thus the absence of loss of ATRX expression on IHC examination does not exclude this diagnosis. However, loss of ATRX is not astrocytoma-specific and is also found in all diffuse hemispheric gliomas, H3 G34-mutated gliomas, some diffuse midline gliomas, H3 K27-altered gliomas and has been described innumerable times in IDH-wildtype glioblastoma (GBM) [16].

Astrocytoma can be classified as grade 2, 3 or 4 according to the diagnostic criteria summarized in Table 3 [7]. The prognostic significance of the stratification of grade 2 and 3 astrocytomas according to mitotic activity has been negated by some studies [3,7,17], however, its significance in IDH-mutated astrocytomas without homozygous CDKN2A/B nucleation has been supported in a recent study by Kros et al. [18], among others. In this study, the mitotic index (with a cutoff of two mitoses per 10 high-magnification fields of view) in astrocytomas without homozygous CDKN2A/B deletion was identified as an independent prognostic factor for disease progression-free survival and is relevant for distinguishing grade 2 (with less than two mitoses) and grade 3 astrocytomas (with two or more mitoses) [18]. Homozygous deletion of CDKN2A/B genes, which are independent predictors of poor prognosis [19,20], has recently been implemented as part of grading and, according to recent recommendations, should be routinely performed in IDH-mutated astrocytomas [16]. Homozygous deletion of CDKN2A/B is relatively rare in primary, therapeutically unaffected IDH-mutated gliomas. Available literature data report the frequency of homozygous deletion for morphologically defined grade 2 in 1.8-3.5%, grade 3 in 3.2-6.7% and grade 4 in 18.8-27% in primary IDH-mutated astrocytomas [19,21]. It is far more frequently detected in recurrent tumours after previous radiotherapy, which explains the need to retest for CDKN2A/B homozygous deletion even in recurrent IDH-mutated gliomas where material is available after eventual reoperation [19,22]. At the same time, post-irradiation acquired CDKN2A/B homozygous deletion represents a potential biomarker predicting resistance to radiotherapy in recurrent IDH-mutated gliomas [22]. Conversely, in IDH-wildtype gliomas, CDKN2A/B homozygous deletion is also frequently found in newly diagnosed, therapeutically untreated tumours [22]. However, a case of astrocytoma and oligodendroglioma with CDKN2A/B homozygous deletion has also been recently described in which the CKDN2A/B homozygous deletion was no longer present in the recurrent tumour [23].

Oligodendroglioma, IDH-mutant, and 1p/19q-codeleted (WHO G2-3)

Oligodendrogliomas are genetically defined by a mutation in the IDH1/2 genes and simultaneous complete codeletion of 1p/19q (Table 4). Characteristic is supratentorial localization, with up to half of the cases affecting the frontal lobes. Only rarely does oligodendroglioma occur infratentorially. Similar to all adult-type diffuse gliomas, oligodendroglioma can infiltrate multiple lobes of the brain, even bilaterally (previously referred to as gliomatosis cerebri), or spread leptomeningeally [3,14]. Compared to astrocytomas, oligodendrogliomas tend to occur in older patients, with the highest incidence in patients between the ages of 40 and 44 years [14]. They are also genetically distinguished from astrocytomas by the presence of a TERT promoter mutation, which is present in most oligodendrogliomas and testing for this mutation can be used to support a diagnosis of oligodendroglioma [16].

The characteristic histological picture includes diffusely growing tumour glial cells of oligodendroglial differentiation with small round nuclei and perinuclear luminosity (resembling an ox eye) in a network of richly branching capillaries (Figure 5). Calcifications are often seen within the tumor. However, the morphological spectrum of genetically defined oligodendroglioma includes tumours with oligoastrocytic or purely astrocytic differentiation [3,4].

Grading of oligodendrogliomas, similar to astrocytoma, combines histological features - significant mitotic activity, presence of necrosis and vascular proliferates - and genetic alteration - homozygous deletion of CDKN2A/B (Table 5) [3]. Similar to astrocytoma, the presence of homozygous deletion of CDKN2A/B is considered a marker of poor prognosis in oligodendroglioma. The prognosis of patients with G3 oligodendroglioma classified on the basis of the demonstration of a homozygous deletion of CDKN2A/B is generally worse than that of patients with G3 oligodendroglioma classified on the basis of histological features alone without a demonstrable CDKN2A/B homozygous deletion; however, this alteration occurs in less than 10% of cases [20]. The median overall survival of G2 oligodendroglioma is 16.6 years, which is the highest among all adult diffuse gliomas [1].

Glioblastoma IDH-wildtype (WHO G4)

Glioblastoma (GBM) is the most common primary adult CNS tumour, accounting for nearly 60% of all gliomas. It is characterized by supratentorial localization, with the most frequent occurrence in the frontal lobes of the brain. GBM is an adult-only tumour and reaches its highest incidence in the 75-84 age group [1]. GBM is characterized by rapid infiltrative growth in the CNS underlying clinical symptomatology often expressed by symptoms of intracranial hypertension. In contrast, distant metastases of GBM are rarely diagnosed clinically and are more often detected postmortem [24,25]. Median survival of patients is around 16 months, despite complex surgical and subsequent oncological therapy [26,27].

The diagnostic criteria for GBM have recently been expanded according to the cIMPACT NOW update 3 and 6 recommendations to include genetic features defining GBM even in the absence of classical morphological features, i.e. necrosis and microvascular proliferations (Figure 6) [5,10]. Thus, according to current recommendations, the genetic features of GBM - TERT promoter mutations, EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10 - must be investigated in all histologically G2/3 IDH-wildtype diffuse gliomas (Table 6) [16]. Based on the identical clinical presentation, disease course, and identically poor prognosis, newly diffuse IDH-wildtype gliomas with genetic features of GBM are classified as GBM [28,29].

According to Zhang et al. [30], GBMs defined by genetic alterations are either under-investigated or early detected/emerging GBMs. In the first group of underscreened GBMs, the failure to detect classical morphological features is due to examination of the tumour margin, outside areas with typical necrosis and vascular proliferations. The second group of early detected/evolving GBMs includes patients in whom the tumour was resected before it "matured" into the characteristic GBM morphology both on imaging and microscopically. However, during the course of the disease, these patients develop recurrences with an already expressed characteristic features on imaging and typical microscopic findings. Classification of GBM based on genetic features led to a more aggressive therapeutic approach and a significant prolongation of patient survival (median survival 23.8 months) in a prospective study by Zhang et al [30] compared to a retrospective control cohort classified as WHO G2 or G3 tumours according to the 2016 WHO CNS, with a median survival of only 16.2 months.

Unlike IDH-mutated gliomas, MGMT promoter methylation should be tested in all GBMs. Temozolomide monotherapy is effective only in patients with MGMT promoter methylation [16]. GBM patients with MGMT promoter methylation have a longer median survival (24 months) compared to patients without MGMT promoter methylation (14 months) [31-33], thus it is both a significant prognostic and predictive marker. In the case of IDH-mutated gliomas, routine testing is not recommended. Some papers have reported that up to 98% of IDH-mutated gliomas have methylation of the MGMT promoter [16]. However, a retrospective analysis of the methylation status of MGMT in IDH-mutated gliomas from 2022 reported a methylation frequency of only 62.3%. The significance of MGMT methylation for IDH-mutated gliomas is also unclear. The aforementioned paper reported that the presence of MGMT promoter methylation is associated with better survival outcomes for IDH-mutated higher-grade gliomas [34].

Conclusion of the integrated diagnostics

Recommendations for the formulation of the diagnosis according to WHO CNS 5 and cIMPACT-NOW are based on the 2014 International Society for Neuro-Oncology consensus recommendation preferring a structured form that includes the final diagnosis according to the integrated diagnosis, histological diagnosis, grading and a list of molecular genetic tests performed including their interpretation and the method used (Table 7) [2,3,5,35].

If molecular genetic tests cannot be performed or their results are not interpretable, it is recommended to classify the tumour based on the morphological diagnosis with the NOS (not otherwise specified) attribute - e.g. high-grade diffuse astrocytic glioma NOS. On the other hand, if even comprehensive molecular genetic examination does not allow to classify the tumour into the defined unit of adult or paediatric-type diffuse glioma according to WHO CNS 5, the tumour is labelled with the NEC (not elsewhere classified) attribute - e.g. diffuse astrocytoma IDH-wildtype, H3-wildtype, NEC [6].

Conclusion

Diagnosis of diffuse gliomas is a constantly evolving process, which is currently undergoing significant changes due to the rapid development and adoption of new methods enabling the study of the molecular genetic and epigenetic background of tumours [36]. It is the study of these changes that has led to the stratification of tumours into groups with a more homogeneous prognosis or therapeutic response, and these findings are reflected in the WHO CNS 2021 classification of gliomas.

Grant support

This work was supported by the Masaryk University Grant Agency (MUNI/A/1379/2022) and the Health Research Agency of the Ministry of Health (NU23-03-00100).

Declaration of Conflict of Interest

The authors declare that they have no conflict of interest in relation to the subject of the paper.

Tables

Table 1. Overview of diffuse gliomas according to WHO 2021 incl. WHO CNS grad assigned to each diagnostic unit and diagnostic genetic alterations.

Overview of diffuse gliomas according to WHO 2021	Grade	Molecular genetic diagnostic features
Adult-type diffuse gliomas
astrocytoma, IDH-mutated	2-4	IDH1/2 mutation, ATRX alteration, TP53 alteration, homozygous deletion CDKN2A/B
oligodendroglioma, IDH-mutated with 1p/19q deletion	2-3	IDH1/2 mutation, 1p/19q deletion, homozygous CDKN2A/B deletion, TERT promoter mutation
glioblastoma, IDH-wildtype	4	EGFR amplification, TERT promoter mutation, CNA +7/-10

1p/19q - combined loss of the short arm of chromosome 1 and loss of the long arm of chromosome 19; ATRX - alpha thalassemia/mental retardation syndrome X-linked; CDKN2A/B - cyclin-dependent kinase inhibitor 2A and 2B; CNA +7/-10 - copy number alteration - combined gain of chromosome 7 and loss of chromosome 10; EGFR - epidermal growth factor receptor; IDH - isocitrate dehydrogenase; TERT - telomerase reverse

Table 2. Diagnostic criteria for IDH-mutated astrocytoma according to WHO.

Home	diffusely growing glioma
	gene mutations
	ARTX gene mutation/ loss of ARTX expression and/or absence of the 1p/19q codon
Supporting	TP53 gene mutations/ strong nuclear expression of p53 in >10% of tumour cells
	methylation profile corresponding to IDH-mutated astrocytoma
	morphologically astrocytic differentiation

1p/19q - combined loss of the short arm of chromosome 1 and loss of the long arm of chromosome 19; ATRX - alpha thalassemia/mental retardation syndrome X-linked; IDH1/2 - isocitrate dehydrogenase 1/2; TP53 - tumor protein p53

Table 3. Grading criteria for IDH-mutated astrocytoma.

Grade 2

failure to meet diagnostic criteria for higher grade

Grade 3

significant mitotic activity defined as

1 or more mitotic figures in stereotatic biopsy
2 or more mitoses/10 HPF (2.4 mm²) in the resection

Grade 4

necrosis and/or vascular proliferation and/or homozygous deletion of CDKN2A/B

CDKN2A/B - cyclin-dependent kinase inhibitor 2A and 2B; HPF - high magnification field of view

Table 4. WHO diagnostic criteria for IDH-mutated oligodendroglioma with 1p/19q deletion.

Home	diffusely growing glioma
	mutations in genes
	codelece 1p/19q
Supporting	methylation profile corresponding to IDH-mutated oligodendroglioma with 1p/19q nucleotide
	absence of ARTX gene mutation/ preserved ARTX expression
	TERT promoter mutations

Table 5: Grading criteria for IDH-mutated oligodendroglioma with 1p/19q deletion.

Grade 2	failure to meet diagnostic criteria for higher grade
Grade 3	Mitotic activity - 6 or more mitoses/10HPF (> 2.5 mitoses/mm²) and/or necrosis and/or vascular proliferation and/or homozygous deletion of CDKN2A/B

CDKN2A/B - cyclin-dependent kinase inhibitor 2A and 2B; HPF - high magnification field of view

Table 6.Diagnostic criteria for glioblastoma IDH-wildtype according to WHO.

Home	diffusely growing glioma
	absence of mutations in IDH1/2 and histone 3 genes (H3F3A or HIST1H3B/C)
	one or more morphological or genetic traits: necrosis microvascular proliferate genetic features of glioblastoma: TERT promoter mutations EGFR amplification combined gain of chromosome 7 and loss of chromosome 10 (CNA +7/-10)
Supporting	Methylation profile corresponding to IDH-wildtype glioblastoma

CNA +7/-10 - copy number alteration - combined gain of chromosome 7 and loss of chromosome 10; EGFR - Epidermal growth factor receptor; H3F3A - H3 histone, family 3A; HIST1H3B/C - histone cluster 1 H3 family member b/c; IDH1/2 - isocitrate dehydrogenase 1/2; TERT - telomerase reverse

Table 7: Example of bioptic conclusion of integrated diagnostics

Integrated diagnosis	IDH-mutated astrocytoma
Histological diagnosis	astrocytoma
WHO CNS grade	4
Molecular information	IDH1 R132H mt (IHC), ATRX mt (IHC), p53 mt (IHC), homozygous deletion of CDKN2A/B (FISH)

ATRX, alpha thalassemia/mental retardation syndrome X-linked; CDKN2A/B, cyclin-dependent kinase inhibitor 2A and 2B; IDH1, isocitrate dehydrogenase 1

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