# Improved prognostic stratification of patients with isocitrate dehydrogenase-mutant astrocytoma

Michael Weller, Jörg Felsberg, Bettina Hentschel, Dorothee Gramatzki, Nadezhda Kubon, Marietta Wolter, Matthias Reusche, Patrick Roth, Dietmar Krex, Ulrich Herrlinger, et al.

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## To cite this version:

Michael Weller, Jörg Felsberg, Bettina Hentschel, Dorothee Gramatzki, Nadezhda Kubon, et al. Improved prognostic stratification of patients with isocitrate dehydrogenase-mutant astrocytoma. Acta Neuropathologica, 2024, Acta Neuropathologica, 147, pp.11. 10.1007/s00401-023-02662-1. hal-04971056

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# Improved prognostic stratification of patients with isocitrate dehydrogenase-mutant astrocytoma

Michael Weller¹,² ID · Jörg Felsberg³ · Bettina Hentschel⁴ · Dorothee Gramatzki¹ · Nadezhda Kubon³ ·

Marietta Wolter³ · Matthias Reusche⁴ · Patrick Roth¹,² · Dietmar Krex⁵ · Ulrich Herrlinger⁶ · Manfred Westphal⁷ ·

Joerg C. Tonn⁸,⁹ · Luca Regli¹⁰,¹¹ · Claude-Alain Maurage¹² · Andreas von Deimling¹³,¹⁴ · Torsten Pietsch¹⁵ ·

Emilie Le Rhun¹,²,¹⁰,¹¹,¹⁶ · Guido Reifenberger³,¹⁷

Received: 16 September 2023 / Revised: 19 November 2023 / Accepted: 20 November 2023
© The Author(s) 2024

## Abstract

Prognostic factors and standards of care for astrocytoma, isocitrate dehydrogenase (IDH)-mutant, CNS WHO grade 4, remain poorly defined. Here we sought to explore disease characteristics, prognostic markers, and outcome in patients with this newly defined tumor type. We determined molecular biomarkers and assembled clinical and outcome data in patients with IDH-mutant astrocytomas confirmed by central pathology review. Patients were identified in the German Glioma Network cohort study; additional cohorts of patients with CNS WHO grade 4 tumors were identified retrospectively at two sites. In total, 258 patients with IDH-mutant astrocytomas (114 CNS WHO grade 2, 73 CNS WHO grade 3, 71 CNS WHO grade 4) were studied. The median age at diagnosis was similar for all grades. Karnofsky performance status at diagnosis inversely correlated with CNS WHO grade (p < 0.001). Despite more intensive treatment upfront with higher grade, CNS WHO grade was strongly prognostic: median overall survival was not reached for grade 2 (median follow-up 10.4 years), 8.1 years (95% CI 5.4–10.8) for grade 3, and 4.7 years (95% CI 3.4–6.0) for grade 4. Among patients with CNS WHO grade 4 astrocytoma, median overall survival was 5.5 years (95% CI 4.3–6.7) without (n = 58) versus 1.8 years (95% CI 0–4.1) with (n = 12) homozygous CDKN2A deletion. Lower levels of global DNA methylation as detected by LINE-1 methylation analysis were strongly associated with CNS WHO grade 4 (p < 0.001) and poor outcome. MGMT promoter methylation status was not prognostic for overall survival. Histomolecular stratification based on CNS WHO grade, LINE-1 methylation level, and CDKN2A status revealed four subgroups of patients with significantly different outcomes. In conclusion, CNS WHO grade, global DNA methylation status, and CDKN2A homozygous deletion are prognostic in patients with IDH-mutant astrocytoma. Combination of these parameters allows for improved prediction of outcome. These data aid in designing upcoming trials using IDH inhibitors.

**Keywords** Brain · CDKN2A · CNS WHO grade · IDH · LINE-1 · Molecular

## Abbreviations

CI Confidence interval

CNS Central nervous system

GGN German Glioma Network

HR Hazard ratio

IDH Isocitrate dehydrogenase

KPS Karnofsky performance status

LINE-1 Long interspersed nuclear element 1

MGMT O⁶-methylguanine-DNA methyltransferase

OS Overall survival

PFS Progression-free survival

RT Radiotherapy

TMZ Temozolomide

WHO World Health Organization

## Introduction

The 2016 revision of the World Health Organization (WHO) classification of tumors of the central nervous system (CNS) had placed major emphasis on the isocitrate dehydrogenase (IDH) mutation status when classifying diffuse gliomas in adults [29]. Patients with diffuse gliomas with seemingly similar histology had very different outcomes when stratified for IDH mutation status [2–4, 8, 24, 52]. However, the diagnostic separation of adult-type diffuse astrocytic gliomas

<footnote>Extended author information available on the last page of the article</footnote>
into IDH-mutant and IDH-wildtype tumors has generated
new challenges regarding the role of grading and molecular
prognosticators within these newly defined tumor types [7].
Detection of an IDH mutation in a diffuse astrocytic glioma
with microvascular proliferation or necrosis is no longer com-
patible with a glioblastoma diagnosis, i.e., such tumors are
now diagnosed as astrocytoma, IDH-mutant, CNS WHO grade
4 [7, 30]. Moreover, homozygous deletion of the CDKN2A/
CDKN2B tumor suppressor gene locus has been introduced
as a molecular biomarker for CNS WHO grade 4 in an IDH-
mutant astrocytoma [30, 41]. Other molecular alterations that
have been associated with aggressive behavior and shorter
survival include high tumor mutational burden and increased
copy number variation load, as well as various aberrations
affecting single genes or chromosomes, such as type of IDH
mutation, PIK3R1 mutation, PDGFRA amplification, copy
number neutral loss of 17p, loss of 19q, and others [42]. In
addition, reduced global DNA methylation, referred to as
glioma CpG island methylator phenotype low (gCIMPlow),
has been associated with worse outcome [11, 41]. Detection
of the global DNA methylation status can be accomplished by
microarray- or sequencing-based methylome analyses [9, 11,
31], with focused methylation analysis of the LINE-1 repeti-
tive element being reported as a valuable surrogate marker for
global DNA methylation level assessment [57].

Histological grading of IDH-mutant astrocytomas is subject to inter-observer variability [29] and its prognostic relevance is a matter of ongoing discussion. While some studies questioned the prognostic role of histological grading, others showed distinct outcomes according to tumor grade (for review see [7]). Further, whether patients diagnosed with IDH-mutant astrocytoma, CNS WHO grade 4, should be treated like IDH-wildtype glioblastoma patients or rather like patients with IDH-mutant astrocytoma, CNS WHO grade 3, remains controversial [47]. The IDH mutation has recently gained clinical importance as a therapeutic target since vorasidenib, an oral brain-penetrant inhibitor of mutant IDH1 and IDH2 enzymes, significantly improved progression-free survival in patients with CNS WHO grade 2 IDH-mutant gliomas [32].

To further define the prognostic roles of clinical features,
CNS WHO grade, and selected molecular biomarkers in
IDH-mutant astrocytoma patients, we assembled a large,
clinically well documented patient cohort with long-term
follow-up data from the German Glioma Network (GGN)
and two institutional cohorts.

**Patients and methods**

Patients

Patients were enrolled in the GGN (n = 212) or followed at
the University Hospitals of Lille, France (n = 32) or Zurich,

Switzerland (n = 14). The GGN is a prospective, non-inter-
ventional cohort study that comprised eight University Hos-
pitals in Germany. All GGN patients gave written informed
consent for participation in the GGN and its translational
research projects. Local ethics approvals were in place in
Lille and Zurich. Patient characteristics, treatment, and out-
come data were collected prospectively within the GGN and
assembled retrospectively following a similar data capture
scheme for patients from Lille and Zurich.

**Central neuropathology review**

Representative tumor specimens from all patients were subjected to central pathology review at the Brain Tumor Reference Center of the German Society for Neuropathology and Neuroanatomy (DGNN) in Bonn (TP) and Düsseldorf (GR). In addition to histological confirmation of a diffuse astrocytic glioma, the tumors were histologically graded according to the World Health Organization (WHO) classification of central nervous system (CNS) tumors [30]. Accordingly, CNS WHO grade 3 tumors were distinguished from CNS WHO grade 2 tumors by the presence of focal or dispersed anaplasia and significant mitotic activity, while CNS WHO grade 4 tumors were distinguished from the CNS WHO grade 2 and 3 tumors by the presence of microvascular proliferation and/or necrosis and/or homozygous CDKN2A/CDKN2B deletion [30]. All tumors were screened for the IDH1-R132H mutation using immunohistochemistry with a mutation-specific monoclonal antibody (clone H09, Dianova, Hamburg, Germany) [10]. Tumors negative for IDH1-R132H by immunohistochemistry were assessed for non-canonical *IDH1* or *IDH2* mutations using Sanger sequencing or pyrosequencing [15, 21, 22]. For the molecular analyses, DNA was extracted from frozen tissue samples using the PureLink™ Genomic DNA Mini Kit (Life Technologies, Carlsbad, CA) or ultracentrifugation [23]. Alternatively, DNA was extracted from formalin-fixed and paraffin-embedded tissue samples using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), the GeneRead DNA FFPE Kit (Qiagen), or the Maxwell® RSC FFPE Plus DNA Kit together with the Maxwell® RSC instrument (Promega, Mannheim, Germany). Tumor tissue samples used for DNA extraction were histologically evaluated to contain a sufficient tumor cell content of usually more than 80%. In four tumors (1 CNS WHO grade 2 and 3 CNS WHO grade 4 tumors), classification as IDH-mutant astrocytoma was based only on array-based DNA methylome analysis using the Heidelberg classifier version v.12.5 (https://www.molecularneuropathology.org/mnp/) without further specification of the specific *IDH1* or *IDH2* mutation by DNA sequencing (Table 1).

In addition to IDH mutation testing, tumors were
investigated for 1p/19q codeletion status using either
microsatellite-based loss of heterozygosity (LOH) analysis [13, 58] or comparative genomic hybridization [51]. In individual cases, the 1p/19q codeletion status was determined based on copy number profiles obtained by array-based

DNA methylome analysis. None of the 258 tumors demonstrated a 1p/19q codeletion.

The *MGMT* promoter methylation status was determined by methylation-specific PCR or DNA pyrosequencing [14,

<table><caption>**Table 1** Patient and disease characteristics by CNS WHO grade</caption><thead><tr><th></th><th>Astrocytoma, IDH-mutant, CNS<br>WHO grade 2<br>n = 114</th><th>Astrocytoma, IDH-mutant, CNS<br>WHO grade 3<br>n = 73</th><th>Astrocytoma, IDH-mutant, CNS WHO<br>grade 4<br>n = 71</th></tr></thead><tbody><tr><td><strong>Age at diagnosis</strong></td><td></td><td></td><td></td></tr><tr><td>Median (years)</td><td>36</td><td>39</td><td>37</td></tr><tr><td>Range (years)</td><td>19–69</td><td>21–80</td><td>23–79</td></tr><tr><td><strong>Sex</strong></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>78 (68.4%)</td><td>46 (63.0%)</td><td>47 (66.2%)</td></tr><tr><td>Female</td><td>36 (31.6%)</td><td>27 (37.0%)</td><td>24 (33.8%)</td></tr><tr><td><strong>KPS at diagnosis</strong></td><td></td><td></td><td></td></tr><tr><td>90–100</td><td>71 (86.6%)</td><td>39 (70.9%)</td><td>33 (47.1%)</td></tr><tr><td>70–80</td><td>11 (13.4%)</td><td>14 (25.5%)</td><td>25 (35.7%)</td></tr><tr><td>&lt;70</td><td>0</td><td>2 (3.6%)</td><td>12 (17.1%)</td></tr><tr><td>No data</td><td>32</td><td>18</td><td>1</td></tr><tr><td><strong>Tumor location</strong></td><td></td><td></td><td></td></tr><tr><td>Frontal</td><td>41 (36.0%)</td><td>27 (37.0%)</td><td>36 (50.7%)</td></tr><tr><td>Temporal</td><td>25 (21.9%)</td><td>16 (21.9%)</td><td>5 (7.0%)</td></tr><tr><td>Parietal</td><td>9 (7.9%)</td><td>4 (5.5%)</td><td>7 (9.9%)</td></tr><tr><td>Cerebellar, brain stem</td><td>0</td><td>0</td><td>2 (2.8%)</td></tr><tr><td>Not localized to one site</td><td>25 (21.9%)</td><td>18 (24.7%)</td><td>20 (28.2%)</td></tr><tr><td>Multifocal</td><td>1 (0.9%)</td><td>0</td><td>0</td></tr><tr><td>Others</td><td>12 (10.5%)</td><td>8 (11.0%)</td><td>1 (1.4%)</td></tr><tr><td>Unknown</td><td>1 (0.9%)</td><td>0</td><td>0</td></tr><tr><td><strong>IDH mutation status</strong></td><td></td><td></td><td></td></tr><tr><td>IDH1<sup>R132H</sup> mutation</td><td>101 (88.6%)</td><td>66 (90.4%)</td><td>62 (87.3%)</td></tr><tr><td>Other IDH1 mutations</td><td>11 (9.6%)<sup>a</sup></td><td>6 (8.2%)<sup>b</sup></td><td>5 (7.0%)<sup>c</sup></td></tr><tr><td>IDH2<sup>R172K</sup> mutation</td><td>1 (0.9%)</td><td>1 (1.4%)</td><td>1 (1.4%)</td></tr><tr><td>IDH mutation type not determined<sup>d</sup></td><td>1 (0.9%)</td><td>0</td><td>3 (4.2%)</td></tr><tr><td><strong>MGMT</strong> promoter methylation status</td><td></td><td></td><td></td></tr><tr><td>Methylated</td><td>72 (65.5%)</td><td>58 (84.1%)</td><td>53 (74.6%)</td></tr><tr><td>Unmethylated</td><td>38 (34.5%)</td><td>11 (15.9%)</td><td>18 (25.4%)</td></tr><tr><td>No data</td><td>4</td><td>4</td><td>0</td></tr><tr><td><strong>CDKN2A</strong> deletion status</td><td></td><td></td><td></td></tr><tr><td>Homozygous deletion</td><td>0</td><td>0</td><td>12 (17.1%)</td></tr><tr><td>No homozygous deletion</td><td>66 (100%)</td><td>61 (100%)</td><td>58 (82.9%)</td></tr><tr><td>No data</td><td>48</td><td>12</td><td>1</td></tr><tr><td><strong>LINE-1</strong> methylation status</td><td></td><td></td><td></td></tr><tr><td>Methylated alleles (%, median)</td><td>83.0</td><td>82.7</td><td>72.8</td></tr><tr><td>Methylated alleles (%, range)</td><td>74.2–86.0</td><td>71.2–86.3</td><td>60.0–79.1</td></tr><tr><td>No data</td><td>62</td><td>15</td><td>1</td></tr></tbody></table>

CI confidence interval, KPS Karnofsky performance status

<footnote><sup>a</sup>Other IDH1 mutations are R132C (n = 4), R132G (n = 3), R132L (n = 2), R132S (n = 2)</footnote>

<footnote><sup>b</sup>Other IDH1 mutations are R132C (n = 3), R132G (n = 2), R132L (n = 1)</footnote>

<footnote><sup>c</sup>Other IDH1 mutations are R132C (n = 2), R132G (n = 1), R132S (n = 2)</footnote>

<footnote><sup>d</sup>Assignment to methylation class astrocytoma, IDH-mutant based on DNA methylome analysis</footnote>
[46] or, in individual cases, by DNA methylome analysis using the STP27 algorithm [1]. A total of 173 of the 212 GGN cases had been included in previous GGN studies [5, 6, 19, 20, 22, 27, 36, 37, 39, 40, 48–51, 53].

## Detection of homozygous deletion of CDKN2A by droplet digital PCR (ddPCR)

We used a commercially available ddPCR assay (Bio-Rad Laboratories) for the detection of CDKN2A homozygous deletions on 9p21 [55]. The loci NCKAP5 and KCNS3 (2p24.2) served as reference loci. The threshold for detection of a homozygous deletion was set to a calculated relative CDKN2A copy number value of <0.5 which was experimentally demonstrated to reliably detect a homozygous deletion when the tumor cell content in the tissue sample used for DNA extraction was ≥75% [55].

## Determination of LINE-1 methylation by pyrosequencing

As a surrogate marker for the global DNA methylation status [57], we determined the methylation level of the LINE-1 repetitive element (GenBank accession number X58075) in the tumor DNA using DNA pyrosequencing of sodium bisulfite converted DNA. The primer pair LINE-1-bisu-F1 5'-taggattttttgagttaggtgtg and LINE-1-bisu-R1 5'-[Bt n] caaaaaatcaaaaaattccctttcc (biotinylated at the 5' -end) was used for amplification of a 156-bp fragment. Pyrosequencing on the PyroMark Q24 (Qiagen, Hilden, Germany) was performed using the sequencing primer LINE-1-bisu-Seq1 5' -taggtgtgggatatagt with the sequence to analyze being "TTY GTGGTGYGTYGTTT". The three investigated CpG sites correspond to the first three CpGs covered by the PyroMark Q96 CpG LINE-1 kit from Qiagen. After pyrosequencing, we calculated the mean value of the methylated allele percentages at the three investigated CpG sites. A ROC analysis was performed to determine an appropriate cut-off value for the LINE-1 methylation levels, i.e., percentage of methylated alleles that distinguished best between CNS WHO grade 4 tumors as opposed to CNS WHO grade 2 or 3 tumors. Thereby, a cut-off value of ≤77% methylated alleles was calculated with an area under the curve (AUC) of 0.98.

## Array-based DNA methylation analyses

Large-scale DNA methylation data obtained by hybridization of tumor DNA to 450 k methylation bead arrays (Illumina, San Diego, CA) were available for 85 patients with IDH-mutant astrocytoma included in this study, comprising 31 CNS WHO grade 2, 31 CNS WHO grade 3, and 23 CNS WHO grade 4 tumors. LINE-1 methylation data were available from 80 of these tumors (29 grade 2, 28 grade 3, and

23 grade 4). 450 k DNA methylation data were generated as described [9] and analyzed with the Heidelberg classifier algorithm version v.12.5 (www.molecularneuropathology.org). Tumors were assigned to the methylation classes "astrocytoma, IDH-mutant, lower grade" or "astrocytoma, IDH-mutant, high-grade" based on calibrated classifier scores of ≥0.9. Principles of the DNA methylation-based classification of central nervous system tumors, the distinction of methylation classes, and the role of calibrated classifier scores have been reported [9].

## Statistical analyses

Progression-free survival (PFS) was calculated from the day of first surgery until tumor progression, death, or end of follow-up. Overall survival (OS) was calculated from the day of first surgery until death or end of follow-up. Kaplan-Meier survival curves, Log-rank test, and Cox regression were used for univariate and multivariate analyses of survival. Chi²-test and Fisher's exact test were used to analyze categorical data. Quantitative data were analyzed by t test and Mann-Whitney U test. A ROC analysis was performed to determine an appropriate cut-off value for the percentage of LINE-1 methylated alleles. Sensitivity and specificity with 95% confidence interval (CI) were calculated.

## Results

### Patient characteristics

The median age was below 40 years for all CNS WHO grades. Less than half (47.1%) of the patients with CNS WHO grade 4 tumors had a KPS 90 or 100, as opposed to 86.6 and 70.9% of the patients with CNS WHO grade 2 or 3 tumors (p < 0.001). IDH-mutant astrocytomas of CNS WHO grade 4 were numerically more often located in the frontal lobes (p = 0.118) (Table 1), and a gross total resection was numerically more often performed in these patients than in patients with CNS WHO grade 2 or 3 tumors (p = 0.177) (Table S1).

### Molecular characteristics

The canonical IDH-R132H mutation was detected in almost 90% of the tumors with equal frequencies across grades. MGMT promoter methylation was detected in more than 70% of all tumors, with the lowest percentage of 65.5% detected in CNS WHO grade 2 tumors (Table 1). CDKN2A homozygous deletions were detected in 12 of 71 patients (16.9%) with CNS WHO grade 4 tumors. Eleven of these tumors also showed histological features of CNS WHO grade 4, i.e., microvascular proliferation or necrosis or both. The LINE-1

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<figure><img src="image_9.png" /><figcaption>**Fig. 1** Outcome of patients with IDH-mutant astrocytoma stratified according to CNS WHO grade and CDKN2A copy number status. (a, b) PFS (a) and OS (b) of the entire cohort of patients with IDH-mutant astrocytomas stratified by CNS WHO grade 2, 3 or 4. (c–f)</figcaption></figure>

PFS (c, e) and OS (d, f) of the entire cohort of patients with IDH-mutant astrocytoma (c, d) or IDH-mutant astrocytoma of CNS WHO grade 4 only (e, f)

levels were significantly lower in tumors assigned to the methylation class “astrocytoma, IDH-mutant, high-grade” (Fig. S5a). The previously determined *LINE-1* cut-off value of ≤77% methylated alleles was applied to assess its value to discriminate the two methylation classes. Overall, sensitivity for detection of the “astrocytoma, IDH mutant, high grade” methylation class by a *LINE-1* methylation level of ≤77% was 94.7% (95% CI 74.0–99.9%) and specificity was 90.2% (95% CI 79.8–96.3%). Among 23 CNS WHO grade 4 tumors with available 450 k DNA methylome data, a similar trend of lower *LINE-1* methylation levels in “astrocytoma, IDH-mutant, high-grade” tumors was observed, albeit the *p*

value remained insignificant likely due to the low number of tumors assigned to the methylation class “astrocytoma, IDH-mutant, lower grade” among the CNS WHO grade 4 tumors (Fig. S5b).

## Prognostic factor analyses

Univariate analyses over the entire cohort revealed that CNS WHO grade, age, KPS, extent of resection, CDKN2A deletion, and *LINE-1* methylation level were prognostic. *MGMT* promoter methylation was prognostic, too, but with a better outcome for patients with tumors lacking *MGMT* promoter

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**Fig. 2** LINE-1 methylation levels in IDH-mutant astrocytomas. LINE-1 methylation levels according to CNS WHO grade 2 (n = 62), CNS WHO grade 3 (n = 58), and CNS WHO grade 4 (n = 70) (a). LINE-1 methylation levels according to CDKN2A deletion status based on 11 tumors with homozygous CDKN2A deletion and 178 tumors without

this alteration (b). PFS (c) and OS (d) of the entire patient cohort according to LINE-1 methylation level stratified into ≤77% methylated alleles versus >77% methylated alleles. PFS (e) and OS (f) of the patients with CNS WHO grade 4 tumors according to LINE-1 methylation levels

methylation (see above). On multivariate analysis, CNS WHO grade 4 extent of resection, and CDKN2A deletion were retained as prognostic factors (Table 2). Similar results were obtained when the same analyses were restricted to patients with IDH-mutant astrocytoma, CNS WHO grade 4 (Table S3).

## Combination of CNS WHO grade and molecular biomarkers for improved prediction of outcome

Next we explored whether our findings might provide a new approach for improved prognostic assessment of patients with IDH-mutant astrocytoma. Stratification according to

LINE-1 methylation levels and CDKN2A copy number status resulted in three groups of patients with distinct overall survival (Fig. 3a). Group assignment remained highly significant upon adjustment for other prognostic factors (Table S4). Since CDKN2A homozygous deletion was a profound negative prognostic factor in our cohort (Figs. 1c–f), we also explored the prognostic significance of the current WHO classification when patients with CDKN2A homozygously deleted tumors were excluded. This resulted in a less distinct separation of outcome of CNS WHO grade 3 and 4 tumors (Fig. 3b). Yet, in tumors without homozygous CDKN2A deletion, lower LINE-1 methylation levels were highly associated with CNS WHO grade 4 (Figs. S6a, b).
<table><caption>**Table 2** Prognostic factors in IDH-mutant astrocytoma: univariate and multivariate analyses</caption><thead><tr><th>Entire cohort, univariate analyses</th><th>Hazard ratio</th><th>p value</th><th>95% CI</th></tr></thead><tbody><tr><td>CNS WHO grade</td><td></td><td></td><td></td></tr><tr><td>2 (ref)</td><td>–</td><td></td><td></td></tr><tr><td>3</td><td>2.35</td><td>&lt;0.001</td><td>1.47–3.76</td></tr><tr><td>4</td><td>4.14</td><td>&lt;0.001</td><td>2.60–6.57</td></tr><tr><td>Age (years)</td><td></td><td></td><td></td></tr><tr><td>&gt; 40 versus ≤ 40 (ref.)</td><td>1.61</td><td>0.013</td><td>1.10–2.34</td></tr><tr><td>KPS</td><td></td><td></td><td></td></tr><tr><td>&lt;80 versus ≥80 (ref.)</td><td>2.55</td><td>&lt;0.001</td><td>1.53–4.25</td></tr><tr><td>Surgery</td><td></td><td></td><td></td></tr><tr><td>No total versus total (ref.)</td><td>1.73</td><td>0.017</td><td>1.10–2.73</td></tr><tr><td>MGMT promoter status</td><td></td><td></td><td></td></tr><tr><td>Methylated versus unmethylated (ref.)</td><td>1.64</td><td>0.043</td><td>1.01–2.64</td></tr><tr><td>IDH mutation status</td><td></td><td></td><td></td></tr><tr><td>IDH1-R132H versus other IDH1 or IDH2 mutations (ref.)</td><td>1.66</td><td>0.169</td><td>0.81–3.40</td></tr><tr><td>CDKN2A deletion status</td><td></td><td></td><td></td></tr><tr><td>Homozygous versus no homozygous (ref.)</td><td>6.04</td><td>&lt;0.001</td><td>2.90–12.56</td></tr><tr><td>LINE-1 methylation status</td><td></td><td></td><td></td></tr><tr><td>≤77 versus &gt;77% methylated alleles (ref.)</td><td>3.54</td><td>&lt;0.001</td><td>2.25–5.56</td></tr><tr><td>Entire cohort, multivariate analyses</td><td>Hazard ratio</td><td>p value</td><td>95% CI</td></tr><tr><td>CNS WHO grade</td><td></td><td></td><td></td></tr><tr><td>2 (ref)</td><td>–</td><td></td><td></td></tr><tr><td>3</td><td>3.09</td><td>0.007</td><td>1.36–7.00</td></tr><tr><td>4</td><td>1.42</td><td>0.577</td><td>0.42–4.80</td></tr><tr><td>Age (years)</td><td></td><td></td><td></td></tr><tr><td>&gt; 40 versus ≤ 40 (ref.)</td><td>1.58</td><td>0.098</td><td>0.92–2.72</td></tr><tr><td>KPS</td><td></td><td></td><td></td></tr><tr><td>&lt;80 versus ≥80 (ref.)</td><td>1.17</td><td>0.627</td><td>0.621–2.21</td></tr><tr><td>Surgery</td><td></td><td></td><td></td></tr><tr><td>No total versus total (ref.)</td><td>2.61</td><td>&lt;0.001</td><td>1.49–4.58</td></tr><tr><td>MGMT promoter status</td><td></td><td></td><td></td></tr><tr><td>Methylated versus unmethylated (ref.)</td><td>1.03</td><td>0.936</td><td>0.54–1.96</td></tr><tr><td>IDH mutations status</td><td></td><td></td><td></td></tr><tr><td>IDH1-R132H versus other IDH1 or IDH2 mutations (ref.)</td><td>1.85</td><td>0.165</td><td>0.78–4.39</td></tr><tr><td>CDKN2A deletion status</td><td></td><td></td><td></td></tr><tr><td>Homozygous versus no homozygous (ref.)</td><td>3.74</td><td>0.008</td><td>1.41–9.92</td></tr><tr><td>LINE-1 methylation status</td><td></td><td></td><td></td></tr><tr><td>≤77 versus &gt;77% methylated alleles (ref.)</td><td>3.39</td><td>0.025</td><td>1.17–9.85</td></tr></tbody></table>

Finally, placing *LINE-1* methylation levels at the apex of the prognostic stratification delineated four subgroups with relevant significant outcome differences (group 2 versus 1, HR = 2.99, *p* = 0.011; group 3 versus 2, HR = 1.68, *p* = 0.095; group 4 versus 3, HR = 3.51, *p* = 0.010).

In the group of patients whose tumors showed high *LINE-1* methylation levels (>77% methylated alleles), outcome was significantly different for patients with CNS WHO grade 2 tumors versus patients with CNS

WHO grade 3 or (rare) grade 4 tumors. In the group of patients whose tumors showed low *LINE-1* methylation levels (≤77%), outcome was significantly different for patients with *CDKN2A* homozygously deleted tumors versus patients whose tumors had no homozygous *CDKN2A* deletion (Fig. 3c). The differences in outcome between the four groups were significant on univariate and multivariate analysis except for group 3 versus 2 (Table S5).

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Fig. 3** Prognostic stratification based on CNS WHO grade, *LINE-1* methylation level, and *CDKN2A* homozygous deletion status. (a) OS stratified by *LINE-1* methylation level (77% cut-off) and *CDKN2A* homozygous deletion status. The three distinct groups of patients correspond to: 1, *LINE-1* methylation levels of >77% without homozygous *CDKN2A* deletion; 2, *LINE-1* methylation levels of ≤77% without homozygous *CDKN2A* deletion; 3, *LINE-1* methylation levels of ≤77% with homozygous *CDKN2A* deletion). (b) OS by CNS WHO grade with omission of patients with *CDKN2A* homozygously deleted</figcaption></figure>

<figure><img src="image_20.png" /></figure>

tumors. (c) OS stratified based on *LINE-1* methylation levels followed by CNS WHO grade 2 versus CNS WHO grade 3 or 4 in tumors with high *LINE-1* methylation levels or followed by *CDKN2A* homozygous deletion status in tumors with low *LINE-1* methylation levels. The four distinct groups of patients correspond to: 1, *LINE-1* methylation levels of >77% and CNS WHO grade 2; 2, *LINE-1* methylation levels of >77% and CNS WHO grade 3 or 4; 3, *LINE-1* methylation levels of ≤77% without homozygous *CDKN2A* deletion; and 4, *LINE-1* methylation levels of ≤77% and homozygous *CDKN2A* deletion

Discussion

The present study provides contemporary data on the pat-
terns of presentation, treatment, and outcome in the newly
defined adult-type diffuse glioma group of IDH-mutant
astrocytomas [7, 30] with a particular focus on astrocy-
toma, IDH-mutant, CNS WHO grade 4. The grading of
IDH-mutant astrocytomas remains a matter of ongoing
controversy [7, 18, 30, 34, 38]. Here, we report that strati-
fication of these tumors into CNS WHO grades 2, 3, or 4
as recently defined in the WHO 2021 classification [30] is
prognostically important (Fig. 1). Currently, the distinction
of CNS WHO grade 2 versus 3 according to the WHO clas-
sification 2021 relies on the presence of focal or dispersed
anaplasia and significant mitotic activity; however, a dis-
tinct cut-off for the mitotic count was not established [30].
A recent study based on patients included in the EORTC
trials 26053 (CATNON) and 22033–26033 supported a
prognostic role of mitotic activity and reported that a cut-
off of two mitoses per ten microscopic high power fields was

linked to significantly longer PFS and marginally longer OS
in patients with IDH-mutant astrocytoma without homozy-
gous CDKN2A/CDKN2B deletion [28]. Another recent study
reported that the combination of <6 mitoses per 3 mm² and
a residual tumor volume of <1 cm² upon postsurgical imag-
ing was indicative of longer time to treatment and overall
survival in patients with IDH-mutant astrocytomas of CNS
WHO grade 2 or 3 [45].

The majority of IDH-mutant astrocytomas, CNS WHO grade 4, present de novo, rather than arising from a pre-existing lower grade astrocytoma [26]. The similar age at diagnosis across the groups defined by CNS WHO grade reported here (Table 1) supports this notion. A recent analysis of pooled data from clinical trials suggested that the canonical R132H mutation may confer an inferior survival compared with the less common, non-canonical mutations in *IDH1* or mutations in *IDH2* [44]. This association was confirmed in a cohort from Italy [17] and a recent meta-analysis [12], while data from the French POLA cohort revealed no clear prognostic association by type of IDH mutation [35].
outcome into cohorts like the GGN and lack of standard-
ized approaches to treatment and follow-up. We also did
not quantitatively evaluate mitotic count as performed in
the recent studies by Kros et al. [28] and Tran et al. [45] that
reported on various cut-offs for mitotic counts predicting
outcome. In addition, the LINE-1 cut-off used in this study
would require independent validation in a distinct patient
cohort. Furthermore, potential diagnostic use in individual
patients would demand the establishment of a validated
assay that is standardized concerning, among others, the
definition of CpG sites to be interrogated, the method for
calculation of methylated allele frequencies, input amount
of DNA and completeness of bisulfite conversion, appro-
priate control samples, and the actual pyrosequencing
protocol. Nevertheless, the present cohort of patients with
IDH-mutant astrocytomas is relatively large and may serve
as a framework for further efforts aiming at characterizing
novel markers for improved prediction of therapy response
and outcome that could also guide treatment strategy and
clinical trial design, notably with the view to defining the
role of IDH inhibitors along the disease trajectory [32]. Fur-
thermore, we provide possible future avenues to improve
histomolecular prognostic assessment of IDH-mutant astro-
cytoma based on CNS WHO grade, global DNA methylation
level, and CDKN2A homozygous deletion (Fig. 3).

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00401-023-02662-1.

**Acknowledgements** The authors would like to thank the staff at the participating clinical centers of the German Glioma Network as well as in Lille, France and Zurich, Switzerland, and the Tumorothèque du C2RC de Lille for their support. Britta Friedensdorf and Heike Seul (Institute of Neuropathology, Heinrich Heine University and University Hospital Düsseldorf, Germany), as well as Ulrike Schoenwiese (Institute for Medical Informatics, Statistics and Epidemiology, University Leipzig, Germany) are acknowledged for excellent technical assistance. The German Glioma Network was supported by the German Cancer Aid (Deutsche Krebshilfe, grant number 70-3163-Wi 3).

**Funding** Open access funding provided by University of Zurich.

**Data availability** Anonymized data may be shared upon appropriate request of qualified investigators for purposes of replicating procedures and results.

Declarations

**Conflict of interest** MiW has received research grants from Quercis and Versameb, and honoraria for lectures or advisory board participation or consulting from Bayer, Curevac, Medac, Novartis, Novocure, Orbus, Philogen, Roche and Sandoz. PR has received honoraria for lectures or advisory board participation from Alexion, Bristol-Myers Squibb, Boehringer Ingelheim, Debiopharm, Midatech Pharma, Novocure, QED, and Roche and research support from Merck Sharp and Dohme and Novocure. DK received honoraria for advisory board participation from Novocure and Bayer and for lectures from Brainlab. UH has received lecture and/or advisory board honoraria from Medac and Bayer. MaW received honoraria for an advisory board from Dou-

bleBond. JCT has received research grants from novocure and Munich
Surgical Imaging and honoraria for lectures or consulting from Seagen
and AAA-Novartis. ELR has received research grants from Bristol
Meyers Squibb (BMS), and honoraria for lectures or advisory board
participation or consulting from Bayer, Janssen, Leo Pharma, Pierre
Fabre, Roche, Seattle Genetics, and Servier. JF, BH, DG, NK, MWO,
MR, LR, CAM, AvD, TP, and GR declare no conflicts of interest.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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## Authors and Affiliations

Michael Weller¹,² ID · Jörg Felsberg³ · Bettina Hentschel⁴ · Dorothee Gramatzki¹ · Nadezhda Kubon³ ·

Marietta Wolter³ · Matthias Reusche⁴ · Patrick Roth¹,² · Dietmar Krex⁵ · Ulrich Herrlinger⁶ · Manfred Westphal⁷ ·

Joerg C. Tonn⁸,⁹ · Luca Regli¹⁰,¹¹ · Claude-Alain Maurage¹² · Andreas von Deimling¹³,¹⁴ · Torsten Pietsch¹⁵ ·

Emilie Le Rhun¹,²,¹⁰,¹¹,¹⁶ · Guido Reifenberger³,¹⁷

<figure><img src="image_21.png" /></figure>

Michael Weller
michael.weller@usz.ch

1 Department of Neurology, University Hospital Zurich, Frauenklinikstrasse 26, 8091 Zurich, Switzerland

2 Department of Neurology, University of Zurich, Zurich, Switzerland

3 Institute of Neuropathology, Heinrich Heine University, Medical Faculty, and University Hospital Düsseldorf, Düsseldorf, Germany

4 Institute for Medical Informatics, Statistics and Epidemiology, University Leipzig, Leipzig, Germany

5 Faculty of Medicine, Department of Neurosurgery, Technische Universität Dresden, University Hospital Carl Gustav Carus, Dresden, Germany

6 Department of Neurology, University of Bonn, Bonn, Germany

7 Department of Neurosurgery, University of Hamburg, Hamburg, Germany

8 Department of Neurosurgery, Ludwig-Maximilians-University Munich, Munich, Germany

9 German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany

10 Department of Neurosurgery, University Hospital Zurich, Zurich, Switzerland

11 Department of Neurosurgery, University of Zurich, Zurich, Switzerland

12 Department of Pathology, Centre Biologie Pathologie, Lille University Hospital, Hopital Nord, Lille, France

13 Department of Neuropathology, University Hospital Heidelberg, Heidelberg, Germany

14 Clinical Cooperation Unit Neuropathology, German Cancer Center (DKFZ), and German Cancer Consortium (DKTK), Partner Site Heidelberg, Heidelberg, Germany

15 Department of Neuropathology, University of Bonn Medical Center, DGNN Brain Tumor Reference Center, Bonn, Germany

16 Department of Neurosurgery, Lille University Hospital, Lille, France

17 German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf, Düsseldorf, Germany

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