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Free Neuropathology 6:10 (2025) Michel Mittelbronn 
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Neurooncology: 2025 update 

Michel Mittelbronn1,2 

1 Department of Life Sciences and Medicine (DLSM); University of Luxembourg; Esch sur Alzette, Luxembourg 

2 Faculty of Science, Technology and Medicine (FSTM), University of Luxembourg, Esch-sur-Alzette, Luxembourg 

Corresponding author: 
Michel Mittelbronn · Department of Life Sciences and Medicine (DLSM) · University of Luxembourg · 6, avenue du Swing · L-4367 Belvaux 
· Luxembourg · michel.mittelbronn@uni.lu 

Submitted: 23 February 2025 · Accepted: 17 March 2025 · Copyedited by: João Gama · Published: 26 March 2025 

Abstract 

This collection of studies highlights groundbreaking advancements in brain tumor research, particularly primary 
CNS tumors and brain metastasis. One major focus is the tumor microenvironment, where alterations in cerebral 
microcirculation and hypoxic-ischemic conditions have been shown to influence metastatic progression. In  
glioblastoma, recurrent tumors exhibit distinct DNA methylation profiles, and global DNA methylation has 
emerged as an independent diagnostic marker for IDH-wildtype glioblastoma. A whole-tumor perspective further 
emphasizes the extensive intratumoral heterogeneity driving glioblastoma evolution. The immune landscape of 
glioblastoma is another key area of research. Cranioencephalic functional lymphoid units have been implicated 
in tumor progression, while time-dependent single-cell phenotyping offers novel insights into immune cell  
dynamics within glioblastoma. Additionally, histone serotonylation has been identified as a critical epigenetic 
regulator in ependymoma tumorigenesis. Diagnostic and prognostic innovations are paving the way for improved 
patient care. Histomorphological features provide enhanced prognosis prediction for glioblastoma patients.  
Confocal laser microscopy enables real-time intraoperative histopathological diagnostics, and sequencing of  
cerebrospinal fluid-derived cell-free DNA presents a promising non-invasive diagnostic approach. Together, 
these top studies of 2024 underscore the complexity of brain tumor biology and the integration of epigenetics, 
immune interactions, and advanced diagnostics into clinical practice. These insights mark significant progress 
toward personalized treatment strategies and improved outcomes in neurooncology. 
 

Keywords: Neurooncology, Neuropathology, Brain tumors, Glioblastoma, Brain metastasis 

Introduction 

Artificial intelligence is currently a hot topic 
and is intended to make work easier for humans. 
However, there are still numerous challenges in  
academic literature research, particularly due to  

hallucinations that lead to the generation of incon-
sistent or completely incorrect references [Albuck 
et al., 2024]. Nevertheless, the author of the current  
article wanted to give it a try to see whether his work 
could be simplified through the use of artificial intel-
ligence, or more specifically, large language models  
 

Review 

https://doi.org/10.17879/freeneuropathology-2025-6316
https://creativecommons.org/licenses/by/4.0/
https://creativecommons.org/publicdomain/zero/1.0/
mailto:michel.mittelbronn@uni.lu


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(LLMs), or at least gain some additional inspiration 
about interesting primary research articles in the 
field of neurooncology published in 2024. Therefore, 
the following very generic question was posed to 
three LLMs (https://chatgpt.com/; https://gem-
ini.google.com/app and https://www.perplexity.ai): 
‘Could you please provide me with the 10 most  
influential primary research papers published in 
neurooncology with a link to neuropathology in the 
year 2024?’. ChatGPT proposed several non-exist-
ent articles, including a review article supposedly  
authored by the writer of the present review.  
However, it altered the author list as follows:  
'Neurooncology: 2023 Update'; Authors: Wisoff JH, 
Sanford RA, Heier LA, et al. Published in: Free  
Neuropathology, 2023 [Mittelbronn, 2023]. In addi-
tion, 5 of the 10 proposed 'primary research papers' 
were actually podcasts. By rephrasing and adjust- 
ing the prompt, some improvements and correct- 
ions were achieved; however, the results improved  
only partially and gradually. PERPLEXITY suggested  
exactly the 10 papers from the Top 10 Series in  
Neurooncology published in Free Neuropathology 
last year but re-dated all articles from 2023 to 2024 
[Mittelbronn, 2024]. After being re-prompted, PER-
PLEXITY explained that it would be inappropriate to 
speculate or provide inaccurate information about 
influential papers from 2024. Only GEMINI – both  
initially and after several attempts or re-prompting 
– refused to provide some suggestions of real scien-
tific papers, instead recommending journals, search 
engines such as PubMed (https://pubmed.ncbi.nlm. 
nih.gov/) or Google Scholar (https://scholar.google. 
com/). As a result, unfortunately, not a single of the 
different LLMs was useful for the compilation of  
influential primary scientific papers in the field of  
neurooncology of this year’s edition. Therefore, the  
author returned to a more traditional approach, 
screening thousands of titles and abstracts related 
to key search terms in the field (e.g., brain tumor, 
glioma, meningioma, brain metastasis, neurooncol-
ogy, etc.). In contrast to last year, however, fewer 
novel treatment approaches in neurooncology were 
identified. Of note, with 29 retractions of papers  
related to the keyword 'glioblastoma' in 2024 (com-
pared to 22 in 2023 and 9 in 2022, and similarly high  
 
 

number of publications), along with multiple correc-
tions, a worrisome increase in potentially sloppy  
scientific behavior in this field was observed. Never-
theless, numerous excellent primary research arti-
cles were identified and subjective selection was a 
challenge. As usual, the goal was to provide a collec-
tion of articles ranging from basic research findings 
to novel diagnostic or prognostic criteria, technical 
and methodological innovations, and therapeutic 
approaches. With this, the “top ten” series in neu-
rooncology for 2024 reads as follows: 

1. Impact of cerebral microcirculation alteration 
and hypoxic-ischemic microenvironment on 
brain metastasis development [Roesler et al., 
2024]. 

2. Global DNA methylation as independent diag-
nostic marker in IDH-wildtype glioblastoma 
[Eckhardt et al., 2024]. 

3. Change in DNA methylation profile in recurrent 
glioblastoma [Drexler et al., 2024]. 

4. Glioblastoma evolution and heterogeneity 
from a whole tumor perspective [Mathur et al., 
2024]. 

5. Cranioencephalic functional lymphoid units in 
glioblastoma [Dobersalske et al., 2024]. 

6. Prognosis prediction via histomorphological 
features in glioblastoma [Kirishima et al., 
2024]. 

7. Confocal laser microscopy for intraoperative 
histopathological diagnostics of intracranial tu-
mors [Wagner et al., 2024]. 

8. Brain tumor diagnostics from cell-free DNA 
from cerebrospinal fluid [Afflerbach et al., 
2024 & Hickman et al., 2024]. 

9. Time-dependent single-cell phenotyping of im-
mune cells in glioblastoma [Kirschenbaum et 
al., 2024]. 

10. Histone serotonylation regulates ependymoma 
tumorigenesis [Chen et al., 2024]. 

https://doi.org/10.17879/freeneuropathology-2025-6316
https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fchatgpt.com%2F&data=05%7C02%7Cmichel.mittelbronn%40ext.uni.lu%7Cba0ec810e19c4ccdec7d08dd64484652%7C445a9c950f9d49539db1bc4a45dd1220%7C0%7C0%7C638776983780334547%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=RshrTTSMI9sswRqDPT0IKVGYhoxt8gcGfkGZFNwSRFU%3D&reserved=0
https://gemini.google.com/app
https://gemini.google.com/app
https://www.perplexity.ai/


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1. Impact of disturbed cerebral micro-
circulation and hypoxic-ischemic mi-
croenvironment on brain metastasis 
development [Roesler et al., 2024] 

Although the basic steps of brain metastasis 
formation, including – amongst others – detach-
ment of cancer cells from the primary tumor and  
intravasation into the blood stream, followed by  
adhesion to brain endothelial cells and finally extra-
vasation into as well as colonization of the brain is 
reiterated, the exact mechanisms and time-depend-
ent sequences are not fully understood. In a previ-
ous groundbreaking work of Kienast et al., the  
metastatic ways of individual cancer cells could be 
traced over several months in a murine model with 
real-time multiphoton laser scanning microscopy 
[Kienast et al., 2010]. With this, the authors could 
identify the initial arrest of cancer cells at cerebral 
blood vessel branches followed by angiogenic pro-
cesses or vessel cooption after early extravasation. 
Using a similar methodological approach, Roesler et 
al. were now able to show a similar intravascular  
tumor cell arrest in brain microvessels before extra-
vasation and formation of micrometastases [Roesler 
et al., 2024]. Of note, as a most novel finding,  
already 24h after intracardiac injection of tumor 
cells, prominent hypoxic-ischemic tissue alterations 
were observed in the brain that were partly well  
corresponding with areas, in which metastasis for-
mation was detected at a later timepoint. In the  
hypoxic-ischemic areas, an upregulation of Ang-2, 
MMP9 and VEGF was observed in brain endothelial 
cells leading to the hypothesis that those hypoxia-
associated molecules might be at least partly re-
sponsible for the seeding of metastatic cells in the 
brain. To corroborate this hypothesis, the authors 
also used a transgenic, endothelial-specific Ang-2 
gain-of-function approach that revealed an increase 
of numbers and volumes of brain metastases com-
pared to wild-type animals thereby indicating that 
extravasation of cancer cells into the brain paren-
chyma is Ang-2 dependent. The findings of the study 
were in line with previous findings showing that 
Ang-2 expression correlated with BBB disruption, 
impaired tight junctions and increased blood vessel 
permeability in a murine triple-negative breast  
cancer model [Avraham et al., 2014]. The findings of 

Roesler et al. indicate that Ang-2 and VEGF are  
crucial for the formation of brain metastases via 
shaping a tumor-supporting pre-metastatic niche, 
therefore being potential treatment targets, espe-
cially in an early phase. With a novel, early pre-met-
astatic inhibition of Ang-2 (via the AMG 386 pepti-
body) and/or VEGF (via aflibercept, a fusion protein 
with VEGFR1 and VEGFR2 domains serving as a  
so-called “VEGF trap”), the metastatic cerebral  
tumor cell load could be considerably reduced (see 
Figure 1). Similar findings were obtained in other 
previous murine brain metastasis models in which a 
combined inhibition of Ang-2 and VEGF reduced the 
number and volume of brain metastases while  
extracranial metastases did not show a positive 
treatment response [Bohn et al., 2017; Kovalchuk 
et al., 2020]. In summary, Roesler et al. present a 
novel mechanism for brain metastatic processes 
linked to an increase of Ang-2 and VEGF in the  
cerebral pre-metastatic niche which are induced by 
focal hypoxic-ischemic events most probably caused 
by occlusion of brain capillaries by tumor cells. 
Showing a significantly reduced cerebral tumor cell 
load by early dual inhibition of Ang-2 and VEGF, this 
treatment approach could be highly promising as a 
prevention strategy in patients suffering from highly 
malignant tumors with a high risk to develop cere-
bral metastases. 

2. Global DNA methylation as inde-
pendent diagnostic marker in IDH-
wildtype glioblastoma [Eckhardt et al., 
2024] 

Large population-based studies still report  
median survival times of slightly more than 6 
months for glioblastoma patients that could be  
considerably increased to a median of 14.8 months 
in cases with maximum treatment including neuro-
surgical resection followed by radiochemotherapy 
[Brodbelt et al., 2015]. Of note, approximately 30 % 
of all glioblastoma patients survive 2 years or longer 
after initial diagnosis. Apart from age, MGMT pro-
moter methylation, extent of resection and follow-
up treatment, there is not much known about glio-
blastoma-intrinsic tumor biological factors that 
might well predict patient prognosis. Nowadays, 
neuropathological diagnostics in the field of neuro- 

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Figure 1: Concept of brain metastasis formation and therapeutic prevention 

 

oncology is most precisely performed by DNA meth-
ylation profiling [Capper et al., 2018]. Within the 
class of IDH wildtype glioblastoma, mainly 3 sub-
classes could be defined: receptor tyrosine kinase I 
(RTK I), RTK II and mesenchymal subclass, however 
without showing any relevant survival differences 
[Drexler et al., 2023b]. In general, only poor data  
is available about the potential prognostic impact  
of methylation differences within distinct tumor  
entities. To address this question, Eckhardt et al.  
assessed 492 primary and 31 relapse IDH wildtype 
glioblastomas that all received similar standard 
treatment regimen. Although no unequivocally clear 
methylation signature associated with patient sur-
vival could be detected, the median methylation 
rate of 0.49 significantly split the cohort in long  
(median methylation above 0.49; median overall 
survival of 18 months) and short (median methyla-
tion level equal or below 0.49; median survival of 13 
months) time survivors. The optimal cut-off level 
was determined as a beta value of 0.458 that  

allowed for identifying a smaller subgroup of 73  
patients with a median overall survival of only 9.2 
months. Of note, the mean methylation level was an 
independent prognostic marker in a multivariate 
analysis together patient age, MGMT promoter 
methylation status and extent of resection, all fac-
tors that are known as prognosticators for a long 
time. The authors further showed that RTK I subclass 
glioblastoma had the worst median survival reach-
ing 14.2 months while displaying significantly lower 
mean methylation levels compared to its counter-
parts that reached 15 months (RTK II) and 18.8 
months (mesenchymal). However, the mean meth-
ylation levels were still significantly associated in 
RTK I and II subclasses. Combined risk classes includ-
ing mean methylation level, age, extent of resection 
and MGMT promoter methylation could further  
increase the prognostic prediction by splitting the 
cohort into a risk factor-free subgroup reaching 39 
months of median survival while the worst subgroup 
only reached 8.3 months (Figure 2). While immune 

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Figure 2: Risk stratification for IDH wildtype glioblastoma patients. Patient age over 65 years, extent of less than 90 % of the contrast-
enhancing tumor area, an unmethylated MGMT promoter and a low mean global methylation beta value of less than 0.49 are considered 
as negative prognostic factors. Median overall survival times are as follows: risk group 1: 39 months; risk group 2: 19 months; risk group 
3: 8.3 months. 

 

cell infiltration could be bioinformatically ruled out 
as underlying explanatory factor, higher mean me-
thylation levels were associated with better radio-
sensitivity, therefore at least providing a hypothesis 
why the patient subgroup with high beta values  
survived longer. These findings were also corrobo-
rated in a glioma cell line panel exhibiting better  
radiosensitivity in cells with a higher beta value. In 
summary, the authors provide a novel, highly im-
portant risk stratification for IDH wildtype glioblas-
toma patients that might also pave the way for new 
treatment stratifications in the future. However, a 
comparison with other tumor entities could not  
confirm high methylation levels as being associated 
with tumor aggressiveness in general. 

3. Change in DNA methylation profile 
in recurrent glioblastoma [Drexler et 
al., 2024] 

DNA methylation profiling has become a highly 
reliable and unbiased reproducible diagnostic tool in 
neuropathological routine diagnostics [Capper et 
al., 2018]. However, only poor data exist about a 
temporal change of DNA methylation upon treat-
ment in malignant brain tumors that might have an 
impact on treatment resistance. To address this 
question, Drexler et al. assessed paired primary  
and recurrent glioblastomas of 47 patients by DNA 
methylation from tumor bulk and plasma, mass 

 

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spectrometry and multiplex bead-based immunoas-
say [Drexler et al., 2024]. A potential change even 
in glioblastoma subclasses might be of importance 
as previous analyses revealed that mesenchymal, 
RTK I and RTK II subclasses differed in patient sur-
vival with 15.5 months, 16 months and 27 months, 
respectively [Dejaegher et al., 2021]. In the primary 
glioblastoma cohort of Drexler et al., DNA methyla-
tion profiling revealed the following subclass distri-
bution: 23.4 % RTK I, 40.4 % RTK II and 36.2 % mes-
enchymal while this composition changed to 12.8 % 
RTK I, 29.8 % RTK II, 40.4 % mesenchymal and 17 % 
with no match with a known class (Figure 3). In total, 
40 % of all recurrent GBM did not match with the 
subclass obtained in the primary tumors (including 
17 % of cases with no match). The highest percent-
age of transitions was towards the mesenchymal 
subclass (72.7 %). Thirteen percent of all patients 
showed a switch of the MGMT promoter methyla-
tion status, mostly from a methylated to a non-

methylated status, a finding that is in line with pre-
vious analyses performed in primary and recurrent 
glioblastoma [Choi et al., 2021]. The authors also 
analyzed multiple clinical -pathological para- 
meters but mostly found incomplete resection of 
contrast-enhancing tumor parts as a significant fac-
tor for subclass transition in the recurrent situation. 
While the cellular composition did not considerably 
vary between the groups with and without subclass 
change in general, the cohort undergoing a switch 
towards a mesenchymal phenotype showed for  
example a decreased number of immune cells in the 
primary tumor tissue, however did not reach the 
level of significance when the analyses were re-
peated for immune cell subtypes. In contrast, higher 
methylation signatures were observed for mono-
cytes, B cells and neutrophils in blood of patients in 
the primary situation later showing a subclass switch 
towards a mesenchymal phenotype compared to 
the cohort with stable glioblastoma subclass in the  

 

 

Figure 3: Temporal molecular change between primary and recurrent glioblastoma 

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recurrent situation. Proteomic analysis revealed a 
stronger metabolic and catabolic processes in the 
primary tumor tissue of patients later undergoing a 
switch to mesenchymal subclass. Of note, no associ-
ation of subclass changes with patient survival was 
observed. This study is of high importance for the 
understanding of temporal molecular changes in gli-
oblastoma and underscores the necessity to re-per-
form full molecular assessment in the recurrent  
situation, especially as also broadly accepted molec-
ular biomarkers such as MGMT promoter methyla-
tion status may change over time. To which degree 
radiochemotherapy induces the temporal switch  
towards a mesenchymal subclass phenotype has to 
be further elucidated. 

4. Glioblastoma evolution and hetero-
geneity from a whole tumor perspec-
tive [Mathur et al., 2024] 

Glioblastoma is a highly heterogenous neo-
plasm composed of both tumor and non-neoplastic 
cells of the microenvironment. In addition, little is 
known about the overall molecular heterogeneity 
including genetic, epigenetic and / or metabolic fea-
tures as diagnostics is mostly performed on smaller 
bulk tissue parts. In a previous large-scale approach, 
a glioblastoma atlas linking classical histomorpho-
logical features with transcriptional profiles, the lat-
ter assessed by in-situ hybridization, revealed a 
strong regional molecular heterogeneity partly re-
lated to microscopic characteristics [Puchalski et al., 
2018]. However, as these previous findings were 
also derived from preselected tissue blocks not  
representing the entire regional spectrum of glio-
blastoma, Mathur et al. sampled multiple tissues 
(n = 103) from the 10 patients using 3D surgical  
neuronavigation to obtain representative areas of 
the tumor core, periphery and contrast-enhancing 
regions [Mathur et al., 2024]. Furthermore, the  
biopsy positions were correlated with preoperative 
MRI scans to generate a representative 3D model of 
the entire glioblastoma. The small biopsy samples 
were assessed by histopathological analyses, high-
throughput chromosome conformation capture  
 
 
 

(Hi-C), whole exome sequencing (WES), RNA sequ- 
encing (RNA-seq) as well as assay for transposase-
accessible chromatin with high-throughput sequ-
encing (ATAC-seq, for tissue and single nucleus) (for 
summary, see Figure 4). The authors detected a vast 
genetic and transcriptional heterogeneity within the 
patients. Only one out of the ten patients showed a 
consistent signature in all biopsies regarding the 
previously published, gene expression-based molec-
ular classification into proneural, neural, classic or 
mesenchymal subtypes while all other patients  
displayed mixed intraindividual signatures [Verhaak 
et al., 2010]. Of note, tumor biopsies from the  
tumor cores more frequently showed a mesenchy-
mal or classic signature while more peripheral  
biopsies revealed rather neural or proneural signa-
tures. The authors also discovered distinct neurode-
velopmental transcriptomic signatures such as for 
example glioblastoma with a gain of chromosome 7 
(with the EGFR, PTPRZ1 and PTN genes) being asso-
ciated with glioblastoma cells in an intermediate 
progenitor cell (IPC) transit-amplifying state or – in 
contrast - glioblastoma with a primitive neuronal 
component, however without a gain of chromo-
some 7, showing transcriptional signatures typically 
observed in cells differentiating from IPCs to neu-
rons. Additional signatures in the tumor center com-
prised an immune-hot microenvironment with in-
terferon signaling and T cell infiltration versus im-
mune-cold areas with a mesenchymal transcript-
omic signature. While most mutations were highly 
heterogenous, chromothripsis seems to be a very 
early event in gliomagenesis with virtually no intra-
individual heterogeneity. Most importantly, the  
authors presented several novel potential diagnos-
tic biomarkers and treatment targets, such as the  
activator protein 1 (AP-1) pathway or several gene 
fusions that were missed by clinical NGS analyses as 
breakpoints were outside exons. With this work, 
Mathur et al. considerably contributed to the under-
standing of glioblastoma evolution and heterogene-
ity and provide promising therapeutic targets for 
further assessment. For more in-depth exploration 
of the findings, a publicly accessible interactive 
online platform is available: https://3d-gbms.shin 
yapps.io/search/. 

 

 

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Figure 4: 3D glioblastoma evolution and heterogeneity: methodological approach and key findings. Abbreviations: oRG: outer radial 
glia; IPC: intermediate progenitor cell; OPC: oligodendrocyte precursor cell; NEUROD1: neuronal differentiation 1 (transcription factor); 
AP-1: activator protein 1 (transcription factor). 

 

5. Cranioencephalic functional lym-
phoid units in glioblastoma [Dober-
salske et al., 2024] 

Bridging veins allow for an immunological com-
munication between CNS and skull bone marrow 
crossing the arachnoid barrier by connecting CNS 
blood vessels with the meningeal blood circulation 
and finally the sinusoidal vasculature of the bone 
marrow [Smyth et al., 2024]. As hematopoietic stem 
and progenitor cells are located in the skull bone 
marrow, however not well studied in the context of 
human diseases, Dobersalske et al. intended to as-
sess the immune cell properties in those areas in  
regional proximity to human glioblastoma, a condi-
tion known for its highly immunosuppressive nature 
[Dobersalske et al., 2024]. Using a CXCL12 analog 

radioligand tracer for CXCR4, a marker for hemato-
poietic and immune cells, the authors found surpris-
ingly high activity in the ipsilateral skull bone mar-
row and meninges related to the glioblastoma local-
ization in usually older glioblastoma patients. In a 
single-cell RNA sequencing approach from corre-
sponding (a) glioblastoma samples, (b) ipsilateral 
bone marrow of the skull, (c) peripheral blood mon-
onuclear cells (PBMCs) and (d) distal hip bone mar-
row, considerable differences in immune cell com-
positions were found. Regarding the myeloid cell 
compartment, mostly naïve monocytes were found 
the ipsilateral skull bone marrow contrasting anti-in-
flammatory monocytes and macrophages, predomi-
nantly found within glioblastoma. Of note, a strong 
accumulation of effector-type, glioma-reactive CD8+ 
T cell activity was detected in the skull of ipsilateral 
bone marrow whereas a lower activity was found in  
 

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Figure 5: Cranioencephalic functional lymphoid units in glioblastoma. Left side: methodological set-up. Right side: major findings 
according to compartment. 

 

intratumor CD8+ T cells. Both CD8+ T cells from 
PBMCs and ipsilateral bone marrow were able to  
experimentally reproduce memory and effector T 
cells, while intratumor T cells failed. Of note, the  
antitumoral activity of skull bone marrow derived 
CD8+ T cells was higher compared to counterparts 
derived from PBMCs, distal hip bone marrow or the 
glioblastoma microenvironment. Similarly high fre-
quencies of tumor-reactive T cells were found in 
skull bone marrow compared to the intratumor  
microenvironment, indicating an immune cell traf-
ficking between both compartments. Using the 
aforementioned radioligand tracer, a significantly 
longer survival benefit for glioblastoma patients 
with stronger intratumor and skull bone marrow im-
mune cell activity was observed already in a rela-
tively small patient cohort (n = 52). Taking the clini-
cal impact into account, these findings imply that 
the skull bone marrow immune cell niches should be 
absolutely protected during neurosurgical and sub-
sequent radiotherapeutic intervention. In summary, 

Dobersalske et al. shed a new light of the local anti-
tumoral immune cell activities in glioblastoma  
patients that needs to be considered for future  
immunotherapies (for summary, see Figure 5). 

6. Prognosis prediction via histomor-
phological features in glioblastoma 
[Kirishima et al., 2024] 

Neuropathological tumor diagnostics is more 
and more shifting away from the previous gold 
standard of microscopic analysis to more unbiased 
molecular methods mostly including genetic and  
epigenetic assessment for both diagnostic precision 
as well as prediction for treatment response and  
patient survival. As molecular, especially epigenetic 
features seem to be more stable than the highly var-
iable morphological features in glioblastoma, it is ex-
tremely important to always sample enough tissue 
from all different tumor areas (e.g. core, periphery, 
contrast-enhancing areas, necroses etc.) [Mikkelsen 

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et al., 2021]. It is very interesting for neuropa-
thologists if complex histomorphological assess-
ments can still compete with modern molecular  
diagnostics. Kirishima et al. undertake the endeavor 
to explore if distinct morphological features may still 
be of prognostic value in glioblastoma. As for today, 
a few studies showed worst survival for some  
glioblastoma subtypes such as epithelioid or 
rhabdoid variants, no unequivocally clear associa-
tion of any specific morphological phenotype with 
clinical prognosis could be detected [Mallick et al., 
2022]. Furthermore, most classical microscopic 
characteristics necessary for the diagnosis of glio-
blastoma, such as pseudopalisading necrosis or mi-
crovascular proliferations, are no longer sufficient 
for this diagnosis as they might also be found in IDH-
mutant astrocytoma [WHO classification of brain 
tumors, 2021]. Therefore, the authors of the pre-
sent study analyze 227 primary glioblastoma accord-
ing to the WHO criteria for the classification of  
tumours of the central nervous system from 2021 
and additionally assessed the following microscopic 
features in 10 % increments: pleomorphic, astro-
cytic, gemistocytic, rhabdoid, oligodendroglioma 
(ODG)-like, small, giant, spindle, epithelioid, primi-
tive neuronal differentiation(PND)-like and lipidized 
[Kirishima et al., 2024; WHO classification of brain 
tumors, 2021]. For histomorphological assessment 
to construct a survival prediction model, the largest, 
most representative areas were analyzed by two 
board-certified pathologists. Of note, also NGS panel 
analysis for genomic profiling, including assessment 
of gene mutations, copy number variation and 
1p/19q co-deletion as well as methylation-specific 
PCR to determine MGMT promoter methylation  
status were performed leading to an integrated final 
diagnosis together with histological and immuno-
histochemical (e.g. for IDH1-R132H) features. As  
major morphological variants, pleomorphic (33 % of 
total tumor area) and astrocytic (29.1 %) differenti-
ation were reported. Most interestingly, if glioblas-
toma showed epithelioid tumor cells in more than 
30 % of total tumor cells, patients displayed an  
unfavorable prognosis independent of clinical pa-
rameters, treatment or molecular alterations. The 
most favorable prognostic values for glioblastoma 
patients could be achieved by calculating a com-
bined score taking into account both gemistocytic 

and epithelioid areas as follows: % area of gemisto-
cytic cells minus 4 x % area of epithelioid cells. A 
value of less than 20 % of this index was an inde-
pendent significant factor for worse survival in a 
multivariate analyses together with age, medullary 
dissemination, chemotherapy and MGMT promoter 
methylation status (for summary see Figure 6). The 
prediction for patient survival could be further  
increased by combining this index with the MGMT 
promoter methylation status, showing worst patient 
survival for the combination of a low index together 
with an unmethylated MGMT promoter. As only 6 
out of 227 displayed an epithelioid phenotype, the 
results certainly have to be considered with caution, 
nevertheless, the prognostic impact of such a differ-
entiation seemed to be strongly associated with 
negative patient survival even in a multivariate anal-
ysis. Although a detailed definition of cell morphol-
ogy is provided by the authors, the reproducibility of 
such an analysis with still suffer from the subjective 
interpretation of individual neuropathologist. If this 
approach should enter the diagnostic routine, future 
automated digital image analysis would be prefera-
ble to undoubtfully recognize and grade the mor-
phological parameters 

7. Confocal laser microscopy for in-
traoperative histopathological diag-
nostics of intracranial tumors [Wagner 
et al., 2024] 

Intraoperative diagnostics performed on fro-
zen sections is time-consuming, often lasting around 
20 minutes from tissue sampling out of the surgical 
cavity to the final transmission of the diagnosis 
[Novis and Zarbo, 1997]. In addition, only single, 
mostly very small tissue fragments can be assessed 
at a time. Even in highly specialized tertiary medical 
centers, a diagnostic shift of 18 % between in-
traoperative and post-operative diagnoses was re-
ported, actually leading to a second subsequent 
neurosurgical intervention in 3 % of all brain tumor 
patients [Harms et al., 2023]. Therefore, there is an 
urgent need for faster methods that can also repeat-
edly analyze larger tissue areas, ideally in real-time 
and/or directly in situ. To address this issue, Wagner 
et al. conducted a phase II clinical study to test the  
 

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Figure 6: Prognostic prediction of histological features in glioblastoma 

 

noninferiority of fluorescein-stained intraoperative 
confocal laser endomicroscopy (CLE) compared to 
the classic assessment of frozen sections by neuro-
pathologists in 210 adult brain tumor patients. CLE 
(using the ZEISS CONVIVO system) is a non-inva-
sive/non-traumatic technique that enables histo-
pathological assessment in vivo almost in real time. 
In addition, fluorescein sodium acts as a contrast-en-
hancing intraoperative dye for an easier identifica-
tion of the tumor-suspicious areas. Of note, no  
serious adverse events were reported, neither in  
relation to CLE nor to fluorescein. Compared to the 
final neuropathological diagnoses, a correct result 
was obtained in 87 % of CLE-assessed samples, while 
classic frozen sections achieved 91 %, demonstrat-
ing the noninferiority of the CLE method (Figure 7). 
The concordance rate between CLE and classic  
frozen sections was 76 %. Most importantly, the  
median time until CLE diagnosis was communicated 
was 3 min compared to 27 min in classic frozen  
sections, resulting in a nearly 10-fold reduction of 

time required for intraoperative neuropathological 
assessment. In summary, in vivo CLE assessment for 
intraoperative neuropathological diagnostics is a 
highly reliable and significantly faster approach that 
could at least partially replace frozen section diag-
nostics in the future. In addition, a considerably 
higher amount of tissue images could be processed 
thereby further increasing its diagnostic validity. 

8. Brain tumor diagnostics from cell-
free DNA from cerebrospinal fluid [Af-
flerbach et al., 2024 and Hickman et 
al., 2024] 

Precise brain tumor diagnostics still relies on 
highly invasive procedures to obtain tissue samples 
for microscopic or molecular diagnostics. As high 
rates of intraoperative adverse events have been  
reported for neurosurgical interventions reaching 
up to 40 %, there is a strong need for non- or less 
invasive diagnostic procedures in neurooncology 

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Figure 7: Diagnostic set-up and major results for clinical phase II non-inferiority study for intraoperative neuropathological assessment 
using fluorescein-stained confocal laser endomicroscopy. 

 

[Drexler et al., 2023a]. Ideally, a non-operative  
tumor classification could even prevent patients 
from invasive follow-up procedures as in the case of 
CNS lymphoma. A second important reason for why 
non-invasive diagnostics would considerably im-
prove patient care would be related to the detection 
of early recurrences or minimal residual tumor load. 
Some studies have shown the suitability of sequenc-
ing techniques to detect tumor-specific mutations 
from cell-free DNA of the CSF in some tumor entities 
such as gliomas [Miller et al., 2019]. For other brain 
tumor entities, such as medulloblastoma, non-inva-
sive molecular diagnostics from cell-free DNA from 
cerebrospinal fluid (CSF) has already been proven to 
be superior to cytological diagnostics with regard to 
both the detection of residual tumor and the predic-
tion of patient prognosis [Liu et al., 2021]. To evalu-
ate the suitability of molecular diagnostics on CSF-
derived cell-free DNA across broader brain tumor 
cohorts, different research teams employed various 
approaches. Consequently, two exceptional papers 

were selected under a single topic. Afflerbach et al. 
analyzed a larger cohort of 110 samples from 99  
patients with suspicion of primary brain tumor using 
Nanopore sequencing (at least 5ng DNA per sample 
was needed), a technique which, in addition, also 
considerably reduces the time needed for sample 
preparation and sequencing [Afflerbach et al., 
2024]. In 45 % of the samples, circulating tumor DNA 
(ctDNA) could be detected in the CSF. Among the 
samples in which ctDNA was successfully detected, 
only the copy number variation (CNV) profile could 
be defined in 56 % (n = 28), only DNA methylation 
profiling in 12 % (n = 6), and both types of infor-
mation in 32 % (n = 16). The CNV profile revealed 
several diagnostically relevant amplifications as well 
as larger chromosomal gains and losses. Of note, 
corresponding cytological analyses detected tumor 
cells in only 9 cases. With their groundbreaking 
study, Afflerbach et al. provided evidence that  
Nanopore sequencing is a fast and reliable diagnos-
tic technique allowing for determining both CNV and 

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DNA methylation profiles from ctDNA from CSF in 
brain tumor patients. In another approach, Hickman 
et al. performed mutational analyses of CSF circulat-
ing ctDNA from 711 brain tumor patients using an 
FDA-authorized targeting next-generation sequenc-
ing platform [Hickman et al., 2024]. Similarly to the 
findings of Afflerbach et al., also Hickman and col-
leagues detected tumor-specific mutations in 53 % 
of all patient samples with clinically documented 
CNS tumors, whereas none of the control samples 
showed a false positive result, therefore constitut-
ing a highly specific diagnostic approach. Consider-
ing the technical limitations of various approaches, 
nanopore-based sequencing is particularly constr-
ained by low genomic coverage, making it unsuita-
ble for the reliable detection of point mutations 
[Emiliani et al., 2025]. Moreover, since short-read 
next-generation sequencing techniques have suc-
cessfully identified tumor-specific mutations with as 
little as 0.1 ng of cell-free DNA from CSF, a method-
ological comparison of different sequencing tech-
niques is essential to determine the most suitable 
approach [Miller et al., 2022]. These approaches will 
considerably change our initial diagnostic approach 
and treatment survey in neurooncology in the fu-
ture. 

9. Time-dependent single-cell pheno-
typing of immune cells in glioblastoma 
[Kirschenbaum et al., 2024] 

Single cell RNA sequencing techniques revolu-
tionized the understanding of individual cell states 
about which one could only speculate from data  
derived from bulk sequencing [Ramsköld et al., 
2012; Tang et al., 2009]. However, it is still challeng-
ing to decipher how cell states change over time  
under distinct conditions, such as for example the 
effect of the tumor microenvironment on infiltrating 
immune cells. To address this question, Kirschen-
baum et al. developed a new method called Zman 
sequencing (“Zman” is the phonetic spelling of the 
Hebrew word for “time”) which combines the use of 
fluorophore pulse labels in the blood as temporal 
stamps with single cell RNA sequencing [Kirschen-
baum et al., 2024]. The authors intravenously ap-
plied four times an identical antibody directed 
against CD45 that, however, was labeled each time 

with a different fluorophore (n = 4 in total) in 12h  
intervals (between 24-60h or 12-48h before mice 
were sacrificed). After having confirmed that the 
fluorophore-coupled antibodies (a) almost exclu-
sively stain immune cells in the blood stream, (b) can 
be reliably detected on immune cells over 96h, (c) 
do not penetrate the brain and (d) are virtually com-
pletely degraded in the blood within 60-90 min, 
transcriptional molecular trajectories of natural 
killer (NK) and myeloid cells in the syngeneic GL261 
murine glioblastoma model were assessed. One can 
assume that CD45-positive immune cells labeled 
with one of the four distinct fluorophores encoun-
tering in the glioma tumor microenvironment 
mostly infiltrated the brain via the blood stream 
within a time frame of +/- 12h before the application 
of the next, subsequent fluorophore-labeled anti-
body. With this approach, a temporal transcriptional 
kinetic profile can be calculated for each immune 
cell type within the glioma microenvironment. Of 
note, primary microglia are mostly not stained by 
this approach as the intravenously injected fluoro-
phore-coupled antibodies did not penetrate the 
brain. With this approach, the authors showed that 
NK cells changed from a cytotoxic to a dysfunctional, 
and myeloid cells from classic monocytes to im- 
munosuppressive tumor-associated macrophages 

(TAM) via activated TGF-1 signaling within 24h and 

36-48h, respectively. As TGF-1 signaling was asso-
ciated with Trem2 activation in immune cells of the 
myeloid lineage, an immunotherapeutic approach 
with an anti-Trem2 antibody was performed in the 
GL261 glioma model. This treatment prevented the 
differentiation to immunosuppressive TAMs leading 
to a pro-inflammatory cell state that is supposed to 
possess a much stronger anti-tumoral properties by 
recruiting T and NK cells [Allen et al., 2017]. With 
their study, Kirschenbaum et al. provide a revolu-
tionary technique allowing for deciphering the  
transcriptional trajectories of immune cells from the 
blood stream. They nicely apply this method to  
assess the impact of the glioma microenvironment 
on infiltrating immune cells showing that the tumor 
mostly escapes from an immune attack by inhibiting 

NK and myeloid cells via TGF-1-activated pathways. 
Finally, they provide a smart initial therapeutic  
approach by preventing myeloid cells to acquire a 
pro-tumorigenic cell state applying an anti-Trem2 
antibody (for summary see Figure 8). 

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Figure 8: Time-dependent immune cells transcriptomics in glioblastoma. Left side: methodological set-up. Right 
side: major findings of NK and myeloid cell/TAM transcriptional trajectories. 

 

10. Histone serotonylation regulates 
ependymoma tumorigenesis [Chen et 
al., 2024] 

Ependymomas constitute a heterogeneous 
group of CNS tumors that are currently classified 
into at least nine distinct molecular subtypes and are 
partly considered an epigenetic disorder [Stuckert 
et al., 2020]. As recent studies have revealed a direct 
epigenetic modification of histone H3 by serotonin, 
a process termed 'serotonylation,' a potential link 
between serotonin-induced histone alterations and 
the development and progression of ependymoma 
has been hypothesized [Farrelly et al., 2019]. To  
address this hypothesis, Chen et al. generated epen-
dymomas by performing in utero electroporation to 
insert the ZFTA-RELA fusion gene and knock out 
Trp53, a tumor suppressor gene. Additionally, a  
designer receptor exclusively activated by designer 

drugs (DREADD) approach was employed to modu-
late neuronal activity in cortical neurons and dorsal 
raphe nucleus neurons. This was achieved by inject-
ing non-naturally occurring receptors into distinct 
brain areas via viral vectors, which were subse-
quently activated by a synthetic ligand. The authors 
showed that the activation of excitatory cortical 
neurons promoted the development of ZFTA-RELA 
fusion-driven ependymomas and identified sero-
tonergic activity in the dorsal raphe nucleus – an 
area remote from the ependymoma – as a suppres-
sive factor for ependymoma progression. However, 
established, both murine and human ependymoma 
cells also displayed a considerable higher amount of 
serotonylated H3 compared to normal CNS tissue 
and histone serotonylation was associated with tu-
mor progression, providing evidence that serotonin 
signaling may have a rather disparate role in epen-
dymoma tumor biology. In the experimental setting,  

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Figure 9: Disparate effects of serotonin in ependymoma tumorigenesis. On the one hand, serotonin signaling 
from the dorsal raphe nucleus inhibits remote ependymoma growth, most likely by reducing neuronal activity in 
the tumor microenvironment. On the other hand, tumor cell-intrinsic serotonylation promotes ependymoma 
growth through epigenetic modification and subsequent ETV5 overexpression, ultimately leading to neuropep-
tide Y (NPY) inhibition. As a result, the inhibitory effect of NPY on brain activity and its tumor-suppressive function 
are abolished. 

ETS transcription variant factor 5 (ETV5), a transcrip-
tion factor with binding sites for both serotonylated 
H3 and the ZFTA-RELA fusion, was identified as a  
tumor-promoting factor. The authors further de-
monstrated that ETV5 was associated with a repres-
sive H3K27 trimethylation signature and decreased 
neuropeptide Y (NPY) expression in ependymoma. 
Moreover, ETV5 overexpression abrogated NPY-me-
diated synaptic inhibition in the tumor microenvi-
ronment, which was linked to reduced tumor 
growth (Figure 9). 

Discussion 

The body of research published in 2024 reveals 
significant advancements in the understanding of 

glioblastoma and brain metastasis, with a particular 
emphasis on tumor heterogeneity, diagnostic ad-
vancements, and therapeutic implications. The stud-
ies covered in this overview contribute to refining 
our approaches to both the molecular and clinical 
management of brain tumors, highlighting critical 
challenges and innovative solutions. A major theme 
emerging from this year's literature is the growing 
recognition of the microenvironment's role in tumor 
progression. Roesler et al. demonstrate the pivotal 
impact of altered cerebral microcirculation and a  
hypoxic-ischemic microenvironment in the develop-
ment of brain metastases [Roesler et al., 2024]. 
Their findings underscore the importance of under-
standing how changes in the vascular network and 
oxygen availability influence tumor spread, high-

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lighting potential targets for therapeutic interven-
tion, especially using prevention strategies inhibit-
ing brain metastasis seeding by combined inhibition 
of VEGF and Ang-2. This approach will change our 
therapeutic view as oncological patients are usually 
treated when symptoms or lesion occur. On the  
primary malignant brain tumor side, Mathur et al. 
explored glioblastoma's evolution and heterogene-
ity from a whole tumor perspective [Mathur et al., 
2024]. The dynamic nature of glioblastoma, with its 
diverse cellular populations and the continuous  
evolution of its genetic and epigenetic landscape, 
presents a substantial challenge for both diagnosis 
and treatment, however there is still a considerable 
debate about how hierarchical or rather reversible 
such an evolution is, the latter more speaking in  
favor of highly dynamic, tumor-intrinsic plasticity 
[Dirske et al., 2019]. DNA methylation continues to 
emerge as a crucial mechanism in brain tumor  
biology. Drexler et al. and Eckhardt et al. both high-
light the significance of DNA methylation profiles in 
glioblastoma [Drexler et al., 2024; Eckhardt et al., 
2024]. Drexler et al. demonstrate the alterations in 
DNA methylation in recurrent glioblastoma, provid-
ing valuable insights into the epigenetic shifts that 
occur during disease progression, especially that 
methylation classifier subclasses have to be consid-
ered with caution as they may considerably change 
over time. Meanwhile, Eckhardt et al. present global 
DNA methylation as an independent diagnostic 
marker for IDH-wildtype glioblastoma, offering a  
potential non-invasive tool for early diagnosis and 
prognosis. Of note, they provide an easily applicable 
risk score that can be integrated into routine diag-
nostic procedures, offering significant prognostic 
value by distinguishing between a high-risk group 
with a median survival of only 8.3 months and a low-
risk group with a median survival of 39 months. 
These findings underscore the growing interest in 
epigenetic biomarkers for glioblastoma, which may 
ultimately improve diagnostic accuracy and allow 
for more personalized therapeutic strategies. In  
addition to DNA methylation, histopathological and 
molecular features are increasingly used to predict 
prognosis. Kirishima et al. demonstrate how histo-
morphological features can provide valuable prog-
nostic information in glioblastoma, supporting the 
role of tissue-based diagnostics in clinical settings 
[Kirishima et al., 2024]. A considerable limitation of 

this study is that morphological criteria – despite  
being carefully and precisely defined – are still sub-
ject to interpretation bias. This will need to be con-
sidered in the future when using artificial intelli-
gence algorithms applied to digital images, which 
are often annotated by human (neuro)pathologists 
during the training phase. The utilization of cutting-
edge technologies such as confocal laser microscopy 
in intraoperative diagnostics, as outlined by Wagner 
et al., further enhances the speed and hopefully also 
the precision in the future of tumor identification 
and delineation, offering real-time guidance during 
surgery and improving patient outcomes [Wagner 
et al., 2024]. This technological advancement, 
alongside the development of non-invasive diagnos-
tic techniques like those reported by Afflerbach et 
al. and Hickman et al., who used sequencing tech-
niques of cell-free DNA from cerebrospinal fluid, 
marks a significant step forward in the non-invasive 
diagnosis and monitoring of brain tumors [Af-
flerbach et al., 2024; Hickman et al., 2024]. Their 
work presents exciting prospects for liquid biopsy as 
a means to detect and track tumor progression in 
real-time. Immune cell dynamics are also a focal 
point of recent studies. Kirschenbaum et al. explore 
the time-dependent phenotyping of immune cells in 
glioblastoma, providing valuable insights into the 
temporal changes in immune cell populations within 
the tumor microenvironment. These findings could 
have significant implications for immunotherapy, as 
the ability to track immune cell profiles over time 
may inform the development of more targeted im-
mune-based therapies [Kirschenbaum et al., 2024]. 
However, at least as important as the scientific find-
ings of this study is the technical development of 
tracking immune cells through time-dependent 
transcriptomics using the novel Zman sequencing 
technique. At present, this approach is primarily 
suited for cells infiltrating other tissue areas, previ-
ously labeled via the bloodstream. An important 
next step in the development of time-dependent 
transcriptomics would be expanding its applicability 
to cells that remain within a distinct cellular collec-
tive, such as brain tumor cells. This ties in with the 
work of Dobersalske et al., who investigated the role 
of cranioencephalic functional lymphoid units in  
glioblastoma, expanding our understanding of how 
immune surveillance and local immune responses 
may influence tumor behavior and response to 

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treatment [Dobersalske et al., 2024]. To further  
decipher the temporal dynamics of 'glymphatic' 
drainage, Zman sequencing could also be of interest 
in expanding upon the findings of the study by 
Dobersalske et al. Finally, the role of post-transla-
tional modifications in tumorigenesis is underscored 
by the study of Chen et al., which examines histone 
serotonylation in ependymoma tumorigenesis 
[Chen et al., 2024]. This work highlights how seroto-
nin-mediated alterations in histone modifications 
can regulate tumor growth and suggests that target-
ing epigenetic regulators may offer a novel thera-
peutic avenue for ependymomas and other brain  
tumors. It remains to be determined if serotonyla-
tion will be a reproducible, important factor in brain 
tumor progression in the future, however there is an 
increasing body of evidence that a crosstalk be-
tween neurons and brain tumor might be implicated 
[Venkataramani et al., 2025]. Together, these stud-
ies reflect a shift toward more comprehensive, mul-
tifactorial approaches to understanding and treating 
brain tumors. In general, there currently seems to 
be a disproportion between scientific studies focus-
ing on the 'bad guys' – the brain tumor cells that  
proliferate, migrate, and infiltrate – and the large 
number of studies examining the tumor microenvi-
ronment. While the microenvironment may contrib-
ute to tumor progression, it lacks the malignant  
oncogenic driver mutations. It may be easier to 
study tumor microenvironmental factors, possibly 
due to superior methodological tools and simpler 

tracking methods, but it is crucial not to lose sight of 
tumor cells as the main players in future research. 
The integration of molecular and immune profiling, 
advanced imaging technologies, and epigenetic 
markers has the potential to revolutionize both the 
diagnosis and treatment of primary and secondary 
brain tumors. As our understanding of the complex 
interplay between tumor biology, the microenviron-
ment, and the immune system deepens, we can  
anticipate more targeted, individualized therapies 
that may significantly improve patient outcomes in 
the future. 

Acknowledgements 

The figures were prepared using https://bio-
render.com figure creation tool. Single images using 
for the figures were freely available tagged with cre-
ative common license. 

Funding Statement 

MM would like to thank the Luxembourg Na-
tional Research Fund (FNR) for the support (FNR 
PEARL P16/BM/11192868 grant). 

Conflict of Interest 

The author does not have any conflict of  
interest to declare. 

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https://doi.org/10.4103/0028-3886.359222
https://doi.org/10.1016/j.cell.2023.12.013
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	Review
	Introduction
	1. Impact of disturbed cerebral microcirculation and hypoxic-ischemic microenvironment on brain metastasis development [Roesler et al., 2024]
	2. Global DNA methylation as independent diagnostic marker in IDH-wildtype glioblastoma [Eckhardt et al., 2024]
	3. Change in DNA methylation profile in recurrent glioblastoma [Drexler et al., 2024]
	4. Glioblastoma evolution and heterogeneity from a whole tumor perspective [Mathur et al., 2024]
	5. Cranioencephalic functional lymphoid units in glioblastoma [Dober-salske et al., 2024]
	7. Confocal laser microscopy for intraoperative histopathological diagnostics of intracranial tumors [Wagner et al., 2024]
	8. Brain tumor diagnostics from cell-free DNA from cerebrospinal fluid [Afflerbach et al., 2024 and Hickman et al., 2024]
	9. Time-dependent single-cell phenotyping of immune cells in glioblastoma [Kirschenbaum et al., 2024]
	10. Histone serotonylation regulates ependymoma tumorigenesis [Chen et al., 2024]
	Discussion
	Acknowledgements
	Funding Statement
	Conflict of Interest
	References

