Integrated analyses reveal two molecularly and clinically distinct subtypes of H3 K27M-mutant diffuse midline gliomas with prognostic significance.

H3 K27M-altered diffuse midline gliomas (DMGs) are highly malignant tumours that arise in the midline structures of the CNS. Most DMGs carry an H3 K27M-mutation in one of the genes encoding for histone H3. Recent studies suggested that epigenetic subgroups of DMGs can be distinguished based on alterations in the MAPK-signalling pathway, tumour localisation, mutant H3-gene, or overall survival (OS). However, as these parameters were studied individually, it is unclear how they collectively influence survival. Hence, we analysed dependencies between different parameters, to define novel epigenetic, clinically meaningful subgroups of DMGs. We collected a multifaceted cohort of 149 H3 K27M-mutant DMGs, also incorporating data of published cases. DMGs were included in the study if they could be clearly allocated to the spinal cord (n = 31; one patient with an additional sellar tumour), medulla (n = 20), pons (n = 64) or thalamus (n = 33), irrespective of further known characteristics. We then performed global genome-wide DNA methylation profiling and, for a subset, DNA sequencing and survival analyses. Unsupervised hierarchical clustering of DNA methylation data indicated two clusters of DMGs, i.e. subtypes DMG-A and DMG-B. These subtypes differed in mutational spectrum, tumour localisation, age at diagnosis and overall survival. DMG-A was enriched for DMGs with MAPK-mutations, medullary localisation and adult age. 13% of DMG-A had a methylated MGMT promoter. Contrarily, DMG-B was enriched for cases with TP53-mutations, PDGFRA-amplifications, pontine localisation and paediatric patients. In univariate analyses, the features enriched in DMG-B were associated with a poorer survival. However, all significant parameters tested were dependent on the cluster attribution, which had the largest effect on survival: DMG-A had a significantly better survival compared to DMG-B (p < 0.001). Hence, the subtype attribution based on two methylation clusters can be used to predict survival as it integrates different molecular and clinical parameters.

Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level.

Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies-mainly studying nucleic acids-has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pG3myc), and synapses/immunological processes (pSHHs, pG3 and pG4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favorable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.

Transcriptomic and epigenetic dissection of spinal ependymoma (SP-EPN) identifies clinically relevant subtypes enriched for tumors with and without NF2 mutation.

Ependymomas encompass multiple clinically relevant tumor types based on localization and molecular profiles. Tumors of the methylation class "spinal ependymoma" (SP-EPN) represent the most common intramedullary neoplasms in children and adults. However, their developmental origin is ill-defined, molecular data are scarce, and the potential heterogeneity within SP-EPN remains unexplored. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations, but neither types and frequency of these alterations nor their clinical relevance have been described in a large, epigenetically defined series. Transcriptomic (n = 72), epigenetic (n = 225), genetic (n = 134), and clinical data (n = 112) were integrated for a detailed molecular overview on SP-EPN. Additionally, we mapped SP-EPN transcriptomes to developmental atlases of the developing and adult spinal cord to uncover potential developmental origins of these tumors. The integration of transcriptomic ependymoma data with single-cell atlases of the spinal cord revealed that SP-EPN display the highest similarities to mature adult ependymal cells. Unsupervised hierarchical clustering of transcriptomic data together with integrated analysis of methylation profiles identified two molecular SP-EPN subtypes. Subtype A tumors primarily carried previously known germline or sporadic NF2 mutations together with 22q loss (bi-allelic NF2 loss), resulting in decreased NF2 expression. Furthermore, they more often presented as multilocular disease and demonstrated a significantly reduced progression-free survival as compared to SP-EP subtype B. In contrast, subtype B predominantly contained samples without NF2 mutation detected in sequencing together with 22q loss (monoallelic NF2 loss). These tumors showed regular NF2 expression but more extensive global copy number alterations. Based on integrated molecular profiling of a large multi-center cohort, we identified two distinct SP-EPN subtypes with important implications for genetic counseling, patient surveillance, and drug development priorities.

Integrated proteomics spotlight the proteasome as a therapeutic vulnerability in embryonal tumors with multilayered rosettes.

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are rare malignant embryonal brain tumors. The prognosis of ETMR is poor and novel therapeutic approaches are desperately needed. Comprehension of ETMR tumor biology is currently based on only few previous molecular studies, which mainly focused on the analyses of nucleic acids. In this study, we explored integrated ETMR proteomics. METHODS: Using mass spectrometry, proteome data were acquired from 16 ETMR and the ETMR cell line BT183. Proteome data were integrated with case-matched global DNA methylation data, publicly available transcriptome data, and proteome data of further embryonal and pediatric brain tumors. RESULTS: Proteome-based cluster analyses grouped ETMR samples according to histomorphology, separating neuropil-rich tumors with neuronal signatures from primitive tumors with signatures relating to stemness and chromosome organization. Integrated proteomics showcased that ETMR and BT183 cells harbor proteasome regulatory proteins in abundance, implicating their strong dependency on the proteasome machinery to safeguard proteostasis. Indeed, in vitro assays using BT183 highlighted that ETMR tumor cells are highly vulnerable toward treatment with the CNS penetrant proteasome inhibitor Marizomib. CONCLUSIONS: In summary, histomorphology stipulates the proteome signatures of ETMR, and proteasome regulatory proteins are pervasively abundant in these tumors. As validated in vitro, proteasome inhibition poses a promising therapeutic option in ETMR.

Molecular refinement of pilocytic astrocytoma in adult patients.

AIM: Pilocytic astrocytomas (PA) in adults are rare and may be challenging to identify based only on histomorphology. Compared to their paediatric counterparts, they are reportedly molecularly more diverse and associated with a worse prognosis. We aimed to describe the characteristics of adult PAs more precisely by comprehensively profiling a series of 79 histologically diagnosed adult cases (≥18 years). METHODS: We performed global DNA methylation profiling and DNA and RNA panel sequencing, and integrated the results with clinical data. We further compared the molecular characteristics of adult and paediatric PAs that had a significant match to one of the established PA methylation classes in the Heidelberg brain tumour classifier. RESULTS: The mean age in our cohort was 33 years, and 43% of the tumours were located supratentorially. Based on methylation profiling, only 39% of the cases received a significant match to a PA methylation class. Sixteen per cent matched a different tumour type and 45% had a Heidelberg classifier score <0.9 with an affiliation to diverse established methylation classes in t-SNE analyses. Although the KIAA1549::BRAF fusion was found in 98% of paediatric PAs, this was true for only 27% of histologically defined and 55% of adult PAs defined by methylation profiling. CONCLUSIONS: A particularly high fraction of adult tumours with histological features of PA do not match current PA methylation classes, indicating ambiguous histology and an urgent need for molecular profiling. Moreover, even in adult PAs with a match to a PA methylation class, the distribution of genetic drivers differs significantly from their paediatric counterparts (p<0.01).

Adult intracranial ependymoma-relevance of DNA methylation profiling for diagnosis, prognosis, and treatment.

BACKGROUND: A methylation-based classification of ependymoma has recently found broad application. However, the diagnostic advantage and implications for treatment decisions remain unclear. Here, we retrospectively evaluate the impact of surgery and radiotherapy on outcome after molecular reclassification of adult intracranial ependymomas. METHODS: Tumors diagnosed as intracranial ependymomas from 170 adult patients collected from 8 diagnostic institutions were subjected to DNA methylation profiling. Molecular classes, patient characteristics, and treatment were correlated with progression-free survival (PFS). RESULTS: The classifier indicated an ependymal tumor in 73.5%, a different tumor entity in 10.6%, and non-classifiable tumors in 15.9% of cases, respectively. The most prevalent molecular classes were posterior fossa ependymoma group B (EPN-PFB, 32.9%), posterior fossa subependymoma (PF-SE, 25.9%), and supratentorial ZFTA fusion-positive ependymoma (EPN-ZFTA, 11.2%). With a median follow-up of 60.0 months, the 5- and 10-year-PFS rates were 64.5% and 41.8% for EPN-PFB, 67.4% and 45.2% for PF-SE, and 60.3% and 60.3% for EPN-ZFTA. In EPN-PFB, but not in other molecular classes, gross total resection (GTR) (P = .009) and postoperative radiotherapy (P = .007) were significantly associated with improved PFS in multivariable analysis. Histological tumor grading (WHO 2 vs. 3) was not a predictor of the prognosis within molecularly defined ependymoma classes. CONCLUSIONS: DNA methylation profiling improves diagnostic accuracy and risk stratification in adult intracranial ependymoma. The molecular class of PF-SE is unexpectedly prevalent among adult tumors with ependymoma histology and relapsed as frequently as EPN-PFB, despite the supposed benign nature. GTR and radiotherapy may represent key factors in determining the outcome of EPN-PFB patients.

Single-nucleus chromatin accessibility profiling highlights distinct astrocyte signatures in progressive supranuclear palsy and corticobasal degeneration.

Tauopathies such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) exhibit characteristic neuronal and glial inclusions of hyperphosphorylated Tau (pTau). Although the astrocytic pTau phenotype upon neuropathological examination is the most guiding feature in distinguishing both diseases, regulatory mechanisms controlling their transitions into disease-specific states are poorly understood to date. Here, we provide accessible chromatin data of more than 45,000 single nuclei isolated from the frontal cortex of PSP, CBD, and control individuals. We found a strong association of disease-relevant molecular changes with astrocytes and demonstrate that tauopathy-relevant genetic risk variants are tightly linked to astrocytic chromatin accessibility profiles in the brains of PSP and CBD patients. Unlike the established pathogenesis in the secondary tauopathy Alzheimer disease, microglial alterations were relatively sparse. Transcription factor (TF) motif enrichments in pseudotime as well as modeling of the astrocytic TF interplay suggested a common pTau signature for CBD and PSP that is reminiscent of an inflammatory immediate-early response. Nonetheless, machine learning models also predicted discriminatory features, and we observed marked differences in molecular entities related to protein homeostasis between both diseases. Predicted TF involvement was supported by immunofluorescence analyses in postmortem brain tissue for their highly correlated target genes. Collectively, our data expand the current knowledge on risk gene involvement (e.g., MAPT, MAPK8, and NFE2L2) and molecular pathways leading to the phenotypic changes associated with CBD and PSP.

Chronic PPARγ Stimulation Shifts Amyloidosis to Higher Fibrillarity but Improves Cognition.

We undertook longitudinal ß-amyloid positron emission tomography (Aß-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone in Aß model mice. We thus tested the hypothesis this treatment would rescue from increases of the Aß-PET signal while promoting spatial learning and preservation of synaptic density. Here, we investigated longitudinally for 5 months PS2APP mice (N = 23; baseline age: 8 months) and App NL-G-F mice (N = 37; baseline age: 5 months) using Aß-PET. Groups of mice were treated with pioglitazone or vehicle during the follow-up interval. We tested spatial memory performance and confirmed terminal PET findings by immunohistochemical and biochemistry analyses. Surprisingly, Aß-PET and immunohistochemistry revealed a shift toward higher fibrillary composition of Aß-plaques during upon chronic pioglitazone treatment. Nonetheless, synaptic density and spatial learning were improved in transgenic mice with pioglitazone treatment, in association with the increased plaque fibrillarity. These translational data suggest that a shift toward higher plaque fibrillarity protects cognitive function and brain integrity. Increases in the Aß-PET signal upon immunomodulatory treatments targeting Aß aggregation can thus be protective.

Diagnostic Yield and Complication Rate of Stereotactic Biopsies in Precision Medicine of Gliomas.

Background: An integrated diagnosis consisting of histology and molecular markers is the basis of the current WHO classification system of gliomas. In patients with suspected newly diagnosed or recurrent glioma, stereotactic biopsy is an alternative in cases in which microsurgical resection is deemed to not be safely feasible or indicated. In this retrospective study, we aimed to analyze both the diagnostic yield and the safety of a standardized biopsy technique. Material and Methods: The institutional database was screened for frame-based biopsy procedures (January 2016 until March 2021). Only patients with a suspected diagnosis of glioma based on imaging were included. All tumors were classified according to the current WHO grading system. The clinical parameters, procedural complications, histology, and molecular signature of the tissues obtained were assessed. Results: Between January 2016 and March 2021, 1,214 patients underwent a stereotactic biopsy: 617 (50.8%) for a newly diagnosed lesion and 597 (49.2%) for a suspected recurrence. The median age was 56.9 years (range 5 months-94.4 years). Magnetic resonance imaging (MRI)-guidance was used in 99.3% of cases and additional positron emission tomography (PET)-guidance in 34.3% of cases. In total, stereotactic serial biopsy provided an integrated diagnosis in 96.3% of all procedures. The most frequent diagnoses were isocitrate dehydrogenase (IDH) wildtype glioblastoma (n = 596; 49.2%), oligodendroglioma grade 2 (n = 109; 9%), astrocytoma grade 3 (n = 108; 8.9%), oligodendroglioma grade 3 (n = 76; 6.3%), and astrocytoma grade 2 (n = 66; 5.4%). A detailed determination was successful for IDH 1/2 mutation in 99.4% of cases, for 1p/19q codeletion in 97.4% of cases, for TERT mutation in 98.9% of cases, and for MGMT promoter methylation in 99.1% of cases. Next-generation sequencing was evaluable in 64/67 (95.5%) of cases and DNA methylome analysis in 41/44 (93.2%) of cases. Thirteen (1.1%) cases showed glial tumors that could not be further specified. Seventy-three tumors were different non-glioma entities, e.g., of infectious or inflammatory nature. Seventy-five out of 597 suspected recurrences turned out to be post-therapeutic changes only. The rate of post-procedural complications with clinical symptoms of the Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or higher was 1.2% in overall patients and 2.6% in the subgroup of brainstem biopsies. There was no fatal outcome in the entire series. Conclusion: Image-guided stereotactic serial biopsy enables obtaining reliable histopathological and molecular diagnoses with a very low complication rate even in tumors with critical localization. Thus, in patients not undergoing microsurgical resection, this is a valuable tool for precision medicine of patients with glioma.

Comprehensive profiling of myxopapillary ependymomas identifies a distinct molecular subtype with relapsing disease.

BACKGROUND: Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown. METHODS: We assembled a cohort of 185 tumors classified as MPE based on DNA methylation. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPE-B, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. RESULTS: MPE-A occurred at a median age of 27 years and were enriched with tumors demonstrating papillary morphology and MGMT promoter hypermethylation. Half of these tumors could not be totally resected, and 85% relapsed within 10 years. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. MPE-B occurred at a median age of 45 years and included many tumors with a histological diagnosis of WHO grade II and tanycytic morphology. Patients within this subtype had a significantly better outcome with a relapse rate of 33% in 10 years (P = 3.4e-06). CONCLUSIONS: We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.

Long-term diazepam treatment enhances microglial spine engulfment and impairs cognitive performance via the mitochondrial 18 kDa translocator protein (TSPO).

Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment.

Extent, pattern, and prognostic value of MGMT promotor methylation: does it differ between glioblastoma and IDH-wildtype/TERT-mutated astrocytoma?

INTRODUCTION: The cIMPACT-NOW update 6 first introduced glioblastoma diagnosis based on the combination of IDH-wildtype (IDHwt) status and TERT promotor mutation (pTERTmut). In glioblastoma as defined by histopathology according to the WHO 2016 classification, MGMT promotor status is associated with outcome. Whether this is also true in glioblastoma defined by molecular markers is yet unclear. METHODS: We searched the institutional database for patients with: (1) glioblastoma defined by histopathology; and (2) IDHwt astrocytoma with pTERTmut. MGMT promotor methylation was analysed using methylation-specific PCR and Sanger sequencing of CpG sites within the MGMT promotor region. RESULTS: We identified 224 patients with glioblastoma diagnosed based on histopathology, and 54 patients with IDHwt astrocytoma with pTERTmut (19 astrocytomas WHO grade II and 38 astrocytomas WHO grade III). There was no difference in the number of MGMT methylated tumors between the two cohorts as determined per PCR, and also neither the number nor the pattern of methylated CpG sites differed as determined per Sanger sequencing. Progression-free (PFS) and overall survival (OS) was similar between the two cohorts when treated with radio- or chemotherapy. In both cohorts, higher numbers of methylated CpG sites were associated with favourable outcome. CONCLUSIONS: Extent and pattern of methylated CpG sites are similar in glioblastoma and IDHwt astrocytoma with pTERTmut. In both tumor entities, higher numbers of methylated CpG sites appear associated with more favourable outcome. Evaluation in larger prospective cohorts is warranted.

The value of stereotactic biopsy of primary and recurrent brain metastases in the era of precision medicine.

Background: Brain metastases (BM) represent the most frequent intracranial tumors with increasing incidence. Many primary tumors are currently treated in protocols that incorporate targeted therapies either upfront or for progressive metastatic disease. Hence, molecular markers are gaining increasing importance in the diagnostic framework of BM. In cases with diagnostic uncertainty, both in newly diagnosed or recurrent BM, stereotactic biopsy serves as an alternative to microsurgical resection particularly whenever resection is not deemed to be safe or feasible. This retrospective study aimed to analyze both diagnostic yield and safety of an image-guided frame based stereotactic biopsy technique (STX). Material and methods: Our institutional neurosurgical data base was searched for any surgical procedure for suspected brain metastases between January 2016 and March 2021. Of these, only patients with STX were included. Clinical parameters, procedural complications, and tissue histology and concomitant molecular signature were assessed. Results: Overall, 467 patients were identified including 234 (50%) with STX. Median age at biopsy was 64 years (range 29 - 87 years). MRI was used for frame-based trajectory planning in every case with additional PET-guidance in 38 cases (16%). In total, serial tumor probes provided a definite diagnosis in 230 procedures (98%). In 4 cases (1.7%), the pathological tissue did not allow a definitive neuropathological diagnosis. 24 cases had to be excluded due to non-metastatic histology, leaving 206 cases for further analyses. 114 patients (49%) exhibited newly diagnosed BM, while 46 patients (20%) displayed progressive BM. Pseudoprogression was seen in 46 patients, a median of 12 months after prior therapy. Pseudoprogression was always confirmed by clinical course. Metastatic tissue was found most frequently from lung cancer (40%), followed by breast cancer (9%), and malignant melanoma (7%). Other entities included gastrointestinal cancer, squamous cell cancer, renal cell carcinoma, and thyroid cancer, respectively. In 9 cases (4%), the tumor origin could not be identified (cancer of unknown primary). Molecular genetic analyses were successful in 137 out of 144 analyzed cases (95%). Additional next-generation sequencing revealed conclusive results in 12/18 (67%) cases. Relevant peri-procedural complications were observed in 5 cases (2.4%), which were all transient. No permanent morbidity or mortality was noted. Conclusion: In patients with BM, frame-based stereotactic biopsy constitutes a safe procedure with a high diagnostic yield. Importantly, this extended to discerning pseudoprogression from tumor relapse after prior therapy. Thus, comprehensive molecular characterization based on minimal-invasive stereotactic biopsies lays the foundation for precision medicine approaches in the treatment of primary and recurrent BM.

Subventricular zone involvement is associated with worse outcome in glioma WHO grade 2 depending on molecular markers.

Neural stem cells within the subventricular zone were identified as cells of origin driving growth of high-grade gliomas, and anatomical involvement of the subventricular zone has been associated with an inferior clinical outcome. Whether the association between poor outcome and subventricular zone involvement also applies to glioma of lower grades is unclear. We therefore analysed a retrospective cohort of 182 patients with glioma grade 2 (according to the WHO 2016 classification) including 78 individuals (43%) with subventricular zone involvement. Patients with and without subventricular zone involvement did not differ in regard to demographics, histopathology, and molecular markers. Notably, subventricular zone involvement was a negative prognostic marker for malignant progression and overall survival on uni- and multivariate analysis. When patients were stratified according to the cIMPACT-NOW update 6, subventricular zone involvement was negatively associated with outcome in IDH-wildtype astrocytomas and 1p19q-codeleted oligodendrogliomas but not in IDH-mutant astrocytomas. Collectively, subventricular zone involvement may represent a risk factor for worse outcome in glioma WHO grade 2 depending on the molecular tumor signature. The present data confirm the relevance of molecular glioma classifications as proposed by the cIMPACT-NOW update 6. These findings warrant evaluation in prospective cohorts.

Pre-therapeutic microglia activation and sex determine therapy effects of chronic immunomodulation.

Modulation of the innate immune system is emerging as a promising therapeutic strategy against Alzheimer's disease (AD). However, determinants of a beneficial therapeutic effect are ill-understood. Thus, we investigated the potential of 18 kDa translocator protein positron-emission-tomography (TSPO-PET) for assessment of microglial activation in mouse brain before and during chronic immunomodulation. Methods: Serial TSPO-PET was performed during five months of chronic microglia modulation by stimulation of the peroxisome proliferator-activated receptor (PPAR)-γ with pioglitazone in two different mouse models of AD (PS2APP, AppNL-G-F ). Using mixed statistical models on longitudinal TSPO-PET data, we tested for effects of therapy and sex on treatment response. We tested correlations of baseline with longitudinal measures of TSPO-PET, and correlations between PET results with spatial learning performance and ß-amyloid accumulation of individual mice. Immunohistochemistry was used to determine the molecular source of the TSPO-PET signal. Results: Pioglitazone-treated female PS2APP and AppNL-G-F mice showed attenuation of the longitudinal increases in TSPO-PET signal when compared to vehicle controls, whereas treated male AppNL-G-F mice showed the opposite effect. Baseline TSPO-PET strongly predicted changes in microglial activation in treated mice (R = -0.874, p < 0.0001) but not in vehicle controls (R = -0.356, p = 0.081). Reduced TSPO-PET signal upon pharmacological treatment was associated with better spatial learning despite higher fibrillar ß-amyloid accumulation. Immunohistochemistry confirmed activated microglia to be the source of the TSPO-PET signal (R = 0.952, p < 0.0001). Conclusion: TSPO-PET represents a sensitive biomarker for monitoring of immunomodulation and closely reflects activated microglia. Sex and pre-therapeutic assessment of baseline microglial activation predict individual immunomodulation effects and may serve for responder stratification.

Molecular diagnostics helps to identify distinct subgroups of spinal astrocytomas.

Primary spinal cord astrocytomas are rare, hence few data exist about the prognostic significance of molecular markers. Here we analyze a panel of molecular alterations in association with the clinical course. Histology and genome sequencing was performed in 26 spinal astrocytomas operated upon between 2000 and 2020. Next-generation DNA/RNA sequencing (NGS) and methylome analysis were performed to determine molecular alterations. Histology and NGS allowed the distinction of 5 tumor subgroups: glioblastoma IDH wildtype (GBM); diffuse midline glioma H3 K27M mutated (DMG-H3); high-grade astrocytoma with piloid features (HAP); diffuse astrocytoma IDH mutated (DA), diffuse leptomeningeal glioneural tumors (DGLN) and pilocytic astrocytoma (PA). Within all tumor entities GBM (median OS: 5.5 months), DMG-H3 (median OS: 13 months) and HAP (median OS: 8 months) showed a fatal prognosis. DMG-H3 tend to emerge in adolescence whereas GBM and HAP develop in the elderly. HAP are characterized by CDKN2A/B deletion and ATRX mutation. 50% of PA tumors carried a mutation in the PIK3CA gene which is seemingly associated with better outcome (median OS: PIK3CA mutated 107.5 vs 45.5 months in wildtype PA). This exploratory molecular profiling of spinal cord astrocytomas allows to identify distinct subgroups by combining molecular markers and histomorphology. DMG-H3 tend to develop in adolescence with a similar dismal prognosis like GBM and HAP in the elderly. We here describe spinal HAP with a distinct molecular profile for the first time.