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).

Significance of molecular diagnostics for therapeutic decision-making in recurrent glioma.

Background: Targeted therapies have substantially improved survival in cancer patients with malignancies outside the brain. Whether in-depth analysis for molecular alterations may also offer therapeutic avenues in primary brain tumors remains unclear. We herein present our institutional experience for glioma patients discussed in our interdisciplinary molecular tumor board (MTB) implemented at the Comprehensive Cancer Center Munich (LMU). Methods: We retrospectively searched the database of the MTB for all recurrent glioma patients after previous therapy. Recommendations were based on next-generation sequencing results of individual patient's tumor tissue. Clinical and molecular information, previous therapy regimens, and outcome parameters were collected. Results: Overall, 73 consecutive recurrent glioma patients were identified. In the median, advanced molecular testing was initiated with the third tumor recurrence. The median turnaround time between initiation of molecular profiling and MTB case discussion was 48 ± 75 days (range: 32-536 days). Targetable mutations were found for 50 recurrent glioma patients (68.5%). IDH1 mutation (27/73; 37%), epidermal growth factor receptor amplification (19/73; 26%), and NF1 mutation (8/73; 11%) were the most detected alterations and a molecular-based treatment recommendation could be made for all of them. Therapeutic recommendations were implemented in 12 cases (24%) and one-third of these heavily pretreated patients experienced clinical benefit with at least disease stabilization. Conclusions: In-depth molecular analysis of tumor tissue may guide targeted therapy also in brain tumor patients and considerable antitumor effects might be observed in selected cases. However, future studies to corroborate our results are needed.

Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

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.

Translocator protein (18kDa) TSPO: a new diagnostic or therapeutic target for stress-related disorders?

Efficient treatment of stress-related disorders, such as depression, is still a major challenge. The onset of antidepressant drug action is generally quite slow, while the anxiolytic action of benzodiazepines is considerably faster. However, their long-term use is impaired by tolerance development, abuse liability and cognitive impairment. Benzodiazepines act as positive allosteric modulators of É£-aminobutyric acid type A (GABAA) receptors. 3α-reduced neurosteroids such as allopregnanolone also are positive allosteric GABAA receptor modulators, however, through a site different from that targeted by benzodiazepines. Recently, the administration of neurosteroids such as brexanolone or zuranolone has been shown to rapidly ameliorate symptoms in post-partum depression or major depressive disorder. An attractive alternative to the administration of exogenous neurosteroids is promoting endogenous neurosteroidogenesis via the translocator protein 18k Da (TSPO). TSPO is a transmembrane protein located primarily in mitochondria, which mediates numerous biological functions, e.g., steroidogenesis and mitochondrial bioenergetics. TSPO ligands have been used in positron emission tomography (PET) studies as putative markers of microglia activation and neuroinflammation in stress-related disorders. Moreover, TSPO ligands have been shown to modulate neuroplasticity and to elicit antidepressant and anxiolytic therapeutic effects in animals and humans. As such, TSPO may open new avenues for understanding the pathophysiology of stress-related disorders and for the development of novel treatment options.

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.

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.

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.

L-type amino acid transporter (LAT) 1 expression in 18F-FET-negative gliomas.

BACKGROUND: O-(2-[18F]-fluoroethyl)-L-tyrosine (18F-FET) is a highly sensitive PET tracer for glioma imaging, and its uptake is suggested to be driven by an overexpression of the L-type amino-acid transporter 1 (LAT1). However, 30% of low- and 5% of high-grade gliomas do not present enhanced 18F-FET uptake at primary diagnosis ("18F-FET-negative gliomas") and the pathophysiologic basis for this phenomenon remains unclear. The aim of this study was to determine the expression of LAT1 in a homogeneous group of newly diagnosed 18F-FET-negative gliomas and to compare them to a matched group of 18F-FET-positive gliomas. Forty newly diagnosed IDH-mutant astrocytomas without 1p/19q codeletion were evaluated (n = 20 18F-FET-negative (tumour-to-background ratio (TBR) < 1.6), n = 20 18F-FET-positive gliomas (TBR > 1.6)). LAT1 immunohistochemistry (IHC) was performed using SLC7A5/LAT1 antibody. The percentage of LAT1-positive tumour cells (%) and the staining intensity (range 0-2) were multiplied to an overall score (H-score; range 0-200) and correlated to PET findings as well as progression-free survival (PFS). RESULTS: IHC staining of LAT1 expression was positive in both, 18F-FET-positive as well as 18F-FET-negative gliomas. No differences were found between the 18F-FET-negative and 18F-FET-positive group with regard to percentage of LAT1-positive tumour cells, staining intensity or H-score. Interestingly, the LAT1 expression showed a significant negative correlation with the PFS (p = 0.031), whereas no significant correlation was found for TBRmax, neither in the overall group nor in the 18F-FET-positive group only (p = 0.651 and p = 0.140). CONCLUSION: Although LAT1 is reported to mediate the uptake of 18F-FET into tumour cells, the levels of LAT1 expression do not correlate with the levels of 18F-FET uptake in IDH-mutant astrocytomas. In particular, the lack of tracer uptake in 18F-FET-negative gliomas cannot be explained by a reduced LAT1 expression. A higher LAT1 expression in IDH-mutant astrocytomas seems to be associated with a short PFS. Further studies regarding mechanisms influencing the uptake of 18F-FET are necessary.

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.

Multifocal high-grade glioma radiotherapy safety and efficacy.

BACKGROUND: Multifocal manifestation of high-grade glioma is a rare disease with very unfavourable prognosis. The pathogenesis of multifocal glioma and pathophysiological differences to unifocal glioma are not fully understood. The optimal treatment of patients suffering from multifocal high-grade glioma is not defined in the current guidelines, therefore individual case series may be helpful as guidance for clinical decision-making. METHODS: Patients with multifocal high-grade glioma treated with conventionally fractionated radiation therapy (RT) in our institution with or without concomitant chemotherapy between April 2011 and April 2019 were retrospectively analysed. Multifocality was neuroradiologically assessed and defined as at least two independent contrast-enhancing foci in the MRI T1 contrast-enhanced sequence. IDH mutational status and MGMT methylation status were assessed from histopathology records. GTV, PTV as well as the V30Gy, V45Gy and D2% volumes of the brain were analysed. Overall and progression-free survival were calculated from the diagnosis until death and from start of radiation therapy until diagnosis of progression of disease in MRI for all patients. RESULTS: 20 multifocal glioma cases (18 IDH wild-type glioblastoma cases, one diffuse astrocytic glioma, IDH wild-type case with molecular features of glioblastoma and one anaplastic astrocytoma, IDH wild-type case) were included into the analysis. Resection was performed in two cases and stereotactic biopsy only in 18 cases before the start of radiation therapy. At the start of radiation therapy patients were 61 years old in median (range 42-84 years). Histopathological examination showed IDH wild-type in all cases and MGMT promotor methylation in 11 cases (55%). Prescription schedules were 60 Gy (2 Gy × 30), 59.4 Gy (1.8 Gy × 33), 55 Gy (2.2 Gy × 25) and 50 Gy (2.5 Gy × 20) in 15, three, one and one cases, respectively. Concomitant temozolomide chemotherapy was applied in 16 cases, combined temozolomide/lomustine chemotherapy was applied in one case and concomitant bevacizumab therapy in one case. Median number of GTVs was three. Median volume of the sum of the GTVs was 26 cm3. Median volume of the PTV was 425.7 cm3 and median PTV to brain ratio 32.8 percent. Median D2% of the brain was 61.5 Gy (range 51.2-62.7) and median V30Gy and V45 of the brain were 59.9 percent (range 33-79.7) and 40.7 percent (range 14.9-64.1), respectively. Median survival was eight months (95% KI 3.6-12.4 months) and median progression free survival after initiation of RT five months (95% CI 2.8-7.2 months). Grade 2 toxicities were detected in eight cases and grade 3 toxicities in four cases consisting of increasing edema in three cases and one new-onset seizure. One grade 4 toxicity was detected, which was febrile neutropenia related to concomitant chemotherapy. CONCLUSION: Conventionally fractionated RT with concomitant chemotherapy could safely be applied in multifocal high-grade glioma in this case series despite large irradiation treatment fields.

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.