PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cell in lung cancer brain metastasis.

BACKGROUND: Lung cancer brain metastasis has a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cell show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD-1 antibodies on CAR T-cell in treating lung cancer brain metastasis. METHODS: We utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy, enabling in vivo characterization of red fluorescent tumor cells and CAR T-cell at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis lung carcinoma cells (EpCAM/tdtLL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cell were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control. RESULTS: Compared to controls receiving T-cell lacking a CAR, mice receiving EpCAM-directed CAR T-cell showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD-1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect. CONCLUSION: CAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD-1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors.

Determinants of long-term survival in patients with IDH-mutant gliomas.

BACKGROUND: Survival times of patients with IDH-mutant gliomas are variable and can extend to decades. Many studies provide progression-free rather than overall survival times and prognostic factors remain ill-defined. Here we explored characteristics of short- and long-term survivors within a cohort of patients with extended follow-up. METHODS: This single-center, case-control study included 86 patients diagnosed between 1998 and 2023 who either died within 6 years after diagnosis or survived at least 15 years. Patient characteristics and prognostic factors were stratified by short- (< 6 years) versus long-term (≥ 15 years) survival. RESULTS: Forty-seven patients (55%) diagnosed with astrocytoma and 39 patients (45%) with oligodendroglioma were included retrospectively. Median follow-up of the survivors was 16.6 years (range 15-28.9). Thirty-four deaths (40%) had been reported at database closure. Long-term survival was associated with CNS WHO grade 2 (p < 0.01), smaller tumor volumes (p = 0.01), lack of contrast enhancement (p < 0.01), wait-and-scan strategies (p < 0.01) and female sex (p = 0.04). In multivariate analyses for oligodendroglioma, larger T2 tumor volumes were associated with shorter survival (HR 1.02; 95% CI 1.01-1.05; p = 0.04). In patients with astrocytoma, lack of contrast enhancement (HR 0.38; 95% CI 0.15-0.94; p = 0.04) and wait-and-scan strategies (HR 5.75; 95% CI 1.66-26.61; p = 0.01) were associated with longer survival. CONCLUSION: Large T2 tumor volume and contrast enhancement may be important risk factors for shorter survival, while age might be of lesser importance. Wait-and-scan strategies may yield excellent long-term survival in some patients with astrocytoma.

Diagnosis of Hirschsprung disease by analyzing acetylcholinesterase staining using artificial intelligence.

OBJECTIVES: Classical Hirschsprung disease (HD) is defined by the absence of ganglion cells in the rectosigmoid colon. The diagnosis is made from rectal biopsy, which reveals the aganglionosis and the presence of cholinergic hyperinnervation. However, depending on the method of rectal biopsy, the quality of the specimens and the related diagnostic accuracy varies substantially. To facilitate and objectify the diagnosis of HD, we investigated whether software-based identification of cholinergic hyperinnervation in digitalized histopathology slides is suitable for distinguishing healthy individuals from affected individuals. METHODS: N = 190 samples of 112 patients who underwent open surgical rectal biopsy at our pediatric surgery center between 2009 and 2019 were included in this study. Acetylcholinesterase (AChE) stained slides of these samples were collected and digitalized via slide scanning and analyzed using two digital imaging software programs (HALO, QuPath). The AChE-positive staining area in the mucosal layers of the intestinal wall was determined. In the next step machine learning was employed to identify patterns of cholinergic hyperinnervation. RESULTS: The area of AChE-positive staining was greater in HD patients compared to healthy individuals (p < 0.0001). Artificial intelligence-based assessment of parasympathetic hyperinnervation identified HD with a high precision (area under the curve [AUC] 0.96). The accuracy of the prediction model increased when nonrectal samples were excluded (AUC 0.993). CONCLUSIONS: Software-assisted machine-learning analysis of AChE staining is suitable to improve the diagnostic accuracy of HD.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Perifocal edema is a risk factor for preoperative seizures in patients with meningioma WHO grade 2 and 3.

BACKGROUND: Patients with intracranial meningiomas frequently suffer from tumor-related seizures prior to resection, impacting patients' quality of life. We aimed to elaborate on incidence and predictors for seizures in a patient cohort with meningiomas WHO grade 2 and 3. METHODS: We retrospectively searched for patients with meningioma WHO grade 2 and 3 according to the 2021 WHO classification undergoing tumor resection. Clinical, histopathological and imaging findings were collected and correlated with preoperative seizure development. Tumor and edema volumes were quantified. RESULTS: Ninety-five patients with a mean age of 59.5 ± 16.0 years were included. Most tumors (86/95, 90.5%) were classified as atypical meningioma WHO grade 2. Nine of 95 tumors (9.5%) corresponded to anaplastic meningiomas WHO grade 3, including six patients harboring TERT promoter mutations. Meningiomas were most frequently located at the convexity in 38/95 patients (40.0%). Twenty-eight of 95 patients (29.5%) experienced preoperative seizures. Peritumoral edema was detected in 62/95 patients (65.3%) with a median volume of 9 cm3 (IR: 0-54 cm3). Presence of peritumoral edema but not age, tumor localization, TERT promoter mutation, brain invasion or WHO grading was associated with incidence of preoperative seizures, as confirmed in multivariate analysis (OR: 6.61, 95% CI: 1.18, 58.12, p = *0.049). Postoperative freedom of seizures was achieved in 91/95 patients (95.8%). CONCLUSIONS: Preoperative seizures were frequently encountered in about every third patient with meningioma WHO grade 2 or 3. Patients presenting with peritumoral edema on preoperative imaging are at particular risk for developing tumor-related seizures. Tumor resection was highly effective in achieving seizure freedom.

A framework for standardised tissue sampling and processing during resection of diffuse intracranial glioma: joint recommendations from four RANO groups.

Surgical resection represents the standard of care for people with newly diagnosed diffuse gliomas, and the neuropathological and molecular profile of the resected tissue guides clinical management and forms the basis for research. The Response Assessment in Neuro-Oncology (RANO) consortium is an international, multidisciplinary effort that aims to standardise research practice in neuro-oncology. These recommendations represent a multidisciplinary consensus from the four RANO groups: RANO resect, RANO recurrent glioblastoma, RANO radiotherapy, and RANO/PET for a standardised workflow to achieve a representative tumour evaluation in a disease characterised by intratumoural heterogeneity, including recommendations on which tumour regions should be surgically sampled, how to define those regions on the basis of preoperative imaging, and the optimal sample volume. Practical recommendations for tissue sampling are given for people with low-grade and high-grade gliomas, as well as for people with newly diagnosed and recurrent disease. Sampling of liquid biopsies is also addressed. A standardised workflow for subsequent handling of the resected tissue is proposed to avoid information loss due to decreasing tissue quality or insufficient clinical information. The recommendations offer a framework for prospective biobanking studies.

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).

Analysing cerebrospinal fluid with explainable deep learning: From diagnostics to insights.

AIM: Analysis of cerebrospinal fluid (CSF) is essential for diagnostic workup of patients with neurological diseases and includes differential cell typing. The current gold standard is based on microscopic examination by specialised technicians and neuropathologists, which is time-consuming, labour-intensive and subjective. METHODS: We, therefore, developed an image analysis approach based on expert annotations of 123,181 digitised CSF objects from 78 patients corresponding to 15 clinically relevant categories and trained a multiclass convolutional neural network (CNN). RESULTS: The CNN classified the 15 categories with high accuracy (mean AUC 97.3%). By using explainable artificial intelligence (XAI), we demonstrate that the CNN identified meaningful cellular substructures in CSF cells recapitulating human pattern recognition. Based on the evaluation of 511 cells selected from 12 different CSF samples, we validated the CNN by comparing it with seven board-certified neuropathologists blinded for clinical information. Inter-rater agreement between the CNN and the ground truth was non-inferior (Krippendorff's alpha 0.79) compared with the agreement of seven human raters and the ground truth (mean Krippendorff's alpha 0.72, range 0.56-0.81). The CNN assigned the correct diagnostic label (inflammatory, haemorrhagic or neoplastic) in 10 out of 11 clinical samples, compared with 7-11 out of 11 by human raters. CONCLUSIONS: Our approach provides the basis to overcome current limitations in automated cell classification for routine diagnostics and demonstrates how a visual explanation framework can connect machine decision-making with cell properties and thus provide a novel versatile and quantitative method for investigating CSF manifestations of various neurological diseases.

Ganglioglioma with adverse clinical outcome and atypical histopathological features were defined by alterations in PTPN11/KRAS/NF1 and other RAS-/MAP-Kinase pathway genes.

Exome-wide sequencing studies recently described PTPN11 as a novel brain somatic epilepsy gene. In contrast, germline mutations of PTPN11 are known to cause Noonan syndrome, a multisystem disorder characterized by abnormal facial features, developmental delay, and sporadically, also brain tumors. Herein, we performed a deep phenotype-genotype analysis of a comprehensive series of ganglioglioma (GG) with brain somatic alterations of the PTPN11/KRAS/NF1 genes compared to GG with common MAP-Kinase signaling pathway alterations, i.e., BRAFV600E. Seventy-two GG were submitted to whole exome sequencing and genotyping and 84 low grade epilepsy associated tumors (LEAT) to DNA-methylation analysis. In 28 tumours, both analyses were available from the same sample. Clinical data were retrieved from hospital files including disease onset, age at surgery, brain localization, and seizure outcome. A comprehensive histopathology staining panel was available in all cases. We identified eight GG with PTPN11 alterations, copy number variant (CNV) gains of chromosome 12, and the commonality of additional CNV gains in NF1, KRAS, FGFR4 and RHEB, as well as BRAFV600E alterations. Histopathology revealed an atypical glio-neuronal phenotype with subarachnoidal tumor spread and large, pleomorphic, and multinuclear cellular features. Only three out of eight patients with GG and PTPN11/KRAS/NF1 alterations were free of disabling-seizures 2 years after surgery (38% had Engel I). This was remarkably different from our series of GG with only BRAFV600E mutations (85% had Engel I). Unsupervised cluster analysis of DNA methylation arrays separated these tumours from well-established LEAT categories. Our data point to a subgroup of GG with cellular atypia in glial and neuronal cell components, adverse postsurgical outcome, and genetically characterized by complex alterations in PTPN11 and other RAS-/MAP-Kinase and/or mTOR signaling pathways. These findings need prospective validation in clinical practice as they argue for an adaptation of the WHO grading system in developmental, glio-neuronal tumors associated with early onset focal epilepsy.

The Acute Superficial Siderosis Syndrome - Clinical Entity, Imaging Findings, and Histopathology.

Superficial siderosis is a consequence of repetitive bleeding into the subarachnoid space, leading to toxic iron and hemosiderin deposits on the surface of the brain and spine. The clinical and radiological phenotypes of superficial siderosis are known to manifest over long time intervals. In contrast, this study defines the "acute superficial siderosis syndrome" and illustrates typical imaging and histopathological findings of this entity. We describe the case of a 61-year-old male patient who was diagnosed with a melanoma metastasis in the right frontal cortex in February 2019. Within a few weeks he developed a progressive syndrome characterized by cerebellar ataxia, gait disturbance, signs of myelopathy, and radiculopathy. MRI revealed ongoing hemorrhage from the metastasis into the lateral ventricle and the subarachnoid space. A semiquantitative assessment of three subsequent MRI within an 8-week period documented the rapid development of superficial siderosis along the surface of the cerebellum, the brain stem, and the lower parts of the supratentorial regions on T2*-weighted sequences. The diagnosis of a superficial siderosis was histopathologically confirmed by identifying iron and hemosiderin deposits on the cortex along with astrogliosis. The recognition of this "acute superficial siderosis syndrome" triggered surgical removal of the hemorrhagic metastasis. Based on a single case presentation, we define the "acute superficial siderosis syndrome" as a clinical entity and describe the radiological and histopathological characteristics of this entity. Early recognition of this syndrome may allow timely elimination of the bleeding source, in order to prevent further clinical deterioration.

Treatment benefit in patients aged 80 years or older with biopsy-proven and non-resected glioblastoma is dependent on MGMT promoter methylation status.

PURPOSE: Glioblastoma is associated with especially poor outcome in the elderly. It is unclear if patients aged ≥80 years benefit from tumor-specific therapy as opposed to receiving best supportive care (BSC) only. METHODS: Patients with IDH-wildtype glioblastoma (WHO 2021), aged ≥80 years, and diagnosed by biopsy between 2010 and 2022 were included. Patient characteristics and clinical parameters were assessed. Uni- and multivariate analyses were performed. RESULTS: 76 patients with a median age of 82 (range 80-89) and a median initial KPS of 80 (range 50-90) were included. Tumor-specific therapy was initiated in 52 patients (68%). 22 patients (29%) received temozolomide monotherapy, 23 patients (30%) were treated with radiotherapy (RT) alone and 7 patients (9%) received combination therapies. In 24 patients (32%), tumor-specific therapy was omitted in lieu of BSC. Overall survival (OS) was longer in patients receiving tumor-specific therapy (5.4 vs. 3.3 months, p < 0.001). Molecular stratification showed that the survival benefit was owed to patients with MGMT promoter methylation (MGMTpos) who received tumor-specific therapy as opposed to BSC (6.2 vs. 2.6 months, p < 0.001), especially to those with better clinical status and no initial polypharmacy. Patients with unmethylated MGMT promoter (MGMTneg) did not benefit from tumor-specific therapy (3.6 vs. 3.7 months, p = 0.18). In multivariate analyses, better clinical status and MGMT promoter methylation were associated with prolonged survival (p < 0.01 and p = 0.01). CONCLUSION: Benefit from tumor-specific treatment in patients with newly diagnosed glioblastoma aged ≥80 years might be restricted to MGMTpos patients, especially to those with good clinical status and no polypharmacy.

MR imaging profile and histopathological characteristics of tumour vasculature, cell density and proliferation rate define two distinct growth patterns of human brain metastases from lung cancer.

PURPOSE: Non-invasive prediction of the tumour of origin giving rise to brain metastases (BMs) using MRI measurements obtained in radiological routine and elucidating the biological basis by matched histopathological analysis. METHODS: Preoperative MRI and histological parameters of 95 BM patients (female, 50; mean age 59.6 ± 11.5 years) suffering from different primary tumours were retrospectively analysed. MR features were assessed by region of interest (ROI) measurements of signal intensities on unenhanced T1-, T2-, diffusion-weighted imaging and apparent diffusion coefficient (ADC) normalised to an internal reference ROI. Furthermore, we assessed BM size and oedema as well as cell density, proliferation rate, microvessel density and vessel area as histopathological parameters. RESULTS: Applying recursive partitioning conditional inference trees, only histopathological parameters could stratify the primary tumour entities. We identified two distinct BM growth patterns depending on their proliferative status: Ki67high BMs were larger (p = 0.02), showed less peritumoural oedema (p = 0.02) and showed a trend towards higher cell density (p = 0.05). Furthermore, Ki67high BMs were associated with higher DWI signals (p = 0.03) and reduced ADC values (p = 0.004). Vessel density was strongly reduced in Ki67high BM (p < 0.001). These features differentiated between lung cancer BM entities (p ≤ 0.03 for all features) with SCLCs representing predominantly the Ki67high group, while NSCLCs rather matching with Ki67low features. CONCLUSION: Interpretable and easy to obtain MRI features may not be sufficient to predict directly the primary tumour entity of BM but seem to have the potential to aid differentiating high- and low-proliferative BMs, such as SCLC and NSCLC.

Tissue Shrinkage of Resected Specimens in Hirschsprung's Disease: Why Pediatric Surgeons Think the Bowel Specimen was Longer Than Indicated in the Pathology Report.

BACKGROUND: Hirschsprung disease (HD) is an aganglionosis of variable length starting at the rectosigmoid colon with surgery as sole therapeutic option. The length of the resected bowel segment is a crucial information for the treating surgeons and influences the prognosis of the patient. It is often artificially altered due to post operative tissue shrinkage. The objective of this study is to quantify the extent tissue shrinkage of HD specimens. MATERIAL AND METHODS: Colorectal HD specimens were measured at the time of surgery and at the time of cut-up, either fresh or after formalin fixation and statistically analyzed. RESULTS: Sixteen colorectal specimens were included. Following formalin fixation the specimen length decreased by 22.7% (P < .001). Without formalin fixation the specimens shrank by an average of 24.9% (P = .05). There was no significant difference in the extent of tissue shrinkage with or without formalin fixation (P = .76). CONCLUSION: This study showed that there is significant tissue shrinkage in HD specimens. The 2 different cohorts revealed that tissue shrinkage is mostly caused by tissue retraction/alteration after organ removal but also to a lesser extent by fixation with formalin. Surgeons and (neuro-)pathologists should be aware of the sizeable shrinking artifact to avoid unnecessary confusion.

Shunt dependency in supratentorial intraventricular tumors depends on the extent of tumor resection.

BACKGROUND: Supratentorial intraventricular tumors (SIVTs) are rare lesions of various entities characteristically presenting with hydrocephalus and often posing a surgical challenge due to their deep-seated localization. We aimed to elaborate on shunt dependency after tumor resection, clinical characteristics, and perioperative morbidity. METHODS: We retrospectively searched the institutional database for patients with supratentorial intraventricular tumors treated at the Department of Neurosurgery of the Ludwig-Maximilians-University in Munich, Germany, between 2014 and 2022. RESULTS: We identified 59 patients with over 20 different SIVT entities, most often subependymoma (8/59 patients, 14%). Mean age at diagnosis was 41 ± 3 years. Hydrocephalus and visual symptoms were observed in 37/59 (63%) and 10/59 (17%) patients, respectively. Microsurgical tumor resection was provided in 46/59 patients (78%) with complete resection in 33/46 patients (72%). Persistent postoperative neurological deficits were encountered in 3/46 patients (7%) and generally mild in nature. Complete tumor resection was associated with less permanent shunting in comparison to incomplete tumor resection, irrespective of tumor histology (6% versus 31%, p = 0.025). Stereotactic biopsy was utilized in 13/59 patients (22%), including 5 patients who received synchronous internal shunt implantation for symptomatic hydrocephalus. Median overall survival was not reached and did not differ between patients with or without open resection. CONCLUSIONS: SIVT patients display a high risk of developing hydrocephalus and visual symptoms. Complete resection of SIVTs can often be achieved, preventing the need for long-term shunting. Stereotactic biopsy along with internal shunting represents an effective approach to establish diagnosis and ameliorate symptoms if resection cannot be safely performed. Due to the rather benign histology, the outcome appears excellent when adjuvant therapy is provided.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood-brain barrier in acute ischemic stroke.

Blood-brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

Molecular matched targeted therapies for primary brain tumors-a single center retrospective analysis.

PURPOSE: Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy. METHODS: Via retrospective analysis, we identified primary brain tumor patients who were treated off-label with a targeted therapy at the University Hospital Frankfurt, Goethe University. We analyzed which types of molecular alterations were utilized to guide molecular off-label therapies and the diagnostic procedures for their assessment during the period from 2008 to 2021. Data on tolerability and outcomes were collected. RESULTS: 413 off-label therapies were identified with an increasing annual number for the interval after 2016. 37 interventions (9%) were targeted therapies based on molecular markers. Glioma and meningioma were the most frequent entities treated with molecular matched targeted therapies. Rare entities comprised e.g. medulloblastoma and papillary craniopharyngeoma. Molecular targeted approaches included checkpoint inhibitors, inhibitors of mTOR, FGFR, ALK, MET, ROS1, PIK3CA, CDK4/6, BRAF/MEK and PARP. Responses in the first follow-up MRI were partial response (13.5%), stable disease (29.7%) and progressive disease (46.0%). There were no new safety signals. Adverse events with fatal outcome (CTCAE grade 5) were not observed. Only, two patients discontinued treatment due to side effects. Median progression-free and overall survival were 9.1/18 months in patients with at least stable disease, and 1.8/3.6 months in those with progressive disease at the first follow-up MRI. CONCLUSION: A broad range of actionable alterations was targeted with available molecular therapeutics. However, efficacy was largely observed in entities with paradigmatic oncogenic drivers, in particular with BRAF mutations. Further research on biomarker-informed molecular matched therapies is urgently necessary.

Immune profile and radiological characteristics of progressive multifocal leukoencephalopathy.

BACKGROUND AND PURPOSE: Progressive multifocal leukoencephalopathy (PML) constitutes a severe disease with increasing incidence, mostly in the context of immunosuppressive therapies. A detailed understanding of immune response in PML appears critical for the treatment strategy. The aim was a comprehensive immunoprofiling and radiological characterization of magnetic resonance imaging (MRI) defined PML variants. METHODS: All biopsy-confirmed PML patients (n = 15) treated in our department between January 2004 and July 2019 were retrospectively analysed. Data from MRI, histology as well as detailed clinical and outcome data were collected. The MRI-defined variants of classical (cPML) and inflammatory (iPML) PML were discriminated based on the intensity of gadolinium enhancement. In these PML variants, intensity and localization (perivascular vs. parenchymal) of inflammation in MRI and histology as well as the cellular composition by immunohistochemistry were assessed. The size of the demyelinating lesions was correlated with immune cell infiltration. RESULTS: Patients with MRI-defined iPML showed a stronger intensity of inflammation with an increased lymphocyte infiltration on histological level. Also, iPML was characterized by a predominantly perivascular inflammation. However, cPML patients also demonstrated certain inflammatory tissue alterations. Infiltration of CD163-positive microglia and macrophage (M/M) subtypes correlated with PML lesion size. CONCLUSIONS: The non-invasive MRI-based discrimination of PML variants allows for an estimation of inflammatory tissue alterations, although exhibiting limitations in MRI-defined cPML. The association of a distinct phagocytic M/M subtype with the extent of demyelination might reflect disease progression.

Short-term fasting in glioma patients: analysis of diet diaries and metabolic parameters of the ERGO2 trial.

PURPOSE: The prospective, randomized ERGO2 trial investigated the effect of calorie-restricted ketogenic diet and intermittent fasting (KD-IF) on re-irradiation for recurrent brain tumors. The study did not meet its primary endpoint of improved progression-free survival in comparison to standard diet (SD). We here report the results of the quality of life/neurocognition and a detailed analysis of the diet diaries. METHODS: 50 patients were randomized 1:1 to re-irradiation combined with either SD or KD-IF. The KD-IF schedule included 3 days of ketogenic diet (KD: 21-23 kcal/kg/d, carbohydrate intake limited to 50 g/d), followed by 3 days of fasting and again 3 days of KD. Follow-up included examination of cognition, quality of life and serum samples. RESULTS: The 20 patients who completed KD-IF met the prespecified goals for calorie and carbohydrate restriction. Substantial decreases in leptin and insulin and an increase in uric acid were observed. The SD group, of note, had a lower calorie intake than expected (21 kcal/kg/d instead of 30 kcal/kg/d). Neither quality of life nor cognition were affected by the diet. Low glucose emerged as a significant prognostic parameter in a best responder analysis. CONCLUSION: The strict caloric goals of the ERGO2 trial were tolerated well by patients with recurrent brain cancer. The short diet schedule led to significant metabolic changes with low glucose emerging as a candidate marker of better prognosis. The unexpected lower calorie intake of the control group complicates the interpretation of the results. Clinicaltrials.gov number: NCT01754350; Registration: 21.12.2012.

TGF-ß activates pericytes via induction of the epithelial-to-mesenchymal transition protein SLUG in glioblastoma.

AIMS: In primary central nervous system tumours, epithelial-to-mesenchymal transition (EMT) gene expression is associated with increased malignancy. However, it has also been shown that EMT factors in gliomas are almost exclusively expressed by glioma vessel-associated pericytes (GA-Peris). In this study, we aimed to identify the mechanism of EMT in GA-Peris and its impact on angiogenic processes. METHODS: In glioma patients, vascular density and the expression of the pericytic markers platelet derived growth factor receptor (PDGFR)-ß and smooth muscle actin (αSMA) were examined in relation to the expression of the EMT transcription factor SLUG and were correlated with survival of patients with glioblastoma (GBM). Functional mechanisms of SLUG regulation and the effects on primary human brain vascular pericytes (HBVP) were studied in vitro by measuring proliferation, cell motility and growth characteristics. RESULTS: The number of PDGFR-ß- and αSMA-positive pericytes did not change with increased malignancy nor showed an association with the survival of GBM patients. However, SLUG-expressing pericytes displayed considerable morphological changes in GBM-associated vessels, and TGF-ß induced SLUG upregulation led to enhanced proliferation, motility and altered growth patterns in HBVP. Downregulation of SLUG or addition of a TGF-ß antagonising antibody abolished these effects. CONCLUSIONS: We provide evidence that in GA-Peris, elevated SLUG expression is mediated by TGF-ß, a cytokine secreted by most glioma cells, indicating that the latter actively modulate neovascularisation not only by modulating endothelial cells, but also by influencing pericytes. This process might be responsible for the formation of an unstructured tumour vasculature as well as for the breakdown of the blood-brain barrier in GBM.

Linking epigenetic signature and metabolic phenotype in IDH mutant and IDH wildtype diffuse glioma.

AIMS: Changes in metabolism are known to contribute to tumour phenotypes. If and how metabolic alterations in brain tumours contribute to patient outcome is still poorly understood. Epigenetics impact metabolism and mitochondrial function. The aim of this study is a characterisation of metabolic features in molecular subgroups of isocitrate dehydrogenase mutant (IDHmut) and isocitrate dehydrogenase wildtype (IDHwt) gliomas. METHODS: We employed DNA methylation pattern analyses with a special focus on metabolic genes, large-scale metabolism panel immunohistochemistry (IHC), qPCR-based determination of mitochondrial DNA copy number and immune cell content using IHC and deconvolution of DNA methylation data. We analysed molecularly characterised gliomas (n = 57) for in depth DNA methylation, a cohort of primary and recurrent gliomas (n = 22) for mitochondrial copy number and validated these results in a large glioma cohort (n = 293). Finally, we investigated the potential of metabolic markers in Bevacizumab (Bev)-treated gliomas (n = 29). RESULTS: DNA methylation patterns of metabolic genes successfully distinguished the molecular subtypes of IDHmut and IDHwt gliomas. Promoter methylation of lactate dehydrogenase A negatively correlated with protein expression and was associated with IDHmut gliomas. Mitochondrial DNA copy number was increased in IDHmut tumours and did not change in recurrent tumours. Hierarchical clustering based on metabolism panel IHC revealed distinct subclasses of IDHmut and IDHwt gliomas with an impact on patient outcome. Further quantification of these markers allowed for the prediction of survival under anti-angiogenic therapy. CONCLUSION: A mitochondrial signature was associated with increased survival in all analyses, which could indicate tumour subgroups with specific metabolic vulnerabilities.