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Children diagnosed with diffuse midline gliomas (DMG) have an extremely poor overall survival: 9-12 months from diagnosis with currently no curative treatment options. Given DMG molecular heterogeneity, surgical biopsies are needed for molecular profiling and as part of enrolment into molecular-based and precision medicine type clinical interventions. In this study, we describe the results of real time profiling and drug testing at the diffuse intrinsic pontine glioma/DMG Research Centre at University Children's Hospital Zurich.Biopsies were taken using a frame based stereotactic robot system (NeuroMate®, Renishaw) at University Children's Hospital Zurich. Tissue samples were evaluated to confirm diagnosis by H3K27M and H3K27 trimethylation loss. Genomic analyses were done using a variety of platforms (INFORM, Oncomine, UCSF500 gene panel). Cell lines were developed by mechanical tissue dissociation and verified by either sequencing or immunofluorescence staining confirming H3K27M mutation and used afterwards for drug testing.Twenty-five robot-assisted primary biopsies were successfully performed. Median hospital stay was 2 days (range 1-4 days). Nine low-passage patient-derived cells were developed, whereas 8 cell lines were used to inform response to clinically relevant drugs. Genome and RNA expression were used to further guide treatment strategies with targeted agents such as dual PI3K/mTOR inhibitor paxalisib.We established a systematic workflow for safe, robot-assisted brainstem biopsies and in-house tissue processing, followed by real-time drug testing. This provides valuable insights into tumour prognostic and individual treatment strategies targeting relevant vulnerabilities in these tumours in a clinically meaningful time frame.

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand–receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation.Significance:DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

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Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand–receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation. Significance: DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

PURPOSE Meningiomas are the most frequent primary intracranial tumors. Patient outcome varies widely from benign to highly aggressive, ultimately fatal courses. Reliable identification of risk of progression for individual patients is of pivotal importance. However, only biomarkers for highly aggressive tumors are established ( CDKN2A/B and TERT), whereas no molecularly based stratification exists for the broad spectrum of patients with low- and intermediate-risk meningioma. METHODS DNA methylation data and copy-number information were generated for 3,031 meningiomas (2,868 patients), and mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNVs), mutations, and WHO grading were analyzed. Prediction power for outcome was assessed in a retrospective cohort of 514 patients, validated on a retrospective cohort of 184, and on a prospective cohort of 287 multicenter cases. RESULTS Both CNV- and methylation family–based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with the WHO classification (c-indexes WHO 2016, CNV, and methylation family 0.699, 0.706, and 0.721, respectively). Merging all risk stratification approaches into an integrated molecular-morphologic score resulted in further substantial increase in accuracy (c-index 0.744). This integrated score consistently provided superior accuracy in all three cohorts, significantly outperforming WHO grading (c-index difference P = .005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (hazard ratio 4.34 [2.48-7.57] and 3.34 [1.28-8.72] retrospective and prospective validation cohorts, respectively). CONCLUSION Merging these layers of histologic and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision making for patients with meningioma on the basis of robust outcome prediction.

Glioblastoma is the most common yet deadliest primary brain cancer1. The neural behavior of glioblastoma, including the formation of synaptic circuitry and tumor microtubes, is increasingly understood to be pivotal for disease manifestation2–8. Nonetheless, the few approved treatments for glioblastoma target its oncological nature, while its neural vulnerabilities remain incompletely mapped and clinically unexploited. Here, we systematically survey the neural molecular dependencies and cellular heterogeneity across glioblastoma patients and diverse model systems. In 27 patient tumour samples taken directly after surgery, we identify a spectrum of cancer cell morphologies indicative of poor prognosis and discover a set of repurposable neuroactive drugs with consistent anti-glioblastoma efficacy. Glioblastoma cells exhibit functional dependencies on highly expressed neuroactive drug targets, while interpretable molecular machine learning (COSTAR) reveals their downstream convergence on AP-1-driven tumour suppression. This drug-target connectivity signature is confirmed by highly accurate in silico drug screening on >1 million compounds using COSTAR, as well as by multi-omic profiling of drug-treated glioblastoma cells. Thus, Ca2+-driven AP-1 pathway induction represents a tumour-intrinsic vulnerability at the intersection of oncogenesis and neural activity-dependent signaling. Opportunities for clinical translation of this neural vulnerability are epitomized by the antidepressant Vortioxetine synergizing with current standard of care treatments in vivo. Together, the results presented here provide a mechanistic foundation and conceptual framework for the treatment of glioblastoma based on its neural origins.

Background and Purpose: The classic presentation of chronic (stage III) hemorrhagic stroke lesions is a fluid-filled cavity. In one of the most commonly used animal models of intracerebral hemorrhage (ICH), we noticed additional solid material within the chronic lesion. We examined the composition of those chronic ICH lesions and compared them with human autopsy cases. Methods: ICH was induced in rats by the injection of collagenase in the striatum. Tissue sections after hematoma resolution corresponding to 3 different chronic time points—28, 42, and 73 to 85 days post-ICH—were selected. Human autopsy reports at the University Hospital of Zurich were searched between 1990 and 2019 for ICH, and 3 chronic cases were found. The rat and human sections were stained with a variety of histopathologic markers. Results: Extensive collagenous material was observed in the chronic lesion after hematoma resolution in both the rat model and human autopsy cases. Additional immunostaining revealed that the material consisted primarily of a loose network of collagen 3 intermingled with occasional GFAP (glial fibrillary acidic protein)-positive processes and collagen 4. Conclusions: A key feature of the chronic ICH lesion is a loose network of collagen 3. The collagenase rat model reproduces the morphology and composition of the chronic human ICH lesion. While identifying new features of ICH lesion pathology, these results are important for treatment and recovery strategies.

A nuclear protein of testis (NUT) carcinoma, also known as NUT midline carcinoma, is a rare subtype of squamous carcinoma known for its aggressive growth behaviour. It can form anywhere in the body. Although, it usually occurs along midline structures (head, neck, lungs). The authors present the first report of intrasellar NUT carcinoma with cavernous sinus infiltration in a 47-year-old patient. MRI showed an inhomogeneous, gadolinium-enhancing lesion with intra- and suprasellar growth, invasion of the cavernous sinus without clear differentiation from normal pituitary tissue. Given the lymphoma diagnosis in the frozen section and invasion of the cavernous sinus, the patient underwent endoscopic, transnasal, and transsphenoidal subtotal resection only. Local tumour and spinal metastases showed a good response to radio-chemotherapy. Despite combined radio-chemotherapy, the patient died of pulmonary insufficiency due to rapid progression of pulmonary metastasis 6 months after the initial diagnosis.

The transforming growth factor (TGF)-β signaling pathway has been recognized as a major factor in promoting the aggressive behavior of glioblastoma, isocitrate dehydrogenase-wildtype. However, there is little knowledge about the expression of TGF-β receptors in glioblastoma. Here, we studied the expression patterns of TGF-β receptor II (TGFβRII), type I receptors activin receptor-like kinase (ALK)-5, and ALK-1, as well as of the transcriptional regulators inhibitor of differentiation (Id) 2, Id3, and Id4 in human glioblastoma. The expression of TGFβRII, ALK-5, and ALK-1 varied greatly, with TGFβRII and ALK-5 being the most abundant and ALK-1 being the least expressed receptor. None of the 3 receptors was preferentially expressed by tumor vasculature as opposed to the tumor bulk, indicating tumor bulk-governed mechanisms of TGF-β signaling with regard to glioblastoma-associated angiogenesis. A positive correlation was found between ALK-1 and Id2, suggesting that Id2, broadly expressed in the tumor cells, is a downstream target of this receptor-dependent pathway. Furthermore, there was a trend for high expression of ALK-5 or Id2 to be associated with inferior overall survival. Hence, we propose that ALK-5 may be used for patient stratification in future anti-TGF-β treatment trials and that Id2 might be a potential target for anti-TGF-β interventions.

Venous thromboembolic events (VTEs) are a major complication in cancer patients, and therefore, also in brain cancer patients, anticoagulants are considered appropriate in the treatment of VTEs.Frequency, risk factors, and treatment of VTEs, as well as associated complications, were assessed in a population-based cohort of glioblastoma patients in the Canton of Zurich, Switzerland. Correlations between clinical data and survival were retrospectively analyzed using the log-rank test and Cox regression models.Four hundred fourteen glioblastoma patients with isocitrate dehydrogenase wild-type status were identified. VTEs were documented in 65 patients (15.7%). Median time from tumor diagnosis to the occurrence of a VTE was 1.8 months, and 27 patients were diagnosed with VTEs postoperatively (within 35 days; 42.2%). History of a prior VTE was more common in patients who developed VTEs than in those who did not (p = 0.004). Bevacizumab treatment at any time during the disease course was not associated with occurrence of VTEs (p = 0.593). Most patients with VTEs (n = 61, 93.8%) were treated with therapeutic anticoagulation. Complications occurred in 14 patients (23.0%), mainly intracranial hemorrhages (n = 7, 11.5%). Overall survival did not differ between patients diagnosed with VTEs and those who had no VTE (p = 0.139). Tumor progression was the major cause of death (n = 283, 90.7%), and only three patients (1.0%) died in association with acute VTEs.Venous thromboembolic events occurred early in the disease course, suggesting that the implementation of primary venous thromboembolism prophylaxis during first-line chemoradiotherapy could be explored in a randomized setting.

BACKGROUND CONTEXT Spinal arachnoid web (SAW) is a rare condition characterized by focal thickening of the arachnoid membrane causing displacement and compression of the spinal cord with progressive symptoms and neurological deficits. Recent reports and clinical experience suggest that SAW is a distinct entity with specific radiological findings and treatment strategies distinguishable from other arachnopathies and potential differential diagnoses. PURPOSE To better define the diagnostic and clinical features, treatment options and outcomes of surgically treated SAW. STUDY DESIGN Multicentric retrospective cohort study. PATIENT SAMPLE Twelve cases of SAW surgically treated at three different centers. OUTCOME MEASURES Self-reported and neurological outcome measurements (pain, sensory-motor deficits, vegetative dysfunctions) were assessed at follow-up timepoints. METHODS Retrospective review of prospectively collected data on all patients surgically treated for SAW from three participating neurosurgical centers between 2014 and 2020. Clinicopathological data, including neurological presentation, radiological and histological findings and outcome data were analyzed. RESULTS Twelve radiologically and surgically confirmed cases of SAW were analyzed. Mean patient age was 54.7 [±12.7], 67% were male. All SAWs were located in the posterior thoracic dural sac. On magnetic resonance imaging (MRI), the "scalpel sign" - a characteristic focal dorsal indentation of the spinal cord resembling a scalpel blade - was identified in all patients. A focal intramedullary syrinx was present in 83%. Preoperative clinical symptoms included signs of myelopathy, pain, weakness and sensory loss, most commonly affecting the trunk/upper back or lower extremities. Laminectomy or laminoplasty with intradural excision of the SAW was the surgical treatment of choice in all cases. Intraoperative ultrasound was valuable to visualize the cerebrospinal fluid (CSF) flow obstruction, confirm the SAW location before dura incision and to control adequacy of resection. After surgery, sensory loss and weakness in particular showed significant improvement. CONCLUSIONS The present study comprises the largest series of surgically treated SAW, underscoring the unique clinical, radiographic, histopathological, and surgical findings. We want to emphasize SAW being a distinct entity of spinal arachnopathy with a favorable long-term outcome if diagnosed correctly and treated surgically. Intraoperative ultrasound aids visualizing the SAW before dural incision, as well as verifying restored CSF flow after resection.

PURPOSE To assess the relationship between F-18-fluoro-ethyl-tyrosine positron emission tomography (FET-PET) parameters of relapsing oligodendroglioma and progression-free survival. MATERIAL AND METHODS The relationship of clinical parameters, FET-PET parameters (SUV$_{max}$, TBR$_{max}$, BTV$_{,}$ time-activity curves) and progression-free survival was analyzed using univariate and multivariate analysis in 42 adult patients with relapsing oligodendroglioma. Kaplan-Meier analysis was used to assess survival. RESULTS Patients who did not undergo surgical resection of their relapsing tumor had significantly lower PFS if the tumor exhibited an SUV$_{max}$ above 3.40 than those with an SUV$_{max}$ below 3.40 (13.1 ± 2.3 months vs. 47.3 ± 6.0 months, respectively, p

PURPOSE To evaluate whether F-fluoroethyltyrosine (FET) PET can discriminate progression from pseudoprogression of brain metastases in patients with non-small cell lung cancer undergoing immunotherapy and radiotherapy to the brain. METHODS Retrospective analysis of F-FET PET scans in cases with documented progression of brain metastases on MRI in a cohort of 53 patients with non-small cell lung cancer receiving immune-checkpoint inhibitors and radiotherapy of brain metastases at the University Hospital of Zurich from June 2015 until January 2019. Response to radiotherapy was assessed by MRI. In case of equivocal findings and/or radiological progression in clinically asymptomatic patients, further assessment with F-FET PET was performed. RESULTS From the cohort of 53 patients, the restaging MRI showed in 30 patients (56.6%) progression of at least 1 treated metastasis. Thereof, F-FET PET was performed in 11 patients, based on the absence of neurological symptoms or presence of systemic response and physicians' decision. F-FET PET correctly identified pseudoprogression in 9 of 11 patients (81.8%). In patients who did not undergo F-FET PET, 5 of 19 (26.3%) were diagnosed with pseudoprogression. CONCLUSIONS Pseudoprogression of brain metastases occurred in 50% of patients diagnosed with progression on MRI. F-FET PET may help differentiate pseudoprogression from real progression in order to avoid discontinuation of effective therapy or unneeded interventions.

Pituicytoma (PITUI), granular cell tumor (GCT), and spindle cell oncocytoma (SCO) are rare tumors of the posterior pituitary. Histologically, they may be challenging to distinguish and have been proposed to represent a histological spectrum of a single entity. We performed targeted next-generation sequencing, DNA methylation profiling, and copy number analysis on 47 tumors (14 PITUI; 12 GCT; 21 SCO) to investigate molecular features and explore possibilities of clinically meaningful tumor subclassification. We detected two main epigenomic subgroups by unsupervised clustering of DNA methylation data, though the overall methylation differences were subtle. The largest group (n = 23) contained most PITUIs and a subset of SCOs and was enriched for pathogenic mutations within genes in the MAPK/PI3K pathways (12/17 [71%] of sequenced tumors: FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1)) and two with accompanying TERT promoter mutation. The second group (n = 16) contained most GCTs and a subset of SCOs, all of which mostly lacked identifiable genetic drivers. Outcome analysis demonstrated that the presence of chromosomal imbalances was significantly associated with reduced progression-free survival especially within the combined PITUI and SCO group (p = 0.031). In summary, we observed only subtle DNA methylation differences between posterior pituitary tumors, indicating that these tumors may be best classified as subtypes of a single entity. Nevertheless, our data indicate differences in mutation patterns and clinical outcome. For a clinically meaningful subclassification, we propose a combined histo-molecular approach into three subtypes: one subtype is defined by granular cell histology, scarcity of identifiable oncogenic mutations, and favorable outcome. The other two subtypes have either SCO or PITUI histology but are segregated by chromosomal copy number profile into a favorable group (no copy number changes) and a less favorable group (copy number imbalances present). Both of the latter groups have recurrent MAPK/PI3K genetic alterations that represent potential therapeutic targets.

Background Invasive aspergillosis of the central nervous system is a rare but increasingly prevalent disease. We present the unusual case of an immunosuppressed patient suffering from unexpected superinfected invasive aspergillosis with cerebral, pulmonal, and adrenal manifestations, mimicking a metastasized bronchial carcinoma. This report reveals the importance of including aspergillosis in the differential diagnosis of a cerebral mass lesion in the light of unspecific clinical findings. Case presentation A 58-year-old immunocompromised female presented to our emergency department with a single tonic-clonic seizure. Imaging showed a ring enhancing cerebral mass with perifocal edema and evidence of two smaller additional hemorrhagic cerebral lesions. In the setting of a mass lesion in the lung, and additional nodular lesions in the left adrenal gland the diagnosis of a metastasized bronchus carcinoma was suspected and the cerebral mass resected. However, histology did not reveal any evidence for a neoplastic lesion but septate hyphae consistent with aspergillus instead and microbiological cultures confirmed concomitant staphylococcal infection. Conclusions A high index of suspicion for aspergillus infection should be maintained in the setting of immunosuppression. Clinical and radiological findings are often unspecific and even misleading. Definite confirmation usually relies on tissue diagnosis with histochemical stains. Surgical resection is crucial for establishing the diagnosis and guiding therapy with targeted antifungal medications.

BACKGROUND Alveolar echinococcosis is a rare condition, but living or working in a rural environment is a substantial risk factor. The liver is the organ primarily affected, with additional extrahepatic manifestations in approximately 25% of cases. Primary extrahepatic disease is rare, and isolated cerebral involvement is extremely unusual. OBSERVATIONS The authors described an illustrative case of isolated cerebral alveolar echinococcosis in an immunocompetent farmer. Magnetic resonance imaging of the brain showed a predominantly cystic lesion with perifocal edema and a “bunch of grapes” appearance in the left frontal lobe. Histology revealed sharply demarcated fragments of a fibrous cyst wall accompanied by marked inflammation and necrosis. Higher magnification showed remnants of protoscolices with hooklets and calcified corpuscles. Immunohistochemistry and polymerase chain reaction (PCR) analysis confirmed the diagnosis of cerebral alveolar echinococcosis. Interestingly, serology and thoracic and abdominal computed tomography results were negative, indicative of an isolated primary extrahepatic manifestation. LESSONS Isolated, primary central nervous system echinococcosis is extremely rare, with only isolated case reports. As in the authors’ case, it can occur in immunocompetent patients, especially persons with a rural vocational history. Negative serology results do not exclude cerebral echinococcosis, which requires histological confirmation. Immunohistochemical staining and PCR analysis are especially useful in cases without classic morphological findings.

BackgroundBased on promising results from radiomic approaches to predict O6-methylguanine DNA methyltransferase promoter methylation status (MGMT status) and clinical outcome in patients with newly diagnosed glioblastoma, the current study aimed to evaluate radiomics in recurrent glioblastoma patients.MethodsPre-treatment MR-imaging data of 69 patients enrolled into the DIRECTOR trial in recurrent glioblastoma served as a training cohort, and 49 independent patients formed an external validation cohort. Contrast-enhancing tumor and peritumoral volumes were segmented on MR images. 180 radiomic features were extracted after application of two MR intensity normalization techniques: fixed number of bins and linear rescaling. Radiomic feature selection was performed via principal component analysis, and multivariable models were trained to predict MGMT status, progression-free survival from first salvage therapy, referred to herein as PFS2, and overall survival (OS). The prognostic power of models was quantified with concordance index (CI) for survival data and area under receiver operating characteristic curve (AUC) for the MGMT status.ResultsWe established and validated a radiomic model to predict MGMT status using linear intensity interpolation and considering features extracted from gadolinium-enhanced T1-weighted MRI (training AUC = 0.670, validation AUC = 0.673). Additionally, models predicting PFS2 and OS were found for the training cohort but were not confirmed in our validation cohort.ConclusionsA radiomic model for prediction of MGMT promoter methylation status from tumor texture features in patients with recurrent glioblastoma was successfully established, providing a non-invasive approach to anticipate patient’s response to chemotherapy if biopsy cannot be performed. The radiomic approach to predict PFS2 and OS failed.

Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

© The Author(s) 2019. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/., Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

Background Despite recent studies, the effect of chemotherapy on programmed death-ligand 1 (PD-L1) expression remains controversial. In this study, we investigated whether PD-L1 expression is affected by platinum-based chemotherapy. Furthermore, we evaluated correlation of PD-L1 expression with oncogenic driver alterations. Materials and Methods We retrospectively evaluated changes in PD-L1 expression by immunohistochemical (IHC) analysis in resected specimens and in biopsies at non–small cell lung cancer recurrence in patients receiving or not adjuvant chemotherapy after surgical resection. Four IHC score groups were defined: TC0 Results Thirty-six patients with adenocarcinoma were included. Twenty (56%) patients underwent adjuvant chemotherapy, and 16 (44%) patients did not receive adjuvant chemotherapy. PD-L1 expression was present in 10 (28%) of 36 initial tumor specimens. From patients receiving adjuvant chemotherapy, 7 (35%) of 20 tumor biopsies showed significant upregulation in PD-L1 expression at recurrence. In contrast, from patients with no adjuvant therapy, only 2 (12.5%) of 16 showed a change in PD-L1 expression. Six (17%) of 36 patients were PD-L1-negative in the primary tumor and turned positive at recurrence. KRAS mutation was present in 70% of patients expressing PD-L1. Conclusion PD-L1 expression in non–small cell lung cancer can change from primary to recurrence, implicating the need for re-biopsy at recurrence. Moreover, chemotherapy might increase expression of PD-L1, supporting a combinatorial therapy with chemotherapy and anti-PD(L)1 treatment.

Rosette-forming glioneuronal tumor (RGNT) is a rare brain neoplasm that primarily affects young adults. Although alterations affecting the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathway have been associated with this low-grade entity, comprehensive molecular investigations of RGNT in larger series have not been performed to date, and an integrated view of their genetic and epigenetic profiles is still lacking. Here we describe a genome-wide DNA methylation and targeted sequencing-based characterization of a molecularly distinct class of tumors (n = 30), initially identified through genome-wide DNA methylation screening among a cohort of > 30,000 tumors, of which most were diagnosed histologically as RGNT. FGFR1 hotspot mutations were observed in all tumors analyzed, with co-occurrence of PIK3CA mutations in about two-thirds of the cases (63%). Additional loss-of-function mutations in the tumor suppressor gene NF1 were detected in a subset of cases (33%). Notably, in contrast to most other low-grade gliomas, these tumors often displayed co-occurrence of two or even all three of these mutations. Our data highlight that molecularly defined RGNTs are characterized by highly recurrent combined genetic alterations affecting both MAPK and PI3K signaling pathways. Thus, these two pathways appear to synergistically interact in the formation of RGNT, and offer potential therapeutic targets for this disease.

Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-programmed cell death receptor 1 therapy in a patient with metastatic melanoma. Histologic analyses revealed robust T cell infiltration and prominent programmed death ligand 1 expression. We identified 209 reported cases in global pharmacovigilance databases (across multiple cancer types) of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4+ T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor repertoire, which we localized to activated memory cytotoxic (CD45RO+GZMB+Ki67+) CD4 cells. We also identified Epstein-Barr virus-specific T cell receptors and EBV+ lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the three cases studied here identify CD4+ and CD8+ T cells as culprits of checkpoint inhibitor-associated immune encephalitis.

BACKGROUND Metastatic tumor spread is a complex multistep process. Due to the blood-brain barrier, metastasis to the central nervous system is restrictive with a distinct predilection for certain tumor types. In melanoma patients, brain metastasis is a common endpoint with the majority showing evidence of widespread disease at autopsy. In a previous murine melanoma model, we have shown that melanoma cells migrate along preexisting vessels into the brain, showing angiotropism/vascular co-option and pericytic mimicry. METHODS Using conventional morphology and immunohistochemistry, we analyze brain metastases from eight autopsy cases. In addition, tissue clearing, which enables three-dimensional visualization over a distance of 100 μm is used. RESULTS We show the angiotropic localization of melanoma deposits in the brains in all eight autopsy cases. Tissue clearing techniques have allowed visualization of melanoma cells in one case exclusively along the abluminal surface of brain blood vessels over a distance of 100 μm, thus showing pericytic mimicry. CONCLUSIONS Our analyses show clear-cut evidence of angiotropism and pericytic mimicry of melanoma cells within the brain over some distance. In addition, these results support the hypothesis of metastasis along pathways other than hematogenous spread, or extravascular migratory metastasis (EVMM). During EVMM, melanoma cells may metastasize to the brain through pericytic mimicry, circumventing the blood-brain barrier.

Treatment of glioblastoma multiforme (GBM), the most aggressive form of primary brain cancer, has essentially not advanced over the past few decades. Numerous challenges hinder the successful development of new therapies, including drug delivery across the blood-brain barrier (BBB), the complexity of the tumor microenvironment (TME), and the lack of clinically predictive cancer models. Here, we present the results of an image-based ex vivo drug-testing platform that addresses these therapeutic roadblocks. To demonstrate the clinical utility of our platform, in a retrospective cohort of 14 GBM patients, we show that ex vivo sensitivity to Temozolomide (TMZ, 1st-line GBM chemotherapy), is associated with longer progression free survival (PFS) and overall survival (OS). Next, by screening 150 clinically approved drugs across 27 GBM surgical patient samples, we identify a set of BBB-permeable neuroactive drugs with anti-glioma activity. These neurological drugs display remarkably consistent on-target killing of cancer cells with minimal toxicity to non-malignant TME cells across both primary and recurrent GBM samples. Single-cell transcriptional profiling of GBM patient samples and functional genetics reveals novel glioma-dependencies on neurological drug-target expression. Furthermore, a drug-target network enrichment analysis uncovers an AP1/BTG/TP53 gene signature associated with the anti-glioma activity of neurological drugs. In silico screening of over 1 million compounds for this common gene signature identified additional drug hits that could be validated in patient samples with 90% accuracy. Multiplexed transcriptomics revealed AP-1 transcription factor family activation to be the common underlying feature of neurological drugs with anti-glioma activity. Among the most promising candidate drugs, we identify the atypical antidepressant Vortioxetine as the strongest inducer of this gene signature, and confirm its efficacy in vivo across multiple mouse models. Vortioxetine in combination with Temozolomide or Lomustine further increased median survival in vivo compared to single agents alone. This study thus provides a clinically predictive and personalized drug-testing platform that identifies new treatment opportunities for GBM, warranting further investigation. Citation Format: Sohyon Lee, Tobias Weiss, Marcel Bühler, Rebekka Wegmann, Julien Mena, Michel Bihl, Sandra Goetze, Audrey van Drogen, Elisabeth J. Rushing, Bernd Wollscheid, Michael Weller, Berend Snijder. Image-based functional precision medicine for repurposing neuroactive drugs in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5325.

Microglia participate in central nervous system (CNS) development and homeostasis and are often implicated in modulating disease processes. However, less is known about the role of microglia in the biology of the neurovascular unit (NVU). In particular, data are scant on whether microglia are involved in CNS vascular pathology. In this study, we use a mouse model of primary familial brain calcification, Pdgfbret/ret, to investigate the role of microglia in calcification of the NVU. We report that microglia enclosing vessel calcifications, coined calcification-associated microglia, display a distinct activation phenotype. Pharmacological ablation of microglia with the CSF1R inhibitor PLX5622 leads to aggravated vessel calcification. Mechanistically, we show that microglia require functional TREM2 for controlling vascular calcification. Our results demonstrate that microglial activity in the setting of pathological vascular calcification is beneficial. In addition, we identify a previously unrecognized function of microglia in halting the expansion of vascular calcification., Science Advances, 7 (9), ISSN:2375-2548

Collagen VI-related myopathies are caused by mutations of COL6A1, COL6A2, and COL6A3 and present with a wide phenotypic spectrum ranging from severe Ulrich congenital muscular dystrophy to mild Bethlem myopathy. Here, we report a consanguineous Kurdish family with 3 siblings affected by autosomal-recessive Bethlem myopathy caused by compound heterozygous mutations of COL6A3. We found the previously described missense mutation c.7447A > G/p.(Lys2483Glu) and a novel large deletion encompassing the exon 1-39 of the COL6A3 gene. Apart from the classical clinical symptoms, all patients had keratoconus, which expands the phenotype of the collagen VI-related myopathies.

Background Brain tumors, whether primary or secondary, have limited therapeutic options despite advances in understanding driver gene mutations and heterogeneity within tumor cells. The cellular and molecular composition of brain tumor stroma, an important modifier of tumor growth, has been less investigated to date. Only few studies have focused on the vasculature of human brain tumors despite the fact that the blood-brain barrier (BBB) represents the major obstacle for efficient drug delivery. Methods In this study, we employed RNA sequencing to characterize transcriptional alterations of endothelial cells (EC) isolated from primary and secondary human brain tumors. We used an immunoprecipitation approach to enrich for EC from normal brain, glioblastoma (GBM), and lung cancer brain metastasis (BM). Results Analysis of the endothelial transcriptome showed deregulation of genes implicated in cell proliferation, angiogenesis, and deposition of extracellular matrix (ECM) in the vasculature of GBM and BM. Deregulation of genes defining the BBB dysfunction module was found in both tumor types. We identified deregulated expression of genes in vessel-associated fibroblasts in GBM. Conclusion We characterize alterations in BBB genes in GBM and BM vasculature and identify proteins that might be exploited for developing drug delivery platforms. In addition, our analysis on vessel-associated fibroblasts in GBM shows that the cellular composition of brain tumor stroma merits further investigation.

Aim of the study Benefit from temozolomide (TMZ) chemotherapy in the treatment of isocitrate dehydrogenase (IDH)–wild-type glioblastoma is essentially limited to patients with O6-methylguanine DNA methyltransferase (MGMT) promoter–methylated tumours. Recent studies suggested that telomerase reverse transcriptase (TERT) promoter hotspot mutations may have an impact on the prognostic role of the MGMT status in patients with glioblastoma. Methods MGMT promoter methylation and TERT promoter mutation status were retrospectively assessed in a prospective cohort of patients with IDH–wild-type glioblastoma of the German Glioma Network (GGN) (n = 298) and an independent retrospective cohort from Dusseldorf, Germany, and Zurich, Switzerland (n = 302). Results In the GGN cohort, but not in the Dusseldorf/Zurich cohort, TERT promoter mutation was moderately associated with inferior outcomes in patients with MGMT promoter–unmethylated tumours (hazard ratio 1.74; 95% confidence interval: 1.07–2.82; p = 0.026). TERT promoter mutations were not associated with better outcomes in patients with MGMT promoter–methylated tumours in either cohort. The two different TERT promoter hotspot mutations (C228T and C250T) were not linked to distinct outcomes. Conclusions Analysis of two independent cohorts of patients with glioblastoma did not confirm previous data, suggesting that TERT promoter mutations confer an enhanced benefit from TMZ in patients with MGMT promoter–methylated glioblastoma. Thus, diagnostic testing for TERT promoter mutations may not be required for prediction of TMZ sensitivity in patients with IDH–wild-type glioblastoma.

Background Only few data are available on treatment-associated behavior of distinct rare CNS embryonal tumor entities previously treated as “CNS-primitive neuroectodermal tumors” (CNS-PNET). Respective data on specific entities, including CNS neuroblastoma, FOXR2 activated (CNS NB-FOXR2), and embryonal tumors with multilayered rosettes (ETMR) are needed for development of differentiated treatment strategies. Methods Within this retrospective, international study, tumor samples of clinically well-annotated patients with the original diagnosis of CNS-PNET were analyzed using DNA methylation arrays (n = 307). Additional cases (n = 66) with DNA methylation pattern of CNS NB-FOXR2 were included irrespective of initial histological diagnosis. Pooled clinical data (n = 292) were descriptively analyzed. Results DNA methylation profiling of “CNS-PNET” classified 58 (19%) cases as ETMR, 57 (19%) as high-grade glioma (HGG), 36 (12%) as CNS NB-FOXR2, and 89(29%) cases were classified into 18 other entities. Sixty-seven (22%) cases did not show DNA methylation patterns similar to established CNS tumor reference classes. Best treatment results were achieved for CNS NB-FOXR2 patients (5-year PFS: 63% ± 7%, OS: 85% ± 5%, n = 63), with 35/42 progression-free survivors after upfront craniospinal irradiation (CSI) and chemotherapy. The worst outcome was seen for ETMR and HGG patients with 5-year PFS of 18% ± 6% and 22% ± 7%, and 5-year OS of 24% ± 6% and 25% ± 7%, respectively. Conclusion The historically reported poor outcome of CNS-PNET patients becomes highly variable when tumors are molecularly classified based on DNA methylation profiling. Patients with CNS NB-FOXR2 responded well to current treatments and a standard-risk CSI-based regimen may be prospectively evaluated. The poor outcome of ETMR across applied treatment strategies substantiates the necessity for evaluation of novel treatments.

Standards of care for meningioma include surgical resection and radiotherapy whereas pharmacotherapy plays almost no role in this disease. We generated primary cultures from surgically removed meningiomas to explore the activity of a novel cyclin-dependent kinase inhibitor, TG02, in meningioma cell cultures. Tumor and cell cultures were characterized by mutation profiling and DNA methylation profiling. DNA methylation data were used to allot each sample to one out of six previously established meningioma methylation classes: benign (ben)-1, 2, 3, intermediate (int)-A, B, and malignant (mal). Four tumors assigned to the methylation class ben-2 showed the same class in culture whereas cultures from five non-ben-2 tumors showed a more malignant class in four patients. Cell cultures were uniformly sensitive to TG02 in the nanomolar range. Assignment of the cell cultures to a more malignant methylation class appeared to be more closely associated with TG02 sensitivity than assignment to a higher WHO grade of the primary tumors. Primary cell cultures from meningioma facilitate the investigation of the anti-meningioma activity of novel agents. TG02, an orally available cyclin-dependent kinase (CDK) inhibitor, warrants further exploration., Highlights • Standards of care for meningioma include surgical resection and radiotherapy whereas pharmacotherapy plays almost no role. • Primary cultures were established to explore the activity of a novel cyclin-dependent kinase inhibitor, TG02. • DNA methylation data were used to assign each sample to one out of six previously established methylation classes. • Cell cultures were uniformly sensitive to TG02 in the nanomolar range. • Methylation class of cell cultures was associated stronger with TG02 sensitivity than WHO grade of the primary tumor.

Coronavirus disease 19 (COVID‐19) is a rapidly evolving pandemic caused by the coronavirus Sars‐CoV‐2. Clinically manifest central nervous system symptoms have been described in COVID‐19 patients and could be the consequence of commonly associated vascular pathology, but the detailed neuropathological sequelae remain largely unknown. A total of six cases, all positive for Sars‐CoV‐2, showed evidence of cerebral petechial hemorrhages and microthrombi at autopsy. Two out of six patients showed an elevated risk for disseminated intravascular coagulopathy according to current criteria and were excluded from further analysis. In the remaining four patients, the hemorrhages were most prominent at the grey and white matter junction of the neocortex, but were also found in the brainstem, deep grey matter structures and cerebellum. Two patients showed vascular intramural inflammatory infiltrates, consistent with Sars‐CoV‐2‐associated endotheliitis, which was associated by elevated levels of the Sars‐CoV‐2 receptor ACE2 in the brain vasculature. Distribution and morphology of patchy brain microbleeds was clearly distinct from hypertension‐related hemorrhage, critical illness‐associated microbleeds and cerebral amyloid angiopathy, which was ruled out by immunohistochemistry. Cerebral microhemorrhages in COVID‐19 patients could be a consequence of Sars‐ CoV‐2‐induced endotheliitis and more general vasculopathic changes and may correlate with an increased risk of vascular encephalopathy., Clinically manifest central nervous system symptoms are common in COVID‐19 patients but their causes are still unknown. We present here four patients who tested positive for Sars‐CoV‐2 with cerebral haemorrhages which were most prominent at the grey and white matter junction of the neocortex and the brainstem. We present evidence of intracerebral endotheliitis in COVID‐19 patients which could predispose to more general vasculopathic changes and may correlate with an increased risk of vascular encephalopathy.

Benefit from temozolomide (TMZ) chemotherapy in the treatment of isocitrate dehydrogenase (IDH)-wild-type glioblastoma is essentially limited to patients with OMGMT promoter methylation and TERT promoter mutation status were retrospectively assessed in a prospective cohort of patients with IDH-wild-type glioblastoma of the German Glioma Network (GGN) (n = 298) and an independent retrospective cohort from Düsseldorf, Germany, and Zurich, Switzerland (n = 302).In the GGN cohort, but not in the Düsseldorf/Zurich cohort, TERT promoter mutation was moderately associated with inferior outcomes in patients with MGMT promoter-unmethylated tumours (hazard ratio 1.74; 95% confidence interval: 1.07-2.82; p = 0.026). TERT promoter mutations were not associated with better outcomes in patients with MGMT promoter-methylated tumours in either cohort. The two different TERT promoter hotspot mutations (C228T and C250T) were not linked to distinct outcomes.Analysis of two independent cohorts of patients with glioblastoma did not confirm previous data, suggesting that TERT promoter mutations confer an enhanced benefit from TMZ in patients with MGMT promoter-methylated glioblastoma. Thus, diagnostic testing for TERT promoter mutations may not be required for prediction of TMZ sensitivity in patients with IDH-wild-type glioblastoma.

HPV-related multiphenotypic sinonasal carcinoma (HMSC) is a recently described distinct tumor entity of the sinonasal tract associated with high-risk subtypes of human papilloma virus (HPV), predominantly type 33. The biological behavior seems to be less aggressive than the often high-grade, highly proliferative morphology implies; however, recurrences are frequent. Most of the cases present as polypoid tumors within the nasal cavity. Microscopic morphology frequently encompasses adenoid cystic-like features or features reminiscent of other salivary gland tumors. Here, we describe four cases of this rare entity, all observed in women. The polypoid tumors were within the nasal cavity, leading to obstruction, facial pain and epistaxis. The morphology was predominantly basaloid, solid and adenoid cystic-like in two of four cases, one with additional glomeruloid features. Another case showed basaloid tumor cells with prominent mature squamous differentiation and extensive keratinization. A single case showed a predominantly solid and reticular growth pattern. All cases were diffusely positive for p16 (100%), expressed SOX10, LEF-1 and partially S-100, and harbored HPV high-risk types 33, 56 (2×) and 82. No recurrences or metastases were detectable after 3-50 months of follow-up. Of note, three of four patients were nurses/nursing assistant. We expand the morphological spectrum by describing a glomeruloid growth pattern and extensive mature keratinization, and add HPV type 82 to the molecular spectrum. The finding of HMSC among predominantly nurses in our cohort warrants further epidemiological studies in larger cohorts.

Immune checkpoint inhibitory (ICI) therapy represents a novel approach in a variety of cancers, with impressive survival benefit. With ICIs, however, a new spectrum of immune related adverse events (irAE) including life threatening hypohysitis has emerged. This autopsy study aimed to investigate inflammatory cells, PD-1 and PD-L1 expression in cases of patients who developed hypophysitis and involvement of other organs. We analysed 6 patients, who were treated with ICIs and developed hypophysitis. Two received an additional MAP-kinase inhibitor, MEK-inhibitor and cytotoxic chemotherapy. Besides the pituitary gland, all investigated adrenal glands (5/5) were affected; three cases had other organs involved (liver (2/6), thyroid (2/6), lung (1/6), myocardium (1/6), colon (1/6). The inflammatory cells of involved organs were further specified and PD1 and PDL-1 expression was analyzed using immunohistochemistry. We observed that patients treated with ICIs alone showed T-cell predominant lymphocytic infiltrates, whereas patients receiving additional therapies demonstrated an increase in B- and T-lymphocytes. Surprisingly, the dominant inflammatory population was not T-cell, but type 2 macrophages. CD25 positive T-regs were sparse or absent. Our study suggests that T cell activation is only partially responsible for irAE. ICI therapy interaction with CTLA-4, PD-1 and PDL-1 in type 2 macrophages appears to result in disturbance of their control. Furthermore, depletion of T-regs seems to contribute significantly. Our findings with simultaneous pituitary and adrenal gland involvement underlines the systemic involvement as well as the importance of monitoring cortisol levels to avoid potentially life threatening hypocortisolism.