Surgeon experience in glioblastoma surgery of the elderly-a multicenter, retrospective cohort study.

PURPOSE: To assess the impact of individual surgeon experience on overall survival (OS), extent of resection (EOR) and surgery-related morbidity in elderly patients with glioblastoma (GBM), we performed a retrospective case-by-case analysis. METHODS: GBM patients aged ≥ 65 years who underwent tumor resection at two academic centers were analyzed. The experience of each neurosurgeon was quantified in three ways: (1) total number of previously performed glioma surgeries (lifetime experience); (2) number of surgeries performed in the previous five years (medium-term experience) and (3) in the last two years (short-term experience). Surgeon experience data was correlated with survival (OS) and surrogate parameters for surgical quality (EOR, morbidity). RESULTS: 198 GBM patients (median age 73.0 years, median preoperative KPS 80, IDH-wildtype status 96.5%) were included. Median OS was 10.0 months (95% CI 8.0-12.0); median EOR was 89.4%. Surgery-related morbidity affected 19.7% patients. No correlations of lifetime surgeon experience with OS (P = .693), EOR (P = .693), and surgery-related morbidity (P = .435) were identified. Adjuvant therapy was associated with improved OS (P < .001); patients with surgery-related morbidity were less likely to receive adjuvant treatment (P = .002). In multivariable testing, adjuvant therapy (P < .001; HR = 0.064, 95%CI 0.028-0.144) remained the only significant predictor for improved OS. CONCLUSION: Less experienced neurosurgeons achieve similar surgical results and outcome in elderly GBM patients within the setting of academic teaching hospitals. Adjuvant treatment and avoidance of surgery-related morbidity are crucial for generating a treatment benefit for this cohort.

Fatal course of undetected Ewing-like sarcoma in a 9-year-old boy with uncharacteristic clinical presentation.

A 9-year-old boy collapsed shortly after complaining of shortness of breath. Despite immediate resuscitation measures, the boy died. A few weeks earlier, he had received antibiotic treatment for respiratory infection. However, the post-mortem examination revealed an advanced tumor mass of the mediastinum with infiltration of vital structures, which was identified as a small blue round neoplasm with aspects of an extramedullary Ewing-like sarcoma by supplementary histological and immunohistochemical examinations.This dramatic clinical course of events shows that the possible presence of serious diseases should always be considered behind harmless symptoms, even in children.

Autoimmune myocarditis is not associated with left ventricular systolic dysfunction.

BACKGROUND: Experimental autoimmune myocarditis (EAM) is a common animal model for the investigation of the pathophysiology of myocarditis. Because of diverging findings from previous studies, we performed serial echocardiographic examinations throughout the course of the disease and investigated the dimensions of the murine heart and left ventricular (LV) systolic function. MATERIALS AND METHODS: Experimental autoimmune myocarditis was induced in male Balb/c mice by subcutaneous injection of a fragment of the α-myosin heavy chain (MyHC-α 614-629: Ac-SLKLMATLFSTYASAD). Transthoracic echocardiography was performed on days 0, 7 and 21 in healthy animals and mice with EAM. RESULTS: Experimental autoimmune myocarditis was associated with a reduction in LV systolic function and an increase in LV internal diameter in diastole (LVIDd) and systole (LVIDs) 7 days postimmunization. After 21 days, EAM led to a significant increase in LV-thickness (1.3-fold increase in LV anterior wall diameter in diastole [LVAWDd]), but there was no difference in LV systolic function between immunized animals and healthy controls. LV-thickness correlated well with the severity of myocarditis in the histopathological examination (LVAWDd: rs = 0.603, P = 0.003, LV anterior wall diameter in systole (LVAWDs): rs = 0.718, P < 0.0001). CONCLUSION: Our results indicate that EAM leads to an initial dilatation of the LV that is followed by ventricular "hypertrophy." On day 21, there was no significant difference in LV systolic function between immunized animals and controls. Furthermore, the ageing of the animals had a major impact on the echocardiographic parameters; therefore, the use of healthy age-matched controls seems warranted when echocardiography is performed in rodents.

Cell Type and Species-specific Patterns in Neuronal and Non-neuronal Methylomes of Human and Chimpanzee Cortices.

Epigenetic changes have likely contributed to the large size and enhanced cognitive abilities of the human brain which evolved within the last 2 million years after the human-chimpanzee split. Using reduced representation bisulfite sequencing, we have compared the methylomes of neuronal and non-neuronal cells from 3 human and 3 chimpanzee cortices. Differentially methylated regions (DMRs) with genome-wide significance were enriched in specific genomic regions. Intraspecific methylation differences between neuronal and non-neuronal cells were approximately 3 times more abundant than interspecific methylation differences between human and chimpanzee cell types. The vast majority (>90%) of human intraspecific DMRs (including DMRs in retrotransposons) were hypomethylated in neurons, compared with glia. Intraspecific DMRs were enriched in genes associated with different neuropsychiatric disorders. Interspecific DMRs were enriched in genes showing human-specific brain histone modifications. Human-chimpanzee methylation differences were much more frequent in non-neuronal cells (n. DMRs = 666) than in neurons (n. DMRs = 96). More than 95% of interspecific DMRs in glia were hypermethylated in humans. Although without an outgroup we cannot assign whether a change in methylation occurred in the human or chimpanzee lineage, our results are consistent with a wave of methylation affecting several hundred non-neuronal genes during human brain evolution.

TERT promoter mutation is associated with worse prognosis in WHO grade II and III meningiomas.

INTRODUCTION: Transcriptional activating mutations in the promoter region of the telomerase reverse transcriptase (TERT) gene occur at high frequency in various types of solid tumors and have also been reported for meningiomas. Especially for atypical and anaplastic meningiomas, the prognostic relevance of TERT promoter mutation is yet unclear. The present study aimed to analyze the frequency of TERT promoter mutation and define its long-term prognostic significance beyond clinical and histological factors in a cohort of meningiomas WHO grade II and III. METHODS: Patients undergoing surgical resection of aggressive meningiomas were included. Analysis for C228T and C250T mutation in the TERT promoter region was performed using PCR method. Patients were stratified into two groups (TERT mutated vs. TERT wild type). Univariate analysis was conducted using molecular and histological factors. RESULTS: 87 patients with atypical (N = 72) and anaplastic meningiomas (N = 15) were included in the study. TERT promoter region was found to be mutated in 4 WHO grade II and 2 WHO grade III meningiomas. TERT promoter mutation was associated with shorter progression free survival than TERT wild type meningiomas (median PFS 12.5 vs. 26 months, p = .004). In the univariate analysis, TERT promoter mutation had a strong prognostic value on overall survival (p = .009) and progression free survival. CONCLUSIONS: Presence of TERT promoter mutation is associated with shorter progression free survival and overall survival in meningiomas WHO grade II and III. In these tumors, TERT promoter mutation should be considered as a clinically relevant prognostic factor to identify high risk patients.

Profiling of methylation and demethylation pathways during brain development and ageing.

Numerous signal pathways are epigenetically controlled during brain development and ageing. Thereby, both 5-methylcytosine (5mC) and the newly described 5-hydroxymethylcytosine (5hmC) are highly exhibited in the brain. As there is an uneven distribution of 5hmC in the brain depending on age and region, there is the need to investigate the underlying mechanisms being responsible for 5hmC generation and decline. The aim of this study was to quantify expression levels of genes that are associated with DNA methylation/demethylation in different brain regions and at different ages. Therefore, we investigated frontal cortex and cerebellum of 40 mice (strain C57BL/6), each eight mice sacrificed at day 0, 7, 15, 30 and 120 after birth. We performed expression profiling of methylation/demethylation genes depending on age and brain region. Interestingly, we see significant expression differences of genes being responsible for methylation/demethylation with a significant reduction of expression levels during ageing. Validating selected expression data on protein level using immunohistochemistry verified the expression data. In conclusion, our findings demonstrate that the regulation of methylation/demethylation pathways is highly controlled depending on brain region and age. Thus our data will help to better understand the complexity and plasticity of the brain epigenome.

Loss of 5-hydroxymethylcytosine and intratumoral heterogeneity as an epigenomic hallmark of glioblastoma.

Glioblastoma (GBM) is the most malignant neoplasm with predominant astrocytic differentiation and the most frequent primary brain tumor of the adult. Here, we investigated 170 human GBM specimens deriving from 162 patients, as well as 66 healthy control tissue specimens deriving from 27 patients, and analyzed the amount of 5-hydroxymethylcytosine (5hmC) in GBMs compared to normal brain and tumor infiltration zones. Additionally, we correlated the amount of 5hmC with two different proliferation markers, Ki67 and H3S10p. Genetic characterization of GBMs enabled us to analyze the effect of isocitrate dehydrogenase 1 (IDH1) mutations, O6-methylguanin-DNA-methyltransferase (MGMT) promoter methylation, and loss of heterozygosity of chromosome 1p and 19q (LOH1p/19q) on 5hmC amount. We found that GBMs show a tremendous loss of 5hmC, and we observed that even the infiltration zones show reduced amounts of 5hmC. Interestingly, the amount of 5hmC was inversely proportional to the two investigated proliferation markers, Ki67 and H3S10p. Correlation of 5hmC amount and molecular genetic markers of GBMs showed that there are no correlations of 5hmC amount and IDH1 mutations, MGMT promoter methylation, and LOH1p/19q. Furthermore, we evaluated the intratumoral distribution of 5hmC in compact and infiltrating areas and found that the quantification of the 5hmC amount is a useful tool in evaluation of tumor infiltration. In summary, our data emphasize that GBMs show a disturbed hydroxymethylome that is disrupted by IDH1 independent pathways, and that loss of 5hmC shows astonishing intratumoral heterogeneity.

5-Hydroxymethylcytosine, the "Sixth Base", during brain development and ageing.

The epigenome is of fundamental importance for development and ageing. The discovery of 5-hydroxymethylcytosine (5hmC), a further base modification of cytosine beyond 5-methylcytosine, might be of high relevance in understanding the complexity of the human brain, as 5hmC is found in great extent in brain tissue. The aim of this study was to investigate the quantity of 5hmC containing nuclei by immunohistochemistry in human and murine brains at several developmental stages. We performed immunohistochemical stainings on frontal cortex, white matter and cerebellar cortex of 15 healthy controls. Three cases each were assigned to five age groups (foetus, adolescent, adult, elderly, aged). Additionally, cortex and cerebellum of 15 mice sacrificed between day 0 and 120 after birth were investigated. We found marked alterations of 5hmC amount during ageing. In human cortex there was an increase of 5hmC of 50%, in white matter we found an increase of even 200% during ageing. In the cerebellum both internal granular cell layer and molecular cell layer showed a significant increase of 5hmC till adulthood. Purkinje cell nuclei showed constantly positive signals for 5hmC. These data were paralleled in murine brains. Co-labelling of 5hmC and markers for mature and immature cells in murine cerebellar cortex at the age of 7 days revealed that 5hmC was found in mature but not in immature cells. In conclusion, the findings described in this study emphasise the importance of 5hmC in brain development and ageing and will help to better understand the complexity and plasticity of the brain.

Long non-coding RNA normalisers in human brain tissue.

The family of long non-coding RNA (lncRNA) is of increasing scientific interest as there is emerging evidence, that lncRNAs are of essential importance for transcriptional and translational control, genomic imprinting and regulation of normal development as well as neuronal plasticity. As the generation of reliable expression profiles requires adequate normalisers, it is of fundamental importance to determine suitable references for lncRNA studies. However, to date no systematic analysis of potential lncRNA normalisers has been performed on human postmortem brain tissue samples. In this study, we investigated three different brain regions (cortex, white matter, and cerebellum) of human postmortem tissue and analysed the expression stability of 90 lncRNAs. Bioinformatical analysis was performed to identify stably expressed lncRNAs. Subsequently, lncRNAs were classified according to their stability values using the NormFinder algorithm. We identified 30 suitable normalisers in cortex, 22 in white matter, and 41 in cerebellum. In addition, there were 13 suitable normalisers for studies comparing cortex and white matter, 25 for studies comparing cortex and cerebellum and 7 for studies comparing white matter and cerebellum. 5 lncRNAs (LUST, IGF2AS (family), 7SK, HOXA6as, NDM29) showed stable expression in all investigated brain regions. A subsequent analysis of the influence of postmortem intervals (PMI) on expression of lncRNAs revealed that expression levels of the newly identified 5 universal lncRNA normalisers are stable within PMI of up to 27 h. Thus, these 5 lncRNAs may be applicable as references for accurate normalisation of lncRNA profiling in multiple brain regions during long PMI, enabling the generation of highly reproducible datasets in lncRNA studies of the human brain.

Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues.

The absolute levels of 5-hydroxymethylcytosine (hmC) and 5-methylcytosine (mC) in human brain tissues at various ages were determined. Additionally, absolute levels of 5-formylcytosine (fC) in adult individuals and cytosine modification levels in sorted neurons were quantified. These data were compared with age-related fC, hmC, and mC levels in mouse brain samples. For hmC, an initial steady increase is observed, which levels off with age to a final steady-state value of 1.2 % in human brain tissue. This level is nearly twice as high as in mouse cerebral cortex. In contrast, fC declines rapidly with age during early developmental stages, thus suggesting that while hmC is a stable epigenetic mark, fC is more likely an intermediate of active DNA demethylation during early brain development. The trends in global cytosine modification dynamics during the lifespan of an organism are conserved between humans and mice and show similar patterns in different organs.

Genome-Wide Methylation Analysis in Two Wild-Type Non-Small Cell Lung Cancer Subgroups with Negative and High PD-L1 Expression.

We conducted a pilot study to analyze the differential methylation status of 20 primary acinar adenocarcinomas of the lungs. These adenocarcinomas had to be wild type in mutation analysis and had either high (TPS > 50%; n = 10) or negative (TPS < 1%; n = 10) PD-L1 status to be integrated into our study. To examine the methylation of 866,895 specific sites, we utilized the Illumina Infinium EPIC bead chip array. Both hypermethylation and hypomethylation play significant roles in tumor development, progression, and metastasis. They also impact the formation of the tumor microenvironment, which plays a decisive role in tumor differentiation, epigenetics, dissemination, and immune evasion. The gained methylation patterns were correlated with PD-L1 expression. Our analysis has identified distinct methylation patterns in lung adenocarcinomas with high and negative PD-L1 expression. After analyzing the correlation between the methylation results of genes and promoters with their pathobiology, we found that tumors with high expression of PD-L1 tend to exhibit oncogenic effects through hypermethylation. On the other hand, tumors with negative PD-L1 expression show loss of their suppressor functions through hypomethylation. The suppressor functions of hypermethylated genes and promoters are ineffective compared to simultaneously activated dominant oncogenic mechanisms. The tumor microenvironment supports tumor growth in both groups.

Pituitary Apoplexy in Macroadenoma After Minor Surgery: An Unusual Case and Literature Review.

Pituitary apoplexy is a rare and severe complication of pituitary adenoma that may present with new-onset headache, ocular palsy, visual disturbances, life-threatening electrolyte imbalance, and endocrinological disturbances due to pituitary hemorrhage and/or infarction. We report the case of a 58-year-old previously healthy patient who developed isolated mild oculomotor nerve palsy of the left eye following osteosynthesis of a traumatic right distal radius fracture. Initial cerebral magnetic resonance imaging showed a pituitary macroadenoma without characteristic signs of pituitary infarction or hemorrhage. The patient presented to the neurology department on the fifth postoperative day with malaise and fatigue due to pituitary insufficiency, deteriorated rapidly and required intensive care monitoring. Clinical stabilization was achieved through the administration of hydrocortisone, and transsphenoidal resection of the pituitary lesion was performed on the 10th day after acute symptom onset. Histological examination revealed a necrotic pituitary adenoma. Pituitary apoplexy may occur after minor surgery in patients with pituitary adenoma. Clinicians should pay particular attention to laboratory signs of pituitary insufficiency in new-onset oculomotor nerve palsy associated with sellar lesions, as cerebral imaging may miss pituitary apoplexy and therefore delay diagnosis and treatment. In our case, delayed decompressive transsphenoidal resection resulted in the normalization of the oculomotor nerve palsy while the pituitary insufficiency persisted. The potential impact of an earlier surgical intervention on the outcome of pituitary function remains uncertain.

Iatrogenic cerebral amyloid angiopathy: A multinational case series and individual patient data analysis of the literature.

BACKGROUND: The transmission of amyloid ß (Aß) in humans leading to iatrogenic cerebral amyloid angiopathy (iCAA) is a novel concept with analogies to prion diseases. However, the number of published cases is low, and larger international studies are missing. AIMS: We aimed to build a large multinational collaboration on iCAA to better understand the clinical spectrum of affected patients. METHODS: We collected clinical data on patients with iCAA from Austria, Croatia, Italy, Slovenia, and Spain. Patients were included if they met the proposed Queen Square diagnostic criteria (QSC) for iCAA. In addition, we pooled data on disease onset, latency, and cerebrospinal fluid (CSF) biomarkers from previously published iCAA cases based on a systematic literature review. RESULTS: Twenty-seven patients (22% women) were included in this study. Of these, 19 (70%) met the criteria for probable and 8 (30%) for possible iCAA. Prior neurosurgical procedures were performed in all patients (93% brain surgery, 7% spinal surgery) at median age of 8 (interquartile range (IQR) = 4-18, range = 0-26 years) years. The median symptom latency was 39 years (IQR = 34-41, range = 28-49). The median age at symptom onset was 49 years (IQR = 43-55, range = 32-70). Twenty-one patients (78%) presented with intracranial hemorrhage and 3 (11%) with seizures. CONCLUSIONS: Our large international case series of patients with iCAA confirms a wide age boundary for the diagnosis of iCAA. Dissemination of awareness of this rare condition will help to identify more affected patients.

Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFß2 in vascular abnormalization.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFß2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFß signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFß2 may represent a new target for vascular normalization therapy.

Extra- and Intracranial Diffuse Large B-Cell Lymphoma (DLBCL) Mimicking Meningioma: A Case Report and Literature Review.

Primary central nervous system lymphomas (PCNLSs) are malignant non-Hodgkin lymphomas solely affecting the central nervous system (CNS). Here, we present a rare case of extra- and intracranial manifestation without adjacent calvarial infiltration. We report a 67-year-old woman who presented with right leg paresis and hypoesthesia, facial hypoesthesia, focal epileptic seizures, and an indolent tumor on the left parietal scalp. MRI showed a left paramedian extra- and intracranial contrast-enhancing tumor with infiltration of the superior sagittal sinus, but without osseous infiltration on CT. The tumor was radiologically suspected to be a meningioma and resection was performed. Histological examination, however, revealed a diffuse large B-cell lymphoma (DLBCL). Thus, the patient received adjuvant treatment according to the MATRix protocol. We provide a detailed analysis of this rare case with a focus on preoperative radiological findings and differential diagnoses. To the best of our knowledge, this is one of only four published cases of DLBCL with extra- and intracranial manifestation without bone affection.

Network topology in brain tumor patients with and without structural epilepsy: a prospective MEG study.

Background: It was proposed that network topology is altered in brain tumor patients. However, there is no consensus on the pattern of these changes and evidence on potential drivers is lacking. Objectives: We aimed to characterize neurooncological patients' network topology by analyzing glial brain tumors (GBTs) and brain metastases (BMs) with respect to the presence of structural epilepsy. Methods: Network topology derived from resting state magnetoencephalography was compared between (1) patients and controls, (2) GBTs and BMs, and (3) patients with (PSEs) and without structural epilepsy (PNSEs). Eligible patients were investigated from February 2019 to March 2021. We calculated whole brain (WB) connectivity in six frequency bands, network topological parameters (node degree, average shortest path length, local clustering coefficient) and performed a stratification, where differences in power were identified. For data analysis, we used Fieldtrip, Brain Connectivity MATLAB toolboxes, and in-house built scripts. Results: We included 41 patients (21 men), with a mean age of 60.1 years (range 23-82), of those were: GBTs (n = 23), BMs (n = 14), and other histologies (n = 4). Statistical analysis revealed a significantly decreased WB node degree in patients versus controls in every frequency range at the corrected level (p1-30Hz = 0.002, pγ = 0.002, pß = 0.002, pα = 0.002, pθ = 0.024, and pδ = 0.002). At the descriptive level, we found a significant augmentation for WB local clustering coefficient (p1-30Hz = 0.031, pδ = 0.013) in patients compared to controls, which did not persist the false discovery rate correction. No differences regarding networks of GBTs compared to BMs were identified. However, we found a significant increase in WB local clustering coefficient (pθ = 0.048) and decrease in WB node degree (pα = 0.039) in PSEs versus PNSEs at the uncorrected level. Conclusion: Our data suggest that network topology is altered in brain tumor patients. Histology per se might not, however, tumor-related epilepsy seems to influence the brain's functional network. Longitudinal studies and analysis of possible confounders are required to substantiate these findings.

Dissecting the Methylomes of EGFR-Amplified Glioblastoma Reveals Altered DNA Replication and Packaging, and Chromatin and Gene Silencing Pathways.

Glioblastoma IDH wildtype is the most frequent brain tumor in adults. It shows a highly malignant behavior and devastating outcomes. To date, there is still no targeted therapy available; thus, patients' median survival is limited to 12-15 months. Epithelial growth factor receptor (EGFR) is an interesting targetable candidate in advanced precision medicine for brain tumor patients. In this study, we performed integrated epigenome-wide DNA-methylation profiling of 866,895 methylation specific sites in 50 glioblastoma IDH wildtype samples, comparing EGFR amplified and non-amplified glioblastomas. We found 9849 significantly differentially methylated CpGs (DMCGs) with Δß ≥ 0.1 and p-value < 0.05 in EGFR amplified, compared to EGFR non-amplified glioblastomas. Of these DMCGs, 2380 were annotated with tiling (2090), promoter (117), gene (69) and CpG islands (104); 7460 are located at other loci. Interestingly, the list of differentially methylated genes allocated eleven functionally relevant RNAs: five miRNAs (miR1180, miR1255B1, miR126, miR128-2, miR3125), two long non-coding RNAs (LINC00474, LINC01091), and four antisense RNAs (EPN2-AS1, MNX1-AS2, NKX2-2-AS1, WWTR1-AS1). Gene ontology (GO) analysis showed enrichment of "DNA replication-dependent nucleosome assembly", "chromatin silencing at rDNA", "regulation of gene silencing by miRNA", "DNA packaging", "posttranscriptional gene silencing", "gene silencing by RNA", "negative regulation of gene expression, epigenetic", "regulation of gene silencing", "protein-DNA complex subunit organization", and "DNA replication-independent nucleosome organization" pathways being hypomethylated in EGFR amplified glioblastomas. In summary, dissecting the methylomes of EGFR amplified and non-amplified glioblastomas revealed altered DNA replication, DNA packaging, chromatin silencing and gene silencing pathways, opening potential novel targets for future precision medicine.

Coexistence of Two Distinct Tumor Types Within One Posterior Fossa Mass Lesion in an Adult Patient Verified by DNA-Methylation Analysis: A Case Report.

We report an 81-year-old patient who underwent microsurgical resection of a posterior fossa mass lesion. Intraoperative findings were suggestive of the presence of two distinctly different tumor types within the lesion, one of which was well-circumscribed and avascular, whereas the other one showed an adhesive growth pattern and extensive vascularisation. Histopathological analysis, including deoxyribonucleic acid (DNA)-methylation-based classification, substantiated the intraoperative impression and confirmed the presence of a subependymoma central nervous system (CNS) World Health Organization (WHO) grade 1 as well as the presence of a hemangioblastoma CNS WHO grade 1. To our knowledge, our patient represents only the second reported case of such a rare constellation. Even though DNA-methylation-based classification is not yet required for the classification of all CNS tumor types by the 2021 WHO classification of tumors of the CNS, it proved to be crucial to verify the final diagnosis in our patient. In the future, DNA-methylation analysis will most likely become an important asset in neuro-oncological diagnostics and further help to guide treatment strategies in complex or rare clinical cases.

Galanin System in the Human Bile Duct and Perihilar Cholangiocarcinoma.

BACKGROUND: Perihilar cholangiocarcinoma (pCCA) is characterised by poor outcomes. Early diagnosis is essential for patient survival. The peptide galanin (GAL) and its receptors GAL1-3 are expressed in various tumours. Detailed characterisation of the GAL system in pCCA is lacking. Our study sought to characterise GAL and GAL1-3 receptor (GAL1-3-R) expression in the healthy human bile duct, in cholestasis and pCCA. METHODS: Immunohistochemical staining was performed in healthy controls (n = 5) and in the peritumoural tissues (with and without cholestasis) (n = 20) and tumour tissues of pCCA patients (n = 33) using validated antibodies. The score values of GAL and GAL1-3-R expression were calculated and statistically evaluated. RESULTS: GAL and GAL1-R were expressed in various bile duct cell types. GAL2-R was only slightly but still expressed in almost all the examined tissues, and GAL3-R specifically in cholangiocytes and capillaries. In a small pCCA patient cohort (n = 18), high GAL expression correlated with good survival, whereas high GAL3-R correlated with poor survival. CONCLUSIONS: Our in-depth characterisation of the GAL system in the healthy human biliary duct and pCCA in a small patient cohort revealed that GAL and GAL3-R expression in tumour cells of pCCA patients could potentially represent suitable biomarkers for survival.

Low values of 5-hydroxymethylcytosine (5hmC), the "sixth base," are associated with anaplasia in human brain tumors.

5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.