DNA methylation-array interlaboratory comparison trial demonstrates highly reproducible paediatric CNS tumour classification across 13 international centres.

AIMS: DNA methylation profiling, recently endorsed by the World Health Organisation (WHO) as a pivotal diagnostic tool for brain tumours, most commonly relies on bead arrays. Despite its widespread use, limited data exist on the technical reproducibility and potential cross-institutional differences. The LOGGIC Core BioClinical Data Bank registry conducted a prospective laboratory comparison trial with 12 international laboratories to enhance diagnostic accuracy for paediatric low-grade gliomas, focusing on technical aspects of DNA methylation data generation and profile interpretation under clinical real-time conditions. METHODS: Four representative low-grade gliomas of distinct histologies were centrally selected, and DNA extraction was performed. Participating laboratories received a DNA aliquot and performed the DNA methylation-based classification and result interpretation without knowledge of tumour histology. Additionally, participants were required to interpret the copy number profile derived from DNA methylation data and conduct DNA sequencing of the BRAF hotspot p.V600 due to its relevance for low-grade gliomas. Results had to be returned within 30 days. RESULTS: High technical reproducibility was observed, with a median pairwise correlation of 0.99 (range 0.94-0.99) between coordinating laboratory and participants. DNA methylation-based tumour classification and copy number profile interpretation were consistent across all centres, and BRAF mutation status was accurately reported for all cases. Eleven out of 12 centres successfully reported their analysis within the 30-day timeframe. CONCLUSION: Our study demonstrates remarkable concordance in DNA methylation profiling and profile interpretation across 12 international centres. These findings underscore the potential contribution of DNA methylation analysis to the harmonisation of brain tumour diagnostics.

Rapid DNA methylation-based classification of pediatric brain tumors from ultrasonic aspirate specimens.

BACKGROUND: Although cavitating ultrasonic aspirators are commonly used in neurosurgical procedures, the suitability of ultrasonic aspirator-derived tumor material for diagnostic procedures is still controversial. Here, we explore the feasibility of using ultrasonic aspirator-resected tumor tissue to classify otherwise discarded sample material by fast DNA methylation-based analysis using low pass nanopore whole genome sequencing. METHODS: Ultrasonic aspirator-derived specimens from pediatric patients undergoing brain tumor resection were subjected to low-pass nanopore whole genome sequencing. DNA methylation-based classification using a neural network classifier and copy number variation analysis were performed. Tumor purity was estimated from copy number profiles. Results were compared to microarray (EPIC)-based routine neuropathological histomorphological and molecular evaluation. RESULTS: 19 samples with confirmed neuropathological diagnosis were evaluated. All samples were successfully sequenced and passed quality control for further analysis. DNA and sequencing characteristics from ultrasonic aspirator-derived specimens were comparable to routinely processed tumor tissue. Classification of both methods was concordant regarding methylation class in 17/19 (89%) cases. Application of a platform-specific threshold for nanopore-based classification ensured a specificity of 100%, whereas sensitivity was 79%. Copy number variation profiles were generated for all cases and matched EPIC results in 18/19 (95%) samples, even allowing the identification of diagnostically or therapeutically relevant genomic alterations. CONCLUSION: Methylation-based classification of pediatric CNS tumors based on ultrasonic aspirator-reduced and otherwise discarded tissue is feasible using time- and cost-efficient nanopore sequencing.

Neurosurgical morbidity in pediatric supratentorial midline low-grade glioma: Results from the German LGG studies.

Surgical resection is a mainstay of treatment for pediatric low-grade glioma (LGG) within all current therapy algorithms, yet associated morbidity is scarcely reported. As supratentorial midline (SML) interventions are particularly challenging, we investigated the frequency of neurosurgical complications/new impairments aiming to identify their risk factors. Records were retrospectively analyzed from 318 patients with SML-LGG from successive German multicenter LGG studies, undergoing surgery between May 1998 and June 2020. Exactly 537 operations (230 resections, 167 biopsies, 140 nontumor procedures) were performed in 318 patients (54% male, median age: 7.6 years at diagnosis, 9.5 years at operation, 11% NF1, 42.5% optic pathway glioma). Surgical mortality rate was 0.93%. Applying the Drake classification, postoperative surgical morbidity was observed following 254/537 (47.3%) and medical morbidity following 97/537 (18.1%) patients with a 40.1% 30-day persistence rate for newly developed neurological deficits (65/162). Neuroendocrine impairment affected 53/318 patients (16.7%), visual deterioration 34/318 (10.7%). Postsurgical morbidity was associated with patient age <3 years at operation, tumor volume ≥80 cm3 , presence of hydrocephalus, complete resection, surgery in centers with less than median reported tumor-related procedures and during the earlier study period between 1998 and 2006, while the neurosurgical approach, tumor location, NF1 status or previous nonsurgical treatment were not. Neurosurgery-associated morbidity was frequent in pediatric patients with SML-LGG undergoing surgery in the German LGG-studies. We identified patient- and institution-associated factors that may increase the risk for complications. We advocate that local multidisciplinary teams consider the planned extent of resection and surgical skills.

Tumor load rather than contrast enhancement is associated with the visual function of children and adolescents with optic pathway glioma - a retrospective Magnetic Resonance Imaging study.

INTRODUCTION: Optic pathway gliomas are often asymptomatic tumors occurring in children with neurofibromatosis type 1 (NF1 + OPG) or sporadically (spOPG). Treatment is usually prompted by visual loss and/or tumor progression on MRI. The aim of this study was to investigate the relationship between visual acuity (VA), tumor growth, and contrast enhancement to provide more distinct indications for the administration of gadolinium-based contrast agents. METHODS: Tumor load was retrospectively measured and enhancement semi-quantitatively scored on 298 MRIs of 35 patients (63% NF1 + OPG). Spearman rank correlation between tumor load and enhancement was calculated and a linear mixed model used to examine the influence of tumor load and enhancement on corresponding VA tests (LogMAR). RESULTS: The optic nerve width in NF1 + OPGs was strongly associated with VA (regression coefficient 0.75; confidence interval 0.61-0.88), but weakly with enhancement (0.06; -0.04-0.15). In spOPGs, tumor volume and optic nerve width were more relevant (0.31; -0.19-0.81 and 0.39; 0.05-0.73) than enhancement (0.09; -0.09-0.27). CONCLUSIONS: Tumor load measures may be more relevant for the surveillance of optic pathway gliomas than enhancement, given that VA is the relevant outcome parameter. Regular contrast administration should therefore be questioned in these patients.

Accurate calling of KIAA1549-BRAF fusions from DNA of human brain tumours using methylation array-based copy number and gene panel sequencing data.

AIMS: KIAA1549-BRAF fusions occur in certain brain tumours and provide druggable targets due to a constitutive activation of the MAP-kinase pathway. We introduce workflows for calling the KIAA1549-BRAF fusion from DNA methylation array-derived copy number as well as DNA panel sequencing data. METHODS: Copy number profiles were analysed by automated screening and visual verification of a tandem duplication on chromosome 7q34, indicative of the KIAA1549-BRAF fusion. Pilocytic astrocytomas of the ICGC cohort with known fusion status were used for validation. KIAA1549-BRAF fusions were called from DNA panel sequencing data using the fusion callers Manta, Arriba with modified filtering criteria and deFuse. We screened DNA methylation and panel sequencing data of 7790 specimens from brain tumour and sarcoma entities. RESULTS: We identified the fusion in 337 brain tumours with both DNA methylation and panel sequencing data. Among these, we detected the fusion from copy number data in 84% and from DNA panel sequencing data in more than 90% using Arriba with modified filters. While in 74% the KIAA1549-BRAF fusion was detected from both methylation array-derived copy number and panel sequencing data, in 9% it was detected from copy number data only and in 16% from panel data only. The fusion was almost exclusively found in pilocytic astrocytomas, diffuse leptomeningeal glioneuronal tumours and high-grade astrocytomas with piloid features. CONCLUSIONS: The KIAA1549-BRAF fusion can be reliably detected from either DNA methylation array or DNA panel data. The use of both methods is recommended for the most sensitive detection of this diagnostically and therapeutically important marker.

Autoantibodies from Patients with Scleroderma Renal Crisis Promote PAR-1 Receptor Activation and IL-6 Production in Endothelial Cells.

BACKGROUND: Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc). Autoantibodies (Abs) against endothelial cell antigens have been implicated in SSc and SRC. However, their detailed roles remain poorly defined. Pro-inflammatory cytokine interleukin-6 (IL-6) has been found to be increased in SSc, but its role in SRC is unclear. Here, we aimed to determine how the autoantibodies from patients with SSc and SRC affect IL-6 secretion by micro-vascular endothelial cells (HMECs). METHODS: Serum IgG fractions were isolated from either SSc patients with SRC (n = 4) or healthy individuals (n = 4) and then each experiment with HMECs was performed with SSc-IgG from a separate patient or separate healthy control. IL-6 expression and release by HMECs was assessed by quantitative reverse transcription and quantitative PCR (RT-qPCR) and immunoassays, respectively. The mechanisms underlying the production of IL-6 were analyzed by transient HMEC transfections with IL-6 promoter constructs, electrophoretic mobility shift assays, Western blots and flow cytometry. RESULTS: Exposure of HMECs to IgG from SSc patients, but not from healthy controls, resulted in a time- and dose-dependent increase in IL-6 secretion, which was associated with increased AKT, p70S6K, and ERK1/2 signalling, as well as increased c-FOS/AP-1 transcriptional activity. All these effects could be reduced by the blockade of the endothelial PAR-1 receptor and/or c-FOS/AP-1silencing. CONCLUSIONS: Autoantibodies against PAR-1 found in patients with SSc and SRC induce IL-6 production by endothelial cells through signalling pathways controlled by the AP-1 transcription factor. These observations offer a greater understanding of adverse endothelial cell responses to autoantibodies present in patients with SRC.

Response to trametinib treatment in progressive pediatric low-grade glioma patients.

INTRODUCTION: A hallmark of pediatric low-grade glioma (pLGG) is aberrant signaling of the mitogen activated protein kinase (MAPK) pathway. Hence, inhibition of MAPK signaling using small molecule inhibitors such as MEK inhibitors (MEKi) may be a promising strategy. METHODS: In this multi-center retrospective centrally reviewed study, we analyzed 18 patients treated with the MEKi trametinib for progressive pLGG as an individual treatment decision between 2015 and 2019. We have investigated radiological response as per central radiology review, molecular classification and investigator observed toxicity. RESULTS: We observed 6 partial responses (PR), 2 minor responses (MR), and 10 stable diseases (SD) as best overall responses. Disease control rate (DCR) was 100% under therapy. Responses were observed in KIAA1549:BRAF- as well as neurofibromatosis type 1 (NF1)-driven tumors. Median treatment time was 12.5 months (range: 2 to 27 months). Progressive disease was observed in three patients after cessation of trametinib treatment within a median time of 3 (2-4) months. Therapy related adverse events occurred in 16/18 patients (89%). Eight of 18 patients (44%) experienced severe adverse events (CTCAE III and/or IV; most commonly skin rash and paronychia) requiring dose reduction in 6/18 patients (33%), and discontinuation of treatment in 2/18 patients (11%). CONCLUSIONS: Trametinib was an active and feasible treatment for progressive pLGG leading to disease control in all patients. However, treatment related toxicity interfered with treatment in individual patients, and disease control after MEKi withdrawal was not sustained in a fraction of patients. Our data support in-class efficacy of MEKi in pLGGs and necessity for upfront randomized testing of trametinib against current standard chemotherapy regimens.

NANOG overexpression and its correlation with stem cell and differentiation markers in meningiomas of different WHO grades.

NANOG, as a key regulator of pluripotency and acting synergistically with other factors, has been described as a crucial transcription factor in various types of cancer. In meningiomas the expression of this marker has not yet been described. With our study, we aimed to identify and localize NANOG and other possible markers of pluripotency, stem cell properties and differentiation in meningioma tissue, to elucidate a possible effect on tumorigenesis. The gene expression levels of NANOG (NANOG1 and NANOGP8), SOX2, OCT4, KLF4, ABCG2, CMYC, MSI1, CD44, NOTCH1, NES, SALL4B, TP53, and EPAS1 were quantitatively examined using RT-qPCR in 33 surgical specimens of low- (WHO grade I) as well as in high-grade (WHO grade II/III) meningiomas with dural tissue as reference. Immunofluorescence co-localization analysis following confocal fluorescence microscopy for NANOG, OCT4, SOX2, Nestin, KI-67, and CD44 was also performed. There was a significant overexpression of NANOG, MSI1, and EPAS1 and a downregulation of NES in all examined tumors. Subgroup analysis (WHO grade I versus grade II/III) revealed differences in the expression of NANOG, CD44, and MSI1. We found 1% NANOG-positive (NANOG+) cells in low-grade and 2% in grade II/III meningiomas co-expressing the other mentioned markers in various compositions. In particular, NANOG+ cells expressing SOX2 and OCT4 were successfully identified (26% low-grade versus 20% high-grade). Our data reveal an overexpression of NANOG and other markers of pluripotency and stemness in meningiomas. Such potentially pluripotent "stem cell-like" cells may have an impact on tumorigenesis and progression in human meningiomas.

LOGGIC Core BioClinical Data Bank: Added clinical value of RNA-Seq in an international molecular diagnostic registry for pediatric low-grade glioma patients.

BACKGROUND: The international, multicenter registry LOGGIC Core BioClinical Data Bank aims to enhance the understanding of tumor biology in pediatric low-grade glioma (pLGG) and provide clinical and molecular data to support treatment decisions and interventional trial participation. Hence, the question arises whether implementation of RNA sequencing (RNA-Seq) using fresh frozen (FrFr) tumor tissue in addition to gene panel and DNA methylation analysis improves diagnostic accuracy and provides additional clinical benefit. METHODS: Analysis of patients aged 0 to 21 years, enrolled in Germany between April 2019 and February 2021, and for whom FrFr tissue was available. Central reference histopathology, immunohistochemistry, 850k DNA methylation analysis, gene panel sequencing, and RNA-Seq were performed. RESULTS: FrFr tissue was available in 178/379 enrolled cases. RNA-Seq was performed on 125 of these samples. We confirmed KIAA1549::BRAF-fusion (n = 71), BRAF V600E-mutation (n = 12), and alterations in FGFR1 (n = 14) as the most frequent alterations, among other common molecular drivers (n = 12). N = 16 cases (13%) presented rare gene fusions (eg, TPM3::NTRK1, EWSR1::VGLL1, SH3PXD2A::HTRA1, PDGFB::LRP1, GOPC::ROS1). In n = 27 cases (22%), RNA-Seq detected a driver alteration not otherwise identified (22/27 actionable). The rate of driver alteration detection was hereby increased from 75% to 97%. Furthermore, FGFR1 internal tandem duplications (n = 6) were only detected by RNA-Seq using current bioinformatics pipelines, leading to a change in analysis protocols. CONCLUSIONS: The addition of RNA-Seq to current diagnostic methods improves diagnostic accuracy, making precision oncology treatments (MEKi/RAFi/ERKi/NTRKi/FGFRi/ROSi) more accessible. We propose to include RNA-Seq as part of routine diagnostics for all pLGG patients, especially when no common pLGG alteration was identified.

MAPK inhibitor sensitivity scores predict sensitivity driven by the immune infiltration in pediatric low-grade gliomas.

Pediatric low-grade gliomas (pLGG) show heterogeneous responses to MAPK inhibitors (MAPKi) in clinical trials. Thus, more complex stratification biomarkers are needed to identify patients likely to benefit from MAPKi therapy. Here, we identify MAPK-related genes enriched in MAPKi-sensitive cell lines using the GDSC dataset and apply them to calculate class-specific MAPKi sensitivity scores (MSSs) via single-sample gene set enrichment analysis. The MSSs discriminate MAPKi-sensitive and non-sensitive cells in the GDSC dataset and significantly correlate with response to MAPKi in an independent PDX dataset. The MSSs discern gliomas with varying MAPK alterations and are higher in pLGG compared to other pediatric CNS tumors. Heterogenous MSSs within pLGGs with the same MAPK alteration identify proportions of potentially sensitive patients. The MEKi MSS predicts treatment response in a small set of pLGG patients treated with trametinib. High MSSs correlate with a higher immune cell infiltration, with high expression in the microglia compartment in single-cell RNA sequencing data, while low MSSs correlate with low immune infiltration and increased neuronal score. The MSSs represent predictive tools for the stratification of pLGG patients and should be prospectively validated in clinical trials. Our data supports a role for microglia in the response to MAPKi.

Long-term follow-up of surgical intervention pattern in pediatric low-grade gliomas: report from the German SIOP-LGG 2004 cohort.

OBJECTIVE: Neurosurgical treatment is an integral part of the treatment algorithms for pediatric low-grade glioma (LGG), yet patterns of surgical procedures are rarely challenged. The objective of this study was to evaluate surgical treatment patterns in pediatric LGG. METHODS: The German Societé Internationale d'Oncologie Pédiatrique (SIOP)-LGG 2004 cohort was analyzed to identify relevant patient and tumor characteristics associated with time to death, next surgery, number of resections, and radiological outcome. RESULTS: A total of 1271 patients underwent 1713 neurosurgical interventions (1 intervention in 947, 2 in 230, 3 in 70, and 4-6 in 24). The median age of the study population was 8.57 years at first surgery, and 46.1% were female. Neurofibromatosis type 1 (NF1) was found in 4.4%, and 5.4% had tumor dissemination. Three hundred fifty-four patients (27.9%) had chemotherapy and/or radiotherapy. The cumulative incidence of second surgery at 10 years was 26%, and was higher for infants, those with spinal and supratentorial midline (SML) tumors, and those with pilomyxoid astrocytomas. The hazard ratio for subsequent surgery was higher given dissemination and noncomplete initial resection, and lower for caudal brainstem and SML tumors. Among 1225 patients with fully documented surgical records and radiological outcome, 613 reached complete remission during the observation period, and 50 patients died. Patients with pilocytic astrocytoma had higher chances for a final complete remission, whereas patients with initial partial or subtotal tumor resection, dissemination, NF1, or primary tumor sites in the spinal cord and SML had lower chances. CONCLUSIONS: Neurosurgery is a key element of pediatric LGG treatment. In almost 50% of the patients, however, at least some tumor burden will remain during long-term follow-up. This study found that most of these patients reached a stable disease status without further surgeries. Multidisciplinary team decisions must balance the goal of complete resection, risk factors, repeated surgeries, and possible treatment alternatives in a wide range of heterogeneous entities. Procedural details and neurological outcome should be recorded to better assess their impact on long-term outcome.

Transcriptional Regulation of Thrombin-Induced Endothelial VEGF Induction and Proangiogenic Response.

Thrombin, the ligand of the protease-activated receptor 1 (PAR1), is a well-known stimulator of proangiogenic responses in vascular endothelial cells (ECs), which are mediated through the induction of vascular endothelial growth factor (VEGF). However, the transcriptional events underlying this thrombin-induced VEGF induction and angiogenic response are less well understood at present. As reported here, we conducted detailed promotor activation and signal transduction pathway studies in human microvascular ECs, to decipher the transcription factors and the intracellular signaling events underlying the thrombin and PAR-1-induced endothelial VEGF induction. We found that c-FOS is a key transcription factor controlling thrombin-induced EC VEGF synthesis and angiogenesis. Upon the binding and internalization of its G-protein-coupled PAR-1 receptor, thrombin triggers ERK1/2 signaling and activation of the nuclear AP-1/c-FOS transcription factor complex, which then leads to VEGF transcription, extracellular secretion, and concomitant proangiogenic responses of ECs. In conclusion, exposure of human microvascular ECs to thrombin triggers signaling through the PAR-1-ERK1/2-AP-1/c-FOS axis to control VEGF gene transcription and VEGF-induced angiogenesis. These observations offer a greater understanding of endothelial responses to thromboinflammation, which may help to interpret the results of clinical trials tackling the conditions associated with endothelial injury and thrombosis.

Assessment of Skin Flaps Above Cranial Defects Following Craniectomy: A Proposed Classification System.

OBJECTIVE: The assessment of the skin flap above cranial defects (SCD) following craniectomy is routine in neurosurgical practice, and a change in the consistency of the skin flap may indicate raised intracranial pressure or the occurrence of a complication necessitating intervention. The purpose of this study was to develop a clinically useful classification system based on clinical assessment of the degree of skin flap bulging or sinking and its firmness. PATIENTS AND METHODS: This was a prospective single-center study. The SCDs of consecutive patients who underwent craniectomy were assessed daily by two trained independent examiners. The consistency of the flap and its bulging or sinking in comparison with the level of the cranium were noted. Testing conditions including the positioning of the patient and examiner were standardized. RESULTS: A total of 520 examinations were conducted in 24 patients during their hospital stay. There was 100% interrater reliability (Cohen's κ = 1.0). In 66.6% of all patients (n = 16/24), a change of the SCD classification in comparison with that recorded on the previous day was noted. CONCLUSIONS: The SCD classification facilitates the reproducible and objective assessment of SCDs, enabling reliable monitoring over time and between individuals.

The cost of alcohol in California.

BACKGROUND: California is the largest alcohol market in the United States. In 2005 alone, Californians consumed almost 14 billion alcoholic drinks, which contributed to many severe and potentially fatal alcohol-related illnesses and conditions. Alcohol use also causes violent and nonviolent crimes, as well as injuries and traffic collisions. While several studies have estimated the magnitude and cost of these problems nationally and others have analyzed underage drinking costs, no overall cost estimate at the state level currently exists for California. We present the first comprehensive estimate of the cost of alcohol consumption in California. METHODS: For each category of alcohol-related problems, we estimated fatal and nonfatal cases attributable to alcohol use. We multiplied alcohol-attributable cases by estimated costs per case to obtain total costs for each problem. Our estimates are presented in 2 sections, the economic costs, estimated using a human capital approach, and quality-of-life costs estimated using a quality adjusted life year framework. RESULTS: Alcohol consumption in California led to an estimated 9,439 deaths and 921,929 alcohol-related problems, such as crime and injury in 2005. The economic cost of these problems is estimated at between $35.4 billion and $42.2 billion. Our main estimate is $38.5 billion, of which $5.4 billion was for medical and mental health spending, $25.3 billion in work losses, and $7.8 billion in criminal justice spending, property damage and public program costs. In addition, alcohol is responsible for severe reductions in individuals' quality of life in California. We estimate that the disability caused by injury, the personal anguish of violent crime victims, and the life years lost to fatality are the largest costs imposed by alcohol. The total value for this reduced quality of life in California is between $30.3 billion and $60.0 billion. Our main estimate for quality-of-life costs is $48.8 billion. CONCLUSIONS: In light of the associated substantial illness, injuries, death, and high cost to society, alcohol consumption in California needs serious attention. In addition, the methods developed in this paper can be expanded to estimate the cost of alcohol in other states.

Bush supersizes effort to weaken the World Health Organization.

Obesity has reached global epidemic levels, and two-thirds of Americans are now either obese or overweight. But the U.S. government, along with the powerful food industry, is trying to thwart the World Health Organization's efforts, through its proposed Global Strategy on Diet, Physical Activity, and Health, to make even the most commonsense recommendations on diet.

Reducing youth exposure to alcohol ads: targeting public transit.

Underage drinking is a major public health problem. Youth drink more heavily than adults and are more vulnerable to the adverse effects of alcohol. Previous research has demonstrated the connection between alcohol advertising and underage drinking. Restricting outdoor advertising in general and transit ads in particular, represents an important opportunity to reduce youth exposure. To address this problem, the Marin Institute, an alcohol industry watchdog group in Northern California, conducted a survey of alcohol ads on San Francisco bus shelters. The survey received sufficient media attention to lead the billboard company, CBS Outdoor, into taking down the ads. Marin Institute also surveyed the 25 largest transit agencies; results showed that 75 percent of responding agencies currently have policies that ban alcohol advertising. However, as the experience in San Francisco demonstrated, having a policy on paper does not necessarily mean it is being followed. Communities must be diligent in holding accountable government officials, the alcohol industry, and the media companies through which advertising occurs.