CIC/ATXN1-rearranged tumors in the central nervous system are mainly represented by sarcomas: A comprehensive clinicopathological and epigenetic series.

CIC fusions have been described in two different central nervous system (CNS) tumor entities. On one hand, fusions of CIC or ATXN1 genes belonging to the same complex of transcriptional repressors, were reported in the CIC-rearranged, sarcoma (SARC-CIC). The diagnosis of this tumor type, which was recently added to the World Health Organization (WHO) Classification of CNS tumors, is difficult mainly because the data concerning its histopathology (as compared to its soft tissue counterpart), immunoprofile, and clinical as well as radiological characteristics are scarce in the literature. On the other hand, a recent study, based on DNA-methylation profiling, has identified a novel high-grade neuroepithelial tumor characterized by recurrent CIC fusions (HGNET-CIC). The aim of this multicentric study was to characterize a cohort of 15 primary CNS tumors harboring a CIC or ATXN1 fusion in terms of clinical, radiological, histopathological, immunophenotypical, and epigenetic characteristics. According to the integrated diagnoses, 14/15 tumors corresponded to SARC-CIC, and only one to HGNET-CIC. The tumors showed similar clinical (mainly pediatric), radiological (mostly supratentorial, cystic, and contrast enhancing), immunophenotypical (common expression of glioneuronal markers), and genetic (similar spectrum of fusions) profiles but their histopathological appearance was clearly distinct. Moreover, we found a novel fusion transcript (CIC::EWSR1) in a SARC-CIC. Most DNA methylation profiles using the Heidelberg Brain Tumor Classifier (v12.8) annotated the samples to the methylation class "SARC-CIC" (9/14 tumors with available data). By using uniform manifold approximation and projection analysis, four other samples were classified as SARC-CIC and another clustered within the methylation class of HGNET-CIC. Our findings confirm that CNS CIC-fused tumors do not represent a single molecular tumor entity. Further analyses are needed to characterize HGNET-CIC in more detail. These results may help to refine the essential diagnostic criteria for SARC-CIC and their terminology (with a suggested consensual name of sarcoma, CIC/ATXN1-complex rearranged).

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.

Intrathecal IgG4 synthesis in IgG4 related spinal hypertrophic pachymeningitis: a case report.

Immunoglobulin G4 (IgG4) related hypertrophic pachymeningitis of the spinal cord is a rare condition, characterized by infiltration of the spinal meninges with IgG4-producing plasma cells and subsequent hypertrophic fibrosis. Here, we report on a 65-year-old woman with IgG4 associated hypertrophic spinal pachymeningitis, in whom cerebrospinal fluid (CSF) analysis was a decisive diagnostic tool. Not only could we demonstrate an intrathecal IgG4 production, but also IgG4 positive plasma cells in CSF. Following decompressive surgery, diagnosis of IgG4 associated hypertrophic pachymeningitis was confirmed histologically. Surgery and immunosuppressive therapy with rituximab were associated with clinical improvement. This case highlights CSF analyses as diagnostic tool for detection of IgG4 related hypertrophic pachymeningitis.

Radiomics and visual analysis for predicting success of transplantation of heterotopic glioblastoma in mice with MRI.

BACKGROUND: Quantifying tumor growth and treatment response noninvasively poses a challenge to all experimental tumor models. The aim of our study was, to assess the value of quantitative and visual examination and radiomic feature analysis of high-resolution MR images of heterotopic glioblastoma xenografts in mice to determine tumor cell proliferation (TCP). METHODS: Human glioblastoma cells were injected subcutaneously into both flanks of immunodeficient mice and followed up on a 3 T MR scanner. Volumes and signal intensities were calculated. Visual assessment of the internal tumor structure was based on a scoring system. Radiomic feature analysis was performed using MaZda software. The results were correlated with histopathology and immunochemistry. RESULTS: 21 tumors in 14 animals were analyzed. The volumes of xenografts with high TCP (H-TCP) increased, whereas those with low TCP (L-TCP) or no TCP (N-TCP) continued to decrease over time (p < 0.05). A low intensity rim (rim sign) on unenhanced T1-weighted images provided the highest diagnostic accuracy at visual analysis for assessing H-TCP (p < 0.05). Applying radiomic feature analysis, wavelet transform parameters were best for distinguishing between H-TCP and L-TCP / N-TCP (p < 0.05). CONCLUSION: Visual and radiomic feature analysis of the internal structure of heterotopically implanted glioblastomas provide reproducible and quantifiable results to predict the success of transplantation.

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.

Effects of Tetrahydrolipstatin on Glioblastoma in Mice: MRI-Based Morphologic and Texture Analysis Correlated with Histopathology and Immunochemistry Findings-A Pilot Study.

BACKGROUND: This study aimed to investigate the effects of tetrahydrolipstatin (orlistat) on heterotopic glioblastoma in mice by applying MRI and correlating the results with histopathology and immunochemistry. METHODS: Human glioblastoma cells were injected subcutaneously into the groins of immunodeficient mice. After tumor growth of >150 mm3, the animals were assigned into a treatment group (n = 6), which received daily intraperitoneal injections of orlistat, and a control group (n = 7). MRI was performed at the time of randomization and before euthanizing the animals. Tumor volumes were calculated, and signal intensities were analyzed. The internal tumor structure was evaluated visually and with texture analysis. Western blotting and protein expression analysis were performed. RESULTS: At histology, all tumors showed high mitotic and proliferative activity (Ki67 ≥ 10%). Reduced fatty acid synthetase expression was measured in the orlistat group (p < 0.05). Based on the results of morphologic MRI-based analysis, tumor growth remained concentric in the control group and changed to eccentric in the treatment group (p < 0.05). The largest area under the receiver operating curve of the predictors derived from the texture analysis of T2w images was for wavelet transform parameters WavEnHL_s3 and WavEnLH_s4 at 0.96 and 1.00, respectively. CONCLUSIONS: Orlistat showed effects on heterotopically implanted glioblastoma multiforme in MRI studies of mice based on morphologic and texture analysis.

Combined Lanreotide Autogel and Temozolomide Treatment of Progressive Pancreatic and Intestinal Neuroendocrine Tumors: The Phase II SONNET Study.

BACKGROUND: In advanced neuroendocrine tumors (NET), antiproliferative treatment options beyond somatostatin analogs remain limited. Temozolomide (TMZ) has shown efficacy in NET alone or combined with other drugs. MATERIALS AND METHODS: SONNET (NCT02231762) was an open, multicenter, prospective, phase II study to evaluate lanreotide autogel 120 mg (LAN) plus TMZ in patients with progressive advanced/metastatic grade 1/2 gastroenteropancreatic (GEP) NET or of unknown primary. Patients could be enrolled at first-line or higher therapy line. The primary endpoint was disease control rate ([DCR], rate of stable disease [SD], partial [PR], and complete response [CR]) at 6 months of LAN and TMZ. Patients with nonfunctioning (NF) NET without progression at 6 months were randomized to 6-month LAN maintenance or watch and wait, patients with functioning (F)-NET with clinical benefit (PR, SD) continued on LAN. RESULTS: Fifty-seven patients were recruited. The majority of patients received the study drug at second or higher treatment line and had an NET G2. DCR at 6 months LAN and TMZ was 73.5%. After 6 months of further LAN maintenance, 54.5% of patients with F-NET and 71.4% with NF-NET had SD or PR vs 41.7% with NF-NET on observation only. LAN and TMZ were effective in all subgroups analyzed. At 12 months of follow-up, median progression-free survival was 11.1 months. Median serum chromogranin A decreased except in NF-NET on observation. O6-methylguanine DNA methyltransferase promoter methylation appeared to better reflect TMZ response than loss of gene expression. During combination therapy, the most frequent treatment-emergent adverse events grade 3/4 reported were nausea (14%), thrombocytopenia (12.3%), and neutropenia (8.8%). Four deaths were reported resulting from severe adverse events not considered related to study medication. CONCLUSIONS: LAN plus TMZ is a treatment option for patients with progressive GEP-NET with more aggressive biological profile showing a manageable safety profile.

LOGGIC/FIREFLY-2: a phase 3, randomized trial of tovorafenib vs. chemotherapy in pediatric and young adult patients with newly diagnosed low-grade glioma harboring an activating RAF alteration.

BACKGROUND: Pediatric low-grade glioma (pLGG) is essentially a single pathway disease, with most tumors driven by genomic alterations affecting the mitogen-activated protein kinase/ERK (MAPK) pathway, predominantly KIAA1549::BRAF fusions and BRAF V600E mutations. This makes pLGG an ideal candidate for MAPK pathway-targeted treatments. The type I BRAF inhibitor, dabrafenib, in combination with the MEK inhibitor, trametinib, has been approved by the United States Food and Drug Administration for the systemic treatment of BRAF V600E-mutated pLGG. However, this combination is not approved for the treatment of patients with tumors harboring BRAF fusions as type I RAF inhibitors are ineffective in this setting and may paradoxically enhance tumor growth. The type II RAF inhibitor, tovorafenib (formerly DAY101, TAK-580, MLN2480), has shown promising activity and good tolerability in patients with BRAF-altered pLGG in the phase 2 FIREFLY-1 study, with an objective response rate (ORR) per Response Assessment in Neuro-Oncology high-grade glioma (RANO-HGG) criteria of 67%. Tumor response was independent of histologic subtype, BRAF alteration type (fusion vs. mutation), number of prior lines of therapy, and prior MAPK-pathway inhibitor use. METHODS: LOGGIC/FIREFLY-2 is a two-arm, randomized, open-label, multicenter, global, phase 3 trial to evaluate the efficacy, safety, and tolerability of tovorafenib monotherapy vs. current standard of care (SoC) chemotherapy in patients < 25 years of age with pLGG harboring an activating RAF alteration who require first-line systemic therapy. Patients are randomized 1:1 to either tovorafenib, administered once weekly at 420 mg/m2 (not to exceed 600 mg), or investigator's choice of prespecified SoC chemotherapy regimens. The primary objective is to compare ORR between the two treatment arms, as assessed by independent review per RANO-LGG criteria. Secondary objectives include comparisons of progression-free survival, duration of response, safety, neurologic function, and clinical benefit rate. DISCUSSION: The promising tovorafenib activity data, CNS-penetration properties, strong scientific rationale combined with the manageable tolerability and safety profile seen in patients with pLGG led to the SIOPe-BTG-LGG working group to nominate tovorafenib for comparison with SoC chemotherapy in this first-line phase 3 trial. The efficacy, safety, and functional response data generated from the trial may define a new SoC treatment for newly diagnosed pLGG. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05566795. Registered on October 4, 2022.

Neurosurgical treatment of pediatric brain tumors - results from a single center multidisciplinary setup.

OBJECTIVE: The challenge of pediatric brain tumor surgery is given due to a relative low prevalence but high heterogeneity in age, localization, and pathology. Improvements of long-term overall survival rates were achieved during the past decades stressing the importance of a multidisciplinary decision process guided by a national treatment protocol. We reviewed the entire spectrum of pediatric brain tumor surgeries from the perspective of an interdisciplinary pediatric neuro-oncology center in Germany. METHODS: Every patient who underwent brain tumor surgery from January 2010 to June 2017 in our Pediatric Neurosurgery department was retrospectively included and evaluated regarding the course of treatment. Perioperative data such as tumor localization, timing of surgery, extent of resection, neuropathological diagnosis, transfusion rates, oncologic and radiation therapy, and neurological follow-up including morbidity and mortality were evaluated. RESULTS: Two hundred ninety-three pediatric brain tumor patients were applicable (age: 8.28 ± 5.62 years, 1.22:1.0 m:f). A total of 531 tumor surgical interventions was performed within these patients (457 tumor resections, 74 tumor biopsies; mean interventions per patient 1.8 ± 1.2). Due to a critical neurologic status, 32 operations (6%) were performed on the day of admission. In 65.2% of all cases, tumor were approached supratentorially. Most frequent diagnoses of the cases were glial tumors (47.8%) and embryonal tumors (17.6%). Preoperative planned extent of resection was achieved in 92.7%. Pre- and postoperative neurologic deficits resolved completely in 30.7%, whereas symptom regressed in 28.6% of surgical interventions. New postoperative neurologic deficit was observed in 10.7%, which resolved or improved in 80% of these cases during 30 days. The mortality rate was 1%. CONCLUSION: We outlined the center perspective of a specialized pediatric neuro-oncological center describing the heterogeneous distribution of cases regarding age-related prevalence, tumor localization, and biology, which requires a high multidisciplinary expertise. The study contributes to define challenges in treating pediatric brain tumors and to develop quality indicators for pediatric neuro-oncological surgery. We assume that an adequate volume load of patients within a interdisciplinary infrastructure is warranted to aim for effective treatment and decent quality of life for the majority of long-term surviving pediatric tumor patients.

NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62.

NEMO is a ubiquitin-binding protein which regulates canonical NF-κB pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identify an NF-κB-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO-encoding IKBKG gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including α-synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at α-synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

A Novel Type of IDH-wildtype Glioma Characterized by Gliomatosis Cerebri-like Growth Pattern, TERT Promoter Mutation, and Distinct Epigenetic Profile.

Diffuse gliomas in adults encompass a heterogenous group of central nervous system neoplasms. In recent years, extensive (epi-)genomic profiling has identified several glioma subgroups characterized by distinct molecular characteristics, most importantly IDH1/2 and histone H3 mutations. A group of 16 diffuse gliomas classified as "adult-type diffuse high-grade glioma, IDH-wildtype, subtype F (HGG-F)" was identified by the DKFZ v12.5 Brain Tumor Classifier . Histopathologic characterization, exome sequencing, and review of clinical data was performed in all cases. Based on unsupervised t -distributed stochastic neighbor embedding and clustering analysis of genome-wide DNA methylation data, HGG-F shows distinct epigenetic profiles separate from established central nervous system tumors. Exome sequencing demonstrated frequent TERT promoter (12/15 cases), PIK3R1 (11/16), and TP53 mutations (5/16). Radiologic characteristics were reminiscent of gliomatosis cerebri in 9/14 cases (64%). Histopathologically, most cases were classified as diffuse gliomas (7/16, 44%) or were suspicious for the infiltration zone of a diffuse glioma (5/16, 31%). None of the cases demonstrated microvascular proliferation or necrosis. Outcome of 14 patients with follow-up data was better compared to IDH-wildtype glioblastomas with a median progression-free survival of 58 months and overall survival of 74 months (both P <0.0001). Our series represents a novel type of adult-type diffuse glioma with distinct molecular and clinical features. Importantly, we provide evidence that TERT promoter mutations in diffuse gliomas without further morphologic or molecular signs of high-grade glioma should be interpreted in the context of the clinicoradiologic presentation as well as epigenetic profile and may not be suitable as a standalone marker for glioblastoma, IDH-wildtype.

Recurrent atypical teratoid/rhabdoid tumors (AT/RT) reveal discrete features of progression on histology, epigenetics, copy number profiling, and transcriptomics.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors manifesting in infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth quantitatively smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). Molecular characteristics of disease recurrence or metastatic spread, which go along with a particularly dismal outcome, are currently unclear. Here, we investigated tumor tissue from 26 patients affected by AT/RT to identify signatures of recurrences in comparison with matched primary tumor samples. Microscopically, AT/RT recurrences demonstrated a loss of architecture and significantly enhanced mitotic activity as compared to their related primary tumors. Based on DNA methylation profiling, primary tumor and related recurrence were grossly similar, but three out of 26 tumors belonged to a different molecular type or subtype after second surgery compared to related primary lesions. Copy number variations (CNVs) differed in six cases, showing novel gains on chromosome 1q or losses of chromosome 10 in recurrences as the most frequent alterations. To consolidate these observations, our cohort was combined with a data set of unmatched primary and recurrent AT/RT, which demonstrated chromosome 1q gain and 10 loss in 18% (n = 7) and 11% (n = 4) of the recurrences (n = 38) as compared to 7% (n = 3) and 0% (n = 0) in the primary tumors (n = 44), respectively. Similar to the observations made by DNA methylation profiling, RNA sequencing of our cohort revealed AT/RT primary tumors and matched recurrences clustering closely together. However, a number of genes showed significantly altered expression in AT/RT-SHH recurrences. Many of them are known tumor driving growth factors, involved in embryonal development and tumorigenesis, or are cell-cycle-associated. Overall, our work identifies subtle molecular changes that occur in the course of the disease and that may help define novel therapeutic targets for AT/RT recurrences.

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.

Biopsies of Caudal Brainstem Tumors in Pediatric Patients-A Single-Center Retrospective Case Series.

OBJECTIVE: The indication for performing biopsies in patients with diffuse lesions in the brain stem is controversial. The possible risks associated with the technically challenging interventions must be balanced against clarifying the diagnosis and the possible therapeutic options. We reviewed the feasibility, risk profile, and diagnostic yield of different biopsy techniques in a pediatric cohort. METHODS: We retrospectively included all patients aged <18 years who had undergone biopsy of the caudal brainstem region (pons, medulla oblongata) at our pediatric neurosurgical center from 2009 to 2022. RESULTS: We identified 27 children. Biopsies were performed using frameless stereotactic (Varioguide; n = 12), robotic-assisted (Autoguide; n = 4), endoscopic (n = 3), and open biopsy (n = 8) techniques. Intervention-related mortality was not observed. Three patients experienced a transient postoperative neurological deficit. No patient experienced intervention-related permanent morbidity. Biopsy yielded the histopathological diagnosis in all 27 cases. Molecular analysis was feasible for 97% of the cases. The most common diagnosis was H3K27M-mutated diffuse midline glioma (60%). Low-grade gliomas were identified in 14% of patients. Overall survival was 62.5% after 24 months of follow-up. CONCLUSIONS: Biopsies of the caudal brainstem in children were feasible and safe in the presented setup. The amount of tumor material acquired allowing for an integrated diagnosis and was obtained at reasonable risk. The selection of the surgical technique depends on the tumor location and growth pattern. We recommend the performance of brainstem tumor biopsies in children at specialized centers to better understand the biology and enable possible novel therapeutic options.

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.

Multiomic neuropathology improves diagnostic accuracy in pediatric neuro-oncology.

The large diversity of central nervous system (CNS) tumor types in children and adolescents results in disparate patient outcomes and renders accurate diagnosis challenging. In this study, we prospectively integrated DNA methylation profiling and targeted gene panel sequencing with blinded neuropathological reference diagnostics for a population-based cohort of more than 1,200 newly diagnosed pediatric patients with CNS tumors, to assess their utility in routine neuropathology. We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%). Discrepant results by neuropathological WHO-based and DNA methylation-based classification (30%) were enriched in histological high-grade gliomas, implicating relevance for current clinical patient management in 5% of all patients. Follow-up (median 2.5 years) suggests improved survival for patients with histological high-grade gliomas displaying lower-grade molecular profiles. These results provide preliminary evidence of the utility of integrating multi-omics in neuropathology for pediatric neuro-oncology.

Proteomics separates adult-type diffuse high-grade gliomas in metabolic subgroups independent of 1p/19q codeletion and across IDH mutational status.

High-grade adult-type diffuse gliomas are malignant neuroepithelial tumors with poor survival rates in combined chemoradiotherapy. The current WHO classification is based on IDH1/2 mutational and 1p/19q codeletion status. Glioma proteome alterations remain undercharacterized despite their promise for a better molecular patient stratification and therapeutic target identification. Here, we use mass spectrometry to characterize 42 formalin-fixed, paraffin-embedded (FFPE) samples from IDH-wild-type (IDHwt) gliomas, IDH-mutant (IDHmut) gliomas with and without 1p/19q codeletion, and non-neoplastic controls. Based on more than 5,500 quantified proteins and 5,000 phosphosites, gliomas separate by IDH1/2 mutational status but not by 1p/19q status. Instead, IDHmut gliomas split into two proteomic subtypes with widespread perturbations, including aerobic/anaerobic energy metabolism. Validations with three independent glioma proteome datasets confirm these subgroups and link the IDHmut subtypes to the established proneural and classic/mesenchymal subtypes in IDHwt glioma. This demonstrates common phenotypic subtypes across the IDH status with potential therapeutic implications for patients with IDHmut gliomas.