Malignant glioma in L-2-Hydroxyglutaric Aciduria: thorough molecular characterization of a case and literature review.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the inactivating homozygous germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

Cerebrospinal fluid cfDNA sequencing for classification of central nervous system glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing (NGS) of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single nucleotide variants (SNVs), and small insertions/ deletions (indels) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), non-malignant (n = 17) and non-diagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/ or cancer specific alterations in 75.0 % (n = 24) of glioblastoma and 52.6 % (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26-48 %) and SNVs at pre-defined gene loci (44 %), followed by SNVs/ indels identified via uninformed variant calling (8-14 %). A molecular-guided tumor classification was possible for 23.5 % (n = 4) of non-diagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma.

Efficacy and toxicity of bimodal radiotherapy in WHO grade 2 meningiomas following subtotal resection with carbon ion boost: Prospective phase 2 MARCIE trial.

BACKGROUND: Novel radiotherapeutic modalities using carbon ions provide an increased relative biological effectiveness (RBE) compared to photons, delivering a higher biological dose while reducing radiation exposure for adjacent organs. This prospective phase 2 trial investigated bimodal radiotherapy using photons with carbon-ion (C12)-boost in patients with WHO grade 2 meningiomas following subtotal resection (Simpson grade 4 or 5). METHODS: A total of 33 patients were enrolled from July 2012 until July 2020. The study treatment comprised a C12-boost (18 Gy [RBE] in 6 fractions) applied to the macroscopic tumor in combination with photon radiotherapy (50 Gy in 25 fractions). The primary endpoint was the 3-year progression-free survival (PFS), and the secondary endpoints included overall survival, safety and treatment toxicities. RESULTS: With a median follow-up of 42 months, the 3-year estimates of PFS, local PFS and overall survival were 80.3%, 86.7%, and 89.8%, respectively. Radiation-induced contrast enhancement (RICE) was encountered in 45%, particularly in patients with periventricularly located meningiomas. Patients exhibiting RICE were mostly either asymptomatic (40%) or presented immediate neurological and radiological improvement (47%) after the administration of corticosteroids or bevacizumab in case of radiation necrosis (3/33). Treatment-associated complications occurred in 1 patient with radiation necrosis who died due to postoperative complications after resection of radiation necrosis. The study was prematurely terminated after recruiting 33 of the planned 40 patients. CONCLUSIONS: Our study demonstrates a bimodal approach utilizing photons with C12-boost may achieve a superior local PFS to conventional photon RT, but must be balanced against the potential risks of toxicities.

Detailed molecular and pathological analyses of primary intracranial embryonal rhabdomyosarcoma with a BRAF mutation: illustrative case.

BACKGROUND: The etiological significance of the RAS and PI3K pathways has been reported in systemic embryonal rhabdomyosarcoma (ERMS) but not in primary intracranial ERMS (PIERMS). Herein, the authors present a unique case of PIERMS with a BRAF mutation. OBSERVATIONS: A 12-year-old girl with progressive headache and nausea was diagnosed with a tumor in the right parietal lobe. Semi-emergency surgery revealed an intra-axial lesion that was histopathologically identical to an ERMS. Next-generation sequencing indicated a BRAF mutation as a pathogenic variation, but the RAS and PI3K pathways showed no alteration. Although there is no established reference class for PIERMS, the DNA methylation prediction was closest to that of ERMS, indicating the possibility of PIERMS. The final diagnosis was PIERMS. The patient underwent local radiotherapy (50.4 Gy) and multiagent chemotherapy, with no recurrence for 12 months after surgery. LESSONS: This may be the first case demonstrating the molecular features of PIERMS, especially the intra-axial type. The results showed a mutation in BRAF but not in the RAS and PI3K pathways, which is different from the existing ERMS features. This molecular difference may cause differences in DNA methylation profiles. Accumulation of the molecular features of PIERMS is necessary before any conclusions can be drawn.

Integrated genetic analyses of immunodeficiency-associated Epstein-Barr virus- (EBV) positive primary CNS lymphomas.

Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, therefore, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV- PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. In addition to novel insights into the pathobiology of EBV+ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

Analysis of recurrence probability following radiotherapy in patients with CNS WHO grade 2 meningioma using integrated molecular-morphologic classification.

Background: The current World Health Organization (WHO) classification of brain tumors distinguishes 3 malignancy grades in meningiomas, with increasing risk of recurrence from CNS WHO grades 1 to 3. Radiotherapy is recommended by current EANO guidelines for patients not safely amenable to surgery or after incomplete resection in higher grades. Despite adequately predicting recurrence probability for the majority of CNS WHO grade 2 meningioma patients, a considerable subset of patients demonstrates an unexpectedly early tumor recurrence following radiotherapy. Methods: A retrospective cohort of 44 patients with CNS WHO grade 2 meningiomas were stratified into 3 risk groups (low, intermediate, and high) using an integrated morphological, CNV- and methylation family-based classification. Local progression-free survival (lPFS) following radiotherapy (RT) was analyzed and total dose of radiation was correlated with survival outcome. Radiotherapy treatment plans were correlated with follow-up images to characterize the pattern of relapse. Treatment toxicities were further assessed. Results: Risk stratification of CNS WHO grade 2 meningioma into integrated risk groups demonstrated a significant difference in 3-year lPFS following radiotherapy between the molecular low- and high-risk groups. Recurrence pattern analysis revealed that 87.5 % of initial relapses occurred within the RT planning target volume or resection cavity. Conclusions: Integrated risk scoring can identify CNS WHO grade 2 meningioma patients at risk or relapse and dissemination following radiotherapy. Therapeutic management of CNS WHO grade 2 meningiomas and future clinical trials should be adjusted according to the molecular risk-groups, and not rely on conventional CNS WHO grading alone.

Clinical outcome following surgical resection and radiotherapy in adult patients with pleomorphic xanthoastrocytoma as defined by DNA methylation profiling.

Background: Molecular brain tumor classification using DNA methylation profiling has revealed that the methylation-class of pleomorphic xanthoastrocytoma (mcPXA) comprised a substantial portion of divergent initial diagnoses, which had been established based on histology alone. This study aimed to characterize the survival outcome in patients with mcPXAs-in light of the diverse selected treatment regimes. Methods: A retrospective cohort of adult mcPXAs were analyzed in regard to their progression-free survival following surgical resection and postoperative radiotherapy. Radiotherapy treatment plans were correlated with follow-up images to characterize the pattern of relapse. Treatment toxicities and molecular tumor characteristics were further analyzed. Results: Divergent initial histological diagnoses were encountered in 40.7%. There was no significant difference in local progression-free (PFS) and overall survival (OS) following gross total or subtotal resection. Postoperative radiotherapy was completed in 81% (22/27) following surgical intervention. Local PFS was 54.4% (95% CI: 35.3-84.0%) and OS was 81.3% (95% CI: 63.8-100%) after 3 years following postoperative radiotherapy. Initial relapses post-radiotherapy were primarily located in the previous tumor location and/or the planning target volume (PTV) (12/13). All patients in our cohort demonstrated the prognostically favorable pTERT-wildtype mcPXA. Conclusion: Our study demonstrated that adult patients with mcPXAs display a worse progression-free survival compared to the reported WHO grade 2 PXAs. Future matched-pair analyses are required with a non-irradiated cohort to elucidate the benefit of postoperative radiotherapy in adult patients with mcPXAs.

Incidentally exploring natural course of a rare entity: representative case for rosette-forming glioneuronal tumors.

Rosette-forming glioneuronal tumors (RGNT) are extremely rare mostly benign tumors of the central nervous system, which are often studied for its histological aspects despite relatively small numbers of clinical especially radiological knowledge.Despite the increasing number of publications on different localizations and treatment protocols, the morphologic and temporal development process of this rare tumor entity is not clear. We were able to coincidentally observe the entire course of the tumor growth of a RGNT on subsequent MRI examinations in a typical case with mild clinical symptoms and no other neurological illnesses, thus possible clinical complications were prevented.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Adult cerebellar glioblastoma categorized into a pediatric methylation class with a unique radiological and histological appearance: illustrative case.

BACKGROUND: Recent studies report that cerebellar glioblastoma (GBM) is categorized into the RTK1 methylation class. GBM pediatric RTK (pedRTK) subtypes are distinct from those of adult GBM. We present a unique adult case of cerebellar GBM classified into the pedRTK subtype. OBSERVATIONS: Magnetic resonance imaging revealed a homogeneous enhancing lesion in the right cerebellum in a 56-year-old woman presenting with ataxia and dizziness. Arterial spin labeling and angiographic findings and the intraoperative orange-colored tumor appearance were reminiscent of hemangioblastoma. She showed an atypical presentation in terms of high glucose metabolism. The histological diagnosis was high-grade glioma with differentiation similar to central nervous system neuroblastoma. The methylation class was GBM pedRTK1. Consistent with this classification, immunoexpression was positive for SOX10 and negative for ANKRD55. She underwent craniospinal radiotherapy (23.4 Gy) with a boost to the tumor bed (total 55.8 Gy). Twelve courses of temozolomide therapy were administered. There was no recurrence 18 months after surgery. LESSONS: Radiological and intraoperative findings, such as hemangioblastoma and high glucose metabolism, were notable characteristics in the present case. Both glial and neuronal differentiation and SOX10 immunoexpression were presenting pathological features. Similar cerebellar GBMs might form a previously unestablished subtype. Establishing effective molecular diagnoses is important.

SMARCB1-deficient and SMARCA4-deficient Malignant Brain Tumors With Complex Copy Number Alterations and TP53 Mutations May Represent the First Clinical Manifestation of Li-Fraumeni Syndrome.

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant central nervous system tumor predominantly affecting infants. Mutations of SMARCB1 or (rarely) SMARCA4 causing loss of nuclear SMARCB1 or SMARCA4 protein expression are characteristic features, but further recurrent genetic alterations are lacking. Most AT/RTs occur de novo, but secondary AT/RTs arising from other central nervous system tumors have been reported. Malignant gliomas, IDH wild-type, arising in patients with Li-Fraumeni syndrome typically show somatic mutations of TP53 as well as complex copy number alterations, but little is known about the loss of SMARCB1 or SMARCA4 protein expression in this context. Here, we report 2 children in whom malignant supratentorial brain tumors with SMARCB1 deficiency, complex copy number alterations, and somatic TP53 mutations lead to the discovery of pathogenic/likely pathogenic TP53 variants in the germline. Screening of the molecularneuropathology.org dataset for cases with similar genetic and epigenetic alterations yielded another case with SMARCA4 deficiency in a young adult with Li-Fraumeni syndrome. In conclusion, SMARCB1-deficient or SMARCA4-deficient malignant brain tumors with complex copy number alterations and somatic TP53 mutations in children and young adults may represent the first clinical manifestation of Li-Fraumeni syndrome and should prompt genetic counseling and investigation for TP53 germline status.

Oligosarcomas, IDH-mutant are distinct and aggressive.

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy-number profile with inactivation of TP53, PTEN, and RB1.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.