New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Malignant meningioma mTOR mutated and precision medicine.

BACKGROUND: WHO grade II and III meningiomas are more invasive than grade I malignancies and determine patients' shorter overall survival. Their tendency to recur after treatment has represented an important therapeutic challenge because of the limited treatment strategies at recurrence. Angiogenesis and mechanistic target of rapamycin (mTOR) activation are two of the main features of higher grade meningiomas, determining invasiveness and tendency to relapse. While these options prove promising, available clinical data on mTOR inhibitors' efficacy are somewhat limited. CASE STUDY: We report a case of a 25-year-old female patient diagnosed with a right parasagittal occipital anaplastic meningioma (grade III WHO) in 2013. The patient underwent multiple treatments and, in 2019, a further recurrence occurred. The patient reported an mTOR mutation, and it is for this reason that the MTB approved treatment with everolimus and bevacizumab. Therapy was administered in May 2019, and partial response and prolonged disease control was obtained in November 2021, when progression took place. The patient's death occurred in March 2022. CONCLUSIONS: This case report provides evidence on the efficacy of mTOR inhibitors as a treatment option in recurrent meningiomas. Furthermore, it highlights the importance of performing a molecular analysis as a preliminary step towards targeting the mTOR pathway.

Paediatric astroblastoma-like neuroepithelial tumour of the spinal cord with a MAMLD1-BEND2 rearrangement.

Astroblastomas are neuroepithelial tumours defined by the presence of MN1 rearrangement and are typically located in the cerebral hemispheres. Rare cases of astroblastoma-like tumours carrying an EWSR1-BEND2 fusion have been recently described in the brain stem and spinal cord. We report a paediatric case of neuroepithelial astroblastoma-like tumour occurring in the spine and carrying a novel MAMLD1-BEND2 fusion. We believe that our case aligns with the rare astroblastoma-like tumours with EWSR1-BEND2 fusion, in terms of non-hemispheric location, pathology, methylation profile and activation of BEND2 transcription. Whether they may represent a distinct entity or a variant of MN1-altered astroblastoma is not clear.

Atypical teratoid/rhabdoid tumor in adults: a systematic review of the literature with meta-analysis and additional reports of 4 cases.

INTRODUCTION: Atypical teratoid/rhabdoid tumor (AT/RT) is a highly aggressive embryonal CNS neoplasm, characterized by inactivation of SMARCB1 (INI1) or rarely of SMARCA4 (BRG1). While it is predominantly a childhood tumor, AT/RT is rare in adults. METHODS: We provide a comprehensive systematic review of literature with meta-analysis; 92 adult cases were found from 74 articles. We additionally present 4 cases of adult AT/RTs (age ranging from 19 to 29 years), located to cerebellum in 2 cases, to ponto-cerebellar angle in 1 case and to spinal cord in the remaining case. RESULTS: Microscopic features of our 4 cases showed a highly cellular tumor with rhabdoid morphology and high mitotic activity. All tumor cells lacked nuclear SMARCB1/INI1 protein expression. In case no. 3 we also performed methylation profiling which clustered the tumor with pediatric AT/RT-MYC subgroup. Prognosis remains poor in both pediatric and adult population with a median overall survival of 11 months. Our review demonstrated median overall survival of 15 months among the adult populations. However, consistent with a recent review, adult AT/RT seems to have highly variable prognosis and some patients reach long term survival with 22.9% of 5-year survival without evidence of disease and mean follow up time of 35.9 months (SD = 36.5). 27.1% of dissemination was also reported among the adult population. CONCLUSIONS: Adult AT/RTs predominantly arise in female patients and in supratentorial location. Midline structures, including the sellar region, are the most affected sites, especially among females aged > 40 years. Male gender is more prevalent between the age of 18 and 40 years and more frequently associated with non-midline tumors. Factors significantly associated with better prognosis are patient's age (< 40 years), combined radio-chemotherapy adjuvant approach and Ki-67 score < 40%.

Comprehensive analysis of the ErbB receptor family in pediatric nervous system tumors and rhabdomyosarcoma.

BACKGROUND: There is a paucity of knowledge regarding pediatric biomarkers, including the relevance of ErbB pathway aberrations in pediatric tumors. We investigated the occurrence of ErbB receptor aberrations across different pediatric malignancies, to identify patterns of ErbB dysregulation and define biomarkers suitable for patient enrichment in clinical studies. PROCEDURE: Tissue samples from 297 patients with nervous system tumors and rhabdomyosarcoma were analyzed for immunohistochemical expression or gene amplification of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). Exploratory analyses of HER3/HER4 expression, and mRNA expression of ErbB receptors/ligands (NanoString) were performed. Assay validation followed general procedures, with additional validation to address Clinical Laboratory Improvement Amendments (CLIA) requirements. RESULTS: In most tumor types, samples with high ErbB receptor expression were found with heterogeneous distribution. We considered increased/aberrant ErbB pathway activation when greater than or equal to two EGFR/HER2 markers were simultaneously upregulated. ErbB pathway dysregulation was identified in ∼20%-30% of samples for most tumor types (medulloblastoma/primitive neuroectodermal tumors 31.1%, high-grade glioma 27.1%, neuroblastoma 22.7%, rhabdomyosarcoma 23.1%, ependymoma 18.8%), 4.2% of diffuse intrinsic pontine gliomas, and no recurrent or refractory low-grade astrocytomas. In medulloblastoma/primitive neuroectodermal tumors and neuroblastoma, this was attributed mainly to high EGFR polysomy/HER2 amplification, whereas EGFR gene amplification was observed in some high-grade glioma samples. EGFR/HER2 overexpression was most prevalent in ependymoma. CONCLUSIONS: Overexpression and/or amplification of EGFR/HER2 were identified as potential enrichment biomarkers for clinical trials of ErbB-targeted drugs.

Cerebellar liponeurocytoma in an elderly patient: DNA methylation profiling as a helpful diagnostic tool.

AIM: Cerebellar liponeurocytoma is a rare primary cerebellar neoplasm that mostly occurs in adults, however, it is rare in the elderly. MATERIALS AND METHODS: We report, in a 79-year-old female, a recurrent vermian cerebellar mass that was previously diagnosed as primary cerebellar tumor with neuroendocrine differentiation. The recurrent lesion showed anaplastic features and lipidization. RESULTS: DNA methylation profiling was performed for the recurrent tumor, which showed a high score match for cerebellar liponeurocytoma. CONCLUSION: This report confirms the usefulness of DNA methylation profiling for the diagnosis of challenging CNS tumors.

Cerebellar liponeurocytoma: clinical, histopathological and molecular features of a series of three cases, including one recurrent tumor.

Cerebellar liponeurocytoma (CL) is an unusual tumor, histologically composed of a mixture of small to medium-sized, rounded neurocytic cells and a variable lipomatous component. Although CL was originally considered as a subtype of medulloblastoma, subsequent molecular studies demonstrated that this tumor was a distinct entity, exhibiting the tumor protein p53 gene (TP53) missense mutations in 20% of cases, chromosome 17 deletion, and the absence of mutations in the adenomatous polyposis coli gene (APC), the protein patched homolog gene (PTCH), the kinase insert domain receptor gene (KDR), and the ß-catenin gene (CTNNB). Apart from these molecular features, little is known about the pathogenesis and the genetic landscape of CL to date. In order to characterize the mutational landscape of CL and identify alterations that are driving tumorigenesis, we report a series of three cases, including one recurrent tumor, analysed by next-generation sequencing (NGS), which identified a total of 22 variants, of which four were missense mutations, nine were synonymous variants, and nine were located on intronic regions. In particular, DNA sequencing identified missense mutations in APC, KDR, and TP53 that could be implicated in promoting tumor progression and angiogenesis of CL. Furthermore, the NGS analysis revealed that recurrent CL did not have additional genetic changes compared with the primary tumor. Moreover, the high frequencies of detected mutations suggested that the identified alterations are germline variants. Indeed, an additional NGS on the genomic DNA obtained from one of the three patients confirmed the presence of the variants in the germline DNA. In conclusion, the obtained data support the hypothesis that CL is a distinct pathological entity that does not show specific somatic alterations driving tumorigenesis.

Paediatric-type diffuse high-grade gliomas in the 5th CNS WHO Classification.

As a relevant element of novelty, the fifth CNS WHO Classification highlights the distinctive pathobiology underlying gliomas arising primarily in children by recognizing for the first time the families of paediatric-type diffuse gliomas, both high-grade and low-grade. This review will focus on the family of paediatric-type diffuse high-grade gliomas, which includes four tumour types: 1) Diffuse midline glioma H3 K27-altered; 2) Diffuse hemispheric glioma H3 G34-mutant; 3) Diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype; and 4) Infant-type hemispheric glioma. The essential and desirable diagnostic criteria as well as the entities entering in the differential will be discussed for each tumour type. A special focus will be given on the issues encountered in the daily practice, especially regarding the diagnosis of the diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype. The advantages and the limits of the multiple molecular tests which may be utilised to define the entities of this tumour family will be evaluated in each diagnostic context.

Characterization of primary glioma cell lines derived from the patients according to 2016 CNS tumour WHO classification and comparison with their parental tumours.

BACKGROUND: Gliomas represent about 80% of primary brain tumours and about 30% of malignant ones, which today don't have a resolution therapy because of their variability. A valid model for the study of new personalized therapies can be represented by primary cultures from patient's tumour biopsies. METHODS: In this study we consider 12 novel cell lines established from patients' gliomas and immunohistochemically and molecularly characterized according to the newly updated 2016 CNS Tumour WHO classification. RESULTS: Eight of these lines were glioblastoma cells, two grade III glioma cells (anaplastic astrocytoma and oligo astrocytoma) and two low grade glioma cells (grade II astrocytoma and oligodendroglioma). All cell lines were analysed by immunohistochemistry for specific glioma markers, respectively VIMENTIN, GFAP, IDH1R132, and ATRX. The methylation status of the MGMT gene promoter was also determined in all lines. The comparison of the immunohistochemical characteristics and of the MGMT methylation status of the lines with the tissues of origin shows that the cells in culture maintain the same characteristics. CONCLUSIONS: Human cancer cell lines represent a support in the knowledge of tumour biology and in drug discovery through its facile experimental manipulation. TRIAL REGISTRATION: NCT04180046.

Diffuse midline glioma H3 K27M-mutant in adults: A report of six cases and literature review.

AIM: Diffuse midline glioma (DMG) H3 K27M-mutant is a specific entity that, as the name indicates, tends to occur in midline structures including the thalamus, brainstem, and spinal cord. DMG predominates in children, is an aggressive tumor with poor prognosis, and is considered a WHO grade IV tumor regardless of histological features. The exact frequency of these mutations in adults diagnosed with glioma in the midline is unknown. MATERIALS AND METHODS: We report a series of 6 more adult cases, and we critically review the current literature on adults with DMG H3 K27M-mutant. RESULTS: There were 5 males and 1 female. The age ranged from 26 to 52 years (median 39 years). All cases showed astrocytic differentiation, with positive staining for H3 K27M protein, and loss of H3 K27me in the tumor cells confirming the diagnosis. CONCLUSION: H3 K27M-mutant midline glioma can occur in adults, affecting midline structures. Increasing awareness of the reporting pathologists of this entity might help in a better determination of the frequency of mutant DMG in adults as well as better diagnosis and patient counseling of the outcome.

Reduced-dose craniospinal irradiation is feasible for standard-risk adult medulloblastoma patients.

INTRODUCTION: Medulloblastoma is the most common malignant brain tumor in children, but accounts for only 1% of brain cancers in adults. For standard-risk pediatric medulloblastoma, current therapy includes craniospinal irradiation (CSI) at reduced doses (23.4 Gy) associated with chemotherapy. Whereas most same-stage adult patients are still given CSI at 36 Gy, with or without chemotherapy, we report here on our use of reduced-dose CSI associated with chemotherapy for older patients. METHODS: We gathered non-metastatic patients over 18 years old (median age 28 years, range 18-48) with minimal or no residual disease after surgery, no negative histological subtypes, treated between 1996-2018 at the Centre Léon Bérard (Lyon) and the INT (Milano). A series of 54 children with similar tumors treated in Milano was used for comparison. RESULTS: Forty-four adults were considered (median follow-up 101 months): 36 had 23.4 Gy of CSI, and 8 had 30.6 Gy, plus a boost to the posterior fossa/tumor bed; 43 had chemotherapy as all 54 children, who had a median 83-month follow-up. The PFS and OS were 82.2 ± 6.1% and 89 ± 5.2% at 5 years, and 78.5 ± 6.9% and 75.2 ± 7.8% at ten, not significantly different from those of the children. CSI doses higher than 23.4 Gy did not influence PFS. Female adult patients tended to have a better outcome than males. CONCLUSION: The results obtained in our combined series are comparable with, or even better than those obtained after high CSI doses, underscoring the need to reconsider this treatment in adults.

Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth.

In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.

EANO-EURACAN clinical practice guideline for diagnosis, treatment, and follow-up of post-pubertal and adult patients with medulloblastoma.

The European Association of Neuro-Oncology (EANO) and EUropean RAre CANcer (EURACAN) guideline provides recommendations for the diagnosis, treatment, and follow-up of post-pubertal and adult patients with medulloblastoma. The guideline is based on the 2016 WHO classification of tumours of the CNS and on scientific developments published since 1980. It aims to provide direction for diagnostic and management decisions, and for limiting unnecessary treatments and cost. In view of the scarcity of data in adults with medulloblastoma, we base our recommendations on adult data when possible, but also include recommendations derived from paediatric data if justified. Our recommendations are a resource for professionals involved in the management of post-pubertal and adult patients with medulloblastoma, for patients and caregivers, and for health-care providers in Europe. The implementation of this guideline requires multidisciplinary structures of care, and defined processes of diagnosis and treatment.

Rosette-forming glioneuronal tumors share a distinct DNA methylation profile and mutations in FGFR1, with recurrent co-mutation of PIK3CA and NF1.

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

Telomere elongation via alternative lengthening of telomeres (ALT) and telomerase activation in primary metastatic medulloblastoma of childhood.

PURPOSE: Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB. METHODS: We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR). RESULTS: H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT. CONCLUSIONS: Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Biological material collection to advance translational research and treatment of children with CNS tumours: position paper from the SIOPE Brain Tumour Group.

Paediatric CNS tumours are the most common cause of childhood cancer-related morbidity and mortality, and improvements in their diagnosis and treatment are needed. New genetic and epigenetic information about paediatric CNS tumours is transforming the field dramatically. For most paediatric CNS tumour entities, subgroups with distinct biological characteristics have been identified, and these characteristics are increasingly used to facilitate accurate diagnoses and therapeutic recommendations. Future treatments will be further tailored to specific molecular subtypes of disease, specific tumour predisposition syndromes, and other biological criteria. Successful biomaterial collection is a key requirement for the application of contemporary methodologies for the validation of candidate prognostic factors, the discovery of new biomarkers, the establishment of appropriate preclinical research models for targeted agents, a quicker clinical implementation of precision medicine, and for other therapeutic uses (eg, for immunotherapies). However, deficits in organisational structures and interdisciplinary cooperation are impeding the collection of high-quality biomaterial from CNS tumours in most centres. Practical, legal, and ethical guidelines for consent, storage, material transfer, biobanking, data sharing, and funding should be established by research consortia and local institutions to allow optimal collection of primary and subsequent tumour tissue, body fluids, and normal tissue. Procedures for the collection and storage of biomaterials and related data should be implemented according to the individual and organisational structures of the local institutions.

Duplications of KIAA1549 and BRAF screening by Droplet Digital PCR from formalin-fixed paraffin-embedded DNA is an accurate alternative for KIAA1549-BRAF fusion detection in pilocytic astrocytomas.

Pilocytic astrocytomas represent the most common glioma subtype in young patients and account for 5.4% of all gliomas. They are characterized by alterations in the RAS-MAP kinase pathway, the most frequent being a tandem duplication on chromosome 7q34 involving the BRAF gene, resulting in oncogenic BRAF fusion proteins. BRAF fusion involving the KIAA1549 gene is a hallmark of pilocytic astrocytoma, but it has also been recorded in rare cases of gangliogliomas, 1p/19q co-deleted oligodendroglial tumors, and it is also a common feature of disseminated oligodendroglial-like leptomeningeal neoplasm. In some difficult cases, evidence for KIAA1549-BRAF fusion is of utmost importance for the diagnosis. Moreover, because the KIAA1549-BRAF fusion constitutively activates the MAP kinase pathway, it represents a target for drugs such as MEK inhibitors, and therefore, the detection of this genetic abnormality is highly relevant in the context of clinical trials applying such new approaches. In the present study, we aimed to use the high sensitivity of Droplet Digital PCR (DDPCR™) to predict KIAA1549-BRAF fusion on very small amounts of formalin-fixed paraffin-embedded tissue in routine practice. Therefore, we analyzed a training cohort of 55 pilocytic astrocytomas in which the KIAA1549-BRAF fusion status was known by RNA sequencing used as our gold standard technique. Then, we analyzed a prospective cohort of 40 pilocytic astrocytomas, 27 neuroepithelial tumors remaining difficult to classify (pilocytic astrocytoma versus ganglioglioma or diffuse glioma), 15 dysembryoplastic neuroepithelial tumors, and 18 gangliogliomas. We could demonstrate the usefulness and high accuracy (100% sensitivity and specificity when compared to RNA sequencing) of DDPCR™ to assess the KIAA1549-BRAF fusion from very low amounts of DNA isolated from formalin-fixed paraffin-embedded specimens. BRAF duplication is both necessary and sufficient to predict this fusion in most cases and we propose that this single analysis could be used in routine practice to save time, money, and precious tissue.