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Single-unit recordings performed in temporal lobe epilepsy patients and in models of temporal lobe seizures have shown that interneurons are active at focal seizure onset. We performed simultaneous patch-clamp and field potential recordings in entorhinal cortex slices of GAD65 and GAD67 C57BL/6J male mice that express green fluorescent protein in GABAergic neurons to analyze the activity of specific interneuron (IN) subpopulations during acute seizure-like events (SLEs) induced by 4-aminopyridine (4-AP; 100 μm). IN subtypes were identified as parvalbuminergic (INPV,n= 17), cholecystokinergic (INCCK),n= 13], and somatostatinergic (INSOM,n= 15), according to neurophysiological features and single-cell digital PCR. INPVand INCCKdischarged at the start of 4-AP-induced SLEs characterized by either low-voltage fast or hyper-synchronous onset pattern. In both SLE onset types, INSOMfired earliest before SLEs, followed by INPVand INCCKdischarges. Pyramidal neurons became active with variable delays after SLE onset. Depolarizing block was observed in ∼50% of cells in each INs subgroup, and it was longer in IN (∼4 s) than in pyramidal neurons (PVand INSOM. We conclude that entorhinal cortex INs are very active at the onset and during the progression of SLEs induced by 4-AP. These results support earlierin vivoandin vivoevidence and suggest that INs have a preferential role in focal seizure initiation and development.SIGNIFICANCE STATEMENTFocal seizures are believed to result from enhanced excitation. Nevertheless, we and others demonstrated that cortical GABAergic networks may initiate focal seizures. Here, we analyzed for the first time the role of different IN subtypes in seizures generated by 4-aminopyridine in the mouse entorhinal cortex slices. We found that in thisin vitrofocal seizure model, all IN types contribute to seizure initiation and that INs precede firing of principal cells. This evidence is in agreement with the active role of GABAergic networks in seizure generation.

LZTR1 is the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase frequently mutated in sporadic and syndromic cancer. We combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations. Unbiased screens converged on EGFR and AXL receptor tyrosine kinases as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome. Pathogenic cancer-associated mutations of LZTR1 failed to promote EGFR and AXL degradation, resulting in dysregulated growth factor signaling. Conditional inactivation of Lztr1 and Cdkn2a in the mouse nervous system caused tumors in the peripheral nervous system including schwannoma-like tumors, thus recapitulating aspects of schwannomatosis, the prototype tumor predisposition syndrome sustained by LZTR1 germline mutations. Lztr1– and Cdkn2a-deleted tumors aberrantly accumulated EGFR and AXL and exhibited specific vulnerability to EGFR and AXL coinhibition. These findings explain tumorigenesis by LZTR1 inactivation and offer therapeutic opportunities to patients with LZTR1-mutant cancer.Significance:EGFR and AXL are substrates of LZTR1-CUL3 ubiquitin ligase. The frequent somatic and germline mutations of LZTR1 in human cancer cause EGFR and AXL accumulation and deregulated signaling. LZTR1-mutant tumors show vulnerability to concurrent inhibition of EGFR and AXL, thus providing precision targeting to patients affected by LZTR1-mutant cancer.This article is highlighted in the In This Issue feature, p. 517

EGFR and AXL protein expression in the brain and immunohistochemical characterization of tumors of Lztr1fl/fl;Cdkn2afl/fl;GFAP-Cre+ mice

LZTR1 is the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase frequently mutated in sporadic and syndromic cancer. We combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations. Unbiased screens converged on EGFR and AXL receptor tyrosine kinases as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome. Pathogenic cancer-associated mutations of LZTR1 failed to promote EGFR and AXL degradation, resulting in dysregulated growth factor signaling. Conditional inactivation of Lztr1 and Cdkn2a in the mouse nervous system caused tumors in the peripheral nervous system including schwannoma-like tumors, thus recapitulating aspects of schwannomatosis, the prototype tumor predisposition syndrome sustained by LZTR1 germline mutations. Lztr1– and Cdkn2a-deleted tumors aberrantly accumulated EGFR and AXL and exhibited specific vulnerability to EGFR and AXL coinhibition. These findings explain tumorigenesis by LZTR1 inactivation and offer therapeutic opportunities to patients with LZTR1-mutant cancer.Significance:EGFR and AXL are substrates of LZTR1-CUL3 ubiquitin ligase. The frequent somatic and germline mutations of LZTR1 in human cancer cause EGFR and AXL accumulation and deregulated signaling. LZTR1-mutant tumors show vulnerability to concurrent inhibition of EGFR and AXL, thus providing precision targeting to patients affected by LZTR1-mutant cancer.This article is highlighted in the In This Issue feature, p. 517

Table S1. List of ubiquitylated peptides from DiGly proteomics assay. Table S2. List of proteins differentially abundant in LZTR1-/- compared toLZTR1+/+ cells quantified by TMT.Table S3. List of top scoring LZTR1 interacting proteins from immunoprecipitation/mass spectrometry.

Supplementary Figure 4. The FGFR3-TACC3 fusion gene and protein are retained in recurrent GBM.

Supplementary Figure 3. Evaluation of the expression of FGFR3-TACC3 fusion elements.

Supplementary Figure 2. Schematics of FGFR3-TACC3 genomic breakpoints.

Supplementary Figure Legends

Supplementary Table 1. Summary of FGFR-TACC fusion transcripts identified in all cancer types.

Supplementary Figure 6. Analysis of SNP6.0 arrays of GBM harboring CNVs of FGFR3 and TACC3 genomic loci.

Purpose: Oncogenic fusions consisting of fibroblast growth factor receptor (FGFR) and TACC are present in a subgroup of glioblastoma (GBM) and other human cancers and have been proposed as new therapeutic targets. We analyzed frequency and molecular features of FGFR–TACC fusions and explored the therapeutic efficacy of inhibiting FGFR kinase in GBM and grade II and III glioma.Experimental Design: Overall, 795 gliomas (584 GBM, 85 grades II and III with wild-type and 126 with IDH1/2 mutation) were screened for FGFR–TACC breakpoints and associated molecular profile. We also analyzed expression of the FGFR3 and TACC3 components of the fusions. The effects of the specific FGFR inhibitor JNJ-42756493 for FGFR3–TACC3–positive glioma were determined in preclinical experiments. Two patients with advanced FGFR3–TACC3–positive GBM received JNJ-42756493 and were assessed for therapeutic response.Results: Three of 85 IDH1/2 wild-type (3.5%) but none of 126 IDH1/2-mutant grade II and III gliomas harbored FGFR3–TACC3 fusions. FGFR–TACC rearrangements were present in 17 of 584 GBM (2.9%). FGFR3–TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. They are mutually exclusive with IDH1/2 mutations and EGFR amplification, whereas they co-occur with CDK4 amplification. JNJ-42756493 inhibited growth of glioma cells harboring FGFR3–TACC3 in vitro and in vivo. The two patients with FGFR3–TACC3 rearrangements who received JNJ-42756493 manifested clinical improvement with stable disease and minor response, respectively.Conclusions: RT-PCR sequencing is a sensitive and specific method to identify FGFR–TACC–positive patients. FGFR3–TACC3 fusions are associated with uniform intratumor expression of the fusion protein. The clinical response observed in the FGFR3–TACC3–positive patients treated with an FGFR inhibitor supports clinical studies of FGFR inhibition in FGFR–TACC–positive patients. Clin Cancer Res; 21(14); 3307–17. ©2015 AACR.See related commentary by Ahluwalia and Rich, p. 3105

Supplementary Figure 1. Genomic PCR images and Sanger sequences of FGFR3-TACC3 genomic breakpoints.

Supplementary Figure 5. PFS and OS of FGFR3-TACC3-positive glioma patients.

Purpose Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs). Methods 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively. Results Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways. Conclusion The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Spinal neurofibromatosis (SNF), a phenotypic subclass of neurofibromatosis 1 (NF1), is characterized by bilateral neurofibromas involving all spinal roots. In order to deepen the understanding of SNF’s clinical and genetic features, we identified 81 patients with SNF, 55 from unrelated families, and 26 belonging to 19 families with at least 1 member affected by SNF, and 106 NF1 patients aged >30 years without spinal tumors. A comprehensive NF1 mutation screening was performed using NGS panels, including NF1 and several RAS pathway genes. The main features of the SNF subjects were a higher number of internal neurofibromas (p < 0.001), nerve root swelling (p < 0.001), and subcutaneous neurofibromas (p = 0.03), while hyperpigmentation signs were significantly less frequent compared with the classical NF1-affected cohorts (p = 0.012). Fifteen patients underwent neurosurgical intervention. The histological findings revealed neurofibromas in 13 patients and ganglioneuromas in 2 patients. Phenotypic variability within SNF families was observed. The proportion of missense mutations was higher in the SNF cases than in the classical NF1 group (21.40% vs. 7.5%, p = 0.007), conferring an odds ratio (OR) of 3.34 (CI = 1.33–10.78). Two unrelated familial SNF cases harbored in trans double NF1 mutations that seemed to have a subclinical worsening effect on the clinical phenotype. Our study, with the largest series of SNF patients reported to date, better defines the clinical and genetic features of SNF, which could improve the management and genetic counseling of NF1.

Glioblastomas (GBMs) in patients harboring somatic or germinal mutations of mismatch-repair (MMR) genes exhibit a hypermutable phenotype. Here, we describe a GBM patient with increased tumor mutational burden and germline MMR mutations, treated using anti-PD1 therapy. A woman with newly diagnosed GBM (nGBM) was treated by surgery, radiotherapy, and temozolomide. The tumor recurred after 13 months leading to a second surgery and treatment with nivolumab. Whole-exome sequencing was performed on the nGBM, recurrent GBM (rGBM), and blood. Immune infiltration was investigated by immunohistochemistry and the immune response in the blood during treatment was analyzed by flow cytometry. High density of infiltrating CD163 + cells was found in both GBM specimens. Large numbers of CD3 + and CD8 + T cells were homogeneously distributed in the nGBM. The infiltration of CD4 + T cells and a different CD8 + T cell density were observed in the rGBM. Both GBM shared 12,431 somatic mutations, with 113 substitutions specific to the nGBM and 1,683 specific to the rGBM. Germline variants included pathogenic mutation in the MSH2 (R359S) gene, suggesting the diagnosis of Lynch syndrome. Systemic immunophenotyping revealed the generation of CD8 + T memory cells and persistent activation of CD4 + T cells. The patient is still receiving nivolumab 68 months after the second surgery. Our observations indicate that the hypermutator phenotype associated with germinal mutations of MMR genes and abundant T-cell infiltration contributes to a durable clinical benefit sustained by a persistent and robust immune response during anti-PD1 therapy.

Background/aim We herein presented a case of pediatric spinal cord pilocytic astrocytoma diagnosed on the basis of histopathological and clinical findings. Materials and methods Given the paucity of data on genetic features for this tumor, we performed exome, array CGH and RNA sequencing analysis from nucleic acids isolated from a unique and not repeatable very small amount of a formalin-fixed, paraffin-embedded (FFPE) specimen. Results DNA mutation analysis, comparing tumor and normal lymphocyte peripheral DNA, evidenced few tumor-specific single nucleotide variants in DEFB119, MUC5B, NUDT1, LTBP3 and CPSF3L genes. Differently, tumor DNA was not characterized by for the main pilocytic astrocytoma gene variations, including BRAFV600E. An inframe trinucleotides insertion involving DLX6 or lnc DLX6-AS1 genes was scored in 44.9% of sequenced reads; the temporal profile of this variation on the expression of DLX-AS1 was investigated in patient's urine-derived exosomes, reporting no significant variation in the one-year molecular follow-up. Array CGH identified a tumor microdeletion at the 6q25.3 chromosomal region, spanning 1,01 Mb and comprising ZDHHC14, SNX9, TULP4 and SYTL3 genes. The expression of these genes did not change in urine-derived exosomes during the one-year investigation period. Finally, RNAseq did not reveal any of the common pilocytic BRAF-KIAA1549 genes fusion events. Conclusion To our knowledge, the present report is one of the first described gene-orphan case studies of a pediatric spinal cord pilocytic astrocytoma.

LZTR1, the substrate-specific adaptor of a CUL3-dependent ubiquitin ligase is among the most frequently mutated ubiquitin ligase coding gene in syndromic and sporadic human cancers including glioblastoma multiforme, in which approximately 27% of cases harbor inactivating mutations and copy number loss. However, both the identity of the protein substrates targeted by LZTR1-mediated ubiquitylation and the biological contexts regulated by specific LZTR1-substrate(s) interactions remain uncertain. Here, we combined biochemical and genetic studies to identify LZTR1 substrates and interrogated their tumor-driving function in the context of LZTR1 loss-of-function mutations and in a new conditional Lztr1 knockout mouse. Multiple screens converged on the receptor tyrosine kinases EGFR and AXL as LZTR1 interactors targeted for ubiquitin-dependent degradation in the lysosome by LZTR1-CUL3 complexes. Pathogenic mutations affecting LZTR1 failed to promote degradation of EGFR and AXL in human tumors. Mice harboring conditional deletion of the LZTR1 gene combined with loss of CDKN2A in the neural progenitor compartment generated peripheral nervous system tumors including schwannoma like and malignant peripheral nervous system tumors (MPNST). Tumors from the LZTR1-mutant mouse model accumulated very high levels of EGFR and AXL and exhibited potent and specific vulnerability to the combinatorial inhibition of EGFR and AXL kinases. These findings explain the mechanism of tumorigenesis associated with LZTR1 inactivation and offer a therapeutic strategy to patients affected by tumors carrying mutations of LZTR1.

We developed an autologous hematopoietic stem cell-based platform designed to deliver IFNa, by a transcriptional and post-transcriptional control mechanism mediated by miRNA target sequences, specifically into the tumor microenvironment (TME) via Tie-2 expressing monocytes (Temferon). As of Feb 2022, 3 escalating doses of Temferon (0.5-2.0x106/kg) were tested across 15 newly diagnosed, unmethylated MGMT GBM patients assigned to 5 cohorts. Follow-up from surgery is 6–28mo (2–25mo after Temferon). To date, no DLTs have been identified. As expected, 1mo after the administration of the highest tested dose, the hematopoietic system of Temferon-treated patients was composed of up to 30% of CD14+ modified cells. Temferon-derived progeny persisted, albeit at lower levels, up to 18mo (longest time of analysis). Despite the substantial proportion of engineered cells, very low concentrations of IFNα were detected in the plasma and in the CSF, indicating tight regulation of transgene expression. SAEs were mostly attributed to conditioning chemotherapy (infections) or disease progression (seizures). 1SUSAR (persistent GGT elevation) occurred. Median OS is 15mo from surgery. Homing of transduced cells to the tumor was demonstrated by the presence of gene-marked cells in the 2nd surgery specimens of 3 out 4 pts belonging to low dose cohorts. Single-cell RNA seq of the TME highlighted a Temferon signature associated with the induction IFNa responsive genes and macrophage repolarization. Potential long-term benefit with Temferon was identified in a patient from cohort 3, who had PD at D+120 with two distant enhancing lesions, and increased tumor necrosis. 1y following Temferon, with no 2nd-line therapy added, there was approximately 40% reduction in enhancing tumor volume compared to D+180 with a stable clinical and imaging picture thereafter. The results provide initial evidence of Temferon’s potential to modulate the TME of GBM patients, and anecdotal evidence for long lasting effects of Temferon in prevention of disease progression.

Increasing clinical use of immune checkpoint inhibitors testifies to the importance of modulating the immune TME to obtain meaningful anti-tumor immune responses. Acting only on T lymphocytes may, however, not be sufficient, e.g. in immunologically-cold tumors or due to de novo or acquired resistance. Moreover, immune-related AEs remain hurdles of T cell therapies. To overcome these limitations and to awaken the immune system in an agnostic way against the tumor, we have developed a genetically modified cell-based autologous hematopoietic stem cell platform (Temferon) delivering immunotherapeutic payloads into the TME through Tie-2 expressing monocytes (TEMs), a subset of tumor infiltrating macrophages. TEM-GBM is an ongoing open-label, Phase 1/2a dose-escalating study evaluating the safety & efficacy of Temferon in up to 21 newly diagnosed patients with glioblastoma & unmethylated MGMT promoter assigned to 7 different cohorts (3 pts each) differing by Temferon dose (0.5-4.0x106/kg) and conditioning regimen (BCNU+ or Busulfan+Thiotepa). By Oct 15th, 2021, 15 pts (cohort 1-5) had received escalating doses of Temferon with a median follow up of 267 days (range: 60-749). Rapid engraftment and hematological recovery from nonmyeloablative conditioning occurred in all pts. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA, were found at increasing proportions in PB and BM, reaching up to 30% at 1 month for the highest cohorts tested (2.0x106/kg) and persisting up to 18 months, albeit at lower levels. Despite the significant proportion of engineered cells, only very low median concentrations of IFNα were detected in the plasma (D+30, 5.9; D+90, 8.8pg/mL) and in the cerebrospinal fluid (D+30, 1.5; D+90, 2.4pg/mL), indicating tight regulation of vector expression. SAEs were mostly attributed to conditioning chemotherapy (e.g. infections) or disease progression (e.g. seizures). 1 SUSAR (persistent GGT elevation) has occurred. Median OS is 14 mth from surgery (11 mth post Temferon). Four pts from the low dose cohorts underwent 2nd surgery. These recurrent tumors contained gene-marked cells and expressed IFN-responsive genes, indicative of local IFNα release by TEMs. In 1 pt, a stable lesion (as defined by MRI) had a higher proportion of T cells & TEMs, an increased IFN-response signature and myeloid re-programming revealed by scRNAseq, as compared to a synchronous, progressing tumor. TCR sequencing of blood and tumor samples showed a post-treatment increase in the cumulative frequency of tumor-associated T cell clones identified in 1st and 2nd surgery specimens (up to 4 out of 9 subjects). These results provide initial evidence for on-target activity of Temferon in GBM, to be consolidated with longer follow up in the higher dose cohorts. Citation Format: Bernhard Gentner, Gaetano Finocchiaro, Francesca Farina, Marica Eoli, Alessia Capotondo, Elena Anghileri, Matteo Barcella, Maria Grazia Bruzzone, Matteo Giovanni Carrabba, Valeria Cuccarini, Giorgio D'Alessandris, Francesco Di Meco, Valeria Ferla, Paolo Ferroli, Filippo Gagliardi, Federico Legnani, Pietro Mortini, Matteo Maria Naldini, Alessandro Olivi, Roberto Pallini, Monica Patanè, Rosina Paterra, Bianca Pollo, Marco Saini, Silvia Snider, Valentina Brambilla, Stefania Mazzoleni, Andrew Zambanini, Carlo Russo, Luigi Naldini, Fabio Ciceri. Genetically modified Tie-2 expressing monocytes target IFN-α2 to the glioblastoma tumor microenvironment (TME): Preliminary data from the TEM-GBM Phase 1/2a study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5213.

GBM with an unmethylated MGMT gene promoter is associated with very poor prognosis. A subset of tumor associated macrophages expressing the angiopoietin receptor Tie2 (TEMs) can be genetically modified for local & tumor restricted release of interferon-α2 (IFN). IFN has antitumor effects, inhibits angiogenesis & modulates the immune system. Temferon consists of autologous HSPCs transduced ex-vivo with an LVV encoding an IFN gene & expression control sequences for TEMs. TEM-GBM is an open-label, Phase I/IIa study (Part A: 3x3x3 dose escalation; Part B: n=12), & Temferon (single dose) is given to patients with first diagnosis of GBM & unmethylated MGMT promoter. Part A 3rd cohort is ongoing & completes dosing in September 2020. Eight patients completed screening; one patient died (disease progression) before Temferon was administered. Six patients received Temferon (3 women, 3 men, mean age 52.3 years). Cohort 1 received Temferon 0.5x106 cells/kg & Cohort 2, 1x106 cells/kg. Neutropenia & thrombocytopenia occurred as expected following conditioning & hematologic recovery (HR) occurred median D+13. Transduced PBMCs were identified by vector copy number (VCN) on myeloid cells at HR & at later timepoints. In general, a dose-ordered increase in VCN was observed (mean VCN D+30 CD14+ Cohort 1: 0.094, cohort 2: 0.125); 1 patient in each cohort had low VCNs. VCN remained detectable up to recent follow up visits (≤ D+180). No dose-limiting toxicities have been reported. Four SAEs occurred in 3 patients who received Temferon (pneumonia, pulmonary embolism, febrile neutropenia, fatigue) but these events were not attributed to Temferon, resolved, & may have been related to the conditioning regimen (carmustine & thiotepa). Disease progression has been confirmed in 3 patients who received Temferon. These preliminary results indicate feasibility of engrafting a pre-determined fraction of Temferon cells in the bone marrow of GBM patients without, so far, causing dose-limiting toxicity.

Background The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT). Methods In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response. Results Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells. Conclusions TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene, we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients, the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed, in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Background Actionable fibroblast growth factor receptor 3 (FGFR3)–transforming acidic coiled-coil protein 3 fusions (F3T3) are found in approximately 3% of gliomas, but their characteristics and prognostic significance are still poorly defined. Our goal was to characterize the clinical, radiological, and molecular profile of F3T3 positive diffuse gliomas. Methods We screened F3T3 fusion by real-time (RT)-PCR and FGFR3 immunohistochemistry in a large series of gliomas, characterized for main genetic alterations, histology, and clinical evolution. We performed a radiological and radiomic case control study, using an exploratory and a validation cohort. Results We screened 1162 diffuse gliomas (951 unselected cases and 211 preselected for FGFR3 protein immunopositivity), identifying 80 F3T3 positive gliomas. F3T3 was mutually exclusive with IDH mutation (P < 0.001) and EGFR amplification (P = 0.01), defining a distinct molecular cluster associated with CDK4 (P = 0.04) and MDM2 amplification (P = 0.03). F3T3 fusion was associated with longer survival for the whole series and for glioblastomas (median overall survival was 31.1 vs 19.9 mo, P = 0.02) and was an independent predictor of better outcome on multivariate analysis. F3T3 positive gliomas had specific MRI features, affecting preferentially insula and temporal lobe, and with poorly defined tumor margins. F3T3 fusion was correctly predicted by radiomics analysis on both the exploratory (area under the curve [AUC] = 0.87) and the validation MRI (AUC = 0.75) cohort. Using Cox proportional hazards models, radiomics predicted survival with a high C-index (0.75, SD 0.04), while the model combining clinical, genetic, and radiomic data showed the highest C-index (0.81, SD 0.04). Conclusion F3T3 positive gliomas have distinct molecular and radiological features, and better outcome.

Background: We developed a macrophage-based treatment relying on ex vivo transduction of autologous hematopoietic stem and progenitor cells (HSPC) to express immune-payloads within the TME. Our ATMP (Temferon) targets IFN-a, an immune-modulatory molecule counteracting also neo-angiogenesis and tumor growth, to a subset of Tie2-expressing, tumor-infiltrating macrophages known as TEMs. Materials and Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed glioblastoma patients with unmethylated MGMT promoter. Key eligibility criteria include age 18-70 years, ECOG 0-1 and KPS >70%, and adequate cardiac, renal, hepatic and pulmonary function. Important exclusion criteria include the presence of active autoimmune disease or receipt of any oral or parenteral chemotherapy or immunotherapy within 2 years of screening. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and transduced ex vivo with a 3 rd generation lentiviral vector encoding for IFN-a2. Transgene expression is confined to TEMs by the Tie2 promoter and post-transcriptional regulation by microRNA-126 thus achieving tumor specificity. The study evaluates safety and biological activity of Temferon in 7 cohorts of three patients each, where escalating doses of Temferon are co-administered with a fixed CD34+ cell dose of non-manipulated supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or + Busulfan). The primary endpoints for this study are: Engraftment of Temferon over the first 90 DaysThe proportion of patients achieving hematologic recovery by Day +30 from ASCTShort-term tolerability of Temferon; stable blood counts and absence of cytopenias, absence of significant organ toxicities (> grade 2); absence of Replication Competent Lentivirus The figure below reports the TEM-GBM study design. Results: As of 28th June 2021, 18 patients have been enrolled; 15 received Temferon (D+0) with follow-up of 30 - 697 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil and platelet engraftment occurred at D+13 and D+12 for patients in cohort 1-3 and D+16 and D+15 for patients assigned to cohort 4 and 5, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted subsequently, albeit at lower levels (up to 18 months). Very low concentrations of IFNa were detected in the plasma (average 7.8 pg/ml at D+30; baseline < LLOQ) and in the cerebrospinal fluid (average 1.6 pg/ml at D+30; baseline < LLOQ), suggesting tight regulation of transgene expression. Seven deaths occurred: six at D+241, +322, +340, +402, +478, +646 after Temferon administration due to disease progression, and one at D+60 due to complications following the conditioning regimen. Nine patients had progressive disease (PD; range D-12 to +239). SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation and poor performance status compatible with autologous stem cell transplantation, concomitant medications and PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present and increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion (as defined by MRI) had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA and TCR sequencing is ongoing. Conclusion: These interim results show that Temferon is generally well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon's potential to activate the immune system and reprogram the tumor microenvironment (TME), as predicted by preclinical studies. Figure 1 Figure 1. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Temferon is an ex vivo gene therapy consisting of autologous HSPCs genetically modified to deliver IFN-α2 within the tumor microenvironment (TME) by Tie-2 expressing macrophages. TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed GBM patients with unmethylated MGMT. Autologous HSPCs are transduced with a LVV encoding for IFN-a2 gene. As of 30th April 2021, 18 patients have been enrolled; 13 received Temferon (D+0) with follow-up of 8 – 662 days. After conditioning and Temferon infusion, a rapid engraftment and hematological recovery occurred, with median neutrophil and platelet engraftment at D+13 and D+12, respectively. No dose limiting toxicities were reported. Temferon-derived cells were found within 14 days post treatment and persisted albeit at lower levels in the long-term. Five deaths occurred: one at +478, three at +322, +340 and +402 days due to PD, and the fourth at +60 due to complications following the conditioning regimen. Eight patients had PD (-12 to +239). SAEs include respiratory tract infections, pulmonary embolism, CMV and C.Diff infections, febrile neutropenia, hemiparesis, seizure, brain abscess, worsening of performance status and respiratory failure compatible with ASCT, concomitant medications and PD. Four patients underwent second surgery. Recurrent tumors had gene-marked cells present and increased expression of ISGs compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. Characterization of T-cell immune repertoire suggests the expansion of tumor-associated clones. TME characterization by scRNA and TCR sequencing is ongoing. Interim results show that Temferon is well tolerated, with no dose limiting toxicities identified to date and provide initial evidence of potential immune system activation within the TME.

Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies.

Gliomatosis cerebri (GC), defined until 2016 as a distinct astrocytic glioma entity, has been removed from the 2016 World Health Organization classification of tumors of the central nervous system. However, its identity is still debated. We retrospectively present 122 patients, including a subgroup with histology confirmation (n = 75, cohort b). Radiological features showed extension limited to 3 lobes in 31%; bilateral, midline, and basal ganglia and subtentorial involvement in 95%, 52%, 84%, and 60%, respectively; and contrast enhancement in 59.5%. Perioperative mortality occurred in 4%. Histology concluded for grades II, III, and IV, respectively, in 31%, 35%, and 22% (not specified in 12%). Thirty-one percent had isocitrate dehydrogenase (IDH) 1 mutation. Treatments included radiotherapy in 51.2% and chemotherapy in 74.5%. Median overall survival was 17 months. Negative prognostic factors for survival were older age, poorer Karnofsky Performance Scale (KPS), subtentorial, midline and disseminated disease, and lack of chemotherapy, at univariate analysis. At multivariate analysis, KPS ≥ 80, chemotherapy, and subtentorial and disseminated disease remained prognostic (p

Recently, we found that temozolomide (TMZ) can upregulate the expression of the multidrug-resistance protein ABCC3 in NK cells from both glioma-bearing mice and glioblastoma patients treated with dendritic cell immunotherapy combined with TMZ, allowing NK cells to escape apoptosis and favoring their role as antitumor effector cells. Here, we demonstrate that CD56dim NK cells expressing CD16+ are predominant in patients surviving more than 12 months after surgery without disease progression. CD56dim CD16+ NK cells co-expressed high levels of ABCC3 and IFN-γ. Notably, not only basal but also TMZ-induced ABCC3 expression was related to a strong, long-term NK cell response and a better prognosis of patients. The identification of the single nucleotide polymorphism (SNP) rs35467079 with the deletion of a cytosine (−897DelC) in the promoter region of the ABCC3 gene resulted associated with a better patient outcome. ABCC3 expression in patients carrying DelC compared to patients with reference haplotype was higher and modulated by TMZ. The transcription factor NRF2, involved in ABCC3 induction, was phosphorylated in CD56dim CD16+ NK cells expressing ABCC3 under TMZ treatment. Thus, ABCC3 protein and the SNP −897DelC can play a predictive role in patients affected by GBM, and possibly other cancers, treated with dendritic cell immunotherapy combined with chemotherapy.

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are often detected in lower-grade gliomas (LGG) and result into 2-hydroxyglutarate (2HG) synthesis. Prior studies showed that 2HG can be detected in vivo using magnetic resonance spectroscopy (MRS), but its accuracy and translational impact are still under investigation. To investigate the clinical feasibility of MRS for in vivo detection and quantification of 2HG on consecutive treatment-naive suspect LGG patients and to compare MRS accuracy with tissue IDH1/2 analysis. MRS spectra at 3 T were acquired with 1H-MRS single-voxel PRESS 2HG-tailored sequences with TE 30 (group 1) or TE 97 (groups 2A and B). Voxel sizes were 1.5 × 1.5 × 1.5 cm3 for group 1 (n = 13) and group 2A (n = 14) and 2 × 2 × 2 cm3 for group 2B (n = 32). Multiple metabolites’ concentrations were analyzed with LCModel. Tumors were assessed for IDH status and main molecular markers. 2HG levels in urine/blood were measured by liquid chromatography–mass spectrometry. The larger voxel TE 97 sequence resulted in highest specificity (100%), sensitivity (79%), and accuracy (87%). Urine and blood 2HG did not result predictive. Our data confirm that 2 × 2 × 2-cm3 voxel TE 97 MRS shows high accuracy for 2HG detection, with good sensitivity and 100% specificity in distinguishing IDH mutant gliomas. Main limits of the technique are small tumor volume and low cellularity. Integrating 2HG-MRS with other metabolites may help non-invasive diagnosis of glioma, prognostic assessment, and treatment planning in clinical setting.

Giant cell glioblastoma (gcGBM) is a rare (5 years), higher than in the standard group. The main findings where partial loss of expression of MMR proteins (overall MSH2 and MSH6) on 30% of cases, mostly related to presence of inflammatory infiltrates, also showing CD28 immunostaining. Microglia IBA1+ was significantly present in patients with longer survival. Correlation with PD-L1 and CTLA4 was found only in 2 cases. POLE sequencing displays mutation F367S on 20% of cases. Our results show that gcGBMs are an histological variant with increased tendency to ultra-mutated phenotype with a better prognosis and suggesting these patients as candidates for immunotherapy.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8+ T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8+ T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy.

In a two-stage phase II study, 24 patients with first diagnosis of glioblastoma (GBM) were treated with dendritic cell (DC) immunotherapy associated to standard radiochemotherapy with temozolomide (TMZ) followed by adjuvant TMZ. Three intradermal injections of mature DC loaded with autologous GBM lysate were administered before adjuvant TMZ, while 4 injections were performed during adjuvant TMZ. According to a two-stage Simon design, to proceed to the second stage progression-free survival (PFS) 12 months after surgery was expected in at least 8 cases enrolled in the first stage. Evidence of immune response and interaction with chemotherapy were investigated. After a median follow up of 17.4 months, 9 patients reached PFS12. In these patients (responders, 37.5%), DC vaccination induced a significant, persistent activation of NK cells, whose increased response was significantly associated with prolonged survival. CD8+ T cells underwent rapid expansion and priming but, after the first administration of adjuvant TMZ, failed to generate a memory status. Resistance to TMZ was associated with robust expression of the multidrug resistance protein ABCC3 in NK but not CD8+ T cells. The negative effect of TMZ on the formation of T cell-associated antitumor memory deserves consideration in future clinical trials including immunotherapy.

Objective: To identify the prognostic significance of TERT promoter mutations (TERTp-mut) and their associations with common molecular alterations in glioblastomas (GBMs). Methods: We sequenced the TERTp-mut in DNA from 395 GBMs and analyzed the results with their respective histology, genetic profile (IDH1 mutation, EGFR amplification, CDKN2A homozygous deletion, loss of chromosome 10, TP53 mutation), and overall survival (OS). Results: TERTp-mut were found in 299 of 395 GBMs (75.7%) and were associated with an older age (median 59.6 years for TERTp-mut vs 53.6 years for TERT promoter wild type [TERTp-wt], p , 0.0001). TERTp-mut was an independent factor of poor prognosis (OS 5 13.8 vs 18.4 months), in both IDH-mutated (OS 5 13.8 vs 37.6 months, p 5 0.022) and IDH-wt GBMs (OS 5 13.7 vs 17.5 months, p 5 0.006). TERTp-mut was associated with IDH-wt, EGFR amplification, CDKN2A deletion, and chromosome 10q loss, but not with MGMT promoter methylation. In the TERTp-wt group, OS was twice longer in EGFR-wt than in EGFR amplification GBMs (OS 5 26.6 vs 13.3 months; p 5 0.005). In the EGFR-wt group, patients with TERTp-wt had a significantly better outcome (OS 5 26.3 vs 12.5 months, p , 0.0001), whereas in the EGFR amplification group, patients with TERTp-mut survived longer (OS 5 15.8 vs 13.3 months, p 5 0.05). Taken together, the absence of both EGFR amplification and TERTp-mut is associated with longer survival in patients with GBM (26.5 months for patients with IDH-wt, 36.7 months for patients with IDH mutation). Conclusions: The analysis of TERTp-mut, in combination with EGFR amplification and IDH mutation status, refines the prognostic classification of GBMs. Neurology® 2014;83:1200–1206

Purpose: Oncogenic fusions consisting of fibroblast growth factor receptor (FGFR) and TACC are present in a subgroup of glioblastoma (GBM) and other human cancers and have been proposed as new therapeutic targets. We analyzed frequency and molecular features of FGFR–TACC fusions and explored the therapeutic efficacy of inhibiting FGFR kinase in GBM and grade II and III glioma. Experimental Design: Overall, 795 gliomas (584 GBM, 85 grades II and III with wild-type and 126 with IDH1/2 mutation) were screened for FGFR–TACC breakpoints and associated molecular profile. We also analyzed expression of the FGFR3 and TACC3 components of the fusions. The effects of the specific FGFR inhibitor JNJ-42756493 for FGFR3–TACC3–positive glioma were determined in preclinical experiments. Two patients with advanced FGFR3–TACC3–positive GBM received JNJ-42756493 and were assessed for therapeutic response. Results: Three of 85 IDH1/2 wild-type (3.5%) but none of 126 IDH1/2-mutant grade II and III gliomas harbored FGFR3–TACC3 fusions. FGFR–TACC rearrangements were present in 17 of 584 GBM (2.9%). FGFR3–TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. They are mutually exclusive with IDH1/2 mutations and EGFR amplification, whereas they co-occur with CDK4 amplification. JNJ-42756493 inhibited growth of glioma cells harboring FGFR3–TACC3 in vitro and in vivo. The two patients with FGFR3–TACC3 rearrangements who received JNJ-42756493 manifested clinical improvement with stable disease and minor response, respectively. Conclusions: RT-PCR sequencing is a sensitive and specific method to identify FGFR–TACC–positive patients. FGFR3–TACC3 fusions are associated with uniform intratumor expression of the fusion protein. The clinical response observed in the FGFR3–TACC3–positive patients treated with an FGFR inhibitor supports clinical studies of FGFR inhibition in FGFR–TACC–positive patients. Clin Cancer Res; 21(14); 3307–17. ©2015 AACR. See related commentary by Ahluwalia and Rich, p. 3105

Melanoma-astrocytoma syndrome (OMIM #155755) is characterized by association of cutaneous malignant melanoma and tumors of central nervous system, generally astrocytomas. The deletion of tumor-suppressor genes on chromosomal region 9p21 (p15/CDKN2B, p16/CDKN2A and p14/ARF) is often reported in families with hereditary predisposition to melanoma and nervous system tumors. We describe a family including a patient with history of melanoma and low grade glioma not associated with mutation on 9 chromosome. The proband is a 40 year old woman who underwent surgery for a cervical cutaneous melanoma. After 4 months, during oncological follow-up, an asymptomatic, left frontal cerebral lesion was detected and subsequently excised. Histopathology indicated a WHO grade II oligoastrocytoma, with 1p/19q deletion and mutation of IDH1 (PCR). In her family there is another case of malignant melanoma (grandfather) and two cases of multiple cutaneus basaliomas (mother and her 33 year-old sister). Array-CGH on lymphocyte DNA was performed, using Cytochip ISCA 4 x 180K platform (Bluegnome). This study revealed the presence of a 1.2 Mb microduplication on 5p15.33, including telomerase reverse transcriptase (TERT) gene. The same duplication was found in the mother DNA. TERT is up-regulated in a variety of human cancers, including melanomas and gliomas. Among gliomas, TERT point mutations, associated to increased expression of the gene, were found in 80% of primary glioblastomas and 78% of oligodendrogliomas. To our knowledge this is the first report of melanoma-astrocytoma syndrome associated to amplification on 5p15.33. Interestingly, genome wide association studies have found rs2736100, also located on 5p15.33, as a susceptibility locus for gliomas.

Autoinflammatory diseases are rare illnesses characterized by apparently unprovoked inflammation without high-titer auto-antibodies or antigen-specific T cells. They may cause neurological manifestations, such as meningitis and hearing loss, but they are also characterized by non-neurological manifestations. In this work we studied a 30-year-old man who had a chronic disease characterized by meningitis, progressive hearing loss, persistently raised inflammatory markers and diffuse leukoencephalopathy on brain MRI. He also suffered from chronic recurrent osteomyelitis of the mandible. The hypothesis of an autoinflammatory disease prompted us to test for the presence of mutations in interleukin-1-pathway genes and to investigate the function of this pathway in the mononuclear cells obtained from the patient. Search for mutations in genes associated with interleukin-1-pathway demonstrated a novel NLRP3 (CIAS1) mutation (p.I288M) and a previously described MEFV mutation (p.R761H), but their combination was found to be non-pathogenic. On the other hand, we uncovered a selective interleukin-6 hypersecretion within the central nervous system as the likely pathogenic mechanism. This is also supported by the response to the anti-interleukin-6-receptor monoclonal antibody tocilizumab, but not to the recombinant interleukin-1-receptor antagonist anakinra. Exome sequencing failed to identify mutations in other genes known to be involved in autoinflammatory diseases. We propose that the disease described in this patient might be a prototype of a novel category of autoinflammatory diseases characterized by prominent neurological involvement.

Cerebellar liponeurocytoma (cLPN) is a very rare central nervous system (CNS) tumour recently recognized as a clinical and pathological entity distinct from medulloblastoma (MB), and included in the WHO classification of CNS tumours under the heading “glioneuronal tumours”. cLPN typically develop in adult age and have a favourable prognosis compared with MB. In this work, we reviewed the clinical and neuroradiological data of two novel cases of adult cLPN diagnosed at our institution; one patient developed distant metastases. We tried to identify novel molecular markers for this malignancy. We found that the transcription factor NEUROG1 (but not ATOH1) is expressed in cLPN, unlike normal adult cerebellum, and that fatty acid binding protein 4 (FABP4), typically found in adipocytes, is significantly overexpressed compared with both normal adult cerebellum and human MB. These findings suggest cLPN occur as a result of transformation of cerebellar progenitors, which are distinct from cerebellar granule progenitors, and aberrantly differentiate into adipocyte-like tumour cells. They also suggest that analysis of FABP4 expression is of help to differentiate cLPN from MB.

Secreted WNT proteins signal through ten receptors of the frizzled (FZD) family. Because of the relevance of the WNT/β-catenin (CTNNB1) signaling pathway in medulloblastomas (MBs), we investigated the expression of all ten members of the FZD gene family (FZD1-10) in 17 human MBs, four MB cell lines and in normal human cerebellum, using real-time PCR. We found that FZD2 transcript was over-expressed in all MBs and MB cell lines. Western blot analysis confirmed the expression of FZD2 at the protein level. Moreover, the levels of FZD2 transcript were found to correlate with those of ASPM transcript, a marker of mitosis essential for mitotic spindle function. Accordingly, ASPM mRNA was expressed at a very low level in the adult, post-mitotic, human cerebellum, at higher levels in fetal cerebellum and at highest levels in MB tissues and cell lines. Unlike FZD2, the other FZDs were overexpressed (e.g., FZD1, FZD3 and FZD8) or underexpressed (e.g., FZD7, FZD9 and FZD10) in a case-restricted manner. Interestingly, we did not find any nuclear immuno-reactivity to CTNNB1 in four MBs over-expressing both FZD2 and other FZD receptors, confirming the lack of nuclear CTNNB1 staining in the presence of increased FZD expression, as in other tumor types. Overall, our results indicate that altered expression of FZD2 might be associated with a proliferative status, thus playing a role in the biology of human MBs, and possibly of cerebellar progenitors from which these malignancies arise.

2056 Background: Several factors influence prognosis of lower grade gliomas, including age, histological diagnosis, 1p/19q chromosome arm deletion (codel 1p/19q), isocitrate dehydrogenase (IDH1/IDH...