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XLS file - 12K, Mutations of PTEN, TP53, and NF1 genes in glioblastoma neurospheres

PDF file - 41K, Table S2: EGFR gene copy number in neurospheres and the corresponding original glioblastomas. Table S3: Immunophenotype of neurospheres. Table S4: EGFR gene amplification, Met expression, and MET gene alterations in glioblastomas and corresponding neurospheres. Table S5: Multiple transcriptional signatures in Met-neg and Met-pos neurospheres. Table S6: Immunohistochemistry on tumors derived from neurospheres. Table S8: Antibodies used in flow-cytometric analysis

PDF file - 2.3MB, Figure S1: Histopathology of primary glioblastomas and genetic lesions of neurospheres. Figure S2: Genome-wide expression profiling and unsupervised hierarchical clustering of neurospheres (classical, mesenchymal and proneural centroids). Figure S3: Expression of MET, EGFR, and a panel of self-renewal markers in neurospheres. Figure S4: Genome-wide expression profiling and unsupervised hierarchical clustering of neurospheres (multiple signatures). Figure S5: Proliferative response, activation of tyrosine kinase receptors, and differentiation pattern in Met-neg-NS and Met-pos-NS. Figure S6: Methigh and Metneg sorting parameters. Figure S7: The Methigh subpopulation of Met-pos-NS retains clonogenic and tumorigenic cells. Figure S8: H&E and Met immunohistochemical staining of histological sections from tumors formed by BT302 neurosphere and its sorted subpopulations. Figure S9: HGF sustains the stem-like and invasive phenotype of Met-pos-NS.

XLS file - 44K, List of primer pairs used for gene sequencing

PDF file - 125K

Thalamic gliomas represent a heterogeneous subset of deep-seated lesions for which surgical removal is advocated, although clear prognostic factors linked to advantages in performance status or overall survival are still lacking. We reviewed our Institutional Cancer Registry, identifying patients who underwent surgery for thalamic gliomas between 2006 and 2020. Associations between possible prognostic factors such as tumor volume, grade, the extent of resection and performance status (PS), and overall survival (OS) were evaluated using univariate and multivariate survival analyses. We found 56 patients: 31 underwent surgery, and 25 underwent biopsy. Compared to biopsy, surgery resulted positively associated with an increase in the OS (hazard ratio, HR, at multivariate analysis 0.30, 95% confidence interval, CI, 0.12–0.75). Considering the extent of resection (EOR), obtaining GTR/STR appeared to offer an OS advantage in high-grade gliomas (HGG) patients submitted to surgical resection if compared to biopsy, although we did not find statistical significance at multivariate analysis (HR 0.53, 95% CI 0.17–1.59). Patients with a stable 3-month KPS after surgery demonstrated to have a better prognosis in terms of OS if compared to biopsy (multivariate HR 0.17, 95% CI, 0.05–0.59). Age and histological grades were found to be prognostic factors for this condition (p = 0.04 and p = 0.004, respectively, chi-square test). Considering the entire cohort, p53 positivity (univariate HR 2.21, 95% CI 1.01–4.82) and ATRX positivity (univariate HR 2.69, 95% CI 0.92–7.83) resulted associated with a worse prognosis in terms of OS. In this work, we demonstrated that surgery aimed at tumor resection might offer a stronger survival advantage when a stable 3-month KPS after surgery is achieved.

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.

Despite the first classification of glioblastoma (GBM) as an immunologically “cold” tumor, evidence suggests that this cancer type is susceptible to T-cell infiltration, providing a glimmer of hope for developing effective immunotherapy approaches. However, whether the “exhausted” phenotype of tumor-infiltrating lymphocytes (TILs) and the immunosuppressive signaling in the GBM microenvironment (ME) can be therapeutically reprogrammed to unlock the anticancer potential of the immune system remains to be elucidated. We performed a histological analysis of GBM specimens collected pre-therapy from 24 newly-diagnosed GBM patients treated with dendritic cell (DC)-immunotherapy as part of the DENDR1 clinical trial (NCT04801147). Our preliminary results suggest an association between the distribution and density of CD8+ TIL subsets and patients’ responses to immunotherapy. TIL spatial patterns revealed either an immune-excluded or an immune-infiltrated scenario with either scattered or widespread TILs. A widespread CD8+ TIL pattern was significantly correlated with a better response to immunotherapy. It was also observed that the frequency of CD8+ TILs was significantly increased at recurrence compared to their primary tumors, suggesting that these patients are more likely to benefit from CD8+ T cell antitumor immunity. In light of these observations, we have hypothesized that tumor-reactive (TR) TILs isolated from GBM can be used as direct personalized treatment mediators exerting an effective antitumor activity with low side effects per se. TR-TILs were isolated from GBM surgical material using CD137 as a key marker, and successfully expanded from 50% of patients’ GBM showing specific reactivity against autologous tumor cells. For the remaining cases, TR-TILs failed to expand, suggesting terminal dysfunction. Single-cell sequencing of the transcriptome was performed on the immune contexture after CD137+ TILs enrichment revealing the presence of a specific signature related to exhausted, cytotoxic, and memory state. A gene signature highly upregulated in regulatory T cells (Treg) was related to a more immune-suppressive ME. Flow cytometry analysis on immune contexture confirmed the presence of FoxP3+ CD4+ T cells, expressing CD137 can be enriched and persist, dysregulating the expansion of TR-TILs during the early stage in culture. Our preliminary findings indicate that a high frequency of Treg can be investigated as a negative predictive indicator of ex-vivo expansion of TR-TILs, which should be taken into consideration when planning a personalized TIL therapy. Citation Format: Irene Sambruni, Silvia Musio, Natalia Di Ianni, Martina Maffezzini, Monica Patanè, Marica Eoli, Antonio Silvani, Bianca Pollo, Francesco DiMeco, Serena Pellegatta. Single-cell analysis of glioblastoma immune contexture identifies a subset of activated and memory tumor-reactive CD8+ TILs and a Treg signature contributing to TIL irreversible dysfunction. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4694.

Background The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM. Methods We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models. Findings B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines. Interpretation We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. Fund Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Purpose Radio-guided surgery with β - decays is a novel technique under investigation. One of the main advantages is its capability to detect small ( ⩽ 0.1 ml) samples after injecting the patient with low activity of radiopharmaceutical. This paper presents an experimental method to quantify this feature based on ex-vivo tests on specimens from meningioma patients. Methods Patients were enrolled on the basis of the standard uptake value (SUV) and the tumour-to-non-tumour activity ratio (TNR) resulted from 68Ga-DOTATOC PET exams. After injecting the patients with 93–167 MBq of 90Y-DOTATOC, 26 samples excised during surgery were analyzed with a β - probe. The radioactivity expected on the neoplastic specimens was estimated according to the SUV found in the PET scan and the correlation with the measured counts was studied. The doses to surgeon and medical personnel were also evaluated. Results Even injecting as low as 1.4 MBq/kg of radiotracer, tumour residuals of 0.1 ml can be detected. A negligible dose to the medical personnel was confirmed. Conclusions Radio-guided surgery with β - decays is a feasible technique with a low radiation dose for both personnel and patient, in particular if the patient is injected with the minimum required activity. A correlation greater than 80% was observed between the measured counts and the expected activity for the lesion samples based on the individual SUV and the TNR. This makes identifiable the minimum injectable radiotracer activity for cases where 90Y is the utilized radionuclide.

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.

Summary In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.

Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog (SHH)-activated MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MBs developing in p53-deficient Ptch+/- SHH mice that, unexpectedly, showed LC/A traits that correlated with mechanistic Target Of Rapamycin Complex 1 (mTORC1) hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/- SHH MBs and CSC-derived MBs resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patients' samples and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.

Simple Summary Active communication between GBM cells and tumor-infiltrating immune components contributes to establishing an immunosuppressive environment where T cells are scarce and exhausted. This condition is particularly exacerbated upon recurrence, which is almost inevitable for GBM patients. Immunotherapeutic approaches, including checkpoint inhibitors, have demonstrated limited efficacy and failed to prolong survival or recurrent GBM patients. Nevertheless, many studies have shown that T cell priming is possible in the GBM microenvironment, and that T cell exhaustion or dysfunction can be reprogrammed. We will revisit data from the literature and report original results obtained from recurrent GBM patients treated with dendritic cells to demonstrate the role of the microenvironment in predicting immunotherapy response and influencing decisions for personalized therapies. Abstract The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response.

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.

Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs.

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

Background: Skull base chordomas are rare tumors arising from notochord. Sphingolipids analysis is a promising approach in molecular oncology, and it has never been applied in chordomas. Our aim is to investigate chordoma behavior and the role of ceramides. Methods: Ceramides were extracted and evaluated by liquid chromatography and mass spectrometry in a cohort of patients with a skull base chordoma. Clinical data were also collected and correlated with ceramide levels. Linear regression and correlation analyses were conducted. Results: Analyzing the association between ceramides level and MIB-1, total ceramides and dihydroceramides showed a strong association (r = 0.7257 and r = 0.6733, respectively) with MIB-1 staining (p = 0.0033 and p = 0.0083, respectively). Among the single ceramide species, Cer C24:1 (r = 0.8814, p ≤ 0.0001), DHCer C24:1 (r = 0.8429, p = 0.0002) and DHCer C18:0 (r = 0.9426, p ≤ 0.0001) showed a significant correlation with MIB-1. Conclusion: Our lipid analysis showed ceramides to be promising tumoral biomarkers in skull base chordomas. Long- and very-long-chain ceramides, such as Cer C24:1 and DHCer C24:1, may be related to a prolonged tumor survival and aggressiveness, and the understanding of their effective biological role will hopefully shed light on the mechanisms of chordoma radio-resistance, tendency to recur, and use of agents targeting ceramide metabolism.

BACKGROUND Meningioma is the most common primary tumor of CNS, usually a benign tumor (grade I) successfully treated by surgical resection, while in the 25–30% of cases is a more aggressive neoplasm with high recurrence rate and poor prognosis (grade II-III). In the last years some studies tried to investigate the genomic profile of meningioma to find additional prognostic factors and potential therapeutic targets. Aim of our study was to use the array-CGH (aCGH) to investigate possible indicators of recurrence risk in meningiomas. MATERIAL AND METHODS We retrospectively selected two group of patients: 34 with meningioma WHO grade I (24 F, 10 M, median age at diagnosis 56 yrs) and 19 with meningioma WHO grade II (9 F, 10 M, median age 59 yrs), in order to compare cases recurrent and non-recurrent in each group. We also analyzed 5/18 recurrences WHO grade I and 10/11 WHO grade II. Genomic profiles were investigated by aCGH. RESULTS We observed an increase in number of genomic imbalances related to grading and in non-recurrent versus recurrent tumors. 9 WHO I meningiomas were without alterations. The mainly alterations observed were deletions and duplications, moreover we also identified chromothripsis of a single chromosome in 2/18 WHO I recurrent and 2/8 WHO II non recurrent meningiomas, and chromothripsis of multiple chromosomes in 5/11 WHO II recurrent meningiomas. As well we detected the presence of mosaicism, mainly in WHO I recurrent and in grade II meningiomas. In 7/14 WHO I meningiomas we found differences in genomic profile analyzing two samples obtained from two distinct areas. 6/15 recurrences showed the same alterations of the primary tumor. We didn’t detected an association between alteration of 1p and 22q and recurrence risk, as previously suggested, as those were the most diffused imbalances in all meningiomas. CONCLUSION In our patients we found a difference in genomic imbalances between grade I and grade II meningiomas, but we also identified additional details of the complexity of alterations in meningioma with the detection of chromothripsis and mosaicism. This latter aspect is a new issue and suggests the presence of more aggressive cell clones that could be predictive of recurrence in grade I meningioma. Moreover, we observed a spatial heterogeneity in half of grade I meningioma analyzed. Our preliminary results suggest that the aCGH, a technique routinely used in many laboratories, could be used to better stratify meningioma patients.

// Cristina Richichi 1 , Daniela Osti 1 , Massimiliano Del Bene 2 , Lorenzo Fornasari 1 , Monica Patane 3 , Bianca Pollo 3 , Francesco DiMeco 2, 4 , Giuliana Pelicci 1, 5 1 Department of Experimental Oncology, European Institute of Oncology, 20139, Milan, Italy 2 Department of Neurosurgery, IRCCS Foundation Neurological Institute “C. Besta”, 20133, Milan, Italy 3 Department of Neuropathology, IRCCS Foundation Neurological Institute “C. Besta”, 20133, Milan, Italy 4 Department of Neurosurgery, Johns Hopkins University, Baltimore, MD 21218, USA 5 Department of Translational Medicine, Piemonte Orientale University “Amedeo Avogadro”, 28100 Novara, Italy Correspondence to: Giuliana Pelicci, email: giuliana.pelicci@ieo.eu Keywords: glioblastoma, tumor-initiating cell frequency, neurosphere, limiting dilution assay, tumorigenicity Received: March 03, 2016 Accepted: August 13, 2016 Published: August 25, 2016 ABSTRACT Glioblastoma (GBM) is maintained by a small subpopulation of tumor-initiating cells (TICs). The arduous assessment of TIC frequencies challenges the prognostic role of TICs in predicting the clinical outcome in GBM patients. We estimated the TIC frequency in human GBM injecting intracerebrally in mice dissociated cells without any passage in culture. All GBMs contained rare TICsand were tumorigenic in vivo but only 54% of them grew in vitro as neurospheres. We demonstrated that neurosphere formation in vitro did not foretell tumorigenic ability in vivo and frequencies calculated in vitro overestimated the TIC content. Our findings assert the pathological significance of GBM TICs. TIC number correlated positively with tumor incidence and inversely with survival of tumor-bearing mice. Stratification of GBM patients according to TIC content revealed that patients with low TIC frequency experienced a trend towards a longer progression free survival. The expression of either putative stem-cell markers or markers associated with different GBM molecular subtypes did not associate with either TIC content or neurosphere formation underlying the limitations of TIC identification based on the expression of some putative stem cell-markers.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

Radio-Guided Surgery (RGS) is a surgical technique aimed at assisting the surgeon to reach as complete a resection of the tumoural lesion as possible. Established methods to date make use of \( \gamma \)-emitting tracers to radio-mark the neoplastic tissue. However, in case of uptake from healthy organs around the lesion the large penetration of photons yields a non-negligible background that can limit the RGS application. The adoption of \( \beta^{ - } \) radiation has been proposed to overcome this limit. To validate the entire RGS procedure, from the evaluation of the tracer uptake of the tumor, to the assumptions on the bio-distribution and the signal detection, tests on ex vivo specimens of meningioma brain tumour were performed. Meningioma was selected due to the well known high receptivity to a \( \beta^{ - } \) emitting radio-tracer already in use in the clinical practice: 90Y-labelled DOTATOC. Patients were enrolled according to the tumour Standard Uptake Value (SUV \( > \) 2) and the expected Tumour to Non-tumour Ratio (TNR \( > \) 10) estimated from 68Ga-DOTATOC PET images. After injecting the patients with 93–167 MBq of \( ^{90} Y - DOTATOC \), 26 samples excised during surgery were examined with a dedicated \( \beta^{ - } \) detecting probe to assess the sensitivity of millimetre-sized tumour remnants in case of administration of low activity value compatible with those injected for diagnostic exams. Even injecting as low as 1.4 MBq/kg of radio-tracer, tumour remnants greater than 0.06 ml would be discriminated by the healthy tissue in few seconds.

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.

BACKGROUND: Skull base chordomas are rare slow-growing neoplasms that arise from notochord. Their morbidity is mainly related to highly aggressive local invasion and resistance to treatments. There isn’t bio-marker representing its heterogeneous appearance. Lipidomics is emerging as a new approach in many cancers and it has never been applied in chordomas. Our aim is to investigate chordoma biological behavior and the role of ceramides production in this context of proliferation and invasion. MATERIAL AND METHODS: A prospective analysis has been performed on 15 frozen and paraffin-embedded skull base chordomas surgically treated at our Institution from 2016 to 2018. Clinical, radiological and pathological data were collected. The expression of Brachyury, cMET, PDGFR-β, cKIT, β-catenin, Filamin-A, GAB1, YKL-40, TERT was assessed by immunohistochemistry. Sphingolipids were extracted from frozen tissues and ceramides and dihydroceramides pattern was evaluated by HPLC/MS-MS. The autophagy and apoptosis pathways were also evaluated by assessing the expression of p62, bcl-2, beclin-1 and LC3-II by immunohistochemistry and western blot; transmission electronic microscopy was also performed in two cases. RESULTS: The histological analysis confirmed the clinical and radiological diagnosis of chordoma in all the cases. Total ceramides and dihydroceramides levels were concomitantly more elevated especially in recurrent tumors that underwent previous surgical resection and radiation therapy in comparison to the primary ones (p

The heterogeneous expression of tumor-associated antigens limits the efficacy of chimeric antigen receptor (CAR)- redirected T cells (CAR-Ts) for the treatment of glioblastoma (GBM). We have found that chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed in 67% of the GBM specimens with limited heterogeneity. CSPG4 is also expressed on primary GBM-derived cells, grown in vitro as neurospheres (GBM-NS), which recapitulate the histopathology and molecular characteristics of primary GBM. CSPG4.CAR-Ts efficiently controlled the growth of GBM-NS in vitro and in vivo upon intracranial tumor inoculation. Moreover, CSPG4.CAR-Ts were also effective against GBM-NS with moderate to low expression of CSPG4. This effect was mediated by the in vivo up-regulation of CSPG4 on tumor cells, induced by tumor necrosis factor-a (TNFα) released by the microglia surrounding the tumor. Overall, the constitutive and TNFα-inducible expression of CSPG4 in GBM may greatly reduce the risk of tumor cell escape observed when targeted antigens are heterogeneously expressed on tumor cells.

Meningiomas are the most frequent primary CNS tumors, usually benign (WHO grade I), but in the 25–30% more aggressive tumors (grade II-III). Histopathological criteria (WHO 2016) are unsatisfactory to recognize meningiomas with tendency to recur. Recent studies have identified some mutations associated with histological features and location (i.e. BAP1 mutation in rhabdoid subtype), while risk stratification schemes based on DNA methylation subgroups have been proposed. Epigenetic modifications of histones play a pivotal role in tumorigenesis, the methylation of lysine 27 (K27) of histone H3 controlled by EZH2 subunit of PRC2 complex. BAP1 could be also involved in epigenetic regulation of PRC2 complex. The deregulation of H3K27 methylation has been found associated with recurrence risk in grade II meningiomas. Aim of the present study was to evaluate histone H3 methylation profile in a group of meningiomas of different grade with follow-up longer than 10 years, comparing non-recurrent to recurrent ones. We performed an immunohistochemical study on 145 meningiomas (50 grade I, 80 grade II and 15 grade III), investigating the expression of trimethylated H3K27 (H3K27me3), EZH2 and BAP1. The results were related to clinical data obtained from our Institutional Tumor Registry and evaluation of clinical reports. We observed a loss of H3K27me3 expression only in grade II and III meningiomas in 18% of cases, without correlation with tendency to recur. EZH2 showed increased percentage of positive nuclei related to the grade. In the group of grade I meningioma EZH2 expression was associated to recurrences. We didn’t observe loss of BAP1 expression. In conclusion, our data suggest that immunohistochemical evaluation of H3K27me3 and EZH2 could be a useful tool to better stratify meningioma with high risk of recurrence. Moreover, EZH2 could be of interest as therapeutic target. Molecular investigations in a larger cohort are needed to confirm these results.

The existence of treatment-resistant cancer stem cells contributes to the aggressive phenotype of glioblastoma. However, the molecular alterations that drive stem cell proliferation in these tumors remain unknown. In this study, we found that expression of the MET oncogene was associated with neurospheres expressing the gene signature of mesenchymal and proneural subtypes of glioblastoma. Met expression was almost absent from neurospheres expressing the signature of the classical subtype and was mutually exclusive with amplification and expression of the EGF receptor (EGFR) gene. Met-positive and Met-negative neurospheres displayed distinct growth factor requirements, differentiated along divergent pathways, and generated tumors with distinctive features. The Methigh subpopulation within Met-pos neurospheres displayed clonogenic potential and long-term self-renewal ability in vitro and enhanced growth kinetics in vivo. In Methigh cells, the Met ligand HGF further sustained proliferation, clonogenicity, expression of self-renewal markers, migration, and invasion in vitro. Together, our findings suggest that Met is a functional marker of glioblastoma stem cells and a candidate target for identification and therapy of a subset of glioblastomas. Cancer Res; 72(17); 4537–50. ©2012 AACR.

Purpose A very promising technique for radio-guided surgery (RGS) based on β -radiation is being developed. The lower background, with respect to the established approach with high penetrant γ radiation, can allow for a large diffusion of such a technique and extending the RGS to cases with a large uptake of surrounding healthy organs, such as abdominal and brain neoplastic disease or tumor of infancy. Methods To validate the β -RGS feasibility study, tests on ex-vivo specimens of brain meningioma and gastro-entero-pancreatic neuroendocrine tumors have been performed. These clinical cases were selected between the tumors known to express receptors to a β -emitting radio-tracer already in use in the clinical routine: 90Y-labelled DOTATOC. Voluntary patients were enrolled according to the standard uptake value (SUV) and the expected tumor-to-non-tumor ratios (TNR) estimated from PET/CT images after administration of 68 Ga-DOTATOC. All of them gave written informed consent to participate in the clinical trial, already approved by the Ethics Committee. After administration of a low activity (about 2.5 mCi) of 90Y-DOTATOC, the extracted ex-vivo specimens and the healthy tissue around the lesions were examined to assess the correlation between the counting rates measured by the developed β -detecting probe and the expected 90Y activity. Results All the neoplastic samples were correctly identified. A dependence of the counting rate both on the volume V and on the total activity of the sample were observed. Even injecting as low as 1.4 MBq/kg of radio-tracer, tumor remnants of 0.1 ml would be detectable in few seconds. Conclusions The results of the clinical tests showed the validity of the underlying assumptions and strengthening the feasibility studies already published. Small radiopharmaceutical activity is required to detect a tumor remnant as expected due to the low background rates. The negligible medical staff exposure was also confirmed.

Purpose: A radio-guided surgery technique with beta- -emitting radio-tracers was suggested to overcome the effect of the large penetration of gamma radiation. The feasibility studies in the case of brain tumors and abdominal neuro-endocrine tumors were based on simulations starting from PET images with several underlying assumptions. This paper reports, as proof-of-principle of this technique, an ex-vivo test on a meningioma patient. This test allowed to validate the whole chain, from the evaluation of the SUV of the tumor, to the assumptions on the bio-distribution and the signal detection. Methods: A patient affected by meningioma was administered 300 MBq of 90Y-DOTATOC. Several samples extracted from the meningioma and the nearby Dura Mater were analyzed with a beta- probe designed specifically for this radio-guided surgery technique. The observed signals were compared both with the evaluation from the histology and with the Monte Carlo simulation. Results: we obtained a large signal on the bulk tumor (105 cps) and a significant signal on residuals of $\sim$0.2 ml (28 cps). We also show that simulations predict correctly the observed yields and this allows us to estimate that the healthy tissues would return negligible signals (~1 cps). This test also demonstrated that the exposure of the medical staff is negligible and that among the biological wastes only urine has a significant activity. Conclusions: This proof-of-principle test on a patient assessed that the technique is feasible with negligible background to medical personnel and confirmed that the expectations obtained with Monte Carlo simulations starting from diagnostic PET images are correct., Comment: 17 pages, 4 Figs, Accepted by Physica Medica

The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas. Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261, creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies. Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0180-0) contains supplementary material, which is available to authorized users.

Mechanisms responsible for telomere maintenance are been recently elucidated: many cancers harbor mutations in alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene resulting in a phenomenon known as alternative lengthening of telomeres (ALT) or point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene leading to an increased telomerase expression. ATRX and TERT mutations have been recently identified also in brain tumors. In gliomas, a molecular stratification based on IDH and ATRX mutational status and 1p/19q codeletion has been suggested. ATRX mutations have been observed mainly in astrocytic tumors and TERT promoter mutations mainly in oligodendroglial tumors. Aim of our study was to evaluate the expression of both ATRX and TERT in WHO grade II gliomas and in their resected recurrences, related to a set of confirmed molecular markers and to clinical data. We performed an immunohistochemical analysis of ATRX and TERT expression in 30 grade II gliomas (astrocytomas, oligoastrocytomas and oligodendroglioma) and in their respective recurrences (grade III-IV). Also evaluation of IDH1 and p53 status and 1p/19q LOH were carried out. Loss of expression of ATRX protein has been associated to the mutated gene and ALT phenotype, while expression of TERT protein could be related to TERT promoter mutations. We found a loss of ATRX expression in 70% of cases; recurrences showed the same pattern of ATRX expression. In the most part of cases ATRX loss was associated to IDH1 and p53 mutation and intact 1p/19q, as previously observed. In the major part of cases with ATRX loss of expression TERT was not detected. TERT high expression was observed in about 30% of grade II gliomas at the first surgery, minor expression of TERT was found in grade III-IV recurrent tumors. In most case high expression of TERT was associated to 1p/19q deletion. We observed a statistically significant correlation between TERT high expression and a better outcome. Our study of protein expression of both ATRX and TERT in low grade gliomas and in their recurrences in relationship with established molecular markers and follow-up data, confirm the possibility of using immunohistochemistry in routine diagnostic procedures for better define this subset of neoplasm.

The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.

Interferon inducible protein 16 (IFI-16) is a member of the p200 human family proteins, having two domains: C-terminus binding dsDNA and N-terminus as a typical protein-protein interaction domain. IFI-16 gene is located at 1q23.1 chromosome, its functions ranging from transcriptional regulation, apoptosis, proliferation and differentiation. IFI-16 is mostly expressed in epithelial, endothelial and hematopoietic tissues, with nuclear localization signal: in the nucleolus it is able to activate and fine-tune the transcription, when in the nucleoplasm probably it is inactivated. In the nucleoli a physical and functional link of IFI-16 to p53 has been reported, as negative regulator of the cell cycle. We have studied IFI-16 expression in 40 low grade and 40 anaplastic gliomas (half of them analysed with their relapse) and 40 glioblastomas using immunohistochemical and molecular techniques, using two antibodies against the C- and N-terminus of IFI-16. Nuclear accumulation of p53, IDH1(R132H), MGMT methylation status and LOH of chromosomes 1p, 19q, 9p, 17p and 10q were also evaluated. We mainly found a nucleolar expression of IFI-16 in astrocytic lower grade gliomas, related to p53: this physical and functional binding was also demonstrated by co-immunoprecipitation and Protein Ligation Assay analysis. In anaplastic gliomas and glioblastomas, IFI-16 is largely expressed in the nucleoplasm, unrelated to p53 expression. This shift of localization suggests a possible loss of its role as protective factor in lower grade astrocytic gliomas. In oligodendrogliomas IFI-16 expression was completely absent and it is strictly related to 1p/19q co-deletion. Our results, related to clinical data, identify IFI-16 as a possible prognostic marker in glioma patients and of interest to define new molecular profiles for their therapy. This is the first study of IFI-16 in gliomas, suggesting an early role of this molecule in proliferation and malignant progression of these tumours.

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.

Medulloblastoma (MDB) is a highly malignant embryonal brain tumor located in the cerebellum with tendency to leptomeningeal dissemination, common in children, rare in adults. Recent genetic analysis suggested classification into four subgroups according to gene expression profiles, including the Wingless signaling pathway-activated group (WNT group), the Sonic Hedgehog signaling pathway-activated group (SHH group), group 3and 4, with distinct molecular, clinicopathological, and prognostic characteristics. AQP4 is the main water channel in the normal central nervous system (CNS) responsible for salt and water balance maintaining. When the blood-brain barrier become disrupted, as in gliomas, water moves from the vasculature into the extracellular space in an AQP4-independent manner, down a hydrostatic gradient to form a vasogenic edema. Moreover, AQPs have a role promoting tumor progression, cell migration and metastasis. Aim of our study was to evaluate for the first time AQP4 expression in adult MDB to focus its role in edema, tumor invasion and dissemination. We analyzed by immunohistochemistry AQP4 expression in 40 adult MDBs, also investigated for GAB1, ß-catenin, filamin-A, NPR3, according to molecular subgroups. Desmoplastic histology and Sonic hedgehog (SHH) pathway activation were more common feature, with GAB-1 and filamin-A expression. Only one case showed nuclear staining for ß-catenin. None expressed NPR3. In classical MDB, AQP4 staining was localized in glial endfoot surrounding tumor vessels. This AQP4 distribution was lost in the desmoplastic type, where the nodular areas were negatives, surrounded by glial elements AQP4 intensely stained. In the molecular layer of the cerebellar cortex in the tumor proximity in classic type MDB it has been observed migrating tumor cells along glial processes AQP4 stained. This finding was mainly observed in patients with early dissemination. These observations could suggest a role for AQP4 in adult MDB, not only in brain tumor edema resolution, but also in leptomeningeal dissemination.

Aquaporins (AQPs) are a family of transmembrane channels, involved in transcellular and transepithelial fluids transport; AQP4 is enhanced in the brain, where is the main water channel, localized in the astrocytic endfoot processes. AQP4 is anchored to the blood-brain barrier (BBB) by the dystrophin-associated complex (DAPC) consisting of α-syntrophin and dystrophin. The AQP4 channel is co-expressed on the membrane with the inwardly rectifying K+ channel (Kir4.1) and the Na+/K+/Cl- cotransporter channel (NKCC1) to maintain extracellular homeostasis. In cancer AQPs promote cellular invasion and migration. Previously, in brain metastasis we observed AQP4 expression mostly in glial cells surrounding tumor, but also in metastatic cells (mainly on NSCLC). Therefore, in this study, our principal aim is to investigate the role of AQP4 pathway in tumor microenvironment, their cross talk with the extracellular matrix and its meaning in the metastatic process. So, we have performed a retrospective analysis about the expression of AQP4 and the molecules and channels associated to AQP4, on 87 brain metastases from different tumors: lung (n=36), breast (n=21), colon (n=7), kidney (n=11), melanoma (n=7), ovary (n=3), thyroid (n=1) and prostate (n=1). Dystrophin, α-syntrophin, Kir4.1 and NKCC1 were first evaluated by immunohistochemistry and then by molecular analysis. On 21 patients we analyzed both the primitive and metastatic tumor. We observed a great AQP4 expression at the metastasis-brain interface; high expression of α-syntrophin and NKCC1 was also observed, while Kir4.1 was lower. Western blot confirmed this expression of AQP4 both in tumor and in the surrounding tissue. qPCR showed a downregulation of AQP4 mRNA except for lung metastases. α-syntrophin showed a very high expression in metastatic cells compared to normal brain, but surprisingly mRNA levels were downregulated; dystrophin showed very low levels of expression both of protein and mRNA in tumor. NKCC1 and Kir4.1 channels were overexpressed compared to the brain. These results showed a deregulation of the whole pathway associated to AQP4 in the presence of the metastases. Besides, confocal images showed a polarization of AQP4 expression mostly in lung and breast metastases. The brain metastasis maintain the same profile of original tumor. Our preliminary studies in primitive neoplasms and their counterpart brain metastases suggest a cross talk from AQP4 pathway with adhesion molecules expression and extracellular matrix in response to the presence of tumor mass. In conclusion, our results confirmed a cross talk between AQP4 and the microenvironment during the metastatic process, having a role in to maintain homeostasis, trying to reduce the edema and to limit the spreading of tumor cells in the parenchyma. This study will provide an important step forward in the knowledge of microenvironment’s involvement in brain metastasis.

In glial tumors the immune-escape is an important mechanism to promote invasion and aggressiveness, thus the aim of our study was to investigate the expression of Interferon inducible protein 16 (IFI16) in human gliomas. IFI16 is a member of the p200 human family proteins that share highly conserved repeats at C-terminus which allow them to bind dsDNA and a typical protein-protein interaction domain at their N-terminus. IFI16 gene is located at 1q23.1 chromosome and encodes for three isoforms of IFI16: its functions ranging from transcriptional regulation, apoptosis, cell growth regulation and differentiation. But, the most intriguing function of IFI16 is its role in the immunomodulation of innate and adaptive immunity. IFI16 protein is located in the nucleolar compartment where it is able to activate and fine-tune the transcription, while when it is located in the nucleoplasm is probably inactive. We have studied IFI16 expression in 120 gliomas (40 grade II, 40 grade III and 40 glioblastomas) by immunohistochemistry, using two antibodies against the C- and N-terminus, and molecular biology techniques. All these cases were also evaluated for p53, IDH1(R132H), MGMT methylation status and LOH of chromosomes 1p, 19q, 9p, 17p and 10q. We found a strong nucleolar expression of IFI16 in astrocytic lower grade gliomas and this expression was strictly related to p53 nuclear expression: this co-localization was demonstrated by confocal analysis and suggests a role of IFI16 as tumor suppressor. In grade III gliomas and glioblastomas, instead, IFI16 was mostly expressed in the nucleoplasm: this shift to the inactive form is related to a shorter survival suggesting a role as prognostic factor. In oligodendrogliomas IFI-16 expression was completely absent and it is strictly related to 1p/19q co-deletion, but with intact IFI16 gene, as showed by array-CGH analysis. As a final point, we have characterized tumor inflammatory infiltrates using antibodies against CD3, CD4, CD8, CD68 and FoxP3 related to IFI16 expression and tumor survival. Particularly in glioblastomas we observed a major expression of CD3+/CD4+ lymphocytes in the cases with strong nucleoplasm IFI16 expression and shorter survival; while CD3+/CD8+ cells were present in glioblastomas with nucleolar expression and longer survival, with also a strong activation of both microglia and macrophages, suggesting an active role of IFI16 in immuno-surveillance. In conclusion, this is the first study of IFI16 in gliomas, and we identify IFI16 as a new potential prognostic factor, highlighting its early role in proliferation, malignant progression and immunomodulation of these tumours.

Molecular and histological classification of anaplastic gliomas have been recently refined by the elucidation of the role of mutations in alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene and in telomerase reverse transcriptase (TERT) promoter. Loss of ATRX expression and the acquisition of alternative lengthening of telomeres (ALT) phenotype is peculiar of a subtype of astrocytic tumours with p53 and IDH1 mutations and better prognosis, while TERT overexpression, due to its promoter's mutations, is strongly associated to oligodendroglial tumours and 1p/19q codeletion, without a significant correlation with survival. Based on these findings, the aim of our study was to better clarify the role of ATRX and TERT expression in the histological classification of anaplastic gliomas, as diagnostic and prognostic markers of survival and relapse. We performed immunohistochemical and molecular analysis of ATRX and TERT expression on 60 grade III gliomas (astrocytomas, oligoastrocytomas and oligodendrogliomas) and the respective recurrences when they were available. Nuclear accumulation of p53, IDH1 status and LOH of chromosomes 1p, 19q, 9p, 17p and 10q were also evaluated. Our results showed a loss of ATRX expression in 60% of anaplastic oligoastrocytomas and 40% of anaplastic astrocytomas, while ATRX was wild-type in oligodendroglial tumours. We then found a strong positive correlation between loss of ATRX expression and p53 nuclear accumulation, while its strong expression was present mostly in tumours with 1p/19q codeletion. Finally, ATRX loss was correlated with a better prognosis both in anaplastic oligoastrocytomas and in anaplastic astrocytomas according to literature. Regarding TERT expression, we did not find a strictly correlation with 1p/19p codeletion and the oligodendroglial histological pattern, but, surprisingly, we found a possible role in relapse of anaplastic gliomas. In our series, tumours with high TERT expression tend to recur within 25 months, while anaplastic gliomas with low o null expression of TERT tend to recur within 60 months. In conclusion, these set of data could allow us in the future to better classify anaplastic gliomas and their tendency to recur adding immunohistochemistry for ATRX and TERT to other molecular markers in routine diagnostic procedures, and to define new molecular profiles for their therapy.

Meningiomas constitute approximately one-third of primary CNS tumors and classified by WHO in meningioma (grade I), with many histologic variants, atypical meningioma (grade II) and anaplastic meningioma (grade III). Most of them have a long survival, 1-3% are anaplastic with poor outcome and high recurrence rate and 15-20% of meningiomas are atypical with an intermediate and variable behavior. At present, only histologic grading is a reliable prognostic factor and we lack markers to identify among atypical meningiomas the most aggressive ones. For the first time in meningiomas, we propose to evaluate Lycopersicon esculentum lectin (LEA, specific for N-acetyl-D-glucosamine and N-acetyl-polylactosamine oligomers) and Aquaporin-4 (AQP4) as new factors involved in tumor invasion. LEA was chosen because it was localized in endothelial cell glycocalix in blood brain barrier (BBB) and it has been demonstrated that altered glycosilation of neoplastic cells induce vascular infiltration of cancer cells through endothelium. AQP4 is the most abundant water channel localized in the glial endfoots of the CNS and has been correlated with peritumoral oedema, that in meningioma can result as a marker of tumor invasion. The present study was performed in 20 grade I, 10 atypical and 5 anaplastic meningiomas in which a part of nervous tissue adjacent the meningioma was present on the surgical specimen. We observed a huge increase of AQP4 expression in the peritumoral nervous tissue in meningioma compared to normal nervous tissue. A thin staining in the leptomeninge and the muscolaris mucosae of the biggest vessels in tumor periphery was noticed, while tumor cells were always negative. Also in atypical meningioma AQP4 clearly define tumor boundaries. So, a minimum infiltration of nervous tissue, poorly identifiable with HE staining, could be simply visible using AQP4 staining. As for the anaplastic meningioma, which shows infiltration of nervous tissue, AQP4 has been localized also in astrocytes trapped into the tumor. In this aggressive meningioma, we found a strong LEA staining in tumor cells membrane, while grade I meningioma showed a less intense positivity. LEA expression was localized in meningothelial tumor cells and tumor vessels, as in the nervous tissue only in the endothelial cells. These observations show a role of AQP4 in peritumoral oedema and point to AQP4 as a useful marker to identify nervous tissue infiltration extension and thus meningioma aggressiveness. Moreover, LEA staining localized both in meningioma cell membranes and in the endothelial cells of the tumor vessels indicates that LEA-affinity oligomers could be implicated in adhesion and invasiveness of tumor cells. Both the glycosidic residues stained by LEA and AQP4 could be potential therapeutic targets.

Introduction: Chordoma is a rare tumor arising from the notochord. Its lethality is largely due to its tendency to recur. Precision medicine with molecular target therapies is emerging as a promising approach. The main goal of the present project is to identify molecular and clinical biomarkers as diagnostic, therapeutic and prognostic factors useful for the management of chordoma patients. Materials and Methods: A retrospective clinical and pathological charts review of all chordoma patients treated at our Institution in the last twelve years has been performed. Clinical data were gathered together with the investigation of histopathological tumor features. Kaplan-Meier analysis was used to determine overall survival (OS). In addition, a preliminary immunohistochemical (IHC) analysis was carried out for the following markers: Brachyury, p53, beta-catenin, filamin-A, GAB‐1, EGFR, PDGFR-beta, c-MET, C-kit, PTEN, YKL-40 and TERT. Sphyngolipidomic analysis will be performed to study the ceramide species in prospectively collected frozen specimens of chordomas and nuclei pulposi, as control group. Results: The study included the last consecutive 48 patients. The average follow-up was 42 months (range 3–139). Sixty surgical operations were performed in the initial or recurrent setting. Thirty patients received post-operative radiation therapy and three patients received post-operative chemotherapy. The mean OS and PFS after surgery was 42 and 49 months, respectively. The Kaplan-Meier analysis predicted OS rates of 70%, 60%, 49% and 49% at 3, 5, 7, 10 years, respectively. The preliminary IHC analysis has been conducted on 18 specimens (eleven primary, seven recurrent). All the classical chordoma expressed Brachyury. The analysis showed a positive trend of activation of the tyrosine-kinase receptors (PDGFR-beta, c-MET, c-Kit) and the Sonic-Hedgehog pathway (GAB-1, filamin-A and beta-catenin patterns). There was a phenotypic change in the expression of PDGFR-beta and GAB-1 between the primary tumor and its recurrence after the gold-standard treatment. Interestingly, TERT expression was significantly expressed in 95% of specimens. Conclusion: Kaplan-Meier analysis confirmed that surgical resection entity represented a statistically significant factor related to OS. In the present small cohort of specimens there was a positive expression of PDGFR-beta, showing that this pathway could potentially be targeted by TRKs inhibitors. TERT expression may play a role in enabling primary tumor replicative immortality and may explain the etiology from embryonic notochordal remnants. Our preliminary IHC data showed that the Sonic-Hedgehog could be a potential pathway involved in chordoma pathogenesis that should be further studied.

Aquaporin4 (AQP4) is the main water channel in the central nervous system and is localized in the astrocytic endfoot processes. AQP4 is involved in important physiological functions, such as water balance and also in neural signal transduction. When the blood-brain barrier become disrupted, as in the case of pathological conditions, such as in brain tumor, water moves from the vasculature into the extracellular space in an AQP4-independent manner, down a hydrostatic gradient to form a vasogenic edema. Moreover, in cancer AQPs have a role promoting tumor progression, invasion and metastasis. The AQP4 contribution to the brain metastasis has not been previously investigated. The aim of our study was to analyze AQP4 distribution in 60 human brain metastases of tumor from lung (n = 20), breast (n = 20), colon (n = 5), kidney (n = 8), melanoma (n = 2), thyroid (n = 1), ovary (n = 3) and prostate (n = 1), using immunohistochemistry and, in some cases western blotting. AQP4 expression on metastasis was related to clinical data of the patients. Our results revealed that: 1) normal brain near metastasis showed an increase of AQP4 expression, accompanied with gliosis; 2) at the metastasis-brain interface, AQP4 staining was increased in all kind of tumor analyzed; 3) AQP4 staining strictly surrounded each metastatic cell, also in some areas in tumor core near vessels, especially in lung and breast; 4) some metastasis from NSCLC showed membrane AQP4 immunostaining. These observations together suggest a beneficial role of AQP4 in the brain edema caused by the metastatic event, indicating that AQP4 expression may restore a normal perivascular rearrangement, also at the periphery of the tumors. Moreover, great AQP4 expression seems to correlate to higher differentiation and a better prognosis. Our findings support to consider AQP4 as a promising therapeutic target.