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Five-year glioblastoma (GBM) survivors (LTS) are the minority of the isocitrate dehydrogenase (IDH)-wild-type GBM patients, and their molecular fingerprint is still largely unexplored. This multicenter retrospective study analyzed a large LTS-GBM cohort from nine Italian institutions and molecularly characterized a subgroup of patients by mutation, DNA methylation (DNAm) and copy number variation (CNV) profiling, comparing it to standard survival GBM. Mutation scan allowed the identification of pathogenic variants in most cases, showing a similar mutational spectrum in both groups, and highlighted TP53 as the most commonly mutated gene in the LTS group. We confirmed DNAm as a valuable tool for GBM classification with a diagnostic refinement by using brain tumor classifier v12.5. LTS were more heterogeneous with more cases classified as diffuse pediatric high-grade glioma subtypes and having peculiar CNVs. We observed a global higher methylation in CpG islands and in gene promoters of LTS with methylation levels of distinct gene promoters correlating with prognosis., Competing Interests: Declaration of competing interest The authors declared that have no competing interest., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: Adult Diffuse midline glioma (DMG) is a very rare disease. DMGs are currently treated with radiotherapy and chemotherapy even if only a few retrospective studies assessed the impact on overall survival (OS) of these approaches., Methods: We carried out an Italian multicentric retrospective study of adult patients with H3K27-altered DMG to assess the effective role of systemic therapy in the treatment landscape of this rare tumor type., Results: We evaluated 49 patients from 6 Institutions. The median age was 37.3 years (range 20.1-68.3). Most patients received biopsy as primary approach (n = 30, 61.2%) and radiation therapy after surgery (n = 39, 79.6%). 25 (51.0%) of patients received concurrent chemotherapy and 26 (53.1%) patients received adjuvant temozolomide. In univariate analysis, concurrent chemotherapy did not result in OS improvement while adjuvant temozolomide was associated with longer OS (21.2 vs. 9.0 months, HR 0.14, 0.05-0.41, p < 0.001). Multivariate analysis confirmed the role of adjuvant chemotherapy (HR 0.1, 95%CI: 0.03-0.34, p = 0.003). In patients who progressed after radiation and/or chemotherapy the administration of a second-line systemic treatment had a significantly favorable impact on survival (8.0 vs. 3.2 months, HR 0.2, 95%CI 0.1-0.65, p = 0.004)., Conclusion: In our series, adjuvant treatment after radiotherapy can be useful in improving OS of patients with H3K27-altered DMG. When feasible another systemic treatment after treatment progression could be proposed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Confocal laser endomicroscopy (CLE) is a US Food and Drug Administration-cleared intraoperative real-time fluorescence-based cellular resolution imaging technology that has been shown to image brain tumor histoarchitecture rapidly in vivo during neuro-oncological surgical procedures. An important goal for successful intraoperative implementation is in vivo use at the margins of infiltrating gliomas. However, CLE use at glioma margins has not been well studied., Methods: Matching in vivo CLE images and tissue biopsies acquired at glioma margin regions of interest (ROIs) were collected from 2 institutions. All images were reviewed by 4 neuropathologists experienced in CLE. A scoring system based on the pathological features was implemented to score CLE and H&E images from each ROI on a scale from 0 to 5. Based on the H&E scores, all ROIs were divided into a low tumor probability (LTP) group (scores 0-2) and a high tumor probability (HTP) group (scores 3-5). The concordance between CLE and H&E scores regarding tumor probability was determined. The intraclass correlation coefficient (ICC) and diagnostic performance were calculated., Results: Fifty-six glioma margin ROIs were included for analysis. Interrater reliability of the scoring system was excellent when used for H&E images (ICC [95% CI] 0.91 [0.86-0.94]) and moderate when used for CLE images (ICC [95% CI] 0.69 [0.40-0.83]). The ICCs (95% CIs) of the LTP group (0.68 [0.40-0.83]) and HTP group (0.68 [0.39-0.83]) did not differ significantly. The concordance between CLE and H&E scores was 61.6%. The sensitivity and specificity values of the scoring system were 79% and 37%. The positive predictive value (PPV) and negative predictive value were 65% and 53%, respectively. Concordance, sensitivity, and PPV were greater in the HTP group than in the LTP group. Specificity was higher in the newly diagnosed group than in the recurrent group., Conclusions: CLE may detect tumor infiltration at glioma margins. However, it is not currently dependable, especially in scenarios where low probability of tumor infiltration is expected. The proposed scoring system has excellent intrinsic interrater reliability, but its interrater reliability is only moderate when used with CLE images. These results suggest that this technology requires further exploration as a method for consistent actionable intraoperative guidance with high dependability across the range of tumor margin scenarios. Specific-binding and/or tumor-specific fluorophores, a CLE image atlas, and a consensus guideline for image interpretation may help with the translational utility of CLE.

Background: First-line therapies for medulloblastoma(MBL) are obtaining higher survival-rates while decreasing late-effects, but treatment at relapse is not standardized. We report here the experience with MBL re-irradiation(re-RT), its timing and outcome in different clinical settings and tumor groups., Methods: Patient's staging/treatment at diagnosis, histotypes/molecular subgroups, relapse site/s, re-treatments outcome are reported., Results: 25 patients were included, with a median age of 11.4 years; 8 had metastases. According to 2016-2021 WHO-classification, 14 had SHH subgroup tumors(six TP53 mutated,one + MYC,one + NMYC amplification), 11 non-WNT/non-SHH (two with MYC/MYCN amplification).Thirteen had received HART-CSI, 11 standard-CSI, one HFRT; all post-radiation chemotherapy(CT), 16 also pre-RT. Median time to relapse (local-LR in nine, distant-DR in 14, LR + DR in two) was 26 months. Fourteen patients were re-operated, in five cases excising single DR-sites, thereafter three received CT, two after re-RT; out of 11 patients not re-operated, four had re-RT as first treatment and seven after CT. Re-RT was administered at median 32 months after first RT: focally in 20 cases, craniospinal-CSI in five. Median post-relapse-PFS/after re-RT was 16.7/8.2 months, while overall survival-OS was 35.1/23.9 months, respectively. Metastatic status both at diagnosis/relapse negatively affected outcome and re-surgery was prognostically favorable. PD after re-RT was however significantly more frequent in SHH (with a suggestive association with TP53 mutation, p = 0.050). We did not observe any influence of biological subgroups on PFS from recurrence while SHH showed apparently worse OS compared to non-WNT/non-SHH group., Conclusions: Re-surgery + reRT can prolong survival; a substantial fraction of patients with worse outcome belongs to the SHH-subgroup., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Confocal laser technology has been recently suggested as a promising method to obtain near real-time intraoperative histological data. We recently demonstrated the accuracy of a newly designed confocal endomiscroscope (CONVIVO) in offering an intraoperative diagnosis during high-grade gliomas (HGGs) removal in an ex vivo study. With this work we aim to perform a standardized, prospective and blinded-to-histological section study for evaluating the potentiality of CONVIVO in offering in-vivo data regarding histological diagnosis and presence of tumor at margins during resection of central nervous system (CNS) tumors., Methods: This prospective, observational, standardized, blinded-to-histological section, clinical trial was approved by the institutional review board in Carlo Besta Neurologic Institute IRCCS Foundation in Milan and is expected to last 24 months. 75 patients will be included, with at least 53 of them being HGGs based on the statistical sample size calculation. Main objectives will be the assessing of the concordance of tumor diagnoses between CONVIVO images and frozen section at the center of all tumor subtypes and the evaluation of the accuracy of CONVIVO in the identification of tumor tissue at the margins, compared to standard histology. For this purpose, "virtual biopsies" and physical biopsies will be performed directly on patient tumor tissue and surrounding brain parenchima during tumor resection, comparing the results of CONVIVO analysis and frozen and histological sections., Results: Despite promising preliminary data on ex vivo usefulness of CLE machines are emerging in literature, still few studies are available when looking at in vivo potentiality of CONVIVO. At this regard, this study will be the first work where a standardized, prospective, and blinded-to-histological section CONVIVO analysis will be performed in an in-vivo setting in neuro-oncological surgery., Conclusions: We hypothesize that this new technique may have a role in offering data regarding presence of tumor tissue, eventually giving an intraoperative diagnosis in neuro-oncological surgery, rendering more fluid the decision-making process in the operating room. Furthermore, the result of this study will provide a solid base for further expanding the clinical applications of confocal machines in neurosurgery.

Background: Altered microRNA profiles have been observed not only in tumour tissues but also in biofluids, where they circulate in a stable form thus representing interesting biomarker candidates. This study aimed to identify a microRNA signature as a non-invasive biomarker and to investigate its impact on glioma biology., Methods: MicroRNAs were selected using a global expression profile in preoperative serum samples from 37 glioma patients. Comparison between serum samples from age and gender-matched controls was performed by using the droplet digital PCR. The ROC curve and Kaplan-Meier survival analyses were used to evaluate the diagnostic/prognostic values. The functional role of the identified signature was assessed by gain/loss of function strategies in glioma cells., Results: A three-microRNA signature (miR-1-3p/-26a-1-3p/-487b-3p) was differentially expressed in the serum of patients according to the isocitrate dehydrogenase (IDH) genes mutation status and correlated with both patient Overall and Progression Free Survival. The identified signature was also downregulated in the serum of patients compared to controls. Consistent with these results, the signature expression and release in the conditioned medium of glioma cells was lower in IDH-wild type cells compared to the mutated counterpart. Furthermore, in silico analysis of glioma datasets showed a consistent deregulation of the signature according to the IDH mutation status in glioma tumour tissues. Ectopic expression of the signature negatively affects several glioma functions. Notably, it impacts the glioma invasive phenotype by directly targeting the invadopodia-related proteins TKS4, TKS5 and EFHD2., Conclusions: We identified a three microRNA signature as a promising complementary or even an independent non-invasive diagnostic/prognostic biomarker. The signature displays oncosuppressive functions in glioma cells and impacts on proteins crucial for migration and invasion, providing potential targets for therapeutic intervention., (© 2023. The Author(s).)

Background: Prognostic factors and role of treatments are not well known in isocitrate dehydrogenase (IDH) wild-type (wt) grade 2 astrocytomas. The aim of this study was to define in these tumors clinical features, molecular characteristics, and prognostic factors, with particular focus on molecular subgroups defined by cIMPACT-NOW update 3., Methods: We analyzed 120 patients with confirmed diagnosis of grade 2 IDHwt astrocytoma according to WHO 2016, collected from seven Italian centers between 1999 and 2017., Results: Median PFS and OS of the whole cohort were 18.9 and 32.6 months. Patients older than 40 years and patients with modest contrast enhancement on MRI had a shorter PFS and OS. Gross total resection yielded superior PFS and OS over non-gross total resection. PFS and OS of patients with either pTERT mutation or EGRF amplification were significantly shorter. The prognostic value of age, contrast enhancement on MRI, and extent of surgery was different within the molecular subgroups. Gross total resection was associated with increased PFS (not reached versus 14 months, p = 0.023) and OS (117.9 versus 20 months, p = 0.023) in patients without EGFR amplification, and with increased OS in those without pTERT mutation (NR vs 53.7 months, p = 0.05). Conversely, for patients with EGFR amplification or pTERT mutation, gross total resection did not yield a significant survival benefit., Conclusion: Patients without EGFR amplification and pTERT mutation could be observed after gross total resection., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pleomorphic xanthoastrocytoma (PXA) in its classic manifestation exhibits distinct morphological features and is assigned to CNS WHO grade 2 or grade 3. Distinction from glioblastoma variants and lower grade glial and glioneuronal tumors is a common diagnostic challenge. We compared a morphologically defined set of PXA (histPXA) with an independent set, defined by DNA methylation analysis (mcPXA). HistPXA encompassed 144 tumors all subjected to DNA methylation array analysis. Sixty-two histPXA matched to the methylation class mcPXA. These were combined with the cases that showed the mcPXA signature but had received a histopathological diagnosis other than PXA. This cohort constituted a set of 220 mcPXA. Molecular and clinical parameters were analyzed in these groups. Morphological parameters were analyzed in a subset of tumors with FFPE tissue available. HistPXA revealed considerable heterogeneity in regard to methylation classes, with methylation classes glioblastoma and ganglioglioma being the most frequent mismatches. Similarly, the mcPXA cohort contained tumors of diverse histological diagnoses, with glioblastoma constituting the most frequent mismatch. Subsequent analyses demonstrated the presence of canonical pTERT mutations to be associated with unfavorable prognosis among mcPXA. Based on these data, we consider the tumor type PXA to be histologically more varied than previously assumed. Histological approach to diagnosis will predominantly identify cases with the established archetypical morphology. DNA methylation analysis includes additional tumors in the tumor class PXA that share similar DNA methylation profile but lack the typical morphology of a PXA. DNA methylation analysis also assist in separating other tumor types with morphologic overlap to PXA. Our data suggest the presence of canonical pTERT mutations as a robust indicator for poor prognosis in methylation class PXA., (© 2022. The Author(s).)

Glioblastoma (GBM) is a fatal tumor whose aggressiveness, heterogeneity, poor blood-brain barrier penetration, and resistance to therapy highlight the need for new targets and clinical treatments. A step toward clinical translation includes the eradication of GBM tumor-initiating cells (TICs), responsible for GBM heterogeneity and relapse. By using patient-derived TICs and xenograft orthotopic models, we demonstrated that the selective lysine-specific histone demethylase 1 inhibitor DDP&#95;38003 (LSD1i) is able to penetrate the brain parenchyma in vivo in preclinical models, is well tolerated, and exerts antitumor activity in molecularly different GBMs. LSD1 genetic targeting further strengthens the role of LSD1 in GBM TIC maintenance. GBM TIC plasticity supports their adaptation and survival under a plethora of environmental stresses, including nutrient deficiency and proteostasis perturbation. By mimicking these stresses in vitro, we found that LSD1 inhibition hampers the induction of the activating transcription factor 4 (ATF4), the master regulator of the integrated stress response (ISR). The resulting aberrant ISR sensitizes GBM TICs to stress-induced cell death, hampering tumor aggressiveness. Functionally, LSD1i interferes with LSD1 scaffolding function and prevents its interaction with CREBBP, a critical ATF4 activator. By disrupting the interaction between CREBBP and LSD1-ATF4 axis, LSD1 inhibition prevents GBM TICs from overcoming stress and sustaining GBM progression. The effectiveness of the LSD1 inhibition in preclinical models shown here places a strong rationale toward its clinical translation for GBM treatment.

Background: A prospective 2002-2014 study stratified 160 patients by resection extent and histological grade, reporting results in 2016. We re-analyzed the series after a median of 119 months, adding retrospectively patients' molecular features., Methods: Follow-up of all patients was updated. DNA copy number analysis and gene-fusion detection could be completed for 94/160 patients, methylation classification for 68., Results: Progression-free survival (PFS) and overall survival (OS) at 5/10 years were 66/58%, and 80/73%. Ten patients had late relapses (range 66-126 mo), surviving after relapse no longer than those relapsing earlier (0-5 y). On multivariable analysis a better PFS was associated with grade II tumor and complete surgery at diagnosis and/or at radiotherapy; female sex and complete resection showed a positive association with OS. Posterior fossa (PF) tumors scoring ≥0.80 on DNA methylation analysis were classified as PFA (n = 41) and PFB (n = 9). PFB patients had better PFS and OS. Eighteen/32 supratentorial tumors were classified as RELA, and 3 as other molecular entities (anaplastic PXA, LGG MYB, HGNET). RELA had no prognostic impact. Patients with 1q gain or cyclin-dependent kinase inhibitor 2A (CDKN2A) loss had worse outcomes, included significantly more patients >3 years old (P = 0.050) and cases of dissemination at relapse (P = 0.007)., Conclusions: Previously described prognostic factors were confirmed at 10-year follow-up. Late relapses occurred in 6.2% of patients. Specific molecular features may affect outcome: PFB patients had a very good prognosis; 1q gain and CDKN2A loss were associated with dissemination. To draw reliable conclusions, modern ependymoma trials need to combine diagnostics with molecular risk stratification and long-term follow-up., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recurrent glycine-to-arginine/valine alterations at codon 34 (G34R/V) within H3F3A gene characterize a subset of hemispheric high-grade gliomas (HGG) affecting children and young adults. These tumors, defined as G34R/V-mutant gliomas, are histologically heterogenous, with microscopic features of either HGG or embryonal tumors (primitve neuroectodermal tumor-like features). To assess the value of immunohistochemistry (IHC) to detect G34R/V-mutated cases, we tested anti-histone G34V (clone 329E5) and anti-histone G34R (clone RM240) antibodies in a series of 28 formalin-fixed and paraffin-embedded samples. A total of 28 cases of hemispheric, IDH-wt HGG mainly affecting children and young adults were evaluated by IHC and by sequencing. The median age of patients at diagnosis was 17 years (0.1 to 26 y). By IHC, 10 of the 28 cases showed nuclear positivity for G34R and 3 of the 28 cases for G34V. Molecular analysis of G34R/V-mutation status was successful in 24 of the 28 cases. Mutation at glycine 34 of the H3F3A gene was identified in 9 of the 24 tumors (37%) by direct sequencing, revealing 7 of 9 positive case by sequencing and 2 of 9 false negative cases by IHC. Two of 15 negative case by sequencing demonstrated a false positivity by IHC. In total, in 4 (16.6%) of 24 cases, IHC and mutational results were discordant: 2 tumors were negative by IHC (false negative) but harbored G34R mutation by sequencing, and 2 cases were positive by IHC (false positive by IHC) but wild type by sequencing. Moreover, most mutated cases showed loss of ATRX expression and/or p53 expression. The positivity by IHC with specific antibody tested is not highly predictive for presence of G34R/V mutation, but confirmation by sequencing is mandatory; G34R/V mutations should be suspected in all hemispheric tumor IDH1/2 wild type, showing loss of OLIG2 and ATRX and/or p53 expression., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

In 2018, the SINch (Italian Society of Neurosurgery) Neuro-Oncology Section, AINO (Italian Association of Neuro-Oncology) and SIN (Italian Association of Neurology) Neuro-Oncology Section formed a collaborative Task Force to look at the diagnosis and treatment of low-grade gliomas (LGGs). The Task Force included neurologists, neurosurgeons, neuro-oncologists, pathologists, radiologists, radiation oncologists, medical oncologists, a neuropsychologist and a methodologist. For operational purposes, the Task Force was divided into five Working Groups: diagnosis, surgical treatment, adjuvant treatments, supportive therapies, and follow-up. The resulting guidance document is based on the available evidence and provides recommendations on diagnosis and treatment of LGG patients, considering all aspects of patient care along their disease trajectory.

Introduction: 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., Materials and Methods: We retrospectively present 122 patients, including a subgroup with histology confirmation (n = 75, cohort b)., Results: 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 < 0.0001). For cohort b, same prognostic factors were confirmed, except for midline location, at univariate analysis; at multivariate analysis, only KPS ≥ 80 and chemotherapy remained prognostic (p < 0.0001)., Conclusion: We described clinical, neuroimaging, management, and histomolecular features of one of the largest GC series. We identified KPS ≥ 80, radiological pattern as subtentorial localization and dissemination, and chemotherapy as prognostic factors, at multivariate analysis. Planning prospective study, associated to focused genetic assays, could help to clarify if GC has specific features that may result in the identification as a separate entity from other gliomas.

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Papillary glioneuronal tumor (PGNT) is a WHO-defined brain tumor entity that poses a major diagnostic challenge. Recently, SLC44A1-PRKCA fusions have been described in PGNT. We subjected 28 brain tumors from different institutions histologically diagnosed as PGNT to molecular and morphological analysis. Array-based methylation analysis revealed that 17/28 tumors exhibited methylation profiles typical for other tumor entities, mostly dysembryoplastic neuroepithelial tumor and hemispheric pilocytic astrocytoma. Conversely, 11/28 tumors exhibited a unique profile, thus constituting a distinct methylation class PGNT. By screening the extended Heidelberg cohort containing over 25,000 CNS tumors, we identified three additional tumors belonging to this methylation cluster but originally histologically diagnosed otherwise. RNA sequencing for the detection of SLC44A1-PRKCA fusions could be performed on 19 of the tumors, 10 of them belonging to the methylation class PGNT. In two additional cases, SLC44A1-PRKCA fusions were confirmed by FISH. We detected fusions involving PRKCA in all cases of this methylation class with material available for analyses: the canonical SLC44A1-PRKCA fusion was observed in 11/12 tumors, while the remaining case exhibited a NOTCH1-PRKCA fusion. Neither of the fusions was found in the tumors belonging to other methylation classes. Our results point towards a high misclassification rate of the morphological diagnosis PGNT and clearly demonstrate the necessity of molecular analyses. PRKCA fusions are highly diagnostic for PGNT, and detection by RNA sequencing enables the identification of rare fusion partners. Methylation analysis recognizes a unique methylation class PGNT irrespective of the nature of the PRKCA fusion.

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.

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.

Background: This prospective study stratified patients by surgical resection (complete = NED vs incomplete = ED) and centrally reviewed histology (World Health Organization [WHO] grade II vs III)., Methods: WHO grade II/NED patients received focal radiotherapy (RT) up to 59.4 Gy with 1.8 Gy/day. Grade III/NED received 4 courses of VEC (vincristine, etoposide, cyclophosphamide) after RT. ED patients received 1-4 VEC courses, second-look surgery, and 59.4 Gy followed by an 8-Gy boost in 2 fractions on still measurable residue. NED children aged 1-3 years with grade II tumors could receive 6 VEC courses alone., Results: From January 2002 to December 2014, one hundred sixty consecutive children entered the protocol (median age, 4.9 y; males, 100). Follow-up was a median of 67 months. An infratentorial origin was identified in 110 cases. After surgery, 110 patients were NED, and 84 had grade III disease. Multiple resections were performed in 46/160 children (28.8%). A boost was given to 24/40 ED patients achieving progression-free survival (PFS) and overall survival (OS) rates of 58.1% and 68.7%, respectively, in this poor prognosis subgroup. For the whole series, 5-year PFS and OS rates were 65.4% and 81.1%, with no toxic deaths. On multivariable analysis, NED status and grade II were favorable for OS, and for PFS grade II remained favorable., Conclusions: In a multicenter collaboration, this trial accrued the highest number of patients published so far, and results are comparable to the best single-institution series. The RT boost, when feasible, seemed effective in improving prognosis. Even after multiple procedures, complete resection confirmed its prognostic strength, along with tumor grade. Biological parameters emerging in this series will be the object of future correlatives and reports., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Medulloblastomas and high-grade gliomas (HGG) are two distinct brain tumor, with different peculiarities in terms of age of onset, localizations and prognosis. The coexistence of the two neoplasms in the same adult patient is an extremely rare event. We present the case of a woman treated with radio-chemotherapy for an HGG, who developed a cerebellar medulloblastoma 7 years later. Considering the poor prognosis of these tumors, the lack of knowledge about the mechanisms of onset as well as effective therapies, it is necessary to determine the exact role of irradiation and the presence of any potential molecular genetic abnormalities in the developing of the two tumors.

Purpose: A radio-guided surgery technique with β(-)-emitting radio-tracers was suggested to overcome the effect of the large penetration of γ 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 300MBq of (90)Y-DOTATOC. Several samples extracted from the meningioma and the nearby Dura Mater were analyzed with a β(-) 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 (105cps) and a significant signal on residuals of ∼0.2ml (28cps). We also show that simulations predict correctly the observed yields and this allows us to estimate that the healthy tissues would return negligible signals (≈1cps). 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., (Copyright © 2016 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Background: The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study., Methods: The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence., Results: We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature., Conclusions: Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.

Objective: The purpose of this study was to assess the capability of contrast-enhanced ultrasound (CEUS) to identify residual tumor mass during glioblastoma multiforme (GBM) surgery, to increase the extent of resection., Methods: The authors prospectively evaluated 10 patients who underwent surgery for GBM removal with navigated ultrasound guidance. Navigated B-mode and CEUS were performed prior to resection, during resection, and after complete tumor resection. Areas suspected for residual tumors on B-mode and CEUS studies were localized within the surgical field with navigated ultrasound and samples were sent separately for histopathological analysis to confirm tumor presence., Results: In all cases tumor remnants were visualized as hyperechoic areas on B-mode, highlighted as CEUS-positive areas, and confirmed as tumoral areas on histopathological analysis. In 1 case only, CEUS partially failed to demonstrate residual tumor because the residual hyperechoic area was devascularized prior to ultrasound contrast agent injection. In all cases CEUS enhanced B-mode findings., Conclusions: As has already been shown in other neoplastic lesions in other organs, CEUS is extremely specific in the identification of residual tumor. The ability of CEUS to distinguish between tumor and artifacts or normal brain on B-mode is based on its capacity to show the vascularization degree and not the echogenicity of the tissues. Therefore, CEUS can play a decisive role in the process of maximizing GBM resection.

MRI grading of grade II and III gliomas may have an important impact on treatment decisions. Occasionally,both conventional MRI (cMRI) and histology fail to clearly establish the tumour grade. Three cMRI features(no necrosis; no relevant oedema; absent or faint contrast enhancement) previously validated in 196 patients with supratentorial gliomas directed our selection of 68 suspected low-grade gliomas (LGG) that were also investigated by advanced MRI (aMRI), including perfusion weighted imaging (PWI), diffusion weighted imaging(DWI) and spectroscopy. All the gliomas had histopathological diagnoses. Sensitivity and specificity of cMRI preoperative diagnosis were 78.5 and 38.5 %, respectively, and 85.7 and 53.8 % when a MRI was included, respectively. ROC analysis showed that cut-off values of 1.29 for maximum rCBV, 1.69 for minimum rADC, 2.1 for rCho/Cr ratio could differentiate between LGG and HGG with a sensitivity of 61.5, 53.8, and 53.8 % and a specificity of 54.7, 43 and 64.3 %, respectively. A significantly longer OS was observed in patients with a maximum rCBV<1.46 and minimum rADC>1.69 (80 vs 55 months, p = 0.01; 80 vs 51 months, p = 0.002, respectively). This result was also confirmed when cases were stratified according to pathology (LGG vs HGG). The ability of a MRI to differentiate between LGG and HGG and to predict survival improved as the number of a MRI techniques considered increased. In a selected population of suspected LGG,classification by cMRI underestimated the actual fraction of HGG. aMRI slightly increased the diagnostic accuracy compared to histopathology. However, DWI and PWI were prognostic markers independent of histological grade.

Purpose: With the rapid discovery of prognostic and predictive molecular parameters for glioma, the status of histopathology in the diagnostic process should be scrutinized. Our project aimed to construct a diagnostic algorithm for gliomas based on molecular and histologic parameters with independent prognostic values., Methods: The pathology slides of 636 patients with gliomas who had been included in EORTC 26951 and 26882 trials were reviewed using virtual microscopy by a panel of six neuropathologists who independently scored 18 histologic features and provided an overall diagnosis. The molecular data for IDH1, 1p/19q loss, EGFR amplification, loss of chromosome 10 and chromosome arm 10q, gain of chromosome 7, and hypermethylation of the promoter of MGMT were available for some of the cases. The slides were divided in discovery (n = 426) and validation sets (n = 210). The diagnostic algorithm resulting from analysis of the discovery set was validated in the latter., Results: In 66% of cases, consensus of overall diagnosis was present. A diagnostic algorithm consisting of two molecular markers and one consensus histologic feature was created by conditional inference tree analysis. The order of prognostic significance was: 1p/19q loss, EGFR amplification, and astrocytic morphology, which resulted in the identification of four diagnostic nodes. Validation of the nodes in the validation set confirmed the prognostic value (P < .001)., Conclusion: We succeeded in the creation of a timely diagnostic algorithm for anaplastic glioma based on multivariable analysis of consensus histopathology and molecular parameters., (© 2015 by American Society of Clinical Oncology.)

Angiosarcoma is a rare vascular malignant neoplasm that mainly occurs in skin and soft tissues. Intracranial localization is very rare and only a few cases have been reported. This report intends to present the clinical, radiological and pathological pictures of a primary central nervous system angiosarcoma along with a review of the literature. A 35-year-old woman presented at our institution with weakness and sensory disturbances of her right hand. Neuroimaging revealed a roughly round, hemorrhagic and moderately enhancing lesion in the left frontal posterior region. The tumor was totally removed under awake anesthesia and continuous monitoring of motor and language functions. Histopathology revealed an epithelioid angiosarcoma. Radical removal, followed by adjuvant radiotherapy and chemotherapy, is able to completely control the disease for a relatively long period., (© 2014 Japanese Society of Neuropathology.)

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.

Background: Prospective follow-up studies of large cohorts of patients with glioblastoma (GBM) are needed to assess the effectiveness of conventional treatments in clinical practice. We report GBM survival data from the Brain Cancer Register of the Fondazione Istituto Neurologico Carlo Besta (INCB) in Milan, Italy, which collected longitudinal data for all consecutive patients with GBM from 1997 to 2010., Methods: Survival data were obtained from 764 patients (aged>16 years) with histologically confirmed primary GBM who were diagnosed and treated over a 7-year period (2004-2010) with follow-up to April 2012 (cohort II). Equivalent data from 490 GBM patients diagnosed and treated over the preceding 7 years (1997-2003) with follow-up to April 2005 (cohort I) were available for comparison. Progression-free survival (PFS) was available from 361 and 219 patients actively followed up at INCB in cohorts II and I, respectively., Results: Survival probabilities were 54% at 1 year, 21% at 2 years, and 11% at 3 years, respectively, in cohort II compared with 47%, 11%, and 5%, respectively, in cohort I. PFS was 22% and 12% at 1 year in cohorts II and I. Better survival and PFS in cohort II was significantly associated with introduction of the Stupp protocol into clinical practice, with adjusted hazard ratios (HRs) of 0.78 for survival and 0.73 for PFS, or a 22% relative decrease in the risk of death and a 27% relative decrease in the risk of recurrence. After recurrence, reoperation was performed in one-fifth of cohort I and in one-third of cohort II but was not effective (HR, 1.05 in cohort I and 1.02 in cohort II). Second-line chemotherapy, mainly consisting of nitrosourea-based chemotherapy, temozolomide, mitoxantrone, fotemustine, and bevacizumab, improved survival in both cohorts (HR, 0.57 in cohort I and 0.74 in cohort II). Radiosurgery was also effective (HR, 0.52 in cohort II)., Conclusions: We found a significant increase in overall survival, PFS, and survival after recurrence after 2004, likely due to improvements in surgical techniques, introduction of the Stupp protocol as a first-line treatment, and new standard protocols for second-line chemotherapy and radiosurgery after tumor recurrence. In both cohorts, reoperation after tumor recurrence did not improve survival.

Object: Fluorescein, a dye that is widely used as a fluorescent tracer, accumulates in cerebral areas where the blood-brain barrier is damaged. This quality makes it an ideal dye for the intraoperative visualization of high-grade gliomas (HGGs). The authors report their experience with a new fluorescein-guided technique for the resection of HGGs using a dedicated filter on the surgical microscope., Methods: The authors initiated a prospective Phase II trial (FLUOGLIO) in September 2011 with the objective of evaluating the safety of fluorescein-guided surgery for HGGs and obtaining preliminary evidence regarding its efficacy for this purpose. To be eligible for participation in the study, a patient had to have suspected HGG amenable to complete resection of the contrast-enhancing area. The present report is based on the analysis of the short- and long-term results in 20 consecutive patients with HGGs (age range 45-74 years), enrolled in the study since September 2011. In all cases fluorescein (5-10 mg/kg) was injected intravenously after intubation. Tumor resection was performed with microsurgical technique and fluorescence visualization by means of BLUE 400 or YELLOW 560 filters on a Pentero microscope., Results: The median preoperative tumor volume was 30.3 cm(3) (range 2.4-87.8 cm(3)). There were no adverse reactions related to fluorescein administration. Complete removal of contrast-enhanced tumor was achieved in 80% of the patients. The median duration of follow-up was 10 months. The 6-months progression-free survival rate was 71.4% and the median survival was 11 months., Conclusions: Analysis of these 20 cases suggested that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows a high rate of complete resection of contrast-enhanced tumor as determined on early postoperative MRI. Clinical trial registration no.: 2011-002527-18 (EudraCT).

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 < 0.001). The CNV assay was validated investigating 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.

Neoplastic meningitis (NM) is diagnosed in 1-2 % of patients with primary brain tumors. Standard treatment of NM includes single-agent or combination chemotherapy, with compounds such as methotrexate, thiotepa, and cytarabine (Ara-C) or its injectable, sustained-release formulation Depocyte(®). In this Report, we reported the data of efficacy and tolerability of an intrathecal Depocyte(®) regimen for patients presenting with NM from primary brain tumors. We described 12 patients with NM confirmed at magnetic resonance imaging (MRI) and with a positive cerebrospinal fluid (CSF) cytology. Patients were treated with repeated courses of intrathecal Depocyte(®) (once every 2 weeks for 1 month of induction therapy and as consolidation therapy on a monthly base in responding patients). Twelve patients (10 males and 2 females) were treated by our Institution. The diagnosis of primitive brain tumor was medulloblastoma in six patients, germinoma in two patients, pylocitic astrocytomas with spongioblastic aspects, teratocarcinoma, meningeal melanoma, and ependimoma in the other four patients. The total number of Depocyte(®) cycles ranged from one to nine. In 7/12 patients, there was clinical and/or radiological response after Depocyte(®), and the toxicity was moderate and transient, mainly due to the lumbar puncture procedure. In the two patients with germinoma, we observed a normalization of MRI Imaging and negativization of CSF with disappearance of the tumor cells. OS was 180 days (range 20-300, CI 95 %).

Background: Fluorescein is widely used as a fluorescent tracer for many applications. Its capability to accumulate in cerebral areas with blood-brain barrier damage makes it an ideal dye for intraoperative visualization of malignant gliomas (MG). We report our preliminary experience in fluorescein-guided removal of grade IV gliomas using a dedicated filter on the surgical microscope., Methods: In September 2011 we started a prospective phase II trial (FLUOGLIO) to evaluate the safety and obtain initial indications about the efficacy of fluorescein-guided surgery for MG. Patients with suspected MG amenable to complete resection of contrast-enhancing areas were eligible to participate in this study. This report is based on a preliminary analysis of the results of 12 patients with grade IV gliomas out of 15 consecutive cases (age range 48-72 years) enrolled since September 2011. Fluorescein was injected intravenously (i.v.) after intubation (5-10 mg/kg). The tumor was removed using a microsurgical technique and fluorescence visualization by BLU 400 or YELLOW 560 filters on a Pentero microscope (Carl Zeiss, Germany). The study was approved by our ethics committee and registered on the European Regulatory Authorities website (EudraCT no. 2011-002527-18)., Results: Histological analysis confirmed grade IV gliomas in 12/15 cases. Median preoperative tumor volume was 33.15 cm(3) (9.6-87.8 cm(3)). No adverse reaction related to the administration of fluorescein was registered. Contrast-enhanced tumor was completely removed in 75 % of the patients., Conclusion: This preliminary analysis suggested that the use of intravenous fluorescein during surgery on grade IV gliomas is safe and allows a high rate of complete resection of contrast-enhanced tumor at the early postoperative MRI.

Object: Locoregional chemotherapy with carmustine wafers, positioned at surgery and followed by radiation therapy, has been shown to prolong survival in patients with newly diagnosed glioblastoma, as has concomitant radiochemotherapy with temozolomide. A combination of carmustine wafers with the Stupp treatment regimen has only been investigated in retrospective studies., Methods: In a single-institution prospective study, the authors assessed 12-month progression-free survival (PFS), toxicity, and overall survival in patients with glioblastoma treated with surgery, carmustine wafers, radiotherapy, and 6-month metronomic temozolomide chemotherapy. Thirty-five patients with de novo glioblastoma, between the ages of 18 and 70 years, and with Karnofsky Performance Scale scores of at least 70, were included in the study. Patients were followed monthly and assessed using MRI every 2 months., Results: After a median follow-up of 15 months, the median time to tumor progression was 12.5 months and median survival was 17.8 months. Due to toxicity (mostly hematological), 7 patients had to prematurely stop temozolomide treatment. Twenty-two patients developed Grade 3 CD4(+) lymphocytopenia. Three patients developed oral-esophageal candidiasis, 2 developed pneumonia, and 1 developed a dorsolumbar zoster. Early intracranial hypertension was observed in 1 patient, and 1 was treated empirically for suspected brain abscess. One patient died of Legionella pneumonia soon after repeat surgery., Conclusions: Overall, this treatment schedule produced promising results in terms of PFS without a marked increase in toxicities as compared with the Stupp regimen. However, the gain in median survival using this schedule was less clear. Only prospective comparative trials will determine whether these preliminary results will translate into a long-term survival advantage with an acceptable toxicity profile.

Background: Atypical teratoid rhabdoid tumor (ATRT) patients display a dismal median overall survival of less than 1 year. A consistent fraction of cases carries de-novo SMARCB1/INI1 constitutional mutations in the setting of the "rhabdoid tumor predisposition syndrome" and the outcome is worst in infant syndromic ATRT patients., Case Presentation: We here describe a patient affected by mosaic Klinefelter syndrome and by rhabdoid tumor predisposition syndrome caused by constitutional SMARCB1/INI1 heterozygous mutation c.118C>T (Arg40X). Patient's ATRT primary tumor occurred at 2 years of age concurrent with metastatic lesions. The patient was rendered without evidence of disease by combined surgery, high-dose poli-chemotherapy and craniospinal irradiation, followed by autologous hematopoietic stem cell transplantation. At the onset of a spinal lesion 5.5 years later, both tumors were pathologically and molecularly evaluated at the national central pathology review board and defined as ATRT in a syndromic patient, with strong evidence of a clonal origin of the two lesions. The patient was then treated according to SIOP guidelines and is now alive without evidence of disease 24 months after the detection of metastatic disease and 90 months after the original diagnosis., Conclusion: The report underscores the current utility of multiple comprehensive approaches for the correct diagnosis and clinical management of patients affected by rare and atypical brain neoplasms. Successful local control of disease and achievement of long-term survival is possible in ATRT patients even in the setting of rhabdoid tumor predisposition syndrome, infant age at diagnosis and metastatic spread of disease, thus justifying the efforts for the management of this severe condition.

Epilepsy occurs in glioma, especially in low-grade glioma (LGG), but also in glioblastoma (GBM). In about 20 % of patients pharmacological treatment with anti-epileptic drugs (AEDs) fails. Refractory epilepsy is a multifactorial phenomenon not yet completely understood. The multidrug resistance phenotype was initially associated to P-glycoprotein (Pgp), an ATP-dependent transporter belonging to the same superfamily of multidrug resistance-associated proteins (MRPs). Glutathione-S-transferase-π (GST-π) is also involved in refractory epilepsy. In the present work we investigated the expression of Pgp, MRP1, MRP3 and GST-π in surgical specimens obtained from 35 patients with glioma and epilepsy. We observed MRP1 expression in tumor and endothelial cells (EC), MRP3 and Pgp expression mainly in ECs and GST-π predominantly in tumor cells (TC). MRP1 and MRP3 were more expressed in high grade glioma (HGG) than in LGG. In 6 cases we could compare tumor and periphery detecting the same MRP1 and Pgp expression, while MRP3 was mainly expressed in the tumor. We observed a trend of a better outcome in seizure control associated with a lower expression of MRP1 and MRP3. MRP3 was statistically more expressed in TCs of HGG than LGG (p = 0.0401) and more expressed in tumor than in periphery, in agreement with recent works that identify MRP3 as a potential target in GBM. Moreover, MRP3 was investigated in association with refractory epilepsy for the first time in our study and it was less expressed in patients with complete response to AEDs (p = 0.0550). Our preliminary data show an association between multidrug resistance transporters and refractory epilepsy in glioma.

The tumors of the central nervous system are classified according to the last international classification published by World Health Organization. The Classification of Tumors of the Central Nervous System was done on 2007, based on morphological features, growth pattern and molecular profile of neoplastic cells, defining malignancy grade. The neuropathological diagnosis and the grading of each histotype are based on identification of histopathological criteria and immunohistochemical data. The histopathology, also consisting of findings with prognostic or predictive relevance, plays a critical role in the diagnosis and treatment of brain tumors. The recent progresses on radiological, pathological, immunohistochemical, molecular and genetic diagnosis improved the characterization of brain tumors. Molecular and genetic profiles may identify different tumor subtypes varying in biological and clinical behavior. To investigate new therapeutic approaches is important to study the molecular pathways that lead the processes of proliferation, invasion, angiogenesis, anaplastic transformation. Different molecular biomarkers were identified by genetic studies and some of these are used in neuro-oncology for the evaluation of glioma patients, in particular combined deletions of the chromosome arms 1p and 19q in oligodendroglial tumors, methylation status of the O-6 methylguanine- DNA methyltransferase gene promoter and alterations in the epidermal growth factor receptor pathway in adult malignant gliomas, isocitrate dehydrogenase 1 (IDH1) and IDH2 gene mutations in diffuse gliomas, as well as BRAF status in pilocytic astrocytomas. The prognostic evaluation and the therapeutic strategies for patients depend on synthesis of clinical, pathological and biological data: histological diagnosis, malignancy grade, gene-molecular profile, radiological pictures, surgical resection and clinical findings (age, tumor location, "performance status").

With recent progress in radiological, pathological, immunohistochemical, molecular and genetic diagnoses, the characterisation of brain tumours has improved. The last World Health Organization (WHO) Classification of Tumours of the Central Nervous System was done in 2007, based on morphological features, growth pattern and molecular profile of neoplastic cells, defined malignancy grade. The neuropathological diagnosis and the grading of each histotype are based on identification of histopathological criteria and immunohistochemical data. Molecular and genetic profiles may identify different tumour subtypes varying in biological and clinical behaviour, indicating prognostic and predictive factors. In order to investigate new therapeutic approaches, it is important to study the molecular pathways responsible for proliferation, invasion, angiogenesis, and anaplastic transformation. Different prognostic and predictive factors for glioma patients were identified by genetic studies, such as the loss of heterozygosis on chromosome 1p and 19q for oligodendrogliomas, proangiogenic factors such as Vascular Endothelial Growth Factor for glioblastomas and the methylation status of gene promoter of MethylGuanine-MethylTransferase. In conclusion, the prognostic evaluation and the therapeutic strategies for patients depend on the synthesis of histological diagnosis, malignancy grade, gene-molecular profile, radiological images, surgical resection and clinical findings (age, tumour location, and "performance status").

Gliomas are very invasive brain tumors with poor prognosis and therefore any attempt to limit tumor cell dissemination in the brain is expected to improve glioma treatment. The recent deorphanization of CXCR7 as additional receptor for CXCL12 and CXCL11 has raised key issues on its interaction with the CXCL12/CXCR4 axis as a mechanism to modulate glioma cell migration. In this work we investigated protein and mRNA expression of the two chemokines CXCL12 and CXCL11, together with their receptors CXCR4 and CXCR7 in human glioma specimens and cell lines by immunohistochemistry, flow cytometry and quantitative real-time PCR. The main purpose of this study was to find out whether and at what extent CXCR4 and CXCR7 are differentially expressed in glioma cells. In human glioma specimens the levels of CXCL11 and CXCR4 mRNA were significantly higher in glioblastomas compared to non-tumor controls or low grade gliomas, whilst no difference was found for CXCL12 and CXCR7 mRNA expression. In cell lines, flow cytometry and immunocytochemical experiments showed CXCR4 was mainly expressed irrespective of its membrane or intracellular localization. In contrast, a predominant intracellular localization together with a negligible membrane expression of CXCR7 was found in all cells examined. In in vitro experiments CXCR4 and CXCR7 antagonists and the silencing of CXCR4 showed complete inhibition of glioma proliferation. Our findings, in agreement with previous data, suggest that in human glioma cells the prevalent intracellular localization of CXCR7 might modulate the functionality of CXCL11/12 either acting as a scavenger for these chemokines or interfering with the signaling pathways activated by the stimulation of CXCR4.

Brain tumour oedema is coupled with blood-brain barrier damage and alteration in water flow. Aquaporin-4 (AQP4) is involved in the development and resolution of brain oedema, and it is strongly upregulated in glioblastoma multiforme (GBM). Here, we evaluated AQP4 expression and content in GBM and correlated with VEGF-VEGFR-2 expression. In the relapse after chemotherapy and radiotherapy, AQP4 content reduced in parallel with VEGF-VEGFR-2, as compared with primary tumours, and in the peripheral areas of relapsed tumours AQP4 mimicked normal findings of perivascular rearrangement. After immunogold electron microscopy, gold particles were attached on the glial membrane facing the perivascular side, likewise AQP4 gold labelling of the vessels of the control areas. In primary tumours the peripheral vessels appeared faintly marked by AQP4, while the perivascular tumour cells showed a strong expression. The vasculature of the inner tumour areas was unlabelled by AQP4, while tumour cells were labelled, in both primary and relapsing tumours. Relapsed tumours after radiotherapy alone showed slight AQP4 reduction and perivascular restoring in the peripheral areas of the tumour. These data indicate that in GBM chemotherapy and radiotherapy induce a down-regulation in AQP4 expression restoring its perivascular rearrangement suggesting its potential role in the resolution of brain oedema.

Brain metastases occur in about 25% of patients who die of cancer. The most common sources of brain metastases in adults are lung, breast, kidney, colorectal cancer and melanoma. The chemokine/receptor system CXCL12/CXCR4 plays a key role in multiple biological functions; among these, homing of neoplastic cells from the primary site to the target and metastasis progression. Recently, an alternative CXCL12 receptor CXCR7 has been discovered. The aim of our study was to investigate the expression of CXCL12 and its receptors CXCR4 and CXCR7 by immunohistochemistry in 56 patients with metastatic brain disease from different non-CNS primary tumors and evaluate their prognostic relevance as well as that of other patient/treatment-related features on patient survival. CXCL12 showed an expression in tumor cells and in tumor vessels; CXCR7 was expressed by tumor and endothelial cells (both within the tumor and in the adjacent brain tissue), while CXCR4 showed a positivity in all samples with a nuclear pattern. Among the investigated immunohistochemical parameters, only CXCL12 expression in tumor endothelial cells showed a statistically significant correlation with shorter survival (p = 0.04 log-rank), perhaps identifying more aggressive tumors. Thus, this is the first study evaluating at the same time the expression of CXCL12 and its two receptors in a cohort of brain metastases.

Purpose: The hypothesis addressed by this study is that a glutamine synthetase (GS) deficiency in neoplastic astrocytes is a possible molecular basis associated with seizure generation in glioblastoma multiforme (GBM)., Methods: Quantitative Western blot analysis of GS was performed in 20 individuals operated for malignant glioma., Results: The levels of GS in patients with GBM and epilepsy were significantly lower (range 0.04-1.15; mean 0.35 +/- 0.36; median 0.25) than in non-epileptic GBM individuals (range 0.78-3.97; mean 1.64 +/- 0.99; median 1.25; P = 0.002). No relationship has been found between histological features (i.e. necrosis, gliosis, stroma, inflammatory cells, giant cells, and haemosiderine) and GS expression or epilepsy., Discussion: Even though the epileptogenesis in glioma is multifactorial, it is conceivable that a down-regulation of GS may have an important pro-epileptogenic role in GBM, through the slowing of glutamate-glutamine cycle. This study suggests that seizures in GBM are coupled with a highly localized enzyme deficiency. The manipulation of GS activity might constitute a novel principle for inhibiting seizures in patients with glioma epilepsy.

Purpose: A prospective study of patients with glioma was carried out. Special attention was paid to the first seizure, to the histology, and to the efficacy and prophylactic role of antiepileptic drugs (AEDs). Prognostic factors were analyzed., Methods: Between February 1st 2004 and April 1st 2006 patients who underwent surgical treatment at the Neurosurgery Department of Bolzano for primary or recurrent glioma were prospectively followed until April 1st 2007. Seizures at onset occurring in the absence of interictal epileptiform abnormalities were regarded as remote symptomatic seizures and not treated., Results: Sixty-four individuals were registered in the study; epilepsy diagnosis was made in 27 cases and in 24 of these seizures were the onset symptom. The correlation with histological grading showed that seizures were more frequent in low-grade glioma and in secondary glioblastoma. Although epilepsy was less frequent in high-grade glioma, in these patients seizures were more difficult to control. Poor seizure control was associated with motor and sensitive focal seizures and presence of neurological deficit. Patients without epilepsy and not taking AEDs never developed seizures during the follow-up., Discussion: Our study clearly shows that epilepsy is more frequent in low-grade gliomas but seizures are more difficult to control in high-grade gliomas. In both cases seizures are a quite exclusive symptom at the onset that never appears during the stable course of the disease. Amongst glioblastoma multiforme (GBM), epilepsy is more frequent in GBM developing through progression from low-grade astrocitoma. Moreover, our study strongly indicates that the prophylactic use of AEDs in glioma is not justified.

Glioblastomas (GBMs) contain transformed, self-maintaining, multipotent, tumour-initiating cancer stem cells, whose identification has radically changed our perspective on the physiology of these tumours. Currently, it is unknown whether multiple types of transformed precursors, which display alternative sets of the complement of properties of true cancer stem cells, can be found in a GBM. If different subsets of such cancer stem-like cells (CSCs) do exist, they might represent distinct cell targets, with a differential therapeutic importance, also depending on their characteristics and lineage relationship. Here, we report the presence of two types of CSCs within different regions of the same human GBM. Cytogenetic and molecular analysis shows that the two types of CSCs bear quite diverse tumorigenic potential and distinct genetic anomalies, and, yet, derive from common ancestor cells. This provides critical information to unravel the development of CSCs and the key molecular/genetic components underpinning tumorigenicity in human GBMs.

Intracerebral haemorrhage may both be a presenting manifestation in unrecognised brain tumour or--more frequently--take place in the disease course of known/suspected brain tumour due to diagnostic/therapeutic procedures, including biopsy, locoregional treatments and anti-angiogenic therapies. Apart from the difficulties inherent to accurate neuroradiological diagnosis in selected cases with small tumour volume, the main clinical problem that neurologists face is represented by decision making in prophylaxis/treatment of venous thromboembolism in these patients. These points are briefly discussed and available evidence on the last point is commented on.

Because of their clinical and neuroradiological features, tumefactive demyelinating lesions, or giant plaques, are easily mistaken for tumors, with a consequent risk of gross errors in the choice of treatment. This article reports a 10-year-old girl who underwent surgery for a left parietal lesion misinterpreted as a glioblastoma which subsequently proved to be a case of giant plaque.

Objective: Enterogenous cysts (ECs) of the central nervous system are developmental malformations that occur in the spinal canal, posterior fossa, or cerebral hemispheres. They are usually benign lesions, and malignant transformation is rare. To date, only three cases of malignant transformation have been reported in the literature. We present a case of a cerebellopontine EC showing foci of epithelial dysplasia and malignant transformation into a low-grade papillary mucinous adenocarcinoma., Clinical Presentation: A 25 year-old man with a 6-year history of hypoacusia presented to our department with facial nerve deficit, visual disturbances, and gait instability. A magnetic resonance imaging scan demonstrated a multiloculated cerebellopontine angle cyst with supratentorial hydrocephalus., Intervention: A retrosigmoidal approach was used to achieve cyst removal. This was followed several months later by ventriculoperitoneal shunt placement. The cyst was adherent to the brainstem, cranial nerves, and vessels, and it resembled a thin encapsulated structure filled with mucinous-like substance. No solid component was identified. Histopathological examination revealed an EC with foci of malignant transformation in a mucinous papillary adenocarcinoma. Magnetic resonance imaging was performed 5 months postoperatively due to progressive clinical worsening; this scan revealed lesion recurrence with severe brainstem compression. Emergency surgery was performed, and a large decompression was achieved. Subsequent follow-up computed tomographic scans showed progression of the residual tumor. The patient's neurological condition rapidly worsened, ultimately resulting in death., Conclusion: The present report suggests that a careful histological examination of all ECs after surgery should be made to exclude dysplastic foci or carcinomatous transformation. Although the clinical behavior of ECs with malignant transformation is unpredictable, surgery remains the treatment of choice. The use of possible adjuvant chemo- or radiotherapy has not been established.

Reliable data on large cohorts of patients with glioblastoma are needed because such studies differ importantly from trials that have a strong bias toward the recruitment of younger patients with a higher performance status. We analyzed the outcome of 676 patients with histologically confirmed newly diagnosed glioblastoma who were treated consecutively at a single institution over a 7-year period (1997-2003) with follow-up to April 30, 2006. Survival probabilities were 57% at 1 year, 16% at 2 years, and 7% at 3 years. Progression-free survival was 15% at 1 year. Prolongation of survival was significantly associated with surgery in patients with a good performance status, whatever the patient's age, with an adjusted hazard ratio of 0.55 (p < 0.001) or a 45% relative decrease in the risk of death. Radiotherapy and chemotherapy improved survival, with adjusted hazard ratios of 0.61 (p = 0.001) and 0.89 (p = 0.04), respectively, regardless of age, performance status, or residual tumor volume. Recurrence occurred in 99% of patients throughout the follow-up. Reoperation was performed in one-fourth of these patients but was not effective, whether performed within 9 months (hazard ratio, 0.86; p = 0.256) or after 9 months (hazard ratio, 0.98; p = 0.860) of initial surgery, whereas second-line chemotherapy with procarbazine, lomustine, and vincristine (PCV) or with temozolomide improved survival (hazard ratio, 0.77; p = 0.008). Surgery followed by radiotherapy and chemotherapy should be considered in all patients with glioblastoma, and these treatments should not be withheld because of increasing age alone. The benefit of second surgery at recurrence is uncertain, and new trials are needed to assess its effectiveness. Chemotherapy with PCV or temozolomide seems to be a reasonable option at tumor recurrence.

Recent studies have shown an anti-tumour activity of cannabinoid receptors CB1 and CB2 in gliomas. This effect was mediated by neurotrophins in breast and prostate carcinoma, while in gliomas this relationship has not yet been considered. The aim of this study was to investigate the expression of cannabinoid receptors CB1 and CB2, neurotrophin NGF and NT-3 and their receptors TrkA and TrkC in glioma and endothelial cells. The analysis was performed in 14 gliomas and 2 non-tumour brain specimens by immunohistochemistry and real-time quantitative-polymerase chain reaction (RTQ-PCR). Gliomas showed a weak immunoreactivity for CB1 and CB2 in tumour and in endothelial cells, and for NGF/TrkA mainly in tumour cells, while a moderate/diffuse immunoreactivity was found for NT-3/TrkC. CB2 was expressed on 3 out of 6 low-grade gliomas and in all high-grade gliomas. Non-tumour brain tissues were weakly positive in astrocytes and endothelium for CB1, CB2, NT-3 and TrkC and negative for NGF and TrkA. By RTQ-PCR, gliomas showed low mRNA levels of NGF/TrkA and moderate levels of CB1, NT-3 and TrkC. CB2 mRNA expression was low or absent. A potential role of cannabinoids, particularly of CB2 agonists devoid of psychotropic side effects, in glioma therapy could have a basis in glioblastomas, because they were all positive, though weakly, to CB2. The presence of neurotrophins and their receptors, mainly NT-3 and TrkC, suggests a possible role of these pathways in glioma growth/invasion, but further investigations are required to verify this hypothesis and a potential relationship between cannabinoids and neurotrophins.

Angiogenesis is a key event in the natural progression of gliomas. Nestin, a marker for multipotential neuroepithelial stem cells, is detected in neuroepithelial tumors and in proliferating endothelial cells (ECs) and is involved in the early stages of lineage commitment, proliferation and differentiation. Nestin expression is correlated with proangiogenic chemokines (CXCL12 and its receptor CXCR4) and growth factors (VEGF, PDGF-B and its receptor PDGFRbeta). VEGF expression upregulates CXCR4 on endothelial cells, binding the chemokine SDF1/CXCL12 (Stromal Derived Factor) that has a role on angiogenesis and chemotaxis of endothelial cells; PDGF (platelet-derived growth factor) and PDGFRbeta are also crucial by increasing the expression of VEGF. We performed a retrospective study on the presence and role of nestin-expressing cells in 102 patients with glioma, relating the findings to VEGF, CXCL12, PDGFRbeta expression and to clinical outcome (time to tumor progression-TTP and survival time-ST). Our results suggest that in gliomas the detection of proliferating ECs expressing nestin correlates to histological malignancy grade and clinical outcome. Also, the expression of CXCL12 in low-grade gliomas was the only factor associated with a significantly shorter TTP, suggesting a role of this chemokine in angiogenic shift and/or disease progression.

Purpose: Recent data suggest that methylation of the DNA repair gene O(6)-methylguanine DNA methyltransferase (MGMT), by increasing the chemosensitivity of glioblastoma multiforme, is significantly associated with improved prognosis. Results in contradiction with these findings, however, are present in the literature and the clinical and genetic context framing MGMT methylation is poorly characterized., Experimental Design: To address these issues, we have investigated the MGMT methylation status, clinical and magnetic resonance imaging characteristics, and relevant genetic features (loss of heterozygosity on 17p and 19q, EGFR amplification, and p53 mutations) in a retrospective study on 86 patients affected by glioblastoma multiforme: 72 patients had a clinical history indicating de novo insurgence of the tumor and the remaining 14 were secondary glioblastoma multiforme., Results: MGMT methylation was detected by methylation-specific PCR in 41 of 86 cases (47.7%; Meth+). Progression-free survival and overall survival were significantly longer in Meth+ than in Meth- patients [10 versus 7 months (P=0.003, log-rank test) and 18 versus 14 months (P=0.0003, log-rank test), respectively]. Mixed-nodular enhancement at magnetic resonance imaging was significantly more frequent in Meth+ and secondary glioblastoma multiforme and ring enhancement in Meth- and primary glioblastoma multiforme (P<0.005). MGMT methylation was more present in secondary glioblastoma multiforme (P=0.006) and associated with loss of heterozygosity on 17p and/or 19q (P=0.005)., Conclusions: These observations suggest that MGMT methylation is part of a genetic signature of glioblastomas that developed from lower-grade gliomas.

Both clinical and biological features have been reported as prognostic factors in low-grade gliomas. Among these, histotype, tumor size, enhancement, age and genetic pattern. Microvessel density (MVD) has been correlated to clinical outcome in astrocytomas, but its impact in oligodendrogliomas and mixed tumors is not sure. The pro-angiogenic chemokine stromal cell-derived factor (SDF-1/CXCL12) and its receptor CXC chemokine receptor 4 (CXCR4) have been described in low-grade gliomas, with a correlation between CXCL12 expression and shorter time to progression (TTP). The intermediate filament Nestin is expressed in proliferating vessels. Platelet-derived growth factor B (PDGF-B) and its receptor PDGFR-beta are also involved in angiogenesis and malignant progression in gliomas.