Search

Glioblastoma multiforme (GBM) is considered the most malignant form of primary brain tumor. Despite multimodal treatment, prognosis remains poor. Ketogenic diet (KD) has been suggested for the treatment of GBM. In this study, the syngenic, orthotopic GL261 mouse glioma model was used to evaluate the effects of KD on the metabolic responses of the tumor using 7T magnetic resonance imaging/spectroscopy. GL261 cells were injected into the caudate nucleus of mice. Following implantation, animals were fed with standard chow or underwent a KD. 18 days after initiating the diet, mice fed with KD displayed significantly higher plasmatic levels of ketone bodies and survived longer than those fed with the standard diet. Decreased concentrations of gamma-aminobutyric acid, N-Acetyl-Aspartate and N-acetylaspartylglutamate were found in tumor tissue after 9 days into the KD, while a huge increase in beta-hydroxybutyrate (bHB) was detected in tumor tissue as compared to normal brain. The accumulation of bHB in the tumor tissue in mice undergoing the KD, may suggest either elevated uptake/release of bHB by tumor cells, or the inability of tumor cells in this context to use it for mitochondrial metabolism.

The presence of redundant copy number variants (CNVs) in groups of patients with neurological diseases suggests that these variants could have pathogenic effect. We have collected array comparative genomic hybridization (CGH) data of about 2,500 patients affected by neurocognitive disorders and we observed that CNVs in 2p16.3 locus were as frequent as those in 15q11.2, being both the most frequent unbalances in our cohort of patients. Focusing to 2p16.3 region, unbalances involving NRXN1 coding region have been already associated with neuropsychiatric disorders, although with incomplete penetrance, but little is known about CNVs located proximal to the gene, in the long noncoding RNA AK127244. We found that, in our cohort of patients with neuropsychiatric disorders, the frequency of CNVs involving AK127244 was comparable to that of NRXN1 gene. Patients carrying 2p16.3 unbalances shared some common clinical characteristics regardless NRXN1 and AK127244 CNVs localization, suggesting that the AK127244 long noncoding RNA could be involved in neurocognitive disease with the same effect of NRXN1 unbalances. AK127244 as well as NRXN1 unbalances seem to have a particular influence on language development, behavior or mood, according with the topographic correlation between NRXN1 expression and prefrontal cortex functions.

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.

We developed anin vitrocontact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 μm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across thein vitroBBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM,αvβ3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

Background Epilepsy is a frequent symptom in patients with glioma. Although treatment with antiepileptic drugs is generally effective in controlling seizures, drug-resistant patients are not uncommon. Multidrug resistance proteins (MRPs) and P-gp are over-represented in brain tissue of patients with drug-resistant epilepsy, suggesting their involvement in the clearance of antiepileptic medications. In addition to their anticonvulsant action, some drugs have been documented for cytotoxic effects. Aim of this study was to evaluate possible in vitro cytotoxic effects of two new-generation antiepileptic drugs on a human glioma cell line U87MG. Methods Cytotoxicity of brivaracetam and lacosamide was tested on U87MG, SW1783 and T98G by MTS assay. Expression of chemoresistance molecules was evaluated using flow cytometry in U87MG and human umbilical vein endothelial cells (HUVECs). To investigate the putative anti-proliferative effect, apoptosis assay, microRNA expression profile and study of cell cycle were performed. Results Brivaracetam and lacosamide showed a dose-dependent cytotoxic and anti-migratory effects. Cytotoxicity was not related to apoptosis. The exposure of glioma cells to brivaracetam and lacosamide resulted in the modulation of several microRNAs; particularly, the effect of miR-195-5p modulation seemed to affect cell cycle, while miR-107 seemed to be implicated in the inhibition of cells migration. Moreover, brivaracetam and lacosamide treatment did not modulate the expression of chemoresistance-related molecules MRPs1-3-5, GSTπ, P-gp on U87MG and HUVECs. Conclusion Based on antineoplastic effect of brivaracetam and lacosamide on glioma cells, we assume that patients with glioma could benefit by the treatment with these two molecules, in addition to standard therapeutic options. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0546-9) contains supplementary material, which is available to authorized users.

5q14.3 deletions including the MEF2C gene have been identified to date using genomic arrays in patients with severe developmental delay or intellectual disability, stereotypic behavior, epilepsy, cerebral malformations and a facial gestalt not really distinctive though characterized by broad and/or high, bulging forehead, upslanting palpebral fissures, flat nasal root and bridge, small, upturned nose, hypotonic small mouth resulting in cupid bow/tented upper lip. MEF2C mutations have been also identified in patients with overlapping phenotype so that it is considered the gene responsible for the 5q14.3 deletion syndrome. To date, one single duplication including MEF2C has been reported in a patient with intellectual disability but its clinical significance remains uncertain also because of the large size of the imbalance. Here we present two further patients with 5q14.3 duplications including MEF2C. Their phenotype indeed suggest the pathogenic effect of the MEF2C duplication although other duplicated genes also brain expressed might contribute to the clinical features. In none of them a clear-cut syndrome can be identified. A comparison between MEF2C deleted/mutated and duplicated patients is also presented.

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.

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.

RATIONAL AND AIMS: Epilepsy is a frequent side effect in patients with glioma. Although treatment with antiepileptic drugs is generally effective in controlling seizures, drug-resistant patient are not uncommon. Multidrug resistance proteins (MRPs) and P-gp are over-represented in brain tissue of drug-resistant epilepsy affected patients suggesting their involvement in the clearance of antiepileptic medications. In addition to the action anticonvulsant drugs has been documented for some a possible cytotoxic effect. METHODS: The in vitro effects of two new generation antiepileptic drugs, Brivaracetam and Lacosamide, were evaluated on two lines of human glioblastoma (A172 and U87). RESULTS: The results show a cytotoxic effect of Brivaracetam and Lacosamide on the cell line U87. However, the cytotoxic mechanism does not appear to be related to apoptosis, evaluated by annexin V binding. Moreover, by flow cytometric analysis, we observed that exposure of the two cell lines to Brivaracetam or Lacosamide at their IC30 does not contribute to the modulation of the chemoresistance molecules MRP1, MRP3, MRP5, GSTπ and P-Gp. Similar results were obtained by evaluating the modulation of the same molecules on human umbilical vein endothelial cells (HUVEC) after exposure to the two drugs. CONCLUSIONS: The data obtained suggest that Brivaracetam and Lacosamide in vitro play a cytotoxic effect on human glioma cells.