Identification, validation and biological characterisation of novel glioblastoma tumour microenvironment subtypes: implications for precision immunotherapy.

BACKGROUND: New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles have failed to direct treatment strategies. We hypothesised that interrogation of the GBM tumour microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision immunotherapy treatment strategies. MATERIALS AND METHODS: A refined and validated microenvironment cell population (MCP) counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition around medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified three novel patient clusters, uniquely characterised by TME composition, functional orientation markers and immune checkpoint proteins. Validation was carried out in three independent GBM-RNA-seq datasets. Neoantigen, mutational and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal Glioma Longitudinal AnalySiS (GLASS) cohort and three immunotherapy clinical trial cohorts [treatment with neoadjuvant/adjuvant anti-programmed cell death protein 1 (PD-1) or PSVRIPO] were further interrogated to assess subtype alterations between primary and recurrent tumours and to assess the utility of TME classifiers as immunotherapy biomarkers. RESULTS: TMEHigh tumours (30%) displayed elevated lymphocyte, myeloid cell immune checkpoint, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 transcripts. TMEHigh/mesenchymal+ patients featured tertiary lymphoid structures. TMEMed (46%) tumours were enriched for endothelial cell gene expression profiles and displayed heterogeneous immune populations. TMELow (24%) tumours were manifest as an 'immune-desert' group. TME subtype transitions upon recurrence were identified in the longitudinal GLASS cohort. Assessment of GBM immunotherapy trial datasets revealed that TMEHigh patients receiving neoadjuvant anti-PD-1 had significantly increased overall survival (P = 0.04). Moreover, TMEHigh patients treated with adjuvant anti-PD-1 or oncolytic virus (PVSRIPO) showed a trend towards improved survival. CONCLUSIONS: We have established a novel TME-based classification system for application in intracranial malignancies. TME subtypes represent canonical 'termini a quo' (starting points) to support an improved precision immunotherapy treatment approach.

Treatment of Nonfunctional Pituitary Adenoma Postoperative Remnants: Adjuvant or Delayed Gamma Knife Radiosurgery?

OBJECTIVE: It is still not clear whether Gamma Knife radiosurgery (GKRS) for nonfunctional pituitary adenomas should be used as a standard adjuvant postoperative therapy or applied when there is documented progression of the remnant on follow-up magnetic resonance imaging. METHODS: We performed a retrospective study of patients with nonfunctional pituitary adenomas who underwent primary surgery and GKRS between 2002 and 2015. Patients were divided into 2 groups on the basis of the GKRS indication: adjuvant treatment (GKRS ≤6 months postoperatively) or delayed treatment (GKRS if documented progression occurred on the follow-up magnetic resonance imaging). RESULTS: Fifty patients were included and grouped based on adjuvant (n = 13) or delayed (n = 37) GKRS following primary surgery. The adjuvant and delayed groups had 10-year actuarial tumor control rates of 92% and 96% (P = 0.408), respectively. The 10-year actuarial endocrinologic control rate was 82% for the adjuvant group and 49% for the delayed group (P = 0.597). None of the patients developed any new neurologic deficit post-GKRS. GKRS-induced complications (intratumoral bleeding and tumoral tissue inflammation) occurred in 6% of the patients, of whom 4% were in the delayed group and 2% in the adjuvant group. CONCLUSION: Adjuvant treatment with GKRS yields the same high long-term tumor control as delayed GKRS. Neither adjuvant nor delayed GKRS induced additional neurologic complications. There is a trend that adjuvant GKRS induces less additional endocrinologic deficits compared with delayed GKRS.

A Systematic Comparison Identifies an ATP-Based Viability Assay as Most Suitable Read-Out for Drug Screening in Glioma Stem-Like Cells.

Serum-free culture methods for patient-derived primary glioma cultures, selecting for glioma stem-like cells (GSCs), are becoming the gold standard in neurooncology research. These GSCs can be implemented in drug screens to detect patient-specific responses, potentially bridging the translational gap to personalized medicine. Since numerous compounds are available, a rapid and reliable readout for drug efficacies is required. This can be done using approaches that measure viability, confluency, cytotoxicity, or apoptosis. To determine which assay is best suitable for drug screening, 10 different assays were systematically tested on established glioma cell lines and validated on a panel of GSCs. General applicability was assessed using distinct treatment modalities, being temozolomide, radiation, rapamycin, and the oncolytic adenovirus Delta24-RGD. The apoptosis and cytotoxicity assays did not unequivocally detect responses and were excluded from further testing. The NADH- and ATP-based viability assays revealed comparable readout for all treatments; however, the latter had smaller standard deviations and direct readout. Importantly, drugs that interfere with cell metabolism require alternative techniques such as confluency monitoring to accurately measure treatment effects. Taken together, our data suggest that the combination of ATP luminescence assays with confluency monitoring provides the most specific and reproducible readout for drug screening on primary GSCs.

In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide.

BACKGROUND: The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. METHODS: Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. RESULTS: The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). CONCLUSION: The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell lines.

Late neurocognitive sequelae in patients with WHO grade I meningioma.

BACKGROUND: Information on neurocognitive outcome following treatment of benign meningiomas is virtually lacking. This is remarkable considering that survival in these patients is the most favourable of all intracranial tumours. The aim of the present study was therefore to document the extent and nature of neurocognitive deficits in patients with World Health Organization (WHO) grade I meningioma after treatment. METHODS: 89 patients with WHO grade I meningioma who underwent surgery with or without adjuvant radiotherapy were individually matched to 89 healthy controls for age, sex and educational level. Neurocognitive functioning of patients was assessed at least 1 year following treatment and compared with that of healthy controls using the Student's t test. Additionally, associations between tumour characteristics (size, lateralisation and localisation), treatment characteristics (radiotherapy) and epilepsy burden (based on seizure frequency and antiepileptic drug use) and neurocognitive functioning were investigated. RESULTS: Compared with healthy controls, patients with meningioma showed significant impairments in executive functioning (p<0.001), verbal memory (p<0.001), information processing capacity (p = 0.001), psychomotor speed (p = 0.001) and working memory (p = 0.006). Patients with skull base meningiomas performed significantly lower on three out of six neurocognitive domains compared with convexity meningiomas. Left-sided as opposed to right-sided meningiomas were related to verbal memory deficits. A higher epilepsy burden was significantly associated with lower executive functioning which primarily could be attributed to antiepileptic drug use. No significant associations were established between neurocognitive status and radiotherapy or tumour volume. CONCLUSIONS: Meningioma patients are characterised by long term deficits in neurocognitive functioning that can partly be attributed to the use of antiepileptic drugs and tumour location but not to the use of radiotherapy.

AKT1(E17K) in human solid tumours.

The serine-threonine kinase AKT1 is a central player in the oncogenic pathway controlled by PI3K. Recently, a somatic mutation in AKT1 (E17K) has been detected in breast, colorectal, lung and ovarian cancers. The E17K change results in constitutive AKT1 activation and induces leukaemia in mice. We determined the occurrence of the E17K variant in a panel of 764 tumour samples. These included breast, lung, ovarian, colorectal and pancreatic carcinomas as well as melanomas and glioblastomas. Despite the fact that these tumours are known to bear alterations in genes involved in the PI3K signalling pathway, AKT1(E17K) was detected only in breast (16/273), colorectal (1/88) and lung (1/155) cancers. Within the neoplasms of breast origin, the AKT1(E17K) variant was mutually exclusive with respect to the PIK3CA(E454K or H1047R) alleles and was present only in ductal and lobular histotypes. Our results, showing that AKT1 mutations seem to occur in a tissue-specific fashion have basic and clinical implications. First, the activity of mutated AKT1 in oncogenic PI3K signalling could be strictly dependent on the cell and tissue milieu. Second, therapeutic efforts aimed at selective targeting the AKT1(E17K) variant could be effective mainly in specific cancer types.

The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids.

Screening of therapeutics relies on representative cancer models. The representation of human glioblastoma by in vitro cell culture models is questionable. We obtained genomic profiles by array comparative genomic hybridization of both short- and long-term primary cell and spheroid cultures, derived from seven glioblastomas and one anaplastic oligodendroglioma. Chromosomal copy numbers were compared between cell cultures and spheroids and related to the parental gliomas using unsupervised hierarchical clustering and correlation coefficient. In seven out of eight short-term cell cultures, the genomic profiles clustered further apart from their parental tumors than spheroid cultures. In four out of eight samples, the genetic changes in cell culture were substantial. The average correlation coefficient between parental tumors and spheroid profiles was 0.89 (range: 0.79-0.97), whereas that between parental tumors and cell cultures was 0.62 (range: 0.10-0.96). In two out of three long-term cell cultures progressive genetic changes had developed, whereas the spheroid cultures were genetically stable. It is concluded that genomic profiles of primary cell cultures from glioblastoma are frequently deviant from parental tumor profiles, whereas spheroids are genetically more representative of the glioblastoma. This implies that glioma cell culture data have to be handled with the highest caution.

Local recurrence and distant metastasis of supratentorial primitive neuro-ectodermal tumor in an adult patient successfully treated with intensive induction chemotherapy and maintenance temozolomide.

Supratentorial primitive neuro-ectodermal tumors (PNET) in adults are very rare. Extraneural metastasis are unusual and the optimal palliative chemotherapy regimen is not established. We present a 26-year-old patient with local recurrence and distant metastasis of supratentorial PNET successfully treated with intensive induction chemotherapy and maintenance temozolomide.

Distribution, characterization and clinical significance of microglia in glioneuronal tumours from patients with chronic intractable epilepsy.

Cells of the microglia/macrophage lineage represent an important component of different brain tumours. However, there is little information about the microglia/macrophage cell system in glioneuronal tumours and its possible contribution to the high epileptogenecity of these lesions. In the present study, the distribution of cells of the microglia/macrophage lineage was studied by immunocytochemistry for CD68 and human leucocyte antigen (HLA)-DR in a group of glioneuronal tumours, including gangliogliomas (GG, n = 30), and dysembryoplastic neuroepithelial tumours (DNT, n = 17), from patients with chronic intractable epilepsy. A significant number of microglia/macrophage cells were observed in the large majority of glioneuronal tumours, both within the tumour and in the peritumoral region. Activated microglial cells positive for HLA-DR were localized around blood vessels and clustered around tumour neuronal cells. The density of activated microglial cells correlated with the duration of epilepsy, as well as with the frequency of seizures prior to surgical resection. These observations indicate that the presence of cells of the microglial/macrophage cell system is a feature of glioneuronal tumours and is functionally related to epilepsy, either directly in epileptogenesis or through activation following seizure activity.

Molecular-genetic characterisation of gliomas that recur as same grade or higher grade tumours.

BACKGROUND: Due to their invasive growth, gliomas usually cannot be removed completely and almost always recur as same grade or higher grade malignancies. OBJECTIVE: To determine whether there were differences in the accumulation of genetic changes between the two types of glioma recurrence. METHODS: We genetically characterised 14 cases of lower grade glioma with a same grade recurrence, 12 cases of glioblastoma recurrence, and 14 cases of lower grade glioma with a higher grade recurrence. We investigated LOH (loss of heterozygosity) at 1p36, 10p15, the PTEN region in 10q23, the DMBT1 region in 10q25, 19q13, 22q13, LOH and mutation of TP53, and EGFR amplification. RESULTS: Genetic heterogeneity in the primary tumour was inferred in 3 cases of lower grade glioma with a higher grade recurrence. The cases of lower grade glioma with a higher grade recurrence displayed increased genetic instability in the recurrence (mean of 2.0 additional genetic changes per case) compared to cases with a same lower grade recurrence or those with a glioblastoma recurrence (mean of 0.6 and 0.8 additional changes per case, respectively). Compared to unselected primary glioblastomas, the glioblastomas that recurred as an operable tumour had infrequent EGFR amplification (8% v 30-40% of cases). CONCLUSIONS: Gliomas recurring as higher grade lesions might be genetically heterogeneous and accumulate more genetic changes than gliomas recurring as same grade lesions (whether originally low or high grade). Primary glioblastomas from patients for which the recurrence is operated because of prognostically more favourable clinical indices have infrequent EGFR amplification.

Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors.

The cell-specific distribution of multidrug resistance extrusion pumps was studied in developmental glioneuronal lesions, including focal cortical dysplasia (15 cases) and ganglioglioma (15 cases) from patients with medically intractable epilepsy. Lesional, perilesional, as well as normal brain regions were examined for the expression of the multidrug resistance gene 1 encoded P-glycoprotein (P-gp) and the multidrug resistance-associated protein 1 (MRP1) by immunocytochemistry. In normal brain MRP1 expression was below detection, whereas P-gp staining was present only in blood vessels. MRP1 and P-gp immunoreactivity was observed in dysplastic neurons of 11/15 cases of focal cortical dysplasia, as well as in the neuronal component of 14/15 ganglioglioma. Glial cells with astrocytic morphology within the lesion showed multidrug-resistant protein immunoreactivity (P-gp>MRP1). Moderate to strong MRP1 and P-gp immunoreactivity was observed in a population of large ballooned neuroglial cells. P-gp appeared to be most frequently expressed in glial fibrillary acidic protein-positive balloon cells (glial type), whereas MRP1 was more frequently expressed in microtubule-associated protein 2-positive balloon cells (neuronal type). In both types of lesions strong P-gp immunoreactivity was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal cells compared with normal cortex. The predominant intralesional cell-specific distribution of multidrug transporter proteins supports the hypothesis of a constitutive overexpression as common mechanism underlying the intrinsic pharmaco-resistance to antiepileptic drugs of both malformative and neoplastic glioneuronal developmental lesions.

The effects of irradiation on cell migration from glioblastoma multiforme biopsy spheroids.

BACKGROUND: Glioblastoma multiforme (GBM) is a primary brain tumour with a very poor prognosis despite aggressive multi-modality treatment. This pre-clinical experimental study focuses on the effect of irradiation on three-dimensional glioma biopsy spheroids in vitro using an outgrowth assay to evaluate cell survival and migrational capacity of the glioma cells. MATERIALS AND METHODS: Tumour tissue of 16 patients with high-grade glioma and two GBM cell lines were used for spheroid preparation. Outgrowth and cell density were the parameters chosen to evaluate cell cytotoxicity and migrational capacity after irradiation (20 Gy and 4 x 5 Gy). RESULTS: Radiation inhibited outgrowth of cell line spheroids, but not of the biopsy spheroids. All biopsy and cell line spheroids showed a significantly lower cell number (95 vs. 24 cells/0.25 mm2) in the outgrowth area after irradiation. CONCLUSION: Irradiation has a cytotoxic effect in GBM biopsy spheroids but it hardly affects cell migration. No correlation was found between patient survival and cell migration nor with cytotoxicity.

Cellular molecular based research.

In recent years significant progress has been made in identifying genetic alterations in glial brain neoplasms. Nowadays, three types of development to glioblastoma multiforme (the most malignant form of primary brain tumours) can be identified using genetic molecular techniques. Moreover, with these techniques patients can be identified who will respond to the treatment with alkylating cytostatic drugs. Future research on the genome level but in particular on the level of gene expression holds promise for better grading systems, tailored treatment based on genetic profiling and new targets for treatment. In this paper the history of genetic research on glioma and the techniques that are used are briefly reviewed.

High concentration of Daunorubicin and Daunorubicinol in human malignant astrocytomas after systemic administration of liposomal Daunorubicin.

The value of chemotherapy in patients with malignant astrocytoma remains controversial. In our laboratories in vitro experiments with organotypic spheroid cultures showed superior effectiveness of anthracyclines. Systemic administration did not provide in therapeutic concentrations so far. Because recent studies on Daunorubicin in liposomes in the treatment of Kaposi sarcoma have shown effectiveness with diminished systemic toxicity, we administered intravenously a single dose of Daunorubicin in liposomes in eight patients at different intervals prior to surgery (12-50 h). In samples taken from tumor, tumor-edge and where possible from adjacent brain, the levels of Daunorubicin and its active metabolite Daunorubicinol were assessed with high performance liquid chromatography. Here we report that high concentrations of Daunorubicin and Daunorubicinol were found in malignant gliomas after systemic administration of liposomal Daunorubicin.

Neurobehavioral status and health-related quality of life in newly diagnosed high-grade glioma patients.

PURPOSE: To evaluate the health-related quality of life (HRQOL) and cognitive functioning of high-grade glioma patients in the postneurosurgical period. PATIENTS AND METHODS: The HRQOL, as assessed by the Short-Form Health Survey-36, tumor-specific symptoms, and objective and subjective neuropsychologic functioning, of 68 newly diagnosed glioma patients were compared with that of 50 patients with non-small-cell lung cancer (NSCLC) and to age- and sex-matched healthy controls. The association between tumor lateralization, extent of resection, and use of medication, and the HRQOL outcomes was also investigated. RESULTS: The HRQOL of the two patient groups was similar but significantly lower than that of the healthy controls. Glioma patients reported significantly more neurologic symptoms and poorer objective and subjective neuropsychologic functioning than the NSCLC patients. Using healthy controls as the reference group, cognitive impairment assessed at the individual patient level was observed in all glioma patients and 52% of the NSCLC patients. Poor performance on timed tasks in the glioma group could be attributed, in large part, to visual and motor deficits. Tumor lateralization was found to affect neuropsychologic functioning in a predictable manner. The extent of resection was not related significantly to neuropsychologic functioning. Corticosteroid use was associated with better recognition memory, whereas antiepileptic drug use was correlated negatively with working memory capacity. CONCLUSION: The general HRQOL of glioma patients is similar to that of patients with NSCLC. However, they suffer from a number of condition-specific neurologic and neuropsychologic problems that have a significant impact on their daily lives in the postsurgical period, before treatment with radiotherapy.

Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumours from patients with intractable epilepsy.

Glioneuronal tumours are an increasingly recognized cause of chronic pharmaco-resistant epilepsy. In the present study the immunocytochemical expression of various glutamate receptor (GluR) subtypes was investigated in 41 gangliogliomas (GG) and 16 dysembryoplastic neuroepithelial tumours (DNT) from patients with intractable epilepsy. Immunocytochemistry with antibodies specific for ionotropic NR1, NR2A/B (NMDA) GluR1, GluR2 (AMPA), GluR5-7 (kainate), and metabotropic mGluR1, mGluR2-3, mGluR5, mGluR7a subtypes demonstrated in both GG and DNT the presence of an highly differentiated neuronal population, containing subunits from each receptor class. More than 50% of tumours contained a high percentage of neuronal cells immunolabelled for NMDA, AMPA and kainate receptor subunits. A high percentage of neurones showed strong expression of NR2A-B, which co-localized with NR1. Group I mGluRs (mGluR1 and mGluR5) were highly represented in the neuronal component of the tumours. Immunolabelling for several GluRs was also present in the glial component. Increased expression of mGluR2-3, mGluR5 and GluR5-7 was observed in reactive astrocytes in the perilesional zone compared to normal cortex. The neurochemical profile of glioneuronal tumours, with high expression of specific GluR subtypes, supports the central role of glutamatergic transmission in the mechanisms underlying the intrinsic and high epileptogenicity of these lesions.

Expression of connexin 43 and connexin 32 gap-junction proteins in epilepsy-associated brain tumors and in the perilesional epileptic cortex.

The expression of the gap-junction proteins connexin (CX) 43 and 32 was evaluated in surgical specimens of brain tumors and perilesional cortex from patients with chronic medically intractable epilepsy. In human normal brain CX32 was expressed in neurons and oligodendrocytes. CX32 immunoreactivity (IR) was observed in the neuronal component of glioneuronal tumors and in all oligodendrogliomas, 50% of which showed strong labeling, independent of the grade of differentiation. CX43, normally expressed in astrocytes, was also detected in most of the human astrocytomas and in the astroglial component of glioneuronal tumors. Whereas most of the low-grade gliomas (>60%) showed strong membranous staining, most high-grade astrocytomas exhibited a reduction of the typical plasma membrane CX43-IR and intracytoplasmic localization. Immunoblot analysis showed different CX43 isoforms in control cortex and in low-grade gliomas. However, only one single isoform (corresponding to the non-phosphorylated form of CX43) appeared to be present in most high-grade gliomas. Increased expression of CX43 protein was present in reactive astrocytes in the epileptic cortex surrounding low-grade tumors as compared to control cortex, indicating the existence of a regulatory pathway involving CX43 in the reorganization of the astrocytic syncytium in regions undergoing reactive gliosis. The high expression of connexin proteins in low-grade tumors and in the peritumoral reactive astrocytes suggests that they could contribute to tumor-related seizures.

GOA, a novel gene encoding a ring finger B-box coiled-coil protein, is overexpressed in astrocytoma.

Serial analysis of gene expression (SAGE) was used to identify a gene named GOA (gene overexpressed in astrocytoma), which codes for a novel Ring finger B-box coiled-coil (RBCC) protein. Northern blot hybridization showed overexpression of GOA in 9 of 10 astrocytomas. Except for kidney, in which high expression was found, expression levels in normal tissues were low and comparable to normal brain. Immunohistochemistry demonstrated presence of GOA, with prominent nuclear staining, in astrocytoma tumor cells and astrocytes of fetal brain, but virtual absence in mature astrocytes. Overexpression was not due to amplification, since amplification of GOA was only found in one of 65 astrocytomas. GOA was localized to 17q24-25, a region that is frequently gained or amplified in a number of other tumor types. GOA contains two LXXLL motifs, which are thought to be important for nuclear receptor binding. Our data suggest an important role of GOA in the process of dedifferentiation that is associated with astrocytoma tumorigenesis and possibly with that of other tumor types as well.

Expression of brain-derived neurotrophic factor and tyrosine kinase B receptor proteins in glioneuronal tumors from patients with intractable epilepsy: colocalization with N-methyl-D-aspartic acid receptor.

Recent evidence suggests that brain-derived neurotrophic factor (BDNF) and its tyrosine kinase B (TrkB) receptor, in addition to promoting neuronal survival and differentiation, modulates synaptic transmission by increasing N-methyl-D-aspartic acid receptor (NMDAR) activity. Overexpression of BDNF may, then, interfere with normal brain function, causing increased excitability. We have examined the immunohistochemical expression of BDNF, full-length TrkB receptor and the NMDAR subunit 1 and subunit 2A/B proteins (NMDAR1 and NMDAR2A/B) in glioneuronal tumors (gangliogliomas, GG, n = 40; dysembryoplastic neuroepithelial tumors, DNT, n = 15), from patients with chronic intractable epilepsy. The great majority of tumors studied were positive for all markers examined, supporting the high level of neurochemical differentiation of these lesions. BDNF and TrkB immunoreactivity (ir) was mainly observed in the neuronal component of the tumors. In GG, more than 90% of tumors contained very intense BDNF-ir ganglion cells. Double labeling confirmed the presence of BDNF-ir and TrkB-ir in neurons which contained NMDAR1. NMDAR2A/B intensely labeled abnormal neurons in both GG and DNT and co-localized with NMDAR1. The presence of BDNF and its receptor in the neuronal component of GG and DNT may suggest a role for this neurotrophin in the development of these lesions, preventing the death of abnormal neuronal cells. In addition, since these neurons contain both NMDAR1 and NMDAR2A/B subunits, the BDNF-TrkB pathway may also contribute through a modulation of glutamatergic transmission to the intrinsic epileptogenicity of glioneuronal tumors.