Loss of cancer cell-derived ADAM15 alters the tumor microenvironment in colorectal tumors.

Tumor progression and response to treatment are highly affected by interactions between cancer cells and the tumor microenvironment (TME). Many of the soluble factors and signaling receptors involved in this crosstalk are shed by a disintegrin and metalloproteinases (ADAMs). Upregulation of ADAM15 has been linked to worse survival in cancer patients and a tumor-promoting function both in vitro and in murine cancer models. Although ADAM15 has been involved in cell-cell and cell-extracellular matrix interactions, its role in the crosstalk between cancer cells and the TME in vivo remains unexplored. Therefore, we aimed to understand how ADAM15 regulates the cell composition of the TME and how it affects tumor progression. Here, we showed an upregulation of ADAM15 in tumor tissues from rectal cancer patients. Subcutaneous injection of wildtype and ADAM15-knockout CT26 colon cancer cells in syngeneic mice confirmed the protumorigenic role of ADAM15. Profiling of tumors revealed higher immune cell infiltration and cancer cell apoptosis in the ADAM15-deficient tumors. Specifically, loss of ADAM15 led to a reduced number of granulocytes and higher infiltration of antigen-presenting cells, including dendritic cells and macrophages, as well as more T cells. Using in vitro assays, we confirmed the regulatory effect of ADAM15 on macrophage migration and identified ADAM15-derived CYR61 as a potential molecular mediator of this effect. Based on these findings, we speculate that targeting ADAM15 could increase the infiltration of immune cells in colorectal tumors, which is a prerequisite for effective immunotherapy.

Meningioma animal models: a systematic review and meta-analysis.

BACKGROUND: Animal models are widely used to study pathological processes and drug (side) effects in a controlled environment. There is a wide variety of methods available for establishing animal models depending on the research question. Commonly used methods in tumor research include xenografting cells (established/commercially available or primary patient-derived) or whole tumor pieces either orthotopically or heterotopically and the more recent genetically engineered models-each type with their own advantages and disadvantages. The current systematic review aimed to investigate the meningioma model types used, perform a meta-analysis on tumor take rate (TTR), and perform critical appraisal of the included studies. The study also aimed to assess reproducibility, reliability, means of validation and verification of models, alongside pros and cons and uses of the model types. METHODS: We searched Medline, Embase, and Web of Science for all in vivo meningioma models. The primary outcome was tumor take rate. Meta-analysis was performed on tumor take rate followed by subgroup analyses on the number of cells and duration of incubation. The validity of the tumor models was assessed qualitatively. We performed critical appraisal of the methodological quality and quality of reporting for all included studies. RESULTS: We included 114 unique records (78 using established cell line models (ECLM), 21 using primary patient-derived tumor models (PTM), 10 using genetically engineered models (GEM), and 11 using uncategorized models). TTRs for ECLM were 94% (95% CI 92-96) for orthotopic and 95% (93-96) for heterotopic. PTM showed lower TTRs [orthotopic 53% (33-72) and heterotopic 82% (73-89)] and finally GEM revealed a TTR of 34% (26-43). CONCLUSION: This systematic review shows high consistent TTRs in established cell line models and varying TTRs in primary patient-derived models and genetically engineered models. However, we identified several issues regarding the quality of reporting and the methodological approach that reduce the validity, transparency, and reproducibility of studies and suggest a high risk of publication bias. Finally, each tumor model type has specific roles in research based on their advantages (and disadvantages). SYSTEMATIC REVIEW REGISTRATION: PROSPERO-ID CRD42022308833.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Expression, prognostic significance and therapeutic implications of PD-L1 in gliomas.

The advent of checkpoint immunotherapy, particularly with programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors, has provided ground-breaking results in several advanced cancers. Substantial efforts are being made to extend these promising therapies to other refractory cancers such as gliomas, especially glioblastoma, which represents the most frequent and malignant glioma and carries an exceptionally grim prognosis. Thus, there is a need for new therapeutic strategies with related biomarkers. Gliomas have a profoundly immunosuppressive tumour micro-environment and evade immunological destruction by several mechanisms, one being the expression of inhibitory immune checkpoint molecules such as PD-L1. PD-L1 is recognised as an important therapeutic target and its expression has been shown to hold prognostic value in different cancers. Several clinical trials have been launched and some already completed, but PD-1/PD-L1 inhibitors have yet to show convincing clinical efficacy in gliomas. Part of the explanation may reside in the vast molecular heterogeneity of gliomas and a complex interplay within the tumour micro-environment. In parallel, critical knowledge about PD-L1 expression is beginning to accumulate including knowledge on expression levels, testing methodology, co-expression with other checkpoint molecules and prognostic and predictive value. This article reviews these aspects and points out areas where biomarker research is needed to develop more successful checkpoint-related therapeutic strategies in gliomas.

Tumour-associated CD204+ microglia/macrophages accumulate in perivascular and perinecrotic niches and correlate with an interleukin-6-enriched inflammatory profile in glioblastoma.

AIMS: Glioblastomas are heterogeneous tumours with a rich tumour microenvironment particularly comprised of tumour-associated microglia/macrophages (TAMs), but also containing a population of dedifferentiated/stem-like glioblastoma cells. Both cell populations contribute to tumour aggressiveness and immune evasion through the actions of various signalling molecules. The scavenger and pattern recognition receptor CD204 is associated with a pro-tumourigenic phenotype of TAMs and has a negative prognostic value. Our objective was to investigate the possible interaction between TAMs and dedifferentiated glioblastoma cells and characterise the myeloid phenotype of CD204-enriched glioblastomas. METHODS: Double immunohistochemistry and cell counting was performed on eight glioblastoma samples to estimate the expression and interaction level between dedifferentiated/stem-like tumour cells and TAMs. Using the NanoString technology, myeloid transcriptome profiling was performed on 46 glioblastomas, which had been selected based on their protein expression levels of CD204 and ionised calcium-binding adaptor molecule-1 (IBA1). The results were validated by immunohistochemistry and in silico gene expression analyses. RESULTS: TAMs especially CD204+ TAMs accumulated in perivascular and perinecrotic niches in close proximity to podoplanin+ glioblastoma cells. Gene profiling revealed that CD204-enriched glioblastoma has a unique signature with upregulation of genes related to hypoxia, angiogenesis and invasion, including interleukin-6. The gene signature favoured a poor prognosis in patients with glioblastoma. CONCLUSIONS: This is the first study to characterise the role of CD204 in the myeloid microenvironment of glioblastoma. Our results support the unfavourable prognostic impact of CD204 and suggest that CD204 and interleukin-6 could serve as targets for re-education of TAMs and potentiation of current anti-glioma therapy.

Implementation of TERT promoter mutations improve prognostication of the WHO classification in meningioma.

TERT promoter mutations have been associated with increased risk of recurrence in meningioma cohorts, thus a potential biomarker for aggressive phenotypes. A main purpose of refining tumour classification is better predictions on the patient level. We compiled data from previous published cohorts to investigate patient-level predictions of recurrence based on TERTp-mut status. Implementation of TERTp-mut into the WHO grading led to better patient prognostication by improved prediction of recurrence. Our results support implementation of TERTp-mut into diagnostics and classification of meningiomas.

Detection of Circulating Peanut Components in Serum after Ingestion.

INTRODUCTION: Detection of specific food allergens brought into circulation following ingestion is complicated by the minute amounts present in serum. We therefore aimed to assess the utility of selective removal of IgE against peanut components using ImmunoCAP combined with the basophil histamine release (HR) assay (BHRA) to identify the absorbed allergen components after ingestion of peanut. METHODS: Serum from six healthy individuals was drawn before and 1 h after ingestion of 100 g peanut. Serum from two peanut allergic patients was depleted for IgE against single allergen components by incubation with ImmunoCAPs coupled with either Ara h 1; 2; 3 or 6 before donor basophils were passively sensitized with the serum preparations. The sensitized cells were challenged with serum obtained from the six individuals before and after ingestion of peanut, and HR was measured after serum provocation. RESULTS: Ara h 2 and Ara h 6 were detected in serum 1 h after ingestion in 6/6 individuals by negative selection. Depletion of specific IgE against Ara h 2 or Ara h 6 almost completely abolished the response to serum provocation, indicating a sequence homology between the two allergen components in serum. Ara h 1 was demonstrated in 5/6 sera and Ara h 3 in 1/6 sera. CONCLUSION: This study is a proof of concept showing that passive sensitization of basophils with sera depleted of component-specific IgE can be used to identify food allergen components present in serum after ingestion.

Somatostatin analogues in treatment-refractory meningioma: a systematic review with meta-analysis of individual patient data.

Treatment-refractory meningiomas have a dismal prognosis and limited treatment options. Meningiomas express high-densities of somatostatin receptors (SSTR), thus potentially susceptible to antitumorigenic effects of somatostatin analogues (SSA). Evidence for SSA in meningiomas is scarce, and it is unclear if published literature would either (1) support wider use of SSA, if (2) more evidence is desirable, or if (3) available evidence is sufficient to discard SSA. We addressed the need for more evidence with a systematic review and meta-analysis. We performed an individual patient data (IPD) meta-analysis. Main outcomes were toxicity, best radiological response, progression-free survival, and overall survival. We applied multivariable logistic regression models to estimate the effect of SSA on the probability of obtaining radiological disease control. The predictive performance was evaluated using area under the curve and Brier scores. We included 16 studies and compiled IPD from 8/9 of all previous cohorts. Quality of evidence was overall ranked "very low." Stable disease was reported in 58% of patients as best radiological response. Per 100 mg increase in total SSA dosage, the odds ratios for obtaining radiological disease control was 1.42 (1.11 to 1.81, P = 0.005) and 1.44 (1.00 to 2.08, P = 0.05) for patients treated with SSA as monodrug therapy vs SSA in combination with everolimus, respectively. Low quality of evidence impeded exact quantification of treatment efficacy, and the association between response and treatment may represent reverse causality. Yet, the SSA treatment was well tolerated, and beneficial effect cannot be disqualified. A prospective trial without bias from inconsistent study designs is warranted to assess SSA therapy for well-defined meningioma subgroups.

Fluorescein-guided resection of cerebral metastases is associated with greater tumor resection.

BACKGROUND: Sodium fluorescein (fluorescein) crosses a disrupted blood-brain barrier similarly to gadolinium contrast in contrast-enhancing cerebral tumors. When exposed to light with 560 nm wavelength during surgery, fluorescein emits a yellow-green fluorescent light that can be visualized through an operating microscope equipped with an appropriate emission filter. The distribution of the fluorescence correlates with the contrast on a gadolinium contrast-enhanced MRI. OBJECTIVE: The objective of this single-center retrospective study was to investigate if the use of fluorescein would increase the extent of resection and to examine if fluorescein guided resection influences postoperative neurological status. METHODS: During the study period from August 2014 to August 2018, 117 patients were operated for cerebral metastases. Of these, 56 operations were guided by fluorescein and 61 by traditional white light. All patients had an early postoperative MRI within 72 h after surgery. RESULTS: The use of fluorescein increased the extent of resection in patients with cerebral metastases. The use of fluorescein was not associated with increased postoperative sequelae or neurological damage regardless of underlying primary cancer. CONCLUSION: Fluorescein is a helpful supplement in the neurosurgical treatment of cerebral metastases.

Proposal of a new grading system for meningioma resection: the Copenhagen Protocol.

INTRODUCTION: The extent of meningioma resection is the most fundamental risk factor for recurrence, and exact knowledge of extent of resection is necessary for prognostication and for planning of adjuvant treatment. Currently used classifications are the EANO-grading and the Simpson grading. The former comprises radiological imaging with contrast-enhanced MRI and differentiation between "gross total removal" and "subtotal removal," while the latter comprises a five-tiered differentiation of the surgeon's impression of the extent of resection. The extent of resection of tumors is usually defined via analyses of resection margins but has until now not been implemented for meningiomas. PET/MRI imaging with 68Ga-DOTATOC allows more sensitive and specific imaging than MRI following surgery of meningiomas. OBJECTIVE: To develop an objective grading system based on microscopic analyses of resection margins and sensitive radiological analyses to improve management of follow-up, adjuvant therapy, and prognostication of meningiomas. Based on the rationale of resection-margin analyses as gold standard and superior imaging performance of 68Ga DOTATOC PET, we propose "Copenhagen Grading" for meningiomas. RESULTS: Copenhagen Grading was described for six pilot patients with examples of positive and negative findings on histopathology and DOTATOC PET scanning. The grading could be traceably implemented and parameters of grading appeared complementary. Copenhagen Grading is prospectively implemented as a clinical standard at Rigshospitalet, Copenhagen. CONCLUSION: Copenhagen Grading provided a comprehensive, logical, and reproducible definition of the extent of resection. It offers promise to be the most sensitive and specific imaging modality available for meningiomas. Clinical and cost-efficacy remain to be established during prospective implementation.

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy-number profile with inactivation of TP53, PTEN, and RB1.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

Multiple formin proteins participate in glioblastoma migration.

BACKGROUND: The prognosis of glioblastoma remains poor, related to its diffuse spread within the brain. There is an ongoing search for molecular regulators of this particularly invasive behavior. One approach is to look for actin regulating proteins that might be targeted by future anti-cancer therapy. The formin family of proteins orchestrates rearrangement of the actin cytoskeleton in multiple cellular processes. Recently, the formin proteins mDia1 and mDia2 were shown to be expressed in glioblastoma in vitro, and their function could be modified by small molecule agonists. This finding implies that the formins could be future therapeutic targets in glioblastoma. METHODS: In cell studies, we investigated the changes in expression of the 15 human formins in primary glioblastoma cells and commercially available glioblastoma cell lines during differentiation from spheroids to migrating cells using transcriptomic analysis and qRT-PCR. siRNA mediated knockdown of selected formins was performed to investigate whether their expression affects glioblastoma migration. Using immunohistochemistry, we studied the expression of two formins, FHOD1 and INF2, in tissue samples from 93 IDH-wildtype glioblastomas. Associated clinicopathological parameters and follow-up data were utilized to test whether formin expression correlates with survival or has prognostic value. RESULTS: We found that multiple formins were upregulated during migration. Knockdown of individual formins mDia1, mDia2, FHOD1 and INF2 significantly reduced migration in most studied cell lines. Among the studied formins, knockdown of INF2 generated the greatest reduction in motility in vitro. Using immunohistochemistry, we demonstrated expression of formin proteins FHOD1 and INF2 in glioblastoma tissues. Importantly, we found that moderate/high expression of INF2 was associated with significantly impaired prognosis. CONCLUSIONS: Formins FHOD1 and INF2 participate in glioblastoma cell migration. Moderate/high expression of INF2 in glioblastoma tissue is associated with worse outcome. Taken together, our in vitro and tissue studies suggest a pivotal role for INF2 in glioblastoma. When specific inhibiting compounds become available, INF2 could be a target in the search for novel glioblastoma therapies.

Alternative lengthening of telomeres is the major telomere maintenance mechanism in astrocytoma with isocitrate dehydrogenase 1 mutation.

PURPOSE: Isocitrate dehydrogenase 1 (IDH1) mutations are associated with improved survival in gliomas. Depending on the IDH1 status, TERT promoter mutations affect prognosis. IDH1 mutations are associated with alpha-thalassemia/mental retardation syndrome X-linked (ATRX) mutations and alternative lengthening of telomeres (ALT), suggesting an interaction between IDH1 and telomeres. However, little is known how IDH1 mutations affect telomere maintenance. METHODS: We analyzed cell-specific telomere length (CS-TL) on a single cell level in 46 astrocytoma samples (WHO II-IV) by modified immune-quantitative fluorescence in situ hybridization, using endothelial cells as internal reference. In the same samples, we determined IDH1/TERT promoter mutation status and ATRX expression. The interaction of IDH1R132H mutation and CS-TL was studied in vitro using an IDH1R132H doxycycline-inducible glioma cell line system. RESULTS: Virtually all ALTpositive astrocytomas had normal TERT promoter and lacked ATRX expression. Further, all ALTpositive samples had IDH1R132H mutations, resulting in a significantly longer CS-TL of IDH1R132H gliomas, when compared to their wildtype counterparts. Conversely, TERT promotor mutations were associated with IDHwildtype, ATRX expression, lack of ALT and short CS-TL. ALT, TERT promoter mutations, and CS-TL remained without prognostic significance, when correcting for IDH1 status. In vitro, overexpression of IDHR132H in the glioma cell line LN319 resulted in downregulation of ATRX and rapid TERT-independent telomere lengthening consistent with ALT. CONCLUSION: ALT is the major telomere maintenance mechanism in IDHR132H mutated astrocytomas, while TERT promoter mutations were associated with IDHwildtype glioma. IDH1R132H downregulates ATRX expression in vitro resulting in ALT, which may contribute to the strong association of IDH1R132H mutations, ATRX loss, and ALT.

A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative analysis of cellular migration and drug response.

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM.

Sodium fluorescein shows high surgeon-reported usability in glioblastoma surgery.

INTRODUCTION: Glioblastoma has a high mortality rate. Current treatment includes largest possible surgical resection of the tumour using neuronavigation and fluorescence to better identify tumour tissue. In recent years, sodium fluorescein has been reintroduced in neurosurgery as a fluorescence to increase the resection rate. In this study we aimed to measure the surgeons experience of using sodium fluorescein to locate and remove tumour tissue. Furthermore we describe a case of sodium fluorescein tissue distribution. MATERIAL AND METHODS: 13 patients with glioblastoma and seven patients with cerebral metastases undergoing surgical resection were included. Surgery was performed using microscope alternating between white light and the YELLOW 560 filter, which visualized sodium fluorescein. Surgeons graded its usability in terms of location and removal on a scale from one to four. The resection rate was determined by neuroradiologists. Tissue samples obtained during surgery were analysed in relation to fluorescence and dysmorphic cells. RESULTS: Surgeons reported high usability in terms of location and removal of tumours using sodium fluorescein with medians of four in all groups, except for sub-total resections which had a median of three. Surgical complications were minimal and both resection rate and survival rate was within international standards. Histological analysis showed a visual correlation between tumorous tissue and intensity of fluorescence. CONCLUSION: Sodium fluorescence is an effective and useful tool for surgeons during fluorescence-guided surgery for the resection of glioblastoma and cerebral metastases.

Nonhypoxic pharmacological stabilization of Hypoxia Inducible Factor 1α: Effects on dopaminergic differentiation of human neural stem cells.

Parkinson's disease is a neurodegenerative disease resulting in degeneration of midbrain dopaminergic neurons. Exploratory studies using human foetal tissue or predifferentiated stem cells have suggested that intracerebral transplantation of dopaminergic precursor cells may become an effective treatment for patients with Parkinson's disease. However, strategies for dopaminergic stem cell differentiation vary widely in efficiency, and better methods still need to be developed. Hypoxia Inducible Factor 1 (HIF-1) is a transcription factor involved in the regulation of genes important for cellular adaption to hypoxia and low glucose supply. HIF-1 is to a large degree regulated by the availability of oxygen as in its presence, the subunit HIF-1α is degraded by HIF prolyl hydroxylase enzymes (HPHs). Stabilization of HIF-1α through inhibition of HPHs has been shown to increase dopaminergic differentiation of stem cells and to protect dopaminergic neurons against neurotoxins. This study investigated the effects of noncompetitive (FG-0041) and competitive (Compound A and JNJ-42041935) HIF-1α stabilizing compounds on the dopaminergic differentiation of human neural stem cells. Treatment with all HPH inhibitors at high oxygen tension (20%) resulted in HIF-1α stabilization as assessed by immunocytochemistry for HIF-1α and detection of increased levels of vascular endothelial growth factor in the conditioned culture medium. Following 10 days of HIF-1α stabilization, the cultures displayed a slightly reduced proliferative activity and significantly increased relative levels of tyrosine hydroxylase-positive dopaminergic neurons. In conclusion, HIF-1α stabilization may represent a promising strategy for the generation of dopaminergic neurons intended for use in experimental in vitro studies and cell replacement therapies.

Rosette-forming glioneuronal tumors share a distinct DNA methylation profile and mutations in FGFR1, with recurrent co-mutation of PIK3CA and NF1.

Rosette-forming glioneuronal tumor (RGNT) is a rare brain neoplasm that primarily affects young adults. Although alterations affecting the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathway have been associated with this low-grade entity, comprehensive molecular investigations of RGNT in larger series have not been performed to date, and an integrated view of their genetic and epigenetic profiles is still lacking. Here we describe a genome-wide DNA methylation and targeted sequencing-based characterization of a molecularly distinct class of tumors (n = 30), initially identified through genome-wide DNA methylation screening among a cohort of > 30,000 tumors, of which most were diagnosed histologically as RGNT. FGFR1 hotspot mutations were observed in all tumors analyzed, with co-occurrence of PIK3CA mutations in about two-thirds of the cases (63%). Additional loss-of-function mutations in the tumor suppressor gene NF1 were detected in a subset of cases (33%). Notably, in contrast to most other low-grade gliomas, these tumors often displayed co-occurrence of two or even all three of these mutations. Our data highlight that molecularly defined RGNTs are characterized by highly recurrent combined genetic alterations affecting both MAPK and PI3K signaling pathways. Thus, these two pathways appear to synergistically interact in the formation of RGNT, and offer potential therapeutic targets for this disease.

Early postoperative MRI after resection of brain metastases-complete tumour resection associated with prolonged survival.

BACKGROUND: This study aimed to investigate the incidence of residual tumour after resection of brain metastases using early postoperative magnetic resonance imaging (MRI) and the influence of residual tumour on overall patient survival. METHODS: Data from 72 consecutive adult patients undergoing surgery for cerebral metastases over an 18-month study period were retrospectively collected. Early postoperative MRI was used to determine the presence of postoperative residual tumour. Patients were divided into three groups according to the presence of tumour remnant on early postoperative MRI: "no residual tumour", "non-measurable residual tumour" and "measurable residual tumour". Survival analysis (mean estimate survival time) was performed using the Kaplan-Meier and log-rank (mantel cox) tests and compared between groups. Surgical reports were evaluated with regard to the surgeon statement about intraoperative extent of resection (EOR) and compared with the presence of tumour remnant found on the early postoperative MRI. RESULTS: Sixty-eight procedures were followed by early postoperative MRI. MRI verified the presence of "measurable residual tumour" following 15 procedures (22%). MRI confirmed complete resection in 57%. Gross total resection was described by the operating surgeon in 85% of the procedures. There was a significant difference in survival time after surgery between the group having no residual tumour on MRI and the group with measurable residual tumour (p = 0.025). This difference could not be explained by the differences in postoperative radiation therapy. The longest survival was found in patients with non-measurable and no residual tumour on early postoperative MRI, who also received postoperative radiotherapy. CONCLUSION: Residual tumour was seen on MRI after 22% of the procedures. The intraoperative assessment of EOR performed by the surgeon diverged from the early postoperative MRI in 40% of procedures. Correct assessment of residual tumour thus requires early postoperative MRI. Measurable residual tumour on early postoperative MRI was associated with shorter overall survival independent on postoperative radiotherapy.

Co-expression of TIMP-1 and its cell surface binding partner CD63 in glioblastomas.

BACKGROUND: We have previously identified tissue inhibitor of metalloproteinases-1 (TIMP-1) as a prognostic marker in glioblastomas. TIMP-1 has been associated with chemotherapy resistance, and CD63, a known TIMP-1-binding protein, has been suggested to be responsible for this effect. The aim of this study was to assess CD63 expression in astrocytomas focusing on the prognostic potential of CD63 alone and in combination with TIMP-1. METHODS: CD63 expression was investigated immunohistochemically in a cohort of 111 astrocytomas and correlated to tumor grade and overall survival by semi-quantitative scoring. CD63 expression in tumor-associated microglia/macrophages was examined by double-immunofluorescence with ionized calcium-binding adapter molecule 1 (Iba1). The association between CD63 and TIMP-1 was investigated using previously obtained TIMP-1 data from our astrocytoma cohort. Cellular co-expression of TIMP-1 and CD63 as well as TIMP-1 and the tumor stem cell-related markers CD133 and Sox2 was investigated with immunofluorescence. TIMP-1 and CD63 protein interaction was detected by an oligonucleotide-based proximity ligation assay and verified using co-immunoprecipitation. RESULTS: The expression of CD63 was widely distributed in astrocytomas with a significantly increased level in glioblastomas. CD63 levels did not significantly correlate with patient survival at a protein level, and CD63 did not augment the prognostic significance of TIMP-1. Up to 38% of the CD63+ cells expressed Iba1; however, Iba1 did not appear to impact the prognostic value of CD63. A significant correlation was found between TIMP-1 and CD63, and the TIMP-1 and CD63 proteins were co-expressed at the cellular level and located in close molecular proximity, suggesting that TIMP-1 and CD63 could be co-players in glioblastomas. Some TIMP-1+ cells expressed CD133 and Sox2. CONCLUSION: The present study suggests that CD63 is highly expressed in glioblastomas and that TIMP-1 and CD63 interact. CD63 does not add to the prognostic value of TIMP-1. Co-expression of TIMP-1 and stem cell markers as well as the wide expression of CD63 might suggest a role for TIMP-1 and CD63 in glioblastoma stemness.

High expression of cystine-glutamate antiporter xCT (SLC7A11) is an independent biomarker for epileptic seizures at diagnosis in glioma.

Epileptic seizures are an important cause of morbidity in glioma patients. Substantial lines of evidence support the concept of the excitatory neurotransmitter glutamate being a crucial mediator of glioma-associated seizures. In gliomas, non-vesicular secretion of glutamate via the cystine-glutamate exchanger (SLC7A11, xCT) constitutes the main mechanism contributing to high extracellular glutamate concentrations. However, a convincing "proof-of-relevance" of this mechanism in patient material is lacking. A cohort of 229 consecutive patients with newly diagnosed glioma was analyzed with respect to presence, time course, and severity of epileptic seizures. 14 patients were excluded due to previous epileptic seizures, insufficient clinical data or insufficient tumor material. The maximal immunohistochemical expression of xCT was determined in 1-3 independent samples from central tumor areas of each tumor using tissue microarrays. In addition to histological grading of the tumors, isocitrate dehydrogenase 1 (IDH1) R132H mutational status was determined by immunohistochemistry. 215 consecutive glioma patients were included in the study (7.4% grade II, 7.0% grade III, 85.6% grade IV). High xCT expression was significantly associated with seizures at onset (p = 0.05) but not with development of seizures or with refractory seizures. Low-grade gliomas (WHO II/III) had lower xCT expression than glioblastoma (p = 0.001), and tumors without IDH1 R132H mutation tended to have higher xCT levels (p = 0.07). In a multivariate analysis, high xCT expression and WHO tumor grade but not IDH1 R132H mutation, were significantly associated with epileptic seizures at diagnosis (odds ratio 2.2, p = 0.02). Further, xCT expression did not correlate with survival (p = 0.27, log-rank test). Thus, high xCT expression is an independent marker for glioma-associated seizures at diagnosis especially in high-grade glioma, but is not associated with worse survival in our cohort.