Glioblastoma multiforme is the most malignant astrocytic glioma and usually resistant to chemotherapy. A small fraction of glioblastomas may contain areas with histological features of ...oligodendroglial differentiation. To determine the molecular genetic alterations in such "glioblastomas with oligodendroglial component", we investigated 13 of these tumors for genetic alterations and/or expression of the TP53, CDKN2A, PTEN, and EGFR genes. In addition, we performed microsatellite analyses for loss of heterozygosity (LOH) on chromosome arms 1p, 19q and 10q. None of tumors showed evidence for LOH on 10q. LOH on 1p was detected in 3 tumors, 1 of which additionally showed LOH on 19q. The 3 tumors with LOH on 1p showed neither TP53 mutations nor nuclear p53 accumulation. In contrast, 9 of 10 tumors without demonstrated losses on 1p showed nuclear p53 accumulation. TP53 mutations were identified in 3 of these cases. Further aberrations detected were epidermal growth factor receptor (EGFR) overexpression (3 of 13 tumors), homozygous CDKN2A deletion (2 of 11 tumors), and PTEN mutation (1 of 13 tumors). Taken together, our results indicate that "glioblastomas with oligodendroglial component" carry heterogeneous genetic alterations. LOH on 10q, PTEN mutation, and homozygous CDKN2A deletion appear to be less common in these tumors as compared to ordinary glioblastomas. Furthermore, a subset of these tumors demonstrates LOH on 1p, i.e., an alteration that has recently been linked to chemosensitivity and good prognosis in anaplastic oligodendrogliomas.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Summary Background The WHO classification of brain tumours describes 15 subtypes of meningioma. Nine of these subtypes are allotted to WHO grade I, and three each to grade II and grade III. Grading ...is based solely on histology, with an absence of molecular markers. Although the existing classification and grading approach is of prognostic value, it harbours shortcomings such as ill-defined parameters for subtypes and grading criteria prone to arbitrary judgment. In this study, we aimed for a comprehensive characterisation of the entire molecular genetic landscape of meningioma to identify biologically and clinically relevant subgroups. Methods In this multicentre, retrospective analysis, we investigated genome-wide DNA methylation patterns of meningiomas from ten European academic neuro-oncology centres to identify distinct methylation classes of meningiomas. The methylation classes were further characterised by DNA copy number analysis, mutational profiling, and RNA sequencing. Methylation classes were analysed for progression-free survival outcomes by the Kaplan-Meier method. The DNA methylation-based and WHO classification schema were compared using the Brier prediction score, analysed in an independent cohort with WHO grading, progression-free survival, and disease-specific survival data available, collected at the Medical University Vienna (Vienna, Austria), assessing methylation patterns with an alternative methylation chip. Findings We retrospectively collected 497 meningiomas along with 309 samples of other extra-axial skull tumours that might histologically mimic meningioma variants. Unsupervised clustering of DNA methylation data clearly segregated all meningiomas from other skull tumours. We generated genome-wide DNA methylation profiles from all 497 meningioma samples. DNA methylation profiling distinguished six distinct clinically relevant methylation classes associated with typical mutational, cytogenetic, and gene expression patterns. Compared with WHO grading, classification by individual and combined methylation classes more accurately identifies patients at high risk of disease progression in tumours with WHO grade I histology, and patients at lower risk of recurrence among WHO grade II tumours (p=0·0096) from the Brier prediction test). We validated this finding in our independent cohort of 140 patients with meningioma. Interpretation DNA methylation-based meningioma classification captures clinically more homogenous groups and has a higher power for predicting tumour recurrence and prognosis than the WHO classification. The approach presented here is potentially very useful for stratifying meningioma patients to observation-only or adjuvant treatment groups. We consider methylation-based tumour classification highly relevant for the future diagnosis and treatment of meningioma. Funding German Cancer Aid, Else Kröner-Fresenius Foundation, and DKFZ/Heidelberg Institute of Personalized Oncology/Precision Oncology Program.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Recent studies show that human CD133(+) (previously known as AC133(+)) cells from mobilized peripheral blood consist of stem cells with either hematopoietic or endothelial potential. To test whether ...this population also contains individual precursors with both capacities, the defining characteristics of the elusive adult hemangioblast, we developed a culture system that allows single-cell analyses of differentiation. In the presence of vascular endothelial growth factor (VEGF), stem cell growth factor (SCGF), and FLT-3 ligand, CD133(+)-enriched cells were first expanded and the amplified cells were transduced with a vector encoding an enhanced green fluorescent protein (EGFP) marker gene. Single EGFP(+) cells were then cocultured with corresponding non-transduced cells from the same experiment, yielding 50-100 marked cells in 8% of the wells after 2 weeks. The resultant cells were divided and differentiated with either granulocyte colony-stimulating factor (G-CSF) or with SCGF and VEGF. These culture conditions resulted in the formation of neutrophil or endothelial cells, respectively, as identified morphologically and by phenotypic staining. Dual differentiation of EGFP(+) cells could be observed in one-quarter of clones from single-seeded cells, suggesting that 2% of EGFP(+) cells were in fact human hemangioblasts. These cells could be expanded for at least 28 days without losing this dual capacity. Hence, this culture system may be of clinical relevance in the development of cellular therapies for disorders involving hematopoiesis and the vascular system. In addition, our results provide important information related to the development of the vasculature and the potential role of hemangioblasts in vasculogenesis in adult humans.
Abstract
INTRODUCTION
Extracellular vesicles (EVs) carry biological information from their cell of origin that is useful for non-invasive detection of tumor biomarkers and disease monitoring. In ...glioblastoma (GBM), blood circulating EVs are elevated and carry GBM-associated proteins. However, it is still challenging to analyze tumor derived EVs for translational purposes. Here, we used imaging flow cytometry (IFCM) as a robust strategy to perform phenotyping of EVs with GBM related surface markers in human plasma.
METHODS
EVs were isolated via differential ultracentrifugation from plasma of (a) 40 GBM patients, pre- and post-surgery, (b) 11matched GBM relapses and (c) 12 healthy donors (HD). EV sizes and concentrations were evaluated by NTA. EV markers (CD9,CD63 and CD81) together with glioma-related markers (integrin beta-1 ITGB1, tenascin C TNC, Profilin-1 PFN1, CD44,GPNMB, SPARC, HLA-II or CD133) were analyzed by IFCM. EV percentages and objects/mL plasma were compared among the groups and correlated with clinical parameters.
RESULTS
CD9 was the predominant tetraspanin in all groups (15-96%), while CD63 had the lowest levels (0-33%) and the strongestdecrease in GBM patients after surgery (fold change FC=-5.4, p<0.01). Among the glioma-related markers, ITGB1 and TNC displayed the most significant differences between the analyzed groups, especially the double positives ITGB1+/CD63+and TNC+/CD63+, which decreased in patients after tumor removal (FC=-3.5 and -12, respectively; p<0.001). Meanwhile,ITGB1+/CD9+and TNC+/CD9+EVs exhibited the highest levels in GBM when compared to HD subjects (FC=8.6 and 17.4;p<0.001) and upon tumor recurrence (FC=3.7 and 10.9, respectively; p<0.01).
SUMMARY/CONCLUSION
We identified EV surface antigens with potential clinical utility as GBM biomarkers. Among them, we highlight ITGB1 and TNC as the most promising markers.
Abstract
Extracellular vesicles (EVs) are secreted by all cell types, including tumor cells, and are found in increased numbers in the plasma of GBM patients. EVs may contain high-value genetic ...material that can be useful for tracking tumor development, as well as membrane proteins that affect other cells. This prompted us to investigate how tumor EVs might influence immune cells in glioma, and in primary and secondary lymphoid organs as well as in the circulation. To this end we used a syngeneic GBM mouse model and tracked tumor EVs from the brain to the meninges, cervical lymph nodes, plasma, bone marrow and spleen. Interestingly, we were able to identify tumor EVs mostly in the cervical lymph nodes by ImageStream imaging flow cytometry just 30min after tumor EV injection into the brain. However, when tumor EVs were produced by a large gliomas transfected with dTomato, we found them mainly in plasma, less frequently in bone marrow and never in the spleen. We confirmed these data by extracting DNA from EVs and detecting specific dTomato sequences using digital droplet PCR. In addition, we detected CD11b+ macrophages in the meninges that likely travel through the lymphatics that have taken up tumor EV or tumor material. We confirm that tumor EVs are capable of eliciting an immune response by activating T cells. However, prolonged contact and large number of EVs could also block antigen recognition by T cells and thus contribute to the propagation of an immunosuppressive environment in GBM.
Abstract
BACKGROUND
Spinal meningiomas account for 1.2-12 % of all meningiomas and 25-45 % of all spinal tumours. 20 % of intracranial, but only 4.6 % of spinal meningiomas recur requiring additional ...treatment. Whereas the classification of intracranial meningiomas has evolved considerably in recent years and uses genetic and epigenetic parameters, the classification of spinal meningiomas is based solely on histopathological findings. By embedding epi-/genetic features, the prognosis of intracranial meningiomas could be significantly improved, which is still lacking for spinal meningiomas. In our work, we integrated genetic and epigenetic parameters into the classification of spinal meningiomas.
METHODS
We performed epi-/genetic profiling of 50 spinal meningiomas. 497 intracranial meningiomas served as a reference cohort. Copy number variations (CNV) were inferred from the methylation data. Principal component (PCA) and t-SNE analysis were conducted. Clinical and histopathological parameters (location, size, recurrence, WHO°, pathological subtype) were correlated with methylation signatures using the DKFZ brain tumour classifier.
RESULTS
The methylation signature of spinal meningiomas matched to that of intracranial meningiomas (50/50), although meningioma subgroup assignment was achieved in only 13/50 cases. PCA and t-SNE analysis showed that most spinal meningiomas separate from cranial meningiomas and form two distinct clusters. Cluster 1 matched the methylation class ben-2, while cases in cluster 2 were heterogenous and had a low MSC score. Cases of cluster 1 were located in the upper spine, are more common in males and had an AKT1E17K mutation. NF2 mutations were found mainly in the second cluster, in line with a chr.22 q loss. Interestingly 4 tumors did not associate with the two spinal meningioma clusters and had a particular higher recurrence rate.
CONCLUSION
Genetic and epigenetic profiling of spinal meningiomas identifies two distinct classes of spinal meningiomas, which may allow an improved prognosis that could lead to a better guidance for adjuvant therapy.
Abstract
OBJECTIVE
Extracellular vesicles (EVs) represent a population of lipid bilayer nanoparticles released by all cell types, including tumor cells that can serve as a noninvasive source for ...liquid biopsy. To date, MRI images have been the established method for monitoring treatment efficacy in brain tumor patients. We investigated the potential of pure EV count for diagnosis, prognosis, and treatment monitoring in gliomas.
METHODS
Plasma samples, differential blood counts, at multiple timepoints before and after surgery of glioblastoma patients (n=101) were collected. Follow-up samples were obtained every 3 months. Healthy donors served as controls (n=29). Plasma EVs concentration was measured by Nanoparticle Tracking Analysis (NTA). EVs were characterized by electron microscopy and imaging flow cytometry. Tumor burden was measured by MRI images. Clinical characteristics were prospectively recorderd. In addition plasma EVs from Mut3 tumor bearing mice were analysed at d3, d5, d7, d10, d12 after tumor injection (n=20). MRI images and differential blood counts were analyzed.
RESULTS
Glioblatoma patients have a 5-fold increase of plasma EVs compared to HD; p < 0.0001). Circulating EVs counts correlated only with FLAIR hyperintensity and with no other MRI or blood-based parameter. Similar results were obtained from Mut3 tumor mice. Dichotomisation of GBM patients in EVhigh and low revealed a significant overall survival and progression free survival benefit for EVlow patients (p=0.004). After surgery, EVs decreased significantly (5-fold, p< 0.0001). A massive drop in EVs was associated with gross-total resection (p < 0.05). At the time of tumor recurrence, the number of circulating EVs increased in all patients during a follow-up (9 months).
CONCLUSION
Our findings highlight the potential of circulating EVs as a biomarker tool for diagnosis, prognosis and treatment monitoring in GBM patietns, as they seem to reflect the presence of a tumor mass and thus may assist in clinical decision making.
Abstract
Immunotherapeutic approaches in cancer aim to booster T cell mediated immune responses. Tumor-specific T cells recognize target cells via human leukocyte antigen (HLA) molecules before cell ...specific lysis is initiated. Malignant gliomas are known for their immunoevasive and immunosuppressive properties which impede effective tumor-specific immune responses, including clinical responses to immune checkpoint inhibition. Our aim was to investigate whether somatic mutation in HLA genes and antigen presentation pathway genes are present in malignant gliomas.
METHODS
We performed next generation sequencing in 138 patients, including matched tumor and blood samples from 100 IDHwt glioblastomas, 25 IDHmut astrocytomas and 13 IDHmut oligodendrogliomas. Sequencing was performed on tumor and peripheral blood for HLA class I (HLA-A, -B, -C), HLA class II (HLA-DR, -DP, -DQ), non-classical HLA molecules (HLA-E, -F, -G, -H), MICA/B, TAP1/2 and beta2 microglobulin (B2M). Identified calls were validated using targeted nanopore sequencing.
RESULTS
In total, we identified 28 mutations based on Illumina sequencing. Mutations were located in HLA-B (2 mutations), HLA-C (1), B2M (2), HLA-DQA1 (4), HLA-DQB1 (4), HLA-DPB1 (1), HLA-E (1), HLA-F (2), HLA-G (3), HLA-H (1), MICA (3), MICB (2), TAP1 (1) and TAP2 (1). No mutations were found in HLA-A and HLA-DRB1. A frequency cutoff of 5% was applied. Additional mutations (n = 46) below this cutoff were identified. The mutations were found in 30 patients (21.7%). Some patients harbored up to 7-8 mutations in multiple HLA and antigen presenting pathway genes. Most mutations were found in the MICA (MHC class I polypeptide–related sequence A) gene locus, which is engaged by the NKG2D ligand which is broadly expressed on NK cells, gd and ab T cells.
CONCLUSION
Our study demonstrates that somatic mutations in HLA and antigen presentation pathway genes are frequently present in malignant gliomas.
Twenty-one unrelated patients with a history of suspected familial Alzheimer disease (FAD) were screened for mutations in PSEN1, PSEN2, and APP, the known FAD genes encoding the presenilins (PS1 and ...PS2) and the amyloid precursor protein (APP). The mutation detection rate was 57%. Of the nine pathogenic mutations found in 12 cases, three were in APP, one in PSEN2, and five in PSEN1, including two novel Greek mutations (L113Q and N135S). Whereas our findings suggest the possibility of single founders for the majority of mutations, we found evidence of recurrence of the APP mutations V717L and V717I.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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