ABSTRACT
Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.
Subject(s)
Dinoprostone/analogs & derivatives , Dinoprostone/metabolism , Hydroxyprostaglandin Dehydrogenases/metabolism , Intra-Abdominal Fat/immunology , T-Lymphocytes, Regulatory/enzymology , T-Lymphocytes, Regulatory/immunology , 3T3 Cells , Animals , Cell Line , Diabetes Mellitus, Type 2/metabolism , HEK293 Cells , Homeostasis/immunology , Humans , Hydroxyprostaglandin Dehydrogenases/genetics , Insulin Resistance/genetics , Intra-Abdominal Fat/cytology , Jurkat Cells , Lymphocyte Activation/immunology , Male , Mice , Mice, Knockout , STAT5 Transcription Factor/metabolismABSTRACT
Wilms tumors (WTs) are histologically diverse childhood cancers with variable contributions of blastema, stroma, and epithelia. A variety of cancer genes operate in WTs, including the tripartite-motif-containing-28 gene (TRIM28). Case reports and small case series suggest that TRIM28 mutations are associated with epithelial morphology and WT predisposition. Here, we systematically investigated the prevalence of TRIM28 inactivation and predisposing mutations in a cohort of 126 WTs with >2/3 epithelial cells, spanning 20 years of biobanking in the German SIOP93-01/GPOH and SIOP2001/GPOH studies. Overall, 44.4% (56/126) cases exhibited loss of TRIM28 by immunohistochemical staining. Of these, 48 could be further analyzed molecularly, revealing TRIM28 sequence variants in each case - either homozygous (~2/3) or heterozygous with epigenetic silencing of the second allele (~1/3). The majority (80%) of the mutations resulted in premature stops and frameshifts. In addition, we detected missense mutations and small deletions predicted to destabilize the protein through interference with folding of key structural elements such as the zinc-binding clusters of the RING, B-box-2, and PHD domains or the central coiled-coil region. TRIM28-mutant tumors otherwise lacked WT-typical IGF2 alterations or driver events, except for rare TP53 progression events that occurred with expected frequency. Expression profiling identified TRIM28-mutant tumors as a homogeneous subset of epithelial WTs that mostly present with stage I disease. There was a high prevalence of perilobar nephrogenic rests, putative precursor lesions, that carried the same biallelic TRIM28 alterations in 7/7 cases tested. Importantly, 46% of the TRIM28 mutations were present in blood cells or normal kidney tissue, suggesting germline events or somatic mosaicism, partly supported by family history. Given the high prevalence of predisposing variants in TRIM28-driven WT, we suggest that immunohistochemical testing of TRIM28 be integrated into diagnostic practice as the management of WT in predisposed children differs from that with sporadic tumors. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Kidney Neoplasms , Wilms Tumor , Child , Humans , Kidney Neoplasms/pathology , Biological Specimen Banks , Wilms Tumor/metabolism , Kidney/pathology , Germ-Line Mutation , Disease Susceptibility/pathology , Tripartite Motif-Containing Protein 28/geneticsABSTRACT
Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.
Subject(s)
Chromatin Assembly and Disassembly/immunology , Forkhead Transcription Factors/immunology , Gene Expression Regulation , Matrix Attachment Region Binding Proteins/immunology , Self Tolerance , T-Lymphocytes, Regulatory/immunology , 3' Untranslated Regions/genetics , 3' Untranslated Regions/immunology , Animals , Cell Differentiation/drug effects , Chromatin Assembly and Disassembly/drug effects , Flow Cytometry , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Genome, Human , Genome-Wide Association Study , Humans , Lentivirus , Lymphocyte Activation/drug effects , Matrix Attachment Region Binding Proteins/genetics , Matrix Attachment Region Binding Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/immunology , MicroRNAs/metabolism , MicroRNAs/pharmacology , RNA Interference , RNA, Small Interfering/immunology , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Self Tolerance/drug effects , Self Tolerance/genetics , Self Tolerance/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Transduction, GeneticABSTRACT
Two distinct genetically defined entities of ependymoma arising in the supratentorial compartment are characterized by the presence of either a C11orf95-RELA or a YAP-MAMLD1 fusion, respectively. There is growing evidence that supratentorial ependymomas without these genetic features exist. In this study, we report on 18 pediatric non-RELA/non-YAP supratentorial ependymomas that were systematically characterized by means of their histology, immunophenotype, genetics, and epigenomics. Comprehensive molecular analyses included high-resolution copy number analysis, methylation profiling, analysis of fusion transcripts by Nanostring technology, and RNA sequencing. Based upon histological and immunohistochemical features two main patterns were identified-RELA-like (n = 9) and tanycytic ependymomas (n = 6). In the RELA-like group histologically assigned to WHO grade III and resembling RELA-fused ependymomas, tumors lacked nuclear expression of p65-RelA as a surrogate marker for a pathological activation of the NF-κB pathway. Three tumors showed alternative C11orf95 fusions to MAML2 or NCOA1. A methylation-based brain tumor classifier assigned two RELA-like tumors to the methylation class "EP, RELA-fusion"; the others demonstrated no significant similarity score. Of the tanycytic group, 5/6 tumors were assigned a WHO grade II. No gene fusions were detected. Methylation profiling did not show any association with an established methylation class. We additionally identified two astroblastoma-like tumors that both presented with chromothripsis of chromosome 22 but lacked MN1 breaks according to FISH analysis. They revealed novel fusion events involving genes in chromosome 22. One further tumor with polyploid cytogenetics was interpreted as PFB ependymoma by the brain tumor methylation classifier but had no relation to the posterior fossa. Clinical follow-up was available for 16/18 patients. Patients with tanycytic and astroblastoma-like tumors had no relapse, while 2 patients with RELA-like ependymomas died. Our data indicate that in addition to ependymomas discovered so far, at least two more supratentorial ependymoma types (RELA-like and tanycytic) exist.
Subject(s)
Ependymoma/genetics , Ependymoma/pathology , Supratentorial Neoplasms/genetics , Supratentorial Neoplasms/pathology , Adaptor Proteins, Signal Transducing , Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male , Transcription Factor RelA , Transcription Factors , YAP-Signaling ProteinsABSTRACT
BACKGROUND: Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ. METHODS: Cell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan-Meier method. RESULTS: Pre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood-brain barrier, diminished proteasome activity and significantly prolonged animal survival. CONCLUSION: BTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.
Subject(s)
Antineoplastic Agents/administration & dosage , Bortezomib/administration & dosage , Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Temozolomide/administration & dosage , Animals , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/enzymology , Cell Line, Tumor , Drug Administration Schedule , Drug Resistance, Neoplasm/drug effects , Glioblastoma/diagnostic imaging , Glioblastoma/enzymology , High-Throughput Nucleotide Sequencing , Humans , Kaplan-Meier Estimate , Methylation , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , O(6)-Methylguanine-DNA Methyltransferase/drug effects , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcription Factor RelA/metabolismABSTRACT
OBJECTIVE: Supra-total glioblastoma resection has gained growing attention with regard to superior long-term disease control. However, aggressive onco-surgical approaches-geared beyond conventional gross total resections (GTR)-are limited by the impairment of adjacent eloquent areas at risk that may entail severe postoperative functional morbidity. Against this backdrop we analyzed our institutional database with regard to potential survival benefits of anterior temporal lobectomy as a paradigm for supra-total resection in patients with precisely temporal-located, non-eloquent glioblastoma. METHODS: Between 2012 and 2017, 38 patients with isolated temporal glioblastoma underwent GTR or temporal lobectomy at the authors' institution. Both groups of differing resection modalities were compared with regard to postoperative Karnofsky performance score (KPS), progression-free survival (PFS), and overall survival (OS). RESULTS: Patients with temporal lobectomy exhibited significantly superior median KPS at the 12 months follow-up compared to the GTR group (median KPS of 80 vs. 60, p = 0.04). Temporal lobectomy was associated with significantly prolonged PFS (p = 0.005) and OS (p = 0.002) coming up to 15 months (95% CI 9.7-22.1) and 23 months (95% CI 14.8-34.5) compared to 7 months (95% CI 3.3-8.3) and 11 months (95% CI 9.2-17.9) for the GTR group. Multivariate analysis revealed temporal lobectomy as the only predictor for both superior PFS (p = 0.037, OR 7.3, 95% CI 1.1-47.4) and OS (p = 0.04, OR 7.8, 95% CI 1.1-55.2). CONCLUSIONS: These results strongly suggest temporal lobectomy as an aggressive supra-total resection policy to constitute the surgical modality of choice for isolated temporal-located glioblastoma.
Subject(s)
Brain Neoplasms/surgery , Glioblastoma/surgery , Neurosurgical Procedures/mortality , Neurosurgical Procedures/methods , Aged , Brain Neoplasms/pathology , Female , Follow-Up Studies , Glioblastoma/pathology , Humans , Male , Middle Aged , Neurosurgical Procedures/classification , Prognosis , Retrospective Studies , Survival RateSubject(s)
Brain Neoplasms , Glioma , Humans , Methylation , Glioma/genetics , Brain Neoplasms/genetics , Isocitrate Dehydrogenase/genetics , Mutation/geneticsABSTRACT
PURPOSE: Magnetic resonance (MR) imaging biomarkers can assist in the non-invasive assessment of the genetic status in glioblastomas (GBMs). Telomerase reverse transcriptase (TERT) promoter mutations are associated with a negative prognosis. This study was performed to identify MR imaging biomarkers to forecast the TERT mutation status. METHODS: Pre-operative MRIs of 64/67 genetically confirmed primary GBM patients (51/67 TERT-mutated with rs2853669 polymorphism) were analyzed according to Visually AcceSAble Rembrandt Images (VASARI) ( https://wiki.cancerimagingarchive.net/display/Public/VASARI+Research+Project ) imaging criteria by three radiological raters. TERT mutation and O6-methylguanine-DNA methyltransferase (MGMT) hypermethylation data were obtained through direct and pyrosequencing as described in a previous study. Clinical data were derived from a prospectively maintained electronic database. Associations of potential imaging biomarkers and genetic status were assessed by Fisher and Mann-Whitney U tests and stepwise linear regression. RESULTS: No imaging biomarkers could be identified to predict TERT mutational status (alone or in conjunction with TERT promoter polymorphism rs2853669 AA-allele). TERT promoter mutations were more common in patients with tumor-associated seizures as first symptom (26/30 vs. 25/37, p = 0.07); these showed significantly smaller tumors [13.1 (9.0-19.0) vs. 24.0 (16.6-37.5) all cm3; p = 0.007] and prolonged median overall survival [17.0 (11.5-28.0) vs. 9.0 (4.0-12.0) all months; p = 0.02]. TERT-mutated GBMs were underrepresented in the extended angularis region (p = 0.03), whereas MGMT-methylated GBMs were overrepresented in the corpus callosum (p = 0.03) and underrepresented temporomesially (p = 0.01). CONCLUSION: Imaging biomarkers for prediction of TERT mutation status remain weak and cannot be derived from the VASARI protocol. Tumor-associated seizures are less common in TERT mutated glioblastomas.
Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Magnetic Resonance Imaging/methods , Telomerase/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor , Female , Humans , Male , Middle Aged , Mutation , Polymorphism, Single Nucleotide , Prognosis , Promoter Regions, GeneticABSTRACT
The value of bevacizumab (BEV) in recurrent glioblastoma is unclear. Imaging parameters and progression-free survival (PFS) are problematic endpoints. Few data exist on clinical factors influencing overall survival (OS) in unselected patients with recurrent glioblastoma exposed to BEV. We retrospectively analyzed 174 patients with recurrent glioblastoma treated with BEV at two German brain tumor centers. We evaluated general patient characteristics, MGMT status, pretreatment, concomitant oncologic treatment and overall survival. Karnofsky performance score, number of prior chemotherapies, number of prior recurrences and combined treatment with irinotecan (IRI) were significantly associated with OS in univariate analysis. We did not find differences in OS related to sex, age, histology, MGMT status, prior surgical treatment or number of prior radiotherapies. Combined treatment with IRI and higher KPS both remained significantly associated with prolonged survival in multivariate analysis, but patients receiving IRI co-treatment had less advanced disease. Grouping into clinically relevant categories revealed an OS of 16.9 months from start of BEV in patients with first recurrence and KPS ≥ 80 % (n = 25). In contrast, in patients with second recurrence and KPS < 80 %, OS was 3.6 months (n = 27). Our observational data support an early use of BEV in patients with good performance status. The benefit of co-treatment with IRI in our cohort seems to be the result of biased patient recruitment.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , Bevacizumab/therapeutic use , Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Neoplasm Recurrence, Local/drug therapy , Adolescent , Adult , Aged , Antineoplastic Agents, Phytogenic/therapeutic use , Brain Neoplasms/diagnosis , Brain Neoplasms/epidemiology , Camptothecin/analogs & derivatives , Camptothecin/therapeutic use , Disease-Free Survival , Drug Therapy, Combination , Female , Glioblastoma/diagnosis , Glioblastoma/epidemiology , Humans , Irinotecan , Karnofsky Performance Status , Male , Middle Aged , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/epidemiology , Prognosis , Retrospective Studies , Treatment Outcome , Young AdultABSTRACT
Melanocytic tumors originating in the central nervous system (MT-CNS) are rare tumors that generally have a favorable prognosis, however malignant tumors do occur. Pathogenetically MT-CNS are not well characterized. Similar to uveal melanoma and blue nevi, they frequently harbor activating GNAQ or GNA11 mutations. Rare NRAS mutations have also been reported. Other mutations have not yet been described. We analyzed 19 MT-CNS, 7 uveal melanomas and 19 cutaneous melanomas using a targeted next generation sequencing approach analyzing 29 genes known to be frequently mutated in other melanocytic tumors (in particular uveal and cutaneous melanomas). In concordance with previous studies, cutaneous melanoma samples showed frequent NRAS or BRAF mutations, as well as mutations in other genes (e.g. NF1, RAC1, PIK3CA, ARID1A). Metastasized uveal melanomas exhibited mutations in GNAQ, GNA11 and BAP1. In contrast, MT-CNS almost exclusively demonstrated mutations in GNAQ (71 %) or GNA11 (12 %). Interestingly both GNA11 mutations identified were detected in MT-CNS diagnosed as intermediate grade melanocytomas which also recurred. One of these recurrent cases also harbored an inactivating BAP1 mutation and was found to have lost one copy of chromosome 3. Our findings show that while MT-CNS do have GNAQ or GNA11 mutations, they rarely harbor other recurrent mutations found in uveal or cutaneous melanomas. Considering chromosome 3 and BAP1 loss are robust markers of poor prognosis in uveal melanoma, it will prove interesting to determine whether these genomic alterations are also of prognostic significance in MT-CNS.
Subject(s)
Biomarkers, Tumor/genetics , Central Nervous System Neoplasms/genetics , High-Throughput Nucleotide Sequencing/methods , Melanoma/genetics , Mutation/genetics , Neoplasm Recurrence, Local/genetics , Adolescent , Adult , Aged , Central Nervous System Neoplasms/pathology , DNA Copy Number Variations , Female , Follow-Up Studies , Humans , Immunoenzyme Techniques , Male , Melanoma/pathology , Meningeal Neoplasms/genetics , Meningeal Neoplasms/pathology , Middle Aged , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Prognosis , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Young AdultABSTRACT
Methylation class "CNS tumor with BCOR/BCOR(L1)-fusion" was recently defined based on methylation profiling and tSNE analysis of a series of 21 neuroepithelial tumors with predominant presence of a BCOR fusion and/or characteristic CNV breakpoints at chromosome 22q12.31 and chromosome Xp11.4. Clear diagnostic criteria are still missing for this tumor type, specially that BCOR/BCOR(L1)-fusion is not a consistent finding in these tumors despite being frequent and that none of the Heidelberger classifier versions is able to clearly identify these cases, in particular tumors with alternative fusions other than those involving BCOR, BCORL1, EP300 and CREBBP. In this study, we introduce a BCOR::CREBBP fusion in an adult patient with a right temporomediobasal tumor, for the first time in association with methylation class "CNS tumor with BCOR/BCOR(L1)-fusion" in addition to 35 cases of CNS neuroepithelial tumors with molecular and histopathological characteristics compatible with "CNS tumor with BCOR/BCOR(L1)-fusion" based on a comprehensive literature review and data mining in the repository of 23 published studies on neuroepithelial brain Tumors including 7207 samples of 6761 patients. Based on our index case and the 35 cases found in the literature, we suggest the archetypical histological and molecular features of "CNS tumor with BCOR/BCOR(L1)-fusion". We also present four adult diffuse glioma cases including GBM, IDH-Wildtype and Astrocytoma, IDH-Mutant with CREBBP fusions and describe the necessity of complementary molecular analysis in "CNS tumor with BCOR/BCOR(L1)-alterations for securing a final diagnosis.
Subject(s)
Brain Neoplasms , Central Nervous System Neoplasms , Glioma , Neoplasms, Neuroepithelial , Adult , Humans , Central Nervous System Neoplasms/diagnostic imaging , Central Nervous System Neoplasms/genetics , Neoplasms, Neuroepithelial/diagnostic imaging , Neoplasms, Neuroepithelial/genetics , Neoplasms, Neuroepithelial/pathology , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Glioma/genetics , Methylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Repressor Proteins/genetics , CREB-Binding Protein/geneticsABSTRACT
The recent identification of isocitrate dehydrogenase 1 (IDH1) gene mutations in gliomas stimulated various studies to explore the molecular consequences and the clinical implications of such alterations. The Cancer Genome Atlas Research Network showed evidence for a CpG island methylator phenotype in glioblastomas that was associated with IDH1 mutations. These alterations were associated with the production of the oncometabolite, 2-hydroxyglutarate, that inhibits oxygenases [ie, ten-eleven translocation (TET) enzymes involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC)]. We investigated 60 gliomas for 5hmC presence, 5-methylcytosine content, TET1 expression, and IDH1 mutation to gain insight into their relationships on a histological level. Of gliomas, 61% revealed no immunoreactivity for 5hmC, and no correlation was observed between IDH1 mutations and loss of 5hmC. Interestingly, expression of TET1 showed remarkable differences regarding overall protein levels and subcellular localization. We found a highly significant (P = 0.0007) correlation between IDH1 mutations and nuclear accumulation of TET1, but not with loss of 5hmC. Of 5hmC-negative gliomas, 70% showed either exclusive or dominant cytoplasmic expression, or no detectable TET1 protein (P = 0.0122). Our data suggest that the loss of 5hmC is a frequent event in gliomas, independent of IDH1 mutation, and may be influenced by the nuclear exclusion of TET1 from the nuclei of glioma cells.
Subject(s)
Brain Neoplasms/pathology , Cell Nucleus/metabolism , Cytosine/analogs & derivatives , DNA-Binding Proteins/metabolism , Glioma/enzymology , Glioma/pathology , Isocitrate Dehydrogenase/metabolism , Proto-Oncogene Proteins/metabolism , 5-Methylcytosine/metabolism , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Cell Line, Tumor , Cytosine/metabolism , DNA Methylation/genetics , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Dioxygenases , Dual-Specificity Phosphatases/genetics , Female , Gene Expression Regulation, Enzymologic , Glioblastoma/enzymology , Glioblastoma/genetics , Glioblastoma/pathology , Glioma/genetics , Humans , Immunohistochemistry , Isocitrate Dehydrogenase/genetics , Male , Mitogen-Activated Protein Kinase Phosphatases/genetics , Mixed Function Oxygenases , Mutation/genetics , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Subcellular Fractions/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
OBJECTIVES: In this study, we analyzed the prognostic value of different MRI progression patterns for survival in patients with recurrent malignant glioma treated with the vascular endothelial growth factor antibody bevacizumab. PATIENTS AND METHODS: Twenty-six adult patients with recurrent malignant glioma treated with bevacizumab or bevacizumab/irinotecan were retrospectively analyzed for the development of contrast-enhanced (T1-weighted MRI) and T2/FLAIR lesions. According to the progression pattern, patients were divided into 3 subgroups: (1) patients with primarily progressive contrast-enhanced lesions in the first MRI after initiation of therapy ('primary PD group'); (2) patients with stable or regressive enhanced lesions but progressive FLAIR lesions ('FLAIR-only PD group'), and (3) patients with stable or regressive contrast-enhanced T1 and FLAIR lesions ('no PD group'). RESULTS: Overall survival (OS) in the 6 patients in the FLAIR-only PD group was not significantly different from the 11 patients in the no PD group (median 311 vs. 254 days, respectively). In contrast, survival in the FLAIR-only PD group was significantly better (p = 0.025) than in the primary PD group. CONCLUSION: FLAIR-only progression is not an independent prognostic factor negatively influencing OS in recurrent glioblastoma treated with bevacizumab and should not lead to discontinuation of bevacizumab therapy.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/pathology , Adult , Analysis of Variance , Bevacizumab , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Disease Progression , Female , Germany/epidemiology , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Irinotecan , Magnetic Resonance Imaging , Male , Middle Aged , Neoplasm Recurrence, Local/mortality , Prognosis , Retrospective Studies , Survival Analysis , Treatment OutcomeABSTRACT
Pediatric glioblastomas recently have been exon sequenced with evidence that approximately 30 % of cases harbour mutations of the histone H3.3 gene. Although studies to determinate their role in risk stratification are on-going, it remains to be determined whether H3.3 mutations could be found in other tumors such as pediatric primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) and whether the presence of H3.3 mutations in glioblastomas could be used as diagnostic tool in their differential diagnosis with CNS-PNETs. We performed a large mutational pyrosequencing-based screening on 123 pediatric glioblastomas and 33 CNS-PNET. The analysis revealed that 39/123 (31.7 %) glioblastomas carry H3.3 mutations. The K27M (AAG â ATG, lysine â methionine) mutation was found in 33 glioblastomas (26 %); the G34R (GGG â AGG, glycine â arginine) was observed in 6 glioblastomas (5.5 %). However, we also identified 4 of 33 cases (11 %) of CNS-PNETs harbouring a H3.3 G34R mutation. Multiplex ligation-dependent probe amplification analysis revealed PDGFR-alpha amplification and EGFR gain in two cases and N-Myc amplification in one case of H3.3 G34R mutated CNS-PNET. None of H3.3 mutated tumors presented a CDKN2A loss. In conclusion, because pediatric patients with glioblastoma and CNS-PNET are treated according to different therapeutic protocols, these findings may raise further concerns about the reliability of the histological diagnosis in the case of an undifferentiated brain tumor harbouring G34R H3.3 mutation. In this view, additional studies are needed to determine whether H3.3 G34 mutated CNS-PNET/glioblastomas may represent a defined tumor subtype.
Subject(s)
Central Nervous System Neoplasms/genetics , Glioblastoma/genetics , Histones/genetics , Mutation/genetics , Neuroectodermal Tumors, Primitive/genetics , Adolescent , Arginine/genetics , Central Nervous System Neoplasms/pathology , Child , Child, Preschool , DNA Mutational Analysis , Female , Glioblastoma/pathology , Glycine/genetics , Humans , Male , Neuroectodermal Tumors, Primitive/pathology , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolismABSTRACT
In a strategy to identify novel genes involved in glioma pathogenesis by molecular characterization of chromosomal translocation breakpoints, we identified the KIAA1797 gene, encoding a protein with an as yet undefined function, to be disrupted by a 7;9 translocation in a primary glioblastoma culture. Array-based comparative genomic hybridization detected deletions involving KIAA1797 in around half of glioblastoma cell lines and glioblastomas investigated. Quantification of messenger RNA levels in human tissues demonstrated highest KIAA1797 expression in brain, reduced levels in all glioblastoma cell lines and most glioblastomas and similar levels in glial and neuronal cells by analysis of different hippocampal regions from murine brain. Antibodies against KIAA1797 were generated and showed similar protein levels in cortex and subcortical white matter of human brain, while levels were significantly reduced in glioblastomas with KIAA1797 deletion. By immunofluorescence of astrocytoma cells, KIAA1797 co-localized with vinculin in focal adhesions. Physical interaction between KIAA1797 and vinculin was demonstrated via co-immunoprecipitation. Functional in vitro assays demonstrated a significant decrease in colony formation, migration and invasion capacity of LN18 and U87MG glioma cells carrying a homozygous KIAA1797 deletion ectopically expressing KIAA1797 compared with mock-transduced cells. In an in vivo orthotopic xenograft mouse model, U87MG tumour lesions expressing KIAA1797 had a significantly reduced volume compared to tumours not expressing KIAA1797. In summary, the frequently deleted KIAA1797 gene encodes a novel focal adhesion complex protein with tumour suppressor function in gliomas, which we name 'focadhesin'. Since KIAA1797 genetic variation has been implicated in Alzheimer's disease, our data are also relevant for neurodegeneration.
Subject(s)
Brain Neoplasms/genetics , Focal Adhesions/genetics , Gene Expression Regulation, Neoplastic/physiology , Genes, Tumor Suppressor/physiology , Glioblastoma/genetics , Animals , Animals, Newborn , Brain/metabolism , Cell Line, Tumor , Cell Movement/genetics , Comparative Genomic Hybridization , Cyclin-Dependent Kinase Inhibitor p15/metabolism , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Disease Models, Animal , Female , Focal Adhesions/immunology , Focal Adhesions/metabolism , Gadolinium , Gene Expression Regulation, Neoplastic/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunoprecipitation , In Vitro Techniques , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Neuroglia/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Transfection , Tumor Stem Cell Assay/methods , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Vinculin/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma.
ABSTRACT
In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.
Subject(s)
5' Untranslated Regions , Astrocytoma/genetics , CpG Islands , DNA Methylation , Neuroendocrine Secretory Protein 7B2/genetics , Neuroendocrine Secretory Protein 7B2/metabolism , Apoptosis , Astrocytoma/metabolism , Astrocytoma/pathology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Base Sequence , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Decitabine , Epigenesis, Genetic , Exons , Gene Expression Regulation, Neoplastic , Humans , Neuroendocrine Secretory Protein 7B2/biosynthesis , Promoter Regions, Genetic , Sequence Analysis, DNA , Transcription, Genetic/drug effectsABSTRACT
Rosette-forming glioneuronal tumors (RGNT) of the fourth ventricle are rare mixed glioneuronal tumors included in the revised WHO classification of central nervous system tumors, showing partial histological similarities to pilocytic astrocytomas. To evaluate potential similarities at the molecular level between these tumors, we analysed a series of 10 RGNT for the presence of KIAA1549-BRAF fusions using interphase fluorescence in situ hybridisation. However, we found no cases showing KIAA1549-BRAF gene fusion or BRAF (V600E) mutation. Our data support the hypothesis that RGNT may represent a distinct entity among the glioneuronal tumors of the central nervous system, with molecular features different from pilocytic astrocytomas.
Subject(s)
Brain Neoplasms/genetics , Fourth Ventricle/pathology , Ganglioglioma/genetics , Oncogene Fusion/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins B-raf/genetics , Adolescent , Adult , Brain Neoplasms/pathology , Cerebral Ventricle Neoplasms/genetics , Child , Female , Ganglioglioma/pathology , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Rosette FormationABSTRACT
The classic medulloblastoma (CMB) and the desmoplastic medulloblastoma (DMB) subtypes represent the major medulloblastoma variants. In contrast to CMB, DMB display high levels of the low-affinity nerve growth factor receptor p75(NTR) . Given the reports of a better clinical course of DMB, we hypothesized that p75(NTR) might act as a tumor suppressor in medulloblastomas. In a large set of medulloblastomas, p75(NTR) was screened for mutations, and its mRNA expression and the DNA methylation status of its 5'-region were assessed. p75(NTR) immunostainings were performed in wild-type murine cerebella and medulloblastomas arising in patched heterozygous mice, and murine cerebellar granule cell precursors (GCP) were analyzed in vitro. Medulloblastoma cells engineered to express p75(NTR) were characterized flow cytometrically and morphologically. One CMB displayed a mutation of the p75(NTR) coding sequence. p75(NTR) mRNA levels clearly delineated DMB and CMB; however, CpG island hypermethylation was excluded as the cause of low p75(NTR) expression in CMB. Sonic Hedgehog-treated GCP showed elevated p75(NTR) expression, and strong expression of p75(NTR) was detected in the external granule cell layer of wild-type mice and in murine ptc(±) medulloblastomas. CMB cells overexpressing p75(NTR) displayed a significant increase in apoptosis. In summary, our data link activated Hedgehog signaling in DMB with p75(NTR) expression and characterize p75(NTR) as a biologically relevant inductor of apoptosis in MB.
Subject(s)
Apoptosis , Cerebellar Neoplasms/pathology , Medulloblastoma/pathology , Receptor, Nerve Growth Factor/physiology , Animals , Blotting, Western , Cerebellar Neoplasms/metabolism , CpG Islands , DNA Methylation , DNA, Neoplasm/genetics , Female , Flow Cytometry , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , Immunoenzyme Techniques , Medulloblastoma/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Multicenter Studies as Topic , Neurons , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, CulturedABSTRACT
Causes underlying inter-individual variations in DNA methylation profiles among normal healthy populations are not thoroughly understood. To investigate the contribution of genetic variation in DNA methyltransferase (DNMT) genes to such epigenetic variation, we performed a systematic search for polymorphisms in all known human DNMT genes [DNMT1, DNMT3A, DNMT3B, DNMT3L and DNMT2 (TRDMT1)] in 192 healthy males and females. One hundred and eleven different polymorphisms were detected. Of these, 24 were located in coding regions and 10 resulted in an amino acid change that may affect the corresponding DNMT protein structure or function. Association analysis between all major polymorphisms (frequency > 1%) and quantitative DNA methylation profiles did not return significant results after correction for multiple testing. Polymorphisms leading to an amino acid change were further investigated for changes in global DNA methylation by differential methylation hybridization. This analysis revealed that a rare change at DNMT3L (R271Q) was associated with significant DNA hypomethylation. Biochemical characterization confirmed that DNMT3L(R271Q) is impaired in its ability to stimulate de novo DNA methylation by DNMT3A. Methylated DNA immunoprecipitation based analysis using CpG island microarrays revealed that the hypomethylation in this sample preferentially clustered to subtelomeric genomic regions with affected loci corresponding to a subset of repetitive CpG islands with low predicted promoter potential located outside of genes.