RNA-Sequencing and immunohistochemistry reveal ZFN7 as a stronger marker of survival than molecular subtypes in G-CIMP-negative glioblastoma

Data de publicació electrónica: 26-10-2020Purpose: Glioblastoma is the most aggressive brain tumor in adults and has few therapeutic options. The study of molecular subtype classifications may lead to improved prognostic classification and identification of new therapeutic targets. The TCGA subtype classification has mainly been applied in USA clinical trials, while the IGS has mainly been applied in European trials. Experimental design: From paraffin-embedded tumor samples of 432 uniformly treated, newly diagnosed glioblastoma patients, we built tissue microarrays for immunohistochemical analysis and applied RNA-Sequencing to the best samples in order to classify them according to the TCGA and IGS subtypes. Results: We obtained transcriptomic results from 124 patients. There was a lack of agreement among the three TCGA classificatory algorithms employed, which was not solely attributable to intratumoral heterogeneity. There was overlapping of the TCGA mesenchymal subtype with IGS cluster 23 and of the TCGA classical subtype with IGS cluster 18. Molecular subtypes were not associated with prognosis, but levels of expression of 13 novel genes were identified as independent prognostic markers in G-CIMP-negative patients, independently of clinical factors and MGMT methylation. These findings were validated in at least one external database. Three of the 13 genes were selected for immunohistochemical validation. In particular, high ZNF7 RNA expression and low ZNF7 protein expression were strongly associated with longer survival, independently of molecular subtypes. Conclusions: The TCGA and IGS molecular classifications of glioblastoma have no higher prognostic value than individual genes and should be refined before being applied to clinical trials

Glioblastoma TCGA mesenchymal and IGS 23 tumors are identifiable by immunohistochemistry and have an immune-phenotype indicating potential benefit from immunotherapy

Data de publicació electrònica: 30-09-2020Purpose: Molecular subtype classifications in glioblastoma may detect therapy sensitivities. Immunohistochemistry would potentially allow the identification of molecular subtypes in routine clinical practice. Experimental design: Paraffin-embedded tumor samples of 124 uniformly treated, newly diagnosed glioblastoma patients were submitted to RNA-Sequencing, immunohistochemistry, and immune-phenotyping to identify differences in molecular subtypes associated with treatment sensitivities. Results: We detected high molecular and immunohistochemical overlapping of the TCGA mesenchymal subtype with IGS cluster 23 and of the TCGA classical subtype with IGS cluster 18. Immunohistochemical patterns, gene fusion profiles, and immune-phenotypes varied across subtypes. Immunohistochemistry revealed that the TCGA classical subtype was identified by high expression of EGFR and low expression of PTEN, while the mesenchymal subtype was identified by low expression of SOX2 and high expression of two antibodies, SHC1 and TCIRG1, selected based on RNA differential transcriptomic expression. The proneural subtype was identified by frequent positive IDH1 expression and high Olig2 and Ki67 expression. Immune-phenotyping showed that mesenchymal and IGS 23 tumors exhibited a higher positive effector cell score, a higher negative suppressor cell score, and lower levels of immune checkpoint molecules. The cell-type deconvolution analysis revealed that these tumors are highly enriched in M2 macrophages, resting memory CD4+ T cells, and activated dendritic cells, indicating that they may be ideal candidates for immunotherapy, especially with anti-M2 and/or dendritic cell vaccination. Conclusions: There is a subset of tumors, frequently classified as mesenchymal or IGS cluster 23, that may be identified with immunohistochemistry and could well be optimal candidates to immunotherapy

Gal-1 expression analysis in the GLIOCAT multicenter study: Role as a prognostic factor and an immune-suppressive biomarker

Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The β-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM

Prognostic impact of CD133 expression in Endometrial Cancer Patients

To assess the impact of CD133 expression on the prognosis of endometrioid endometrial carcinoma (EEC). We retrospectively assessed CD133 expression in tissue microarray of 116 surgically treated FIGO I-III EEC. Tumors with ≥10% of CD133-expressing cells were considered CD133-positive (CD133+). On the basis of CD133 expression, clinical and pathological parameters, progression-free survival (PFS) and overall survival (OS) were evaluated. Of the EEC studied 85.2% showed CD133-expressing cells. Only 61% (n = 66) of EEC presented ≥10% of CD133 expressing cells and were considered CD133+. The mean OS for CD133+ tumour patients was 161 months (95% CI, 154-168) as compared with 146 months (95% CI, 123-160) for those with CD133- tumors (p = 0.012). The mean PFS for CD133+ tumour was 159 months (95% CI, 149-168) as compared with 147 months (95% CI, 132-161) in those with a CD133-tumour (p = 0.014). CD133+ tumours were less likely to have vascular invasion (p = 0.010) and more likely to be well differentiated (p = 0.034). C133+ tumours predicted favorable OS and PFS of EEC patients, with a Hazard Ratio 4.731 (95% CI, 1.251-17.89; p = 0.022). CD133+ tumor status correlates with favorable prognosis of EEC. Our findings are in agreement with studies addressing brain and colorectal tumours

Posttranslational nitro-glycative modifications of albumin in Alzheimer's disease: implications in cytotoxicity and amyloid-β peptide aggregation

Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.This work was supported by the Spanish Ministry of Science and Innovation (SAF2012-38140; BIO2011-25039); Fondo de Investigación Sanitaria (PI10/00587; PI11/3035; and Red HERACLES RD06/0009, RD12/0042/0014); FEDER Funds; Generalitat de Catalunya (SGR05-266; SGR09-760); and Fundació la Marató de TV3 (100310). M.A.V. is the recipient of an ICREA Academia Award

A comparison of RNA-Seq results from paired formalin-fixed paraffin-embedded and fresh-frozen glioblastoma tissue samples

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserve

Nitro-oxidative stress after neuronal ischemia induces protein nitrotyrosination and cell death

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia

Posttranslational nitro-glycative modifications of albumin in Alzheimer's disease: implications in cytotoxicity and amyloid-β peptide aggregation

Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.This work was supported by the Spanish Ministry of Science and Innovation (SAF2012-38140; BIO2011-25039); Fondo de Investigación Sanitaria (PI10/00587; PI11/3035; and Red HERACLES RD06/0009, RD12/0042/0014); FEDER Funds; Generalitat de Catalunya (SGR05-266; SGR09-760); and Fundació la Marató de TV3 (100310). M.A.V. is the recipient of an ICREA Academia Award

Nitro-oxidative stress after neuronal ischemia induces protein nitrotyrosination and cell death

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia