Blood Of Glioma Patients Research Articles

The Profiles of Tet-Mediated DNA Hydroxymethylation in Human Gliomas.

Gliomas are the most common primary malignant intracranial brain tumors. Their proliferative and invasive behavior is controlled by various epigenetic mechanisms. 5-hydroxymethylcytosine (5-hmC) is one of the epigenetic DNA modifications that employs ten-eleven translocation (TET) enzymes to its oxidation. Previous studies demonstrated altered expression of 5-hmC across gliomagenesis. However, its contribution to the initiation and progression of human gliomas still remains unknown. To characterize the expression profiles of 5-hmC and TET in human glioma samples we used the EpiJET 5-hmC and 5-mC Analysis Kit, quantitative real-time PCR, and Western blot analysis. A continuous decline of 5-hmC levels was observed in solid tissue across glioma grades. However, in glioblastoma (GBM), we documented uncommon heterogeneity in 5-hmC expression. Further analysis showed that the levels of TET proteins, but not their transcripts, may influence the 5-hmC abundance in GBM. Early tumor-related biomarkers may also be provided by the study of aberrant DNA hydroxymethylation in the blood of glioma patients. Therefore, we explored the patterns of TET transcripts in plasma samples and we found that their profiles were variously regulated, with significant value for TET2. The results of our study confirmed that DNA hydroxymethylation is an important mechanism involved in the pathogenesis of gliomas, with particular reference to glioblastoma. Heterogeneity of 5-hmC and TET proteins expression across GBM may provide novel insight into define subtype-specific patterns of hydroxymethylome, and thus help to interpret the heterogeneous outcomes of patients with the same disease.

Myeloid Diagnostic and Prognostic Markers of Immune Suppression in the Blood of Glioma Patients

BackgroundAlthough gliomas are confined to the central nervous system, their negative influence over the immune system extends to peripheral circulation. The immune suppression exerted by myeloid cells can affect both response to therapy and disease outcome. We analyzed the expansion of several myeloid parameters in the blood of low- and high-grade gliomas and assessed their relevance as biomarkers of disease and clinical outcome.MethodsPeripheral blood was obtained from 134 low- and high-grade glioma patients. CD14+, CD14+/p-STAT3+, CD14+/PD-L1+, CD15+ cells and four myeloid-derived suppressor cell (MDSC) subsets, were evaluated by flow cytometry. Arginase-1 (ARG1) quantity and activity was determined in the plasma. Multivariable logistic regression model was used to obtain a diagnostic score to discriminate glioma patients from healthy controls and between each glioma grade. A glioblastoma prognostic model was determined by multiple Cox regression using clinical and myeloid parameters.ResultsChanges in myeloid parameters associated with immune suppression allowed to define a diagnostic score calculating the risk of being a glioma patient. The same parameters, together with age, permit to calculate the risk score in differentiating each glioma grade. A prognostic model for glioblastoma patients stemmed out from a Cox multiple analysis, highlighting the role of MDSC, p-STAT3, and ARG1 activity together with clinical parameters in predicting patient’s outcome.ConclusionsThis work emphasizes the role of systemic immune suppression carried out by myeloid cells in gliomas. The identification of biomarkers associated with immune landscape, diagnosis, and outcome of glioblastoma patients lays the ground for their clinical use.

Telomerase-positive circulating tumor cells are associated with poor prognosis via a neutrophil-mediated inflammatory immune environment in glioma

BackgroundGliomas are the most common aggressive cancer in the central nervous system. Considering the difficulty in monitoring glioma response and progression, an approach is needed to evaluate the progression or survival of patients with glioma. We propose an application to facilitate clinical detection and treatment monitoring in glioma patients by using telomerase-positive circulating tumor cells (CTCs) and to further evaluate the relationship between the immune microenvironment and CTCs in glioma patients.MethodsFrom October 2014 to June 2017, 106 patients newly diagnosed with glioma were enrolled. We used the telomerase reverse transcriptase CTC detection method to detect and analyze the CTC statuses of glioma patients before and after surgery. FlowSight and FISH confirmed the CTCs detected by the telomerase-based method. To verify the correlation between CTCs and the immune response, peripheral white blood cell RNA sequencing was performed.ResultsCTCs were common in the peripheral blood of glioma patients and were not correlated with the pathological classification or grade of patients. The results showed that the presence of postoperative CTCs but not preoperative CTCs in glioma patients was a poor prognostic factor. The level of postoperative CTCs, which predicts a poor prognosis after surgery, may be associated with neutrophils. RNA sequencing suggested that postoperative CTCs were positively correlated with innate immune responses, especially the activation of neutrophils and the generation of neutrophil extracellular traps, but negatively correlated with the cytotoxic response.ConclusionsOur results showed that telomerase-positive CTCs can predict a poor prognosis of patients with glioma. Our results also showed a correlation between CTCs and the immune macroenvironment, which provides a new perspective for the treatment of glioma.

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma

Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid–Siglec interactions between glioma cells and MDSCs in the tumor microenvironment.

P06.08 BRAINTuNE: Brain Tumor Neoepitope Evaluation for personalized glioma immunotherapy

Abstract Background: Checkpoint inhibitors for cancer immunotherapy have been shown to unleash a subdued endogenous T-cell response against mutated tumor antigens, so-called neoepitopes. However, tumors with a low burden of non-synonymous mutations - including gliomas - are poorly presented to the immune system, and therefore unlikely to respond to checkpoint inhibition alone. It is therefore important to identify the signature of relevant immunogenic neoepitopes and the T cell receptors (TCRs) recognizing these epitopes to employ a more specific immunotherapy. Limited availability of tumor-infiltrating lymphocytes (TILs) in turn prompts the identification of neoepitope-specific T-cells in peripheral blood. Aim: This study aimed at establishing a platform for the identification of immunogenic neoepitopes and corresponding neoepitope-specific T cells in the peripheral blood of glioma patients. Methods and Results: Putative neoepitopes were predicted from next generation sequencing mutanome data of tumor samples from three glioma patients. Patient-specific neoepitopes were synthesized as codon-optimized tandem mini gene vector (TMG) optimized for neoepitope presentation, with up to 10 neoeptiopes per TMG. Autologous PBMC-derived dendritic cells were transfected with in vitro transcribed TMG RNA or loaded with peptides to test T-cell neoepitope-reactivity in co-culture assays. On probing the reactivity of T-cells from peripheral blood, we found patient-specific reactivity against neoepitopes. Conclusion and scope: We investigated the T-cell reactivity towards patient specific neoepitopes in the peripheral blood of 3 glioma patients and the phenotypic identity of these neoepitope-reactive T-cells. We are currently probing CD8+ T-cell subpopulations to characterize the neoepitope-reactive T-cell phenotype. Ongoing studies aim to further characterize the neoepitope-specific T-cells with respect to their TCR sequences and expand this platform to a larger cohort of glioma patients to provide a proof-of-principle for a minimally invasive approach to identify tumor-reactive T-cells for personalized glioma immunotherapy.

Immunomethylomic approach to explore the blood neutrophil lymphocyte ratio (NLR) in glioma survival

BackgroundDifferentially methylated regions (DMRs) within DNA isolated from whole blood can be used to estimate the proportions of circulating leukocyte subtypes. We use the term “immunomethylomics” to describe the application of these immune lineage DMRs to studying leukocyte profiles. Here, we applied this approach to peripheral blood DNA from 72 glioma patients with molecularly defined brain tumors, representing common patient groups with defined characteristic survival times and risk factors. We first estimated the proportions of leukocyte subtypes in samples using deconvolution algorithms with reference DMR libraries from isolated leukocyte populations and Illumina 450K DNA methylation data. Then, we calculated the neutrophil to lymphocyte ratio (NLR) using methylation-derived cell composition estimates (mdNLR). The NLR is considered an indicator of immunosuppressive cells in cancer patients.ResultsElevated mdNLR scores were observed in glioma patients compared to mdNLR values of published controls. Significantly decreased survival times were associated with mdNLR ≥ 4.0 in Cox proportional hazards models adjusted for age, gender, tumor grade, and molecular subtype (HR 2.02, 95% CI, 1.11–3.69). We also identified five myeloid-related CpGs that were highly correlated with the mdNLR (adjusted R2 ≥ 0.80). Each of the five myeloid CpG loci was associated with survival when adjusted for the above covariates and offer a simplified approach for utilizing fresh or archived peripheral blood samples for interrogating a very small number of methylation markers to estimate myeloid immune influences in glioma survival.ConclusionsThe mdNLR (based on DNA methylation) is a novel candidate methylation biomarker that represents immunosuppressive myeloid cells within the blood of glioma patients with potential application in clinical trials and future epidemiologic studies of glioma risk and survival.

CBM-03MOLECULAR CHARACTERIZATION OF GLIOMA CIRCULATING TUMOR CELLS (gCTCs) AT SINGLE CELL RESOLUTION

Glioblastoma multiforme (GBM), the most aggressive and most common malignant adult brain tumor, accounts for ∼50% of all gliomas and it is classified histologically as grade IV. Growing evidence for intratumor heterogeneity shows that single-site biopsies or selected profiling of a portion of a resected tumor falls short of revealing the complete genomic landscape of the entire tumor. EGFR amplification occurs in ∼50% of primary GBM tumors, and is frequently associated with the expression of a constitutively active, ligand-independent mutant form of the receptor, EGFRvIII. Glioma circulating tumor cells (gCTCs) have recently been identified in the blood of glioma patients by our research group. In this study, we report that gCTCs can be captured, enumerated and genetically characterized for EGFRvIII. To achieve this goal, we have thus far a) detected gCTCs through a Telomerase promoter–based assay, b) isolated individual gCTCs using a vacuum-assisted capillary microdissection approach, c) performed whole transcriptome amplification and d) analyzed the expression of EGFRviii by qPCR under optimized conditions. Furthermore, our group is conducting an IRB-approved pilot study with the purpose of examining the feasibility of this approach (to support initiation of an expanded study). In this study, we seek to characterize the genetic heterogeneity of individual gCTCs from high grade glioma patients. This approach would have major impact as it will clarify the biologically-relevant question of heterogeneity of gCTCs in a given patient with glioma, predict whether a patient would respond or be resistant to a molecularly targeted therapy, unravel the overall mechanistic underpinnings of a given patient's brain tumor, and lastly, track the biological evolution of the tumor over the patient's treatment course and post-treatment period. Our ultimate goal is to streamline predictive testing and effectively utilize a Neurooncology targeted biomarker to benefit patients.

CBM-02THE PROGNOSTIC VALUE OF PERIPHERAL BLOOD ACTIVATED CD38+HLA-DR+CD8+T CELLS IN PATIENTS WITH GLIOMAS

BACKGROUND: Cytotoxic CD8+ T cells play an important role in antitumor immunity. However, the impact of activated CD8+ T cells on the prognosis of glioma patients remains uncertain. We examined the activated CD38+ HLA-DR+ CD8+ T cells in preoperative peripheral blood and determined whether these cells could predict the prognosis of patients with glioma. METHODS: This prospective study included 72 patients with gliomas (grade II n = 14; grade III n = 15; grade IV n = 43; mean age at diagnosis, 52.3 ± 14.0 years) and 17 healthy participants from June 2013 to December 2014. The CD38+ HLA-DR+ CD8+ subsets were retrieved from peripheral blood of glioma patients before tumor resection and were analyzed by conducting flow cytometry using anti-CD8, anti-CD38, anti-HLA-DR, anti-CCR5, anti-TNFR2, anti-Tim3 and anti-Ki67 antibodies. We evaluated immune profiles and clinical characteristics of recruited patients. The Wald-Wolfowitz runs test, multivariable linear regression model, and survival analysis were performed for data analyses. RESULTS: The activated phenotype (CD38+/HLA-DR+) CD8+ T cells were increased in grade IV patients compared to healthy controls (p <0.05). The activated CD8+ T cells expressed elevated level of CCR5 in low grade and high grade gliomas (all p <0.05). The proportion of the activated CD8+ T cells was associated with tumor size, age, and clinical outcomes. In grade IV patients, higher level of activated CD8+ T cells yielded better progression free survival (p <0.05). CONCLUSIONS: The findings of this study support that the expression of activation markers CD38+/HLA-DR+ on CD8+ T cells may predicted the prognosis of patients with gliomas, especially in high grade gliomas. Additional investigations are warranted to understand the associations between crucial immune mediators and the activated CD8+ T cells in glioma immunology.

Liquid biopsies in patients with diffuse glioma.

Diffuse gliomas are the most common malignant primary tumors of the central nervous system. Like other neoplasms, these gliomas release molecular information into the circulation. Tumor-derived biomarkers include proteins, nucleic acids, and tumor-derived extracellular vesicles that accumulate in plasma, serum, blood platelets, urine and/or cerebrospinal fluid. Recently, also circulating tumor cells have been identified in the blood of glioma patients. Circulating molecules, vesicles, platelets, and cells may be useful as easily accessible diagnostic, prognostic and/or predictive biomarkers to guide patient management. Thereby, this approach may help to circumvent problems related to tumor heterogeneity and sampling error at the time of diagnosis. Also, liquid biopsies may allow for serial monitoring of treatment responses and of changes in the molecular characteristics of gliomas over time. In this review, we summarize the literature on blood-based biomarkers and their potential value for improving the management of patients with a diffuse glioma. Incorporation of the study of circulating molecular biomarkers in clinical trials is essential for further assessment of the potential of liquid biopsies in this context.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1399-y) contains supplementary material, which is available to authorized users.

A Study on the Biochemical and Cytogenetic Status in the Blood of Glioma Patients

Malignant gliomas, the most common subtype of primary brain tumors, are aggressive, highly invasive and neurologically destructive tumors, considered being the deadliest of human cancers. As an attempt to understand the biology of glial tumor, a study on macromolecules like proteins, matrix metalloproteinases, lipids antioxidants and Deoxyribonucleic acid, in the blood of glioma patients was made. Biochemical assessment of significant pathophysiological enzymes, antioxidants and marker enzymes was performed. MMP expression was determined using gelatin zymography. Karyotyping analysis was done to determine chromosomal aberrations. A marked rise was observed in the proteins and lipids of glioma patients as compared to the normal cases. The antioxidant status of the patients was found to be lowered. Karyotypic analysis of the peripheral blood chromosomes presented various chromosomal aberrations in glioma patients. The biochemical parameters were significantly increased in the patient population (p<0.01, p<0.001) when compared to those of normal. Zymographic analysis showed the presence of MMP-2 and MMP-9 in the patient sample. Karyotypic investigation showed alterations in the chromosomal pattern of the glioma patients. The study provides baseline information on the biochemical alterations in the blood of glioma patients which can be further exploited for detailed investigations.