Glucagon is best known for its contribution to glucose regulation through activation of the glucagon receptor (GCGR), primarily located in the liver. However, glucagon's impact on other organs may also contribute to its potent effects in health and disease. Given that glucagon-based medicine is entering the arena of anti-obesity drugs, elucidating extrahepatic actions of glucagon are of increased importance. It has been reported that glucagon may stimulate secretion of arginine-vasopressin (AVP)/copeptin, growth hormone (GH) and adrenocorticotrophic hormone (ACTH) from the pituitary gland. Nevertheless, the mechanisms and whether GCGR is present in human pituitary are unknown. In this study we found that intravenous administration of 0.2 mg glucagon to 14 healthy subjects was not associated with increases in plasma concentrations of copeptin, GH, ACTH or cortisol over a 120-min period. GCGR immunoreactivity was present in the anterior pituitary but not in cells containing GH or ACTH. Collectively, glucagon may not directly stimulate secretion of GH, ACTH or AVP/copeptin in humans but may instead be involved in yet unidentified pituitary functions.

Outer radial glial cells (oRGs) give rise to neurons and glial cells and contribute to cell migration and expansion in developing neocortex. HOPX has been described as a marker of oRGs and possible actor in glioblastomas. Recent years' evidence points to spatiotemporal differences in brain development which may have implications for the classification of cell types in the central nervous system and understanding of a range of neurological diseases. Using the Human Embryonic/Fetal Biobank, Institute of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark, HOPX and BLBP immunoexpression was investigated in developing frontal, parietal, temporal and occipital human neocortex, other cortical areas and brain stem regions to interrogate oRG and HOPX regional heterogeneity. Furthermore, usage of high‐plex spatial profiling (Nanostring GeoMx ® DSP) was tested on the same material. HOPX marked oRGs in several human developing brain regions as well as cells in known gliogenic areas but did not completely overlap with BLBP or GFAP. Interestingly, limbic structures (e.g. olfactory bulb, indusium griseum, entorhinal cortex, fimbria) showed more intense HOPX immunoreactivity than adjacent neocortex and in cerebellum and brain stem, HOPX and BLBP seemed to stain different cell populations in cerebellar cortex and corpus pontobulbare. DSP screening of corresponding regions indicated differences in cell type composition, vessel density and presence of apolipoproteins within and across regions and thereby confirming the importance of acknowledging time and place in developmental neuroscience.

BACKGROUND:CNS immune privilege has been challenged in recent years. Glioblastoma (GBM) immune dysfunction includes complex interactions with the immune system outside the CNS. The aim of this study was to determine diagnostic and prognostic potential of immune-related proteins in plasma in GBM and interrogate biomarker presence in the brain tumor microenvironment (TME). METHODS:One hundred and fifty-eight patients with glioma WHO grade II-IV were included. Plasma collected at surgery was screened for 92 proteins using proximity extension assay technology and related to clinical outcome. Secretion and expression of candidate prognostic biomarkers were subsequently analyzed in 8 GBM cell lines and public RNAseq data. RESULTS:Plasma levels of 20 out of 92 screened proteins were significantly different in patients with GBM compared to patients with astrocytoma WHO grade II-III. High plasma interleukin-8 (IL-8) (hazard ratio [HR] = 1.52; P = .0077) and low CD244 (HR = 0.36; P = .0004) were associated with short progression-free survival and high plasma IL-8 (HR = 1.40; P = .044) and low ICOS ligand (ICOSLG) (HR = 0.17; P = .0003) were associated with short overall survival (OS) in newly diagnosed patients with GBM. A similar trend was found for ICOSLG (HR = 0.34; P = .053) in recurrent GBM. IL-8 was mostly secreted and expressed by mesenchymal GBM cell lines and expressed by vascular cells and immune cells in the TME. This was also the case for ICOSLG, although less consistent, and with additional expression in tumor-associated oligodendrocytes. CONCLUSIONS:High plasma IL-8 and low ICOSLG at surgery are associated with short OS in newly diagnosed GBM. Source of plasma ICOSLG may be found outside the TME.

Glioblastomas (GBM) are heterogeneous highly vascular brain tumors exploiting the unique microenvironment in the brain to resist treatment and anti-tumor responses. Anti-angiogenic agents, immunotherapy, and targeted therapy have been studied extensively in GBM patients over a number of decades with minimal success. Despite maximal efforts, prognosis remains dismal with an overall survival of approximately 15 months. Bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, underwent accelerated approval by the U.S. Food and Drug Administration in 2009 for the treatment of recurrent GBM based on promising preclinical and early clinical studies. Unfortunately, subsequent clinical trials did not find overall survival benefit. Pursuing pleiotropic targets and leaning toward multitarget strategies may be a key to more effective therapeutic intervention in GBM, but preclinical evaluation requires careful consideration of model choices. In this study, we discuss bevacizumab resistance, dual targeting of pro-angiogenic modulators VEGF and YKL-40 in the context of brain tumor microenvironment, and how model choice impacts study conclusions and its translational significance.

Background: Complex local and systemic immune dysfunction in glioblastoma (GBM) may affect survival. Interleukin (IL)-6 and YKL-40 are pleiotropic biomarkers present in the tumor microenvironment and involved in immune regulation. We therefore analyzed plasma IL-6, YKL-40, and genetic variation in YKL-40 and explored their ability to distinguish between glioma subtypes and predict survival in GBM. Methods: One hundred fifty-eight patients with glioma WHO grade II-IV were included in the study. Plasma collected at surgery was analyzed for IL-6 and YKL-40 (CHI3L1) by ELISA. CHI3L1 rs4950928 genotyping was analyzed on whole-blood DNA. Results: Neither plasma IL-6 nor YKL-40 corrected for age or rs4950928 genotype could differentiate GBM from lower grade gliomas. GC and GG rs4950928 genotype were associated with lower plasma YKL-40 levels (CC vs. GC, p = 0.0019; CC vs. GG, p = 0.01). Only 10 and 14 out of 94 patients with newly diagnosed GBM had elevated IL-6 or YKL-40, respectively. Most patients received corticosteroid treatment at time of blood-sampling. Higher pretreatment plasma IL-6 was associated with short overall survival (OS) [HR = 1.19 (per 2-fold change), p = 0.042] in univariate analysis. The effect disappeared in multivariate analysis. rs4950928 genotype did not associate with OS [HR = 1.30, p = 0.30]. In recurrent GBM, higher YKL-40 [HR = 2.12 (per 2-fold change), p = 0.0005] but not IL-6 [HR = 0.99 (per 2-fold change), p = 0.92] were associated with short OS in univariate analysis. Conclusion: In recurrent GBM high plasma YKL-40 may hold promise as a prognostic marker. In newly diagnosed GBM perioperative plasma IL-6, YKL-40, and genetic variation in YKL-40 did not associate with survival. Corticosteroid use may complicate interpretation of results.

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and malignant primary brain tumor with limited treatment options and a dismal prognosis. Immunotherapy is advancing in other types of cancer but has not yet proven effective in glioblastoma. Prognostic and predictive biomarkers and new treatment targets are urgently needed. MATERIAL AND METHODS 158 patients with glioma WHO grade II-IV were included in the study. Plasma was analyzed for interleukin (IL)-6, YKL-40 (Gene: CHI3L1) and 91 other immune-related protein biomarkers by ELISA or/and Olink technology. CHI3L1 rs4950928 genotyping was analyzed on DNA extracted from whole-blood. RESULTS Fourteen of 92 immune-related biomarkers in plasma detected with the Olink immuno-oncology array were related with tumor grade or/and type (p< 0.05) (corrected for age), whereas plasma IL-6 and YKL-40 did not change with tumor grade. In univariate analysis of plasma from 94 patients with newly diagnosed GBM higher baseline IL-6 was associated with short OS (HR=1.19 (per 2-fold change in IL-6), p=0.04), YKL-40 showed a similar trend (HR=1.20 (per 2-fold change in YKL-40), p=0.056) and high/low baseline levels of additional 15 immune-related biomarkers (e.g. CD244, Granzyme B, ICOS ligand, IL-8 and Pleiotrophin) were associated with short OS (p< 0.05). Genetic variation in YKL-40 was associated with plasma YKL-40 levels (+/+ vs. -/+ vs. -/-, p=0.0004) but not with OS in patients with GBM (HR= 0.78 (+/+ vs. -/+), p=0.32). CONCLUSION High levels of several immune-related biomarkers in plasma including IL-6, IL-8, CD244, Granzyme B, ICOS ligand, and Pleiotrophin from patients with glioblastoma were related or inversely related to OS.

The astroglial lineage consists of heterogeneous cells instrumental for normal brain development, function and repair. Unfortunately, this heterogeneity complicates research in the field, which suffers from lack of truly specific and sensitive astroglial markers. Nevertheless, single astroglial markers are often used to describe astrocytes in different settings. We therefore investigated and compared spatiotemporal patterns of immunoreactivity in developing human brain from 12 to 21 weeks post conception and publicly available RNA expression data for four established and potential astroglial markers - GFAP, S100, AQP4 and YKL-40. In the hippocampal region, we also screened for C3, a complement component highly expressed in A1-reactive astrocytes. We found diverging partly overlapping patterns of the established astroglial markers GFAP, S100 and AQP4, confirming that none of these markers can fully describe and discriminate different developmental forms and subpopulations of astrocytes in human developing brain, although AQP4 seems to be the most sensitive and specific marker for the astroglial lineage at midgestation. AQP4 characterizes a brain-wide water transport system in cerebral cortex with regional differences in immunoreactivity at midgestation. AQP4 distinguishes a vast proportion of astrocytes and subpopulations of radial glial cells destined for the astroglial lineage, including astrocytes determined for the future glia limitans and apical truncated radial glial cells in ganglionic eminences, devoid of GFAP and S100. YKL-40 and C3d, previously found in reactive astrocytes, stain different subpopulations of astrocytes/astroglial progenitors in developing hippocampus at midgestation and may characterize specific subpopulations of 'developmental astrocytes'. Our results clearly reflect that lack of pan-astrocytic markers necessitates the consideration of time, region, context and aim when choosing appropriate astroglial markers.

ABSTRACT Purpose: Antihypertensive therapy may improve bevacizumab efficacy in cancer patients. We examined efficacy and toxicity of angiotensin system inhibitors (ASI) and other antihypertensive drugs in bevacizumab treated recurrent glioblastoma patients. Methods: We retrospectively combined a national prescription registry with a clinical database with recurrent glioblastoma patients (n = 243). Results: Patients who initiated ASI after bevacizumab (n = 26) showed a tendency towards improved progression-free survival and overall survival (OS) with hazard rate (HR) reductions (HR = 0.70 and HR = 0.79, respectively). Calcium antagonists during bevacizumab therapy significantly improved OS (HR = 0.57). Conclusions: Overall the study supports a potential beneficial effect of antihypertensive treatment on prognosis of bevacizumab treated glioblastoma patients.

Glioblastoma (GBM) is a devastating tumor in adults characterized by extensive vascularization and poor survival. New treatment approaches include the targeting of angiogenesis with the hope of improving the outcome of GBM patients. In GBM, the EGFR receptor is often overexpressed or mutated and its activation was found to correlate with poor prognosis. Notch signaling is known for its role in angiogenesis during foetal development and in other cancers, but the importance of Notch signaling in GBM angiogenesis is still under investigation. GBMs often display dysregulation of the EGFR and Notch pathways and the putative crosstalk between these two pathways in regards to angiogenesis underlines their importance in gliomagenesis and therapeutic resistance. In this study, we aimed at elucidating the effect of combined targeting of both, the Notch and the EGFR signalling and its effect on angiogenesis and GBM tumor growth. In vitro experiments were performed using two patient-derived GBM cell cultures, both expressing EGFR and Notch. We demonstrated that a combined inhibition of Notch (γ-secretase inhibitor; DAPT) and EGFR (Iressa) downregulated expression of pro-survival genes (Akt and ERK), reduced expression and secretion of the angiogenic cytokine - VEGF; and displayed additive inhibition of cell viability assessed by MTT assay. Further, the abrogation of EGFR/Notch signaling in GBM cells reduced the degree of angiogenesis. Finally, DAPT and Iressa single-therapy reduced tumor-growth in vivo, however no significant benefit could be found upon combinational therapy. The present study implies that EGFR- and Notch signaling are important for GBM tumorigenesis and angiogenesis.

The secreted glycoprotein YKL-40 is elevated (compared to normal) both in plasma and tumor tissue in several types of cancers including glioblastoma (GBM), which is a highly vascularized aggressive malignant brain tumor with a devastating prognosis and limited treatment options. YKL-40 regulates Vascular Endothelial Growth Factor (VEGF) and long-term inhibition of VEGF induces YKL-40, thereby making these pro-angiogenic factors important for the development of GBM vascular phenotype. When evaluated independently, both plasma and tumor tissue YKL-40 levels have proven prognostic in patients with GBM. However, no direct comparison of the two parameters has been reported yet. In search of reliable biomarkers in GBM, this study aimed at exploring the putative correlation between tumor/plasma YKL-40 and VEGF levels in matched primary GBM tissue biopsies and plasma samples and their impact on GBM patient survival. Biopsies and plasma samples from 50 patients with GBM were collected from 2005 to 2015 at Rigshospitalet, Copenhagen, Denmark in relation to neurosurgery, and analyzed for YKL-40 and VEGF by immunohistochemistry and commercial enzyme-linked immunosorbent assay. Clinical data including age, sex, steroid dose, performance status, progression-free survival, overall survival etc. have been compiled in a clinical database. Preliminary results evaluating the correlation between above-mentioned markers in plasma and tumor tissue and their impact on GBM patient survival will be presented. We sincerely hope that our data will provide some insights into the regulation of the GBM angiogenic phenotype and its contribution to poor prognosis in this fatal disease.

Abstract Background: Recurrent glioblastoma multiforme (GBM) is an aggressive and deadly disease with limited treatment options. In responding patients bevacizumab-containing therapy prolongs survival and improves quality of life, although the beneficial effect varies greatly. The impact of prognostic factors in recurrent GBM patients has not been studied in detail and results are inconsistent. More importantly, no validated predictive baseline markers associated with a clinical response to bevacizumab therapy have been identified. Furthermore, GBM is a very heterogenic tumor and has been shown to change molecular pattern with time and as a result of treatment. The primary goal of this study was to describe molecular characteristics in GBM tumors at initial diagnosis and at recurrence and relate these data, including clinical baseline factors, to clinical outcome with the aim of identifying predictive factors for clinical bevacizumab response. Based on these data we will also elucidate to what extent molecular markers change expression from initial GBM diagnosis to time of relapse. Materials and Methods: For generation of prognostic models 219 recurrent GBM patients treated with bevacizumab plus irinotecan according to a previous published clinical protocol were included. For biomarker analysis, routine molecular analyses were available for 147 of the included patients at time of initial diagnosis and 81 patients at time of relapse prior to bevacizumab therapy. The candidate biomarkers constituted an immunohistochemistry (IHC) panel of EGFR, P53, MGMT and IDH1 and polymerase chain reaction analysis of 1p19q-status. Tumor samples from initial diagnosis and from time of relapse were analyzed by the χ2 test and the Wilcoxon test in order to evaluate changes in IHC expression patterns. Multiple candidate prognostic factors were screened by univariate logistic regression and Cox regression analysis modeling response and survival endpoints and variables with P-values of less than 0.10 were considered for multivariate analysis. Factors with a P-value of less than 0.05 in multivariate analysis were included in the prognostic models for response, progression-free survival (PFS) and overall survival (OS). Results: In multivariate analysis, corticosteroid use had a negative predictive impact on response at first evaluation (OR = 0.45; 95% CI: 0.22-0.93; P = 0.030) and at best response (OR = 0.51; 95% CI: 0.26-1.02; P = 0.056). Independent prognostic factors (P<0.05) negatively associated with PFS and OS were corticosteroid use, neurocognitive deficit and multifocal disease. Based on the three identified factors a prognostic model for overall survival at 6 and 12 months was developed. Significant prognostic and potentially predictive molecular biomarkers will be added to the models and results will be presented. Citation Format: Thomas Urup, Signe Regner Michaelsen, Camilla Bjørnbak Holst, Anders Toft, Ib Jarle Christensen, Kirsten Grunnet, Michael Kosteljanetz, Helle Broholm, Ulrik Lassen, Hans Skovgaard Poulsen. A prognostic model for clinical response to bevacizumab in recurrent glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4306. doi:10.1158/1538-7445.AM2015-4306