Tumor volume and cancer stem cell expression as prognostic markers for high-dose loco-regional failure in head and neck squamous cell carcinoma - A DAHANCA 19 study.

BACKGROUND AND PURPOSE: Reliable and accessible biomarkers for patients with Head and Neck Squamous Cell Carcinoma (HNSCC) are warranted for biologically driven radiotherapy (RT). This study aimed to investigate the prognostic value of putative cancer stem cell (CSC) markers, hypoxia, and tumor volume using loco-regional high-dose failure (HDF) as endpoint. MATERIALS AND METHODS: Tumor tissue was retrieved from patients treated with primary chemo-(C-)RT and nimorazole for HNSCC in the Danish Head and Neck Cancer Study Group (DAHANCA) 19 study. Tumor volume, hypoxic classification, and expression of CSC markers CD44, SLC3A2, and MET were analyzed. For patients with eligible data on all parameters (n = 340), the risk of HDF following primary chemo-(C-)RT were analyzed by these biomarkers as a whole and stratified for p16-positive oropharynx (p16 + OPSCC) vs p16-negative (p16-) tumors (oral cavity, p16- oropharynx, hypopharynx and larynx). RESULTS: Higher risk of HDF was seen for patients with larger primary and nodal volume (>25 cm3, Hazard Ratio (HR): 3.00 [95 % CI: 1.73-5.18]), high SLC3A2 (HR: 2.99 [1.28-6.99]), CD44 (>30 % positive, HR: 2.29 [1.05-5.00]), and p16- tumors (HR: 2.53 [1.05-6.11]). p16- tumors had a higher CSC marker expression than p16 + OPSCC. The factors associated with the highest risk of HDF were larger volume (HR: 3.29 [1.79-6.04]) for p16- tumors (n = 178) and high SLC3A2 (HR: 6.19 [1.58-24.23]) for p16 + OPSCC (n = 162). CONCLUSION: Tumor volume, p16, and CSC markers are potential biomarkers for HDF for patients with HNSCC treated with (C-)RT. Lower expression of CSC in p16 + OPSCC may contribute to better tumor control.

Critically deviating vital signs among patients with non-specific diagnoses-A register-based historic cohort study.

BACKGROUND: One third of ambulance patients receive non-specific diagnoses in hospital. Mortality is 3-4%, however due to the high patient volume this group accounts for 20% of all deaths at day 30. Non-specific diagnoses do not provide much information on causes for death. Vital signs at first contact with ambulance personnel can act as a proxy for the patient's condition. Thus, we aimed to describe the prevalence of abnormal vital signs, as determined by a modified NEWS2, in ambulance patients who received a non-specific hospital diagnosis. Secondly, we examined the association between vital signs, NEWS2 scores, type of non-specific diagnosis, and mortality among these patients. METHODS: Register-based historic cohort study of ambulance patients aged 16+ in the North Denmark Region during 2012-2016, who received a non-specific diagnosis (ICD-10 chapters R or Z) at hospital. We used NEWS2 scores to determine if first vital signs were normal or deviating (including critical). Mortality was estimated with the Kaplan-Meier estimator. Association between vital signs and mortality was evaluated by logistic regression. RESULTS: We included 41,539 patients, 20.9% (N = 8,691) had normal vital signs, 16.3% (N = 6,766) had incomplete vital sign registration, 62.8% (N = 26,082) had deviating vital signs, and of these 6.8% (N = 1,779) were critical. If vital signs were incompletely registered or deviating, mortality was higher compared to normal vital signs. Patients with critical vital signs displayed the highest crude 48-hour and 30-day mortality (7.0% (5.9-8.3) and 13.4% (11.9-15.1)). Adjusting for age, sex, and comorbidity did not change that pattern. Across all vital sign groups, despite severity, the most frequent diagnosis assigned was Z039 observation for suspected disease or condition unspecified. CONCLUSIONS: Most ambulance patients with non-specific diagnoses had normal or non-critical deviating vital signs and low mortality. Around 4% had critical vital signs and high mortality, not explained by age or comorbidity.

D-2-hydroxyglutarate regulates human brain vascular endothelial cell proliferation and barrier function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) genes result in excessive production of (D)-2-hydroxyglutarate (D-2HG) which intrinsically modifies tumor cell epigenetics and impacts surrounding noncancerous cells through nonepigenetic pathways. However, whether D-2HG has a paracrine effect on endothelial cells in the tumor microenvironment needs further clarification. We quantified microvessel density by immunohistochemistry using tissue sections from 60 high-grade astrocytic gliomas with or without IDH mutation. Microvessel density was found to be reduced in tumors carrying an IDH mutation. Ex vivo experiments showed that D-2HG inhibited endothelial cell migration, wound healing, and tube formation by suppressing cell proliferation but not viability, possibly through reduced activation of the mTOR/STAT3 pathway. Further, D-2HG reduced fluorescent dextran permeability and decreased paracellular T-cell transendothelial migration by augmenting expression of junctional proteins thereby collectively increasing endothelial barrier function. These results indicate that D-2HG may influence the tumor vascular microenvironment by reducing the intratumoral vasculature density and by inhibiting the transport of metabolites and extravasation of circulating cells into the astrocytoma microenvironment. These observations provide a rationale for combining IDH inhibition with antitumor immunological/angiogenic approaches and suggest a molecular basis for resistance to antiangiogenic drugs in patients whose tumors express a mutant IDH allele.

Alcohol use and hepatocellular carcinoma risk in patients with alcohol-related cirrhosis.

OBJECTIVES: Insights into risk factors for hepatocellular carcinoma (HCC) among patients with alcohol-related cirrhosis (ALD cirrhosis) are important for decisions about HCC surveillance. We studied the effects of continued hazardous alcohol use in ALD cirrhosis on HCC risk. METHODS: Within a nationwide registry-based cohort of patients with ALD cirrhosis, we compared HCC risk between patients with a continued hazardous alcohol use and matched comparators. We used Fine-Gray regression to compare the risk of HCC and Cox regression to compare all-cause mortality. We also included patients with ALD cirrhosis in a clinical case-control study. Cases had HCC, and controls did not. Alcohol use was quantified using the AUDIT-C-questionnaire. Logistic regression was used to analyze the association between hazardous alcohol use and HCC risk. RESULTS: In the registry-based study, we included 8,616 patients with continued hazardous alcohol use and 8,616 matched comparators. Patients with a continued hazardous alcohol use had a lower HCC risk (subdistribution hazard ratio: 0.64, 95% confidence interval [CI]: 0.57 - 0.72) and higher mortality (hazard ratio: 1.62, 95% CI: 1.56 - 1.67). In the clinical study, we included 146 patients with ALD cirrhosis of whom 53 had newly diagnosed HCC. Hazardous alcohol use was insignificantly associated with a lower HCC risk (odds ratio: 0.61, 95% CI: 0.25 - 1.46). CONCLUSIONS: Hazardous alcohol use in patients with ALD cirrhosis is associated with higher mortality and, consequently, a lower HCC risk. Even if alcohol is carcinogenic, HCC surveillance will therefore likely be more effective in patients with ALD cirrhosis without a hazardous alcohol use.

Increased demand of urine cultures from Danish general practice: a five-year register-based study.

OBJECTIVE: To characterise and explore the development in the number and content of urine samples sent from general practice in the North Denmark Region to the Department of Clinical Microbiology (DCM) at Aalborg University Hospital during a five-year period. DESIGN: A register-based study. SETTING: General practice. SUBJECTS: Urine samples received at DCM, Aalborg University Hospital from general practice between 2017 and 2022. MAIN OUTCOME MEASURES: Number and content of urine samples. RESULTS: A total of 255,271 urine samples from general practice were received at DCM, with 76.1% being from female patients. Uropathogens were identified in 43.0% of the samples. During the five-year period, a 23.0% increase in the number of urine samples per person (incidence rate ratio (IRR) 1.23, 95% CI 1.21-1.25) was observed. A slight increase in the proportion of positive cultures (risk ratio (RR) 1.03, 95% CI 1.01-1.05) was seen. No notable change in the patient population (age, gender) was observed. Overall, Escherichia coli was the most identified uropathogen (60.4%) followed by Klebsiella spp. (8.7%) and Enterococcus spp. (7.7%). Distribution of the various uropathogens differed slightly depending on patient gender and age, importantly E. coli was less frequently observed in males aged >65 years. CONCLUSION: During the past five years an increasing amount of urine cultures have been requested at DCM from general practice. Importantly, the cause(s) of this increasing demand needs to be explored further in future studies.

Appropriate diagnostics of urinary tract infections can reduce the use of antibiotics in general practice.From 2017 to 2022 a 23% increase per person in requested urine cultures from general practice was observed.A slight increase in positive cultures was found, but no notable change in the patient population (age, gender) was seen.E. coli was the most identified uropathogen independent of gender and age, however, the proportion differed within the various groups.

Differential expression of checkpoint markers in the normoxic and hypoxic microenvironment of glioblastomas.

Glioblastoma is the most common primary malignant brain tumor in adults with an overall survival of only 14.6 months. Hypoxia is known to play a role in tumor aggressiveness but the influence of hypoxia on the immune microenvironment is not fully understood. The aim of this study was to investigate the expression of immune-related proteins in normoxic and hypoxic tumor areas by digital spatial profiling. Tissue samples from 10 glioblastomas were stained with a panel of 40 antibodies conjugated to photo-cleavable oligonucleotides. The free oligo-tags from normoxic and hypoxic areas were hybridized to barcodes for digital counting. Differential expression patterns were validated by Ivy Glioblastoma Atlas Project (GAP) data and an independent patient cohort. We found that CD44, Beta-catenin and B7-H3 were upregulated in hypoxia, whereas VISTA, CD56, KI-67, CD68 and CD11c were downregulated. PD-L1 and PD-1 were not affected by hypoxia. Focusing on the checkpoint-related markers CD44, B7-H3 and VISTA, our findings for CD44 and VISTA could be confirmed with Ivy GAP RNA sequencing data. Immunohistochemical staining and digital quantification of CD44, B7-H3 and VISTA in an independent cohort confirmed our findings for all three markers. Additional stainings revealed fewer T cells and high but equal amounts of tumor-associated microglia and macrophages in both hypoxic and normoxic regions. In conclusion, we found that CD44 and B7-H3 were upregulated in areas with hypoxia whereas VISTA was downregulated together with the presence of fewer T cells. This heterogeneous expression should be taken into consideration when developing novel therapeutic strategies.

Albumin-corrected Zn and available free Zn-binding capacity as indicators of Zn status - potential for clinical implementation.

We have established and describe two measurement procedures to diagnose possible zinc (Zn) deficiency; albumin-corrected Zn concentration and available free Zn-binding capacity. Reference intervals for both biomarkers were established in healthy adults from the Danish population. The clinical usefulness of the measurement procedures was investigated in patients with cirrhosis and in patients given parenteral nutrition due to short bowel syndrome. The results of both methods indicate that there is a risk of overdiagnosing Zn deficiency based on low plasma Zn concentrations. Needless Zn supplementation may thus be avoided by using the albumin-corrected Zn concentration or available free Zn-binding capacity.

Stage-dependent expression of fibrogenic markers in alcohol-related liver disease.

Alcohol-related liver disease (ALD) is the primary cause of liver-related mortality and morbidity. Liver fibrosis remains the best validated surrogate marker for patient prognosis and is usually assessed in liver biopsies using histochemical stains detecting collagen as the major extracellular matrix (ECM) component in ALD. Distinction of collagen subtypes is of clinical interest, as they, including their cleavage products, have different functions. Changes in production, distribution, and composition of specific collagen subtypes and other extracellular matrix (ECM) components as well as markers for their main cellular source (activated hepatic stellate cells (aHSCs) and myofibroblasts (MFBs)) have not been fully elucidated in ALD. Our aims were to investigate the stage-dependent expression of collagen subtypes and markers for aHSCs and MFBs in ALD. We included liver biopsies from 49 ALD patients with fibrosis stages F0-F4. Biopsies were classified according to the semi-quantitative non-alcoholic fatty liver disease (NAFLD) activity score (NAS-CRN). Slides were stained with H&E, Sirius Red, and immunohistochemically with antibodies against collagen types I, III, IV and VI, and aHSC/MFB markers α-SMA, osteonectin, and CD271. Expression was examined using automated digital imaging analysis (DIA), by calculating the positive area relative to the total liver biopsy area (proportionate area). Likewise, collagen-proportionate area (CPA) was assessed using DIA of Sirius Red stained slides. CPA correlated highly with fibrosis stage (rs = 0.88, P = 5.9E-17) and moderately with activity score (rs = 0.58, P = 0.00001). Collagen type I proportionate area increased from 0.3% (± 0.1) at F1 to 10.2% (± 1.6) at F4 (P < 0.001). Collagen type III proportionate area increased from 0.6% (± 0.2) at F0 to 11.9% (± 1.7) at F4 (P < 0.001). Collagen type IV proportionate area increased from 17.0% (± 2.5) at F0 to 27.0% (± 3.9) at F4 (P = 0.079). Collagen type VI proportionate area increased from 14.8% (± 1.4) at F0 to 31.4% (± 2.4) at F4 (P < 0.001). Proportionate areas for α-SMA and CD271 increased from 10.7% (± 1.6) and 6.5% (± 1.0) at F0 to 29.5% (± 3.4) and 22.1% (± 2.2) at F4 (P < 0.001). Proportionate area for osteonectin increased from 1.4-1.8% at F0-F2 to 3.1% (± 0.5) at F3 and 3.2% (± 0.6) at F4 (P < 0.05). In conclusion, our data indicate that collagen types I, III and VI and the aHSC/MFB markers α-SMA and CD271 show a stage-dependent increase in ALD. In early ALD, collagen types IV and VI are important components of the perisinusoidal and pericellular fibrosis. Immunohistochemistry is a valuable additional tool to characterize the ECM changes in ALD. Further research is needed to explore the functional role of CD271 and the stage-dependent expression of other collagen subtypes in ALD.

Tumour-associated CD204+ microglia/macrophages accumulate in perivascular and perinecrotic niches and correlate with an interleukin-6-enriched inflammatory profile in glioblastoma.

AIMS: Glioblastomas are heterogeneous tumours with a rich tumour microenvironment particularly comprised of tumour-associated microglia/macrophages (TAMs), but also containing a population of dedifferentiated/stem-like glioblastoma cells. Both cell populations contribute to tumour aggressiveness and immune evasion through the actions of various signalling molecules. The scavenger and pattern recognition receptor CD204 is associated with a pro-tumourigenic phenotype of TAMs and has a negative prognostic value. Our objective was to investigate the possible interaction between TAMs and dedifferentiated glioblastoma cells and characterise the myeloid phenotype of CD204-enriched glioblastomas. METHODS: Double immunohistochemistry and cell counting was performed on eight glioblastoma samples to estimate the expression and interaction level between dedifferentiated/stem-like tumour cells and TAMs. Using the NanoString technology, myeloid transcriptome profiling was performed on 46 glioblastomas, which had been selected based on their protein expression levels of CD204 and ionised calcium-binding adaptor molecule-1 (IBA1). The results were validated by immunohistochemistry and in silico gene expression analyses. RESULTS: TAMs especially CD204+ TAMs accumulated in perivascular and perinecrotic niches in close proximity to podoplanin+ glioblastoma cells. Gene profiling revealed that CD204-enriched glioblastoma has a unique signature with upregulation of genes related to hypoxia, angiogenesis and invasion, including interleukin-6. The gene signature favoured a poor prognosis in patients with glioblastoma. CONCLUSIONS: This is the first study to characterise the role of CD204 in the myeloid microenvironment of glioblastoma. Our results support the unfavourable prognostic impact of CD204 and suggest that CD204 and interleukin-6 could serve as targets for re-education of TAMs and potentiation of current anti-glioma therapy.

The Epigenetic Regulator Jumonji Domain-Containing Protein 6 (JMJD6) Is Highly Expressed but Not Prognostic in IDH-Wildtype Glioblastoma Patients.

Patients with IDH-wildtype glioblastoma (GBM) generally have a poor prognosis. However, there is an increasing need of novel robust biomarkers in the daily clinico-pathological setting to identify and support treatment in patients who become long-time survivors. Jumonji domain-containing protein 6 (JMJD6) is involved in epigenetic regulation of demethylation of histones and has been associated with GBM aggressiveness. We investigated the expression and prognostic potential of JMJD6 tumor fraction score in 184 IDH-wildtype GBMs. Whole-slides were double-stained with an antibody against JMJD6 and an exclusion-cocktail consisting of 4 antibodies (CD31, SMA, CD45, and Iba-1), enabling evaluation of tumor cells only. Stainings were quantified with a combined software- and scoring-based approach. For comparison, IDH-mutated WHO grade II, III and IV astrocytic gliomas were also stained, and the JMJD6 tumor fraction score increased with increasing WHO grade, although not significantly. In multivariate analysis including age, gender, performance status and post-surgical treatment high JMJD6 tumor fraction score was associated with longer overall survival in IDH-wildtype GBMs (p = 0.03), but the effect disappeared when MGMT promoter status was included (p = 0.34). We conclude that JMJD6 is highly expressed in IDH-wildtype GBM but it has no independent prognostic value.

Prognostic role of Ki-67 in glioblastomas excluding contribution from non-neoplastic cells.

Survival of glioblastoma patients varies and prognostic markers are important in the clinical setting. With digital pathology and improved immunohistochemical multiplexing becoming a part of daily diagnostics, we investigated the prognostic value of the Ki-67 labelling index (LI) in glioblastomas more precisely than previously by excluding proliferation in non-tumor cells from the analysis. We investigated the Ki-67 LI in a well-annotated population-based glioblastoma patient cohort (178 IDH-wildtype, 3 IDH-mutated). Ki-67 was identified in full tumor sections with automated digital image analysis and the contribution from non-tumor cells was excluded using quantitative double-immunohistochemistry. For comparison of the Ki-67 LI between WHO grades (II-IV), 9 IDH-mutated diffuse astrocytomas and 9 IDH-mutated anaplastic astrocytomas were stained. Median Ki-67 LI increased with increasing WHO grade (median 2.7%, 6.4% and 27.5%). There was no difference in median Ki-67 LI between IDH-mutated and IDH-wildtype glioblastomas (p = 0.9) and Ki-67 LI was not associated with survival in glioblastomas in neither univariate (p = 0.9) nor multivariate analysis including MGMT promoter methylation status and excluding IDH-mutated glioblastomas (p = 0.2). Ki-67 may be of value in the differential diagnostic setting, but it must not be over-interpreted in the clinico-pathological context.

Expression and Prognostic Value of the Immune Checkpoints Galectin-9 and PD-L1 in Glioblastomas.

Immunotherapeutic targeting of the PD-1/PD-L1 axis has been widely implemented for treatment of several cancer types but shown disappointing results in glioblastomas (GBMs), potentially due to compensatory mechanisms of other expressed immune checkpoints. Galectin-9 is an immune-checkpoint protein that facilitates T-cell exhaustion and apoptosis and could be a potential target for immune-checkpoint inhibition. A total of 163 GBMs IDH wildtype were immunostained with anti-Galectin-9 and PD-L1 antibodies. Software-based quantitation of immunostainings was performed and co-expression was investigated using double immunofluorescence. Both Galectin-9 and PD-L1 protein expression were found in all 163 tumors and showed a significant positive correlation (p = 0.0017). Galectin-9 expression varied from 0.01% to 32% (mean = 6.61%), while PD-L1 membrane expression ranged from 0.003% to 0.14% (mean = 0.048%) of total tumor area. Expression of Galectin-9 and PD-L1 was found on both microglia/macrophages and tumor cells, and colocalization of both markers was found in 88.3% of tumors. In multivariate analysis, neither Galectin-9 (HR = 0.99), PD-L1 (HR = 1.05), nor their combinations showed prognostic value. Galectin-9 and PD-L1 were expressed in all investigated GBMs and the majority of patients had co-expression, which may provide rationale for multi-targeted immune checkpoint inhibition.

Role of B-cells in Mycosis Fungoides.

Mycosis fungoides is the most common type of cutaneous T-cell lymphoma. The inflammatory micro-environment in mycosis fungoides is complex. There is accumulating evidence that the neoplastic T-cells take control of the microenvironment and thereby promote their own expansion by suppressing cellular immunity. B-cells have proved to be upregulated in large-cell transformed mycosis fungoides, and could potentially play a role in disease progression. To investigate the presence of B-cells in mycosis fungoides compared with controls, this study analysed 85 formalin-fixed and paraffin-embedded mycosis fungoides biopsies. MS4A1 gene expression was significantly upregulated in mycosis fungoides compared with controls (p < 0.0001) and further upregulated in disease progression, (p = 0.001). Digital quantification of PAX5+/CD20+ cells confirmed the increased presence of B-cells in mycosis fungoides compared with controls. No co-labelling of CD3/CD20 was observed in the neoplastic T-cells. This study found a significantly increased presence of B-cells in the tumour-associated microenvironment in mycosis fungoides. These findings could potentially lead to new treatment strategies for mycosis fungoides.

Gene expression profiling of morphologic subtypes of pancreatic ductal adenocarcinoma using surgical and EUS-FNB specimens.

BACKGROUND/OBJECTIVES: Various classifications of pancreatic ductal adenocarcinoma (PDAC) based on RNA profiling resulted in two main subtypes. Kalimuthu and coworkers proposed a morphology-based classification that concurred with these subtypes. Immune therapy approaches in PDAC were so far disappointing. Morphologic PDAC subtypes may differ regarding key immune-oncology pathways. We aimed to examine the reproducibility and prognostic value of Kalimuthu's morphologic classification, and to evaluate differences between subtypes regarding gene expression related to tumor biology and immune-oncology. METHODS: PDAC specimens from 196 patients were included, 108 consecutive chemotherapy-naïve surgical specimens and 88 endoscopic ultrasound-guided fine needle biopsies (EUS-FNBs). The specimens were evaluated as per Kalimuthu by two pancreatic pathologists, resulting in Group A and Group B tumors. Digital mRNA expression profiling was performed, on the surgical specimens using the NanoString IO360 panel of 770 key tumor biology related and 30 custom-genes, and on the EUS-FNBs using a targeted panel of 123 genes. RESULTS: Morphologic subtyping reached substantial interobserver agreement between the two pathologists. In the surgical and EUS-FNB cohorts, 44.4% and 38.6% were Group A tumors, which were associated with improved survival. Group A showed higher expression of immune-related genes and cytokine/chemokine/interleukin signaling and Group B of genes related to cancer cell proliferation and cell cycle regulation. Hierarchical clustering based on significant differences in gene expression levels between Groups A and B revealed clusters with prognostic value. CONCLUSIONS: Morphologic subtyping according to Kalimuthu is reproducible and holds prognostic value, in surgical as well as EUS-FNB specimens. As upregulation of immune-related genes was found in Group A, future studies should evaluate the potential of immune therapy approaches with special emphasis on this subtype of PDAC.

Microglia-Secreted Factors Enhance Dopaminergic Differentiation of Tissue- and iPSC-Derived Human Neural Stem Cells.

Microglia have recently been established as key regulators of brain development. However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1ß, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate in vitro derivation of dopaminergic neurons.

Drivers of carbon stocks in forest edges across Europe.

Forests play a key role in global carbon cycling and sequestration. However, the potential for carbon drawdown is affected by forest fragmentation and resulting changes in microclimate, nutrient inputs, disturbance and productivity near edges. Up to 20% of the global forested area lies within 100 m of an edge and, even in temperate forests, knowledge on how edge conditions affect carbon stocks and how far this influence penetrates into forest interiors is scarce. Here we studied carbon stocks in the aboveground biomass, forest floor and the mineral topsoil in 225 plots in deciduous forest edges across Europe and tested the impact of macroclimate, nitrogen deposition and smaller-grained drivers (e.g. microclimate) on these stocks. Total carbon and carbon in the aboveground biomass stock were on average 39% and 95% higher at the forest edge than 100 m into the interior. The increase in the aboveground biomass stock close to the edge was mainly related to enhanced nitrogen deposition. No edge influence was found for stocks in the mineral topsoil. Edge-to-interior gradients in forest floor carbon changed across latitude: carbon stocks in the forest floor were higher near the edge in southern Europe. Forest floor carbon decreased with increasing litter quality (i.e. high decomposition rate) and decreasing plant area index, whereas higher soil temperatures negatively affected the mineral topsoil carbon. Based on high-resolution forest fragmentation maps, we estimate that the additional carbon stored in deciduous forest edges across Europe amounts to not less than 183 Tg carbon, which is equivalent to the storage capacity of 1 million ha of additional forest. This study underpins the importance of including edge influences when quantifying the carbon stocks in temperate forests and stresses the importance of preserving natural forest edges and small forest patches with a high edge-to-interior surface area.

The presence of TIM-3 positive cells in WHO grade III and IV astrocytic gliomas correlates with isocitrate dehydrogenase mutation status.

Diffuse gliomas are aggressive brain tumors that respond poorly to immunotherapy including immune checkpoint inhibition. This resistance may arise from an immunocompromised microenvironment and deficient immune recognition of tumor cells because of low mutational burden. The most prominent genetic alterations in diffuse glioma are mutations in the isocitrate dehydrogenase (IDH) genes that generate the immunosuppressive oncometabolite d-2-hydroxyglutarate. Our objective was to explore the association between IDH mutation and presence of cells expressing the immune checkpoint proteins galectin-9 and/or T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Astrocytic gliomas of World Health Organization (WHO) grades III or IV (36 IDH-mutant and 36 IDH-wild-type) from 72 patients were included in this study. A novel multiplex chromogenic immunohistochemistry panel was applied using antibodies against galectin-9, TIM-3, and the oligodendrocyte transcription factor 2 (OLIG2). Validation studies were performed using data from The Cancer Genome Atlas (TCGA) project. IDH mutation was associated with decreased levels of TIM-3+ cells (p < 0.05). No significant association was found between galectin-9 and IDH status (p = 0.10). Most TIM-3+ and galectin-9+ cells resembled microglia/macrophages, and very few TIM-3+ and/or galectin-9+ cells co-expressed OLIG2. The percentage of TIM-3+ T cells was generally low, however, IDH-mutant tumors contained significantly fewer TIM-3+ T cells (p < 0.01) and had a lower interaction rate between TIM-3+ T cells and galectin-9+ microglia/macrophages (p < 0.05). TCGA data confirmed lower TIM-3 mRNA expression in IDH-mutant compared to IDH-wild-type astrocytic gliomas (p = 0.013). Our results show that IDH mutation is associated with diminished levels of TIM-3+ cells and fewer interactions between TIM-3+ T cells and galectin-9+ microglia/macrophages, suggesting reduced activity of the galectin-9/TIM-3 immune checkpoint pathway in IDH-mutant astrocytic gliomas.

Expression Profiling of Primary and Recurrent Glioblastomas Reveals a Reduced Level of Pentraxin 3 in Recurrent Glioblastomas.

Glioblastomas (GBM) are highly infiltrative tumors and despite intensive treatment tumor recurrence is inevitable. The immune microenvironment in recurrent GBM is poorly characterized, but it is potentially influenced by therapeutic interventions with surgery, radiotherapy, and chemotherapy. The aim of this study was to obtain a deeper insight in the immune microenvironment in primary and recurrent GBM. Primary and recurrent glioblastoma samples from 18 patients were identified and expression profiling of 770 myeloid innate immune-related markers was performed. Leukemia inhibitory factor and pentraxin 3 were expressed at lower levels in recurrent tumors. Using in silico data and immunohistochemical staining, this was validated for pentraxin 3. Both high leukemia inhibitory factor and pentraxin 3 expression appeared to be associated with shorter survival in primary and recurrent GBM using in silico data. In primary GBM, gene set analysis also showed higher expression of genes involved in metabolism, extracellular matrix remodeling and complement activation, whereas genes involved in T cell activation and checkpoint signaling were expressed at higher levels in recurrent GBM. The reduced level of pentraxin 3 in recurrent glioblastomas and the gene set analysis results suggest an altered microenvironment in recurrent GBM that might be more active.

Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas.

Most glioblastoma patients have a dismal prognosis, although some survive several years. However, only few biomarkers are available to predict the disease course. EGR1 and EGR3 have been linked to glioblastoma stemness and tumour progression, and this study aimed to investigate their spatial expression and prognostic value in gliomas. Overall 207 gliomas including 190 glioblastomas were EGR1/EGR3 immunostained and quantified. A cohort of 21 glioblastomas with high P53 expression and available tissue from core and periphery was stained with double-immunofluorescence (P53-EGR1 and P53-EGR3) and quantified.EGR1 expression increased with WHO-grade, and declined by 18.9% in the tumour periphery vs. core (P = 0.01), while EGR3 expression increased by 13.8% in the periphery vs. core (P = 0.04). In patients with high EGR1 expression, 83% had methylated MGMT-promoters, while all patients with low EGR1 expression had un-methylated MGMT-promoters. High EGR3 expression in MGMT-methylated patients was associated with poor survival (HR = 1.98; 95%CI 1.22-3.22; P = 0.006), while EGR1 high/EGR3 high, was associated with poor survival vs. EGR1 high/EGR3 low (HR = 2.11; 95%CI 1.25-3.56; P = 0.005). EGR1 did not show prognostic value, but could be involved in MGMT-methylation. Importantly, EGR3 may be implicated in cell migration, while its expression levels seem to be prognostic in MGMT-methylated patients.

SoilTemp: A global database of near-surface temperature.

Current analyses and predictions of spatially explicit patterns and processes in ecology most often rely on climate data interpolated from standardized weather stations. This interpolated climate data represents long-term average thermal conditions at coarse spatial resolutions only. Hence, many climate-forcing factors that operate at fine spatiotemporal resolutions are overlooked. This is particularly important in relation to effects of observation height (e.g. vegetation, snow and soil characteristics) and in habitats varying in their exposure to radiation, moisture and wind (e.g. topography, radiative forcing or cold-air pooling). Since organisms living close to the ground relate more strongly to these microclimatic conditions than to free-air temperatures, microclimatic ground and near-surface data are needed to provide realistic forecasts of the fate of such organisms under anthropogenic climate change, as well as of the functioning of the ecosystems they live in. To fill this critical gap, we highlight a call for temperature time series submissions to SoilTemp, a geospatial database initiative compiling soil and near-surface temperature data from all over the world. Currently, this database contains time series from 7,538 temperature sensors from 51 countries across all key biomes. The database will pave the way toward an improved global understanding of microclimate and bridge the gap between the available climate data and the climate at fine spatiotemporal resolutions relevant to most organisms and ecosystem processes.