Common opioids and stimulants in autopsy and DUID cases: A comparison of measured concentrations.

Quantitative results from toxicological analyses of autopsy material are widely compared to ranges in reference works to determine if drug concentrations are in relevant levels for establishing intoxication. This study compares concentrations of commonly used opioids and stimulants from drug addict autopsies and driving under the influence of drugs (DUID) cases to supplement current knowledge of the possible span and overlaps of measured concentrations. The study included whole-blood results from forensic autopsies of drug addicts performed from 2015 to 2020 (n = 220) and DUID cases from 2015 to 2019 (n = 7088). The focus was on heroin/morphine, methadone, cocaine, amphetamine and MDMA concentrations because these drugs are commonly encountered in both fatal intoxications and DUID cases and the potential for abuse is well known. In the DUID group, the opioids heroin/morphine and methadone and the stimulants amphetamine and MDMA were often seen in concentrations above the reported lower comatose-fatal level whereas cocaine was almost always below. Thus, based on our data, the potential for false assessment of intoxication cases when comparing to reported comatose-fatal limits appears greatest on lower end concentrations of heroin/morphine, methadone, amphetamine and MDMA, whereas false assessment of cocaine appears less likely because most control cases are below reported comatose-fatal levels.

THC-influenced drivers in the new Danish 3-level offense system.

Objective: The objectives of the study were to describe the distribution of tetrahydrocannabinol (THC)-influenced drivers in a new Danish 3-level offense system and discuss the consequences of the changed legislation.Methods: By request of the police, blood of individuals suspected of driving under the influence of drugs (DUID) in Funen and Southern Jutland was sampled by medical staff and shipped to the Section of Forensic Toxicology of the University of Southern Denmark in Odense. Samples from individuals suspected for driving under the influence of cannabis (DUIC) were stored at 5 °C prior to immediate analysis, and THC content in whole-blood samples was established by gas chromatography-mass spectrometry analysis. Quantitative results for blood THC levels were available from 2017 and 2018, resulting in 2,206 eligible cases. Data before and after the legal change on December 15, 2017, were extracted from the department's laboratory information management system.Results: With the new graduated sanctions introduced in December 2017, 70% of the DUIC suspects faced milder sanctions. The number of DUIC cases has been increasing and has almost doubled in the last 4 years, from 648 cases in 2015 to 1,206 in 2018. Correspondingly, the total number of DUID cases increased by 80% from 898 cases in 2015 to 1,614 cases in 2018. The concentration of THC in blood was above the legal limit of 0.001 mg/kg in 73% of the cases; 18% had concentrations categorized as low, 32% as medium, and 22% as high.Conclusion: We found that more than two-thirds of the THC offenders faced milder sanctions compared to before the new legislation, suggesting that the new graduated legislation has had a significant impact. The current trend is a steadily increasing number of DUID cases. We believe that the continual increase is caused by a combination of factors, including increased police awareness and training of police personnel along with improved roadside screening abilities.

Expression and prognostic value of the WEE1 kinase in gliomas.

High-grade gliomas have an aggressive clinical course and new clinical biomarkers and therapeutic targets are highly needed. WEE1 is a regulator of the G2 checkpoint in glioblastoma (GBM) cells. Inhibition of this kinase has, in experimental glioma studies, been suggested to enhance sensitivity to irradiation and temozolomide. However, expression level and prognostic potential of WEE1 protein in gliomas remain uninvestigated. In this study, glioma samples from 235 patients across all four WHO grades were analyzed by immunohistochemistry. Using image analysis, we calculated the area fraction of WEE1 positive nuclei. We found that WEE1 protein was localized in tumor cell nuclei and expressed in all glioma types and grades. Although WEE1 protein levels are higher in GBMs (mean 24.5%) relative to grade III (mean 14,0%, p < 0.05) and grade II (mean 6.8%, p < 0.001) gliomas, high WEE1 protein was associated with better survival in GBMs (p = 0.002). This was confirmed in multivariate analysis (HR 0.60, p = 0.003) even when adjusted for MGMT status (HR 0.60, p = 0.005). In conclusion, we report a nuclear expression of WEE1 protein in all glioma grades and types. The WEE1 positive nuclear area was correlated with malignancy grade but it was inversely associated with prognosis in GBM. Although WEE1 is a frequently occurring protein and has been proposed as a novel target in GBM, the role of WEE1 in glioma patient survival appears to be connected to the MGMT status and is more complex than previously anticipated.

miR-21 Is Linked to Glioma Angiogenesis: A Co-Localization Study.

MicroRNA-21 (miR-21) is the most consistently over-expressed microRNA (miRNA) in malignant gliomas. We have previously reported that miR-21 is upregulated in glioma vessels and subsets of glioma cells. To better understand the role of miR-21 in glioma angiogenesis and to characterize miR-21-positive tumor cells, we systematically stained consecutive serial sections from ten astrocytomas for miR-21, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), phosphatase and tensin homolog (PTEN), octamer-binding transcription factor 4 (Oct4), sex-determining region Y box 2 (Sox2) and CD133. We developed an image analysis-based co-localization approach allowing global alignment and quantitation of the individual markers, and measured the miR-21 in situ hybridization signal against the immunohistochemical staining of the six different markers. miR-21 significantly co-localized with the hypoxia- and angiogenesis-associated markers HIF-1α (p=0.0020) and VEGF (p=0.0096), whereas the putative miR-21 target, PTEN, was expressed independently of miR-21. Expression of stem cell markers Oct4, Sox2 and CD133 was not associated with miR-21. In six glioblastoma cultures, miR-21 did not correlate with the six markers. These findings suggest that miR-21 is linked to glioma angiogenesis, that miR-21 is unlikely to regulate PTEN, and that miR-21-positive tumor cells do not possess stem cell characteristics.

High levels of c-Met is associated with poor prognosis in glioblastoma.

The tyrosine kinase receptor c-Met has been suggested to be involved in crucial parts of glioma biology like tumor stemness, growth and invasion. The aim of this study was to investigate the prognostic value of c-Met in a population-based glioma patient cohort. Tissue samples from 238 patients with WHO grade I, II, III and IV tumors were analyzed using immunohistochemical staining and advanced image analysis. Strong c-Met expression was found in tumor cells, blood vessels, and peri-necrotic areas. At the subcellular level, c-Met was identified in the cytoplasm and in the cell membrane. Measurements of high c-Met intensity correlated with high WHO grade (p = 0.006) but no association with survival was observed in patients with WHO grade II (p = 0.09) or III (p = 0.17) tumors. High expression of c-Met was associated with shorter overall survival in patients with glioblastoma multiforme (p = 0.03). However the prognostic effect of c-Met in glioblastomas was time-dependent and only observed in patients who survived more than 8.5 months, and not within the first 8.5 months after diagnosis. This was significant in multivariate analysis (HR 1.99, 95 % CI 1.29-3.08, p = 0.002) adjusted for treatment and the clinical variables age (HR 1.01, 95 % CI 0.99-1.03, p = 0.30), performance status (HR 1.34, 95 % CI 1.17-1.53, p < 0.001), and tumor crossing midline (HR 1.28, 95 % CI 0.79-2.07, p = 0.29). In conclusion, this study showed that high levels of c-Met holds unfavorable prognostic value in glioblastomas.

MicroRNA biomarkers in glioblastoma.

Recent research suggests that deregulation of microRNAs (miRNAs) is involved in initiation and progression of many cancers, including gliomas and that miRNAs hold great potential as future diagnostic and therapeutic tools in cancer. MiRNAs are a class of short non-coding RNA sequences (18-24 nucleotides), which base-pair to target messenger RNA (mRNA) and thereby cause translational repression or mRNA degradation based on the level of complementarity between strands. Profiling miRNAs in clinical glioblastoma samples has shown aberrant expression of numerous miRNAs when compared to normal brain tissues. Understanding these alterations is key to developing new biomarkers and intelligent treatment strategies. This review presents an overview of current knowledge about miRNA alterations in glioblastoma while focusing on the clinical future of miRNAs as biomarkers and discussing the strengths and weaknesses of various methods used in evaluating their expression.

What is the clinical value of cancer stem cell markers in gliomas?

Recent data indicate that cancer stem cells (CSCs) are responsible for resistance of glioblastomas to radiotherapy and chemotherapy, thereby contributing to the poor survival of these patients. In order to identify novel prognostic markers in gliomas, several CSC markers have been investigated. This review summarizes current reports on putative glioma CSC markers and reviews the prognostic value of the individual immunohistochemical markers reported in the literature. Using the Pubmed database, twenty-seven CSC studies looking at membrane markers (CD133, podoplanin, CD15, and A2B5), filament markers (nestin), RNA-binding proteins (Musashi-1) and transcription factors (BMI1, SOX2, Id1 and Oct-4) qualified for this review. The level of CD133 and nestin increased with increasing malignancy grade, and for both markers a prognostic significance was identified in the majority of the studies. Moreover, the co-expression of CD133 and nestin was shown to have an even more powerful prognostic value than just single markers. Regarding podoplanin and Musashi-1, there was a trend towards a prognostic value when summarizing all studies. Especially the co-expression of Musashi-1 and MIB1 seemed promising. For the remaining markers CD15, A2B5, BMI1, SOX2, Id1 and Oct4, no prognostic value was found regarding overall survival in this review. In conclusion we find that CD133, nestin, CD133/nestin, podoplanin, Musashi-1 and Musashi-1/MIB1 are the most promising markers for future investigation. Evaluation in larger cohorts with known clinical data and known status of important biomarkers like MGMT and IDH1 is necessary to reveal their full clinical potential.

MiR-21 expression in the tumor cell compartment holds unfavorable prognostic value in gliomas.

High-grade gliomas are some of the most lethal forms of human cancer, and new clinical biomarkers and therapeutic targets are highly needed. MicroRNAs (miRNAs), a group of short noncoding RNAs, hold great potential as new biomarkers and targets as they are commonly deregulated in a variety of diseases including gliomas. MicroRNA-21 (miR-21) is the most consistently overexpressed miRNA in several cancers including gliomas and is therefore very promising as a useful clinical biomarker and therapeutic target. To better understand the role of miR-21 in gliomas, paraffin-embedded glioma tissue samples from 193 patients with grade I, II, III, and IV tumors were analyzed by in situ hybridization (ISH) using LNA-DNA chimeric probes. We found miR-21 expression in tumor cells and tumor-associated blood vessels, whereas no expression was seen in adjacent normal brain parenchyma. Using advanced image analysis we obtained quantitative estimates reflecting the miR-21 expression levels in each of these compartments. The miR-21 levels correlated significantly with grade [p = 0.027, r (s) = 0.161, 95 % confidence interval (CI), 0.015-0.301] with the highest levels measured in glioblastomas. Only tumor cell miR-21 was associated with poor prognosis when adjusting for known clinical parameters (age, grade, and sex) in a multivariate analysis [p = 0.049, hazard ratio (HR) = 1.545, 95 % CI, 1.002-2.381]. In conclusion, we have shown that miR-21 is located in both tumor cells and tumor blood vessels and that its level in the tumor cell compartment holds unfavorable prognostic value in gliomas.