Simon Kjær Hermansen

Hair testing is a powerful tool in forensic toxicology, particularly for identifying past substance exposure when blood or urine samples are collected too late. Its extended window for substance detection enables the identification of previously consumed substances and patterns of exposure. Hair, from the root to the tip, has frequently been used in forensic toxicology to assess both a single dose and long‐term exposures in drug‐facilitated crimes, as well as in investigations of the cause of death. The ongoing development and increasing use of hair testing underscore the need for a fresh assessment of its evolving potential. This focus article provides insight into the field of hair testing, focusing on recent applications, emerging trends, and advancements in the hair testing workflow from sampling to interpretation. Recent trends to address factors influencing substance levels in hair involve new protocols for micro‐segmentation, longitudinal hair scanning, assessment of melanin content and verification of cosmetic hair treatments. Advancing the field of hair testing requires the continuous development and refinement of processes to reveal forensic relevant substances such as psychoactive substances, toxins, doping agents, and environmental contaminants and their distribution in the hair. It is generally agreed that standardized hair testing procedures are necessary to promote a shared framework for interpretation that can assist forensic toxicologists in evaluating substances in hair. This may enhance reliability and broaden the applications of hair testing. This article is categorized under: Toxicology > Analytical Toxicology > Hair Toxicology > Drug Analysis

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.

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06–23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93–33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45–0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.

Astrocytomas are the most frequent primary brain tumors in adults, and despite aggressive treatment patients often experience recurrence. Survival decreases with increasing tumor grade, and especially patients with grade IV glioblastoma have poor prognosis due to the aggressive character of this tumor. Matrix metalloproteinase-2 (MMP-2) is an extracellular matrix degrading enzyme which has been shown to play important roles in different cancers. The aim of this study was to investigate the expression and prognostic potential of MMP-2 in astrocytomas. Tissue samples from 89 patients diagnosed with diffuse astrocytoma, anaplastic astrocytoma and glioblastoma were stained immunohistochemically using a monoclonal MMP-2 antibody. The MMP-2 intensity in cytoplasm/membrane was quantified by a trained software-based classifier using systematic random sampling in 10% of the tumor area. We found MMP-2 expression in tumor cells and blood vessels. Measurements of MMP-2 intensity increased with tumor grade, and MMP-2 expression was found to be significantly higher in glioblastomas compared to normal brain tissue (p<0.001), diffuse astrocytomas (p<0.001) and anaplastic astrocytomas (p<0.05). MMP-2 expression was associated with shorter overall survival in patients with grade II-IV astrocytic tumors (HR 1.60; 95% CI 1.03–2.48; p = 0.036). In glioblastoma, high MMP-2 was associated with poorer prognosis in patients who survived longer than 8.5 months independent of age and gender (HR 2.27; 95% CI 1.07–4.81; p = 0.033). We found a positive correlation between MMP-2 and tissue inhibitor of metalloproteinases-1 (TIMP-1), and combined MMP-2 and TIMP-1 had stronger prognostic value than MMP-2 alone also when adjusting for age and gender (HR 2.78; 95% CI 1.30–5.92; p = 0.008). These findings were validated in bioinformatics databases. In conclusion, this study indicates that MMP-2 is associated with aggressiveness in astrocytomas and may hold an unfavorable prognostic value in patients with glioblastoma.

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.

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.

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.

SUMMARY Aim: Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. Materials & methods: Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. Results: MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. Conclusion: MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.