Use of Multiple Machine Learning Approaches for Selecting Urothelial Cancer-Specific DNA Methylation Biomarkers in Urine.

Diagnosing urothelial cancer (UCa) via invasive cystoscopy is painful, specifically in men, and can cause infection and bleeding. Because the UCa risk is higher for male patients, urinary non-invasive UCa biomarkers are highly desired to stratify men for invasive cystoscopy. We previously identified multiple DNA methylation sites in urine samples that detect UCa with a high sensitivity and specificity in men. Here, we identified the most relevant markers by employing multiple statistical approaches and machine learning (random forest, boosted trees, LASSO) using a dataset of 251 male UCa patients and 111 controls. Three CpG sites located in ALOX5, TRPS1 and an intergenic region on chromosome 16 have been concordantly selected by all approaches, and their combination in a single decision matrix for clinical use was tested based on their respective thresholds of the individual CpGs. The combination of ALOX5 and TRPS1 yielded the best overall sensitivity (61%) at a pre-set specificity of 95%. This combination exceeded both the diagnostic performance of the most sensitive bioinformatic approach and that of the best single CpG. In summary, we showed that overlap analysis of multiple statistical approaches identifies the most reliable biomarkers for UCa in a male collective. The results may assist in stratifying men for cystoscopy.

Biomonitoring of polycyclic aromatic hydrocarbons in firefighters at fire training facilities and in employees at respiratory protection and hose workshops.

Introduction: Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic to humans and are formed by incomplete combustion. PAHs are always present during firefighting operations, and fire department members can be exposed to them in the workplace. Methods: In this study, we analyzed 1-hydroxypyrene (1-OHP) in 36 urine samples from nine firefighters, collected before and after fire training sessions, and 32 urine samples from eight employees at respiratory protection and hose workshops. To assess breakthrough PAH exposure through personal protective equipment and potential dermal uptake, some of the workshop employees wore cotton garments under their regular workwear. Cotton samples were then examined for the presence of 17 semi-volatile and low-volatility PAHs. Results: After firefighting exercises, we observed approximately a fivefold increase in mean 1-OHP concentrations in samples from firefighters, from 0.24 µg/L to 1.17 µg/L (maximum: 5.31 µg/L). In contrast, 1-OHP levels in workshop employees were found to be low, with the majority of urine samples yielding concentrations below the limit of quantification (LOQ: 0.05 µg/L, maximum: 0.11 µg/L). Similarly, low PAH levels were found on the workshop employees' cotton undergarments, with maximum concentrations of 250 and 205 ng/g for pyrene and benzo[a]pyrene, respectively. Discussion: In conclusion, significant increases in 1-OHP in urine were observed in firefighters after training sessions, whereas work-related exposure remained low among workshop employees.

Quantitative Metabolism and Urinary Elimination Kinetics of Seven Neonicotinoids and Neonicotinoid-Like Compounds in Humans.

Reverse dosimetry, i.e., calculating the dose of hazardous substances that has been taken up by humans based on measured analyte concentrations in spot urine samples, is critical for risk assessment and requires metabolic and kinetic data. We quantitatively studied the metabolism of seven major neonicotinoid and neonicotinoid-like compounds (NNIs) after single oral doses in male volunteers and determined key kinetic parameters and urinary elimination for NNIs together with their metabolites. Complete and consecutive urine samples were collected over 48 h. All samples were analyzed by tandem mass spectrometry, following liquid or gas chromatographic separation. Single- and group-specific NNI metabolites were quantified, i.e., hydroxylated and N-dealkylated NNIs and NNI-associated carboxylic acids and their glycine derivatives. Large, substance-dependent variations of key toxicokinetic parameters were observed. Mean times of concentration maxima (tmax) in urine varied between 2.0 (imidacloprid) and 25.8 h (N-desmethyl-clothianidin), whereas mean urinary elimination half-times (t1/2) were between 2.5 (acetamiprid) and 49.5 h (sulfoxaflor). Mean 48 h excretion fractions (Fue's) were between 0.03% (2-chloro-1,3-thiazole-5-carboxylic acid glycine) and 84% (clothianidin). In contrast, the interindividual differences of Fue's between the volunteers for each of the NNIs and their metabolites remained low (below a factor of 2 between the maximum and minimum derived Fue with the exception of 6-chloronicotinic acid in the acetamiprid dose study). The obtained quantitative data enabled choosing appropriate biomarkers for exposure assessment and, at the same time, for risk assessment by reverse dosimetry at current environmental exposures, i.e., comparing the calculated doses that have been taken up to currently available acceptable daily intakes of NNIs.

Impact of diesel exhaust exposure on urinary 1-hydroxypyrene in underground salt and potash workers.

BACKGROUND: Diesel engine exhaust (DEE) and some of the polycyclic aromatic hydrocarbons (PAH) it contains are carcinogenic to humans (for example benzo(a)pyrene) and can cause lung cancer in workers. The objective of this study was to assess exposures to DEE and its component PAH and the potential associations between these two health hazards in a salt and potash mining population. METHODS: Between 2017 and 2019, 1003 underground workers (mining n = 801, maintenance n = 202) and 243 above-ground facility workers from two German mines participated. Personal exposure to DEE was assessed in air as elemental carbon for diesel particulate matter (EC-DPM), whereas exposure to PAH was assessed in pre- and post-shift urine samples in terms of 1-hydroxypyrene (1-OHP). Associations between EC-DPM and 1-OHP were studied using linear regression models. RESULTS: The highest EC-DPM exposures were measured in mining workers (median 0.06 mg/m³) followed by workers in the maintenance (0.03 mg/m3) and facility areas (<0.02 mg/m3). Exposures above the current German occupational threshold level of 0.05 mg/m3 were observed in 56%, 17%, and 5% of mining, maintenance and facility workers, respectively. 1-OHP increased statistically significantly across a work shift in underground workers but not in facility workers. Regression analyses revealed an increase of post-shift 1-OHP by almost 80% in mining and 40% in maintenance compared with facility workers. 1-OHP increased with increasing EC-DPM among underground workers. However, internal exposure of 1-OHP mainly remained at levels similar to those of the German general population in more than 90% of the urine samples. CONCLUSIONS: While exposures to DEE above the current German OEL for EC-DPM are quite common in the studied population of underground salt and potash miners (39.5% overall), urinary concentrations of 1-OHP did not reflect these findings.

Quantitation of 6-chloronicotinic acid and 2-chloro-1,3-thiazole-5-carboxylic acid and their glycine conjugates in human urine to assess neonicotinoid exposure.

Neonicotinoids and neonicotinoid-like compounds (NNIs) are widely used insecticides and their ubiquitous occurrence in the environment requires methods for exposure assessment in humans. The majority of the NNIs can be divided into 6-chloropyridinyl- and 2-chlorothiazolyl-containing compounds, suggesting the formation of the group-specific metabolites 6-chloronicotinic acid (6-CNA), 2-chloro-1,3-thiazole-5-carboxylic acid (2-CTA), and their respective glycine derivatives (6-CNA-gly, 2-CTA-gly). Here, we developed and validated an analytical method based on gas chromatography coupled to mass spectrometry (GC-MS/MS) to simultaneously analyze these four metabolites in human urine. As analytical standards for the glycine conjugates were not commercially available, we synthesized 6-CNA-gly, 2-CTA-gly, and their 13C2,15N-labeled analogs for internal standardization and quantitation by stable isotope dilution. We also ensured chromatographic separation of 6-CNA and its isomer 2-CNA. Enzymatic cleavage during sample preparation was proven unnecessary. The limits of quantitation were between 0.1 (6-CNA) and 0.4 µg/L (2-CTA-gly) and the repeatability was satisfactory (coefficient of variation was <19% over the calibration range). We analyzed 38 spot urine samples from the general population and were able to quantify 6-CNA-gly in 58% of the samples (median 0.2 µg/L). In contrast, no 6-CNA could be detected. The results are in line with well-known metabolic pathways specific in humans, that, compared to rodents, favor the formation and excretion of phase-II-metabolites (glycine derivatives) rather than phase-I metabolites (free carboxylic acids). Nevertheless, the exact source of exposure (i.e., the specific NNI) remains elusive in the general population, may even vary quantitatively between different NNIs, and also might be regional specific based on the respective use of individual NNIs. In sum, we developed a robust and sensitive analytical method for the determination of four group-specific NNI metabolites.

Exposure to polycyclic aromatic hydrocarbons assessed by biomonitoring of firefighters during fire operations in Germany.

BACKGROUND: Firefighters are exposed to a variety of hazardous substances including carcinogens such as polycyclic aromatic hydrocarbons (PAH) during firefighting. In order to minimize the uptake of such substances into the body, firefighters wear personal protective equipment. Only few data exist from real-life firefighting missions and under common although highly variable exposure scenarios such as fighting fires in residential buildings, outdoor, and vehicle fires. The aim of this study is to assess the levels of 1-Hydroxypyrene (1-OHP) as marker for incorporated PAH during firefighting operations in Germany using biomonitoring methods. METHODS: We analyzed urine samples for 1-OHP from 77 firefighters who reported firefighting operations (with and without creatinine adjustment). Urine samples were collected before (baseline) and, where applicable, after firefighting operations at three time points subsequent (2-4, 6-8, and 12 h). RESULTS: Compared to the baseline measurements, mean 1-OHP concentrations after firefighting missions were doubled (0.14 vs. 0.31 µg/L urine, 0.13 µg/g vs. 0.27 µg/g creatinine) and this increase was observed 2-4 h after firefighting. Firefighting in residential buildings (N = 54) and of outdoor and vehicle fires (N = 17) occurred most frequently, whereas blazes, vegetation fires, and fires in underground facilities (N = 6) were rarely encountered. For residential building fires, a 3-fold increase in mean 1-OPH concentrations was observed, whereas no increase could be observed for outdoor and vehicle fires. The highest increase was observed for firefighters with interior attack missions (0.11 µg/L vs. 0.48 µg/L 1-OHP) despite the use of self-contained breathing apparatus (SCBA). During the suppression of outdoor or vehicle fires using SCBA, again, no increase was observed. Although PAH are taken up during certain firefighting missions, the 1-OHP levels almost entirely remained (in 64 of the 77 reported missions) within the normal range of the German general population, i.e., below the reference levels (95th percentiles) of smokers (0.73 µg/g creatinine) and non-smokers (0.30 µg/g creatine). CONCLUSION: Under study conditions, properly applied protective clothing and wearing of SCBA led to a significant reduction of PAH exposure levels. But there are individual situations in which PAH are increasingly incorporated since the incorporation depends on several factors and can be extremely variable. In contrast to many workplaces with high occupational exposure levels, firefighters are not exposed to PAH on a daily basis. Nevertheless, the possibility of an individual increased cancer risk for a particular firefighter cannot completely be ruled out.

Rapid quantification of seven major neonicotinoids and neonicotinoid-like compounds and their key metabolites in human urine.

Neonicotinoids and neonicotinoid-like compounds (NNIs) are frequently used insecticides worldwide and exposure scenarios can vary widely between countries and continents. We have developed a specific and robust analytical method based on liquid chromatography-electrospray tandem mass spectrometry coupled to online-SPE (online-SPE-LC-ESI-MS-MS) to analyze the seven most important NNIs from a global perspective together with nine of their key metabolites in human urine. The method also includes the neonicotinoid-like flupyradifurone (FLUP), an important future substitute for classical neonicotinoids, and two of its major human metabolites, 5-hydroxy- and N-desfluoroethyl-FLUP. Validation of the method was carried out using pooled urine samples from low-dose human metabolism studies and spiked urine samples with a wide range of creatinine concentrations. Depending on the analyte, the limits of quantitation were between 0.06 and 2.1 µg L-1, the inter-day and intra-day imprecisions ≤6%, and the mean relative recoveries between 89% and 112%. The method enabled us to successfully quantify NNIs and their metabolites at current environmental exposures in 34 individuals of the German general population and 43 pregnant women from Brazil with no known occupational exposures to NNIs.

Human biomonitoring of neonicotinoid exposures: case studies after the use of a spray-agent to ornamental plants and a topical medication to pets.

Acetamiprid (ACE) and imidacloprid (IMI) are insecticides of global importance and are used as spray and watering agents for ornamental plants to control biting and sucking insects or as topical medications on pets to remove and control fleas. Human biomonitoring data on ACE and IMI exposures when applying these products are limited. We investigated exposures to ACE and IMI in male volunteers after the domestic application of either an ACE-containing agent or an IMI-containing spot-on medication. Complete and consecutive urine samples were collected for up to 56 h after application. Urine samples were analyzed for ACE, IMI, and their respective metabolites (N-desmethyl-ACE, IMI-olefin, and sum of 4-/5-hydroxy-IMI) by liquid chromatography-tandem mass spectrometry. Fairly uniform concentrations of N-desmethyl-ACE could be observed before and after orchid treatment, so that an ACE exposure associated with orchid treatment can most likely be excluded. In contrast, after the application of the IMI-containing medication, elevated concentrations of IMI, 4-/5-hydroxy-IMI, and IMI-olefin were quantified in urine samples post-20 h with maximum concentrations of 3.1, 14.9, and 8.0 µg/g creatinine, respectively, well above general background levels. Nevertheless, the IMI intake (10.6 µg/kg bw), calculated from the excreted amounts, was around five times below the current European acceptable daily intake. Based on the case results here, household exposures to ACE and IMI after spray treatment of ornamental plants and anti-flea treatment of dogs can be regarded as low and safe. However, people regularly applying neonicotinoid-containing formulations, such as professional gardeners and employees in animal shelters, should be studied in more detail.

The Carcinogenic Properties of Overlooked yet Prevalent Polycyclic Aromatic Hydrocarbons in Human Lung Epithelial Cells.

The WHO classified air pollution as a human lung carcinogen and polycyclic aromatic hydrocarbons (PAHs) are components of both indoor (e.g., tobacco smoke and cookstoves) and outdoor (e.g., wildfires and industrial and vehicle emissions) air pollution, thus a human health concern. However, few studies have evaluated the adverse effects of low molecular weight (LMW) PAHs, the most abundant PAHs in the environment. We hypothesized that LMW PAHs combined with the carcinogenic PAH benzo[a]pyrene (B[a]P) act as co-carcinogens in human lung epithelial cell lines (BEAS-2B and A549). Therefore, in this paper, we evaluate several endpoints, such as micronuclei, gap junctional intercellular communication (GJIC) activity, cell cycle analysis, anti-BPDE-DNA adduct formation, and cytotoxicity after mixed exposures of LMW PAHs with B[a]P. The individual PAH doses used for each endpoint did not elicit cytotoxicity nor cell death and were relevant to human exposures. The addition of a binary mixture of LMW PAHs (fluoranthene and 1-methylanthracene) to B[a]P treated cells resulted in significant increases in micronuclei formation, dysregulation of GJIC, and changes in cell cycle as compared to cells treated with either B[a]P or the binary mixture alone. In addition, anti-BPDE-DNA adducts were significantly increased in human lung cells treated with B[a]P combined with the binary mixture of LMW PAHs as compared to cells treated with B[a]P alone, further supporting the increased co-carcinogenic potential by LMW PAHs. Collectively, these novel studies using LMW PAHs provide evidence of adverse pulmonary effects that should warrant further investigation.

Human metabolism and urinary excretion of seven neonicotinoids and neonicotinoid-like compounds after controlled oral dosages.

Few human data on exposure and toxicity are available on neonicotinoids and neonicotinoid-like compounds (NNIs), an important group of insecticides worldwide. Specifically, exposure assessment of humans by biomonitoring remains a challenge due to the lack of appropriate biomarkers. We investigated the human metabolism and metabolite excretion in urine of acetamiprid (ACE), clothianidin (CLO), flupyradifurone (FLUP), imidacloprid (IMI), sulfoxaflor (SULF), thiacloprid (THIAC) and thiamethoxam (THIAM) after single oral dosages at the currently acceptable daily intake levels of the European Food Safety Authority. Consecutive post-dose urine samples were collected up to 48 h. Suspect screening of tentative metabolites was carried out by liquid chromatography-high-resolution mass spectrometry. Screening hits were identified based on their accurate mass, isotope signal masses and ratios, product ion spectra, and excretion kinetics. We found, with the exception of SULF, extensive metabolization of NNIs to specific metabolites which were excreted next to the parent compounds. Overall, 24 metabolites were detected with signal intensities indicative of high metabolic relevance. Phase-I metabolites were predominantly derived by mono-oxidation (such as hydroxy-FLUP, -IMI, and -THIAC) and by oxidative N-desalkylation (such as N-desdifluoroethyl-FLUP and N-desmethyl-ACE, -CLO and -THIAM). IMI-olefin, obtained by dehydration of hydroxylated IMI, was identified as a major metabolite of IMI. SULF was excreted unchanged in urine. Previously reported metabolites of NNIs such as 6-chloronicotinic acid or 2-chlorothiazole-4-carboxylic acid and their glycine derivatives were detected either at low signal intensities or not at all and seem less relevant for human biomonitoring. Our highly controlled approach provides specific insight into the human metabolism of NNIs and suggests suitable biomarkers for future exposure assessment at environmentally relevant exposures.

Mismatch Repair Protein Expression and Microsatellite Instability in Cutaneous Squamous Cell Carcinoma.

There exist relatively sparse and conflicting data on high-level microsatellite instability (MSI-H) and deficient mismatch repair (dMMR) in cutaneous malignancies. We aimed to determine the expression profiles of MMR proteins (MSH2, MSH6, MLH1, and PMS2) in different progression stages of cutaneous squamous cell carcinoma (cSCC, 102 patients in total) by immunohistochemistry, and search for MSI-H in patients with low-level MMR or dMMR using multiplex-PCR. Low-level MMR protein expression was observed in five patients: One patient with primary cSCC < 2 mm thickness and low-level MLH1, three patients with primary cSCC > 6 mm (including one with low-level MSH2, as well as MSH6 expression, and two with low-level PMS2), and one patient with a cSCC metastasis showing low-level MSH2 as well as MSH6. Intergroup protein expression analysis revealed that MLH1 and MSH2 expression in actinic keratosis was significantly decreased when compared to Bowen's disease, cSCC < 2 mm, cSCC > 6 mm, and cSCC metastasis. In cases with low-level MMR, we performed MSI-H tests revealing three cases with MSI-H and one with low-level MSI-L. We found low-level MMR expression in a small subset of patients with invasive or metastatic cSCC. Hence, loss of MMR expression may be associated with tumour progression in a small subgroup of patients with non-melanoma skin cancer.

Expression of Mismatch Repair Proteins in Merkel Cell Carcinoma.

We aimed to assess for the first time the mismatch repair (MMR) protein expression in Merkel cell carcinoma (MCC). Immunohistochemistry was performed for MLH1, MSH2, MSH6, and PMS2 on patients' tumor tissue (n = 56), including neighbored healthy control tissue. In cases with low-level MMR expression (<10th percentile), we performed multiplex PCR in combination with high-resolution capillary electrophoresis in order to confirm microsatellite instability (MSI). Microscopic evaluation revealed a high median expression for all MMR proteins studied (91.6-96.3%). However, six patients (56/10.7%) had low-level MLH1 expression, six (55/10.9%) had low-level MSH2 expression, five (56/8.9%) had low-level MSH6 expression, and six (54/11.1%) had low-level PMS2 expression. Together, we observed nine (56/16.1%) patients who had low-level MMR expression of at least one protein. Of the patients with low-level MMR expression, MSI evaluation was possible in five cases, revealing one case with high-level MSI. In all MMR proteins assessed, low-level expression was significantly (p = 0.0004 to p < 0.0001) associated with a negative Merkel cell polyomavirus (MCPyV) status. However, the expression profiles of the MMR proteins did not correlate with clinical outcome measures such as disease relapse or death (p > 0.05). MCC appears to be a malignancy characterized by low-level MMR rather than completely deficient MMR in a subset of cases, predominantly affecting MCPyV-negative tumors. Future studies will establish whether this subset of MCC patients respond better to immune checkpoint inhibitor therapy.

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer.

We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this "bladder-specific" region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.

Expression of Programmed Cell Death Proteins in Kaposi Sarcoma and Cutaneous Angiosarcoma.

Not only for cutaneous angiosarcoma (CAS) patients but also for advanced and therapy-refractory patients with classic Kaposi sarcoma (CKS) and human immunodeficiency virus (HIV)-associated Kaposi sarcoma (HIV-KS) there is a high need for more effective treatment modalities. The aim of this work was to study programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) protein expression and related immune parameters in CKS, HIV-KS, and CAS and correlate it with other immunologic parameters and clinical data. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tumor tissue of 19 CKS, 7 HIV-KS, and 12 CAS patients using antibodies against the following (and they are): PD-1, PD-L1, CD4, CD8, CD56, and FOXP3. PD-1 expression significantly correlated with PD-L1 expression Moreover, PD-1 and PD-L1 expression significantly correlated with CD56 and FOXP3 expression. High intratumoral FOXP3 expression was significantly associated with disease relapse (P=0.029). CD4 and FOXP3 expression was significantly higher in CKS and CAS, as compared with HIV-KS. All in all, PD-1 and PD-L1 expression was relatively weak and did not significantly differ between CKS, HIV-KS, and CAS patients. Nevertheless, PD-1 was positive in 31.6% of CKS, 28.6% of HIV-KS, and 33.3% of CAS patients. PD-L1 was expressed in 36.6% of CKS, 28.6% of HIV-KS, and 41.7% of CAS patients. We have provided evidence that PD-1/PD-L1 signalling is of importance in angiosarcomas such as CKS, HIV-KS, and CAS. Our results support the notion that the use of PD-1/PD-L1 inhibitors may represent an effective strategy against these tumors.

Toward noninvasive follow-up of low-risk bladder cancer - Rationale and concept of the UroFollow trial.

BACKGROUND: Follow-up recommendations for patients with nonmuscle invasive bladder cancer (NMIBC) are largely based upon expert opinion. A growing body of evidence suggests that current follow-up strategies for bladder cancer patients with low and intermediate risk represent overdiagnosis and may lead to overtreatment. The goal of this study is to explore the options of a noninvasive follow-up in patients with pTa G1-2/low-grade NMIBC. METHODS: The risks and options for a urine marker-guided, noninvasive follow-up of patients with pTa G1-2/low-grade NMIBC were defined and the study design for a prospective randomized trial (UroFollow) was developed based upon the current literature. RESULTS: The investigators postulated that follow-up of patients with pTa G1-2/low-grade NMIBC requires a high sensitivity of urinary tumor markers. However, data from prospective studies with prediagnostic urine samples are scarce, even for approved markers, and cross-sectional studies with symptomatic patients overestimate the sensitivity. So far, cell-based markers (e.g., uCyt+ and UroVysion) in urine appeared to have higher sensitivities and specificities in low-grade NMIBC than urine cytology and markers analyzing soluble tumor-associated antigens. Marker panels are more sensitive than single-marker approaches at the expense of a lower specificity. Given a prospective randomized comparison with a marker sensitivity of 80% compared to usual care with cystoscopy, the sample size calculation yielded that 62 to 185 patients under study per arm are needed depending on different recurrence rates. CONCLUSIONS: Based upon these findings the UroFollow trial has been designed as a prospective randomized study comparing a noninvasive marker-based (UroVysion, NMP22, urine cytology, and ultrasound) follow-up with the current standard of care over a period of 3 years.

TGFBI Protein Is Increased in the Urine of Patients with High-Grade Urothelial Carcinomas, and Promotes Cell Proliferation and Migration.

Here, we discovered TGFBI as a new urinary biomarker for muscle invasive and high-grade urothelial carcinoma (UC). After biomarker identification using antibody arrays, results were verified in urine samples from a study population consisting of 303 patients with UC, and 128 urological and 58 population controls. The analyses of possible modifying factors (age, sex, smoking status, urinary leukocytes and erythrocytes, and history of UC) were calculated by multiple logistic regression. Additionally, we performed knockdown experiments with TGFBI siRNA in bladder cancer cells and investigated the effects on proliferation and migration by wound closure assays and BrdU cell cycle analysis. TGFBI concentrations in urine are generally increased in patients with UC when compared to urological and population controls (1321.0 versus 701.3 and 475.6 pg/mg creatinine, respectively). However, significantly increased TGFBI was predominantly found in muscle invasive (14,411.7 pg/mg creatinine), high-grade (8190.7 pg/mg) and de novo UC (1856.7 pg/mg; all p < 0.0001). Knockdown experiments in vitro led to a significant decline of cell proliferation and migration. In summary, our results suggest a critical role of TGFBI in UC tumorigenesis and particularly in high-risk UC patients with poor prognosis and an elevated risk of progression on the molecular level.

Noninvasive diagnosis of urothelial cancer in urine using DNA hypermethylation signatures-Gender matters.

Urothelial cancer (UCa) is the most predominant cancer of the urinary tract and noninvasive diagnosis using hypermethylation signatures in urinary cells is promising. Here, we assess gender differences in a newly identified set of methylation biomarkers. UCa-associated hypermethylated sites were identified in urine of a male screening cohort (n = 24) applying Infinium-450K-methylation arrays and verified in two separate mixed-gender study groups (n = 617 in total) using mass spectrometry as an independent technique. Additionally, tissue samples (n = 56) of mixed-gender UCa and urological controls (UCt) were analyzed. The hypermethylation signature of UCa in urine was specific and sensitive across all stages and grades of UCa and independent on hematuria. Individual CpG sensitivities reached up to 81.3% at 95% specificity. Albeit similar methylation differences in tissue of both genders, differences were less pronounced in urine from women, most likely due to the frequent presence of squamous epithelial cells and leukocytes. Increased repression of methylation levels was observed at leukocyte counts ≥500/µl urine which was apparent in 30% of female and 7% of male UCa cases, further confirming the significance of the relative amounts of cancerous and noncancerous cells in urine. Our study shows that gender difference is a most relevant issue when evaluating the performance of urinary biomarkers in cancer diagnostics. In case of UCa, the clinical benefits of methylation signatures to male patients may outweigh those in females due to the general composition of women's urine. Accordingly, these markers offer a diagnostic option specifically in males to decrease the number of invasive cystoscopies.

Toxicokinetics of N-ethyl-2-pyrrolidone and its metabolites in blood, urine and amniotic fluid of rats after oral administration.

The toxicokinetics of N-ethyl-2-pyrrolidone (NEP), an embryotoxic organic solvent, has been studied in Sprague-Dawley rats after oral exposure. NEP and its metabolites 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI) were measured in plasma of pregnant and non-pregnant rats, and fetuses after NEP administration by gavage for 14 consecutive days at 50 mg/kg/day, and in plasma of non-pregnant rats after a single NEP administration. Additionally, amniotic fluid and 24-h urine samples of the pregnant rats were analyzed for NEP metabolites. Furthermore, 24-h urine samples from a repeated dose 28-day oral toxicity study in female (non-pregnant) and male rats administered developmentally non-toxic (0, 5, and 50 mg/kg/day) or toxic (250 mg/kg/day) doses of NEP were analyzed. Median peak plasma concentrations in non-pregnant rats after a single dose and repeated doses were 551 and 611 (NEP), 182 and 158 (5-HNEP), and 63.8 and 108 µmol/L (2-HESI), respectively; whereas in pregnant rats and fetuses 653 and 619 (NEP), 80.5 and 91.7 (5-HNEP) and 77.3 and 45.7 µmol/L (2-HESI) were detected. Times to reach maximum plasma concentrations for NEP, 5-HNEP, and 2-HESI were 1, 4, and 8 h, respectively, and were comparable to N-methyl-2-pyrrolidone (NMP) and its corresponding metabolites. In pregnant rats, plasma elimination of NEP and metabolite formation/elimination was much slower compared to non-pregnant rats and efficient placental transfer of NEP was observed. Our data, overall, suggest differences in the toxicokinetics of chemicals between pregnant and non-pregnant rats which need to be addressed in risk assessment, specifically when assessing developmental toxicants such as NEP.

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.