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.

Periinterventional Management of Edoxaban in Major Procedures: Results from the DRESDEN NOAC REGISTRY.

Background Edoxaban is a non-vitamin K dependent oral anticoagulant (NOAC) licensed for venous thromboembolism (VTE) treatment or stroke prevention in atrial fibrillation. Major surgical procedures are not uncommon in anticoagulated patients but data on perioperative edoxaban management are scarce. Patients and Methods Using data from the prospective DRESDEN NOAC REGISTRY, we extracted data on major surgical procedures in edoxaban patients. Periinterventional edoxaban management patterns and rates of outcome events were evaluated until day 30 after procedure. Results Between 2011 and 2021, 3,448 procedures were identified in edoxaban patients, including 287 (8.3%) major procedures. A scheduled interruption of edoxaban was observed in 284/287 major procedures (99%) with a total median edoxaban interruption time of 11.0 days (25-75th percentile: 5.0-18.0 days). Heparin bridging was documented in 183 procedures (46 prophylactic dosages, 111 intermediate and 26 therapeutic dosages). Overall, 7 (2.4%; 95% CI: 1.2-4.9%) major cardiovascular events (5 VTE, 2 arterial thromboembolic events) and 38 major bleedings (13.2%; 95% CI: 9.8-17.7%) were observed and 6 patients died (2.1%; 95% CI: 1.0-4.5%). Rates of major cardiovascular events with or without heparin bridging were comparable (4/137; 2.9%; 95% CI: 1.1-7.3% vs. 3/82; 3.7%; 95% CI: 1.3-10.2%). Major bleedings occurred numerically more frequent in patients receiving heparin bridging (23/137; 16.8%; 95% CI: 11.5-23.9%) versus procedures without heparin bridging (9/82; 11.0%; 95% CI: 5.9-19.6%). Conclusion Within the limitations of our study design, real-world periprocedural edoxaban management seems effective and safe. Use of heparin bridging seems to have limited effects on reducing vascular events but may increase bleeding risk.

Direct Oral Anticoagulants: Laboratory Challenges and Antidotes.

The use of direct oral anticoagulants (DOACs) is increasing in patients needing treatment of venous thromboembolism (VTE) and stroke prevention in atrial fibrillation (SPAF). This is due to the net clinical benefit in comparison to vitamin K antagonists (VKAs). The rise in DOAC use is accompanied by a remarkable reduction in heparin and VKA prescriptions. However, this rapid change in anticoagulation patterns brought new challenges to patients, prescribers, laboratories, and emergency physicians. Patients have new liberties concerning nutritional habits and comedication and no longer need frequent monitoring or dose adjustments. Still, they have to comprehend that DOACs are potent anticoagulants that may cause or contribute to bleeding. Challenges for the prescriber include decision pathways for choosing the right anticoagulant and dosage for a specific patient and to change bridging practice in case of invasive procedures. Laboratory personnel are challenged by DOAC due to limited 24/7 availability of specific DOAC quantification tests and by the impact of DOAC on routine coagulation assays and thrombophilia tests. Challenges for the emergency physician result from the increasing age of DOAC anticoagulated patients, the difficulties to establish last intake of DOAC type and dosage, to interpret coagulation test results in emergency situations, and to make decisions for or against DOAC reversal strategies in acute bleeding or urgent surgery. In conclusion, although DOACs make long-term anticoagulation safer and more convenient for patients, DOACs pose challenge to all healthcare providers involved in anticoagulation decisions. The key to correct patient management and optimal outcome therefore lies in education.

Effectiveness and safety of edoxaban therapy in daily-care patients with atrial fibrillation. Results from the DRESDEN NOAC REGISTRY.

BACKGROUND: Edoxaban is a non-vitamin K dependent oral anticoagulant (NOAC) licensed for stroke prevention in atrial fibrillation (SPAF). Outcome data on clinical effectiveness and safety in routine care are increasing. PATIENTS AND METHODS: In the prospective, non-interventional DRESDEN NOAC REGISTRY a network of 230 physicians enrolled >5000 NOAC patients who received prospective central follow. All reported outcome events (stroke/transient ischemic attack/systemic embolism; ISTH bleeding; death) were adjudicated using standard definitions. RESULTS: Between 2016 and 2021, 1258 SPAF patients receiving edoxaban were followed for 927.1 ± 562.2 days with a mean edoxaban exposure of 790.3 ± 577.2 days. Edoxaban was discontinued by 274 patients (10.1/100 patient-years; 95% CI 8.9-11.3). The combined endpoint of stroke/TIA/systemic embolism occurred at a rate of 1.7/100 patient-years (95% CI 1.3-2.3) in the intention-to-treat analysis and at 1.3/100 patient-years (95% CI 0.9-1.9) in the on-treatment analysis (censored 3 days after last edoxaban intake). On-treatment rates of ISTH major bleeding were comparable for patients receiving edoxaban 30 mg OD (3.6/100 patient-years; 95% CI 2.2-5.5) or 60 mg OD (2.5/100 patient-years; 95% CI 1.8-3.2). A total of 151 patients (12.0%) died (4.7/100 patient-years; 95% CI 4.0-5.5), with non-stroke cardiovascular events (n = 50), infection/sepsis (n = 40) and terminal malignant disease (n = 31) being the leading causes of death. CONCLUSION: Overall rates of effectiveness and safety outcomes were in line with latest real-world data (such as ETNA-AF registry) and confirm findings of the phase-III ENGAGE-AF trial. Non-thrombotic cardiovascular events and infectious diseases were the leading causes of death, whereas fatal stroke and fatal bleeding were rare.

Real-time quantitative PCR method for detection and quantification of Clostridioides difficile cells and spores.

A quantitative PCR method was developed for the detection of Clostridioides difficile Chaperonin-60 gene. The method had a specificity for C. difficile strains of clinical and epidemiological importance and allowed for quantification of C. difficile cells and spores.

Thrombophilia, Thrombosis and Thromboprophylaxis in Pregnancy: For What and in Whom?

Compared with nonpregnant women, pregnancy carries a four- to fivefold higher risk of venous thromboembolism (VTE). Despite increasing use of heparin prophylaxis in identified high-risk patients, pulmonary embolism still is the leading cause of maternal mortality in the western world. However, evidence on optimal use of thromboprophylaxis is scarce. Thrombophilia, the hereditary or acquired tendency to develop VTE, is also thought to be associated with complications in pregnancy, such as recurrent miscarriage and preeclampsia. In this review, the current evidence on optimal thromboprophylaxis in pregnancy is discussed, focusing primarily on VTE prevention strategies but also discussing the potential to prevent recurrent pregnancy complications with heparin in pregnant women with thrombophilia.

Genetic Variation in Toll-Like Receptor 5 and Colonization with Flagellated Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency-defined by a dominant negative stop codon polymorphism, rs5744168-is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

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.

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.

Improved yield and accuracy for DNA extraction in microbiome studies with variation in microbial biomass.

A major challenge for microbiome studies is maintaining an even and accurate DNA extraction in the presence of samples with a wide range of bacterial content. Here we compare five DNA extraction methods using replicate stool samples that were diluted to create high and low biomass samples. Our results indicate greater variation in microbiome composition between high and low biomass samples than variation between methods. Many of the extraction methods had reduced yield from low biomass samples; however, our adapted plate column-based extraction method was evenly efficient and captured the largest number of high-quality reads. Based on these results, we have identified a DNA extraction method that ensures adequate yield in metagenomic microbiome studies that have samples with a broad range of bacterial content.

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.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

Effectiveness and safety of dabigatran therapy in daily-care patients with atrial fibrillation. Results from the Dresden NOAC Registry.

The effectiveness and safety of dabigatran for stroke prevention in atrial fibrillation (SPAF) demonstrated in RE-LY needs to be confirmed in daily care. To evaluate treatment persistence, effectiveness and safety of dabigatran therapy in SPAF patients in daily care, we used data from an ongoing, prospective, non-interventional registry of more than 2,500 patients on novel oral anticoagulants in daily care. Between October 1, 2011 and February 28, 2013, a total of 341 SPAF patients receiving dabigatran were enrolled. The combined endpoint of stroke/transient ischaemic attack/systemic embolism occurred at a rate of 2.93/100 patient-years in the intention-to-treat analysis (95%-CI 1.6-4.9) and at 1.9/100 patient-years in the on treatment analysis (events within three days after last intake). On-treatment rates were higher in patients selected for 110 mg dabigatran (n=183) BID compared to the 158 patients selected for 150 mg BID (2.88 [95% CI 1.16- 5.93] vs 0.86/100 patient-years [95% CI 0.10, 3.12]). On treatment, major bleeding occurred at a rate of 2.3/100 patient-years and numerically more often in patients receiving the 110 mg BID dose compared to the 150 mg BID dose (2.9 vs 1.7/100 patient-years). Dabigatran treatment discontinuation occurred in a total of 124 patients during follow-up (25.8 per 100 patient-years in Kaplan Meier analysis). Main reasons for treatment discontinuation were non-bleeding side effects. Our data contribute to the confirmation of effectiveness and relative safety of dabigatran in unselected patients in daily care. However, discontinuation rates are not lower than those reported for patients treated with vitamin K antagonists.

Automated quantification of FISH signals in urinary cells enables the assessment of chromosomal aberration patterns characteristic for bladder cancer.

Targeting the centromeres of chromosomes 3, 7, 17 (CEP3, 7, 17) and the 9p21-locus (LSI9p21) for diagnosing bladder cancer (BC) is time- and cost-intensive and requires a manual investigation of the sample by a well-trained investigator thus overall limiting its use in clinical diagnostics and large-scaled epidemiological studies. Here we introduce a new computer-assisted FISH spot analysis tool enabling an automated, objective and quantitative assessment of FISH patterns in the urinary sediment. Utilizing a controllable microscope workstation, the microscope software Scan^R was programmed to allow automatic batch-scanning of up to 32 samples and identifying quadruple FISH signals in DAPI-scanned nuclei of urinary sediments. The assay allowed a time- and cost-efficient, automated and objective assessment of CEP3, 7 and 17 FISH signals and facilitated the quantification of nuclei harboring specific FISH patterns in all cells of the urinary sediment. To explore the diagnostic capability of the developed tool, we analyzed the abundance of 51 different FISH patterns in a pilot set of urine specimens from 14 patients with BC and 21 population controls (PC). Herein, the results of the fully automated approach yielded a high degree of conformity when compared to those obtained by an expert-guided re-evaluation of archived scans. The best cancer-identifying pattern was characterized by a concurrent gain of CEP3, 7 and 17. Overall, our automated analysis refines current FISH protocols and encourages its use to establish reliable diagnostic cutoffs in future large-scale studies with well-characterized specimens-collectives.

Rates, management, and outcome of rivaroxaban bleeding in daily care: results from the Dresden NOAC registry.

Worldwide, rivaroxaban is increasingly used for stroke prevention in atrial fibrillation and treatment of venous thromboembolism, but little is known about rivaroxaban-related bleeding complications in daily care. Using data from a prospective, noninterventional oral anticoagulation registry of daily care patients (Dresden NOAC registry), we analyzed rates, management, and outcome of rivaroxaban-related bleeding. Between October 1, 2011, and December 31, 2013, 1776 rivaroxaban patients were enrolled. So far, 762 patients (42.9%) reported 1082 bleeding events during/within 3 days after last intake of rivaroxaban (58.9% minor, 35.0% of nonmajor clinically relevant, and 6.1% major bleeding according to International Society on Thrombosis and Haemostasis definition). In case of major bleeding, surgical or interventional treatment was needed in 37.8% and prothrombin complex concentrate in 9.1%. In the time-to-first-event analysis, 100-patient-year rates of major bleeding were 3.1 (95% confidence interval 2.2-4.3) for stroke prevention in atrial fibrillation and 4.1 (95% confidence interval 2.5-6.4) for venous thromboembolism patients, respectively. In the as-treated analysis, case fatality rates of bleeding leading to hospitalizations were 5.1% and 6.3% at days 30 and 90 after bleeding, respectively. Our data indicate that, in real life, rates of rivaroxaban-related major bleeding may be lower and that the outcome may at least not be worse than that of major vitamin K antagonist bleeding, and probably better. This trial was registered at www.clinicaltrials.gov as identifier #NCT01588119.

Safety of switching from vitamin K antagonists to dabigatran or rivaroxaban in daily care--results from the Dresden NOAC registry.

AIM: Vitamin-K antagonists (VKA) and non-vitamin-K dependent oral anticoagulants (NOAC) have been approved for anticoagulation in venous thromboembolism (VTE) and atrial fibrillation and patients previously treated with VKA are switched to NOAC therapy. Safety data for this switching are urgently needed. METHODS: Using data from a large regional prospective registry of daily care NOAC patients, we evaluated the safety of switching anticoagulation from VKA to dabigatran or rivaroxaban. Switching procedures and cardiovascular and bleeding events occurring within 30 days after switching were centrally adjudicated. RESULTS: Between 1 October 2011 and 18 June 2013, 2231 patients were enrolled. Of these, 716 patients were switched from VKA to NOAC. Only 410 of the 546 evaluable patients (75.1%) had a recorded INR measurement within the 10 days preceding or following the end of VKA treatment (mean INR 2.4). As of day 30, major bleeding complications were rare (0.3%; 95% CI 0.0, 1.0) with an overall bleeding rate of 12.2% (95% CI 9.8, 14.8). Major cardiovascular events occurred in 0.8% (95% CI 0.3, 1.8). There was no significant difference in outcome event rates between the subgroups of patients with or without INR testing. CONCLUSION: In daily care, only 75% of VKA patients have an INR measurement documented before NOAC are started. On average, NOAC are started within 2 to 5 days after the last intake of VKA. However, at 30 days follow-up cardiovascular events or major bleedings were rare both in patients with and without INR testing. However, switching procedures need to be further evaluated in larger cohorts of patients.

Peri-interventional management of novel oral anticoagulants in daily care: results from the prospective Dresden NOAC registry.

AIMS: Patients receiving novel oral anticoagulants (NOACs) frequently undergo interventional procedures. Short half-lives and rapid onset of action allow for short periods of NOAC interruption without heparin bridging. However, outcome data for this approach are lacking. We evaluated the peri-interventional NOAC management in unselected patients from daily care. METHODS AND RESULTS: Effectiveness and safety data were collected from an ongoing, prospective, non-interventional registry of >2100 NOAC patients. Outcome events were adjudicated using standard event definitions. Of 2179 registered patients, 595 (27.3%) underwent 863 procedures (15.6% minimal, 74.3% minor, and 10.1% major procedures). Until Day 30 ± 5 post-procedure, major cardiovascular events occurred in 1.0% of patients [95% confidence interval (95% CI) 0.5-2.0] and major bleeding complications in 1.2% (95% CI 0.6-2.1). Cardiovascular and major bleeding complications were highest after major procedures (4.6 and 8.0%, respectively). Heparin bridging did not reduce cardiovascular events, but led to significantly higher rates of major bleeding complications (2.7%; 95% CI 1.1-5.5) compared with no bridging (0.5%; 0.1-1.4; P = 0.010). Multivariate analysis demonstrated diabetes [odds ratio (OR) 13.2] and major procedures (OR 7.3) as independent risk factors for cardiovascular events. Major procedures (OR 16.8) were an independent risk factor for major bleeding complications. However, if major and non-major procedures were separately assessed, heparin bridging was not an independent risk factor for major bleeding. CONCLUSION: Continuation or short-term interruption of NOAC is safe strategies for most invasive procedures. Patients at cardiovascular risk undergoing major procedures may benefit from heparin bridging, but bleeding risks need to be considered.

Analyses in human urothelial cells identify methylation of miR-152, miR-200b and miR-10a genes as candidate bladder cancer biomarkers.

Urinary miRNAs are discussed as potential biomarkers for bladder cancer. The majority of miRNAs, however, are downregulated, making it difficult to utilize reduced miRNA signals as reliable diagnostic tools. Because the downregulation of miRNAs is frequently associated with hypermethylation of the respective regulative sequences, we studied whether DNA hypermethylation might serve as an improved diagnostic tool compared to measuring downregulated miRNAs. miRNA expression arrays and individual qPCR were used to identify and confirm miRNAs that were downregulated in malignant urothelial cells (RT4, 5637 and J82) when compared to primary, non-malignant urothelial cells (HUEPC). DNA methylation was determined by customized PCR-arrays subsequent to methylation-sensitive DNA-restriction and by mass spectrometry. miRNA expression and DNA methylation were determined in untreated cells and in cultures treated with the demethylating agent 5-Aza-2'-deoxycytidine. miR-200b, miR-152 and miR-10a displayed differential expression and methylation among untreated cancer cell lines. In addition, reduced miRNA expression of miR-200b, miR-152, and miR-10a was associated with increased DNA methylation in malignant cells versus HUEPC. Finally, the demethylation approach revealed a causal relationship between both parameters for miR-152 in 5637 and also suggests a causal connection of both parameters for miR-200b in J82 and miR-10a in 5637. In conclusion, our studies in multiple bladder cancer cell lines and primary non-malignant urothelial cells suggest that hypermethylation of miR-152, miR-10a and miR-200b regulative DNA sequences might serve as epigenetic bladder cancer biomarkers.