Search

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.

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 (&ge, 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 &ldquo, bladder-specific&rdquo, 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 &gt, 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.

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.

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.

Background The histopathology of lichen sclerosus (LS) suggests abnormalities in extracellular matrix (ECM) composition. Objectives We aimed to investigate the expression pattern of ECM proteins and related growths factors and Smad signal transducers in LS as compared with healthy skin. Methods To assess the expression of decorin, biglycan, versican, perlecan, fibronectin, dermatopontin, extracellular matrix protein 1 (ECM-1), matrix metalloproteinase 1, tissue inhibitor of metalloproteinase 1, connective tissue growth factor (CTGF), transforming growth factor β1, and Smad-3 protein, real-time RT-PCR and immunohistochemistry were performed on skin specimens obtained from the genital region of healthy subjects (n = 10) as well as LS patients (n = 26). Results Median mRNA as well as mean protein expression of biglycan, versican, fibronectin, and ECM-1 was significantly higher in LS when compared with healthy controls. Both mRNA and protein CTGF expression observed in LS was significantly higher than in controls. CTGF mRNA expression significantly correlated with mRNA expression of biglycan, versican and fibronectin. Conclusions Expression of ECM proteins (e.g. proteoglycans, ECM-1) and CTGF is altered in LS. TGF-s/Smad-3 independent up-regulation of CTGF may induce accumulation of ECM proteins and maintain fibrosis in chronic LS.

β-Cell regeneration declines with aging, but the molecular mechanisms controlling β-cell replication in humans are not well understood. We compared the expression of selected cell cycle proteins in prenatal and adult tissue and examined the association of these proteins with β-cell replication. Pancreatic tissue from a total of 20 human fetuses and adults was stained for Ki67, cyclin D3, p16 and p27, and insulin. The β-cellular expression of these cell cycle proteins was determined. The frequency of β-cell replication was lower in adult compared with prenatal β-cells ( 0.75, respectively). The strong expression of cyclin D3 in adult human β-cells and its correlation to p27 and p16 suggest a positive role in human β-cell cycle regulation. p16 and p27 appear to restrict β-cell replication with aging. The age dependency of cell cycle regulation in human β-cells might explain the reduced β-cell regeneration in adult humans.

Introduction Insulin therapy has been suggested to preserve beta-cell mass in patients with diabetes through the mechanisms of beta-cell rest as well as direct effects on beta-cell proliferation. However, data about the effects of hyperinsulinism on beta-cell mass and turnover in humans are sparse. Patients and methods Pancreatic tissue specimens from five patients with pancreatic insulinomas and ten non-diabetic control subjects were examined. Pancreatic sections were stained for insulin, Ki67 (replication) and TUNEL (apoptosis), and quantitative morphometric analyses were performed. Results Fractional beta-cell area was 1.11% ± 0.67% in the tumor-free pancreatic tissue of the insulinoma patients and 0.78% ± 0.26% in the control group (p = 0.19). There also were no differences in islet size (p = 0.62) or beta-cell nuclear diameter (p = 0.20). Beta-cell replication and apoptosis were infrequently detected, without any measurable differences between the groups. There were also no differences in percentage of duct cells expressing insulin (p = 0.47), a surrogate marker for islet neogenesis. Conclusions Beta-cell area and turnover are not significantly altered in the proximity of intra-pancreatic insulinomas. Future in vivo studies, ideally employing larger animal models, are warranted to further evaluate the impact of exogenous insulin on beta-cell turnover.

The scattered and limited data on hnRNAs (pre-mRNAs) of cytochromes P450 (CYP) are compiled and discussed for the first time. The methods for determination and quantification of hnRNAs are compared. In most cases, CYP hnRNA levels were determined as a parameter of transcriptional activity. It is known, however, that some CYPs, in particular CYP2E1, are in addition specifically and extensively regulated by post-transcriptional processes. Obviously, these processes also influence the processing of CYP hnRNAs so that their levels cannot be considered a mere result of transcription. The underlying mechanisms of post-transcriptional CYP hnRNA and mRNA regulation are not well understood. It is our aim therefore to bring together available data on CYP hnRNA and to discuss them in the light of recent advances in knowledge concerning pre-mRNA processing and interactions between RNA and low molecular weight interfering RNAs. By doing this, we hope to drive research in a direction which appears promising in providing some long-awaited answers with respect to mechanisms of post-transcriptional CYP regulation.

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.

Introduction Genital lichen sclerosus (LS) is considered a potential precursor lesion of squamous cell carcinoma. We aimed to investigate the expression pattern of cell cycle regulators, tumour suppressor proteins and proliferation markers in genital LS as compared to extragenital LS (ELS) and healthy controls (HC). Methods In order to assess the expression of minichromosome maintenance protein 3 (MCM3), MCM7, Ki-67, cyclin D1, cyclin E, p16, p21, and p53, immunohistochemistry and immunofluorescence were performed on skin specimens obtained from the genital region of LS patients (short-standing LS, n = 19; long-standing LS, n = 15), patients with ELS (n = 10), and HC (n = 8). Results Median protein expression of MCM3 and Ki-67 was significantly higher in LS when compared to ELS and HC. In patients with long-standing LS, the expression profiles of MCM3 and Ki-67 significantly correlated. Moreover, long-standing LS lesions showed significantly increased expression of p53 when compared to short-standing LS, ELS, and HS. Immunoreactivity of MCM7, p16, p21, cyclin D1 and cyclin E did not significantly differ between the groups. Conclusions Tumour suppressor proteins such as p53 are significantly overexpressed in genital LS when compared to extragenital disease and healthy skin. The significant p53 overexpression, particularly in long-standing genital lesions, may reflect the increased risk of malignant transformation and/or oxidative stress associated with LS. Moreover, we have demonstrated that proliferation markers such as Ki-67 and MCM3 are significantly up-regulated in genital LS as compared to controls. With regard to cell cycle regulation and proliferation rates, ELS significantly differs from its genital counterpart.

Introductionβ-cell regeneration is an area under active investigation for the future treatment of diabetes, but little is known about the patterns and dynamics of prenatal β-cell development in humans. In particular, the quantitative changes in β-cell mass in the developing pancreas have not been elucidated in detail. We addressed the following questions in prenatal humans: i) what is the timing of β-cell occurrence and islet growth? ii) What are the dynamics of β-cell replication and apoptosis?MethodsPancreatic tissue was obtained from 65 human embryos and foetuses aged between 8 weeks post conception (p.c.) and birth. Sections were stained for insulin, glucagon, Ki67 (proliferation marker), TUNEL (apoptosis marker) and CD31 (blood vessel marker), and morphometric analyses were performed.Resultsβ-cells were detected from gestational week 9 onward, whereas glucagon expression was detected already at week 8. The fractional β-cell area of the pancreas increased in a linear fashion until birth (r=0.60, PConclusionsβ-cell differentiation in humans occurs from week 9 p.c. onward. The first endocrine cells are closely associated with the ductal epithelium suggesting differentiation from precursor cells. High rates of β-cell replication suggest that this mechanism plays an important role in the prenatal expansion of β-cell mass.

Introduction Beta-cell-regeneration is considered as a future option for the therapy of diabetes, but the detection of beta-cell replication in human pancreas is still challenging. Therefore, the expression of Ki67, PCNA and MCM-7 in human pancreatic tissue was quantified in order to validate their use as beta-cell replication markers. Methods Human pancreatic tissue samples were stained for Ki67, PCNA, MCM7, insulin and nuclei, and the expression of replication markers was quantified. Co-expression of the markers was assessed by four-colour fluorescence-staining. Results All three markers could be detected in endocrine and exocrine tissue. There was a significant correlation between the expression frequencies of all three markers in the exocrine tissue (r > 0.49, respectively) and in beta-cells (r > 0.95, respectively). A subset of beta-cells with differential expression of the three replication markers was identified. Quantitative analyses revealed that only 36–55% of all exocrine cells expressed two markers at the same time. Conclusions The expression of Ki67, MCM-7 and PCNA in adult human pancreas is highly correlated, but the labelling of individual cells differs between the markers. The analysis of a combination of markers, preferably MCM7 and Ki67, appears to yield the most reliable results for the determination of beta-cell replication and may allow for a differentiation of cell cycle stages.