An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer

Background: Urinary biomarkers are needed to improve the care and reduce the cost of managing bladder cancer. Current biomarkers struggle to identify both high and low-grade cancers due to differing molecular pathways. Changes in microRNA (miR) expression are seen in urothelial carcinogenesis in a phenotype-specific manner. We hypothesised that urinary miRs reflecting low- and high-grade pathways could detect bladder cancers and overcome differences in genetic events seen within the disease. Methods: We investigated urinary samples (n ¼ 121) from patients with bladder cancer (n ¼ 68) and age-matched controls (n ¼ 53). Fifteen miRs were quantified using real-time PCR. Results: We found that miR is stable within urinary cells despite adverse handling and detected differential expression of 10 miRs from patients with cancer and controls (miRs 15a/15b/24-1/27b/100/135b/203/212/328/1224, ANOVA Po0.05). Individually, miR-1224-3p had the best individual performance with specificity, positive and negative predictive values and concordance of 83%, 83%, 75% and 77%, respectively. The combination of miRs-135b/15b/1224-3p detected bladder cancer with a high sensitivity (94.1%), sufficient specificity (51%) and was correct in 86% of patients (concordance). Conclusion: The use of this panel in patients with haematuria would have found 94% of urothelial cell carcinoma, while reducing cystoscopy rates by 26%. However, two invasive cancers (3%) would have been missed

Integrated Epigenome Profiling of Repressive Histone Modifications, DNA Methylation and Gene Expression in Normal and Malignant Urothelial Cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20–30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5–10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer

Changes in circulating microRNA levels associated with prostate cancer

BACKGROUND: The aim of this study was to investigate the hypothesis that changes in circulating microRNAs (miRs) represent potentially useful biomarkers for the diagnosis, staging and prediction of outcome in prostate cancer. METHODS: Real-time polymerase chain reaction analysis of 742 miRs was performed using plasma-derived circulating microvesicles of 78 prostate cancer patients and 28 normal control individuals to identify differentially quantified miRs. RESULTS: A total of 12 miRs were differentially quantified in prostate cancer patients compared with controls, including 9 in patients without metastases. In all, 11 miRs were present in significantly greater amounts in prostate cancer patients with metastases compared with those without metastases. The association of miR-141 and miR-375 with metastatic prostate cancer was confirmed using serum-derived exosomes and microvesicles in a separate cohort of patients with recurrent or non-recurrent disease following radical prostatectomy. An analysis of five selected miRs in urine samples found that miR-107 and miR-574-3p were quantified at significantly higher concentrations in the urine of men with prostate cancer compared with controls. CONCLUSION: These observations suggest that changes in miR concentration in prostate cancer patients may be identified by analysing various body fluids. Moreover, circulating miRs may be used to diagnose and stage prostate cance

Inboard and outboard radial electric field wells in the H- and I-mode pedestal of Alcator C-Mod and poloidal variations of impurity temperature

We present inboard (HFS) and outboard (LFS) radial electric field (E[subscript r]) and impurity temperature (T[subscript z]) measurements in the I-mode and H-mode pedestal of Alcator C-Mod. These measurements reveal strong Er wells at the HFS and the LFS midplane in both regimes and clear pedestals in T[subscript z], which are of similar shape and height for the HFS and LFS. While the H-mode E[subscript r] well has a radially symmetric structure, the E[subscript r] well in I-mode is asymmetric, with a stronger ExB shear layer at the outer edge of the E[subscript r] well, near the separatrix. Comparison of HFS and LFS profiles indicates that impurity temperature and plasma potential are not simultaneously flux functions. Uncertainties in radial alignment after mapping HFS measurements along flux surfaces to the LFS do not, however, allow direct determination as to which quantity varies poloidally and to what extent. Radially aligning HFS and LFS measurements based on the T[subscript z] profiles would result in substantial inboard-outboard variations of plasma potential and electron density. Aligning HFS and LFS E[subscript r] wells instead also approximately aligns the impurity poloidal flow profiles, while resulting in a LFS impurity temperature exceeding the HFS values in the region of steepest gradients by up to 70%. Considerations based on a simplified form of total parallel momentum balance and estimates of parallel and perpendicular heat transport time scales seem to favor an approximate alignment of the E[subscript r] wells and a substantial poloidal asymmetry in impurity temperature.United States. Dept. of Energy (Cooperative Agreement DE-FC02-99ER54512)Swiss National Science Foundatio

Competing mortality in patients diagnosed with bladder cancer: evidence of undertreatment in the elderly and female patients

Background: Bladder cancer (BC) predominantly affects the elderly and is often the cause of death among patients with muscleinvasive disease. Clinicians lack quantitative estimates of competing mortality risks when considering treatments for BC. Our aim was to determine the bladder cancer-specific mortality (CSM) rate and other-cause mortality (OCM) rate for patients with newly diagnosed BC. Methods: Patients (n ¼ 3281) identified from a population-based cancer registry diagnosed between 1994 and 2009. Median follow-up was 48.15 months (IQ range 18.1–98.7). Competing risk analysis was performed within patient groups and outcomes compared using Gray’s test. Results: At 5 years after diagnosis, 1246 (40%) patients were dead: 617 (19%) from BC and 629 (19%) from other causes. The 5-year BC mortality rate varied between 1 and 59%, and OCM rate between 6 and 90%, depending primarily on the tumour type and patient age. Cancer-specific mortality was highest in the oldest patient groups. Few elderly patients received radical treatment for invasive cancer (52% vs 12% for patients o60 vs 480 years, respectively). Female patients with high-risk non-muscle-invasive BC had worse CSM than equivalent males (Gray’s Po0.01). Conclusion: Bladder CSM is highest among the elderly. Female patients with high-risk tumours are more likely to die of their disease compared with male patients. Clinicians should consider offering more aggressive treatment interventions among older patients

MicroRNA-375 plays a dual role in prostate carcinogenesis

Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, aswellaswithregionallymph nodesmetastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.Research Center of Portuguese Oncology Institute - Porto (CI-IPOP 4–2012) and by the Federal funds through Programa Operacional Temático Factores de Competitividade (COMPETE) with co-participation from the European Community Fund (FEDER) and by the National funds through Fundação para a Ciência e Tecnología (FCT) under the projects EXPL/BIM-ONC/0556/2012. FQV and JRC were or are supported by FCT-Fundação para a Ciência e a Tecnologia grants (SFRH/BD/70564/2010 and SFRH/BD/71293/2010, respectively)

Identification of a hypoxia-regulated miRNA signature in bladder cancer and a role for miR-145 in hypoxia-dependent apoptosis

Background: Hypoxia leads to the stabilisation of the hypoxia-inducible factor (HIF) transcription factor that drives the expression of target genes including microRNAs (miRNAs). MicroRNAs are known to regulate many genes involved in tumourigenesis. The aim of this study was to identify hypoxia-regulated miRNAs (HRMs) in bladder cancer and investigate their functional significance. Methods: Bladder cancer cell lines were exposed to normoxic and hypoxic conditions and interrogated for the expression of 384 miRNAs by qPCR. Functional studies were carried out using siRNA-mediated gene knockdown and chromatin immunoprecipitations. Apoptosis was quantified by annexin V staining and flow cytometry. Results: The HRM signature for NMI bladder cancer lines includes miR-210, miR-193b, miR-145, miR-125-3p, miR-708 and miR-517a. The most hypoxia-upregulated miRNA was miR-145. The miR-145 was a direct target of HIF-1a and two hypoxia response elements were identified within the promoter region of the gene. Finally, the hypoxic upregulation of miR-145 contributed to increased apoptosis in RT4 cells. Conclusions: We have demonstrated the hypoxic regulation of a number of miRNAs in bladder cancer. We have shown that miR- 145 is a novel, robust and direct HIF target gene that in turn leads to increased cell death in NMI bladder cancer cell lines

Interaction of Copper-Amine With Southern Pine: Retention and Migration

The retention and leachability of copper in copper-amine (Cu-EA)-treated southern pine (SP) are influenced by the formulation and the composition of copper-amine treating solutions. The sources of copper used, Cu(OH)2, CuCO3, CuSO4, and Cu(NO3)2, in the copper-amine complex formulation affect the leachability of copper. Data show that copper-amine from CuSO4- and Cu(NO3)2-treated wood has less copper loss during laboratory water leaching than that from Cu(OH)2- and CuCO3-treated wood. Increasing the amine-to-copper molar ratio increases the copper retention by wood, but reduces the leach resistance of copper. The nature of amine ligands, such as monoethanolamine (primary amine), 2-methylamino-ethanol (secondary amine), and N, N-dimethyl-ethanolamine (tertiary amine), has some effect on copper retention and copper leaching. As the molecular weight of amine ligands increases, copper loss during leaching decreases

Relationship of EMAST and Microsatellite Instability Among Patients with Rectal Cancer

Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature identified in 60% of sporadic colon cancers and may be linked with heterogeneous expression of the DNA mismatch repair (MMR) protein hMSH3. Unlike microsatellite instability-high (MSI-H) in which hypermethylation of hMLH1 occurs followed by multiple susceptible gene mutations, EMAST may be associated with inflammation and subsequent relaxation of MMR function with the biological consequences not known. We evaluated the prevalence of EMAST and MSI in a population-based cohort of rectal cancers, as EMAST has not been previously determined in rectal cancers. We analyzed 147 sporadic cases of rectal cancer using five tetranucleotide microsatellite markers and National-Cancer-Institute-recommended MSI (mononucleotide and dinucleotide) markers. EMAST and MSI determinations were made on analysis of DNA sequences of the polymerase chain reaction products and determined positive if at least two loci were found to have frame-shifted repeats upon comparison between normal and cancer samples from the same patient. We correlated EMAST data with race, gender, and tumor stage and examined the samples for lymphocyte infiltration. Among this cohort of patients with rectal cancer (mean age 62.2 ± 10.3 years, 36% female, 24% African American), 3/147 (2%) showed MSI (three males, two African American) and 49/147 (33%) demonstrated EMAST. Rectal tumors from African Americans were more likely to show EMAST than Caucasians (18/37, 49% vs. 27/104, 26%, p = 0.014) and were associated with advanced stage (18/29, 62% EMAST vs. 18/53, 37%, non-EMAST p = 0.02). There was no association between EMAST and gender. EMAST was more prevalent in rectal tumors that showed peri-tumoral infiltration compared to those without (30/49, 60% EMAST vs. 24/98, 25% non-EMAST, p = 0.0001). EMAST in rectal cancer is common and MSI is rare. EMAST is associated with African-American race and may be more commonly seen with metastatic disease. The etiology and consequences of EMAST are under investigation, but its association with immune cell infiltration suggests that inflammation may play a role for its development