HumanMethylation450K Array-Identified Biomarkers Predict Tumour Recurrence/Progression at Initial Diagnosis of High-risk Non-muscle Invasive Bladder Cancer.

BACKGROUND: High-risk non-muscle invasive bladder cancer (HR-NMIBC) is a clinically unpredictable disease. Despite clinical risk estimation tools, many patients are undertreated with intra-vesical therapies alone, whereas others may be over-treated with early radical surgery. Molecular biomarkers, particularly DNA methylation, have been reported as predictive of tumour/patient outcomes in numerous solid organ and haematologic malignancies; however, there are few reports in HR-NMIBC and none using genome-wide array assessment. We therefore sought to identify novel DNA methylation markers of HR-NMIBC clinical outcomes that might predict tumour behaviour at initial diagnosis and help guide patient management. PATIENTS AND METHODS: A total of 21 primary initial diagnosis HR-NMIBC tumours were analysed by Illumina HumanMethylation450 BeadChip arrays and subsequently bisulphite Pyrosequencing. In all, 7 had not recurred at 1 year after resection and 14 had recurred and/or progressed despite intra-vesical BCG. A further independent cohort of 32 HR-NMIBC tumours (17 no recurrence and 15 recurrence and/or progression despite BCG) were also assessed by bisulphite Pyrosequencing. RESULTS: Array analyses identified 206 CpG loci that segregated non-recurrent HR-NMIBC tumours from clinically more aggressive recurrence/progression tumours. Hypermethylation of CpG cg11850659 and hypomethylation of CpG cg01149192 in combination predicted HR-NMIBC recurrence and/or progression within 1 year of diagnosis with 83% sensitivity, 79% specificity, and 83% positive and 79% negative predictive values. CONCLUSIONS: This is the first genome-wide DNA methylation analysis of a unique HR-NMIBC tumour cohort encompassing known 1-year clinical outcomes. Our analyses identified potential novel epigenetic markers that could help guide individual patient management in this clinically unpredictable disease.

Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer.

High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P<0.05), and similarly significant increases in mean levels of methylation in high-grade tumors for the ATP5G2, VAX2, INSRR, PRDM14, VSX1, TFAP2b, PRRX1, and HIST1H4F genes (P<0.05). Although inappropriate promoter methylation was not invariantly associated with reduced transcript expression, a significant association was apparent for the ARHGEF4, PON3, STAT5a, and VAX2 gene transcripts (P<0.05). Herein, we present the first genome-wide DNA methylation analysis in a unique HG-NMIBC cohort, showing extensive and discrete methylation changes relative to normal bladder and low-intermediate-grade tumors. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination, with more conventional therapeutic options in the treatment of this clinically unpredictable disease.

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer.

INTRODUCTION: Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis. METHODS: Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases. RESULTS: The ISL1 genes' promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM). CONCLUSIONS: In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.

Elevated NCOR1 disrupts PPARalpha/gamma signaling in prostate cancer and forms a targetable epigenetic lesion.

The loss of anti-proliferative responsiveness in prostate cancer cell lines toward ligands for vitamin D receptor, retinoic acid receptors/retinoid X receptors and peroxisome proliferator activated receptor (PPAR)alpha/gamma may entail underlying epigenetic events, as ligand insensitivity reflects significantly altered messenger RNA expression of corepressors and histone-modifying enzymes. Expression patterns were dependent on phases of the cell cycle and associated with repressed basal gene expression of vitamin D receptor and PPARalpha/gamma target genes, for example CDKN1A [encodes p21((waf1/cip1))]. Elevated nuclear corepressor 1 (NCOR1) and nuclear corepressor 2/silencing mediator of retinoic acid and thyroid hormone receptor protein levels were detected in prostate cancer cell lines compared with non-malignant counterparts. Knockdown of the corepressor NCOR1 significantly elevated basal expression of a cohort of target genes, including CDKN1A. Both chemical [histone deacetylases inhibitor (HDACi)] and NCOR1 knockdown targeting enhanced anti-proliferative sensitivity toward PPARalpha/gamma ligands in prostate cancer cell lines. Pursuing PPARalpha/gamma signaling, microarray approaches were undertaken to identify pathways and genes regulated uniquely by a combination of PPARalpha/gamma activation and HDAC inhibition. Again, HDACi and knockdown approaches demonstrated that elevated NCOR1 expression and activity distorted PPARalpha/gamma gene targets centered on, for example cell cycle control, including CDKN1A and TGFBRAP1. Quantitative real time polymerase chain reaction validation and chromatin immunoprecipitation assays both confirmed that elevated NCOR1 disrupted the ability of PPARalpha/gamma to regulate key target genes (CDKN1A and TGFBRAP1). Interrogation of these relationships in prostate cancer samples using principal component and partial correlation analyses established significant interdependent relationships between NCOR1-PPARalpha/gamma and representative target genes, independently of androgen receptor expression. Therefore, we conclude that elevated NCOR1 distorts the actions of PPARalpha/gamma selectively and generates a potential epigenetic lesion with diagnostic and prognostic significance.

Nanotechnology and its relevance to the urologist.

OBJECTIVES: We review important aspects of nanotechnology, and discuss the wide range of research and clinical applications of nanomedicine in the field of urology. There is particular emphasis on key clinical and pre-clinical studies to provide an update on recent and potential applications in the care of urological patients. METHODS: A directed Medline literature review of nanotechnology was performed. Important publications that have shaped our understanding of nanotechnology were selected for review and were augmented by manual searches of reference lists. RESULTS: Nanotechnology is the study, design, creation, synthesis, manipulation, and application of functional materials, devices, and systems through control of matter at the nanometer scale. Studies demonstrate a number of important concepts. These include nanovectors, nanotubes, and nanosensors for targeted drug delivery; nanowires and nanocantilever arrays for early detection of precancerous and malignant lesions; and nanopores for DNA sequencing. These advances will lead to significant applications relevant to the diagnosis, management, and treatment of all urological conditions. CONCLUSIONS: This review is designed for the urologist to provide an overview and update on nanotechnology and its applications in the field of urology. In the future, it is widely expected that nanotechnology and nanomedicine will have a significant impact on urological research and clinical practice, allowing urologists to intervene at the cellular and molecular level. With structured, safe implementation, nanotechnologies have the potential to revolutionise urological practice in our lifetime.

Current challenges in Wilms' tumor management.

Wilms' tumor is a renal cancer that predominantly affects children during the first 2 years of life. The continuing success of clinical trials in Wilms' tumor patients over the past 30 years has led to an overall survival of 85%, and treatment-related morbidity has been reduced. Less-aggressive chemotherapeutic regimes are available for patients with validated good prognostic factors, such as low stage and favorable histology. It is becoming increasingly apparent that treatment can be optimized through stratification of patients according to tumor stage and histology. Established treatments for Wilms' tumor include perioperative vincristine and actinomycin, with or without doxorubicin or radiotherapy. Relapsed patients have the option of salvage chemotherapy with ifosfamide, carboplatin and etoposide, as well as high-dose chemotherapy regimes and autologous hemopoietic stem-cell rescue. Further research is required to refine these regimes and identify further the role of additional prognostic factors in this childhood disease. In this article we discuss the most-debated issues and advances that have been made in the management of Wilms' tumor.

Basic principles of real-time quantitative PCR.

Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.

New phosphodiesterase inhibitors in the treatment of erectile dysfunction.

Sildenafil revolutionised the treatment of erectile dysfunction (ED) on its introduction in 1998. Not only is it effective, but is, perhaps even more importantly, an extremely acceptable manner in which to treat ED (i.e., oral pharmacotherapy), compared to the other treatments that were available at that time (e.g., intracavernosal injection). Whilst sildenafil remains the market leader for ED treatment, it does have some shortcomings: its clinical efficacy is diminished in 'difficult to treat' patient groups, such as diabetics, side effects, such as blue-tinged vision and headache, and the need to time the ingestion of sildenafil to 1 h prior to expected time of sexual intercourse. Recently, newer phosphodiesterase inhibitors ('sons of viagra') have become available, and potentially to improve upon, what has become regarded as the gold standard for ED treatment. This review article discusses the differences between vardenafil, tadalafil and sildenafil, in order to allow the clinician to make an informed decision as to which phosphodiesterase inhibitor to prescribe when dealing with the ever expanding number of patients seeking help for ED.

Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells.

We completed a multicenter study of the effects of pomegranate cold-pressed (Oil) or supercritical CO(2)-extracted (S) seed oil, fermented juice polyphenols (W), and pericarp polyphenols (P) on human prostate cancer cell xenograft growth in vivo, and/or proliferation, cell cycle distribution, apoptosis, gene expression, and invasion across Matrigel, in vitro. Oil, W, and P each acutely inhibited in vitro proliferation of LNCaP, PC-3, and DU 145 human cancer cell lines. The dose of P required to inhibit cell proliferation of the prostate cancer cell line LNCaP by 50% (ED(50)) was 70 microg/mL, whereas normal prostate epithelial cells (hPrEC) were significantly less affected (ED(50) = 250 g/mL). These effects were mediated by changes in both cell cycle distribution and induction of apoptosis. For example, the androgen-independent cell line DU 145 showed a significant increase from 11% to 22% in G(2)/M cells (P <.05) by treatment with Oil (35 microg/mL) with a modest induction of apoptosis. In other cell lines/treatments, the apoptotic response predominated, for example, in PC-3 cells treated with P, at least partially through a caspase 3-mediated pathway. These cellular effects coincided with rapid changes in mRNA levels of gene targets. Thus, 4-hour treatment of DU 145 cells with Oil (35 microg/mL) resulted in significant 2.3 +/- 0.001-fold (mean +/- SEM) up-regulation of the cyclin-dependent kinase inhibitor p21((waf1/cip1)) (P <.01) and 0.6 +/- 0.14-fold down-regulation of c-myc (P <.05). In parallel, all agents potently suppressed PC-3 invasion through Matrigel, and furthermore P and S demonstrated potent inhibition of PC-3 xenograft growth in athymic mice. Overall, this study demonstrates significant antitumor activity of pomegranate-derived materials against human prostate cancer.

Allogeneic stem-cell transplantation for renal-cell cancer.

Metastatic renal-cell carcinoma (RCC) is resistant to chemotherapy, and patients with this disease have a poor outlook. Immunotherapy by use of cytokines and vaccines against tumour antigens has shown encouraging results in a small group of patients. Advances in the understanding of the graft-versus-tumour effect in haematological malignant disorders have led to the use of stem-cell transplantation for treatment of solid-organ malignant diseases such as RCC. Techniques of bone-marrow ablation have been superseded by safer conditioning regimens, with occasional complete remission and partial remission in some patients. Graft-versus-host disease, engraftment failure, and disease progression remain important obstacles to the widespread use of new techniques for metastatic RCC. Here, we summarise important issues surrounding immunotherapy for RCC, the problems encountered with use of immunotherapy, and the present use of non-myeloablative techniques for treatment of this disease.

Altered SMRT levels disrupt vitamin D3 receptor signalling in prostate cancer cells.

We hypothesized that key antiproliferative target genes for the vitamin D receptor (VDR) were repressed by an epigenetic mechanism in prostate cancer cells resulting in apparent hormonal insensitivity. To explore this possibility, we examined nuclear receptor corepressor expression in a panel of nonmalignant and malignant cell lines and primary cultures, and found frequently elevated SMRT corepressor mRNA expression often associated with reduced sensitivity to 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)2D3). For example, PC-3 and DU-145 prostate cancer cell lines had 1.8-fold and twofold increases in SMRT mRNA relative to normal PrEC cells (P<0.05). Similarly, 10/15 primary tumour cultures (including three matched to normal cells from the same donors) had elevated SMRT mRNA levels; generally NCoR1 and Alien were not as commonly elevated. Corepressor proteins often have associated histone deacetylases (HDAC) and reflectively the antiproliferative action of 1alpha,25(OH)2D3 can be 'restored' by cotreatment with low doses of HDAC inhibitors such as trichostatin A (TSA, 15 nM) to induce apoptosis in prostate cancer cell lines. To decipher the transcriptional events that lead to these cellular responses, we undertook gene expression studies in PC-3 cells after cotreatment of 1alpha,25(OH)2D3 plus TSA after 6 h. Examination of known VDR target genes and cDNA microarray analyses revealed cotreatment of 1alpha,25(OH)2D3 plus TSA cooperatively upregulated eight (out of 1176) genes, including MAPK-APK2 and GADD45alpha. MRNA and protein time courses and inhibitor studies confirmed these patterns of regulation. Subsequently, we knocked down SMRT levels in PC-3 cells using a small interfering RNA (siRNA) approach and found that GADD45alpha induction by 1alpha,25(OH)2D3 alone became very significantly enhanced. The same distortion of gene responsiveness, with repressed induction of GADD45alpha was found in primary tumour cultures compared and to matched peripheral zone (normal) cultures from the same donor. These data demonstrate that elevated SMRT levels are common in prostate cancer cells, resulting in suppression of target genes associated with antiproliferative action and apparent 1alpha,25(OH)2D3-insensitivity. This can be targeted therapeutically by combination treatments with HDAC inhibitors.

Epigenetic repression of transcription by the Vitamin D3 receptor in prostate cancer cells.

Normal prostate epithelial cells are acutely sensitive to the antiproliferative action of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), whilst prostate cancer cell lines and primary cultures display a range of sensitivities. We hypothesised that key antiproliferative target genes of the Vitamin D receptor (VDR) were repressed by an epigenetic mechanism in 1alpha,25(OH)(2)D(3)-insensitive cells. Supportively, we found elevated nuclear receptor co-repressor and reduced VDR expression correlated with reduced sensitivity to the antiproliferative action of 1alpha,25(OH)(2)D(3). Furthermore, the growth suppressive actions of 1alpha,25(OH)(2)D(3) can be restored by co-treatment with low doses of histone deacetylation inhibitors, such as trichostatin A (TSA) to induce apoptosis. Examination of the regulation of VDR target genes revealed that co-treatment of 1alpha,25(OH)(2)D(3) plus TSA co-operatively upregulated GADD45alpha. Similarly in a primary cancer cell culture, the regulation of appeared GADD45alpha repressed. These data demonstrate that prostate cancer cells utilise a mechanism involving deacetylation to suppress the responsiveness of VDR target genes and thus ablate the antiproliferative action of 1alpha,25(OH)(2)D(3).

Luteinising hormone releasing hormone analogues in the treatment of prostate cancer.

The use of the luteinising hormone releasing hormone (LHRH) analogues--goserelin (Zoladex, AstraZeneca) and leuprorelin (Prostap, Wyeth)--is well established and forms the backbone of the treatment of locally advanced and metastatic prostate cancer. Comparable efficacy with orchidectomy and, historically, diethylstilbestrol (DES) is accepted, with the advantages of reversibility and limited thromboembolic and cardiovascular toxicity, respectively. Side effects such as hot flushes, loss of libido, lethargy and decreased bone mineral density have recently stimulated more interest in the use of non-steroidal anti-androgens such as bicalutamide (Casodex, AstraZeneca) in locally advanced disease. Although better tolerated, bicalutamide has significant problems with gynaecomastia and breast pain. Maximal androgen blockade using LHRH analogues and their adjuvant use with radiotherapy are discussed, as well as their experimental application in intermittent androgen suppression therapy. Similar side effect profiles are reported for the LHRH analogues but injection tolerability differs with the smaller 23G needle for Prostap 3 compared to the 16G needle for Zoladex LA. There is no evidence to suggest a difference in the efficacy between the LHRH analogues goserelin and leuprorelin, although no direct comparison has yet been undertaken.