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Prostate cancer (PCa) is the fifth leading cause of cancer related deaths worldwide, in part due to a lack of molecular stratification tools that can distinguish primary tumours that will remain indolent from those that will metastasise. Amongst potential molecular biomarkers, microRNAs (miRs) have attracted particular interest because of their high stability in body fluids and fixed tissues. These small non-coding RNAs modulate several physiological and pathological processes, including cancer progression. Herein we explore the prognostic potential and the functional role of miRs in localised PCa and their relation to nodal metastasis. We define a 7-miR signature that is associated with poor survival independently of age, Gleason score, pathological T state, N stage and surgical margin status and that is also prognostic for disease-free survival in patients with intermediate-risk localised disease. Within our 7-miR signature, we show that miR-378a-3p (hereafter miR-378a) levels are low in primary tumours compared to benign prostate tissue, and also lower in Gleason score 8–9 compared to Gleason 6–7 PCa. We demonstrate that miR-378a impairs glucose metabolism and reduces proliferation in PCa cells through independent mechanisms, and we identify glucose transporter 1 (GLUT1) messenger RNA as a direct target of miR-378a. We show that GLUT1 inhibition hampers glycolysis, leading to cell death. Our data provides a rational for a new PCa stratification strategy based on miR expression, and it reveals that miR-378a and GLUT1 are potential therapeutic targets in highly aggressive glycolytic PCa.

Prostate cancer (PCa) is the fifth leading cause of cancer related deaths worldwide, in part due to a lack of molecular stratification tools that can distinguish primary tumours that will remain indolent from those that will metastasise. Amongst potential molecular biomarkers, microRNAs (miRs) have attracted particular interest because of their high stability in body fluids and fixed tissues. These small non-coding RNAs modulate several physiological and pathological processes, including cancer progression. Herein we explore the prognostic potential and the functional role of miRs in localised PCa and their relation to nodal metastasis. We define a 7-miR signature that is associated with poor survival independently of age, Gleason score, pathological T state, N stage and surgical margin status and that is also prognostic for disease-free survival in patients with intermediate-risk localised disease. Within our 7-miR signature, we show that miR-378a-3p (hereafter miR-378a) levels are low in primary tumours compared to benign prostate tissue, and also lower in Gleason score 8-9 compared to Gleason 6-7 PCa. We demonstrate that miR-378a impairs glucose metabolism and reduces proliferation in PCa cells through independent mechanisms, and we identify glucose transporter 1 (GLUT1) messenger RNA as a direct target of miR-378a. We show that GLUT1 inhibition hampers glycolysis, leading to cell death. Our data provides a rational for a new PCa stratification strategy based on miR expression, and it reveals that miR-378a and GLUT1 are potential therapeutic targets in highly aggressive glycolytic PCa.

Background Our prior Systemic Treatment Options for Cancer of the Prostate systematic reviews showed improved survival for men with metastatic hormone-naive prostate cancer when abiraterone acetate plus prednisolone/prednisone (AAP) or docetaxel (Doc), but not zoledronic acid (ZA), were added to androgen-deprivation therapy (ADT). Trial evidence also suggests a benefit of combining celecoxib (Cel) with ZA and ADT. To establish the optimal treatments, a network meta-analysis (NMA) was carried out based on aggregate data (AD) from all available studies. Methods Overall survival (OS) and failure-free survival data from completed Systemic Treatment Options for Cancer of the Prostate reviews of Doc, ZA and AAP and from recent trials of ZA and Cel contributed to this comprehensive AD-NMA. The primary outcome was OS. Correlations between treatment comparisons within one multi-arm, multi-stage trial were estimated from control-arm event counts. Network consistency and a common heterogeneity variance were assumed. Results We identified 10 completed trials which had closed to recruitment, and one trial in which recruitment was ongoing, as eligible for inclusion. Results are based on six trials including 6204 men (97% of men randomised in all completed trials). Network estimates of effects on OS were consistent with reported comparisons with ADT alone for AAP [hazard ration (HR) = 0.61, 95% confidence interval (CI) 0.53–0.71], Doc (HR = 0.77, 95% CI 0.68–0.87), ZA + Cel (HR = 0.78, 95% CI 0.62–0.97), ZA + Doc (HR = 0.79, 95% CI 0.66–0.94), Cel (HR = 0.94 95% CI 0.75–1.17) and ZA (HR = 0.90 95% CI 0.79–1.03). The effect of ZA + Cel is consistent with the additive effects of the individual treatments. Results suggest that AAP has the highest probability of being the most effective treatment both for OS (94% probability) and failure-free survival (100% probability). Doc was the second-best treatment of OS (35% probability). Conclusions Uniquely, we have included all available results and appropriately accounted for inclusion of multi-arm, multi-stage trials in this AD-NMA. Our results support the use of AAP or Doc with ADT in men with metastatic hormone-naive prostate cancer. AAP appears to be the most effective treatment, but it is not clear to what extent and whether this is due to a true increased benefit with AAP or the variable features of the individual trials. To fully account for patient variability across trials, changes in prognosis or treatment effects over time and the potential impact of treatment on progression, a network meta-analysis based on individual participant data is in development.

Previous research has seen difficulties in establishing clear discrimination by principal component analysis (PCA) between drug-treated cells analysed by single point SR-FTIR spectroscopy, relative to multisampling cell monolayers by conventional FTIR. It is suggested that the issue arises due to signal mixing between cellular-response signatures and cell cycle phase contributions in individual cells. Consequently, chemometric distinction of cell spectra treated with multiple drugs is difficult even with supervised methods. In an effort to separate cell cycle chemistry from cellular response chemistry in the spectra, renal carcinoma cells were stained with propidium iodide and fluorescent-activated cell sorted (FACS) after exposure to a number of chemotherapeutic compounds; 5-fluorouracil (5FU) and a set of novel gold-based experimental compounds. The cell spectra were analysed separately by PCA in G1, S or G2/M phase. The mode of action of established drug 5FU, known to disrupt S phase, was confirmed by FACS analysis. The chemical signature of 5FU-treated cells discriminated against both the control and gold-compound (KF0101)-treated cell spectra, suggesting a different mode of action due to a difference in cellular response.

Over the course of the past 30 years progress in identification of risk factors, improvements in therapy regimens and advances in treatment delivery have all served to render testis cancer a treatable and survivable malignancy. Institution of specialised oncology centres in the UK to address treatment options for urological cancers has proven to be an effective approach to managing testis cancer by drawing on interdisciplinary expertise and applying insights gained from ongoing RD. This strategy could become a model for treating other cancers as well.

Here we report on investigations into using Raman optical tweezers to analyse both live and chemically fixed prostate and bladder cells. Spectra were subjected to chemometric analysis to discriminate and classify the cell types based on their spectra. Subsequent results revealed the potential of Raman tweezers as a potential clinical diagnostic tool.

To evaluate the use of local anaesthesia (LA) in 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) for superficial transitional cell carcinoma (TCC) of the bladder, and to provide further toxicity and tolerability data on this new method within the context of a phase 1 trial.ALA PDT was administered to 19 patients with recurrent superficial TCC (stage Ta/carcinoma in situ, grades 1-3) using escalating doses of ALA (3-6%) and 633 nm laser light (25-50 J/cm2) under various LA (lignocaine) protocols. Pain was assessed using a linear analogue scale from 0 to 10. The endpoints of tolerability and toxicity were assessed for the different LA, light and ALA doses, with lignocaine levels.ALA PDT is painful and requires some form of anaesthesia. The discomfort was immediate, associated with bladder spasm, and was a function of the ALA concentration rather than the total light dose given. Simple passive diffusion (PD) of 2% lignocaine instilled for 40 min before PDT gave adequate anaesthesia with 3% ALA (n=8; median pain score 1, range 0-2). With 6% ALA the pain was dramatically increased using PD (n=6; median pain score 8, range 5-10) and therefore the more potent LA technique of electromotive drug administration (EMDA) of 2% lignocaine was used, with excellent results (n=3; median pain score 1, range 0-2). All patients had transient bladder irritability that typically lasted 9-12 days, with no subjective/objective change in long-term bladder function. No other toxicity was reported. Serum lignocaine levels were minimal.Bladder ALA PDT is both safe and feasible under LA. At a dose of 3% ALA, the procedure was well-tolerated using PD of lignocaine. At higher doses (6% ALA) more effective anaesthesia is required and this can be obtained satisfactorily with EMDA of lignocaine. With refinement, ALA PDT may be feasible as an outpatient treatment for superficial bladder TCC.

To determine the E-cadherin and beta-catenin expression phenotype in untreated primary prostate cancer and corresponding bone metastases.Paired bone metastasis and primary prostate specimens were obtained from 14 men with untreated metastatic prostate carcinoma. The tumours were histologically graded by an independent pathologist. Expression of mRNA for E-cadherin and beta-catenin was detected within the tumour cells using in-situ hybridization with a 35S-labelled cDNA probe. The expression of E-cadherin and beta-catenin were graded as uniform, heterogeneous or negative.The mRNA for E-cadherin was expressed in 13 of 14 primary carcinomas and 11 bone metastases; beta-catenin was expressed by 13 and nine, respectively. Of the primary tumours, nine expressed E-cadherin and beta-catenin uniformly; in contrast, all metastases had down-regulated E-cadherin and/or beta-catenin.The down-regulation of E-cadherin and beta-catenin are a feature of the metastatic phenotype, which may be a significant factor in the genesis of bone metastases. However, this does not appear to be reflected in the expression of these molecules in the primary tumours.

Prostate cancer in bone is generally thought to progress more rapidly than in its primary site, a supposition that is supported by studies of prostate-specific antigen velocity. However, descriptions of proliferative rates in metastases have relied on inferred data from in vitro studies of cell lines derived from metastases. The aim of this study was to determine directly the proliferative rate within bone metastases arising from prostate cancer.10 bone biopsies containing metastatic deposits of untreated prostatic cancer were obtained. These were immunohistochemically stained for the Ki-67 protein with the monoclonal antibody MIB-1, using the streptavidin-biotin complex technique. Benign prostatic tissue was used as the control. Using an image analyser, the Ki-67 index (% of cells staining positively) in each specimen was determined.In the 10 specimens the Ki-67 index ranged from 0.15 to 7.82%. Wide overlap was seen between groups of differing tumour differentiation.The proliferative rate as determined by the Ki-67 index in bone metastases of prostate cancer is similar to that reported in primary tumours. There does not appear to be a relationship between tumour grade and proliferative index in these specimens.

To present the results of the staged management of complex entero-urinary fistulae.Ten patients with complex entero-urinary fistulae were reviewed; all patients were referred to a national intestinal failure unit after failed treatment in other centres. Each patient was treated in three stages. The acute stage involved proximal defunctioning and distal drainage of both the gastrointestinal and urinary tracts to isolate the fistula, together with the eradication of sepsis. The recovery stage involved total parenteral nutrition, organ support, radiological planning of surgical reconstruction and intensive nursing. The reconstructive stage followed when the patient was stable, nutritionally replenished and intra-abdominal sepsis was controlled. Surgery was undertaken jointly by urological and gastrointestinal surgeons.The fistulae were treated successfully in all patients, with functional restoration in four, and/or diversion of the gastrointestinal and urological tracts in six. The mean (range) time to reconstruction was 5 (1-20) months. There were no postoperative deaths.A staged multidisciplinary approach with delayed reconstruction can achieve a successful outcome in the management of complex entero-urinary fistulae.

To assess (i) the optical properties and depth of penetration of varying wavelengths of light in ex-vivo human bladder tissue, using specimens of normal bladder wall, transitional cell carcinoma (TCC) and bladder tissue after exposure to ionizing radiation; and (ii) to estimate the depth of bladder wall containing cancer that could potentially be treated with intravesical photodynamic therapy (PDT), assuming satisfactory tissue levels of photosensitizer. Materials and methods The study included 11 cystectomy specimens containing invasive TCC (five from patients who had previously received external-beam bladder radiotherapy, but with recurrent TCC) and three 'normal' bladders removed from patients treated by exenteration surgery for extravesical pelvic cancer. Full-thickness bladder wall and tumour samples were taken from these specimens and using an 'intravesical' and a previously validated interstitial model, the optical penetration depths (i.e. the tissue depth at which the light fluence is 37% of incident) were calculated at wavelengths of 633, 673 and 693 nm.There were no significant differences in light penetration between normal and tumour-affected bladder tissue at each wavelength. There were significant differences in light penetration among wavelengths; light at 693 nm penetrated approximately 40% further than light at 633 nm (P0.002). The light currently used in bladder PDT (633 nm) has a mean (SEM) optical penetration depth of 4.0 (0.1) mm within TCC. In addition, at this wavelength, there was 29% greater light penetration in previously irradiated than in unirradiated bladder wall (P = 0.001). This did not occur in the tumour-affected bladder.Bladder tissue is relatively more translucent than other human tissues and there is therefore great potential for PDT in the treatment of bladder cancer. As there is no difference in light penetration between TCC and normal bladder tissue, a tumour-specific response with diffuse illumination of the bladder will depend on drug localization within the tumour. The currently used wavelength of 633 nm can be expected to exert a PDT effect within bladder tumour up to a depth of 20 mm. Increasing the wavelength will allow deeper pathology to be treated.

To determine whether the calcium-dependent cell adhesion molecule E-cadherin is expressed in metastatic deposits of prostate cancer in bone.Ten bone biopsies containing metastatic deposits of untreated prostatic cancer were obtained and immunohistochemically stained for E-cadherin with the monoclonal antibody HECD-1, using the streptavidin-biotin complex technique. Benign prostatic tissue was used as the control.Of the 10 specimens, nine showed positive expression of E-cadherin, graded as strong in four. E-cadherin expression was strongest in well-differentiated metastases and decreased with increasing tumour grade. In some specimens there were mixed patterns of expression.E-cadherin is strongly expressed in prostatic bone metastases and the degree of expression appears to reflect local tumour grade. This suggests that loss of E-cadherin expression may not be critically linked to metastatic potential.

Prostate cancer metastases form selectively in the bone marrow. Previously we demonstrated motility was important for the formation of primary prostatic epithelial cell colonies in bone marrow stroma (BMS) co-culture. In this study we looked at the influence of motility factors on the colony formation of epithelial cells derived from benign (bPEC) or malignant (mPEC) prostate tissue. After 7 days co-culture we found that anti-scatter factor consistently inhibited prostate epithelial cell colony formation on BMS (7/7 mPEC and 4/7 bPEC samples showed significant inhibition). Antibodies against bFGF and 5T4 did not significantly affect colony formation. Addition of fibroblast conditioned media (derived from benign prostates) to co-cultures stimulated the colony formation of bPEC (170%) and mPEC (252%). This stimulation was eliminated by depletion of SF from the conditioned media. Immunohistochemical staining found c-Met expression in 5/6 bPEC cultures and 7/9 mPEC cultures. When grown in BMS co-culture expression of c-Met was positive in 3/6 bPEC and 2/7 mPEC samples. In conclusion, scatter factor influences the in vitro formation of prostate epithelial cell colonies on BMS co-culture.

Metastases of prostate cancer form selectively within the skeleton. To understand this metastatic spread, we studied the ability of prostate epithelial cells to grow and proliferate within the bone marrow, using primary coculture.Prostate epithelia and fibroblasts were prepared from men with benign prostatic hyperplasia (n = 13) and cancer of the prostate (n = 10). Confluent cultures of bone-marrow stroma or fibroblast controls were prepared in 96-well plates, and identical plates were treated with detergent to expose the extracellular matrix of the cells. Epithelial cells were seeded onto either cells or matrix, and their growth characteristics were determined by counting increases in colony size and number over time. Further experiments evaluated the effects on epithelial growth when cells were exposed to media conditioned by these stroma, using an MTT assay.Results showed that for epithelial cells derived from malignant (or benign) tissue, the median value of the total area of colonies formed on bone-marrow stroma was 2.1 (benign, 2.6) mm2, in contrast to 0.3 (benign, 0.4) mm2 or 0.25 (benign, 0) mm2 when these cells were cocultured with fibroblasts from benign or malignant prostates, respectively. Statistics indicated that growth was significantly greater on bone-marrow stroma than on control stroma (P0.005). However, no significant stimulation of epithelial cell growth was seen when these epithelial cells were cultured on extracellular matrix from bone-marrow stroma or when exposed to bone-marrow stroma-conditioned media in comparison to fibroblast controls. No statistical differences were found between the formation of colonies from malignant tissue in comparison to benign.This system allows the investigation of bone-marrow stroma colonization by primary prostate epithelial cells, and could be developed for the study of metastasis.

Prostate cancer selectively metastasises to the bone. To investigate the importance of prostate epithelial cell adhesion to bone marrow cells in this process we examined the binding of human primary prostatic epithelial cells (PEC) to human bone marrow stromal cultures (BMS). We found that PEC derived from both malignant and benign tissue showed greater adhesion to BMS than to benign prostatic fibroblasts (median difference was 340% and 200% respectively), skin fibroblasts or plastic tissue culture plates. Adhesion to BMS grown from the bone marrow of patients with prostatic skeletal metastases was no different from those grown from normal bone marrow. The role of integrin molecules in these cell interactions was determined. Collagen type I and fibronectin were found to increase PEC adhesion whereas vitronectin and laminin did not. Inhibition studies demonstrated that although there was heterogeneity between samples, antibodies against the integrins alpha2 and beta1 consistently inhibited PEC binding to BMS. This result was more marked for PEC derived from malignant tissue. However studies investigating the effects of disintegrins and anti-alpha3 and anti-alpha5 integrins indicated that for a percentage of patients these integrins and RGD (arginine, glycine, aspartamine)-dependent binding pathways were also involved. In summary, the results indicate that BMS are adherent to primary PEC derived from both malignant and benign tissue. The integrin alpha2beta1 is a major contributor to this interaction.