Prognostic role of ECOG performance status in patients with urothelial carcinoma of the upper urinary tract: an international study

Study Type – Prognosis (inception cohort series) Level of Evidence 2a What's known on the subject? and What does the study add? ECOG Performance Status has gained wide popularity as an integral part of the assessment of patients with upper urinary tract carcinoma. Our findings indicate that ECOG‐PS is strongly associated with perioperative and overall survival and should be considered carefully in our decision‐making process.

OBJECTIVE

• ? To evaluate the prognostic role of ECOG Performance status (ECOG‐PS) in a large multi‐institutional international cohort of patients treated with radical nephroureterectomy for upper tract urothelial carcinoma.

MATERIALS AND METHODS

• ? Data of 427 patients treated with radical nephroureterectomy at five international institutions in Asia, Europe and Northern America were collected retrospectively from 1987 to 2008.
• ? Logistic and Cox regression models were used for univariable and multivariable analyses.

RESULTS

• ? ECOG‐PS was 0 in 272 of 427 (64%) patients. The median follow‐up of the whole cohort was 32 months.
• ? The five‐year recurrence‐free (RFS), cancer‐specific (CSS) and overall (OS) survival estimates were 71.7%, 74.9% and 68.5%, respectively, in patients with ECOG‐PS 0 compared with 60.1%, 67.8%, and 51.4% respectively, in patients with ECOG‐PS ≥1 (P value 0.08 for RFS, 0.43 for CSS, and <0.001 for OS, respectively).
• ? On multivariable Cox regression analyses, ECOG‐PS was not an independent predictor of either RFS (hazard ratio 1.4; P = 0.107) or CSS (hazard ratio 1.2; P = 0.426) but was an independent predictor of OS (hazard ratio 1.5; P = 0.03).

CONCLUSIONS

• ? In this large multicentre international study, ECOG‐PS was not significantly associated with RFS and CSS.
• ? Conversely we find a strong association with survival 1‐month after surgery and OS. Further research is needed to ascertain the additive prognostic role of ECOG‐PS in well‐designed prospective multicentre studies.

     

Use of fluorescein isothiocyanate-human serum albumin for the intravesical photodiagnosis of non-muscle-invasive bladder cancer: an in vitro study using multicellular spheroids composed of normal human urothelial and urothelial cell carcinoma cell lines

OBJECTIVE

• ? To evaluate human serum albumin (HSA), fluorescently labelled with fluorescein isothiocyanate (FITC), as a potential intravesical photodiagnostic method for the early detection of non‐muscle‐invasive bladder cancer.

PATIENTS AND METHODS

• ? By using multicellular spheroids prepared from normal human urothelial (NHU) cells and from different urothelial cell carcinoma (UCC) cell lines (T24, J82), we simulated three‐dimensionally the normal urothelium and non‐muscle‐invasive UCCs present in the bladder of patients.
• ? The distribution of FITC‐HSA in these spheroids was investigated.

RESULTS

• ? Our data showed that fluorescently labelled albumin is quite evenly dispersed throughout the spheroids. However, in the case of the 10 mg/mL incubations, the fluorescence intensity seems to increase slightly towards the spheroid core.
• ? Using 1 mg/mL, the penetration of FITC‐HSA in T24 differed significantly from the penetration in NHU spheroids, but this was not the case for J82 spheroids.
• ? When the concentration of FITC‐HSA was increased 10‐fold, all UCC spheroids exhibited a significantly different accumulation of FITC‐HSA.

CONCLUSIONS

• ? As spheroids represent a suitable in vitro model for predicting the in vivo behaviour of compounds, our data suggest that FITC‐HSA could be used for the early detection of non‐muscle‐invasive bladder cancer.
• ? Human serum albumin conjugates of new or already available intravesical drugs could be generated to create alternative bladder cancer therapies with increased selectivity.

     

Reversing bladder outlet obstruction attenuates systemic and tissue oxidative stress

What's known on the subject? and What does the study add? Oxidative damage in bladder tissue and systemic oxidative biomarkers were both found to be increased in rabbits with partial bladder outlet obstruction. It is shown that the reversal of partial bladder outlet obstruction will attenuate the systemic oxidative stress.

OBJECTIVE

• ? To investigate whether partial bladder outlet obstruction (PBOO) increases systemic oxidative stress and whether relief of PBOO could attenuate this stress.

MATERIALS AND METHODS

• ? Surgically created PBOO in male New Zealand white rabbits was assessed after 4 weeks in one group of rabbits (n = 4), and was relieved in two additional groups of rabbits (n = 4 each) that were assessed at 4 and 8 weeks after relief of PBOO.
• ? Four sham‐operated rabbits served as controls. The assessed oxidative stress biomarkers included urinary and plasma 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and plasma malondialdehyde (MDA), total anti‐oxidant capacity (TAC) and glutathione (GSH).
• ? In addition, the copy number of mitochondrial DNA and the 8‐OHdG content in bladder tissues from these rabbits were also determined at the beginning and at indicated time points in the experiments.

RESULTS

• ? There were significant increases in both the 8‐OHdG levels of urine, plasma and bladder tissue and the plasma MDA after induction of PBOO.
• ? There were also significant decreases in the TAC, in GSH levels and in mitochondrial DNA copy number in bladder tissues after PBOO.
• ? Most importantly, all of the values returned toward the control levels after the PBOO was reversed at 8 weeks.

CONCLUSION

• ? PBOO increases systemic and oxidative stress and its reversal results in a progressive reduction of both systemic and tissue oxidative stress.

     

Current status of molecular markers for prognostication and outcome in invasive bladder cancer

What's known on the subject? and What does the study add? Currently, prognostication of patients with invasive BC is hampered owing to the inadequacy of standard clinicopathological risk factors to predict accurately individual treatment outcomes. This review provides a comprehensive albeit brief overview on current studies elucidating the potential role of different molecular markers to close this gap of evidence. It focusses on biostatistical considerations in the interpretation of study results which are essential to provide meaningful clinical conclusions for an individual patient.

OBJECTIVE

• ? To improve prognostication and the management of patients with invasive bladder cancer (BC).

METHODS

• ? Standard clinicopathological risk factors are not reliably enough to accurately predict outcomes in patients after radical treatment and guide clinicians for recommending selectively the use of adjuvant therapies.
• ? With detailed insights into the molecular pathology of BC, biomarkers have come to the fore of researchers as a potential tool to close this gap of evidence.
• ? However, their definitive role in the diagnostic and therapeutic management of patients with invasive BC has not clearly been addressed so far.

RESULTS

• ? Invasive BC are an extremely heterogenenous group of malignancies which are characterized by multiple genetic alterations involved in the carcinogenesis and development of metastatic spread. Thus, it is questionable whether any single marker will provide superior prognostication compared with a combination of markers.
• ? Current studies evaluating the predictive value of a multitude of markers have used high‐throughput technologies and investigated the gain in predictive accuracy within new nomograms which encompass well‐established clinicopathological and novel putative molecular parameters. p53 overexpression was found to be associated with increased risk of recurrence in urothelial and non‐urothelial cancer. In pT1 disease, the combination of p53, p21 and p16 as well as epigenetic alterations of myopodin expression has been shown to provide improved prognostication, and this might help to advocate more selectively the use of early radical treatment.
• ? After the bladder‐sparing approach, p53 and p21 overexpression indicate decreased probability of long‐term bladder preservation. Additionally, altered retinoblastoma expression is associated with improved survival after adjuvant chemotherapy.
• ? To provide meaningful conclusions for individual prognosis and the need of adjuvant treatment, biostatistical pitfalls in the analysis and interpretation of results have to be taken into account.

CONCLUSIONS

• ? Different molecular markers have the potential to improve prognostication of patients with invasive BC and provide improved evidence for targeted therapy in the neoadjuvant, adjuvant and metastatic setting.
• ? However, in order to advocate their routine clinical use on a sound scientific basis prospective data are still necessary.

     

Risk group stratification to predict recurrence after transurethral resection in Japanese patients with stage Ta and T1 bladder tumours: validation study on the European Association of Urology guidelines

Study Type – Therapy (case series)
Level of Evidence 4 What’s known on the subject? and What does the study add? EAU guidelines on non‐muscle‐invasive bladder tumours have been widely used for the prediction of recurrence after TUR. However, there are substantial differences in bladder cancer incidence and mortality rates between European countries and Japan. This study provides useful factors for predicting recurrence and validation of EAU guidelines on the risk group stratification to predict recurrence in Japanese patients with stage Ta and T1 bladder tumours.

OBJECTIVE

• ? To validate the European Association of Urology (EAU) guidelines on risk group stratification to predict recurrence in Japanese patients with stage Ta and T1 bladder tumours.

PATIENTS AND METHODS

• ? A cohort of 592 Japanese patients who were treated with transurethral resection (TUR) and histopathologically diagnosed with Ta and T1 urothelial carcinoma of the bladder were enrolled in this retrospective study.
• ? The primary endpoint of the present study was recurrence‐free survival, and the median follow‐up duration was 37 months in recurrence‐free survivors.

RESULTS

• ? Multivariate Cox proportional hazards regression analysis showed that the Eastern Cooperative Oncology Group performance status (ECOG PS), prior recurrence rate, number of tumours and T category were independent predictors of time to recurrence (P < 0.05). According to the EAU guidelines for predicting recurrence, the vast majority of Japanese patients were classified into intermediate risk.
• ? The intermediate‐risk patients were further divided into intermediate‐low‐risk and intermediate‐high‐risk subgroups based on the European Organization for Research and Treatment of Cancer risk table, and a significant difference in the recurrence‐free survival rates was found between these subgroups (P < 0.001).
• ? It was also found that patients with high risk combined with intermediate‐high risk had significantly poorer recurrence‐free survival rates than those with low risk combined with intermediate‐low risk (P < 0.001).

CONCLUSIONS

• ? This is the first report on the ECOG PS as a potentially useful predictor for bladder tumour recurrence.
• ? The risk group stratification of the EAU guidelines for recurrence might not be applicable to Japanese patients with Ta and T1 bladder tumours, but the subgroup classification of intermediate risk could be appropriate.

     

Temporal diabetes and diuresis-induced alteration of nerves and vasculature of the urinary bladder in the rat

What’s known on the subject? and What does the study add? Diabetes mellitus seriously affects urinary bladder function. Diabetes induces time‐dependent bladder hypertrophy and altered tissue composition. However, the alterations of the density of nerves and vasculatures in bladder under diabetes remains poorly studied. Diabetes induced time‐dependent changes of density of the nerves and vasculatures in the bladder tissues. In addition, diabetes‐related polyuria plays an important role in this alteration.

OBJECTIVE

• ? To characterize the temporal changes of the nerves and vasculature of the bladder in diabetic rats.

MATERIALS AND METHODS

• ? A total of 36 Sprague–Dawley rats were divided into three groups: streptozotocin‐induced diabetics, 5% sucrose‐induced diuretics and age‐matched controls.
• ? The characteristics of the nerves and vasculature in the equatorial cross‐sectional areas of the bladder were examined by immunofluorescence staining of their specific markers, neurofilament 200 (NF200) and CD31, at 1, 9 or 20 weeks after induction.
• ? The distributions of the nerves and blood vessels were observed and the densities were quantified.

RESULTS

• ? Diabetes caused a significant reduction in body weight. Bladder weight increased in diabetic and diuretic rats, but not in controls.
• ? The total cross‐sectional wall area and detrusor muscle area at the equatorial midline were greater in bladders of diabetic and diuretic rats than in controls.
• ? Neurofilament 200‐immunoreactive (NF200‐IR) nerves were mainly distributed in the detrusor muscle. CD31‐immunoreactive blood vessels were mainly distributed in the mucosa/submucosa.
• ? There were no significant differences in the NF200‐IR nerve terminal area among control, diabetic and diuretic groups. However nerve density was decreased at 9 and 20 weeks in the muscle, and at 20 weeks in the mucosa/submucosa in diabetic and diuretic animals.
• ? Blood vessel density decreased in the diabetic and diuretic groups at 20 weeks in the muscle.

CONCLUSIONS

• ? Diabetes induced time‐dependent changes in the density of the nerves and vasculature in the bladder tissues.
• ? Diabetes‐related polyuria plays an important role in these changes.

     

Evans blue as a selective dye marker for white-light diagnosis of non-muscle-invasive bladder cancer: an in vitro study

What's known on the subject? and What does the study add? We found that Evans blue preferentially accumulate in spheroids prepared from urothelial cell carcinoma (UCC) cells as compared to spheroids composed of normal human urothelial (NHU) cells. The present findings could be important for future developments in clinical diagnostics for early bladder cancer detection staging and grading involving white light cystocopy.

OBJECTIVE

• ? To develop a diagnostic method relying on the preferential accumulation of a dye in non‐muscle‐invasive bladder cancer (NMIBC) that is visible in conjunction with white‐light cystoscopy (WLC).

MATERIALS AND METHODS

• ? We investigated in detail the permeation of Evans blue in urothelial cell carcinoma (UCC) spheroids prepared from T24, J82 and RT‐112 human cell lines and spheroids composed of normal human urothelial (NHU) cells.
• ? To gain more insight into the differential accumulation, all spheroids were investigated ultrastructurally using transmission electron microscopy (TEM).

RESULTS

• ? We found that, after exposure to Evans blue for 2 h, UCC spheroids accumulated dramatically more dye than spheroids composed of NHU cells.
• ? Using TEM it was found that the malignant spheroids contain similar ultrastructural characteristics, i.e. a wide intercellular space and a decreased number of desmosome‐like cell attachments, to those from clinical samples of non‐papillary carcinoma in situ of the bladder.

CONCLUSION

• ? We believe the present findings could be important for future developments in clinical diagnostics for early bladder cancer detection, staging and grading involving WLC.

     

Maintenance therapy with bacillus Calmette-Guérin Connaught strain clearly prolongs recurrence-free survival following transurethral resection of bladder tumour for non-muscle-invasive bladder cancer

Study Type – Therapy (RCT) Level of Evidence 1b

OBJECTIVE

• ? To confirm the recurrence‐preventing efficacy and safety of 18‐month bacillus Calmette‐Guérin (BCG) maintenance therapy for non‐muscle‐invasive bladder cancer.

PATIENTS AND METHODS

• ? The enrolled patients had been diagnosed with recurrent or multiple non‐muscle‐invasive bladder cancer (stage Ta or T1) after complete transurethral resection of bladder tumours (TURBT).
• ? The patients were randomized into three treatment groups: a maintenance group (BCG, 81 mg, intravesically instilled once weekly for 6 weeks as induction therapy, followed by three once‐weekly instillations at 3, 6, 12 and 18 months after initiation of the induction therapy), a non‐maintenance group (BCG, 81 mg, intravesically instilled once weekly for 6 weeks) and an epirubicin group (epirubicin, 40 mg, intravesically instilled nine times). The primary endpoint was recurrence‐free survival (RFS).

RESULTS

• ? Efficacy analysis was performed for 115 of the full‐analysis‐set population of 116 eligible patients, including 41 maintenance group patients, 42 non‐maintenance group patients and 32 epirubicin group patients.
• ? At the 2‐year median point of the overall actual follow‐up period, the final cumulative RFS rates in the maintenance, non‐maintenance and epirubicin groups were 84.6%, 65.4% and 27.7%, respectively.
• ? The RFS following TURBT was significantly prolonged in the maintenance group compared with the non‐maintenance group (generalized Wilcoxon test, P= 0.0190).

CONCLUSION

• ? BCG maintenance therapy significantly prolonged the post‐TURBT RFS compared with BCG induction therapy alone or epirubicin intravesical therapy.

     

Survival of patients with small cell carcinoma of the prostate during 1973-2003: a population-based study

Study Type – Therapy (inception cohort) Level of Evidence 2a What's known on the subject? and What does the study add? Small cell carcinoma of the prostate is a lethal disease. Survival data is currently based on case reports and single institution case series which give limited information on its prognostic factors. In this large population‐based study, we provide more robust estimates of survival and have defined the prognostic factors.

OBJECTIVE

• ? To describe the survival of patients with primary small cell carcinoma (SCC) of the prostate and assess prognostic factors based on a large population sample.

PATIENTS AND METHODS

• ? A total of 241 cases of SCC of the prostate were reported to the Surveillance, Epidemiology, and End Results (SEER) registries from 1973 to 2003 of which 191 cases were included in our study.
• ? We used the Kaplan–Meier method for estimating survival, and Cox proportional hazard regression modelling to evaluate prognostic variables.

RESULTS

• ? The overall age‐adjusted incidence rate was 0.278 per 1 000 000 (95% confidence interval, 0.239–0.323).
• ? In all, 60.5% presented as metastatic disease compared with 39.5% who presented as local/regional disease (P= 0.012).
• ? The 12, 24, 36, 48 and 60 months observed survival rates were 47.9%, 27.5%, 19%, 17% and 14.3% respectively.
• ? On univariate analyses, age <60, concomitant low‐grade prostatic adenocarcinoma, absence of metastasis, prostatectomy and radiation therapy were favourable prognostic factors.
• ? In multivariate regression modelling, age, pathology and stage were strong predictors of survival.

CONCLUSIONS

• ? Using the SEER database, we present the largest study describing the epidemiology of primary SCC of the prostate.
• ? We found age, concomitant low‐grade prostatic adenocarcinoma, and stage of the disease to be the strongest predictors of survival for patients with prostatic SCC.
• ? Future studies evaluating a broader range of clinical and molecular markers are needed to refine the prognostic model of this relatively rare disease.

     

Role of Rho-kinase and protein kinase C during contraction of hypertrophic detrusor in mice with partial urinary bladder outlet obstruction

What's known on the subject? and What does the study add? Intracellular signaling via Rho‐kinase and protein kinase C modulate contraction of urinary bladder smooth muscle. These systems can be involved in the bladder overactivity and may constitute pharmalogical targets for treatment of OAB. This study describes the integrated action of these two cellular pathways in BOO and normal bladders, using a mouse model. It is also shown that the activation via membrane depolarization could specifically be inhibited in BOO bladders using Rho‐kinase blockers.

OBJECTIVE

• ? To study muscarinic/purinergic receptor activation and Rho‐kinase/protein kinase C (PKC) signalling during smooth muscle contraction in normal and hypertrophic mouse urinary bladders.

METHODS

• ? Partial urinary outflow obstruction was induced in adult female (10–12 weeks) C57Bl/6 mice and comparisons were made with sham‐operated controls.
• ? Bladder preparations were examined in vitro.
• ? Expression of signalling proteins was examined using Western blot analysis.

RESULTS

• ? Obstructed bladders increased more than threefold in weight and were found to have enhanced muscarinic and attenuated purinergic components during nerve‐induced contractions.
• ? The contractile response to carbachol was shifted towards lower concentrations of carbachol for the peak response and had a markedly enhanced sustained component. The amplitude of the α,β‐methylene ATP‐induced responses was lowered. Rho‐kinase inhibitor Y27632 (10 µm ) inhibited peak and sustained contractile responses to carbachol in control bladders (peak by 38%; plateau 57%) and obstructed bladders (peak 37% plateau 47%).
• ? PKC inhibitor GF109203X (1 µm ) inhibited carbachol contractions in controls (peak by 29%; plateau 29%) and obstructed bladders (peak 17%; plateau 12%). Inhibition by a similar extent was observed after nerve stimulation.
• ? Sensitivity to Ca²⁺ in high‐K⁺ depolarized intact tissues increased in obstructed bladders. This increased receptor‐independent Ca²⁺‐sensitivity was abolished by Y27632.
• ? Tissue contents of the myosin‐binding phosphatase subunit MYPT‐1 and catalytic phosphatase subunit PP1β, were decreased and the contents of RhoGDI, RhoA and CPI‐17 increased. A decrease in the Rho‐kinase isoform ROCK‐1 was observed.

CONCLUSION

• ? Based on these results, one can speculate that Rho‐kinase inhibition would preferentially target the pathological phasic activity in the urinary bladder rather than inhibit the physiological receptor‐mediated bladder emptying.

     

Prognostic value of molecular markers, sub-stage and European Organisation for the Research and Treatment of Cancer risk scores in primary T1 bladder cancer

Study Type – Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? The stakes are high when making treatment decisions in T1 bladder cancer (BC). Conservative management may lead to progression and possibly death from BC. Conversely, radical cystectomy could be over‐treatment of non‐progressive disease. The problem for clinicians is that reliable prognostic indices are lacking. We performed a head‐to‐head comparison of two substaging systems, European Organisation for the Research and Treatment of Cancer (EORTC) risk scores and four molecular markers in T1 carcinomas of the bladder treated conservatively with BCG. T1 sub‐stage according to a new system (micro‐invasive [T1m] and extensive‐invasive [T1e]) was the most important clinical variable for predicting progression to carcinoma invading bladder muscle. The performance of the EORTC risk scores was disappointing for this T1 sub‐group. Molecular markers were not significant in multivariable analysis for predicting progression. Future studies may lead to the incorporation of sub‐stage (T1m/T1e) in the TNM classification system for urinary BC to guide clinical decision‐making in T1 BC.

OBJECTIVE

• ? To evaluate the prognostic significance of four molecular markers, sub‐stage and European Organisation for the Research and Treatment of Cancer (EORTC) risk scores in primary T1 bladder cancer (BC) treated with adjuvant bacille Calmette–Guérin.

PATIENTS AND METHODS

• ? The slides of 129 carcinomas of the bladder from two university hospitals were reviewed and the T1 diagnosis was confirmed.
• ? T1 sub‐staging was done in two separate rounds, using a new system that identifies micro‐invasive (T1m) and extensive‐invasive (T1e) T1BC, and then according to invasion of the muscularis mucosae (T1a/T1b/T1c).
• ? The EORTC risk scores for recurrence and progression were calculated.
• ? Uni‐ and multivariable analyses for recurrence and progression were performed using clinicopathological variables, T1 sub‐stage, EORTC risk scores and molecular markers (fibroblast growth factor receptor 3 gene mutation and Ki‐67, P53, P27 expression).

RESULTS

• ? The median follow‐up was 6.5 years. Forty‐two patients remained recurrence‐free (33%). Progression to T2 or metastasis was observed in 38 (30%) patients.
• ? In multivariable analysis for recurrence, multiplicity was significant. In multivariable analysis for progression, female gender, sub‐stage (T1m/T1e) and carcinoma in situ (CIS) were significant.
• ? Molecular markers were significant in univariable and in multivariable analyses for recurrence.
• ? EORTC risk scores were not significant.

CONCLUSIONS

• ? CIS, female gender and sub‐stage (T1m/T1e) were the most important variables for progression.
• ? The additional value of molecular markers was modest.
• ? Sub‐stage (T1m/T1e) could potentially be incorporated in future tumour‐node‐metastasis classifications.

     

Confocal laser endomicroscopy for the diagnosis of urothelial bladder neoplasia: a technology of the future?

Study Type – Diagnostic (exploratory cohort)
Level of Evidence 2b What’s known on the subject? and What does the study add? Bladder cancer is initially diagnosed by white light cystoscopy followed by histopathological evaluation after transurethral resection of tissue suspicious for cancer. Difficulties may occur especially in the assessment of flat lesions or in discrimination between CIS and inflammatory disease. Confocal endomicroscopy during diagnostic colonoscopy has been a valuable tool for in vivo microscopic visualization and detection of colonic neoplasias and has contributed to optimized detection rates of up to 99%. We evaluated resected bladder urothelium of 18 patients by confocal endomicroscopy and correlated microscopic findings with standard histopathology. Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible. As nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue, confocal endomicroscopy is a promising novel tool for the in vivo microscopic visualization of bladder cancer.

OBJECTIVE

• ? To evaluate bladder urothelium by confocal laser endomicroscopy (CLE) and correlate microscopic findings with standard histopathology.

PATIENTS AND METHODS

• ? Fresh bladder urothelium tissue specimens were topically stained with acriflavine for instantaneous microscopic imaging.
• ? A single‐line laser in a handheld CLE probe delivered an excitation wavelength of 488 nm providing a high resolution of 0.7 µm and an adjustable imaging depth of 0–250 µm.
• ? Resection specimens of 18 patients were investigated with 1000‐fold magnification and ex vivo findings were compared with targeted histopathology (haematoxylin and eosin staining).

RESULTS

• ? Typical tumour growth patterns such as altered nuclear to cytoplasmic ratio, pseudopapillar tissue stratification and neoangiogenesis were readily visible.
• ? Nuclear and subnuclear architecture of healthy bladder tissue could be discriminated against neoplastic tissue.

CONCLUSIONS

• ? In addition to white light cystoscopy, CLE is a promising novel tool for the in vivo microscopic visualization of bladder cancer; first results of the present study show its potential to define microscopic characteristics of bladder cancer tissue.
• ? Further in vivo studies are necessary to determine sensitivity and specificity of the technique.

     

Is joint hypermobility associated with vesico‐ureteral reflux? An assessment of 50 patients

Study Type – Aetiology (individual cohort) Level of Evidence 2b What's known on the subject? and What does the study add? Recent studies have already shown associations between generalized joint hypermobility (GJH) and voiding and defecation dysfunction and/or slow transit constipation. Changes in extracellular matrix composition in vesico‐ureteric junction of vesico‐ureteral reflux (VUR) patients were also observed previously. This study is the first to assess joint mobility as a parameter for connective tissue composition in vesico‐ureteral reflux. We convincingly demonstrate that VUR patients have significantly more hypermobile joints compared to controls and this provides a new angle to the intriguing subjects of development of VUR and susceptibility to VUR.

OBJECTIVE

• ? To assess whether there is an increased prevalence of joint hypermobility in patients with vesico‐ureteric reflux (VUR).

MATERIALS AND METHODS

• ? We studied 50 patients with primary VUR and matched controls drawn from a reference population.
• ? Joint mobility was assessed using the Bulbena hypermobility score.

RESULTS

• ? We identified significantly more patients with VUR with generalized joint hypermobility than controls (24% vs 6.7%, P= 0.007).

CONCLUSION

• ? Our findings confirm our clinical observation of an increased rate of joint hypermobility in patients with VUR. We speculate that an altered composition of the connective tissue may contribute to the severity of the (pre‐existing) VUR phenotype.

     

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways

What’s known on the subject? and What does the study add? Diabetes is a common precursor for ladder pathology, including detrusor overactivity and cystopathy. There is preliminary, but increasing evidence, suggesting that oxidative stress plays a significant role in the development of diabetic complications including its affect on the bladder. In the present study we investigated the effect of streptozotocin induced‐diabetes in rats on the global expression of genes in the rat bladder using microarray analysis, and combined this data with our previously reported study looking at changes in protein levels using proteomics. This analysis demonstrated that markers of oxidative stress were significantly increased in the diabetic bladder. Overall, our work adds to the growing body of evidence that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways which may contribute to a deterioration in bladder function.

OBJECTIVE

• ? To investigate the role that oxidative stress plays in the development of diabetic cystopathy.

MATERIALS AND METHODS

• ? Comparative gene expression in the bladder of non‐diabetic and streptozotocin (STZ)‐induced 2‐month‐ old diabetic rats was carried out using microarray analysis.
• ? Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S‐transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins.
• ? The activity of protein degradation pathways was assessed using Western blot analysis.

RESULTS

• ? Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ‐induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P= 1.27 × 10^?10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder.
• ? It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis.
• ? This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up‐regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0‐fold, respectively, and the apoptosis inducing protein, BAX, was up‐regulated by 6.76 ± 0.76‐fold.

CONCLUSION

• ? Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function.

     

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy

What's known on the subject? and What does the study add? Unlike most other cancers mutations of the p53 gene (TP53), typically indicated by increased p53 expression, are rare in renal cell carcinomas (RCC) and there is no evidence that mutation of TP53 is associated with outcome or treatment response. However, whilst TP53 mutations are not linked with outcome, p53 expression is as we show here. Our study is the first to demonstrate simultaneously that patients with increased p53 expression (significantly associated with MDM2 expression), have reduced disease specific survival even though the expressed p53 is rarely mutated. We therefore identify increased expression of wild‐type p53 and MDM2 in RCC as targets for future therapeutic approaches.

OBJECTIVE

• ? To resolve much debated issues surrounding p53 function, expression and mutation in renal cell carcinoma (RCC), we performed the first study to simultaneously determine p53/MDM2 expression, TP53 mutational status (in p53‐positive patients) and outcome in RCC.

PATIENTS AND METHODS

• ? In total, 90 specimens obtained from patients with RCC, who were treated by radical nephrectomy, were analyzed by immunohistochemistry for p53 and MDM2 on a tissue microarray, and p53 was functionally and genetically analyzed in p53 positive samples.
• ? Outcome analysis was by the Kaplan–Meier method and univariate analysis was used to identify variables for subsequent multivariate analysis of correlations between clinical parameters and biomarker expression.

RESULTS

• ? Up‐regulation of p53 in RCC is strongly linked with MDM2 up‐regulation (P < 0.001).
• ? Increased coexpression of p53 and MDM2 identifies those patients with a significantly reduced disease‐specific survival by univariate (P= 0.036) and Cox multiple regression analysis (P= 0.027; relative risk, 3.20).
• ? Functional (i.e. functional analysis of separated alleles in yeast) and genetic analysis of tumours with increased p53 expression shows that most patients (86%) retain wild‐type p53.

CONCLUSIONS

• ? Coexpression of p53/MDM2 identifies a subset of patients with poor prognosis, despite all of them having organ‐confined disease.
• ? Up‐regulated p53 is typically wild‐type and thus provides a mechanistic explanation for the association between p53 and MDM2 expression: up‐regulated wild‐type p53 likely promotes the observed MDM2 coexpression.
• ? The results obtained in the present study suggest that the p53 pathway is altered in a tissue/disease‐specific manner and that therapeutic strategies targeting this pathway should be investigated to determine whether the tumour suppressive function of p53 can be rescued in RCC.

     

Automatic evaluation of ultrasonography-estimated bladder weight and bladder wall thickness in community-dwelling men with presumably normal bladder function

Study Type – Diagnostic (exploratory cohort) Level of Evidence 2b What's known on the subject? and What does the study add? The diagnostic potential of ultrasound derived measurements of bladder wall thickness and bladder weight in men with LUTS and varying degrees of BOO have been explored. However, there is a paucity of such measurements in the asymptomatic population with which to compare such patients. This study investigates these measurements in community‐dwelling men with presumably normal bladder function.

OBJECTIVE

• ? To identify measurements of ultrasonography (US)‐derived bladder wall thickness (BWT) and bladder weight in community‐dwelling men with presumably normal bladder function.

SUBJECTS AND METHODS

• ? A total of 100 male volunteers underwent transabdominal US measurements of BWT and bladder weight, using the BVM 9500 bladder scanner (Verathon Medical, Bothell, WA, USA), at a variety of bladder filling volumes.
• ? The data were explored for any correlation between measurements of BWT and US‐estimated bladder weight (UEBW) with subject age, height, weight, body mass index (BMI), International Consultation on Incontinence Questionnaire – Male Lower Urinary Tract Symptoms (ICIQ M‐LUTS) score, International Prostate Symptom Score (IPSS) and IPSS Quality of Life index (IPSS QoL).

RESULTS

• ? Several statistically significant but weak correlations were observed: BWT and weight (r= 0.216, P= 0.032); BWT and BMI (r= 0.246, P= 0.014); UEBW and weight (r= 0.304, P= 0.002); and UEBW and BMI (r= 0.260, P= 0.009).
• ? Bladder filling volume appeared to have a greater effect on BWT than on UEBW, although this could not be determined accurately.
• ? There was a substantial difference in measurements of BWT and UEBW in the assessment of inter‐ and intra‐observer reliability testing.

CONCLUSION

• ? Further studies are required to validate automated measurements of BWT and UEBW and to investigate such measurements in the symptomatic and asymptomatic male population.

     

Expression of integrin proteins in non‐muscle‐invasive bladder cancer: significance of intravesical recurrence after transurethral resection

Study Type – Aetiology (case control)
Level of Evidence 3b What’s known on the subject? and What does the study add? A number of studies have reported several clinicopathological factors closely associated with intravesical recurrence of non‐muscle invasive bladder cancer (NMIBC). In addition, various types of molecular markers have been shown to be useful for predicting intravesical recurrence of NMIBC following transurethral resection (TUR). Of six subunits of integrin proteins, including α2, α3, α5, α6, β1 and β4, the expression level of the β4 subunit in NMIBC, in addition to pathological T stage and concomitant carcinoma in situ appeared to be independently related to intravesical recurrence. Therefore, consideration of the expression levels of integrins, particularly that of the β4 subunit, in TUR specimens would contribute to further accurate prediction of intravesical recurrence of NMIBC.

OBJECTIVES

• ? To evaluate the expression of integrin proteins, a family of transmembrane heterodimers, in non‐muscle‐invasive bladder cancer (NMIBC).
• ? To assess the significance of these proteins as prognostic indicators in patients undergoing transurethral resection (TUR).

PATIENTS AND METHODS

• ? The present study comprised 161 patients diagnosed as having NMIBC after TUR.
• ? Expression levels of six subunits of integrin proteins, including α2, α3, α5, α6, β1 and β4, were measured in TUR specimens by immunohistochemical staining.

RESULTS

• ? Of the six proteins, expression levels of α2‐, α3‐, α6‐ and β4‐subunits were significantly associated with the incidence of intravesical recurrence. Univariate analysis identified expression levels of α3‐, α6‐ and β4‐subunits as important predictors of intravesical recurrence, while tumour size, pathological T stage and concomitant carcinoma in situ (CIS) were also important.
• ? Multivariate analysis showed that the expression level of the β4 subunit, pathological T stage and concomitant CIS are independently related to intravesical recurrence.
• ? There were significant differences in intravesical recurrence‐free survival for patients who were positive for the three independent risk factors; intravesical recurrence occurred in 10 of 49 (20.4%) patients who were negative for all risk factors, 31 of 68 who were positive for one risk factor (45.6%), and 30 of 44 who were positive for two or three risk factors (68.2%).

CONCLUSIONS

• ? Consideration of the expression levels of integrins, particularly those of the β4 subunit, in TUR specimens, in addition to conventional variables, would contribute to accurate prediction of intravesical recurrence after TUR for NMIBC patients.

     

IL-23 gene therapy for mouse bladder tumour cell lines

OBJECTIVES

• ? To evaluate the antitumour effects of IL‐23 gene transfer into mouse bladder carcinoma (MBT2) cells.
• ? To investigate the mechanisms underlying the subsequent constitutive secrection of IL‐23 by the MBT2 cells

MATERIALS AND METHODS

• ? An expression vector containing IL‐23 gene was introduced into MBT2 cells by liposome‐mediated gene transfer, and secretion of IL‐23 was confirmed by ELISA.
• ? The in vivo antitumour effect of IL‐23‐secreting MBT2 cells (MBT2/IL‐23) was examined by injecting the cells into syngeneic C3H mice.
• ? A tumour vaccination study using mitomycin C (MMC)‐treated IL‐23‐secreting MBT2 cells was carried out, and the usefulness of in vivo CD25 depletion for an additional vaccine effect was also investigated.
• ? The mechanisms underlying the antitumour effects were investigated by antibody depletion of CD8 or CD4 T cells, or natural killer cells, and cells infiltrating the tumour sites in vivo were assessed using immunohistochemistry.

RESULTS

• ? Stable transformants transduced with MBT2/IL‐23 secreted IL‐23 into the culture supernatant.
• ? Genetically engineered IL‐23‐secreting MBT2 cells were rejected in syngeneic mice.
• ? MBT2/IL‐23‐vaccinated mice inhibited the tumour growth of parental MBT2 cells injected at a distant site and this vaccine effect was enhanced by combination with in vivo CD25 depletion by an antibody.
• ? The main effector cells for the direct antitumour effect of MBT2/IL‐23 were CD8 T cells, which was shown by in vivo depletion and immunohistochemical study.

CONCLUSIONS

• ? IL‐23‐secreting MBT2 cells were rejected in syngeneic mice by the activation of CD8 T cells.
• ? MMC‐treated MBT2/IL‐23 can have a tumour vaccine effect for parental MBT2 cells, and this effect was enhanced by combination with in vivo CD25 depletion.

     

Leukotriene D4 receptor antagonist montelukast alleviates protamine sulphate‐induced changes in rat urinary bladder

What's known on the subject? and What does the study add? The mastocytosis in detrusor muscle and the leaky epithelium in interstitial cystitis were the most studied features. In this study the leaky epithelium was shown using the ruthenium red staining in electron microscopy and uroplakin distribution in light microscopy besides the mast cell concentration in detrusor muscle using tryptase immunohistochemistry.

OBJECTIVE

• ? To study the effects of montelukast (ML), a leukotriene receptor antagonist which has been shown to be effective in inhibiting the action of cysteinyl‐containing leukotrienes, on protamine sulphate (PS)‐induced changes in rat urinary bladder.

MATERIALS AND METHODS

• ? Wistar female rats were catheterized and intravesically infused with PBS (control group) or PS (PS group) dissolved in PBS twice in 24 h.
• ? In the PS‐applied and ML‐treated group (PS + ML group) after the 10 mg/kg PS instillation, ML was injected i.p. twice daily for 3 days.
• ? The urinary bladder was investigated for general morphology under a light microscope.
• ? Tryptase immunohistochemistry was used to observe mast cell distribution and activation. Uroplakin distribution was also identified with immunohistochemistry.

RESULTS

• ? Alterations of glycosaminoglycan (GAG) and urothelial permeability were seen with ruthenium red (RR) staining techniques under a transmission electron microscope, and topographical changes of luminal urothelial structure were seen with a scanning electron microscope.
• ? Biochemically malondialdehyde (MDA) and gluthatione (GSH) concentrations were analysed. In the PS group, there was degenerated urothelium with irregular uroplakin distribution, increased inflammatory cell infiltration, increased number of both granulated and activated mast cells, irregularity of GAG and penetration of RR into the intercellular spaces and dilated tight junctions.
• ? In PS + ML group, there was relatively regular uroplakin distribution, a decrease in inflammatory cell infiltration, a decreased number of both activated and granulated mast cells in the mucosa, regular GAG and no penetration of RR into the intercellular areas, and regular tight junctions in most regions.
• ? The significant decrease in MDA and the increased GSH concentrations in the PS + ML group was in accordance with the histological findings.

CONCLUSION

• ? Montelukast appears to have a protective function in the bladder injury model via the anti‐inflammatory effects of this leukotriene receptor antagonist.

     

Surveillance guidelines based on recurrence patterns after radical cystectomy for bladder cancer: the Canadian Bladder Cancer Network experience

Study Type – Prognosis (cohort) Level of Evidence 2a What's known on the subject? and What does the study add? Radical cystectomy with pelvic lymph node dissection is recognized as the standard of care for carcinoma invading bladder muscle and for refractory non‐muscle‐invasive bladder cancer. Owing to high recurrence and progression rates, a two‐pronged strict surveillance regimen, consisting of both functional and oncological follow‐up, has been advocated. It is also well recognized that more aggressive tumours with extravesical disease and node‐positive disease recur more frequently and have worse outcomes. This study adds to the scant body of literature available regarding surveillance strategies after radical cystectomy for bladder cancer. In the absence of any solid evidence supporting the role of strict surveillance regimens, this extensive examination of recurrence patterns in a large multi‐institutional project lends further support to the continued use of risk‐stratified follow‐up and emphasizes the need for earlier strict surveillance in patients with extravesical and node‐positive disease.

OBJECTIVES

• ? To review our data on recurrence patterns after radical cystectomy (RC) for bladder cancer (BC).
• ? To establish appropriate surveillance protocols.

PATIENTS AND METHODS

• ? We collected and pooled data from a database of 2287 patients who had undergone RC for BC between 1998 and 2008 in eight different Canadian academic centres.
• ? Of the 2287 patients, 1890 had complete recurrence information and form the basis of the present study.

RESULTS

• ? A total of 825 patients (43.6%) developed recurrence.
• ? According to location, 48.6% of recurrent tumours were distant, 25.2% pelvic, 14.5% retroperitoneal and 11.8% to multiple regions such as pelvic and retroperitoneal or pelvic and distant.
• ? The median (range) time to recurrence for the entire population was 10.1 (1–192) months with 90 and 97% of all recurrences within 2 and 5 years of RC, respectively.
• ? According to stage, pTxN+ tumours were more likely to recur than ≥pT3N0 tumours and ≤pT2N0 tumours (5‐yr RFS 25% vs. 44% vs. 66% respectively, P < 0.001). Similarly, pTxN+ tumours had a shorter median time to recurrence (9 months, range 1–72 months) than ≥pT3N0 tumours (10 months, range 1–70 months) or ≤pT2N0 tumours (14 months, range 1–192 months, P < 0.001).

CONCLUSIONS

• ? Differences in recurrence patterns after RC suggest the need for varied follow‐up protocols for each group.
• ? We propose a stage‐based protocol for surveillance of patients with BC treated with RC that captures most recurrences while limiting over‐investigation.