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.

     

Evidence‐based clinical practice guidelines for bladder cancer (Summary – JUA 2009 Edition)

In Japan, until now, the treatment of bladder cancer has been based on guidelines from overseas. The problem with this practice is that the options recommended in overseas guidelines are not necessarily suitable for Japanese clinical practice. A relatively large number of clinical trials have been conducted in Japan in the field of bladder cancer, and the Japanese Urological Association (JUA) considered it appropriate to formulate their own guidelines. These Guidelines present an overview of bladder cancer at each clinical stage, followed by clinical questions that address problems frequently faced in everyday clinical practice. In this English translation of a shortened version of the original Guidelines, we have abridged each overview, summarized each clinical question and its answer, and only included the references we considered of particular importance.     

Cyclooxygenase-2 promotes angiogenesis in pTa/T1 urothelial bladder carcinoma but does not predict recurrence

OBJECTIVE: To investigate the effect of cyclooxygenase-2 (COX-2) on microvessel density (MVD) and on the clinical prognosis in patients with non-muscle invasive urothelial carcinoma of the bladder, as COX-2 expression is significantly greater in epithelial tumours and there is increasing evidence that COX-2 might contribute to tumour neovascularization. PATIENTS AND METHODS: We assessed tumour samples from 110 patients undergoing transurethral resection for primary pTa/pT1 bladder carcinoma (pTa, 84; pT1, 26; grade 1, 22; grade 2, 81; grade 3, seven). Paraffin sections were assessed immunohistochemically using antibodies against COX-2, CD34 (endothelial cells) and CD105 (proliferating vessels). COX-2 expression was quantified by the number of stained cells (negative, +, ++) and the MVD calculated as vessels per field. RESULTS: Of the 110 tumours, 45 (41%) had no immunostaining for COX-2, 40 had faint staining with at least isolated positive cells (+) and 25 stained ++. COX-2 positive tumours had significantly greater vascularization for proliferating vessels. In COX-2 negative tumours the MVD was 22.1, identified by CD34 immunostaining, and 3.4 for proliferating vessels (CD105), whereas COX-2 positive tumours had a MVD of 18.3 (CD34), and of 5.8, respectively (CD105). Complete follow-up data were available in 91 patients; after a mean follow-up of 25 months, 18 (20%) had tumour recurrences. There was no significant difference in the recurrence rates or disease-free survival between COX-2-positive (19%, 25.6 months) or -negative patients (21%, 25.2 months). CONCLUSION: These results confirm the involvement of COX-2 in angiogenesis in bladder cancer, as COX-2 promoted blood vessel proliferation in the tumour zone, and indicate the usefulness of COX-2-inhibiting drugs in preventing and treating superficial bladder cancer.     

Pharmacokinetic study to optimize the intravesical administration of gemcitabine

Study Type – Therapy (case control)
Level of Evidence 3b OBLECTIVES To assess in a phase II pharmacokinetic study whether different pH levels, dilution volumes and exposure times affect intracellular bioavailability and systemic absorption of gemcitabine. SUBJECTS AND METHODS Six arms of three patients each with a non‐muscle‐invasive bladder cancer (NMIBC) were planned to receive six combinations of two different dilution volumes (50 mL vs 100 mL), two pH levels (2.5–3.5 vs 5.5) and two exposure times (1 h vs 2 h) of the study drug. Blood samples were taken before, during and 1 h after drug instillation. Cold biopsy specimens from the exophytic tumor, its base of implant and a macroscopically healthy mucosa were taken during transurethral resection. High‐pressure liquid chromatography/high‐resolution mass spectrometry (HPLC/HRMSn) analysis of plasma and tissue samples was used to determine concentrations of gemcitabine (dFdC) and its inactive metabolite (dFdU). RESULTS The arm at pH 5.5 in 50 mL was withdrawn as 2000 mg dFdC are insoluble in these conditions. The different instillation conditions resulted in negligible plasma dFdC concentrations but significant differences in intracellular content and metabolism of dFdC. The lowest intratissue concentration of dFdC was detected in a 50 mL solution at a pH of 2.5–3.5 kept in the bladder for 1 h (standard arm). A pH 5.5 solution in 100 mL with a 2‐h exposure favored the maximal intratumoral dFdC absorption which was 90 times higher than that recorded in the standard arm. CONCLUSIONS The most commonly reported administration scheme of gemcitabine produced the lowest tissue bioavailability of dFdC. Other combinations of pH, dilution volume and duration of instillation proved more advantageous and merit testing in clinical trials.     

Guideline of guidelines: non‐muscle‐invasive bladder cancer

Non‐muscle‐invasive bladder cancer (NMIBC) represents the vast majority of bladder cancer diagnoses, but this definition represents a spectrum of disease with a variable clinical course, notable for significant risk of recurrence and potential for progression. Management involves risk‐adapted strategies of cystoscopic surveillance and intravesical therapy with the goal of bladder preservation when safe to do so. Multiple organizational guidelines exist to help practitioners manage this complicated disease process, but adherence to management principles among practising urologists is reportedly low. We review four major organizational guidelines on NMIBC: the American Urological Association (AUA)/Society of Urologic Oncology (SUO), European Association of Urology (EAU), National Comprehensive Cancer Network (NCCN), and National Institute for Health and Care Excellence (NICE) guidelines.     

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.

     

Sexual function of patients under surveillance for bladder cancer

OBJECTIVE

To describe the prevalence of sexual dysfunction and evaluate risk factors in patients just diagnosed with non‐muscle‐invasive bladder cancer (NMI UC), who have the prospect of an intensive surveillance scheme by cysto‐urethroscopy to detect tumour recurrences.

PATIENTS AND METHODS

We conducted a cross‐sectional survey on 150 patients just diagnosed with primary or recurrent NMI UC. Patients were participating in a randomized clinical multicentre trial (CEFuB), comparing two surveillance schemes. Patients were asked to complete questionnaires at study entry 3 months before the start of the study‐surveillance scheme (demographic characteristics, a validated visual analogue scale, and validated subset of questions on sexual function and performance derived from QLQ‐BLS‐24). The results were compared with those from an age‐and gender‐matched healthy population.

RESULTS

The response rate was 95% (142/150); 61% (87/142) of the respondents were sexually active in the previous 4 weeks after diagnosis, 66% (70/105) of men and 46% (17/37) of women. Although libido was not negatively affected, 54% (47/87) of the patients had a sexual dysfunction, and 23% (17/73) were afraid to inflict harm on their partner by sexual contact. Sexually active patients perceived a higher state of general health (P = 0.03).

CONCLUSIONS

The prevalence of sexual dysfunction in patients with NMI UC is very high (54%) compared with an age‐ and gender‐matched healthy population (20–45%). No predictors for sexual dysfunction were found. These patients and partners would benefit from proper sexual information in the outpatient clinic.     

The results of concurrent chemo‐radiotherapy for recurrence after treatment with bacillus Calmette‐Guérin for non‐muscle‐invasive bladder cancer: is immediate cystectomy always necessary?

OBJECTIVES

To report our original experience in patients in whom bacille Calmette‐Guérin (BCG) therapy has failed for T1 bladder cancer with subsequent progression to T2 disease treated with chemo‐radiotherapy, as the management of recurrent high‐grade T1 bladder cancer after failed BCG therapy is challenging, and radical cystectomy is the standard treatment because there are no well established second‐line bladder‐preserving therapies.

PATIENTS AND METHODS

From 1988 to 2002, 18 patients with T2 recurrence after failure of BCG therapy for T1 bladder cancer were treated with chemo‐radiotherapy at the authors’ institution. Patients received a visibly complete transurethral resection of the bladder tumour (TURBT) and concurrent chemo‐radiotherapy with a mid‐treatment evaluation after 40 Gy. Patients with less than a complete response had a prompt cystectomy; the others completed radiotherapy to 64–65 Gy. The primary treatment outcome was freedom from cystectomy due to recurrence not treatable by conservative measures; secondary outcomes included disease‐specific (DSS) and overall survival (OS).

RESULTS

With a median follow‐up of 7.0 years, only one patient had persistent tumour at re‐staging TURBT and had an immediate cystectomy. Of the remaining 17 patients, 10 (59%) were free of any bladder recurrence. The actuarial 7‐year DSS and OS were 70% and 58%, respectively. At 7 years, 54% of patients were alive with intact bladders and free of invasive recurrence.

CONCLUSIONS

In this study we specifically evaluated patients with apparently small muscle‐invasive recurrences after BCG treatment for T1 bladder cancer. Selective bladder preservation with chemo‐radiotherapy is possible, with low morbidity and a high chance of long‐term bladder control. If successful in treating T2 recurrences after BCG therapy, it now seems timely to critically evaluate chemo‐radiotherapy as an alternative to immediate cystectomy in the management of patients with T1 recurrences after BCG.     

A chemosensitivity test to individualize intravesical treatment for non‐muscle‐invasive bladder cancer

OBJECTIVE

To describe the design of a new chemosensitivity assay based on the expression of genes involved in the resistance to standard intravesical regimens, to allow individualization of therapy for high‐risk non‐muscle‐invasive bladder cancer.

PATIENTS AND METHODS

To date, 35 patients with high‐risk no‐nmuscle‐invasive bladder cancer have been enrolled, all candidates for transurethral resection of the bladder (TURB) followed by intravesical treatment. The intravesical regimen was chosen according to the risk profile of each patient. All patients were evaluated by cystoscopy 3 and 6 months after TURB. According to the molecular characterization of each tumour, our team of molecular oncologists determined for each patient a molecular profile of chemosensitivity to BCG, mitomycin c, anthracyclines and gemcitabine. This profile was then correlated to the response to intravesical therapy 6 months after TURB.

RESULTS

This chemosensitivity test was able to predict response to treatment in 96% of patients. The assay is easy to perform, inexpensive and quick.

CONCLUSION

Our results, although preliminary, are encouraging for the future of an individualized therapeutic approach, with the aim to provide a higher treatment success rate while sparing patients unnecessary toxicity from drugs that are not suited for their tumours.     

A 1‐year maintenance after early adjuvant intravesical chemotherapy has a limited efficacy in preventing recurrence of intermediate risk non‐muscle‐invasive bladder cancer

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

OBJECTIVE

To evaluate the efficacy of 1‐year maintenance after a 6‐week cycle of early intravesical chemotherapy, as the role of maintenance in intravesical chemotherapy is debated.

PATIENTS AND METHODS

Between May 2002 and August 2003, 577 patients with non‐muscle‐invasive bladder cancer (NMI‐BC) underwent transurethral resection (TUR) and early intravesical chemotherapy (epirubicin, 80 mg/50 mL). They were randomized between a 6‐week induction cycle and the induction cycle plus maintenance with 10 monthly instillations. In all, 95 patients with T1G3, Tis or single and primary Ta–T1 G1–G2 tumours were excluded; 482 patients at intermediate risk of recurrence continued the study. All patients had cytology and cystoscopy at 3‐monthly intervals for the first 2‐years and 6‐monthly thereafter.

RESULTS

The tumours’ characteristics were equally distributed between the two arms. Treatment interruption for toxicity was required in 39 patients. One death due to toxicity of early instillation occurred. The median follow‐up was 48 months. Ten patients (2.5%) progressed and 117 patients (29.6%) recurred. No statistically significant difference in the recurrence‐free rate (RFS) was detected between the two arms (P = 0.43). An advantage in favour of the maintenance arm was evident only at 18 months after TUR (P = 0.03). A trend for a higher benefit from maintenance in primary and multiple tumours was detected.

CONCLUSIONS

In patients with intermediate risk NMI‐BC treated by TUR and early adjuvant chemotherapy, adding a maintenance regimen with monthly instillations for 1 year is of limited efficacy in preventing recurrence.     

Intravesical gemcitabine therapy for non-muscle invasive bladder cancer (NMIBC): a systematic review

? Intravesical immunotherapy or chemotherapy for non-muscle invasive bladder cancer is a well-established treatment for preventing or delaying tumour recurrence after tumour resection. However, up to 70% of patients may fail and new intravesical agents with improved effectiveness are needed. Gemcitabine is a relatively new anticancer drug that has shown activity against bladder cancer. ? To systematically review the literature on the effectiveness and toxicity of intravesical gemcitabine for non-muscle invasive bladder cancer (NMIBC). ? MEDLINE, EMBASE, CINAHL, the Cochrane database of systematic reviews, LILACS, SCOPUS, BNI, Biomed Central, Web of Science and BIOSIS were searched to identify trials of intravesical gemcitabine for the treatment of NMIBC. Also searched were meeting proceedings, international guidelines and trial registries. Data on authors, study design, patient characteristics, interventions and outcome data relating to tumour recurrence, disease progression, survival and adverse events were extracted from relevant studies. ? Six relevant randomised trials were identified with the number of patients randomised in each trial varying from 30 to 341 (total 704). All trials compared gemcitabine to active controls and varied in the reporting of outcomes. ? The first was a marker lesion study which reported greater tumour response rates when intravesical gemcitabine (2 g) was given as three bi-weekly doses (36%) or six weekly doses (40%) compared with a single dose (9%). ? One study compared a single postoperative instillation of intravesical gemcitabine with a saline placebo in 341 patients and found no significant difference in the rates of tumour recurrence (28% vs 39%, respectively) or recurrence-free survival (hazard ratio 0.95, 95% confidence interval 0.64-1.39, P= 0.77). The rate of progression to invasive disease was greater with gemcitabine (2.4% vs 0.8%). ? A further trial compared gemcitabine with intravesical mitomycin C (MMC) and reported that the rates of recurrence (28% vs 39%) and progression (11% vs 18%) were lower with gemcitabine but did not reach statistical significance. The overall incidence of adverse events was significantly less with gemcitabine (38.8% vs 72.2%, P= 0.02). ? Three trials compared gemcitabine with intravesical bacille Calmette-Guérin (BCG) but a meta-analysis was not possible due to clinical heterogeneity. ? In untreated patients at intermediate risk of recurrence (primary Ta-T1, no carcinoma in situ) one trial showed that gemcitabine and BCG were similar with respective recurrence rates of 25% and 30% (P= 0.92) and overall progression equal. Dysuria (12.5% vs 45%, P < 0.05) and frequency (10% vs 45%, P < 0.001) were significantly less with gemcitabine. ? In a second trial of high-risk patients the recurrence rate was significantly greater with gemcitabine compared with BCG (53.1% vs 28.1%, P= 0.04%) and the time to recurrence significantly shorter with gemcitabine (25.5 vs 39.4 months, P= 0.042). ? Finally, in a third trial of high-risk patients who had failed previous intravesical BCG therapy, gemcitabine was associated with significantly fewer recurrences (52.5% vs 87.5%, P= 0.002) and a longer time to recurrence (3.9 vs 3.1 months, P= 0.9) compared with BCG. Progression rates were similar in both groups (33% vs 37.5%, P= 0.12) with no significant differences in grade 2 or 3 toxicities. ? The data from several observational studies confirm the pharmacology of gemcitabine as an intravesical agent whilst others report the activity of gemcitabine in terms of tumour recurrence. However, these studies are inherently biased and these data should be interpreted appropriately. ? In conclusion a single study suggests that in NMIBC multiple doses of intravesical gemcitabine reduce tumour recurrences to a greater extent than a single dose. ? In contrast, a single dose immediately after surgery is ineffective based on one study. Gemcitabine may be more active than MMC with a lower toxicity profile. ? Compared with intravesical BCG therapy, gemcitabine had similar effects in intermediate-risk patients, less effective in high-risk patients and superior in BCG-refractory patients. However, each randomised trial identified represents a different clinical setting in NMIBC and therefore the evidence base is limited. Consequently these data should be interpreted with caution until further corroborative evidence becomes available. ? Intravesical gemcitabine is a promising drug that may add to the urologist's options in treating patients with NMIBC.