Intravesical meglumine gamma-linolenic acid in superficial bladder cancer: an efficacy study.

OBJECTIVES: Gamma-linolenic acid (GLA) is known to be cytotoxic to malignant cells. We assessed the efficacy of the novel intravesical formulation, meglumine gamma-linolenic acid (MeGLA), in a phase II trial, in patients with recurrent, superficial bladder cancer. PATIENTS AND METHODS: Thirty patients with recurrent, superficial transitional cell carcinoma (TCC) were recruited. The tumour pattern was recorded at flexible cystoscopy. Patients received a single intravesical instillation of 50ml of either 50mg (1mg/ml) (15 patients), or 125mg (2.5mg/ml) (15 patients) of MeGLA in water, retained for one hour. At subsequent cystoscopy, the tumour patterns were recorded, prior to undertaking routine cystodiathermy. Biopsies were obtained for histological assessment. Responses were divided into complete, partial or none. RESULTS: All 30 patients retained the drug for 1 hour without significant local or systemic side effects. There were 4 (13%) complete responses, 9 (30%) partial responses, and 17 (57%) non-responders. Histology showed no evidence of damage to surrounding urothelium. CONCLUSIONS: Our data confirms the safety and tolerability of MeGLA, which is consistent with findings from a previous phase I trial. A response rate of 43% also indicates that MeGLA has a significant cytotoxic effect against TCC and the results are similar to those obtained using standard, single-dose, intravesical regimens.

The effects of meglumine gamma linolenic acid (MeGLA) on an organ culture model of superficial bladder cancer.

The objective of this research was to assess the effects of the novel intravesical drug MeGLA in a physiologically representative model of superficial bladder cancer. Petri dishes were used to culture 5 mm square explants of rat bladder in minimal volumes of supplemented culture medium. Parental and resistant MGH-U1 urothelial cancer cells were transfected with a green fluorescent protein (GFP) vector. Transfectants were purified by flow cytometry. Cells were seeded onto the prepared organ cultures and imaging was performed using confocal microscopy. Confirmation of the tumour colonies was done using scanning electron microscopy. MeGLA was added at various concentrations to the colonies and its effects noted over several days. Results showed that colonies of GFP-MGH-U1 cells established themselves on the explants and could be identified by confocal microscopy. The colonies could then be followed over several days. The colonies were able to survive high concentrations of the drug of up to 1 mg/ml, 400 times the IC > 90% for monolayers and equivalent to doses in clinical use. We conclude that MeGLA is less effective in this system than on monolayer cell lines. However, it showed cytotoxic effects which were comparable to those seen with conventional agents in the same system.

Cellular resistance to mitomycin C is associated with overexpression of MDR-1 in a urothelial cancer cell line (MGH-U1).

OBJECTIVE: To compare multidrug resistance (MDR)-1 and MDR-3 gene expression in a new urothelial cancer cell line (MGHU-1, with resistance to mitomycin C) against controls and the established (epirubicin-resistant) MDR clone, and to correlate MDR with cytotoxicity data. MATERIALS AND METHODS: Resistance to mitomycin C was induced by the long-term exposure of wild-type MGHU-1 cells to increasing concentrations (20-400 nmol/L) of mitomycin C. The cytotoxicity of mitomycin C or epirubicin was then compared in MGHU-1, MGHU-MMC (mitomycin C-resistant) and MGHU-1R (established MDR) cells, using the tetrazolium biomass assay. The expression of MDR-1 and -3 was investigated by the reverse transcriptase-polymerase chain reaction, using cDNA-specific primers after titration, and compared with DNA and negative controls. RESULTS: MDR-1 and -3 were significantly and equally overexpressed in MGHU-1R, and associated with a dramatic increase in the 50% inhibitory drug concentration (P < 0.001) for mitomycin C and epirubicin against controls. In MGHU-MMC, the overexpression of MDR-1 was three times greater than that of MDR-3. The cytotoxicity profile for both agents was very similar to that of MGHU-1R. Trace amounts of MDR-1, but not MDR-3, were identified in the MGHU-1 wild-type. CONCLUSIONS: Urothelial cancer cell resistance to mitomycin C is associated with cross-resistance to epirubicin and overexpression of MDR-1, suggesting that mitomycin C falls within the MDR category. Clinical application of this methodology may allow patients to be identified who are unlikely to benefit from intravesical chemotherapy.

A model of superficial bladder cancer using fluorescent tumour cells in an organ-culture system.

OBJECTIVE: To develop a reproducible in vitro simulation of superficial bladder cancer for testing cytotoxic agents at clinically relevant concentrations. MATERIALS AND METHODS: Square explants (5 mm) of rat bladder were cultured in Petri dishes in minimal volumes of Waymouth's MB 752/1 medium supplemented with 10% fetal calf serum, antibiotics and glutamine. Parental and resistant MGH-U1 urothelial cancer cells were transfected with a green fluorescent protein (GFP) vector. Transfectants were purified by flow cytometry. Cells were seeded onto the prepared organ cultures and images obtained using confocal microscopy. The tumour colonies were confirmed using scanning electron microscopy. Conventional intravesical cytotoxic agents including epirubicin, mitomycin-C and estramustine were tested in the system. RESULTS: Colonies of GFP-MGH-U1 cells became established on the explants and were identified by confocal microscopy; the development of the colonies was then followed over several days. Staining the explant for viability allowed imaging of normal urothelium on the explant surface or surrounding skirt of urothelial cells. The conventional cytotoxic agents epirubicin, mitomycin C and estramustine showed the expected differential responses to parental and resistant cell types. The colonies were able to survive high concentrations of the drug, equivalent to those in clinical use. The colonies were imaged serially over a period of several days. CONCLUSION: This system provides a more realistic model for testing cytotoxic agents for use in intravesical therapy, by allowing clinically relevant concentrations of drugs to be tested. The differential properties of the parental and resistant cells are maintained. The model also enables the same tumour colony to be imaged over several days in culture. The model may also be adapted for use in testing the effects of drugs on normal urothelium and the study of the effects of growth factors.

Nonhomologous bioinjectable materials in urology: 'size matters'?

OBJECTIVE: To assess the factors that influence how particles might become fixed in tissues or migrate from them, by measuring the size of the injectable particles, their susceptibility to phagocytosis and their affinity for fibroblast attachment in culture. MATERIALS AND METHODS: The particle size of three types of particulate unphysiological bioinjectable material, i.e. Urocol (Genesis Medical, Ltd., London), Macroplastiquetrade mark (Uroplasty Ltd., Reading, UK) and Urethrin (Mentor Medical Systems, Wantage, UK) was analysed using phase-contrast light microscopy and confocal microscopy. Human monocytes from peripheral blood were incubated with the three materials in phagocytic studies, where ingestion was determined by confocal microscopy. A fibroblast cell line was used to ascertain the ability of the particles to act as a substrate for cell attachment in culture. RESULTS: The mean (SEM) maximum particle diameters of Macroplastique, Urethrin and Urocol were 209 (5.10) microm, 49 (1.52) microm and 14 (0.39) microm, respectively. Rat peritoneal macrophages and human peripheral blood monocytes commonly ingested Urocol particles; the phagocytosis of Urethrin was rare and that of Macroplastique was not detected. Fibroblasts adhered to Urocol paste and Urethrin particles, but not to Macroplastique. CONCLUSION: Published reports of particle size and phagocytosis are confusing, but a relationship clearly exists. Macroplastique is the largest particle and is least likely to be phagocytosed by human mononuclear phagocytes. Urocol paste is the slowest to dissipate in culture conditions; the flat surfaces of Urethrin, but not Macroplastique, can serve as a substrate for fibroblast anchorage.

Estramustine reversal of resistance to intravesical epirubicin chemotherapy.

OBJECTIVE: Failure of epirubicin treatment of superficial bladder cancer implies multidrug resistance (MDR) which is common. MDR is characterised by decreased cellular levels of drug. TCC cell lines sensitive to epirubicin and resistant to both epirubicin and mitomycin C were used to investigate augmented therapy by adding the MDR reversing agent estramustine to an in vitro model. METHODS: Cells were cultured as monolayers. Fluorescence analysis was performed by flow cytometry and confocal microscopy. Cells were exposed to epirubicin 20 microg/ml for 2 h and increasing amounts of estramustine. Cytotoxicity was determined under similar exposure conditions and MTT culture (dye reduction by live cells) allowed viable biomass to be read optically. RESULTS: Resistant cells accumulated eight times less epirubicin than sensitive cells. Confocal microscopy confirmed this for nuclear uptake. Accumulation in resistant cells can be increased to near-sensitive cell levels using 40 microg/ml estramustine. Image analysis of confocal fluorescence showed a shift from cytoplasm to nucleus. This correlated with increased cytotoxicity. CONCLUSION: Estramustine plus epirubicin chemotherapy can overcome MDR and may achieve more successful tumour killing in vivo. It may also prevent emergence of resistance. Primary TCC culture examination permits detection of sensitive and resistant cells and may predict outcome allowing a more logical treatment selection.

A prospective trial of flexible cystodiathermy for recurrent transitional cell carcinoma of the bladder.

PURPOSE: The flexible cystoscope has a proved role in the followup of patients with transitional cell carcinoma of the bladder but the full extent of its therapeutic role has yet to be defined. We analyzed 171 flexible cystodiathermies to assess patient tolerance and treatment success. Potential cost savings were also analyzed. MATERIALS AND METHODS: All patients with single or multiple small papillary (Ta) recurrences at followup flexible cystoscopy were treated with flexible cystodiathermy. Plain lubricating gel was used and no other analgesia was prescribed. A visual analog pain scale was completed by the patient after the procedure and an observer rating of discomfort was recorded. Followup for efficacy of treatment was performed. RESULTS: A total of 103 patients were treated with cystodiathermy during the last 3 years. Median followup was 21 months (range 12 to 42). Of the patients 52 (50.5%) had no recurrence of transitional cell carcinoma after treatment and 51 (49.5%) required treatment for recurrence. Only 13 patients (12.6%) had recurrences at or close to the original tumor site. Pain scales indicated that the procedure was well tolerated and all patients agreed to undergo it in the future if required. Estimated cost savings were approximately $66,500 per 100 patients. CONCLUSIONS: Flexible cystodiathermy is a well tolerated and efficacious treatment for recurrent small papillary (Ta) transitional cell carcinoma of the bladder. Since transitional cell carcinoma of the bladder frequently occurs in an elderly and often unfit population treatment that avoids general anesthetic offers considerable advantages.

Water as a tumoricidal agent in bladder cancer. In vitro studies in parental and resistant cell lines.

OBJECTIVE: Water is widely used as a tumoricidal agent during transurethral resection of bladder tumour (TURBT). Despite this, recurrences are common. One possible explanation may be that while the parental bladder cancer cells are sensitive to water, their multidrug resistant (MDR) clones which occur commonly, may be resistant to it. If so, is this a preserve of the MDR phenotype or do other mechanisms confer resistance to water? METHODS: The parental human urothelial cancer cell lines (MGHU-1 and RT112) and their drug-resistant variants (MDR and non-MDR) were exposed to short pulses of water. The MDR status of these cells was verified using confocal microscopy to detect intracellular accumulation of fluorescent drug (epirubicin). A clonogenic assay was used to establish tumoricidal efficacy on cells in suspension. A thiozolyl blue (MTT) assay was used on adherent cells to assess the residual viable biomass. RESULTS: In the MDR clone of cells, colony counts were reduced by 20% after treatment with water as compared to a 60% reduction in the parental cell line. There was a similar reduction in colony counts between the parental RT112 and its non-MDR cisplatin resistant variant. CONCLUSION: Water is less effective on MDR cells. Resistance to water may be a function of the MDR phenotype.

Intravesical chemotherapy with gamma linolenic acid becomes a realistic prospect in serum-free applications: in vitro cytotoxicity and systemic absorption studies.

PURPOSE: To assess the cytotoxicity of Meglumine gamma linolenic acid (MeGLA) in serum-free application on 2 urothelial cancer cell lines, to examine whether the instant kill action of MeGLA is retained in a serum free environment, and to study the pharmacokinetics of intravesical instillation of gamma linolenic acid (GLA). MATERIALS AND METHODS: The 2 human urothelial cancer cell lines (MGH-U1 & RT112) were utilized in classical cytotoxicity assays in which drug exposure lasted 2 hours in serum or in serum-free application. The thiozolyl blue (MTT) assay was used to quantify the residual viable biomass 5 days later. Immediate cytotoxicity was also compared in serum and serum-free application. Four Wistar rats were used to study the intravesical absorption profile of tritiated GLA (3H-GLA). RESULTS: There was a 10-fold enhancement of the lytic efficacy of MeGLA in serum-free application and this enhancement was also observed in experiments assessing instant kill. There was a similar enhancement of efficacy seen in the multi-drug resistant (MDR) clone of cells. The absorption profile showed < 2% of instilled counts were absorbed and the commonest destination for the absorbed GLA was the liver. CONCLUSIONS: The cytotoxic action of MeGLA was enhanced in serum free application. This enhancement was maintained when cells expressed the MDR phenotype. There was limited absorption from the bladder. MeGLA is a feasible intravesical agent for use in superficial bladder cancer.

Bladder cancer recurrence by implantation of exfoliated cells: is gamma-linolenic acid an effective tumoricidal agent?

OBJECTIVE: To compare the tumoricidal efficacy of meglumine gamma-linolenic acid (MeGLA), mitomycin C, epirubicin and water on two urothelial cell lines, and to establish the effect of serum protein levels derived from bladder cancer resection craters on the action of these agents. MATERIALS AND METHODS: The human urothelial cell lines MGHU-1 and RT112 and their drug-resistant variants were exposed to short pulses of aqueous MeGLA, mitomycin, epirubicin and water. Both adherent and suspended cells were exposed to these agents. The MTT viable biomass assay and a clonogenic assay were used to establish tumoricidal efficacy. These experiments were then repeated to assess the effect of added serum proteins on the test results. Estimates of protein in the waste irrigation fluid from 10 patients undergoing transurethral resection of bladder tumour (TURBT) were used to select the quantity of protein used in the study, to establish the clinical relevance. RESULTS: MeGLA caused > 95% reduction in the residual viable biomass of adherent cells, compared with < 50% reduction with any other agent. Both epirubicin and mitomycin were as effective as MeGLA in preventing colony formation from suspended drug-sensitive (parental) cells. However, using multidrug-resistant (MDR) cell lines, only MeGLA prevented any colony formation, although counts were greatly reduced by mitomycin and epirubicin. Water was least effective as a tumoricidal agent on both adherent and suspended cells. On the latter, water was markedly inactivated by adding 5% serum. TURBT waste irrigation fluid was found frequently to contain such quantities of serous fluid contamination, as shown by albumin estimates in waste fluid from 10 consecutive patients undergoing this procedure. CONCLUSION: MeGLA is an effective tumoricidal agent against both parental and MDR cell lines. Its efficacy is maintained in the presence of clinically relevant serum contamination.

Is gamma-linolenic acid an effective intravesical agent for superficial bladder cancer? In vitro cytotoxicity and in vivo tolerance studies.

The essential fatty acid gamma-linolenic acid (GLA) is an effective cytotoxic agent when applied topically and for prolonged periods to tumour cells. Topical application, by intravesical therapy, is firmly established in the treatment of superficial bladder cancer. However, this form of therapy is limited to a maximum duration of 2 h. At such a short drug exposure time, does GLA retain its cytotoxicity? We have examined this question by exposing the superficial bladder cancer cell lines MGH-U1 and RT112 to meglumine-GLA (MeGLA) for time intervals ranging from 30 min to 2 h, at drug concentrations ranging from 1000 to 1.95 microg/ml. The MTT viable biomass assay was used to assess cell kill. Greater than 90% inhibition was observed at a concentration of 125 microg/ml (IC > 90), at 2 h drug exposure. At shorter drug exposure times, higher drug concentrations were needed to induce the same effect. At 1 h drug exposure, the IC > 90 was recorded at 500 microg/ml. In vivo intravesical tolerance studies were conducted in rats. Rats exposed to 2.5 mg/ml MeGLA intravesically for 2 h or less remained well and bladder histology showed minimal changes. This study confirms that GLA retains its cytotoxicity at short drug exposure times and is well tolerated by normal bladder mucosa in vivo. Bladder mucosa tolerated > 10x the concentration required for the IC > 90 in vitro. MeGLA is therefore a feasible intravesical agent for superficial bladder cancer.

Collecting duct carcinoma. An unusual renal tumour.

Collecting duct carcinoma is a rare form of renal tumour. Its recognition is important both clinically and histopathologically since it can mimic other renal neoplasms in appearance. Nephrectomy is the treatment of choice and, as the tumour usually pursues an aggressive course, adjuvant chemotherapy or immunotherapy may also be considered appropriate. We report two such cases.

Primary nocturnal enuresis: a urodynamic study spanning three generations.

The familial incidence of primary nocturnal enuresis is well recognised. Twin studies suggest there to be a significant genetic component to the aetiology of this disorder. However, family studies to date have been based on symptomatic enquiry alone although detrusor instability is a recognised feature of primary noctural enuresis in 70-80% of cases. For the first time we describe a family of adults with urodynamically proven instability spanning three generations. The pattern of inheritance lends support to the proposition that such detrusor instability is transmitted as an autosomal dominant characteristic.

Adenocarcinoma of the prostate and Eaton-Lambert syndrome. A previously unreported association.

Eaton-Lambert myasthenic syndrome is rare. We report the first case of adenocarcinoma of prostate associated with this syndrome. Subsequent treatment by orchidectomy caused tumour regression and remission of the syndrome. The patient remains well 9 months after the treatment.

Case report: multiple epidermoid cysts of the testes--the ultrasound appearances.

Epidermoid cysts are uncommon tumours of the testes representing approximately 1% of all testicular tumours. Their importance lies in the fact that they are completely benign and can be treated by local excision, thereby saving the patient an orchidectomy. We describe a case in which the patient had multiple epidermoid cysts involving both testes and faced possible bilateral orchidectomy.

An assessment of resedation following flumazenil-induced antagonism of intravenous midazolam: comparison of psychomotor and amnesic recovery with a non-sedated reference group.

The specific benzodiazepine antagonist flumazenil can enhance patient recovery following local anaesthetic day-case surgery performed under sedation. However, in view of its short elimination half-life, concerns have been expressed about the risk of resedation following its use. An open, randomised, parallel group study was designed to explore this question. Eighty-five patients were studied. Group A (n=43) patients underwent local anaesthetic cystoscopy with intravenous (i.v.) midazolam sedation. Following cystoscopy, and 30 min after the injection of midazolam, a bolus dose of flumazenil (0.5 mg i.v.) was given. Group B (n=42) patients underwent no operation and received no drugs but, in all other respects, were treated in an identical fashion to patients in group A. Tests of psychomotor function and memory were administered at baseline and again at 0.5, 1, 2, 3 and 4 h (or equivalent times for group B patients) following the injection of flumazenil. The test results showed no evidence of resedation, but there was evidence of incomplete reversal, as shown by significant differences in critical flicker fusion and delayed word recall at the 0.5-h test point. Group B patients showed no evidence of practice effects but did demonstrate an impairment in test performance possibly related to motivational factors. In conclusion, this study provides no evidence of resedation when using flumazenil to reverse the acute effects of midazolam. Incomplete reversal of amnesia need not delay patient discharge but has important implications with respect to the timing and nature of information imparted to patients prior to their release from hospital.

Absorption characteristics of lignocaine following intravesical instillation.

The systemic absorption of topical lignocaine applied intravesically was studied in 11 patients with indwelling catheters. Group A patients (n = 9) had lignocaine 400 mg (as 40 ml of a 1% solution) instilled into the bladder for 1 hour whilst in Group B patients (n = 2) the application was for 2 hours. The mean maximum lignocaine concentration measured in Group A patients was 121 ng ml-1 with a time to maximum concentration of 60-90 minutes. The two patients in Group B had corresponding values of 410 and 1580 ng ml-1 respectively at 120 minutes. Transitional cell epithelium, like intact skin, constitutes a significant barrier to the uptake of topically applied lignocaine which, from the bladder, is both slow and limited. Intravesical application is safe (maximum levels in Group A patients being x 30 less than the minimal toxic level of lignocaine) but its clinical efficacy remains to be determined.

Walk-in, walk-out day case genito-scrotal surgery with sedation reversal. A survey of patient attitudes and morbidity.

OBJECTIVES: To survey patient attitudes and assess the extent of patient morbidity in the first 24 h following walk-in, walk-out day case genito-scrotal surgery with sedation reversal. PATIENTS AND METHODS: One hundred patients who were undergoing genito-scrotal surgery were eligible for inclusion in the study. All patients walked into theatre, positioned themselves on the operating table and were sedated with intravenous (i.v.) midazolam (Hypnovel, Roche Products Ltd, Welwyn, UK) following which the appropriate local anaesthetic block was performed. One minute before the end of surgery sedation was reversed with an i.v. injection of 0.5 mg flumazenil (Anexate, Roche Products Ltd). When conscious and alert (approx. 2 min), patients were allowed to get themselves up off the operating table and walk from theatre. All patients were given a questionnaire to complete and were reviewed 2 weeks after surgery. RESULTS: Eighty-six of 100 patients returned questionnaires. Fifty-five patients were asymptomatic and no patient experienced nausea or vomiting in the first 24 h post-operatively. Other symptoms such as headache (2%), drowsiness (20%) and dizziness (11%) were uncommon and significantly less frequent than seen with general anaesthetic procedures. Seventeen patients described minor wound complications and one patient with a scrotal haematoma was readmitted for overnight observation. Nearly all patients (98%) expressed their satisfaction with the technique. CONCLUSION: Walk-in, walk-out day case surgery as described is a well-tolerated technique with a low morbidity and towards which patients have a positive attitude.