Metallothionein isoform 1 and 2 gene expression in the human bladder: evidence for upregulation of MT-1X mRNA in bladder cancer.

The goals of this study were to determine the expression of metallothionein isoform 1 and 2 proteins (MT-1 and MT-2) in bladder cancer and then to determine which MT isoform-specific genes promoted the expression of these proteins. Immunohistochemical analysis disclosed no immunoreactivity for MT-1 and MT-2 (designated as MT-1/2 to reflect the nonspecificity of the antibody for the two isoforms) in cells comprising the normal bladder or in nonmalignant bladder disorders, such as cystitis and interstitial cystitis. Immunohistochemical analysis demonstrated that MT-1/2 was overexpressed in all samples of carcinoma in situ and in high-grade bladder cancer, with variable overexpression in low-grade bladder cancer and dysplastic lesions. The intensity and frequency of MT-1/2 staining correlated with the grade of the tumor. The MT-1 and MT-2 proteins are encoded by a family of eight genes (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-IH, MT-1X, and MT-2A), and reverse transcriptase-polymerase chain reaction was used to determine which genes were expressed in the normal bladder and in bladder cancer. This analysis demonstrated that both normal and cancerous bladder tissue expressed mRNA for the MT-2A and MT-1X genes. The expression of MT-1E mRNA was variable in both normal bladder and bladder cancer specimens. Comparison of expression relative to that of beta-actin demonstrated that the level of MT-1X mRNA was overexpressed greatly in bladder cancer as compared to the level in normal bladder tissue. In contrast, the level of MT-2A mRNA was similar in both the normal and the bladder cancer specimens. The level of MT-1X expression did not vary with tumor grade. These studies suggest that the overexpression of MT-1/2 protein in bladder cancer is a result of the overexpression of the MT-1X gene.

Enhanced immunocompetence by garlic: role in bladder cancer and other malignancies.

Of the many beneficial actions of garlic, inhibition of the growth of cancer is perhaps the most remarkable. Our previous animal studies demonstrated that aged garlic extract was highly effective, and unlike the approved immunotherapy for human bladder cancer, bacillus Calmette--Guérin (BCG), garlic was effective when added to the diet. To elucidate the mechanism of this antitumor effect, the literature describing antitumor and immune-enhancing effects of garlic is reviewed. Garlic can detoxify carcinogens by stimulation of cytochrome P(450) enzymes, antioxidant activity or sulfur compound binding. Studies demonstrate a direct toxic effect of garlic to sarcoma and gastric, colon, bladder and prostate cancer cells in tissue culture, but these effects cannot explain the inhibition of growth of transplanted cancer in animal models. The most likely explanation of this effect is immune stimulation. Comparison of the effects of garlic to BCG immunotherapy reveals many similarities. Both stimulate proliferation of lymphocytes and macrophage phagocytosis, induce the infiltration of macrophages and lymphocytes in transplanted tumors, induce splenic hypertrophy, stimulate release of interleukin-2, tumor necrosis factor-alpha and interferon-gamma, enhance natural killer cell, killer cell and lymphokine-activated killer cell activity. These activities represent effective stimulation of the immune response. Studies suggest that garlic may be useful in preventing the suppression of immune response that is associated with increased risk of malignancy. Data suggest that maintenance of immune stimulation can significantly reduce the risk of cancer. Clinical trials should be initiated to test the hypothesis that the immune stimulation and other beneficial effects of garlic are able to reduce the incidence of cancer.

Immunotherapy for bladder cancer.

The primary role of immunotherapy for bladder cancer is to treat superficial transitional cell carcinomas (ie, carcinoma in situ, Ta, and T1). Immunotherapy in the form of bacille Calmette-Guérin (BCG), interferon, bropirimine, keyhole limpet hemocyanin, and gene therapy is intended to treat existing or residual tumor, to prevent recurrence of tumor, to prevent progression of disease, and to prolong survival of patients. Presently, BCG is commonly used and is the most effective immunotherapeutic agent against superficial transitional cell carcinoma. Data support that BCG has a positive impact on tumor recurrence, disease progression, and survival. Proper attention to maintenance schedules, route of administration, dosing, strains, and viability is essential to obtain the maximum benefits of BCG immunotherapy. This review highlights and summarizes the recent advances concerning immunotherapy, with special emphasis on BCG therapy for transitional cell carcinoma.

Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer.

In the United States, bladder cancer is the fourth most common human malignancy. In the past decade, the incidence of bladder cancer has increased by 36%. However, mortality has declined by 8%. Intravesical chemotherapy was considered to be partially responsible for this improvement in survival, but a recent review of clinical studies shows no reduction in disease progression with intravesical chemotherapy. Fortunately, the results of immunotherapy with bacille Calmette-Guérin (BCG) are quite different, and it is expected that patients treated with optimal BCG treatment regimens will have a long-term reduction in tumor recurrence, tumor progression, and cancer mortality.

Keyhole limpet hemocyanin immunotherapy of bladder cancer: laboratory and clinical studies.

BACKGROUND: Since the serendipitous observation by Olsson in 1974 that patients immunized with 5 mg of keyhole limpet hemocyanin (KLH) had a marked reduction in recurrence of superficial bladder cancer, multiple laboratory and clinical studies have confirmed the efficacy of KLH immunotherapy. RESULTS: In 1981, we reported that KLH immunotherapy reduced tumor growth and prolonged survival in the MBT-2 murine model of transitional cell carcinoma (TCC), and in 1988, Jurincic and co-workers demonstrated that KLH was superior to mitomycin C chemotherapy in preventing bladder tumor recurrence. Subsequent studies using Immucothel (Biosyn), crude KLH, and endotoxin-free KLH confirmed the efficacy of KLH immunotherapy in the MBT-2 murine bladder cancer model (p < 0.05), and resulted in up to 100% survival. CONCLUSIONS: To evaluate the efficacy of KLH immunotherapy in patients, a multicenter clinical trial was performed. Sixty-four patients with CIS or residual stage T(a), T(1) TCC, or both were enrolled in a phase I-II trial of escalating doses of weekly KLH given intravesically for 6 weeks. Patients were followed with cystoscopic examination, urine cytology, and bladder biopsy. Complete response was seen in 50% of patients with CIS, 20% of patients with residual T(a), T(1) TCC, and 33% of patients with both CIS and residual T(a), T(1) TCC. Responses occurred at all doses tested: 0.4, 2, 10 and 50 mg. No significant difference in response according to dose was noted, but optimal overall complete response was seen with a dose of only 2 mg. The toxicity of KLH is minimal. KLH appears to be a safe and highly effective immunotherapy for superficial bladder cancer.

Management of patients with Bacilli Calmette-Guérin-refractory carcinoma in situ of the urinary bladder: cost implications of a clinical trial for valrubicin.

OBJECTIVE: This study was undertaken to identify the expected first- and second-year clinical costs associated with intravesical valrubicin therapy, using a decision analytic model, for patients with Bacilli Calmette-Guérin (BCG)-refractory carcinoma in situ (CIS) of the urinary bladder. BACKGROUND: Cancer of the urinary bladder is the fourth most common malignancy in men and the sixth most common noncutaneous carcinoma overall. One histopathologic stage of bladder cancer is CIS, for which BCG intravesical immunotherapy is the first-line therapy. Radical cystectomy has been recommended for patients with CIS who do not respond to or become refractory to therapy with BCG. Surgery, however, may not be appropriate for all patients, especially those who are ineligible for the lengthy procedure because of advanced age or comorbidities and those who prefer alternative nonsurgical management. For these groups, intravesical valrubicin therapy is a plausible alternative. METHODS: Models were developed and populated with data from 1 open-label study of 90 patients, information from the medical literature, and input from clinical experts. The analysis was conducted from the payor perspective for direct costs only. RESULTS: Our data indicate that first- and second-year expected costs for valrubicin therapy are $19,912 and $23,496, respectively. Expected cost for radical cystectomy was also evaluated, since some patients may have no other option if drug therapy fails. CONCLUSION: Our cost-consequence analysis and clinical data provide decision-makers with tools to aid in global budgetary projections of fractional and total expected health care costs associated with the management BCG-refractory CIS of the urinary bladder.

Metallothionein isoform 3 as a potential biomarker for human bladder cancer.

The goal of the present study was to determine if the expression of metallothionein isoform 3 (MT-3) might serve as a biomarker for human bladder cancer. To accomplish this goal, we defined the localization and expression of MT-3 protein and mRNA using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders. We used immunohistochemistry, immunoblot, and RT-PCR analysis to define the localization and expression of MT-3 protein and mRNA. Immunohistochemical analysis disclosed no immunoreactivity for MT-3 in normal bladder cells. The absence of MT-3 expression in the normal bladder was further confirmed by demonstrating that MT-3 mRNA could not be detected using reverse transcriptase-polymerase chain reaction (RT-PCR) or MT-3 protein using immunoblot. Immunohistochemistry also disclosed no immunoreactivity for MT-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders. Immunohistochemical analysis demonstrated that MT-3 was expressed in carcinoma in situ (CIS), high-grade bladder cancer, low-grade bladder cancer, and dysplastic lesions. MT-3 immunostaining was intense in both CIS and high-grade bladder cancer, and low to moderate in low-grade bladder cancer and dysplastic lesions. We determined MT-3 mRNA expression in a subset of these bladder cancer specimens; expression was elevated as compared to that of the housekeeping gene, ss-actin. The cDNA from the RT-PCR reaction primed for MT-3 contained a FokI restriction site, a site unique for MT-3 as compared to other MT family members. In conclusion, this study demonstrates that MT-3 is up-regulated in human bladder cancer and that this up-regulation increases with increasing tumor grade. The finding that MT-3 expression is minimal in normal bladder suggests that MT-3 might be developed into an effective biomarker for bladder cancer.

The potential application of Allium sativum (garlic) for the treatment of bladder cancer.

Additional studies are needed to identify the active ingredients in Allium Sativum (garlic) that are responsible for the observed antitumor activity and immune stimulation. Garlic seems to detoxify chemical carcinogens and prevent carcinogenesis and can also directly inhibit the growth of cancer cells. Current data suggest that low molecular weight sulfur compounds and protein F4 have immune-stimulation properties. Garlic is reported to stimulate immunity, including macrophage activity, natural killer and killer cells, and LAK cells, and to increase the production of IL-2, TNF, and interferon-gamma. These cytokines are associated with the beneficial Th1 antitumor response, which is characteristic of effective cancer immunotherapies. As is true of BCG, garlic stimulates the proliferation of macrophages and lymphocytes and protects against the suppression of immunity by chemotherapy and ultraviolet radiation. Garlic is clearly not a panacea for cancer, but its broad range of beneficial effects are worthy of serious consideration in clinical trials for the prevention and treatment of cancer.

Maintenance bacillus Calmette-Guerin immunotherapy for recurrent TA, T1 and carcinoma in situ transitional cell carcinoma of the bladder: a randomized Southwest Oncology Group Study.

PURPOSE: Bacillus Calmette-Guerin (BCG) immunotherapy has been widely accepted as the optimal treatment for carcinoma in situ and high grade superficial transitional cell carcinoma. However, controversy remains regarding the role of maintenance therapy, and its long-term effect on recurrence and progression. MATERIALS AND METHODS: All patients in the study had transitional cell carcinoma of the bladder with carcinoma in situ or an increased risk of recurrence. The criteria for increased risk were 2 or more episodes of tumor within the most recent year, or 3 or more tumors within 6 months. At least 1 week following biopsy of carcinoma in situ and resection of any stage Ta or T1 transitional cell tumors 660 patients were started on a 6-week induction course of intravesical and percutaneous Connaught BCG. Three months following initiation of BCG induction therapy 550 consenting patients were stratified by purified protein derivative skin test and the presence of carcinoma in situ, and then randomized by central computer to receive BCG maintenance therapy (maintenance arm) or no BCG maintenance therapy (no maintenance arm). Maintenance therapy consisted of intravesical and percutaneous BCG each week for 3 weeks given 3, 6, 12, 18, 24, 30 and 36 months from initiation of induction therapy. The 384 eligible patients who were disease-free at randomization constitute the primary intent to treat analytic group because they could be followed for disease recurrence. All patients were followed for adverse effects of treatment, recurrence, disease worsening and survival. RESULTS: No toxicities above grade 3 were noted in the 243 maintenance arm patients. The policy of withholding maintenance BCG from patients with increased side effects may have diminished the opportunity to observe severe toxicity. Estimated median recurrence-free survival was 35.7 months (95% confidence interval 25.1 to 56.8) in the no maintenance and 76.8 months (64.3 to 93.2) in the maintenance arm (log rank p<0.0001). Estimated median time for worsening-free survival, defined as no evidence of progression including pathological stage T2 disease or greater, or the use of cystectomy, systemic chemotherapy or radiation therapy, was 111.5 months in the no maintenance and not estimable in the maintenance arm (log rank p = 0.04). Overall 5-year survival was 78% in the no maintenance compared to 83% in the maintenance arm. CONCLUSIONS: Compared to standard induction therapy maintenance BCG immunotherapy was beneficial in patients with carcinoma in situ and select patients with Ta, T1 bladder cancer. Median recurrence-free survival time was twice as long in the 3-week maintenance arm compared to the no maintenance arm, and patients had significantly longer worsening-free survival.

Diet and nutrition in urologic cancer.

Cancer is a major cause of mortality and morbidity throughout the world and is projected to become the leading cause of death in the United States and other developed countries in the next few years. There is a large body of evidence linking diet and nutrition with the development of urologic cancers. This is an area where intervention and education can have a major preventive effect on the occurrence of cancer on a worldwide basis. With bladder cancer, a significant protective effect is conferred by a combination of high doses of vitamins A, B6, C and E plus zinc. For prostate cancer, reduced fat intake has a protective effect. A lesser benefit is also suggested with intake of vitamins D and C. Evidence for chemoprevention against renal cell cancer is supported mainly by epidemiologic studies with animal studies indicating possible benefit of vitamin D supplementation. Further research is needed before vitamins and other nutritional supplements can be advocated as standard therapy. Current data support increased intake of vitamins A, B6, C, D and E, reduction of animal fat and increased intake of fruits and vegetables.

Preventing progression and improving survival with BCG maintenance.

BCG immunotherapy provides a superior reduction in tumour recurrence compared with chemotherapy. Unlike chemotherapy, however, it also appears to reduce disease progression. The benefit of BCG is long-term, but protection from disease progression without maintenance therapy is lost when comparisons are made after 15 years. Data suggest that optimal maintenance therapy with BCG provides even better protection from tumour recurrence, reduces disease progression and improves survival. Maintenance BCG schedules using single instillations at 1-3 month intervals have not been proved to be better than induction alone. However, a Southwest Oncology Group (SWOG) study using three, weekly instillations of Connaught BCG, found this regimen to be markedly superior. Before the introduction of BCG, carcinoma in situ (CIS) would progress to muscle invasion in 52% of patients. In the SWOG study, the additional instillations increased the complete response rate in CIS from the expected 68% to 84%. With maintenance BCG, long-term (7 years) tumour recurrence in high-risk patients reduced from the expected 52% with a single 6-week course to only 25% (p < 0.000001). Worsening-free survival, defined as the absence of evidence of disease progression, including pathologic stage T2 or greater disease, or the need for systemic chemotherapy, radiation therapy or cystectomy, significantly increased (p < 0.049, log rank test). In 391 randomized patients, the already excellent 86% survival at 4 years observed with induction therapy improved to 92% in patients receiving maintenance BCG (p < 0.04). There is thus increasingly good evidence that BCG maintenance therapy, at the optimal treatment schedule, provides superior protection from tumour progression and recurrence, and improves long-term survival.

Quinolone antibiotics: a potential adjunct to intravesical chemotherapy for bladder cancer.

OBJECTIVES: Despite complete transurethral resection of superficial bladder tumors, the recurrence rate averages 88% at 15 years. Intravesical chemotherapy decreases the recurrence rate, particularly if given immediately after tumor resection. Anticancer drugs such as doxorubicin target topoisomerase II as do the quinolone antibiotics. We evaluated two fluoroquinolones independently and in combination with doxorubicin for cytotoxic effects against bladder cancer cells in vitro. METHODS: Three human transitional carcinoma cell lines, T24 (grade I), HTB9 (grade II), and TccSup (grade IV), were exposed to either ciprofloxacin or ofloxacin in concentrations ranging from 0 (control) to 1000 microg/mL for 24, 48, and 96 hours. In a separate experiment, a 30% cytotoxic dose (IC30) of doxorubicin was applied to the cell cultures for 1 hour and washed off, followed by exposure to ciprofloxacin or ofloxacin for 48 and 96 hours. Cytotoxicity was evaluated using the MTT colorimetric assay. RESULTS: At 96 hours, significant cytotoxicity (P <0.05) for ciprofloxacin was seen starting at 12.5 microg/mL (HTB9, TccSup) and 50 microg/mL (T24) and for ofloxacin at 12.5 microg/mL (HTB9) and 50 microg/mL (TccSup, T24). Maximum cytotoxicity with ciprofloxacin was 95.4+/-0.4% (HTB9, 400 microg/mL) and with ofloxacin was 95.2+/-0.3% (HTB9, 800 microg/mL). Exposure to doxorubicin (IC30, 1 hour) resulted in cell kill rates of 30.9+/-5.2% (T24), 50.7+/-2.7% (HTB9), and 25.4+/-10.6% (TccSup). The addition of as little as 25 microg/mL of ciprofloxacin increased kill rates to 78.5+/-1.2% (T24), 61.2+/-1.6% (HTB9), and 74.2+/-2.4% (TccSup); P < 0.05 relative to doxorubicin alone. Similarly, 50 microg/mL of ofloxacin significantly increased kill rates to 81.8+/-1.6% (T24), 63.3+/-2.5% (HTB9), and 67.8+/-2.0% (TccSup). Both drugs showed even greater synergism at higher concentrations. CONCLUSIONS: Ciprofloxacin and ofloxacin exhibit significant time- and dose-dependent cytotoxicity against transitional carcinoma cells and significantly enhance the cytotoxicity of doxorubicin. These effects occur at concentrations achievable in the urine of patients after oral administration. This suggests that quinolone antibiotics might be useful as an adjunct to intravesical chemotherapy and might reduce seeding of cancer cells after transurethral resection of bladder tumors.

Chemoprevention of urological cancer.

PURPOSE: Cancer is a major cause of mortality and morbidity throughout the world, and ranks as the second leading cause of death in the United States. Most cancers have a latent period of 10 to 20 years, which provides ample time for preventive measures. Transitional cell carcinoma of the bladder and adenocarcinoma of the prostate have protracted courses and may be ideal for chemopreventive strategies. We review the biochemistry and epidemiology of chemopreventive agents, and the laboratory and clinical studies of their role in urological cancer. MATERIALS AND METHODS: We performed a computerized MEDLINE search and manual bibliographical review of relevant peer reviewed studies and reports from 1966 to 1998. These reports were analyzed and scrutinized, and the important findings are summarized. RESULTS: Neoplastic lesions of the bladder and prostate are uniquely suited to the development and evaluation of chemopreventive agents. Epidemiological reports provide the strongest evidence of a protective role for dietary agents in cancer of the bladder, prostate and kidney. Observational and recent experimental trials support these findings in cases of adenocarcinoma of the prostate and transitional cell carcinoma of the bladder. There is strong evidence for a protective effect of vitamin A in bladder cancer. Superior protection has been reported with a combination of high doses of vitamins A, B6, C and E plus zinc. For prostate cancer strong evidence exists for a preventive effect of reduced fat intake, vitamin E, selenium and soy proteins. A lesser benefit is also suggested with intake of vitamins D and C. Evidence of chemoprevention against renal cell cancer is supported mainly by epidemiological studies, and animal studies indicate possible benefit of vitamin D supplementation. CONCLUSIONS: Although there is no incontrovertible proof, numerous studies implicate dietary and nutritional factors in the onset and progression of cancer of the bladder, prostate and kidney. It is possible that the preventive effect of dietary constituents may be in part from consumption with other nutrients and bioactive compounds in whole foods. Further research is needed before vitamins and other nutritional supplements can be advocated as standard therapy but the preponderance of evidence supports increased intake of vitamins A, B6, C, D and E, reduction of animal fat, and increased consumption of fruits and vegetables.

Bladder cancer: twenty years of progress and the challenges that remain.

The most obvious improvements in the care of patients with bladder cancer over the past 20 years are bacille Calmette-Guérin immunotherapy for superficial bladder cancer and cisplatin and methotrexate-based combination chemotherapy for advanced disease. The challenges that remain are prevention, early detection, and improved treatment of both superficial and advanced disease. Meeting these challenges requires research, financial investment, and public education.