Intravesical chemotherapy and immunotherapy for superficial tumors

Urologic Oncology 4 (1998) 121–129

Review article

Intravesical chemotherapy and immunotherapy for superficial tumors: Basic mechanism of action and future direction H. Akaza, M.D. a,*, K.H. Kurth, M.D. b, R. Williams, M.D. c, S. Hinotsu, M.D. d, M.A.S. Jewett, M.D. e, K. Naito, M.D. f, K. Okada, M.D. g, P.F. Schellhammer, M.D. h, R.F. van Velthoven, M.D. i, J.A. Witjes, M.D. j a

Department of Urology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1, Tennodai, Tsukuba-city, Ibaraki, 305, Japan b University of Amsterdam, Department of Urology, Meibergdreef 9, Amsterdam, 1105AZ, The Netherlands c University of Iowa Hospital, Department of Urology, 200 Hawkins Dr., Iowa City, IA, 52242, USA d Tokyo University, 7-3-1, Hongo, Bunkyo-ward, Tokyo, 113-0033, Japan e 200 Elizabeth St., Toronto, Ontario, M5G 2C4, Canada f Yamaguchi University School of Medicine, Department of Urology, 1144, Kogushi, Ube, Yamaguchi, 755-8505, Japan g Fukui Medical School, Department of Urology, 23-3 Shimoaitsuki, Matsuokacyo, Yoshida-gun, Fukui, 910-1193, Japan h Eastern Virginia Graduate School of Medicine, 600 Gresham Dr., Norfolk, VA, 23507, USA i Institute Jules Bordet, Department of Surgery, 1 rue Heger-Bordet, Brussels, 1000, Belgium j University Hospital Nijmegen, Department of Urology, P.O. Box 9101, Nijmegen, 6500HB, The Netherlands Manuscript accepted February 5, 1999

The previous four Consensus Conferences (Antwerp, 1985; Hakone, 1987; Williamsburg, 1990; Antwerp, 1993) have provided detailed discussions of the current status, significance, and limitations of the therapeutic methods for prophylaxis of recurrence of superficial bladder cancer [1–3]. However, those discussions have failed to take into consideration the factors involved in recurrence after transurethral resection of superficial bladder tumors (TUR-Bt), and recurrence has been handled as a single entity. To date, various research efforts have elucidated many of the details of the various factors influencing recurrence of superficial bladder cancer, and there is a strong awareness of the need to develop strategies to prevent recurrence in relation to each of those various factors. This article first looks at the various factors involved in the recurrence of superficial bladder cancer and then proceeds to a discussion of the current status and future directions of recurrence prevention strategies in relation of each of those factors. In addition, we investigated the usefulness of hazard curves of recurrence as a method for understanding the status of recurrence. 1. Recurrence factors after TUR-Bt for superficial bladder cancer In general, the following mechanisms are surmised as possible explanations of intravesical tumor recurrence: (1) * Corresponding author. Tel. and Fax: (181) 298.53.3196 or (181) 298.53.3223; E-mail: [email protected].

implantation (dispersion) of tumor cells in the epithelium from another site [4], (2) growth of co-existing microscopic lesions [5], (3) growth of tumor cells left behind at the time of incomplete TUR-Bt, and (4) new occurrence of a tumor (i.e., a second primary cancer or subsequent primary cancer). In principle, the first three mechanisms listed above represent tumor cells or tissues already present at the time when TUR-Bt is performed in relation to the main lesion. On the other hand, the fourth mechanism refers to tumors that develop anew after an interval from the performance of TUR-Bt. Strictly speaking, the first three mechanisms represent recurrence, whereas the fourth mechanism should perhaps be defined as representing the development of a second primary cancer (or subsequent primary cancer). However, at present, tumors arising from all of these mechanisms are usually grouped together and labeled as “recurrence.” 2. Second primary cancer As with other malignant tumors, bladder cancers demonstrate alterations in many kinds of oncogenes (i.e., mutation, over-expression, and deletion). The present understanding of molecular changes in human bladder cancer was well summarized by Shirai et al. at the fourth Consensus Conference [6]. In head and neck cancer, molecular analyses of premalignant and malignant tissues have produced strong evidence that clonal genetic alterations occur during the early stage of carcinogenesis [7]. The concepts of multistep and field carcinogenesis are potential mechanisms for the development of a second primary cancer. With regard to the new

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occurrence of a bladder tumor after TUR-Bt (i.e., the fourth mechanism noted above), one hypothesis holds that such patients are constantly exposed to a carcinogenic stimulus and that new cancers will appear at fixed time intervals. A second hypothesis holds that exposure to a carcinogenic stimulus causes some epithelial cells to undergo initiation with repeated cell division at a constant rate. The initiating events alone are not sufficient to culminate in carcinogenesis. A secondary stimulus (promotion) is required. Future research will have to determine whether either of or both of these hypotheses, or some other hypothesis, accurately explains the etiology of “second primary cancers.” The importance of both an initiator and a promoter in bladder carcinogenesis in rats was investigated by Hicks [8]. After initiation with the chemical carcinogen FANFT, tumors developed in only approximately 20% of animals. However, when initiation was followed by administration of saccharin, a promoter, the incidence of bladder tumors rose to 95%. Moreover, even when the administration of saccharin was performed 6 weeks after the initiation with FANFT, tumors developed in 72% of the animals. Hicks confirmed that the same phenomenon occurred when tryptophan was used as the promoter. In addition, Kawai et al. [9] performed studies that used a different rat carcinogenesis system and reported findings similar to those of Hicks. They demonstrated that an inflammatory response induced with killed Escherichia coli (KEC) as a promoter of bladder carcinogenesis even when there

was a time interval between initiation and KEC administration. Both Hicks and Kawai et al. confirmed that, in each of their experimental systems, administration of only the promoter did not cause any bladder tumors to develop. In Japan’s Wakayama Prefecture, where occupational bladder cancer in former workers in dye factories has been manifested, bladder tumors are now developing after a mean interval of 25 years since exposure to carcinogens [10]. These facts support the possibility that carcinogenesis results from cells that have been initiated and are subsequently stimulated by a different promotor after a period of latent inactivity (Fig. 1). The bottom level of Figure 1 speculates on the mechanism by which initiated cells exist in humans. Once a superficial bladder tumor has developed, in advance to visible lesions (Ta or T1), there exist initiated cells and precancerous cells. Treatment of visible Ta and T1 cancers by TUR-Bt is possible, but the sites of initiated cells cannot be identified visually, and it is thus technically impossible to eradicate all potential cancer. In most cases of carcinoma in situ (CIS), treatment by TUR-Bt monotherapy is difficult because the full extent of the lesion cannot be identified. However, microscopic lesions that contribute to the recurrence resulting from the the first three mechanisms listed above may be eliminated by intravesical instillation of anticancer agents or bacillus Calmette-Guérin (BCG). On the other hand, although initiated cells may have undergone some genetic change, their biological properties are likely to be more sim-

Fig. 1. A hypothesis of bladder carcinogenesis and tumor recurrence. KEC, killed Escherichia coli; CIS, carcinoma in situ.

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ilar to normal epithelial cells and will not respond to administered anticancer agents. At a later time, under the influence of promoter, they could develop into cancerous cells. This would result in the formation of a second primary cancer. Various factors can act as promoters, including chemicals in tobacco, saccharin, cystitis, injury caused to the bladder epithelium by the TUR-Bt procedure, and chemical compounds [6]. 3. Investigation of recurrence factors on the basis of hazard curves of recurrence Figure 2 shows a profile of the hazard ratio of tumor recurrence after TUR-Bt. This method of plotting data was first used by Akaza [11] for superficial bladder cancer using SAS septer [12,13], and is regarded as appropriate for depicting tumor recurrence. Up to this point, it has been elucidated that most tumor recurrences are concentrated in the comparatively early period (1–2 years) after TUR-Bt (Fig. 2). This finding indicates that early adjuvant therapy administered at the time of the initial TUR [14,15] will lower the recurrence. Intravesical instillation of BCG has been found to reduce recurrence [16] and support the use of potent adjuvant therapy. 4. Current status of therapy for prevention of recurrence Intravesical chemotherapeutic regimens are currently being applied by many investigators [3]. This approach can be considered representative of the treatments being administered to prevent recurrence of superficial bladder cancers secondary to the first three mechanisms described above. The hazard ratio can be understood as the risk of tumor recurrence at a given time point after TUR-Bt as described on Figure 2. It is seen from the plots that the hazard ratio is high during the 3- to 6-month period (at the longest within 1 year) after TUR-Bt, whereas the hazard ratio later becomes lower and fairly constant on a long-term basis. It also can be seen in Figure 2 that intravesical chemotherapy clearly reduced the hazard ratio during the early period after

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Table 1 Effect of intravesical chemotherapy on recurrence in controlled studies Percent recurrence Drug

No. of cases

Control (A)

Treatment (B)

(A)2(B)

Thiotepa Doxorubicin Mitomycin C Epirubicin

1,007 1,241 1,157 399

56 47 50 41

44 34 35 29

12 13 15 12

TUR-Bt by reducing the hazard of implanted cells and microscopic tumor lesions remaining immediately after the TUR-Bt. However, the hazard ratio was not reduced thereafter, and if we assume that this represents the risk of recurrence due to the proposed fourth mechanism, then this perhaps can be interpreted as indicating that intravesical chemotherapy is ineffective against this mechanism of recurrence. Table 1 presents data that represents a part of the consensus reached in 1993 by the working group of the Fourth International Consensus Conference on Bladder Cancer (Antwerp, Belgium) in relation to the efficacy of intravesical chemotherapy [3]. A 12 to 15% lower tumor recurrence rate compared with the control group (no treatment was administered to prevent recurrence) was noted, but no prophylactic efficacy was found in relation to progression of the stage of the recurrent tumor (Table 2). Once a tumor has invaded the bladder muscle layer (i.e., grade T2 and higher), the prognosis of the patient becomes very poor [17,18]. For this reason, in addition to reduction of recurrences, prevention of stage and grade progression are important outcomes of prophylactic therapy. With regard to stage progression, no advantage of intravesical chemotherapy has been confirmed [19–21]. Comparing the long-term follow-up of 1,938 treated patients with a group of 2,607 untreated controls, treatment with intravesical chemotherapy demonstrated no significant progression or survival advantage [19]. A combination of controlled studies (1,423 patients) also failed to demonstrate any effect on progression: It was 6.6% for treated patients and 7.2% for controls. Although the reason why reduction of recurrence does not influence progression remains a question, an explanation might be that progression originates in the second primary tumor recurrence and not in the recurrent tumor of the first three mechanisms listed above. On the other hand, BCG seems to be a promisTable 2 Effect of intravesical chemotherapy on stage progression in controlled studies

Fig. 3. Hazard function estimate (primary multiple tumor).

Control

Treatment

Drug

No. of cases

Progression

No. of cases

Progression (%)

Thiotepa Doxorubicin Mitomycin C Epirubicin

314 389 336 194

14 15.2 3.9 3.6

199 183 191 205

12 12.6 7.3 2.4

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ing drug that reduces tumor progression and even bladder cancer death [19,22] especially if used with a maintenance schedule. For example, Lamm [19] observed an improved progression-free survival in patients treated with maintenance therapy in responders to an initial 6-week course. These results were confirmed with long-term follow-up [23].

5. Concept of marker tumor A drug administered intravesically can be most accurately assessed by its effect against marker tumor(s). For this reason, the marker lesion model is an important experimental tool. However, the ethics and/or safety of purposely leaving in the bladder a tumor that could be easily resected by TUR-Bt is questionable [24]. In the European Organization for the Research and Treatment of Cancer (EORTC) phase II trials of mitomycin and epirubicin, which used a marker lesion, progression of the marker tumor was very low [25]. In Japan, it has been necessary to demonstrate the direct antitumor efficacy of a drug against a marker lesion to gain the approval of the Japanese Ministry of Health and Welfare. A phase II study of BCG (Tokyo 172 strain) proved that a complete response (CR) rate (complete disappearance of tumor[s]) against Ta/T1 marker tumor(s) was 68.8%. No disease progression was observed during the trial period [26]. Those data confirm the feasibility and safety of the marker lesion model for the objective evaluation of the antitumor activity of intravesically administered drugs for superficial bladder cancer.

6. Efficacy of BCG in prevention of superficial bladder cancer recurrence At present, it is said that BCG demonstrates efficacy in preventing tumor recurrence. This raises the question of the mechanism of efficacy. Does BCG show more potent effects than chemotherapy in relation to the types of lesions caused by the first three mechanisms listed above, which remain after TUR-Bt? Is BCG also effective against the fourth type of recurrent bladder cancer lesions (i.e., second primary cancers)? It is necessary to determine whether intravesicallyinstilled BCG achieves its efficacy by inducing cytotoxic T lymphocytes (CTL) against self tumors (even those that would later recur anew) and creating an immunologic surveillance mechanism. The effects of BCG are not seen only in the prophylaxis of recurrence of CIS and Ta and T1 bladder tumors [27]. In a multicenter collaborative study in Japan, intravesical BCG therapy was able to completely eliminate the tumors from 84.4% of CIS cases and 68.8% of Ta and T1 cases [26]. Moreover, this effect was maintained, especially in the cases that achieved a CR [16]. Figure 3 shows the time-course plots of the hazard ratio for recurrence in cases that achieved CR or partial response (PR) after undergoing intravesical instillation of BCG. One

Fig. 2. Hazard for tumor recurrence after bacillus Calmette-Guerin (BCG) (complete response 1 partial response with subsequent transurethral resection of the prostate).

plot is for cases that received additional BCG instillation as maintenance therapy after achieving CR or PR, that the second plot is for cases that did not receive any maintenance BCG. Even when BCG maintenance therapy was not administered, there was no increase in the hazard ratio immediately after TUR-Bt, which was comparable to that seen when only TUR-Bt was performed (Fig. 2). Furthermore, the hazard ratio remained very low thereafter. These findings indicate that intravesically-instilled BCG is very effective not only against bladder tumor recurrence of the types represented by the first three mechanisms listed above, but also against recurrence due to the fourth mechanism. Table 3 presents a summary of the data on the prophylactic effect of BCG on bladder tumor recurrence, which were reported at the international consensus meeting held in Antwerp in 1993 [3]. 7. Mechanism of expression of efficacy by BCG immunotherapy Although intravesical BCG therapy for superficial bladder cancer is effective immunotherapy in the treatment of superficial bladder cancer, uncertainties still exist with regard to patient selection, optimal treatment protocol, reduction of side effects, and mechanism of action. The goal of several groups worldwide has been to clarify the mechanisms of action of BCG. The immunostimulating effect of BCG has been well known for decades. It is related to the immunologic response against this highly resistant and antigenic organism. Studies performed by several investigators have shown that

Table 3 Effect of intravesical bacillus Calmette-Guérin on recurrence in controlled studies Percent recurrence No. of cases

Control (A)

Treatment (B)

(A)2(B)

402

75

31

44

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intravesical application induces an array of immunologic changes locally, in the bladder. Chronic bladder wall infiltration of mononuclear cells (predominantly CD41 helper/ inducer T lymphocytes; CD81 T cells, macrophages, and B cells are present to a lesser extent) has been described [28,29]. The BCG-induced infiltrates showed human leukocyte antigen (HLA)-DR and interleukin-(IL) 2 receptor expression, indicating an activated status of T cells. During the first hours after each BCG instillation, large amounts of cytokines are detectable in the urine, including IL-1, IL-2, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNFa), and interferon gamma (IFNg) [30–35]. These cytokines are primarily secreted in the urine during the first 6-hour period following instillation of BCG. The general trend is the level of cytokine to increase with repeated instillations. However, cytokine production may persist much longer: Establishment of cytokine producing (granulomatous) infiltrates up to 21 months has been reported [36]. In line with the observations in humans, the generation of a Th1-type (i.e., IFNgproducing) response at the bladder site after intravesical BCG therapy was found in a mouse. The cells infiltrating the bladder wall may be the major producers of the urinary cytokines. However, urothelial tumor cells themselves also can react to the intravesical therapy. Probably as a result of BCG-induced cytokines such as IFNg and/or TNFa, major histocompatibility complex (MHC) class II and ICAM-1 molecules on normal and tumor urothelial cells are upregulated [28,29,37,38]. Because these molecules are fundamental to many immune functions, including antigen presentation and cell-mediated cytotoxicity, it could be speculated that upregulation might enable bladder tumor cells to present (BCG/tumor) antigens and/or render them more sensitive to cytotoxic cells. Furthermore, urothelial (tumor) cells may actively participate in the response to BCG by the production of cytokines. IL-6, TNFa, and IL-8 release can be upregulated in bladder tumor cells after exposure to BCG [39,40]. In addition, bladder carcinoma cells are capable of adherence, ingestion, and degradation of the bacilli in vitro [41–44]. Controversy exists with regard to the occurrence of BCG internalization in vivo, as well as to the role of fibronectin-mediated attachment in patients [41–43,45]. Few studies have provided insight in the actual effector mechanism in the BCG-mediated reaction against bladder cancer. Mouse models have indicated that natural killer (NK) cells were not the primary effector cells in BCG therapy, but rather that BCG-induced antitumor activity is T (CD41 and CD81) lymphocyte dependent, and not the result of detectable tumor-specific immunity. The effector cell remained unknown [46]. Theoretically, lymphokine activated killer cells (LAK) or LAK-like cells, generated through BCG induced cytokines such as IL2 could play a role in the working mechanism of BCG. In vitro BCG seems to operate preferentially via monocytes TH1-like cells. These cells secrete IL-2 and IFN, thereby activating CD81/CD561 T-cytotoxic/NK effector cells with cytolytic ability toward bladder tumor cells [47].

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The in vivo role of this kind of effector cell in intravesical BCG therapy for superficial bladder carcinoma, however, remains to be elucidated. Notably, lymphocytes with NK/LAK/ BAK phenotype were not major infiltrating cells, which was revealed by immunohistochemical studies [28,29]. The main therapeutic goals with regard to the optimization of BCG therapy for superficial bladder cancer are to improve clinical response rates, decrease toxicity, and predict which patients are not going to respond to BCG therapy. Efforts to identify prognostic indicators have been pursued by many investigators. Because an immune, T-cell– mediated mechanism seemed to be of importance in the mode of action of BCG [47], efforts have focused on identifying immune parameters such as granulomatous inflammation in the bladder wall and purified protein derivative (PPD) skin test responsiveness. However, these tests did not provide the necessary sensitivity or specificity. Subsequent studies focused on urinary cytokine induction. Measurement of these cytokines may be more relevant because it reflects the local immunologic reaction in the entire bladder, and BCGinduced cytokines may be involved in the generation of killer cells. Despite complicating factors, including stability in urine, pleiotropic effects, and multiplicity of sources [33], data are presently emerging that indicate an association between cytokine induction and clinical response to BCG therapy [33,48]. However, evaluation of large patient numbers is required to obtain definitive evidence on the prognostic value of BCG-induced urinary cytokines. Recent studies have indicated the advantage of maintenance therapy [49]. Measurement of urinary cytokines can provide information on the immunologic consequences of BCG applications according to maintenance schedules. Interestingly, during a second 6-week course of BCG instillations, accelerated IL-2 induction and a trend for a decrease in IL-2 during the final instillation were observed. These observations suggest that the number of weekly instillations during maintenance regimens (i.e., after the 6-week induction course) could be less than six [50]. In fact, these observations support the clinical experience of applying three weekly BCG instillations every 3 months during maintenance therapy. However, a major drawback of maintenance BCG therapy is that more intensive treatment with the live BCG organisms implies a greater risk for serious systemic side effects. Therefore, studies most recently undertaken and focusing on nonliving mycobacterial preparations are of interest. Mycobacterial cell walls as an alternative to live BCG in the treatment of bladder cancer have shown promising results [51,52]. In addition, it has been reported that released products of BCG contain chemotactic activity as well as activity to enhance lymphocyte mediated bladder tumor cell killing [53,54]. However, for successful clinical application of these alternatives to live BCG, it is necessary to insure contact and attachments of tumor cells to provoke its antitumor effects [44,45]. In conclusion, intravesical BCG therapy seems to generate a situation of “immunopotentiation”: The immunologic

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changes suggest recognition of bladder tumor cells as the basis of tumor elimination as a result of the therapy. Furthermore, a local immune response to BCG seems to be required for the efficacy of BCG therapy. The effector mechanism that eventually eradicates the bladder tumor cells is not known.

8. Limitations of intravesical instillation therapy To date the therapies that have been used to prevent recurrence of superficial bladder cancer have employed instillation of drugs directly into the bladder. Accordingly, these methods do not overcome the inherent limitations of local therapy. The therapeutic approach of instilling anticancer drugs directly into the bladder aims to increase the local concentration of the drugs while avoiding the development of systemic adverse effects. In addition, BCG cannot achieve efficacy if a live or lyophilized BCG preparation was not instilled into the bladder [44]. However, periodic intravesical instillation therapy is not necessarily acceptable to patients, and it does not address the problem of extravesical disease and/or metastasis. One attempt to develop a systemic treatment modality is currently being carried out on an international scale through clinical trials with oral administration of bropirimine, which is an interferon inducer. Based on the results of a phase I clinical study performed by Sarosdy and colleagues [55], it is anticipated that oral bropirimine will show a high direct antitumor efficacy rate in the treatment of CIS. The Food and Drug Administration (FDA) did not approve this drug for BCG failure. However, a Japanese trial indicates oral bropirimine not only has potential as a therapeutic agent for CIS [56], but also may prove useful for the prevention of recurrence after TUR-Bt of Ta and T1 superficial bladder tumors [57].

9. Bropirimine, an interferon inducer In a Japanese phase II study, 20 patients with histologically proven recurrent Ta,T1 transitional cell carcinoma (TCC) were studied. At least one marker lesion was left behind after TUR-Bt. Bropirimine (750 mg) was given orally three times at 2-hour intervals on 3 consective days every week for 12 weeks. Of 17 evaluable patients, one did not complete the treatment protocol. There were five responders including two with a CR and three with a PR. The response rate was 31.3% (5 of 16 patients; 95% confidence limit 5 11 258.7%) for the 16 completely treated patients. Adverse reactions occurred in 70.6% of the evaluable patients. Flu-like symptoms were most common, including malaise, headache, and elevation of body temperature. However, all reactions were tolerable [57]. Bropirimine was deemed useful for the prophylaxis of recurrence of superficial bladder cancer after TUR-Bt be-

cause of its efficacy against marker tumors, its efficacy against CIS, and its oral route of administration with a good safety profile.

10. The future of recurrence prevention therapy What approach should be taken to more effectively prevent bladder tumors of the type represented by second primary cancers? Lamm et al. [58] reported findings that indicate that large doses of vitamins are effective in the prevention of second primary cancers. In our own research, we demonstrated that a preparation of oral Lactobacillus casei (biolactis preparation; BLP) inhibited chemical carcinogenesis of bladder cancer in rats and mice [59,60]. In a double-blind, placebo-controlled clinical study, we found that BLP was effective in preventing bladder tumor recurrence [61]. In consideration of the fact that the incidence of bladder cancer is higher in men than women, inhibition of bladder carcinogenesis by suppression of androgen activity is under study in the animal model [62].

11. Efficacy of BLP in preventing bladder tumor recurrence BLP is a lyophilized preparation, 1 g of which contains ca. 1 3 1010 live cells of L. casei YIT 9018. There have been numerous reports presenting experimental data showing that, even in humans, oral administration of BLP causes changes in the bacterial flora of the intestines and results in inhibition of activation of carcinogens and immunopotentiation. Tomita and colleagues [59] reported a study in which they first administered N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to rats and then administered DL-tryptophan, which is a known promoter of bladder cancer. Then, using concanavalin A (an agglutinin of plant origin), which has the property of selectively agglutinating malignant cells, they performed studies on BLP as an inhibitor of BBN carcinogenesis. Their findings indicated that, in comparison with the control group (i.e., no BLP treatment), the BLPtreated group showed inhibition of BBN-induced carcinogenesis in the early stage of the process. These authors then studied actual BBN-induced carcinogenesis in Wistar rats. BLP was found to show statistically significant inhibition of the tumor weight per bladder when it was administered throughout the carcinogenic process and during the promotion stage thereof, and statistically significant inhibition of the weight of the entire bladder when it was administered throughout the carcinogenic process and during the initiation stage thereof [59]. Similar studies were conducted in mice, and it was again confirmed that BLP shows efficacy in inhibiting carcinogenesis [60]. BLP has been shown to have immunologic effect suppressing tumor growth in mice [63]. Indole and phenolic compounds, which are metabolites produced from amino acids by enteric bacteria, are known

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to act as promoters of carcinogenesis. It has been shown that BLP administration reduces the amounts of these compounds excreted in the urine [64]. In addition, studies using the Ames test to screen for carcinogens excreted in the urine of people who have consumed roasted meat have demonstrated that BLP administration significantly effects inhibition of those substances [65]. Thus, it can be surmised that BLP might be able to inhibit the carcinogenesis of bladder cancer by exerting a significant inhibitory effect on the formation of carcinogens in the intestinal tract, which might be reabsorbed into circulating blood and excreted into urine. A double-blind, placebo-controlled, multicenter collaborative study [61] on BLP was performed. In medium-risk groups (i.e., a primary and multiple bladder tumor group and a recurrent and single bladder tumor group), BLP showed a statistically significant effect in preventing recurrence. Conversely, in the high-risk group (i.e., the recurrent and multiple bladder tumor group), BLP could not be proven to show prophylactic efficacy. Even when BLP itself was instilled into the bladder, it did not show a direct antitumor effect. Assuming that the mechanisms of bladder tumor recurrence in the high-risk group are the first three mechanisms noted earlier, it can be surmised that combined administration of oral BLP with intravesical instillation of anticancer agents would be useful in this patient group.

12. Conclusion Numerous and varied steps are required to culminate in the development of a cancer. The overall process of carcinogenesis involves the initial exposure to a carcinogen, activation of the carcinogen, binding of the activated carcinogen to DNA, loss of the ability to repair DNA alterations, expression of a cancer gene(s), inactivation of a tumor suppressor gene(s), promotion by a growth factor(s) or hormone(s), and finally escape from immunologic surveillance. Only after completion of this complex process does a cancer become clinically recognizable. The anticancer agents that are currently being used target clinical cancer. In this sense, intravesical instillation of anticancer agents in the treatment of superficial bladder cancer is only one aspect of local therapy. However, in the treatment of superficial bladder cancer, an equally important problem is prevention of recurrence, and it has been pointed out—both clinically and theoretically—that intravesical instillation of anticancer agents is insufficient in this regard. As dicussed, BCG intravesical instillation therapy shows a potent direct antitumor effect, and it has the potential for specific activation of the immunologic surveillance system. In addition, basic and clinical studies have indicated that BLP is potentially involved in counteracting carcinogenic stimuli in the initial stage of cancer recurrence. At present, from a cost-benefit point of view, it is probably impossible to achieve prevention of bladder cancer in the

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population at large. However, it is extremely important to devise strategies to prevent recurrence in the patient population that has a history of superficial bladder cancer (i.e., prevention of recurrence after TUR-Bt). When therapy for inhibiting the formation of second primary cancers (i.e., cancers due to the previously-mentioned fourth mechanism) is developed, it also will likely be effective for preventing primary bladder cancer among subjects at high risk due to environmental exposure, life style, and genetic predisposition.

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