Prevention of Recurrence With Epirubicin and Lactobacillus Casei After Transurethral Resection of Bladder Cancer

Article Outline

• Abstract
• Materials and Methods
• Results
• Discussion
• Conclusions
• References
• Copyright

Purpose

A prospective, randomized, controlled trial was done to evaluate whether oral administration of a preparation of the probiotic agent Lactobacillus casei (Yakult Honsha, Tokyo, Japan) could enhance the prevention of recurrence by intravesical instillation of epirubicin after transurethral resection for superficial bladder cancer.

Materials and Methods

Between August 1999 and December 2002, 207 patients clinically diagnosed with superficial bladder cancer were included as study candidates and underwent transurethral resection, followed by intravesical instillation of 30 mg epirubicin/30 ml saline twice during 1 week. After histological confirmation of superficial bladder cancer they were again included as study participants with 102 randomized to receive treatment with 6 additional intravesical instillations of epirubicin during the 3-month period after transurethral resection (epirubicin group) and 100 randomized to intravesical chemotherapy on the same schedule as the epirubicin group plus oral administration of 3 gm Lactobacillus casei preparation per day for 1 year (epirubicin plus Lactobacillus casei group). Patients were evaluated for intravesical recurrence, disease progression, prognosis and adverse drug reactions.

Results

The 3-year recurrence-free survival rate was significantly higher in the epirubicin plus Lactobacillus casei group than in the epirubicin group (74.6% vs 59.9%, p = 0.0234), although neither progression-free nor overall survival differed between the groups. The incidence of adverse drug reactions did not significantly differ between the groups and there were no serious adverse drug reactions.

Conclusions

Intravesical instillation of epirubicin plus oral administration of Lactobacillus casei preparation is a novel, promising treatment for preventing recurrence after transurethral resection for superficial bladder cancer.

Key Words: bladder, bladder neoplasms, neoplasm recurrence, local, epirubicin, Lactobacillus casei

Abbreviations and Acronyms: BCG, bacillus Calmette-Guerin, EPI-ADM, epirubicin, LC, Lactobacillus casei, TUR, transurethral resection

Although superficial bladder cancer can be treated with TUR, the high frequency of intravesical recurrence is a concern. It was reported that intravesical recurrence develops in 50% to 70% of patients within 5 years after TUR for superficial bladder cancer and the risk of progression to invasive cancer is 5% to 20%.¹ The recurrence risk peaks in the early postoperative phase at 100 to 120 days after TUR for multiple tumors and at 350 to 440 days after TUR for a solitary tumor, and then it decreases to and continues at a stable level for a long period.² Intravesical instillation therapy using anticancer agents or BCG has been developed to prevent intravesical recurrence of superficial bladder cancer after TUR. Intravesical instillation of mitomycin C, doxorubicin or EPI-ADM was reported to decrease the short-term (1 to 3-year) recurrence rate by about 20%.³ On the other hand, intravesical instillation of BCG has stronger efficacy for preventing recurrence than anticancer agents, although the incidence and severity of adverse effects are higher with it than with chemotherapy.⁴ Therefore, BCG is reserved for patients with high risk superficial bladder cancer, while anticancer agents are used in patients with intermediate risk cancer.

The LC preparation used in the study was a powdered preparation containing about 1 × 10¹⁰ cells of LC Shirota strain per gm. In Japan LC preparation has been safely used as a probiotic agent for more than 30 years. When it is orally administered, the LC preparation was reported to act as an immunomodulator through the intestinal tract and potentiate antitumor responses in mice.⁵ Intravesical instillation of heat killed cells of the LC Shirota strain was also shown to exert antitumor effects in mice with bladder cancer and prevent bladder cancer.⁶ In a randomized, comparative clinical trial Aso et al reported that the 50% recurrence-free interval after TUR for superficial bladder cancer was significantly prolonged by oral LC preparation to 1.8 times that in a control group.⁷ They also performed a placebo controlled, double-blind clinical trial and noted that treatment with LC preparation was safe and effective for preventing intravesical recurrence after TUR for superficial bladder cancer.⁸ These reports suggest that LC preparation can prevent intravesical recurrence after TUR for superficial bladder cancer through a mechanism different from that of intravesical chemotherapy.

Therefore, we planned a randomized, controlled trial in patients with superficial bladder cancer at intermediate risk for recurrence to evaluate whether postoperative oral administration of LC preparation could enhance the prevention of recurrence by intravesical instillation chemotherapy with EPI-ADM after TUR for superficial bladder cancer.

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Materials and Methods 

This study was a multicenter, prospective, nonblinded, randomized, controlled trial. Patients were considered eligible if they had clinical stage Ta or T1, grade 1 or 2 primary or recurrent transitional cell carcinoma and the tumors appeared to have been eliminated completely by TUR. Patients meeting any of certain criteria were excluded from study, including a primary single Ta grade 1 tumor, grade 3 tumor, multiple recurrent tumors, history of urothelial carcinoma of the upper urinary tract, history of intravesical instillation of EPI-ADM or BCG, history of intravesical instillation of any agents during the 4-week period preceding the study, other active neoplasms or serious medical conditions. LC strain is contained in a fermented milk drink such as Yakult (Yakult Honsha, Tokyo, Japan) or a fermented milk product such as yogurt as an intestinal remedy. Therefore, patients regularly ingesting such beverage, food or drug containing LC strains were also excluded.

Before treatment patients provided a history and underwent physical examination, urinalysis, urine cytology examination, complete blood count, blood urea nitrogen and serum creatinine determination, liver function tests and electrocardiography. Chest x-rays and excretory urography were also performed. At study entry no patients had evidence of residual tumor on endoscopic examination and urine cytology.

Patients were added to the study via fax using a 2-step method at the Kyushu University Urological Oncology Group Data Center. After providing informed consent in writing patients were documented as study candidates before TUR for superficial bladder cancer. A 30 mg dose of EPI-ADM dissolved in 30 ml physiological saline was instilled into the bladder through a sterile catheter immediately after (within 2 hours) and 1 week after TUR. Patients were instructed not to void for 2 hours after instillation. When confirmed to be eligible based on the results of histopathological examination of resected tumor specimens, patients were again included as study participants and randomly assigned to receive treatment with 6 additional intravesical instillations of EPI-ADM during the 3-month period after TUR (EPI-ADM group) or intravesical chemotherapy on the same schedule as the EPI-ADM group plus 3 gm oral LC preparation per day (EPI-ADM plus LC group). Intravesical instillation of EPI-ADM was administered 3, 4, 6, 8, 10 and 12 weeks after TUR. The LC preparation dose was determined according to trials by Aso et al, in which the efficacy and safety of 3 gm oral LC per day were observed.⁷, ⁸ Figure 1 shows the group treatment schedules. Administration of the LC preparation was begun within 2 weeks after randomization and continued for 1 year.

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• Fig. 1. 

  Treatment schedule of EPI-ADM and EPI-ADM plus LC groups. In EPI-ADM group 30 mg EPI-ADM/30 ml saline were given immediately after (within 2 hours), and 1, 3, 4, 6, 8, 10 and 12 weeks (W) after TUR. In EPI-ADM plus LC group same intravesical instillation schedule was used as in EPI-ADM group plus oral administration of 3 gm LC preparation per day, which was begun within 2 weeks after randomization and continued for 1 year.

In each groups urinalysis and cytological examination of urine samples were performed monthly for 3 months after TUR, every 3 months in the first 2 years and at 6-month intervals thereafter. Cystoscopy was performed every 3 months in the first 2 years and at 6-month intervals thereafter. Local and systemic toxicity was also monitored. Routine laboratory tests (hematology and biochemistry) were performed before, and 1, 3, 6, 9 and 12 months after TUR. All patients were followed at least 3 years and the treatment method after the first recurrence was at the discretion of each investigator. The severity of adverse reactions was assessed according to Common Terminology Criteria for Adverse Events, version 2.0.

The primary end point of this trial was the intravesical recurrence-free survival rate. Recurrence was defined as positive findings on cystoscopy or consecutive positive findings on urine cytology. Positive findings on cystoscopy were confirmed histologically by biopsy or TUR. Secondary end points were the progression-free survival rate, the overall survival rate, and the incidence and severity of adverse drug reactions. Progression was defined as muscle invasive bladder cancer or metastasis. Intravesical recurrence-free, progression-free and overall survival was defined as the interval from TUR to each event or the last followup without an event. Observation was concluded on January 31, 2006.

Sample size was calculated as described. Assuming that the 3-year recurrence-free survival rate would be 55% and 75% in the EPI-ADM and EPI-ADM plus LC groups, respectively, 92 patients per group were required to detect a 20% difference between the groups with a power of 80% and a 2-tailed significance level of 5%. Considering possible study dropouts due to inability to undergo the study, or violation of eligibility criteria or protocol treatment 200 study participants (100 per group) were set as the target sample size.

Statistical analysis was performed with an intent to treat design in all 202 eligible patients using the SAS® system package. The significance of differences in patient background factors, and the incidence and severity of toxicity between the 2 groups was examined by the chi-square test. Differences in time to first intravesical recurrence, progression or death were assessed using nonrecurrence, progression-free and overall survival curves, respectively, as calculated by the Kaplan-Meier method with the determination of statistical significance by the log rank test. The Cox proportional hazard model was used to adjust for possible bias in background factors. Differences were considered significant at p <0.05. The risk of recurrence was defined as the fraction of patients with recurrence in each 6-month period. The HR in each period was calculated using maximum likelihood estimates derived from a piecewise exponential model.⁹ The protocol was approved by the local institutional review board.

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Results 

Between August 1999 and December 2002, 207 patients at 25 institutions were included as study candidates. They underwent TUR and subsequent intravesical instillation of EPI-ADM twice during 1 week. Five patients in whom histopathological review revealed invasive bladder cancer were eliminated from the study as ineligible. The remaining 202 patients were again documented as study participants and randomly assigned to the EPI-ADM group (102) or the EPI-ADM plus LC group (100). There were no significant differences between the groups in the sex ratio, patient age, smoking habit, number of tumors, T stage, grade or tumor size (table 1).

Table 1. Patient characteristics

	No. EPI-ADM	EPI-ADM + LC	p Value (chi-square test)
Overall	102	100	0.2510
Sex:
M	86	78
F	16	22
Age:			0.8955
Younger than 70	55	53
70 or Older	47	47
Smoking habit:			0.6650
No	53	55
Yes	49	45
Tumor multiplicity:			0.9903
Primary/solitary	40	40
Primary/multiple	52	50
Recurrent/solitary	10	10
T stage:			0.9955
Ta	53	52
T1	49	48
Tumor grade:			0.9425
1	21	21
2	81	79
Tumor size (cm):			0.8363
Less than 1	33	31
1 or Greater	69	69

Participants were observed for recurrence for 0.6 to 79.9 (median 26.9) and 0.2 to 75.0 months (median 43.6) in the EPI-ADM and EPI-ADM plus LC groups, respectively. For progression they were observed for 0.6 to 79.9 (median 49.9) and 0.2 to 75.7 months (median 48.8), respectively. For any cause of death they were observed for 0.6 to 79.9 (median 49.9) and 0.2 to 75.7 months (median 49.1), respectively.

More than 80% of patients in the EPI-ADM plus LC group ingested 80% or greater of the prescribed LC preparation throughout the 1-year treatment period, indicating high compliance with treatment with LC preparation. A total of 31 patients could not complete the protocol treatment irrespective of recurrence. One EPI-ADM group patient and 10 EPI-ADM plus LC group patients, including 4 during intravesical chemotherapy and 6 during treatment with LC preparation after intravesical chemotherapy, discontinued visiting the study institutions during the study period. Six and 14 patients in the EPI-ADM and EPI-ADM plus LC groups, respectively, discontinued study participation due to withdrawal of informed consent or exacerbation of other disease.

During the observation period bladder cancer recurred in 42 (41.2%) and 26 (26.0%) patients in the EPI-ADM and EPI-ADM plus LC groups, respectively. The 3-year recurrence-free survival rate was 59.9% (95% CI 49.9–69.8) in the EPI-ADM group and 74.6% (95% CI 65.5–83.6) in the EPI-ADM plus LC group. The EPI-ADM plus LC group showed a significantly higher recurrence-free survival rate than the EPI-ADM group (log rank test p = 0.0234, fig. 2).

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• Fig. 2. 

  Recurrence-free survival curves after TUR for superficial bladder cancer in 2 groups. EPI-ADM (epi) plus LC group showed significantly higher recurrence-free survival than EPI-ADM group (p = 0.0234).

To eliminate biases due to differences in background factors univariate analysis of 8 variables possibly affecting bladder cancer recurrence was performed using a Cox proportional-hazard model. Of the 8 variables group, multiplicity, tumor size and T stage each significantly affected the recurrence-free survival rate (table 2). On multivariate analysis the significance of the difference in the recurrence-free survival rate between the EPI-ADM and EPI-ADM plus LC groups was adjusted for the 3 other factors, ie multiplicity, tumor size and tumor stage. A significant difference between groups was also detected on multivariate analysis (table 3).

Table 2. Univariate analysis of factors influencing recurrence

Table 3. Multivariate adjusted recurrence HRs

Variables + Models	HR	95% CI	p Value
Univariate	0.5714	0.3500–0.9328	0.0230
Adjusted for:
Tumor multiplicity	0.5734	0.3510–0.9368	0.0241
Tumor size	0.5549	0.3396–0.9066	0.0169
Tumor stage	0.5721	0.3504–0.9341	0.0233
Tumor multiplicity, size + stage	0.5654	0.3450–0.9265	0.0216

Figure 3 shows the risk of tumor recurrence during followup after TUR for superficial bladder cancer. In the EPI-ADM group the risk of tumor recurrence was increased in the first 2 years and it peaked around 6 months after TUR. However, in the EPI-ADM plus LC group the risk of tumor recurrence was clearly suppressed, especially in year 1 after TUR.

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• Fig. 3. 

  Risk of tumor recurrence in EPI-ADM (epi) plus LC group was clearly decreased, especially in year 1 after TUR, compared to that in EPI-ADM group.

One and 3 patients in the EPI-ADM and EPI-ADM plus LC groups, respectively, showed disease progression. Death occurred during followup in 3 EPI-ADM group patients due to bladder cancer in 1 and other causes in 2, and in 4 EPI-ADM plus LC group patients due to bladder cancer in 1 and other causes in 3. There were no significant differences between the groups in progression-free or overall survival (data not shown).

Table 4 shows the incidence and severity of adverse reactions. The most common adverse reactions involved local toxicity due to intravesical chemotherapy, such as pain on micturition, urinary frequency and gross hematuria. In the EPI-ADM plus LC group 6% and 2% of patients had constipation and diarrhea possibly related to LC preparation treatment, respectively. There were no significant differences in the incidence of adverse reactions between the groups. Neither serious (grade 3 or 4) adverse reactions nor abnormal laboratory findings were observed in either group. No patients elected to discontinue protocol treatment due to adverse drug reactions.

Table 4. Adverse drug reaction incidence + severity

	No. EPI-ADM (%)	No. EPI-ADM + LC (%)	p Value (chi-square test)
Pain on micturition:
Grade 1	34 (33.3)	24 (24.0)	0.929
Grade 2	8 (7.8)	7 (7.0)
Urinary frequency:
Grade 1	22 (21.6)	19 (19.0)	0.905
Grade 2	9 (8.8)	6 (6.0)
Gross hematuria:
Grade 1	15 (14.7)	14 (14.0)	0.836
Grade 2	4 (4.0)	2 (2.0)
Constipation:
Grade 1	2 (2.0)	4 (4.0)	0.895
Grade 2	2 (2.0)	2 (2.0)
Diarrhea:
Grade 1	0	1 (1.0)	1.000
Grade 2	0	1 (1.0)

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Discussion 

Intravesical instillation chemotherapy with EPI-ADM is effective for preventing recurrence after TUR for superficial bladder cancer. Several reports suggest that sufficient preventive effects of EPI-ADM can be achieved even with relatively few instillations. In fact, Ali-El-Dein et al reported that single dose instillation of EPI-ADM immediately after TUR for superficial bladder cancer was as effective as multiple instillations of EPI-ADM for preventing tumor recurrence.¹⁰ Okamura et al also reported that maintenance intravesical instillation of EPI-ADM did not improve the tumor recurrence rate.¹¹ However, in our previous study comparing 9 short-term and 19 long-term intravesical instillations of EPI-ADM long-term treatment was significantly more effective for preventing intravesical recurrence after TUR for superficial bladder cancer.¹² Thus, the optimal treatment schedule for intravesical EPI-ADM is still controversial. When considering cost-effectiveness and the risk of adverse reactions, a smaller number of intravesical instillations may be more beneficial. Therefore, 8 instillations of EPI-ADM, representing relatively short-term treatment, were performed in this trial.

Probiotics are viable bacteria that show beneficial health effects by improving the balance of intestinal bacterial flora.¹³ The LC strain Shirota has been recognized as a typical probiotic strain.¹⁴ A case-control study indicated that routine intake of Lactobacillus beverages may prevent bladder cancer.¹⁵ Therefore, patients ingesting food, beverages or drugs containing Lactobacillus strains regularly were excluded from this study.

However, it is possible that EPI-ADM group patients began ingesting Lactobacillus products during this study, which may have resulted in underestimation of recurrence prevention by LC preparation. Regardless of this the EPI-ADM plus LC group showed significantly higher recurrence-free survival and the difference between the groups was still significant on multivariate analysis using factors found to be significant on univariate analysis.

Intravesical instillation of BCG is indicated to prevent recurrence after TUR for high risk superficial bladder cancer, although it sometimes causes severe toxicity. Since BCG was not used in this study, patients with a high risk tumor, such as a grade 3 tumor, or multiple recurrent tumors were excluded from study. However, it may be worthwhile to investigate the efficacy of LC preparation for preventing recurrence after TUR for such high risk superficial bladder cancers.

In the EPI-ADM plus LC group the risk of recurrence decreased in the early phase, especially in year 1 after TUR for superficial bladder cancer, in which the LC preparation was administered. The period of a clear suppression of the risk of tumor recurrence closely corresponded with that of the oral administration of LC preparation. Therefore, if LC preparation oral administration were continued in the late phase after TUR, further improvement in recurrence-free survival may be achieved. Neither progression-free nor overall survival differed significantly between the groups, probably due to the small number of events. However, since intravesical recurrence may require hospital treatment, improvement in recurrence-free survival may be beneficial for decreasing costs for patients who must undergo TUR to treat superficial bladder cancer.

The LC strain contained in the LC preparation used is highly tolerant of gastric acid, bile acids and other components of digestive juices, and it can multiply in the intestinal tract to normalize intestinal flora. LC prevents the production and promotes the elimination of carcinogens and mutagens by intestinal bacteria, and it prevents the excretion of mutagens in urine in humans who have consumed fried ground beef.¹⁶ Thus, LC is believed to prevent tumor development by improving intestinal flora. LC is also a biological response modifier that enhances immune system activity in humans.¹⁷ It would be of great interest to clarify the mechanisms by which the combination of intravesical instillation of EPI-ADM and oral administration of LC preparation prevents intravesical recurrence after TUR for superficial bladder cancer.

The safety of LC preparation is established, although it may cause gastrointestinal symptoms in rare cases. While constipation and diarrhea developed in patients receiving LC preparation in the current study, the incidence was low, severity was mild and compliance with LC preparation treatment was favorable. There were no differences between the groups in the incidence or severity of adverse reactions, including those associated with micturition. Although the number of patients who could not complete the protocol treatment was higher in the EPI plus LC than in the EPI group (24 vs 7), this difference was not due to adverse reactions to LC preparation. Thus, the combination of intravesical instillation of EPI-ADM plus long-term oral LC preparation appeared highly tolerable.

Our findings reveal that adding oral treatment with LC preparation with a high degree of safety may enhance the efficacy of intravesical EPI-ADM for preventing intravesical recurrence after TUR for superficial bladder cancer. We are considering a prospective, randomized trial of this treatment vs BCG to prevent recurrence after TUR for superficial bladder cancer.

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Conclusions 

Oral LC preparation enhanced the efficacy of intravesical EPI-ADM for preventing recurrence after TUR for superficial bladder cancer. To our knowledge this is the first report of the additive effect of an oral nonchemotherapy for preventing tumor recurrence by intravesical chemotherapy.

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