Efficacy of bacillus Calmette-Guérin Strains for Treatment of Nonmuscle Invasive Bladder Cancer: A Systematic Review and Network Meta-Analysis

Author's Accepted Manuscript Efficacy of Bacillus Calmette-guerin Strains for the Treatment of non-muscle Invasive Bladder Cancer: a Systematic Review and Network Meta-analysis Brock E. Boehm , John E. Cornell , Hanzhang Wang , Neelam Mukherjee , Jacob S. Oppenheimer , Robert S. Svatek

PII: DOI: Reference:

S0022-5347(17)39246-7 10.1016/j.juro.2017.01.086 JURO 14612

To appear in: The Journal of Urology Accepted Date: 5 January 2017 Please cite this article as: Boehm BE, Cornell JE, Wang H, Mukherjee N, Oppenheimer JS, Svatek RS, Efficacy of Bacillus Calmette-guerin Strains for the Treatment of non-muscle Invasive Bladder Cancer: a Systematic Review and Network Meta-analysis, The Journal of Urology® (2017), doi: 10.1016/ j.juro.2017.01.086. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.

Embargo Policy All article content is under embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date. Questions regarding embargo should be directed to [email protected].

ACCEPTED MANUSCRIPT

Efficacy of Bacillus Calmette-Guerin Strains for the Treatment of Non-Muscle Invasive Bladder Cancer: A systematic review and network meta-analysis Brock E. Boehm1, John E. Cornell2, Hanzhang Wang1, Neelam Mukherjee1, Jacob S.

RI PT

Oppenheimer, and Robert S. Svatek1* Departments of 1Urology and 2Biostatistics at the University of Texas Health Science Center San Antonio, San Antonio Texas

Running Title: Effect of Bacillus Calmette-Guerin Strains on the Treatment Efficacy of Non-

SC

Muscle Invasive Bladder Cancer

Word count No. Figures: 4 No. Tables: 2 No. Supplementary Figures: 2 No. Supplementary Tables: 2

TE D

Grant support:

M AN U

Key words: bladder cancer, Bacillus Calmette-Guerin, Non-Muscle Invasive, Immunotherapy,

1. Max and Minnie Tomerlin Voelcker Fund 2. NIH 5K23CA178204-03

*Correspondence:

EP

3. The Roger L. and Laura D. Zeller Charitable Foundation Chair in Urologic Cancer

AC C

Robert S. Svatek MD, MSCI Assistant Professor, Urology The University of Texas Health Science Center San Antonio 7703 Floyd Curl Dr. MC 7845 San Antonio, Tx 78229

Phone: (210)-567-5676 Fax: (210)-567-6868 Email: [email protected]

ACCEPTED MANUSCRIPT

Abstract Purpose: To determine the efficacy of genetically distinct Bacillus Calmette-Guérin (BCG) strains in preventing disease recurrence for patients with non-muscle invasive bladder cancer (NMIBC).

RI PT

Experimental Design: We conducted a systematic review and network meta-analysis of trials evaluating BCG strains against all possible comparators (different BCG strains, chemotherapy, and non-BCG biologic therapies) and intravesical chemotherapy as the common comparator. MEDLINE served as the primary data source, with search from inception to October 2016 for clinical trials involving patients with NMIBC receiving BCG. Primary outcome measure was

SC

bladder cancer recurrence defined as recurrent bladder tumor of any grade or stage. Randomeffects network meta-analysis provided estimates for outcomes and is presented as odds ratios

M AN U

(ORs).

Results: Across all possible comparators (n=65 trials, 12,246 patients, 9 strains) there were 2,177 (38.6%) recurrences among 5,642 treated patients compared with 2,316 (42.6%) recurrences among 5,441 comparators. With chemotherapy as the common comparator (n=28 trials, 5,757 patients, 5 strains), Tokyo 127 (0.39 OR 95% CI 0.16-0.93), Pasteur (0.49 OR 95% CI 0.28-0.86), and TICE (0.61 OR 95% CI 0.40-0.93) strains were significantly better than chemotherapy at preventing recurrence. No BCG strain demonstrated significant superiority

TE D

when compared against any other strain at preventing recurrence in our network meta-analysis. Conclusions: BCG strains exhibit significant differences in efficacy when compared against chemotherapy. However, no definitive conclusions are reached regarding strain superiority and head-to-head trials are greatly needed to further understand the importance of strain selection in

AC C

EP

determining BCG efficacy.

ACCEPTED MANUSCRIPT

Translational Relevance

The primary treatment for early stage bladder cancer is Bacillus Calmette-Guérin (BCG), a live attenuated mycobacterium that is instilled into the urinary bladder via a urethral catheter. BCG

RI PT

significantly decreases the rate of bladder cancer recurrence but response is heterogeneous and some patients are unresponsive. BCG represents a family of daughter strains that were originally derived from virulent Mycobacterium bovis. BCG strains have considerable genotypic diversity and elicit variable immune responses. It is unclear, however, if these genetic

differences influence BCG’s oncologic efficacy. Understanding the influence of BCG strains on

SC

bladder cancer recurrence is critical because manufacturing shortages have resulted in

episodes of diminished global availability of specific BCG strains. In addition, understanding the

M AN U

importance of BCG strains on oncology efficacy could elucidate mechanisms underlying responsiveness to BCG.

Introduction

Urinary bladder cancer is the fourth most common malignancy in men (1). In 2016, there will be an estimated 76,960 new cases and bladder cancer will account for 16,390 deaths in the United

TE D

States (1). The majority of patients diagnosed with bladder cancer present with non-muscle invasive bladder cancer (NMIBC) including carcinoma in-situ (CIS), Ta, and T1 stages. For these patients, the standard initial therapy is complete removal of all papillary lesions via transurethral resection and bladder preservation is possible in most cases. Due to the high

EP

rates of disease recurrence, adjuvant therapy with intravesical instillation of Bacillus CalmetteGuérin (BCG) immunotherapy is indicated for all high-grade and some low-grade non-muscle

AC C

invasive bladder tumors (2).

The term BCG comprises a family of related strains derived from a virulent strain of Mycobacterium bovis (M. bovis). Following 13 years of serial in-vitro passaging, Calmette and Guérin developed the original BCG strain, which was attenuated yet provided protection against virulent strains including Mycobacterium tuberculosis. Subsequent to its first use as a tuberculosis vaccine in 1921, BCG cultures were distributed globally for use in vaccine manufacturing. As a result of continuous passages under different conditions in various laboratories throughout the world, BCG strains began to diverge genetically, until the introduction of freeze-dried seed lots in the 1960’s. As a result, existing BCG vaccine strains

ACCEPTED MANUSCRIPT

have extensive genotypic diversity, including deletions and duplications, which can be linked to their dissemination patterns (3). In some studies, genetically distinct strains have been associated with differences in elicited immune responses including reactogenicity and immunogenicity (4); however, it is not known if such changes influence BCG’s efficacy in the

RI PT

treatment of bladder cancer.

Studies comparing BCG strains in head-to-head trials suggest that strains can influence clinical outcomes (5-9). Unfortunately, however, these trials have been relatively small, lacking

statistical power to be able to reliably assess any effect related to strain differences. Therefore,

SC

we initiated a systematic review and network meta-analysis to determine if differences in BCG strains are associated with response to BCG in bladder cancer. A network meta-analysis

M AN U

integrates data from direct comparisons of treatments within trials with indirect comparisons of interventions assessed against a common comparator in different trials, to compare all investigated interventions.

Materials and Methods

TE D

Evidence Acquisition

We conducted a systematic review of Medline (http://www.ncbi.nlm.nih.gov/pubmed), from 1966 to October 2016 in adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (10, 11) using the following search criteria:

EP

1. Medical Subject Headings (MeSH) terms: (“Urinary Bladder Neoplasms”[MeSH]) AND (“BCG Vaccine”[MeSH] OR “Mycobacterium bovis”[MeSH] OR “BCG Connaught” [Supplementary Concept])

AC C

2. Ovid text work terms: bladder cancer or BCG or TICE or Connaught 3. Linkage: 1 and 2, limited to human adults and publications in English

Selection Criteria

All publications involving patients with carcinoma in-situ (CIS), Ta or T1 stage bladder cancer that compared transurethral resection plus intravesical BCG (any strain) to either transurethral resection alone, transurethral resection plus intravesical chemotherapy, transurethral resection plus intravesical BCG (either different strain or different dose), transurethral resection plus a

ACCEPTED MANUSCRIPT

non-BCG biologic (defined as either: Interferon-α2b (INTRON® A), Bropirimine, autologous intravesical macrophage cell therapy (BEXIDEM®), or Maltose Tetrapalmitate), or transurethral resection plus a combination of intravesical treatments were reviewed. Patients included in this analysis were BCG naïve. Selection of studies to include in the network meta-analysis was

RI PT

based on several criteria: the number of studies available for a given comparator, similarity with respect to patient populations and study characteristics (transitivity), and the availability of headto-head comparisons. For final analysis, only the subset of trials with chemotherapy as the common comparator met these criteria.

SC

Data collection

M AN U

Literature search and data extraction from each publication was conducted independently by one of three investigators (BEB, HW, and RSS) and included: sample size, number of patients per treatment group, exposure and outcome definition, study location, type of control, study design, strain type and dose, treatment schedule and summary of results. Any discrepancies were resolved by consensus in meeting with the presence of the moderator (JEC). Exclusion criteria were studies with fewer than 10 patients, case reports, non-randomized studies, or those studies not reporting any data on recurrence. Data from cohorts represented in more than one

TE D

publication were only reported once, with the most up-to-date information represented. Maintenance treatment with intravesical BCG was defined as any additional treatment(s) following an initial induction treatment. Standard doses for all BCG strains are listed in Table 1.

EP

Any dose less than the standard dose was designated as “low-dose”.

Risk of Bias Assessment and Reporting

AC C

All of the extracted data was evaluated for internal consistency and contrasted with the trial’s protocol and all published reports. Data was extracted independently by two authors (BEB and JSO). Data was checked for validity, missing values, and completeness across all variables according to the criteria in the Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 (12). The method of randomization and concealment was also assessed according to the Cochrane Handbook. All disagreements were resolved upon discussion with the moderator (RSS). The result of this meta-analysis was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (11).

ACCEPTED MANUSCRIPT

Statistical Evaluation

The primary study endpoint was disease recurrence, rather than time to recurrence, defined as pathologically confirmed low- and/or high-grade bladder cancer at any time following BCG

RI PT

treatment. We did not interpret time to recurrence as the OR fails to account for heterogeneity in exposure times among patients randomized to each study arm. It essentially ignores

censoring and assumes that the OR remains constant over time, an assumption that may or may not be true and is essentially untestable. Our initial approach to evaluate the comparative effectiveness among all unique BCG strains (n=10) was to perform a series of standard pairwise

SC

random-effect meta-analyses stratified by comparator and strain within comparator (13). The odds ratio (OR) was used to measure the relative effectiveness of the BCG strains in reducing

M AN U

recurrence. We grouped the meta-analyses by comparison type: BCG vs. chemotherapy, BCG + surgery vs. surgery only, BCG induction + maintenance vs. BCG induction only, BCG standard dose vs. BCG low dose. We used the standard 0.5 zero-cell continuity correction to account for zero-event arms. The standard ߯ ଶ test for heterogeneity and the ‫ ܫ‬ଶ index were used to assess statistical heterogeneity in treatment effects across studies. A significance level < 0.15 and/or ‫ ܫ‬ଶ > 50% were used to determine the presence of statistical heterogeneity (14). We performed a frequentist random-effects network meta-analysis on a subset of 28 trials with

TE D

chemotherapy as the common comparator (15-17). It is the only subset of sufficient size that includes head-to-head direct comparisons, as well as indirect comparisons, to assess the comparative effectiveness among 5 BCG strains (Tokyo 127, Pasteur, TICE, Connaught, RIVM). Two BCG strains (Glaxo, Armand-Frappier), each represented by a single trial, were

EP

excluded from these analyses: (1) A fair-quality trial comparing the Glaxo BCG strain with the Pasteur BCG strain(6) (no chemotherapy comparator) and (2) A very small fair-quality trial comparing Armand-Frappier BCG strain against chemotherapy(18). The 28 included trials are

AC C

sufficiently similar with respect to patient populations and comparators to assure sufficient transitivity (similarity) across trials to meaningfully combine direct and indirect effect estimates of the relative effectiveness among the 5 BCG strains. Network meta-analysis also assumes consistency in effect estimates derived from direct head-to-head comparisons among the BCG strains and indirect comparisons derived based on the contrast between each BCG strain and chemotherapy. To assess inconsistency between direct and indirect comparison, the method described by Lu and Andes (2006) (19) was used.

ACCEPTED MANUSCRIPT

Results from our network meta-analysis are summarized graphically and in league tables. Primary assessment of comparative effectiveness among the 5 BCG strains is expressed as odds ratios with 95% confidence intervals. Relative rankings among the 5 BCG strains were obtained by computing the Surface Under the Cumulative Ranking Curve (SUCRA) (20).

RI PT

SUCRA scores vary between 0 and 1, and they track with the mean rank for each strain based on the probability that a particular strain is ranked best among the comparator strains (rank = 1), second best (rank = 2), and so forth. The higher the SUCRA score the higher the overall mean rank for a particular BCG strain. There is a great deal of uncertainty associated with these

computed form the network of trials (10, 21).

M AN U

Results

SC

relative rankings, and they must be interpreted within the context of the actual effect estimates

Clinical Trials and Patient Characteristics

The PRISMA flowchart describing the search process is shown in Figure 1. Initially, our search yielded 262 potentially relevant publications. The titles of all 262 results were reviewed, and if the publication could not be eliminated based on title alone, the abstract was subsequently

TE D

reviewed. The remaining 132 manuscripts were reviewed in full to determine eligibility. Finally, 65 studies contained data in accordance with the eligibility criteria. From these 65 studies, 12,246 participants were available for meta-analysis. For the 7,326 patients for which gender was identified, 6,076 (82.9%) and 1,250 (17.1%) were male and female, respectively.

EP

Regarding the 8,554 patients for which tumor stage was provided, 3,973 (46.4%), 3,479 (40.7%), and 1,102 (12.9%) were identified as Ta, T1, and CIS, respectively.

AC C

A final meta-analysis was performed with chemotherapy as the common comparator, with 28 studies included in this analysis. There were 1195 recurrent events out of n=3311 patients randomized to the BCG arm, and there were 1160 recurrent events out of n=2677 patients randomized to the chemotherapy arm. This yeilds a total of 2355 recurrent events out of n=5988 patients enrolled in these 28 studies.

Trial Characteristics

ACCEPTED MANUSCRIPT

Characteristics of trials are detailed in Supplemental Table 1. The trial publication years ranged from 1978 to 2016. The trials did not consistently list follow-up as mean or median, and as such, an average was computed for the mean (24 months) and median (51.7 months). A wide range of chemotherapy and immunotherapy control groups were identified (the number in

RI PT

parentheses denotes number of studies in which each agent was utilized): Mitomycin C (17), Epirubicin (10), Interferon-α2b (3), Doxorubicin (3), Gemcitabine (3), Thiotepa (2), Maltose Tetrapalmitate (1), BEXIDEM® (1), Bropirimine (1), and Ofloxacin (1). In all, a total of 10

different strains of BCG were represented: Connaught (19), Pasteur (17), TICE (16), Tokyo 127 (5), RIVM (4), Danish 1331 (3), Armand-Frappier (2), Moreau (1), Glaxo (1) and Evans (1).

SC

Lastly, a total of 21 studies utilized doses of BCG strains that differed from the standard dose (

M AN U

Pasteur (11), Connaught (7), Danish 1331 (2), Tokyo 127 (1)).

Risk of Bias

Risk of bias in contributing to the primary outcome of recurrence following BCG therapy was generally moderate (Figure 2). A large portion of the studies demonstrate an unclear risk of selection, performance, and detection bias (Supplemental Table 2). The vast majority of publications do not explicitly state any information concerning blinding, allocation concealment,

TE D

and randomization. Nevertheless, > 85% of the studies were considered “low risk” of bias when evaluating attrition and reporting bias. In addition, no major inclination was noted for small

Recurrence

EP

studies to over- or under-estimate treatment effects.

BCG significantly reduced recurrence compared to chemotherapy (OR = 0.68 95% CI 0.54-

AC C

0.85, ‫ ܫ‬ଶ = 72.6%) and surgery (OR = 0.48 95% CI 0.28-0.80, ‫ ܫ‬ଶ = 46.3%). Pairwise metaanalysis of BCG versus chemotherapy broken down by BCG strain demonstrated Pasteur (OR = 0.51, 95% CI 0.32-0.80, ‫ ܫ‬ଶ = 45.8%) and TICE (OR = 0.66, 95% CI 0.45-0.98, ‫ ܫ‬ଶ = 73.2%) strains as demonstrating significant decreases in recurrence (Figure 3). TICE strain was associated with an increased risk of recurrence compared to Connaught strain (OR = 1.93, 95% CI 1.19-3.11) based on one randomized trial. RIVM strain was associated with an increased risk of recurrence compared to TICE strain (OR = 1.29, 95% CI 1.01-1.64) based upon one randomized trial.

ACCEPTED MANUSCRIPT

Networks considering the impact of BCG on disease recurrence with eligible comparisons considering all possible comparators are shown in Figure 4a, demonstrating predominately indirect comparisons of BCG against chemotherapy and limited number of direct, head-to-head, comparisons among BCG strains. Networks considering chemotherapy as the common

RI PT

comparator is shown in Figure 4b. In this final network meta-analysis, Tokyo 127 (OR = 0.39, 95% CI 0.16 - 0.93), Pasteur (OR = 0.50, 95% CI 0.28 - 0.86), and TICE (OR = 0.61, 95% CI 0.40 - 0.93) were associated with a decreased risk of recurrence compared to chemotherapy alone (Table 2). Tokyo strain was associated with a non-significant decrease in disease

recurrence compared to all BCG strains while Connaught strain was associated with a non-

SC

significant increase in disease recurrence compared to Pasteur, TICE, and Tokyo 127 BCG strains. SUCRA identified Tokyo 127 strain as having the highest probability for superiority;

M AN U

followed by Pasteur, TICE, Connaught and RIVM (Supplemental Figures 1and 2).

Discussion

In the U.S., there are three BCG strains that are FDA approved for use in patients with NMIBC, including Armond-Frappier, Connaught, and TICE strains. However, TICE stain is the only BCG strain currently marketed in the U.S. and the manufacturer of Connaught recently announced

TE D

that it would cease production of BCG indefintiely. Ongoing manufacturing problems are a result of challenges in sustaining lots of BCG (22). Resulting global shortages and BCG crises highlight the importance of understanding strain differences in determining treatment response since alternative strains are available. While it has been demonstrated that these genetic

EP

differences between BCG vaccine strains can influence phenotypic properties such as immunogenicity and reactogenicity (3), it is unclear if such strain differences influence the

AC C

clinical efficacy of BCG in treating bladder cancer.

This study provides evidence of marked efficacy in preventing disease recurrence across BCG strains using both direct and indirect comparisons from network metanalysis. These findings are important as regulatory agencies including the US Food and Drug Administration (FDA) grapple with handling of global BCG shortages due to manufacturing problems. In recent years, the FDA has not approved pleas for allowance of non-FDA approved BCG strains even in times of national BCG shortages due to lack of data regarding the oncologic efficacy of various daughter BCG strains. Importantly, we identified no evidence to support a statistically signficant difference in clinical efficacy across BCG strains.

ACCEPTED MANUSCRIPT

The aforementioned genetic differences among the various strains can be traced back to the early 20th century, 1921, when Albert Calmette (physician) and Camille Guérin (veterinarian) from the Pasteur Institute in Paris, France, cultivated an oral vaccine against tuberculosis

RI PT

through numerous serial passages of Mycobacterium bovis on potatoes enriched with glycerol (3). M. bovis was chosen as it is considered attenuated with respect to Mycobacterium

tuberculosis due to loss of RD4 – RD11, the region of difference gene groups. This deletion leads to M. bovis, and all subsequently derived BCG strains, demonstrating resistance to

pyrazinamide antibiotics. One further deletion, RD1, remains the essential difference between

SC

BCG and its parent, M. bovis, responsible for encoding the protein secretion system ESX-1. This original vaccine was distributed worldwide later that year, 1921, and continued to undergo

M AN U

serial passages using the method perfected by Calmette and Guérin, leading to several additional mutations that created the vast assortment of BCG strains. This process continued until the early 1960’s with the introduction of freeze-dryed seed lots.

Despite these demonstrated genetic differences among BCG strains, very few trials have been conducted to compare the efficacy of different BCG strains. We identified 5 randomized headto-head trials comparing different BCG strains (5-9). Four of these trials did not involve

TE D

maintenance BCG therapy (5-9). Because maintenance BCG provides superior recurrence-free survival benefit compared to BCG therapy without maintenance (23), results from these four trials may not be applicable to practices where maintenance BCG is standard of care. As an example, a provocative study found 5-year recurrence-free survival of patients treated with BCG

EP

Connaught (74.0%) significantly improved compared to those treated with BCG TICE (48.0%) (7). However, patients in this study were not routinely treated with maintenance BCG as is standard of care in the U.S. Interestingly, the effect of specific BCG strains could be modified

AC C

by the use of maintenance therapy. Outcomes across strains were recently reported in a large observational cohort of 2099 patients with primary T1G3 bladder cancer treated with BCG TICE (N=599) and Connaught (n=1546)(24). When no maintenance was given, Connaught was more effective than TICE for time to first recurrence (HR=1.34, P=0.004). However, when maintenance was given, TICE was more effective than Connaught for time to first recurrence (HR=0.64, P=0.01). In accordance with these findings, our analysis suggests that Connaught strain is not superior to any other BCG strain. In fact, based upon SUCRA, Tokyo 127 was more than twice as likely to be the best treatment for NMIBC compared to other BCG strains while Connaught ranked lower than Pasteur and TICE. Thus, studies evaluating differences in

ACCEPTED MANUSCRIPT

BCG strains should consider standard of care treatment patterns including the use of maintenance which has consistently demonstated superiority over induction alone (23, 25). As previously mentioned, Tokyo, Pasteur, and TICE appear to rank 1st, 2nd, and 3rd overall

RI PT

based on SUCRA values; but the lack of evidence that one is clearly superior, coupled with the wide confidence intervals for the odds ratios suggests prudence in attempting to draw definitive conclusions based on the existing evidence. The absence of sufficient numbers of head-tohead trials among these BCG strains also supports our conclusions that the evidence is

insufficient to make definitive statements or recommendations about the relative superiority of

SC

one BCG strain versus another.

M AN U

Results from other head-to-head trials could be limited in ability to detect differences in efficacy due to insufficient sample size. . A randomized controlled trial was initiated to compare Tokyo 127 versus Connaught, but was closed before complete accrual (8). Outcomes at a median follow-up of 2.4 years were reported on 129 patients including 66 treated with Tokyo and 63 treated with Connaught BCG strains. The study was powered to detect a recurrence free survival difference of 0.2 (alpha 0.05, Beta = 0.2) with a sample size of 96 patients per arm. The complete response rate was 90.3% and 85.0% in patients given the Tokyo and Connaught

TE D

strains, respectively, which did not differ significantly (p = 0.896). The 2-year recurrence-free survival rate was 73.2% and 68.8%, respectively.

Our analysis is subject to potential limitations. This analysis does not correct for important

EP

confounders such as interinstituitional bias, schedule of maintenance (3 weeks versus 1 week) and repeat transurethral resection for high-grade T1 which has now become standard practice. Because of non-standardized and scant reporting effects of BCG strains on adverse events we

AC C

were unable to evaluate the tolerability of BCG strains and the extent to which this could influence ability to give complete doses. Further, treatment toxicity is an important component to be considered for BCG immunotherapy, however, a comparative-effectiveness analysis is outside the scope of this meta-analysis.Complete response to CIS is a more definitive outcome parameter to evaluate but our results are based on recurrence for both CIS and papillary lesions because there were too few studies to parcel out CIS patients from which to draw meaningful conclusiosns. Many trials were included despite not utilizing routine maintenance BCG and as discussed this could influence the overall interpretations of oncologic efficacy between strains. The risk of bias was unclear in many of the studies concerning selection bias due to under-

ACCEPTED MANUSCRIPT

reporting of blinding and concealment procedures. Because many published reports provided only the number of patients experiencing disease recurrence, the odds of recurrence was determined without considering censoring or the time to event. This analysis considered the effect of BCG strains but did not consider the effect of combination therapy. This could be

RI PT

addressed in future work as the efficacy of BCG may be improved through use of additional agents(26). Conclusion

SC

The specific strain of BCG could influence bladder cancer recurrence rates. Tokyo 127, Pasteur, and TICE BCG strains all demonstrated a significant decrease in recurrence when compared

M AN U

with chemotherapy. However, no strain was significantly superior to another BCG strain during network analysis that incorporates direct and indirect comparisons. Tokyo 172 strain was identified as having a high probability for superiority and should be compared to more commonly

Figure Legend

TE D

used TICE and Connaught BCG strains.

Figure 1. Flowchart Detailing Inclusion of Trials

EP

Figure 2. Risk of Bias Graph

Figure 3. Pairwise Meta-Analysis Comparing BCG Strains Against Chemotherapy: OR (95% CI)

AC C

Figure 4. Network maps. Network maps were created for bladder cancer Recurrence Based Upon (a) All Possible Comparators and (b) chemotherapy as common comparator

ACCEPTED MANUSCRIPT

References:

AC C

EP

TE D

M AN U

SC

RI PT

1. Siegel RL, Miller KD and Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016; 66: 7-30. 2. Hall MC, Chang SS, Dalbagni G et al: Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, T1, and Tis): 2007 update. J Urol 2007; 178: 2314. 3. Gan C, Mostafid H, Khan MS, Lewis DJ. BCG immunotherapy for bladder cancer—the effects of substrain differences. Nat Rev Urol. 2013; 10: 580-8. 4. Ritz N, Hanekom WA, Robins-Browne R, Britton WJ, Curtis N. Influence of BCG vaccine strain on the immune response and protection against tuberculosis. FEMS Microbiol Rev. 2008; 32: 821-41. 5. Fellows GJ, Parmar MK, Grigor KM, Hall RR, Heal MR, Wallace DM. Marker tumour response to Evans and Pasteur bacille Calmette-Guérin in multiple recurrent pTa/pT1 bladder tumours: report from the Medical Research Council Subgroup on Superficial Bladder Cancer (Urological Cancer Working Party). Br J Urol 1994; 73: 639-44. 6. Mukherjee A, Persad R, Smith PJ. Intravesical BCG treatment for superficial bladder cancer: longterm results using two different strains of BCG. Br J Urol. 1992; 69: 147-50. 7. Rentsch CA, Birkhäuser FD, Biot C, Gsponer JR, Bisiaux A, Wetterauer C, et al. Bacillus CalmetteGuérin strain differences have an impact on clinical outcome in bladder cancer immunotherapy. Eur Urol 2014; 66: 677-88. 8. Sengiku A, Ito M, Miyazaki Y et al: A prospective comparative study of intravesical bacillus Calmette-Guérin therapy with the Tokyo or Connaught strain for nonmuscle invasive bladder cancer. J Urol 2013; 190: 50. 9. Witjes WP, Witjes JA, Oosterhof GO, Debruyne MJ. Update on the Dutch Cooperative Trial: mitomycin versus bacillus Calmette-Guérin-Tice versus bacillus Calmette- Guérin RIVM in the treatment of patients with pTA-pT1 papillary carcinoma and carcinoma in situ of the urinary bladder. Dutch South East Cooperative Urological Group. Semin Urol Oncol, suppl., 1996; 14: 10-6. 10. Hutton B, Salanti G, Caldwell DM, Chaimani A, Schmid CH, Cameron C, et al. The PRISMA extension statement for reporting of systematic reviews incorporating network meta-analyses of health care interventions: checklist and explanations. Ann Intern Med 2015; 162: 777-84. 11. Savitski MM, Mathieson T, Becher I, Bantscheff M. H-score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples. J Proteome Res. 2010; 9: 5511-6. 12. Higgins JPT and Green S: Cochrane Handbook for Systematic Reviews of Interventions, Version 5.1.0. London: The Cochrane Collaboration 2011. Available at www.handbook.cochrane.org. 13. DerSimonian R and Laird N: Meta-analysis in clinical trials. Control Clin Trials 1986; 7: 177-88. 14. Higgins JP and Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med 2002; 21: 1539-58. 15. Salanti G. Indirect and mixed-treatment comparison, network, or multiple-treatments metaanalysis: many names, many benefits, many concerns for the next generation evidence synthesis tool. Res Synth Methods 2012; 3: 80-97. 16. Chaimani A, Higgins JP, Mavridis D, Spyridonos P, Salanti G. Graphical tools for network metaanalysis in STATA. PLoS One 2013; 8: e76654. 17. Dias S, Welton NJ, Sutton AJ et al: NICE DSU Technical Support Document 2: A Generalised Linear Modelling Framework for Pairwise and Network Meta-Analysis of Randomised Controlled Trials. Updated September 2016. Sheffield, United Kingdom: National Institute for Health and Clinical Excellence (NICE) Decision Support Unit (DSU) 2011. Available at http: //www.nicedsu.org.uk. 18. Cookson MS, Herr HW, Zhang ZF, Soloway S, Sogani PC, Fair WR. The treated natural history of high risk superficial bladder cancer: 15-year outcome. J Urol. 1997; 158: 62-7.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

19. Lu G and Ades A: Assessing evidence inconsistency in mixed treatment comparisons. J Am Stat Assoc 2006; 101: 447-59. 20. Salanti G, Ades AE and Ioannidis JP: Graphical methods and numerical summaries for presenting results from multiple-treatment meta-analysis: an overview and tutorial. J Clin Epidemiol 2011; 64: 16371. 21. Trinquart L, Attiche N, Bafeta A, Porcher R, Ravaud P. Uncertainty in Treatment Rankings: Reanalysis of Network Meta-analyses of Randomized Trials. Ann Intern Med 2016; 164: 666-73. 22. Mostafid AH, Palou Redorta J, Sylvester R, Witjes JA. Therapeutic options in high-risk nonmuscle-invasive bladder cancer during the current worldwide shortage of bacille Calmette-Guérin. Eur Urol 2015; 67: 359-60. 23. Lamm DL, Blumenstein BA, Crissman JD, Montie JE, Gottesman JE, Lowe BA, et al. 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. The Journal of urology. 2000; 163: 1124-9. 24. Witjes JA, Dalbagni G, Karnes RJ, Shariat S, Joniau S, Palou J, et al: The efficacy of BCG TICE and BCG Connaught in a cohort of 2,099 patients with T1G3 non-muscle-invasive bladder cancer. Urol Oncol 2016; 34: 484.e19. 25. Oddens J, Brausi M, Sylvester R, Bono A, van de Beek C, van Andel G, et al. Final results of an EORTC-GU cancers group randomized study of maintenance bacillus Calmette-Guérin in intermediateand high-risk Ta, T1 papillary carcinoma of the urinary bladder: one-third dose versus full dose and 1 year versus 3 years of maintenance. Eur Urol 2013; 63: 462-72. 26. Svatek RS, Zhao XR, Morales EE, Jha MK, Tseng TY, Hugen CM, et al. Sequential intravesical mitomycin plus Bacillus Calmette-Guérin for non-muscle-invasive urothelial bladder carcinoma: translational and phase I clinical trial. Clin Cancer Res 2015; 21: 303-11.

ACCEPTED MANUSCRIPT

Table 1. Standard Dose of BCG Daughter Strains

Standard Dose

Armand Frappier Connaught Danish 1331 Evans

120 mg (milligrams)

AC C

EP

SC

M AN U

TE D

Glaxo Moreau Pasteur RIVM TICE Tokyo

81 mg 120 mg 1-5 X 109 CFU (Colony Forming Units) 1-3 X 109 CFU 80 mg 150 mg 2 X 108 CFU 50 mg 80 mg

RI PT

BCG Strain

ACCEPTED MANUSCRIPT

TOKYO

TICE

TOKYO

2.59 (1.07,6.22)

-

-

TICE

1.64 (1.08,2.50)

0.63 (0.24,1.66)

-

RIVM

1.02 (0.54,1.94)

0.40 (0.13,1.17)

PASTEUR

2.02 (1.16,3.51)

0.78 (0.28,2.20)

CONNAUGHT

1.38 (0.91,2.10)

0.54 (0.22,1.31)

PASTEUR -

-

-

0.62 (0.31,1.28)

-

-

1.23 (0.62,2.46)

1.97 (0.85,4.58)

-

0.84 (0.48,1.48)

1.35 (0.63,2.88)

0.68 (0.73, 1.37)

M AN U

-

TE D EP

RIVM

SC

CHEMOTHERAPY

AC C

TREATMENT

RI PT

Table 2. League Table of Network Meta-Analysis Comparing BCG Strains: OR (95% CI)

ACCEPTED MANUSCRIPT

Figure 1

RI PT

Initial Search = 262 potential publications

M AN U

Manuscripts reviewed in full = 132

SC

Eliminated based on title or abstract = 130

Non-randomized = 7 Duplicate reports = 56

EP

Figure 2

TE D

Trials included in meta-analysis = 65

Risk of Bias

AC C

Random Sequence Generation (Selection Bias)

Allocation Concealment (Selection Bias)

Low Risk of Bias

Blinding of Participants, Personnel, and Outcome…

Unclear Risk of Bias High Risk of Bias

Incomplete Outcome Data (Attrition Bias)

Selective Outcome Reporting (Reporting Bias)

0% 20% 40% 60% 80%100%

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Figure 3.

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

Figure 4a

AC C

EP

TE D

Figure 4b

ACCEPTED MANUSCRIPT

Supplemental Figure 1. SUCRA (surface under the cumulative ranking curve) Utilizing Chemotherapy as Common Comparator

AC C

EP

TE D

M AN U

SC

RI PT

Different BCG strains are represented as different columns. On the vertical axis is the possible rank of each treatment from best to worst in preventing recurrence. Top number in each entry represents the probability that a treatment is ranked best, 2nd, 3rd, 4th, 5th, or worst among the six treatment options. Bottom number represents the standard error (SE) of this estimate. The bottom two rows report the mean rank (SE) for each treatment, and the bottom row gives the Surface Under the Cumulative Ranking Curve (SUCRA). SUCRA is a function of the mean rank, standardized to range from 0 to 1. The higher the SUCRA the greater the likelihood that a BCG strain is ranked the best among the 6 strains included in our network meta-analysis.

ACCEPTED MANUSCRIPT

Supplemental Figure 2. Network Rank-O-Gram for Recurrence With Chemotherapy as Common Comparator

AC C

EP

TE D

M AN U

SC

RI PT

The horizontal axis depicts the spectrum of possible ranks (best to worst) and the vertical axis shows the corresponding cumulative probability of a treatment over each rank. Six different rankings are included and represented by small vertical bars along the curves.

ACCEPTED MANUSCRIPT

Supplemental Table 1 Trial Characteristics 65

RI PT

1978 2016

51.7 months 24 months 24 3 38

AC C

EP

TE D

M AN U

SC

No. trials Publication date: Oldest Most recent Followup duration: Median Max Disease type: Ta or T1 Ca in situ Ta or T1 + Ca in situ Treatment comparisons: BCG vs. TURBT alone BCG vs. MMC + BCG BCG vs. MMC BCG vs. MMC vs. MMC BCG vs. MMC vs. TURBT BCG vs. BCG vs. MMC (same strain, different dose) BCG vs. BCG vs. MMC (different strains) BCG vs. MMC + Doxorubicin BCG + MMC vs. MMC BCG + MMC vs. MMC + BCG + INFα BCG vs. Epirubicin BCG vs. Epirubicin vs. TURBT BCG vs. BCG + Epirubicin BCG vs. Epirubicin + INFα BCG maintenance vs. BCG non-maintenance vs. Epirubicin BCG vs. BCG + Isoniazid vs. Epirubicin BCG vs. INFα BCG vs. Gemcitabine BCG vs. Doxorubicin BCG vs. Doxorubicin vs. Thiotepa BCG vs. Thiotepa BCG vs. Bropirimine BCG vs. Maltose Tetrapalmitate vs. TURBT BCG vs. BEXIDEM BCG vs. BCG + Ofloxacin BCG vs. BCG vs. BCG (different doses) BCG (maintenance) vs. BCG (no maintenance) BCG vs. BCG (different strains) BCG vs. BCG (12 weeks vs. 6 weeks) BCG vs. BCG (different doses) BCG (intravesical + percutaneous)(maintenance) vs. BCG (intravesical + percutaneous)(no maintenance)

2 (1, 2) 3 (3-5) 5 (6-10) 1 (11) 1 (12) 1 (13) 1 (14) 1 (15) 3 (16-18) 1 (19) 4 (20-23) 1 (24) 3 (25-27) 1 (28) 1 (29) 1 (30) 1 (31) 3 (32-34) 2 (35, 36) 1 (37) 1 (38) 1 (39) 1 (40) 1 (41) 1 (42) 1 (43) 5 (44-48) 4 (49-52) 1 (53) 4 (54-57) 1 (58)

ACCEPTED MANUSCRIPT

16 (1, 2, 10, 11, 14, 15, 18, 26, 35, 49, 51-54, 57, 63)

? ? ?

Blinding of Participants, Personnel, and Outcome Assessors (Performance & Detection Bias) ? ? ?

? ? ?

Selective Outcome Reporting (Reporting Bias)

M AN U Allocation Concealment (Selection Bias)

AC C

Random Sequence Generation (Selection Bias)

EP

Risk of Bias

19 16 17 5 4 3 2 1 1 1

Incomplete Outcome Data (Attrition Bias)

Supplemental Table 2

7 (29, 44-48, 58)

TE D

Maintenance vs. no maintenance BCG strain: Connaught TICE Pasteur Tokyo 127 RIVM Danish 1331 Armand-Frappier Moreau Glaxo Evans

Agrawal 2007 Akaza 1994 Ali-El-Dein 1999

42 (3-9, 12, 13, 16, 17, 19-25, 27, 28, 30-34, 36-43, 50, 55, 56, 59-62, 64, 65)

SC

No maintenance

3 (59-61) 1 (62) 1 (63) 1 (64) 1 (65)

RI PT

BCG (intravesical + percutaneous) vs. BCG (intravesical) BCG (intravesical + percutaneous) vs. BCG (oral) BCG (intravesical + dermal scarification) vs. BCG (intravesical) BCG (intravesical) vs. BCG (oral) vs. TURBT BCG (3 year maintenance) vs. BCG (3 year maintenance) vs. BCG (1 year maintenance) vs. BCG ( 1 year maintenance) (different doses) BCG maintenance: Maintenance

? + +

+ + +

ACCEPTED MANUSCRIPT

+ + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + +

RI PT

SC

? ? ? + ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

M AN U

+ ? ? ? + ? ? ? ? + ? ? ? ? + + + + + ? ? ? ?

TE D

+ ? ? ? + ? ? ? ? + + ? + + + + ? ? ? ? -

? ? ? ? ? ? ?

? ? ? ? ? ? -

+ + + + + +

+ + + + + + +

? ? ? ? +

+ ? ? ? ? ?

? ? ? ? ? ?

+ + + + +

? + + + + +

-

?

?

+

+

-

? ?

? ?

+ +

+ +

-

?

?

+

+

EP

? ? ? + ? + +

AC C

Badalament 1987 Bilen 2000 Brosman 1982 Burger 2010 Cai 2008 Cheng 2005 Colombel 2006 Cookson 1997 De Reijke 2005 Di Lorenzo 2010 Di Stasi 2006 Fellows 1994 Friedrich 2007 Gardmark 2007 Gontero 2013 Gruenwald 1997 Hemdan 2014 Hinotsu 2010 Hinotsu 2006 Hudson 1987 Ibrahiem 1988 Jarvinen 2012 Jarvenin 2009 Jiminez-Cruz 1997 Kaasinen 2003 Kaasinen 2000 Koga 2010 Krege 1996 Kumar 2002 Lamm 2000 Lamm 1991 (NEJM) Lamm 1991 (JU) Lamm 1995 Lamm 1990 Lamm 1985 Luftenegger 1996 Marinez-Pineiro 2002 Marinez-Pineiro 1990 Melekos 1996 (O) Melekos 1996 (IUN) Melekos 1993

ACCEPTED MANUSCRIPT

References:

+ + + + + + + + + + + ? + + + + + + + ? +

RI PT

? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

SC

? + ? ? ? ? ? ? ? ? ? ? ? + ? ? ? ? ? ? ?

M AN U

? ? ? ? ? ? ? ? + ? ? ? ? + ? ? ? ? + ? ?

TE D

Mukherjee 1992 Netto Junior 1991 Oddens 2013 Ojea 2007 Oosterlinck 2011 Pagano 1991 Pagano 1995 Palou 2001 Porena 2010 Rentsch 2014 Rintala 1991 Rintala 1996 Sekine 2001 Sengiku 2013 Sylvester 2010 Witjes 1999 Witjes 1998 (JU) Witjes 1998 (AU) Witjes 1993 Witjes 1996 Yalcinkaya 1998

+ ? + + + + + + + + + + + + + + + + + + ?

AC C

EP

1. Cookson MS, Herr HW, Zhang ZF, Soloway S, Sogani PC, Fair WR. The treated natural history of high risk superficial bladder cancer: 15-year outcome. The Journal of urology. 1997;158:62-7. 2. Pagano F, Bassi P, Milani C, Meneghini A, Maruzzi D, Garbeglio A. A low dose bacillus CalmetteGuerin regimen in superficial bladder cancer therapy: is it effective? The Journal of urology. 1991;146:32-5. 3. Di Stasi SM, Giannantoni A, Giurioli A, Valenti M, Zampa G, Storti L, et al. Sequential BCG and electromotive mitomycin versus BCG alone for high-risk superficial bladder cancer: a randomised controlled trial. The Lancet Oncology. 2006;7:43-51. 4. Kaasinen E, Wijkstrom H, Malmstrom PU, Hellsten S, Duchek M, Mestad O, et al. Alternating mitomycin C and BCG instillations versus BCG alone in treatment of carcinoma in situ of the urinary bladder: a nordic study. European urology. 2003;43:637-45. 5. Oosterlinck W, Kirkali Z, Sylvester R, da Silva FC, Busch C, Algaba F, et al. Sequential intravesical chemoimmunotherapy with mitomycin C and bacillus Calmette-Guerin and with bacillus CalmetteGuerin alone in patients with carcinoma in situ of the urinary bladder: results of an EORTC genitourinary group randomized phase 2 trial (30993). European urology. 2011;59:438-46.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

6. Gardmark T, Jahnson S, Wahlquist R, Wijkstrom H, Malmstrom PU. Analysis of progression and survival after 10 years of a randomized prospective study comparing mitomycin-C and bacillus CalmetteGuerin in patients with high-risk bladder cancer. BJU international. 2007;99:817-20. 7. Jarvinen R, Kaasinen E, Sankila A, Rintala E, FinnBladder G. Long-term efficacy of maintenance bacillus Calmette-Guerin versus maintenance mitomycin C instillation therapy in frequently recurrent TaT1 tumours without carcinoma in situ: a subgroup analysis of the prospective, randomised FinnBladder I study with a 20-year follow-up. European urology. 2009;56:260-5. 8. Lamm DL, Blumenstein BA, David Crawford E, Crissman JD, Lowe BA, Smith JA, Jr., et al. Randomized intergroup comparison of bacillus calmette-guerin immunotherapy and mitomycin C chemotherapy prophylaxis in superficial transitional cell carcinoma of the bladder a southwest oncology group study. Urologic oncology. 1995;1:119-26. 9. Rintala E, Jauhiainen K, Alfthan O, Hansson E, Juusela H, Kanerva K, et al. Intravesical chemotherapy (mitomycin C) versus immunotherapy (bacillus Calmette-Guerin) in superficial bladder cancer. European urology. 1991;20:19-25. 10. Witjes JA, v d Meijden AP, Collette L, Sylvester R, Debruyne FM, van Aubel A, et al. Long-term follow-up of an EORTC randomized prospective trial comparing intravesical bacille Calmette-GuerinRIVM and mitomycin C in superficial bladder cancer. EORTC GU Group and the Dutch South East Cooperative Urological Group. European Organisation for Research and Treatment of Cancer GenitoUrinary Tract Cancer Collaborative Group. Urology. 1998;52:403-10. 11. Friedrich MG, Pichlmeier U, Schwaibold H, Conrad S, Huland H. Long-term intravesical adjuvant chemotherapy further reduces recurrence rate compared with short-term intravesical chemotherapy and short-term therapy with Bacillus Calmette-Guerin (BCG) in patients with non-muscle-invasive bladder carcinoma. European urology. 2007;52:1123-29. 12. Krege S, Giani G, Meyer R, Otto T, Rubben H. A randomized multicenter trial of adjuvant therapy in superficial bladder cancer: transurethral resection only versus transurethral resection plus mitomycin C versus transurethral resection plus bacillus Calmette-Guerin. Participating Clinics. The Journal of urology. 1996;156:962-6. 13. Ojea A, Nogueira JL, Solsona E, Flores N, Gomez JM, Molina JR, et al. A multicentre, randomised prospective trial comparing three intravesical adjuvant therapies for intermediate-risk superficial bladder cancer: low-dose bacillus Calmette-Guerin (27 mg) versus very low-dose bacillus CalmetteGuerin (13.5 mg) versus mitomycin C. European urology. 2007;52:1398-406. 14. Witjes WP, Witjes JA, Oosterhof GO, Debruyne MJ. Update on the Dutch Cooperative Trial: mitomycin versus bacillus Calmette-Guerin-Tice versus bacillus Calmette-Guerin RIVM in the treatment of patients with pTA-pT1 papillary carcinoma and carcinoma in situ of the urinary bladder. Dutch South East Cooperative Urological Group. Seminars in urologic oncology. 1996;14:10-6. 15. Sekine H, Ohya K, Kojima SI, Igarashi K, Fukui I. Equivalent efficacy of mitomycin C plus doxorubicin instillation to bacillus Calmette-Guerin therapy for carcinoma in situ of the bladder. International journal of urology : official journal of the Japanese Urological Association. 2001;8:483-6. 16. Jarvinen R, Kaasinen E, Rintala E, Group TF. Long-term results of maintenance treatment of mitomycin C or alternating mitomycin C and bacillus Calmette-Guerin instillation therapy of patients with carcinoma in situ of the bladder: a subgroup analysis of the prospective FinnBladder 2 study with a 17-year follow-up. Scandinavian journal of urology and nephrology. 2012;46:411-7. 17. Rintala E, Jauhiainen K, Kaasinen E, Nurmi M, Alfthan O. Alternating mitomycin C and bacillus Calmette-Guerin instillation prophylaxis for recurrent papillary (stages Ta to T1) superficial bladder cancer. Finnbladder Group. The Journal of urology. 1996;156:56-9; discussion 9-60. 18. Witjes JA, Caris CT, Mungan NA, Debruyne FM, Witjes WP. Results of a randomized phase III trial of sequential intravesical therapy with mitomycin C and bacillus Calmette-Guerin versus mitomycin C

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

alone in patients with superficial bladder cancer. The Journal of urology. 1998;160:1668-71; discussion 71-2. 19. Kaasinen E, Rintala E, Pere AK, Kallio J, Puolakka VM, Liukkonen T, et al. Weekly mitomycin C followed by monthly bacillus Calmette-Guerin or alternating monthly interferon-alpha2B and bacillus Calmette-Guerin for prophylaxis of recurrent papillary superficial bladder carcinoma. The Journal of urology. 2000;164:47-52. 20. Cheng CW, Chan SF, Chan LW, Chan CK, Ng CF, Cheung HY, et al. Twelve-year follow up of a randomized prospective trial comparing bacillus Calmette-Guerin and epirubicin as adjuvant therapy in superficial bladder cancer. International journal of urology : official journal of the Japanese Urological Association. 2005;12:449-55. 21. de Reijke TM, Kurth KH, Sylvester RJ, Hall RR, Brausi M, van de Beek K, et al. Bacillus CalmetteGuerin versus epirubicin for primary, secondary or concurrent carcinoma in situ of the bladder: results of a European Organization for the Research and Treatment of Cancer--Genito-Urinary Group Phase III Trial (30906). The Journal of urology. 2005;173:405-9. 22. Melekos MD, Zarakovitis IE, Fokaefs ED, Dandinis K, Chionis H, Bouropoulos C, et al. Intravesical bacillus Calmette-Guerin versus epirubicin in the prophylaxis of recurrent and/or multiple superficial bladder tumours. Oncology. 1996;53:281-8. 23. Melekos MD, Zarakovitis I, Dandinis K, Fokaefs E, Chionis H, Dauaher H, et al. BCG versus epirubicin in the prophylaxis of multiple superficial bladder tumours: results of a prospective randomized study using modified treatment schemes. International urology and nephrology. 1996;28:499-509. 24. Melekos MD, Chionis HS, Paranychianakis GS, Dauaher HH. Intravesical 4'-epi-doxorubicin (epirubicin) versus bacillus Calmette-Guerin. A controlled prospective study on the prophylaxis of superficial bladder cancer. Cancer. 1993;72:1749-55. 25. Ali-El-Dein B, Nabeeh A, Ismail EH, Ghoneim MA. Sequential bacillus Calmette-Guerin and epirubicin versus bacillus Calmette-Guerin alone for superficial bladder tumors: a randomized prospective study. The Journal of urology. 1999;162:339-42. 26. Bilen CY, Ozen H, Aki FT, Aygun C, Ekici S, Kendi S. Clinical experience with BCG alone versus BCG plus epirubicin. International journal of urology : official journal of the Japanese Urological Association. 2000;7:206-9. 27. Cai T, Nesi G, Tinacci G, Zini E, Mondaini N, Boddi V, et al. Can early single dose instillation of epirubicin improve bacillus Calmette-Guerin efficacy in patients with nonmuscle invasive high risk bladder cancer? Results from a prospective, randomized, double-blind controlled study. The Journal of urology. 2008;180:110-5. 28. Hemdan T, Johansson R, Jahnson S, Hellstrom P, Tasdemir I, Malmstrom PU, et al. 5-Year outcome of a randomized prospective study comparing bacillus Calmette-Guerin with epirubicin and interferon-alpha2b in patients with T1 bladder cancer. The Journal of urology. 2014;191:1244-9. 29. Hinotsu S, Akaza H, Naito S, Ozono S, Sumiyoshi Y, Noguchi S, et al. Maintenance therapy with bacillus Calmette-Guerin Connaught strain clearly prolongs recurrence-free survival following transurethral resection of bladder tumour for non-muscle-invasive bladder cancer. BJU international. 2011;108:187-95. 30. Sylvester RJ, Brausi MA, Kirkels WJ, Hoeltl W, Calais Da Silva F, Powell PH, et al. Long-term efficacy results of EORTC genito-urinary group randomized phase 3 study 30911 comparing intravesical instillations of epirubicin, bacillus Calmette-Guerin, and bacillus Calmette-Guerin plus isoniazid in patients with intermediate- and high-risk stage Ta T1 urothelial carcinoma of the bladder. European urology. 2010;57:766-73.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

31. Jimenez-Cruz JF, Vera-Donoso CD, Leiva O, Pamplona M, Rioja-Sanz LA, Martinez-Lasierra M, et al. Intravesical immunoprophylaxis in recurrent superficial bladder cancer (Stage T1): multicenter trial comparing bacille Calmette-Guerin and interferon-alpha. Urology. 1997;50:529-35. 32. Di Lorenzo G, Perdona S, Damiano R, Faiella A, Cantiello F, Pignata S, et al. Gemcitabine versus bacille Calmette-Guerin after initial bacille Calmette-Guerin failure in non-muscle-invasive bladder cancer: a multicenter prospective randomized trial. Cancer. 2010;116:1893-900. 33. Gontero P, Oderda M, Mehnert A, Gurioli A, Marson F, Lucca I, et al. The impact of intravesical gemcitabine and 1/3 dose Bacillus Calmette-Guerin instillation therapy on the quality of life in patients with nonmuscle invasive bladder cancer: results of a prospective, randomized, phase II trial. The Journal of urology. 2013;190:857-62. 34. Porena M, Del Zingaro M, Lazzeri M, Mearini L, Giannantoni A, Bini V, et al. Bacillus CalmetteGuerin versus gemcitabine for intravesical therapy in high-risk superficial bladder cancer: a randomised prospective study. Urologia internationalis. 2010;84:23-7. 35. Hinotsu S, Akaza H, Isaka S, Kanetake H, Kubota Y, Kuroda M, et al. Sustained prophylactic effect of intravesical bacille Calmette-Guerin for superficial bladder cancer: a smoothed hazard analysis in a randomized prospective study. Urology. 2006;67:545-9. 36. Lamm DL, Blumenstein BA, Crawford ED, Montie JE, Scardino P, Grossman HB, et al. A randomized trial of intravesical doxorubicin and immunotherapy with bacille Calmette-Guerin for transitional-cell carcinoma of the bladder. The New England journal of medicine. 1991;325:1205-9. 37. Martinez-Pineiro JA, Jimenez Leon J, Martinez-Pineiro L, Jr., Fiter L, Mosteiro JA, Navarro J, et al. Bacillus Calmette-Guerin versus doxorubicin versus thiotepa: a randomized prospective study in 202 patients with superficial bladder cancer. The Journal of urology. 1990;143:502-6. 38. Brosman SA. Experience with bacillus Calmette-Guerin in patients with superficial bladder carcinoma. The Journal of urology. 1982;128:27-30. 39. Witjes WP, Konig M, Boeminghaus FP, Hall RR, Schulman CC, Zurlo M, et al. Results of a European comparative randomized study comparing oral bropirimine versus intravesical BCG treatment in BCG-naive patients with carcinoma in situ of the urinary bladder. European Bropirimine Study Group. European urology. 1999;36:576-81. 40. Ibrahiem EH, Ghoneim MA, Nigam V, Brailovsky C, Elhilali MM. Prophylactic maltose tetrapalmitate and bacillus Calmette-Guerin immunotherapy of recurrent superficial bladder tumors: preliminary report. The Journal of urology. 1988;140:498-500. 41. Burger M, Thiounn N, Denzinger S, Kondas J, Benoit G, Chapado MS, et al. The application of adjuvant autologous antravesical macrophage cell therapy vs. BCG in non-muscle invasive bladder cancer: a multicenter, randomized trial. Journal of translational medicine. 2010;8:54. 42. Colombel M, Saint F, Chopin D, Malavaud B, Nicolas L, Rischmann P. The effect of ofloxacin on bacillus calmette-guerin induced toxicity in patients with superficial bladder cancer: results of a randomized, prospective, double-blind, placebo controlled, multicenter study. The Journal of urology. 2006;176:935-9. 43. Agrawal MS, Agrawal M, Bansal S, Agarwal M, Lavania P, Goyal J. The safety and efficacy of different doses of bacillus Calmette Guerin in superficial bladder transitional cell carcinoma. Urology. 2007;70:1075-8. 44. Akaza H, Hinotsu S, Aso Y, Kakizoe T, Koiso K. Bacillus Calmette-Guerin treatment of existing papillary bladder cancer and carcinoma in situ of the bladder. Four-year results. The Bladder Cancer BCG Study Group. Cancer. 1995;75:552-9. 45. Badalament RA, Herr HW, Wong GY, Gnecco C, Pinsky CM, Whitmore WF, Jr., et al. A prospective randomized trial of maintenance versus nonmaintenance intravesical bacillus CalmetteGuerin therapy of superficial bladder cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 1987;5:441-9.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

46. Hudson MA, Ratliff TL, Gillen DP, Haaff EO, Dresner SM, Catalona WJ. Single course versus maintenance bacillus Calmette-Guerin therapy for superficial bladder tumors: a prospective, randomized trial. The Journal of urology. 1987;138:295-8. 47. Koga H, Ozono S, Tsushima T, Tomita K, Horiguchi Y, Usami M, et al. Maintenance intravesical bacillus Calmette-Guerin instillation for Ta, T1 cancer and carcinoma in situ of the bladder: randomized controlled trial by the BCG Tokyo Strain Study Group. International journal of urology : official journal of the Japanese Urological Association. 2010;17:759-66. 48. Palou J, Laguna P, Millan-Rodriguez F, Hall RR, Salvador-Bayarri J, Vicente-Rodriguez J. Control group and maintenance treatment with bacillus Calmette-Guerin for carcinoma in situ and/or high grade bladder tumors. The Journal of urology. 2001;165:1488-91. 49. Fellows GJ, Parmar MK, Grigor KM, Hall RR, Heal MR, Wallace DM. Marker tumour response to Evans and Pasteur bacille Calmette-Guerin in multiple recurrent pTa/pT1 bladder tumours: report from the Medical Research Council Subgroup on Superficial Bladder Cancer (Urological Cancer Working Party). British journal of urology. 1994;73:639-44. 50. Mukherjee A, Persad R, Smith PJ. Intravesical BCG treatment for superficial bladder cancer: longterm results using two different strains of BCG. British journal of urology. 1992;69:147-50. 51. Rentsch CA, Birkhauser FD, Biot C, Gsponer JR, Bisiaux A, Wetterauer C, et al. Bacillus CalmetteGuerin strain differences have an impact on clinical outcome in bladder cancer immunotherapy. European urology. 2014;66:677-88. 52. Sengiku A, Ito M, Miyazaki Y, Sawazaki H, Takahashi T, Ogura K. A prospective comparative study of intravesical bacillus Calmette-Guerin therapy with the Tokyo or Connaught strain for nonmuscle invasive bladder cancer. The Journal of urology. 2013;190:50-4. 53. Gruenwald IE, Stein A, Rashcovitsky R, Shifroni G, Lurie A. A 12 versus 6-week course of bacillus Calmette-Guerin prophylaxis for the treatment of high risk superficial bladder cancer. The Journal of urology. 1997;157:487-91. 54. Kumar A, Dubey D, Bansal P, Mandhani A, Naik S. Urinary interleukin-8 predicts the response of standard and low dose intravesical bacillus Calmette-Guerin (modified Danish 1331 strain) for superficial bladder cancer. The Journal of urology. 2002;168:2232-5. 55. Martinez-Pineiro JA, Flores N, Isorna S, Solsona E, Sebastian JL, Pertusa C, et al. Long-term follow-up of a randomized prospective trial comparing a standard 81 mg dose of intravesical bacille Calmette-Guerin with a reduced dose of 27 mg in superficial bladder cancer. BJU international. 2002;89:671-80. 56. Pagano F, Bassi P, Piazza N, Abatangelo G, Drago Ferrante GL, Milani C. Improving the efficacy of BCG immunotherapy by dose reduction. European urology. 1995;27 Suppl 1:19-22. 57. Yalcinkaya F, Kamis L, Ozteke O, Gunlusoy B, Yigitbasi O, Unal S. Prospective randomized comparison of intravesical BCG therapy with standard dose versus low doses in superficial bladder cancer. International urology and nephrology. 1998;30:41-4. 58. Lamm DL, Blumenstein BA, Crissman JD, Montie JE, Gottesman JE, Lowe BA, et al. 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. The Journal of urology. 2000;163:1124-9. 59. Lamm DL, DeHaven JI, Shriver J, Sarosdy MF. Prospective randomized comparison of intravesical with percutaneous bacillus Calmette-Guerin versus intravesical bacillus Calmette-Guerin in superficial bladder cancer. The Journal of urology. 1991;145:738-40. 60. Lamm DL. Bacillus Calmette-Guerin immunotherapy for bladder cancer. The Journal of urology. 1985;134:40-7. 61. Witjes JA, Fransen MP, van der Meijden AP, Doesburg WH, Debruyne FM. Use of maintenance intravesical bacillus Calmette-Guerin (BCG), with or without intradermal BCG, in patients with recurrent

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

superficial bladder cancer. Long-term follow-up of a randomized phase 2 study. Urologia internationalis. 1993;51:67-72. 62. Lamm DL, DeHaven JI, Shriver J, Crispen R, Grau D, Sarosdy MF. A randomized prospective comparison of oral versus intravesical and percutaneous bacillus Calmette-Guerin for superficial bladder cancer. The Journal of urology. 1990;144:65-7. 63. Luftenegger W, Ackermann DK, Futterlieb A, Kraft R, Minder CE, Nadelhaft P, et al. Intravesical versus intravesical plus intradermal bacillus Calmette-Guerin: a prospective randomized study in patients with recurrent superficial bladder tumors. The Journal of urology. 1996;155:483-7. 64. Netto Junior NR, Levi d'Ancona CA, Claro JF, Ilari O. Immunoprophylaxis of superficial bladder cancer: a prospective and randomized comparison of oral versus intravesical Bacillus Calmette-Guerin. Archivos espanoles de urologia. 1991;44:1025-8. 65. Oddens J, Brausi M, Sylvester R, Bono A, van de Beek C, van Andel G, et al. Final results of an EORTC-GU cancers group randomized study of maintenance bacillus Calmette-Guerin in intermediateand high-risk Ta, T1 papillary carcinoma of the urinary bladder: one-third dose versus full dose and 1 year versus 3 years of maintenance. European urology. 2013;63:462-72.

ACCEPTED MANUSCRIPT

Key of Definitions for Abbreviations

BCG - Bacillus Calmette-Gurein

RI PT

NMIBC - Non-muscle invasive bladder cancer M. bovis - Mycobacterium bovis

PRISMA - Preferred Reporting Items for Systematic Reviews and Meta-Analyses

AC C

EP

TE D

M AN U

SC

OR - odds ratio