Comparing Clinical Efficacy of Different Bacillus Calmette-Guérin Strains in Patients With T1 High Grade Bladder Cancer

Background/Aim: This study aimed to compare the clinical efficacy of two different Bacillus Calmette-Guérin (BCG) strains, TICE strain (OncoTICE) and Connaught strain (ImmuCyst), as a first line intravesical instillation therapy in patients with T1 high grade bladder cancer. Patients and Methods: Patients with newly diagnosed T1 high-grade bladder cancer who underwent transurethral resection of bladder tumor (TURBT) followed by intravesical instillation therapy were enrolled. The effects of BCG strain on recurrence, progression, and side effects were analyzed using Kaplan-Meier and Cox proportional hazards models. Results: Among 147 patients, 53 patients received Connaught strain and 94 patients received TICE strain. The completion rate of induction instillation was 92.45% in the Connaught group and 91.49% in the TICE group (p=1.00). The three-year recurrence-free survival rate was 71.7% in the Connaught group and 63.83% in the TICE group (p=0.33), whereas the three-year progression-free survival rate was 96.23% in the Connaught group and 89.36% in the TICE group (p=0.21). On Cox regression test, carcinoma in situ and ≥eight lesions were significant predictors for recurrence. No significant difference was observed in recurrence and progression between the two BCG regimens. The complication rates according to the Cleveland Clinic grading system showed no significant difference between the two groups (p=0.13). Conclusion: Both the Connaught and TICE strains of BCG demonstrated comparable three-year recurrence-free survival rates and three-year progression-free survival rates for T1 high grade bladder cancer, as well as comparable adverse events. Due to the global BCG shortage, further strain comparisons are essential for clinical validation.

TURBT, residual tumors are detected in a substantial subset of patients, ranging from 26 to 83% (5), underscoring the limitations of TURBT alone therapy.
Apart from TURBT therapy, post-operative intravesical chemotherapy is offered to patients with BC.Mitomycin C (MMC), a prominent chemotherapy agent, has exhibited promising outcomes.One randomized controlled trial conducted by Friedrich et al. showed the efficacy of MMC in reducing BC recurrence rate (6).Another research revealed that intravesical chemotherapy instillation significantly reduced the 5-year recurrence rate compared with TURBT alone -59% vs. 45%, respectively (7).
Fortunately, scientists even found that bacille Calmette-Guérin (BCG) strains can have a great effect on BC.BCGs were initially designed as a tuberculosis vaccine; however, in 1959, the study by Old et al. showed that BCGs can also be used against cancers (8).Moreover, it was shown that intravesical BCG instillation, following TURBT, is the most effective treatment for NMIBC.Based on the American Urological Association/Society of Urologic Oncology (AUA/SUO) and EUA guidelines, BCG is recognized as gold standard for intermediate and high-risk NMIBC (9,10), and it is considered that BCG instillation can reduce the incident rate of BC recurrence and progression.There are various strains of BCG, such as TICE, RIVM, Connaught, Tokyo 172 and others.Studies have demonstrated varying efficacies for these different strains (11)(12)(13).As for the side effects, there were neither significant variations due to different doses of BCG nor differences between the various brands of BCG (14,15).
Because of the prevalence of BC, Connaught strain (ImmuCyst), had been widely utilized to treat NMIBC.Nonetheless, there was a worldwide shortage of Connaught strain so that we have to introduce TICE strain (OncoTICE) in our institution.As a consequence, in order to determine the efficacy of the new drug, we retrospectively compared the difference between the TICE strain and Connaught strain regarding demography, tumor recurrence, tumor progression and side effects (SE).

Patients and Methods
This study involved a retrospective review and analysis of data collected from 2008 to 2022.Ethical approval for the study was obtained from the Institutional Review Board (IRB No. CE13240A-3) at Taichung Veterans General Hospital.Informed consent was obtained from all participants, and all experiments were conducted in strict adherence to applicable guidelines and regulations.Patients diagnosed with BCG-naïve T1 high-grade bladder urothelial cell carcinoma, who had undergone a diagnostic TURBT, were recruited for this study.Subsequently, these patients received treatment using either the Connaught strain or the TICE strain of BCG.According to EAU guidelines (4), low-risk NMIBC was defined as primary, single, low-grade Ta and T1 tumor with a diameter of <3 cm, without carcinoma in situ (CIS) in a patient <70 years.Tumor without CIS with at most one additional clinical risk factors (age >70, multiple papillary tumors or tumor diameter >3 cm) was also classified as low-risk NMIBC.Conversely, other types of BC would be categorized into intermediate-or high-risk NMIBC.
Concerning the availability date of different BCG strains, the Connaught strain was administrated form 2008 -2012, whereas the TICE strain was given as alternative from 2013 -2022 as the result of the shortage of the Connaught strain.The exclusion criteria were as follows: 1) low-risk NMIBC patients, 2) tumor stage beyond T1, 3) patients who had received intravesical chemotherapy instillation within one year before BCG treatment, 4) cases with unknown BCG cycle or strain instilled in other hospitals.
The demographic the data included age, sex, smoking, body mass index (BMI), hypertension (HTN), diabetes mellitus (DM), herbal use, hair dye use, creatinine levels, end stage renal disease (ESRD) and hemodialysis were all recorded and analyzed.In addition, tumor circumstances diagnosed by the initial TURBT were documented through tumor stage, size, EAU risk classification and number of lesions.Furthermore, immediate intravesical chemotherapy instillation with MMC or epirubicin, within 24 h post-TURBT was also documented.After the intravesical BCG induction therapy, follow-up TURBT or cystoscopy would be conducted to examine tumor recurrence/progression.Maintenance BCG is given once per week for three weeks at 3, 6, and 12 months after the initial BCG treatment.It is recommended for one year for patients at intermediate risk of recurrence and for three years for higher risk for recurrence.The follow up protocol included three monthly urine cytology, cystoscopy and upper tract imaging including compute tomography (CT) or intravenous pyelogram (IVP).Recurrence was defined as the reappearance of bladder cancer with the same or lower tumor stage than before.Progression was defined as muscleinvasive bladder cancer (MIBC) or metastasis.Besides, the patients with radical or partial cystectomy, after having tumor recurrence/progression, were also recorded.
The primary outcomes were the efficacy of two different BCG strains by comparing survival rates, tumor recurrence, tumor progression and conduction of cystectomy from the date of TURBT using the Kaplan-Meier method.In addition, the secondary outcome was SE.Base on the Cleveland Clinic Approach of BCG Toxicity, SE of BCG instillation was documented and classified into grade 1 -3 severity.
Statistical analysis.All discontinuous variables are presented as number (percentage), while continuous variables are shown as the mean (standard deviation).For nonparametric analysis, Fisher's exact test was used when the values of the factors were less than 5, whereas the Chi-Square test was performed for values of 5 or greater.After conducting the normality test for the continuous variables, the Mann-Whitney U-test was used.As for survival analysis, Kaplan-Meier estimates were compared with log-rank test.Additionally, potential covariates were assessed for tumor recurrence and progression, and Cox regression model was established for identifying the predictors through their hazard ratio (HR) with a 95% confidence interval (CI) and corresponding pvalues.Statistical tests were performed by using the SAS Academic Software, Release: 3.81 (SAS institute, Cary, NC, USA).

Results
From 2008 to 2022, 169 patients with NMIBC received either Connaught strain (n=55), TICE strain (n=107), or unknown BCG strain (n=7) instillation.However, 7 patients administered with unknown BCG strain were excluded from our data.Moreover, 4 patients in the TICE group having received intravesical chemotherapy within one year before BCG treatment were also excluded.Those who were diagnosed with low grade tumor (two in the Connaught group and six in the TICE group), and those received BCG by unknown hospitals (three in TICE group) were all excluded.
Finally, a total of 147 patients were enrolled in this study; 53 in the Connaught group and 94 in the TICE group.The median follow-up time of the Connaught and TICE groups were 89 months (range=3-171 months) and 28 months (range=5-116 months), respectively.Patient characteristics.The patient demographic data exhibited no significant differences between the TICE and Connaught groups regarding factors, such as age, sex, body mass index (BMI), hypertension (HTN), diabetes mellitus (DM), end stage renal disease (ESRD), smoking, hemodialysis status, hair dye used, or Creatinine at diagnose (Table I).Compared to the Connaught, the TICE group had a lower likelihood of using herbal drugs with a p-value of 0.015.Regarding multiple lesions, the TICE group may have a higher overall count than the Connaught (p=0.041).In addition, other tumor features, such as CIS, tumor size, or EAU risk classification were no statistically significantly different between the two groups.However, the TICE group received immediate chemotherapy instillation more frequently than the Connaught group (the TICE group vs. the Connaught group, MMC 38.30% vs. 37.74%, epirubicin 25.53% vs. 7.55%, p=0.012).Moreover, the follow-up time for the Connaught and the TICE group were 90.85±50.12months and 36.93±24.34months, respectively (p<0.0001), indicating a significant difference in the follow-up duration between the two groups.II), the BCG induction completion rate showed no significant differences between the TICE group (91.49%) and the Connaught group (92.45%) (p=1.00).The Chi-Square test analyzed for three-year recurrence-free survival  III).On multivariate adjustment, CIS (HR=2.12;95%C=1.17-3.87;p=0.014) and ≥eight multiple lesions (HR=3.88;95%CI=1.42-10.63;p=0.0083) remained significant predictors for tumor recurrence.Kaplan-Meier survival plot (Figure 1) showed no significant difference in RFS between the TICE and the Connaught group (p=0.22).

Primary outcomes. Regarding the clinical efficacy (Table
In terms of PFS (Figure 2), a notably prolonged PFS was observed in the Connaught group when compared to the TICE group (p=0.048).However, the median duration of PFS remained indeterminable in both groups.The COX regression analysis presented in Table IV indicated that neither the TICE nor the Connaught induction regimens exhibited significant predictive value for tumor progression.Instead, only the presence of creatinine at the time of diagnosis emerged as a predictive factor for tumor progression (HR=1.29;95%CI=1.00-1.67;p=0.048).
Secondary outcome: adverse events.The incidence of side effects, including cystitis, urinary tract infection (UTI), and hematuria, showed significant differences between the two groups (Table VI).The TICE group had lower frequency of side effects compared to the Connaught group, with respective

Discussion
Our principal finding is that both the Connaught strain and TICE strain BCG exhibited comparable 3-year RFS rates, and 3-year PFS rates.Besides, we found that adverse events occurred more often in the Connaught group, although most were not severe.Therefore, in the edge of BCG shortage, TICE strain is indeed another viable alternative to Connaught strain.However, further investigations comparing these strains are necessary so that medical professionals can confirm their effectiveness.Nowadays, based on EAU guidelines, the principal BC treatment is post-TURBT intravesical BCG instillation.Nonetheless, alternative therapeutic options, including intravesical chemotherapy instillation with agents like MMC and epirubicin, persist.Although, these therapies exhibit some effects on BC, they are not as good as BCG.One metaanalysis showed that there was no significant difference in recurrence risk between BCG and MMC (Relative risk (RR)=0.95,95%CI=0.81-1.11);however, BCG with a maintenance course had lower recurrence risk than MMC (RR=0.79,95%CI=0.71-0.87).Furthermore, BCG also displayed a lower recurrence risk than epirubicin (RR=0.54,95%CI=0.40-0.74)(16).Besides, compared to TURBT alone therapy, adjuvant BCG therapy reduced the risk of BC recurrence (RR=0.56,95%CI=0.43-0.71,I2=0%) and progression (RR=0.39,95%CI=0.24-0.64,I2=40%).Several other meta-analyses had similar findings, consistently highlighting that BCG is better than MMC, TURBT alone and epirubicin (17,18).Therefore, BCG indeed play an optimal role in the management of BC.
Nevertheless, owing to the shortage of the Connaught strain, our institution had no choice but to switch to an alternate BCG strain, namely TICE strain, for BC treatment.Consequently, we had to compare the differences between them.Prior study underscored that the TICE strain, when administered with a maintenance schedule, provided longer RFS in NMIBC patients (HR=0.58,95%CI=0.39-0.86)compared to the Connaught strain (19).Another analysis showed the same spectrum between TICE and Connaught strains with maintenance schedule; interestingly, in the absence of maintenance, the Connaught strain appears more effective on BC for time to first recurrence (HR=1.48,95%CI=1.20-1.82)than the TICE strain ( 12).Yet, a study conducted by Chen et al. demonstrated that there were no differences regarding three-year RFS and PFS in patients with at least 1 maintenance course, which meant nine times of BCG therapy (20).The aforementioned reports demonstrated that the TICE strain with maintenance program could hold an advantageous position over Connaught strain.Conversely, in the context of an induction program, the TICE strain would be inferior to Connaught strain.Despite the above, we focused on the patients whether they can tolerate entire induction cycle or not (Table II), and the comparison between the two groups did not reveal any significant differences.Moreover, our investigation illustrated that the analyzed three-year RFS and PFS rates were somewhat higher for the Connaught group compared to the TICE group.Over a five-year period, the TICE group exhibited slightly higher overall death and cancer-specific death compared to the Connaught group.The results indicated that there were no significant differences in terms of clinical efficacy between the TICE and the Connaught groups, used in patients with T1 high grade BC.While our findings imply that the Connaught group exhibits an extended PFS in comparison to the TICE group, it should be noted that the median PFS duration could not be definitively determined.This observation suggests that the data might be incomplete or insufficient, a conclusion that is further substantiated by the COX regression analysis.
In our study population, the TICE group underwent immediate intravesical chemotherapy instillation more frequently compared to the Connaught group (MMC 38.30% vs. 37.74%, epirubicin 25.53% vs. 7.55%; p=0.012).According to a research and guideline from the EAU, immediate intravesical chemotherapy is recommended for reducing recurrence rates in certain low-risk patients (4,21).However, the patients in our study belong to a high-grade (high-risk) category, which makes them less likely to be affected by the benefits of immediate intravesical chemotherapy.
Earlier studies have indicated comparable SE in patients receiving both TICE and Connaught strains, ranging from 13.5% to 42% in frequency (22).A meta-analysis also showed that reducing the amount of BCG administered results in a lower likelihood of experiencing SE when compared to the standard BCG dosage.Furthermore, their results also uncovered that lower doses of BCG might be less effective in preventing cancer recurrence (23).
The aforementioned studies showed the relationship between different BCG strains and SE.Moreover, our results are in agreement with the aforementioned studies.Compared to the Connaught group, the TICE group exhibited a significantly lower frequency of adverse effects, including cystitis, UTI, and hematuria.However, differences in BCG infection and the complications category were not statistically significant between the two groups.Consequently, we could conclude that the TICE strain had a lower incidence of SE than the Connaught strain, although there was no distinction in the severity of SE.
Our observations were in alignment with the conclusions drawn in prior studies.Nevertheless, several limitations are present within this study.Primarily, the sample size in both the Connaught and TICE groups was relatively small, with the TICE group's size being nearly twice as large as that of the Connaught group.Besides, the duration of follow-up was uneven with a p-value <0.0001.The incorporation of the TICE strain into our institution was prompted by the worldwide scarcity of the Connaught strain.Given the prevailing clinical circumstances, the relatively shorter follow-up duration for the TICE group was considered reasonable.Nonetheless, the median follow-up period for TICE was 28 months, a duration deemed adequately extensive to detect occurrences of recurrence and progression among patients diagnosed with T1 high grade BC.Second, according to the research conducted by Witjes et al., the clinical efficacy of the TICE strain would be different when cooperating with maintenance course in comparison to Connaught strain (12).However, in our experiment, the results did not show any significantly different predictors of recurrence and progression between the TICE strain and Connaught strain.Additionally, we did not differentiate the patients based on whether they achieved maintenance or not.Instead, we just classified the patients into categories based on completion or non-completion of the entire BCG induction cycle.Therefore, this factor could potentially hinder our ability to differentiate between the superior strain concerning tumor recurrence and progression.Alternatively, it remains plausible that both strains of BCG demonstrated comparable outcomes in terms of recurrence and progression.Moreover, another point that should be considered is the difference in dosage between Connaught and TICE, which could affect their effectiveness and SE.However, surprisingly, seldomly researchers had been interested in addressing this issue.
According to a previous study by Rausch et al. (24), the estimated Glomerular filtration rate (<60 ml/min) was one of the significant predictive determinants for recurrence and overall progression.Typically, the more severe the tumor, the higher the likelihood of causing obstruction that affects kidney function, consequently leading to progression.As for our study, the data also showed a similar tendency for tumor progression; the results of univariate regression showed that creatinine would impact the occurrence of tumor progression (p=0.0048;Table V).Although creatinine values were not significantly different between the two strains of BCG (p=0.097)(Table I), the mean value for the TICE group (1.50) exceeded the normal range (0.7-1.2), whereas the mean value for the Connaught group (1.16) did not.Therefore, this result might influent the overall tumor progression causing a statistically significant difference between the two groups with the p-value of 0.048 (Figure 2).Finally, novel agents such as 5 aminolevulinic acid for photodynamic diagnosis in TURBT for non-muscle-invasive bladder cancer addition to BCG administration shows better diagnostic accuracy and evaluation, but it could potentially improve the treatment protocol in the future (25).

Conclusion
In our retrospective, single-center analysis of T1 high-grade bladder cancer, both the Connaught and TICE BCG strains demonstrated comparable completion rates, three-year RFS rates, and three-year PFS rates.Adverse events were more frequent in the Connaught group; however, the majority were of low grade.Due to the global BCG shortage, further strain comparisons are needed for clinician validation.

Table I .
Yu et al: Different BCG Strains in Bladder Cancer 1301 Characteristics of patients with bladder urothelial carcinoma receiving Bacillus Calmette-Guérin (n=147).
a Mann-Whitney U-test.b Chi-Square test.c Fisher's Exact test.*p<0.05.Continuous data are expressed as mean±SD.Categorical data are expressed as number and percentage.BMI: Body mass index; HTN: hypertension; DM: diabetes mellitus; ESRD: end stage renal disease; Crea: creatinine; CIS: carcinoma in situ; EAU: European Association of Urology; MMC: mitomycin C.

Table III ,
Table IV, Table V).The univariate regression

Table II .
Outcome of patients with bladder urothelial carcinoma receiving Bacillus Calmette-Guérin (n=147).Square test.b Fisher's Exact test.Continuous data are expressed mean±SD.Categorical data are expressed number and percentage.RFS: Recurrence-free survival rate; PFS: progression-free survival.

Table III .
Uni-multi variants predict tumor recurrence from transurethral resection of bladder tumor.

Table IV .
Uni-multi variants predict tumor progression from transurethral resection of bladder tumor.

Table V .
Uni-multi variants predict radical/partial cystectomy from transurethral resection of bladder tumor.