Stesura Seveso Archivio Italiano di Urologia e Andrologia 2023; 95, 2 95 ORIGINAL PAPER progression, intravesical Bacillus Calmette-Guérin (BCG) is the gold standard adjuvant therapy (4). The use of intravesical instillation of BCG for high risk NMIBC demonstrates a role for immunotherapy in UC. BCG, originally used as a vaccine against tuberculosis (TB), contains live-attenuated Mycobacterium bovis (4). The specific mechanism of BCG in NMIBC treatment continues to be studied, however, its role is attributed to both local immunological efforts and systemic immune responses (5). Some work has suggested that BCG vacci- nation may be associated with a lower rate of bladder can- cer incidence (6). Despite this relationship, research on BCG immunization as a possible predictive factor in NMIBC has been limited. In the present study, we evalu- ated the relationship between history of infantile BCG vaccination with the depth of invasion and the grade in patients with NMIBC. METHODS Data were retrospectively collected between 2017 and 2022. Inclusion criteria included all patients with a new diagnosis of NMIBC at the Thunder Bay Regional Health Sciences Centre (TBRHSC), for whom complete clinical, lab, and pathological data could be retrieved. Data collect- ed included the history of infantile BCG as well as the patients’ age, sex, comorbid status, CBC, vaccination, his- tory of intravesical BCG instillation, pathological data, recurrence, and progression. Vaccination status was corre- lated with these variables. Institutional ethical approval was obtained from the TBRHSC research ethics board (RP- 741). Correlations between continuous variables were done using Student’s t-test. Categorical variables were compared using Fisher Exact test. A p-value of < 0.05 was used to define significance. RESULTS A total of 188 patients met the inclusion criteria for our study. No patients were lost to follow up. The mean fol- low-up time was 26 ± 7 months. Of the 188 individuals meeting the eligibility criteria, 113 individuals had received the infantile BCG immunization and 75 did not. Objective: To evaluate the utility of infantile BCG vaccination history in predicting stage and grade of tumours in non-muscle invasive bladder cancer (NMIBC). Materials and methods: We retrospectively analyzed data from patients from a single center who were diagnosed with new NMIBC and underwent transurethral resection of bladder tumour (TURBT) between 2017 and 2022. We assessed BCG immunization status with various demographics and comorbidi- ties, as well as tumour recurrence, progression, stage, and grade. Results: A total of 188 patients met the inclusion criteria for our study. The mean age of patients at the time of diagnosis was significantly lower in those that had been immunized with BCG (71 ± 9) than those who had not (77 ± 10) (p < 0.0001). History of BCG immunization did not correlate with sex, history of diabetes mellitus (DM), prior history of intravesical BCG treat- ment, and tumour recurrence, progression, stage, and grade. Conclusions: History of infantile BCG vaccination did not corre- late with the depth of invasion and/or the grade in patients with non-muscle invasive bladder cancer. Patients that received infantile BCG vaccination were significantly younger at the time of diagnosis of NMIBC. KEY WORDS: BCG; Non-muscle invasive bladder cancer (NMIBT); Bladder cancer. Submitted 11 April 2023; Accepted 26 May 2023 INTRODUCTION Bladder cancer is the tenth most common malignancy worldwide, with increasing incidence, particularly in developed nations (1). Approximately 80% of bladder can- cers arise in individuals aged 65 or older with the mean age being 73 years old (2, 3). This is thought to reflect a disease process requiring many decades of development following exposure to risk factors, such as tobacco (2). Urothelial car- cinoma (UC) accounts for 90% of bladder cancers (4). At the time of presentation, approximately 70% of UC cases are non-muscle invasive (NMIBC), while 30% are mus- cle invasive (MIBC) (4). Initial management of NMIBC is transurethral resection of bladder tumor (TURBT) (4). For those with NMIBC who are deemed to be at high risk for History of infantile BCG immunization did not predict lamina propria invasion and/or high-grade in patients with non-muscle invasive bladder cancer Anastasia MacDonald 1, Vahid Mehrnoush 1, Asmaa Ismail 1, Livio Di Matteo 2, Ahmed Zakaria 1, Waleed Shabana 1, Ashraf Shaban 1, Mohammed Bassuony 1, Hazem Elmansy 1, Walid Shahrour 1, Owen Prowse 1, Ahmed Kotb 1 1 Northern Ontario School of Medicine, Thunder Bay, ON, Canada; 2 Department of Economics, Lakehead University, Thunder Bay, ON, Canada. DOI: 10.4081/aiua.2023.11380 Summary Archivio Italiano di Urologia e Andrologia 2023; 95, 2 A. MacDonald, V. Mehrnoush, A. Ismail, et al. 96 A statistically significant difference was identified between the age of individuals who had received the immunization and those who did not (p < 0.0001). The mean age at the time of diagnosis for those immunized was 71 ± 9 years, and 77 ± 10 in the non-immunized group. There was no statistically significant difference found between immunization status and other patient characteristics including sex, history of diabetes mellitus (DM), or history of intravesical BCG treatment. History of immunization did not correspond with tumour recur- rence, progression, stage, or grade in this population. The results are summarized in Table 1. DISCUSSION Studies assessing the relationship between NMIBC and BCG immunization have been limited. One scoping liter- ature review identified a 35-37% lower age-standardized rate of bladder cancer incidence in individuals with BCG immunizations, suggesting an association between the two (6). We demonstrated that BCG immunization did not corre- late with tumour characteristics in NMIBC, including stage, grade, and risk stratification. This may be explained by the routes of administration and subsequent immune responses elicited. The anti-tumour activity of intravesical BCG therapy is attributed to non-specific immune mech- anisms related to the direct interaction with urothelial cells, as well as a contribution of systemic immune response, though specific mechanisms have yet to be fully elucidated (7). BCG immunization is also associated with non-specific immune mechanisms that provide protec- tion against tuberculosis, however, given the nature of vaccinations, this response is exclusively systemic (8). Interestingly, this generalized immune response from immunization has been shown to confer protection against other respiratory infections through a mechanism referred to as trained immunity (TI) (8, 9). It was demon- strated that BCG immunotherapy in NMIBC patients induced TI and provided protection against respiratory infections (9). This suggests that intravesical BCG thera- py can produce similar systemic immune responses as the BCG vaccination. Given that our data demonstrated that immunization status did not impact the tumour progres- sion characteristics and risk stratification, this may sug- gest increased importance in the role of the local immune response in intravesical BCG in preventing the progres- sion of NMIBC. Our findings may also be explained by the waning protec- tion from immunization over time. While bladder cancer incidence increases in the elderly (10), individuals immu- nized with BCG are typically immunized as infants. It has been well documented that protection from this immu- nization against TB infections wanes over time (7). Studies have identified that a positive purified protein derivative (PPD) skin test, an indication of BCG immunity, was asso- ciated with a better response to intravesical BCG therapy than those with a negative reaction (11, 12). Niwa et al. (2017) demonstrated that the recurrence-free survival (RFS) in patients with a slightly positive or negative PPD skin test reaction was significantly diminished compared to the RFS in those with a strongly positive response. This may suggest that a reduced immune response from BCG immunization does not generate the same benefit in BCG treatment. Given that the mean age of those vaccinated with BCG in our study was 71 years, and our study specif- ically looked at infantile BCG immunization, this may also explain why individuals with waning immunity from remote immunization did not influence tumour character- istics or risk stratification in patients with NMIBC. In our study, the mean age of patients diagnosed with new NMIBC was significantly lower in individuals who received the infantile BCG vaccination (71 ± 9) compared to those who did not (77 ± 10) (p < 0.0001). Increased age is a risk factor for developing UC, largely attributed to a disease course that develops decades after exposure to risk factors (2). Countries with the lowest incidence of bladder cancer are typically those found to be below aver- age on the human development index (HDI), which may be attributable to less industrial chemical exposure and access to tobacco, major risk factors for UC (1). Interestingly, such countries tend to have a higher inci- dence of tuberculosis and subsequently higher rates of infantile BCG immunization (13). This may imply that non-immunized individuals were likely raised in coun- tries with low TB incidence, yet above-average HDI. Such individuals would likely have had a higher risk of expo- sure to industrial chemicals and tobacco, leading to the slow development of bladder cancer and presentation at a later age. Those immunized and likely raised in countries with less exposure to common risk factors may have developed UC earlier on due to other reasons, such as genetics, diets, or other lifestyle factors. Older age at presentation has been shown to be a poor prognostic factor in NMIBC (4). Consequently, the older age of presentation with NMIBC in non-immunized indi- viduals poses a significant healthcare concern. The inci- dence of bladder cancer has been steadily increasing, par- ticularly in developed countries (1). Such countries do not tend to implement routine immunization against TB Table 1. Correlation of clinical and tumour data with history of infantile BCG immunization. No infantile BCG Infantile BCG P value Age (mean + SD) 77 + 10 71 + 9 0.0001 Sex Males 60 77 0.09 Females 15 36 Recurrence No 34 57 0.5 Yes 41 56 Progression No 72 108 1 Yes 3 5 Intravesical BCG No 44 68 0.9 Yes 31 45 DM No 61 86 0.5 Yes 14 27 Stage Ta 58 86 1 T1 17 27 Grade Low 54 81 1 High 21 32 SD: standard deviation. BCG: Bacillus Calmette–Guérin. DM: Diabetes Mellitus. Archivio Italiano di Urologia e Andrologia 2023; 95, 2 97 Infantile BCG vaccination in patients with NMIBC given the low incidence. Consequently, there is a growing population of non-immunized individuals presenting with NMIBC and potentially at older ages. This may result in overall more complicated patients with poorer prog- nostics. Further research in this area would be of utility given the growing aging population and potentially increased demands on healthcare systems. There are several limitations to our study. First, it is a ret- rospective study completed at a single center. Therefore, selection bias was inevitable, and our study represents a relatively small sample size of patients. This study also limited by the relatively short follow up period (26 + 7 months) for assessing recurrence and progression. Additionally, we did not account for the various demo- graphic factors that may influence the risk factors for developing NMIBC. CONCLUSIONS Infantile BCG immunization was not associated with higher risk stratification in patients with NMIBT. The mean age of patients diagnosed with NMIBC was signifi- cantly lower in patients who received the infantile BCG vaccination. REFERENCES 1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin. 2018; 68:394. 2. Mushtaq J, Thurairaja R, Nair, R. Bladder cancer Surgery (Oxf). 2019; 37:529. 3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019 CA Cancer J Clin. 2019; 69:7. 4. Saginala K, Barsouk A, Aluru JS, et al. Epidemiology of Bladder Cancer Med Sci. 2020; 8:15. 5. Taniguchi K, Koga S, Nishikido M, et al. Systemic immune response after intravesical instillation of Bacille Calmette-Guérin (BCG) for superficial bladder cancer Clin Exp Immunol. 1999; 115:131. 6. Trigo S, Gonzalez K, Di Matteo L, et al. Bacillus Calmette-Guerin vaccine and bladder cancer incidence: Scoping literature review and preliminary analysis Arch Ital Urol. 2021; 93:1. 7. Mukherjee N, Julián E, Torrelles JB, Svatek RS. Effects of Mycobacterium bovis Calmette et Guérin (BCG) in oncotherapy: Bladder cancer and beyond Vaccine. 2021; 39:7332. 8.Covián C, Fernández-Fierro A, Retamal-Díaz A, et al. BCG- Induced Cross-Protection and Development of Trained Immunity: Implication for Vaccine Design Front Immunol. 2019; 10:2806. 9. van Puffelen J H, Novakovic B, van Emst L, et al. Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections J Immunother Cancer. 2023; 11:e005518. 10. Martinez L, Cords O, Liu Q, et al. Infant BCG vaccination and risk of pulmonary and extrapulmonary tuberculosis throughout the life course: a systematic review and individual participant data meta- analysis Lancet Glob Health. 2022: 10:e1307. 11. Niwa N, Kikuchi E, Matsumoto K, et al. Purified protein deriva- tive skin test reactions are associated with clinical outcomes of patients with nonmuscle invasive bladder cancer treated with induc- tion bacillus Calmette-Guérin therapy Urol Oncol. 2018; 36e15. 12. Biot C, Rentsch CA, Gsponer JR, et al. Preexisting BCG-specific T cells improve intravesical immunotherapy for bladder cancer Sci Transl Med. 2012; 4:72. 13. Centers for Disease Control and Prevention. BCG Vaccine Fact Sheet. 2016. Retrieved from https://www.cdc.gov/tb/publications/ factsheets/prevention/bcg.htm Correspondence Anastasia MacDonald, MD anamacdonald@nosm.ca Vahid Mehrnoush, MD vahidmehrnoush7@gmail.com Asmaa Ismail, MD asmaaismail0782@gmail.com Ahmed Zakaria, MD aszakaria81@yahoo.com Waleed Shabana, MD waleed.shabana@gmail.com Ashraf Shaban, MD ashraf.shaban@tbh.net Mohammed Bassuony, MD mohammed.bassuony@tbh.net Hazem Elmansy, MD hazem.elmansy@tbh.net Walid Shahrour, MD walid.shahrour@tbh.net Owen Prowse, MD owen.prowse@tbh.net Ahmed Kotb, MD, PhD, FRCSC, FRCS Urol, FEBU (Corresponding Author) Associate Professor drahmedfali@gmail.com Northern Ontario School of Medicine (NOSM) and Thunder Bay Regional Health Research Institute (TBRHRI) Thunder Bay, ON, Canada Livio Di Matteo, MD ldimatte@lakeheadu.ca Department of Economics, Lakehead University, Thunder Bay, ON Conflict of interest: The authors declare no potential conflict of interest.