Bacillus Calmette-Guérin (BCG) therapy lowers the incidence of Alzheimer's disease in bladder cancer patients.

BACKGROUND: Alzheimer's disease (AD) affects one in ten people older than 65 years. Thus far, there is no cure or even disease-modifying treatment for this disease. The immune system is a major player in the pathogenesis of AD. Bacillus Calmette-Guérin (BCG), developed as a vaccine against tuberculosis, modulates the immune system and reduces recurrence of non-muscle invasive bladder cancer. Theoretical considerations suggested that treatment with BCG may decrease the risk of AD. We tested this hypothesis on a natural population of bladder cancer patients. METHODS AND
FINDINGS: After removing all bladder cancer patients presenting with AD or developing AD within one-year following diagnosis of bladder cancer, we collected data on a total of 1371 patients (1134 males and 237 females) who were followed for at least one year after the diagnosis of bladder cancer. The mean age at diagnosis of bladder cancer was 68.1 years (SD 13.0). Adjuvant post-operative intra-vesical treatment with BCG was given to 878 (64%) of these patients. The median period post-operative follow-up was 8 years. During follow-up, 65 patients developed AD at a mean age of 84 years (SD 5.9), including 21 patients (2.4%) who had been treated with BCG and 44 patients (8.9%) who had not received BCG. Patients who had been treated with BCG manifested more than 4-fold less risk for AD than those not treated with BCG. The Cox proportional hazards regression model and the Kaplan-Meier analysis of AD free survival both indicated high significance: patients not treated with BCG had a significantly higher risk of developing AD compared to BCG treated patients (HR 4.778, 95%CI: 2.837-8.046, p = 4.08x10-9 and Log Rank Chi-square 42.438, df = 1, p = 7.30x10-11, respectively). Exposure to BCG did not modify the prevalence of Parkinson's disease, 1.9% in BCG treated patients and 1.6% in untreated (Fisher's Exact Test, p = 1).
CONCLUSIONS: Bladder cancer patients treated with BCG were significantly less likely to develop AD at any age than patients who were not so treated. This finding of a retrospective study suggests that BCG treatment might also reduce the incidence of AD in the general population. Confirmation of such effects of BCG in other retrospective studies would support prospective studies of BCG in AD.

Introduction

Alzheimer’s disease (AD) affects over 5 million Americans. Its prevalence is expected to triple by 2030 and its cost to rise accordingly [1, 2]. While cardiovascular mortality was cut by more than a half in the past 30 years and cures were found for many types of cancer, no cure or even disease-modifying treatment has been found for AD. Any treatment that decreases the incidence of AD or delays its onset will have a substantial impact on the individual and on society.

AD is marked by three major pathological features: accumulation of amyloid β (Aβ) plaques, neurofibrillary tangles (hyperphosphorylated tau protein) and sustained innate neuroinflammation [3]. Since the late 1980, Aβ has been the target of most clinical trials. A series of well conducted trials with anti-Aβ agents (or with tau-aggregation inhibitors) have all failed to reduce cognitive or functional decline in patients with mild-to moderate and even “prodromal” AD [4-6]. These failures have led to a reduced effort in drug-discovery for AD by major pharmaceutical companies [7]. It seems that once dementia is present, disease progression becomes Aβ-independent and probably less susceptible to interventions [8]. In retrospect, this may not be surprising since Aβ deposition takes place years before symptoms appear.

“Setbacks in Alzheimer research demand new strategies, not surrender” is the tittle of a recent Editorial in PLOS Medicine [7]. Can modulation of the immune system and prevention, instead of treatments targeting (Aβ) or tau, be one of these new strategies?

It is now believed that inflammation is not just a response of the immune system to neuronal loss but a major player in AD pathogenesis. Neuro-inflammation contributes to neurodegeneration and to AD pathogenesis [8, 9]. The currently recognized role of the immune system in AD is as follows: the presence of Aβ activates microglia cells; the cells migrate and phagocytize the Aβ. Early in AD development, this process clears the Aβ but later the microglia are no longer able to process the Aβ. This leads them to a condition of sustained activation termed "reactive microgliosis" producing pro-inflammatory cytokines and neurotoxins. The ability of the microglia to process Aβ decreases but their state of activation persists resulting in neurotoxicity. This creates a vicious cycle of response to AD neuropathology and neurotoxicity. Toxic neuroinflammation is amplified by the recruitment of macrophages at a later stage of the disease. The immune system involvement in AD involves many classes of cells and cytokines, some promoting neurotoxicity while others are neuroprotective. It has been suggested that systemic regulatory immune cells such as CD4+ CD25+Foxp3+ (Tregs) are involved in protecting the brain from AD pathology, and their failure may contribute to disease progression [10]. Tregs regulate the immune responses critical for maintenance of self-tolerance. Their population can be expanded by administration of a relatively low dose of IL-2 [11]. Other key cytokines participating in AD include the pro-inflammatory TNFα, IL-1β and the double action cytokine IL-6, which at low levels reduces microglia activity and at high levels is pro-inflammatory [3,10]. Another prominent anti-inflammatory cytokine is IL-10. It is released by microglia and astrocytes and inhibits the production of pro-inflammatory cytokines. However, clinical trials with IL-10 therapy did not support a protective role [3].

Intra-vesical Bacillus Calmette-Guérin (BCG) has been used since 1972 for preventing recurrence of non-muscle invasive bladder cancer (NMIBC). Numerous clinical trials have proved its efficacy [12]. The exact mechanism of BCG anticancer activity has not been deciphered, but it is well recognized that BCG manifests immune effects; it binds fibronectin in the bladder wall and stimulates Th1 cells to secrete multiple cytokines including: IL-1, 2, 5, 6, 8, 10, 12 and 18, as well as IFNγ, TNFα and GM-CSF. It is presumed that these cytokines induce cell-mediated cytotoxic mechanisms that eliminate cancer cells.

It was recently hypothesized that administration of BCG might decrease the prevalence of AD in elderly persons through modulation of the immune system [13] possibly by expanding the Treg population [10]. This hypothesis was based on the inverse relation between BCG vaccination against tuberculosis and the prevalence of AD/ dementia; moreover, BCG immunization has shown beneficial effects on the course of other CNS immune-related disorders like multiple sclerosis [14] and on experimental autoimmune encephalomyelitis [15]. In addition, BCG vaccination improves both brain pathology and cognitive performance in the APP/PS1 transgenic mouse model of AD [16].

In the present report, we studied the effects of BCG administration on the risk to develop AD in a population of bladder cancer patients. According to oncological guidelines, some bladder cancer patients are treated with BCG and others are not exposed to BCG. Thus, we could compare the effects of BCG on AD in these two groups. Since most cases of bladder cancer are not acutely lethal, long-term follow-up is available in many patients.

Methods

Patients

The study is based on data retrieved from the computerized archives of the Hadassah-University Hospitals. This secondary and tertiary medical center is divided into two campuses located at the opposing edges of Jerusalem serves about 1.5 million people, from both the Jewish and the Arab populations.

The archives of the Hadassah-University medical hospitals provide a unique source of long-term follow-up on many types of pathologies. The records of patients with bladder cancer were crosschecked with the hospital archive database of patients diagnosed with AD (ICD-9-cm codes 294.20 or 331.0) and Parkinson’s disease (PD) (ICD-9-cm code 332.0).

The Institutional Review Board Committee (IRB number 0037-17HMO) approved the study. All data were fully anonymized before analysis and the ethics committee waived the requirement for informed consent.

Patient management

Patients diagnosed with bladder cancer underwent transurethral resection of the tumor and an immediate instillation of a chemotherapeutic agent. Intra-vesical immunotherapy with OncoTICE BCG 12.5mg per vial containing 2–8 x 108 CFU Tice BCG was liberally used, especially in the years 1990 and 2010. During that time period BCG was offered to patients with any stage T1, any high-grade tumor, CIS, low-grade tumors larger than 3cm, more than 3 low-grade tumors and low-grade tumors that recurred within 2 years. A six-week induction course was given to these patients and maintenance doses up to three years to patients with high-grade disease [12]. For study purposes, any dose of BCG qualified the patient in the “BCG group”. Intravesical chemotherapy instillation (commonly used nowadays for low-grade tumors) was rarely prescribed during the study. Follow up was maintained for five years with periodic cystoscopies. Afterwards, annual check-up and ultrasonography of the urinary system was performed indefinitely [17].

Statistical analysis

We used the Cox Proportional Hazards regression model (Cox PH) to estimate cause-specific hazard ratios (HRs) with 95% confidence intervals (CIs) for risk of AD comparing bladder cancer patients who did not receive BCG as a treatment to BCG treated bladder cancer patients. The effects of BCG, tumor grade, stage, and gender on the risk of AD were evaluated with the Cox PH with SPSS version 25.0 (IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). Kaplan-Meier survival analysis with the log-rank test (Mantel-Cox) were used to compare the rates of AD-free survivals in BCG treated and untreated patients. Continuous variables were compared using two-sample t-test. Fisher’s Exact Test was used to compare categorical variables. All statistical tests were two tailed and a p value smaller than 0.05 was considered significant.

Results

From 1966 to 2018, 1522 patients were treated for bladder cancer in Hadassah-University medical hospitals. For the purpose of analysis, pathological stage was dichotomized to non-muscle invasive (patients were excluded from the analysis either because they suffered from AD while being diagnosed with bladder cancer, they died in less than one year following the diagnosis of bladder cancer or they developed AD earlier than one year after diagnosis of bladder cancer. This left 1371 patients (1134 males and 237 females) for analysis (S1 File Excel, supporting information). Clinical characteristics of the patients are presented in Table 1 and S1, S2 and S3 Tables (supporting information). Median postoperative follow-up was 8 years (IQR 3–14 years).

Table 1

Characteristics of patients in the observed population.

	Given BCG	Not Given BCG	Total
Number of patients	878 (64%)	493 (36%)	1371
Mean age at diagnosis (SD)	67.5 (12.6)	69.0 (13.7)	68.1 (13.0)a
Male/Female	730/148 (83.1%/16.9%)	404/89 (81.9%/18.1%)	1134/237 (82.7%/17.3%)
Stage <T2 / ≥T2	867/11 (98.7%/1.3%)	312/181 (63.3%/36.7%)	1179/192 (86.0%/14.0%)
Grade Low/High	452/426 (51.5%/48.5%)	281/212 (57%/43%)	733/638 (53.5%/46.5%)
Mean age at the end of Follow-up (SD)	78.7 (10.8)	75.9 (13.4)	77.7 (11.8)b
Parkinson Yes/No	13/865 (1.5%/98.5%)	6/487 (1.2%/98.8%)	19/1352 (1.4%/98.6%)

ap = 0.0449

bp<0.0001

The mean age at diagnosis of bladder cancer was 68.1 years (SD 13.0). Patients given BCG were marginally younger than patients not given BCG (67.5±12.6 vs. 69.0±13.7, p = 0.0449). The mean age by the end of follow-up of patients given BCG was significantly older compared to patients not given (78.7±10.8 vs. 75.9±13.4, p<0.0001) reflecting the fact that BCG was not given to patients with stage ≥ T2. During follow-up, AD was diagnosed in 65 patients (4.7%) at a mean age of 84 years (SD 5.9), including 21 patients (2.4%) given BCG and 44 patients (8.9%) not given BCG. Among the 730 males who were given BCG, 18 were diagnosed with AD (2.47%) whereas 37 cases of AD (9.16%) were diagnosed among the 404 patient who did not receive BCG. The female population was much less represented with 7 cases of AD (7.87%) among 89 patients non-BCG treated and 3 cases of AD (2.03%) among 148 BCG treated patients (S1, S2 and S3 Tables).

The Cox PH analysis was run for 1371 patients out of which 303 patients (22.1%) were censored cases before the earliest event of AD (occurring at age 71 years), leaving 1068 patients (93.9% censored cases) and 65 AD cases (6.1%). In the resulting 1068 patients’ population, the mean age at detection of AD for BCG treated patients was 82.57 years (SD = 7.23) slightly but significantly older than 81.53 years (SD = 6.87), the mean age of the patients without BCG (p = 0.0231) (Table 2).

Table 2

Characteristics of the 1068 patients analyzed by Cox PH and K-M.

	Given BCG (%within row)(%within column)	Not Given BCG (%within row)(%within column)	Total (%within row)(%within column)	p value
Number of patients	702 (65.7%)	366 (34.3%)	1068
Mean age (SD) at AD	82.57 (7.23)	81.53 (6.87)	82.2 (7.1)	0.0231
Male/Female	587/115 (66.3%/63.2%) (83.6%/16.4%)	299/67 (33.7%/36.8%) (81.7%/18.3%)	886/182(65.7%/34.3%) (83.0%/17.0%)	0.441
Stage <T2 / ≥T2	696/8 (74.9%/5.8%) (98.9%/1.1%)	233/131 (25.1%/(94.2%) (63.7%/35.8%)	929/139(65.9%/34.1%) (87.0%/ 13.0%)	1.52 x 10−58
Grade Low/High	357/345 (62.9%/69.0%) (50.9%/49.1%)	211/155 (37.1%/31.0%) (57.7%/42.3%)	568/500(65.7%/34.3%) (53.2%/46.8%)	0.0387
Parkinson Yes/No	13/689 (68.4%/65.7%) (1.9%/98.1%)	6/360 (31.6%/34.3%) (1.6%/98.4%)	19/1049(65.7%/34.3%) (1.8%/98.2%)	1

Cox PH analysis (93.9% censored cases) showed that BCG treatment reduced dramatically the risk of developing AD (Fig 1). This effect was highly significant (HR 4.778, 95%CI: 2.837–8.046, p = 4.08x10-9). The covariates grade and gender, were found to be not significant when run with BCG (p = 0.216 and p = 0.395 respectively) and the stage covariate was discarded as by definition patients with stage BCG therapy and patients with stage ≥T2 were not. Exposure to BCG did not change the risk of Parkinson’s disease (1.9% in BCG treated patients and 1.6% in untreated, p = 1 by Fisher’s Exact Test, Table 2).

Fig 1

Cox PH survival curves of AD-free bladder cancer patients treated or not with BCG according to agea.

aHR 4.778, 95%CI: 2.837–8.046, p = 4.08x10-9.

The Kaplan–Meier analysis performed on the population defined in the Cox PH regression (1068 patients; Table 2) showed that the AD-free survival curve of the BCG recipients was significantly different from the survival curve of non BCG treated patients (Log Rank: Chi-square 42.438,df = 1, p = 7.30x10-11) with overall 93.9% censored cases (Fig 2). When stratifying the population by gender, the difference remained highly significant (males: Log Rank Chi-Square of 35.162, df = 1 and p = 3.03x10-9; females: Log Rank Chi-Square of 6.735, df = 1 and p = 0.00945) (S1 and S2 Figs). The higher p value for females is probably due to the small size of the female population. There were no significant differences in the AD-free survival between male and female patients not given BCG (Log Rank: Chi-square 0.390, df = 1, p = 0.533). This was also true between BCG treated females and males (Log Rank: Chi-square 0.433, df = 1, p = 0.511) (S3 and S4 Figs). The Cohen effect sizes [] were large and similar, 0.82 and 0.80 for females and males respectively. This result lends support to the finding from the Cox PH model that gender is not a significant co-variate (p = 0,395) and mitigates against any marked introduction of bias caused by combining males and females data in the general analysis [18].

Fig 2

Kaplan-Meier survival curves of AD-free bladder cancer patients treated or not with BCG according to agea.

aLog Rank Chi-square 42.438, df = 1, p = 7.30E-11 between BCG treated and BCG not treated patients (93.9% of the 1068 patients were censored cases).

Discussion

Bladder cancer patients typically present in their 6th to 8th decades of life making them an appropriate population for investigations in AD. In this study, we followed a cohort of these patients for a median period of 8 years after diagnosis of bladder cancer. About 60% of them were given adjuvant treatment with intravesical instillations of BCG and the rest were not. During follow-up, 4.7% of the patients developed AD, a rate similar to the 4.66% reported in a recent European meta-analysis [19]. Cox PH and Kaplan-Meier analyses documented respectively that patients not treated with BCG had a significantly higher risk of developing AD and a significantly different AD-free survival (HR 4.778, 95%CI: 2.837–8.046, p = 4.08x10-9 and Log Rank Chi-square 42.438, df = 1, p = 7.30x10-11) compared to BCG treated patients (Figs 1 and 2). This happened despite the significantly older age of patients given BCG (Table 1). Exposure to BCG did not change the risk of developing Parkinson’s disease.

The incidence of AD (after deleting all the patients censored before the first case of AD; Table 2) rose to 6%, which is in the range reported in the general population at this age [19].

Male and Female populations differ in both the incidence of bladder cancer and AD. AD affects women more commonly while bladder cancer is more common in men [20]. Stratifying the population for gender, we found a highly significant effect of BCG in both men (Log Rank Chi-Square of 35.162, df = 1, p = 3.03x10-9) and women AD-free survival (Log Rank Chi-Square of 6.735, df = 1, p = 0.00945) (S1 and S2 Figs). AD incidence was not higher in females compared to males (10/182 = 5.5% vs 55/886 = 6.2%). This may indicate that this population is unique. However, the small number of female patients and likewise the small number of AD cases among females preclude a definite conclusion.

BCG, the tuberculosis vaccine, has been used by urologists for many years as an anti-cancer medication, but it also appears to show activity against other diseases. It is well recognized nowadays that immunization with living organisms has what is termed “off-target” effects, which in different populations can be dramatic. Aaby et al., in a randomized trial conducted in Guinea-Bissau, showed that immunization with BCG reduced neonatal mortality in low birthweight neonates by 50% [21]. This effect is not fully understood, but induction of innate immune memory and heterologous lymphocyte activation have been mentioned as possible explanations [22].

How can an anti-cancer agent instilled into the bladder exert an impact outside the bladder? Does BCG given intravesically provoke a systemic immune response? The answer is yes; a tenfold rise in the serum levels of IL-2 and a fivefold increase in IFNγ were documented at 5–6 weeks after intravesical BCG instillation. This was accompanied by a threefold increase in BCG induced killer-cell activity manifested by peripheral blood mononuclear cells [23].

Is there any other evidence connecting BCG immunization to AD prevention? Yes, there are epidemiological observations as well as experiments in an animal model. A strong inverse association was found between AD prevalence and BCG vaccination in countries where it is routinely used [13]. BCG administration was stopped in the U.S. in the early 60's. Might this have contributed to the rise in AD we currently notice? [13]. APP/PS1 mice provide a transgenic model of AD. These mice develop the deposition of amyloid plaques in the brain at six weeks of age. BCG immunization inhibits both brain pathology and cognitive dysfunction in this model [16]. BCG treatment even reversed cognitive decline. BCG-treated APP/PS1 mice showed recruitment of inflammation-resolving monocytes across the choroid plexus and perivascular spaces to cerebral sites of plaque pathology, increased circulating IFNγ levels, upregulation of cerebral anti-inflammatory cytokine levels and elevated expression of neutrophic factors in the brain. In closer proximity to bladder cancer, a recent large Surveillance, Epidemiology, and End Results (SEER) based study reviewed the records of 23, 932-bladder cancer patients [24]. They found that BCG treated patients had better disease specific survival (HR, 0.9; CI, 0.8 to 1.01), but the effect of BCG on overall survival was even greater (HR, 0.87; CI, 0.83 to 0.92). The authors acknowledge that this could be due to an "unmeasured factor" and could represent an "off target" effect of BCG.

How might BCG given intravesically decrease the occurrence of AD? The exact mechanism of the BCG anti AD effect is obviously unknown. It is however, well appreciated that intra-vesical BCG increases systemic IL-2 levels [23], which expands the populations of the neuroprotective Treg cells [11] In addition, BCG increases anti-inflammatory cytokines in the brain and therefore reduces neuro-inflammation which is one of the three major pathological features of AD [9, 16]. In the APP/PS1 mouse model, amplification of these cells by peripheral IL-2 decreased plaque formation and restored cognitive function [16]. Changes in the blood-cerebrospinal fluid barrier found in patients with mild cognitive impairment may facilitate the entry of systemic IL-2 into the nervous system [25]. Neonatal BCG vaccination has been shown to induce on the one hand anti-inflammatory meningeal macrophage M2 polarization and neurotrophic factor expression that were T lymphocytes and Il-10 dependent and on the other hand neuroprotective Tregs in models of neurodegenerative diseases [10, 26, 27].

Does this effect result from the direct activity of BCG on the immune system or indirectly by inducing changes in the microbiota? To the best of our knowledge, there is no definitive data on the effect of BCG on the microbiota in animals or in man. Some data on the bladder ecosystem were recently reported pointing to the importance of Proteobacteria in bladder cancer recurrence [28]. It has been shown in a Specific Pathogen Free (SPF) APP/PS1 transgenic mouse model that there is a shift in the gut microbiota with an increase of Proteobacteria compared to non-transgenic wild-type mice [29]. In addition, Germ Free APP/PS1 mice did not seem to develop the same level of Aβ amyloid as their SPF counterpart. When repopulated with the microbiota of the conventionally raised APP/PS1 transgenic mice, their cerebral level of Aβ amyloid increased substantially [30].

Limitations of the study

The study presented here has several potential confounding caveats:

Selection bias. More fit patients may have been given BCG more often than were the less fit patients. One cannot rule out the possibility that the treating urologist ruled out BCG treatment in the frailer (and possibly more prone to AD) patients.

The large differences in the AD risk of bladder cancer patients reported here and in the USA may be due to the fact that 60% of the bladder cancer patients at Hadassah Hospital received BCG whereas in the USA probably less than 20% of all bladder cancer patients received BCG [31], masking its effect in large retrospective studies.

Bias due to high percentage of censored data. Only 4.7% of the patients developed AD in the observed population meaning that 95.3% of the cases were censored in the Cox PH analysis. This number declined to 93.9% in the Kaplan-Meier analysis after removing the 303 cases that were censored at an age younger than the age of the first AD event (71 years). A high percentage of censored events may increase the risk of bias [32]. On the other hand, the Kaplan-Meier survival curves never crossed each other mitigating against bias from this source (Figs 1 and 2).

Bias due to a lower risk of AD in cancer survivors. Several reports including a large cohort study and two prospective studies showed that the risk of AD is reduced by 33–35% in cancer patients [33, 34]. In one study, late-stage cancer was associated with an even lower risk of AD (HR = 0.50, 95%CI: 0.27, 0.94) [35]. This may result from metabolic differences between AD and cancer patients: Cancer = upregulation of glycolysis; AD = upregulation of oxidative phosphorylation [36]. The beneficial effect of BCG in our study was much stronger: HR = 4.778 for AD risk in non-BCG treated patients compared to treated patients. Since all patients in the current study were cancer survivors, we can hypothesize that BCG had a significant additive role in lowering the AD risk. Still one cannot rule out the possibility that BCG survivors have a yet unexplained reduced risk for AD that is not directly due to the effect of BCG.

Bias due to the retrospective design of the study and reliance on the ICD coding system. The diagnosis of AD in the current study was based on hospital ICD coding and not on more accurate measures such as mental tests or imaging studies. This shortcoming should act similarly on both recipients and non-recipients of BCG and should not modify the results largely.

Bias due to the different sensitivity for diagnosis of AD in different hospital departments. Diagnosing AD is often difficult especially in emergency admissions or when patient’s history is not available. Some AD cases could have been missed in this way. This bias should also act similarly on recipients and non-recipients of BCG.

Men and women presented similar rates of incidence of AD. This differs from the standard reported higher rate of AD in women. It could be due to the lesser representation of women in the study but could also suggest some other unique characteristics of the studied population.

A dose-dependent relationship between the number of BCG instillations and the risk of AD was not established. This was due to the small number of patients with AD in the BCG group. The absence of dose-dependent relations decreases the strength of our conclusions.

A Mantoux test or other tests of lymphocyte responsiveness to BCG were not done. These tests are not part of the routine management of bladder cancer patients today. Therefore, we do not know which patient responded immunologically to BCG.

In summary, we showed that BCG given intravesically to patients with bladder cancer lowered their risk of developing AD by more than fourfold. This is only a single observation found retrospectively in a unique population. There is however, a plausible biological explanation for this phenomenon and supporting findings are found in epidemiological studies and in an animal model of AD. We hope that the results presented here will stimulate studies in other populations and further work on the mechanism of BCG protection will be done. A prospective BCG vaccination study of elderly subjects can then be envisioned with different doses of BCG and a continuous follow-up of cognitive capacities. Obviously, a preventive strategy would not be via a bladder administered vaccination. The intra dermic route or an oral route with an appropriate formulation should be considered [37].

AD and Age distribution of patients (Male and Female) not treated or treated with BCG.

(DOCX)

Click here for additional data file.

AD and Age distribution of patients (Male only) not treated or treated with BCG.

(DOCX)

Click here for additional data file.

AD and Age distribution of patients (Female only) not treated or treated with BCG.

(DOCX)

Click here for additional data file.

Kaplan–Meier survival curves of the AD-free female patients according to treatment (BG vs. No BCG) and to agea.

aLog Rank: Chi-Square 6.735, df = 1, p = 0.00945.

(DOCX)

Click here for additional data file.

Kaplan–Meier survival curves of the AD-free male patients according to treatment (BCG vs. No BCG) and to agea.

(DOCX)

Click here for additional data file.

Kaplan–Meier survival curves of the AD-free male and female patients not treated with BCGa.

aLog Rank: Chi-Square 35.162, df = 1, p = 3.03x10-9.

(DOCX)

Click here for additional data file.

Kaplan–Meier survival curves of the AD-free male and female patients treated with BCGa.

aLog Rank: Chi-square 0.433, df = 1, p = 0.511.

(DOCX)

Click here for additional data file.

Excel.

(XLSX)

Click here for additional data file.

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19 Sep 2019

PONE-D-19-23671

Bacillus Calmette-Guérin (BCG) therapy lowers the incidence of Alzheimer’s disease in bladder cancer patients

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Reviewer #1: The authors reported that Bacillus Calmette-Guérin (BCG) could decrease the risk of Alzheimer’s disease, through modulating immune system. The observed data are from a total of 1371 bladder cancer patients. It is very interesting, and will provide valuable clue for immunotherapy in Alzheimer’s disease.

1. Due to the different sensitivity for bladder cancer patients, BCG therapy will fail in 37% to 45% of patients treated over a 2-year span (PMID: 28801026). Is this affect the conclusion in the present study ? Could you give results of cytokine levels in the BCG treated patients? Such as IFN-gamma, IL-2, TNF-a, chemotactic factors and so on.

2. The dose and injection times of BCG should be presented and analyzed in this study.

3. Could you add some potential mechanisms for BCG-related reducation of AD risk in bladder cancer patients in the disscussion section ?

Reviewer #2: This study was conducted in 1371 non-muscle invasive bladder cancer patients with or without BCG treatment followed by long time follow up. The data reveal a significant benefit of BCG treatment in bladder cancer patients in prevention of AD development. The findings and conclusion are clear and straight forward. Moreover, the discussion and statistical analysis are complete and comprehensive.

1. The findings support current understanding on the beneficial effects of BCG in prevention of AD development.

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2. Our internal editors have looked over your manuscript and determined that it may be within the scope of our Early Diagnosis and Treatment of Alzheimer's Disease Call for Papers. This collection of papers is headed by a team of Guest Editors for PLOS ONE: Michael Weiner, Roberta Brinton, Jussi Tohka and Yona Levites. With this Collection we hope to bring together researchers working on a wide range of disciplines, from molecular and preclinical work, through to patient-centered studies, including clinical trials.   Additional information can be found on our announcement page: https://collections.plos.org/s/alzheimersdisease. If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. Agreeing to be part of the call-for-papers will not affect the date your manuscript is published. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.

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4. Thank you for stating the following in the Competing Interests/Financial Disclosure * (delete as necessary) section:

"CLG got a grant from the Alzheimer's Germ Quest, Inc. https://alzgerm.org. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We note that you received funding from a commercial source: [Name of Company]

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

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8 Oct 2019

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS ONE

Dear Editor

Thank you for your letter and the comments regarding PONE-D-19-23671, ”Bacillus Calmette-Guérin (BCG) therapy lowers the incidence of Alzheimer’s disease in bladder cancer patients” and for the opportunity to submit a revised version. Here are our responses to the comments:

Comment 1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Response: We wish to thank the reviewers.

Comment 2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Response: We wish to thank the reviewers.

Comment 3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Response: We wish to thank the reviewers.

Comment 4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Response: We wish to thank the reviewers.

Comment 5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors reported that Bacillus Calmette-Guérin (BCG) could decrease the risk of Alzheimer’s disease, through modulating immune system. The observed data are from a total of 1371 bladder cancer patients. It is very interesting, and will provide valuable clue for immunotherapy in Alzheimer’s disease.

Comment 1. Due to the different sensitivity for bladder cancer patients, BCG therapy will fail in 37% to 45% of patients treated over a 2-year span (PMID: 28801026). Is this affect the conclusion in the present study ? Could you give results of cytokine levels in the BCG treated patients? Such as IFN-gamma, IL-2, TNF-a, chemotactic factors and so on.

Response: Indeed, the response to BCG is not complete and there are patients with disease recurrence or progression after BCG and nobody knows if it relates to the immune response per se to BCG. Nevertheless, as shown in the graph below, the overall survival of patient given BCG was significantly better (p<0.0001 Log-Rank test) than that of patients not given (Fig. #1 below). This was mainly due to better disease specific survival (p<0.0001 Log-Rank test, Fig. #2, below), since patients with muscle-invasive disease were not candidate for BCG. The final result is that patients given BCG were older at the end of the observational period (mean 78.8±10.8 vs. 75.9±13.4, p=0.0005) and therefore, were at a higher risk of developing AD compared to untreated patients. This important point strengthens the conclusions and was added to the results section (to Table 1) and to the discussion. As our study was retrospective (starting 20 years ago), cytokine levels were never evaluated in bladder cancer patients. In future prospective studies, the determination of levels of relevant cytokines should definitely be included.

Fig.#1

Fig. #2

Comment 2. The dose and injection times of BCG should be presented and analyzed in this study.

Response: OncoTICE BCG 12.5mg per vial containing 2-8 x 108 CFU Tice BCG was used throughout the study. This was added to the “patient’s management” section. treatment schedule is presented in this section.

Comment 3. Could you add some potential mechanisms for BCG-related reduction of AD risk in bladder cancer patients in the discussion section?

Response: We added two references [25 and 26] to the discussion of the potential impact of BCG on AD course. Laćan et al (ref #26) showed that BCG induces Tregs cells. In addition, BCG increases anti-inflammatory cytokine levels in the brain and therefore reduces neuro-inflammation which is one of the three major pathological features of AD [16]. Furthermore, neonatal BCG vaccination has been shown on one hand to induce anti-inflammatory meningeal macrophage M2 polarization and neurotrophic factor expression that were T lymphocytes and Il-10 dependent and on the other hand neuroprotective Tregs in models of neurodegenerative diseases [ref #10, 25, 26].

Reviewer #2: This study was conducted in 1371 non-muscle invasive bladder cancer patients with or without BCG treatment followed by long time follow up. The data reveal a significant benefit of BCG treatment in bladder cancer patients in prevention of AD development. The findings and conclusion are clear and straight forward. Moreover, the discussion and statistical analysis are complete and comprehensive.

Comment 1. The findings support current understanding on the beneficial effects of BCG in prevention of AD development.

Response: We wish to thank the reviewer.

Comment 2. The figure legends of Fig. 1 and 2 are not shown.

Response: The legends appear below the figures (as requested for an initial draft). We will change this in the final submission.

Comment 3. A few grammar errors: e.g. p. 11 line 105 “effects and on the course.”

Response: A thorough revision of the manuscript by a native English speaker (Irun R. Cohen) was done again.

________________________________________

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Reviewer #1: Yes: Zhibin Yao and Fangfang Qi

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Response: This was done.

We would appreciate receiving your revised manuscript by December, 2020 !!!!. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Response: See below what is stated about the funding body.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: This was done

2. Our internal editors have looked over your manuscript and determined that it may be within the scope of our Early Diagnosis and Treatment of Alzheimer's Disease Call for Papers. This collection of papers is headed by a team of Guest Editors for PLOS ONE: Michael Weiner, Roberta Brinton, Jussi Tohka and Yona Levites. With this Collection we hope to bring together researchers working on a wide range of disciplines, from molecular and preclinical work, through to patient-centered studies, including clinical trials. Additional information can be found on our announcement page: https://collections.plos.org/s/alzheimersdisease. If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. Agreeing to be part of the call-for-papers will not affect the date your manuscript is published. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.

Response: We agree for including the manuscript in the collection.

3. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study. Specifically, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Response: All data were fully anonymized before analysis and the ethics committee waived the requirement for informed consent. This was added to the “Methods”.

4. Thank you for stating the following in the Financial Disclosure section:

Response: Charles L. Greenblatt got a grant from the Alzheimer's Germ Quest, Inc. https://alzgerm.org. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We note that you received funding from a commercial source: [Name of Company]

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

Response: Alzheimer’s Germ Quest, Inc. is a privately held company, founded in 2017, and controlled by Dr. Leslie Norins and his wife, Rainey. This company provides small grants (10,000 $ in our case) to investigate the idea of the possibility that AD was actually an infection of an unusual type without any request to the granted researchers. It is incorporated in Florida, as a public benefit corporation. “Public benefit” indicates that one of its charter purposes is to help the citizenry, in this case accelerating and intensifying the search for infectious agents as root causes of AD. This firm is completely independent and not affiliated with or endorsed by any government agency, nonprofit group, pharmaceutical company, or other entity. Donations from the public are neither solicited nor accepted. The firm’s capital and operating funds come from its founders. Therefore, there is no Competing Interests and this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Response: This was done.

Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

Response: As stated, there are no Competing Interests.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests.

Finally, our colleague who performed most of the statistics is an Emeritus Professor who does not want to appear in publications in general. Therefore, we added him (with his agreement) in the Acknowledgments. His last request, after seeing that the paper was under revision, was to remain anonymous and therefore we changed slightly the Acknowledgments to respect his will as follow:

We wish to thank a member of the Department of Developmental Biology and Cancer Research, expert in mathematical biological models, Faculty of Medicine, Hebrew University of Jerusalem, who advised us on sampling size, performed the Kaplan-Meier and Cox Proportional Hazards analyses and reviewed the article. We thank Naseem Maalouf for the preparation of the graphs done on GraphPad Prism software version 6.0.

Sincerely

Ofer N. Gofrit and Herve Bercovier

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Oct 2019

Bacillus Calmette-Guérin (BCG) therapy lowers the incidence of Alzheimer’s disease in bladder cancer patients

PONE-D-19-23671R1

Dear Dr. Gofrit,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS ONE

30 Oct 2019

PONE-D-19-23671R1

Bacillus Calmette-Guérin (BCG) therapy lowers the incidence of Alzheimer’s disease in bladder cancer patients

Dear Dr. Gofrit:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stephen D Ginsberg

Section Editor

PLOS ONE