RevIews

Mechanisms of BCG immunotherapy

and its outlook for bladder cancer
Caroline Pettenati1 and Molly A. Ingersoll 2,3
*
Abstract | BCG immunotherapy is the gold-standard treatment for non-muscle-invasive bladder
cancer at high risk of recurrence or progression. Preclinical and clinical studies have revealed that
a robust inflammatory response to BCG involves several steps: attachment of BCG;
internalization of BCG into resident immune cells, normal cells, and tumour urothelial cells;
BCG-mediated induction of innate immunity, which is orchestrated by a cellular and cytokine
milieu; and BCG-mediated initiation of tumour-specific immunity. As an added layer of
complexity, variation between clinical BCG strains might influence development of tumour
immunity. However, more than 40 years after the first use of BCG for bladder cancer, many
questions regarding its mechanism of action remain unanswered. Clearly, a better understanding
of the mechanisms underlying BCG-mediated tumour immunity could lead to improved efficacy,
increased tolerance of treatment, and identification of novel immune-based therapies. Indeed,
enthusiasm for bladder cancer immunotherapy, and the possibility of combining BCG with other
therapies, is increasing owing to the availability of targeted immunotherapies, including
checkpoint inhibitors. Understanding of the mechanism of action of BCG immunotherapy has
advanced greatly, but many questions remain, and further basic and clinical research efforts are
needed to develop new treatment strategies for patients with bladder cancer.

1
Department of Urology,
Hôpital Européen Georges
Pompidou, AP-HP,
Paris Descartes University,
Paris, France.
2
Unit of Dendritic Cell
Immunobiology,
Department of Immunology,
Institut Pasteur,
Paris, France.
3
Inserm U1223,
Paris, France.
*e-mail: molly.ingersoll@
pasteur.fr
https://doi.org/10.1038/
s41585-018-0055-4
Bladder cancer is the ninth most common malignancy
worldwide and the fifth most common in Europe and
the USA, with the highest incidence found in southern
European countries, such as Spain and Italy1,2. Bladder
cancer shows a strong sex bias, with approximately 75%
of patients being men, although this figure varies from
country to country2. Approximately 20% of patients are
diagnosed with muscle-invasive disease, which repre-
sents a diverse group of molecularly distinct subtypes
of cancer3–6. Treatment options include chemotherapy
and radical cystectomy to remove the bladder, and
although patient prognosis can be poor in the case
of non-organ-confined or metastatic disease, new
­immunotherapy approaches are improving outcomes7–10.
The vast majority of patients, ~80%, present with
non-muscle-invasive bladder cancer (NMIBC) 11.
Prognosis of these patients is considerably better than
that of patients with muscle-invasive bladder cancer
(MIBC), but NMIBC is associated with risks of recur-
rence (31–78%) and progression to MIBC (1–45%) at
5 years12,13. Treatment of NMIBC includes endoscopic
surgical removal of the tumour and/or tumours, and,
depending on the grade and other risk-associated fac-
tors (such as tumour size or multifocality) subsequent
adjuvant intravesical treatment can be administered
to reduce the potential for recurrence or progression.
The risk of recurrence and progression are influenced
by many factors, including tumour grade, size, stage
and multiplicity, recurrence rate, and the presence
of carcinoma in situ (CIS). Risk tables, such as those
developed by the European Organisation for Research
and Treatment of Cancer (EORTC), help physicians to
­estimate the risk of recurrence and progression13.
Use of intravesical immunotherapy with BCG, a live,
attenuated strain of Mycobacterium bovis used for vacci-
nation against tuberculosis, was first described in humans
in 1976 by Morales and colleagues (Fig. 1). BCG immuno­
therapy is the gold-standard adjuvant treatment for
NMIBC with a high risk of progression (that is, stage T1
tumours, high-grade carcinoma, CIS, and multiple and
recurring stage Ta tumours >3 cm) and is also recom-
mended for treatment of intermediate-risk NMIBC14–16.
Despite many unknowns regarding its mechanism of
action, BCG immunotherapy induces a durable and effec-
tive antitumour immune response and results in positive
clinical outcomes. As new, targeted immunotherapies
for many tumour types emerge and gain approval for use
in humans, revisiting the mechanisms of BCG-induced
cancer immunity could guide the development of specific
therapies to effectively harness the immune system.
In this Review, we describe current knowledge of
the induction of durable adaptive immune responses by

Nature Reviews | Urology

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews
Key points
• BCG immunotherapy is the gold-standard treatment for high-risk non-muscle-invasive
bladder cancer (NMIBC) to prevent disease recurrence and progression.
• BCG induces a robust innate immune response over several weeks that leads to
lasting antitumour adaptive immunity.
• Different BCG substrains are in clinical use around the world, but whether these
strains have varying efficacies in the induction of tumour immunity is unclear.
• Efforts to improve BCG immunotherapy have largely failed, and to date, no existing
therapy outperforms BCG for treatment of high-risk NMIBC.
• New approaches, which incorporate novel immunotherapies such as checkpoint
inhibitor antibodies, might successfully synergize with BCG to improve patient
outcomes.
BCG immunotherapy derived from clinical trials and
preclinical research. We discuss efforts to augment BCG
immunotherapy, including the development of combi-
nation treatment protocols, and highlight avenues for
future research.
Gold standard for high-risk NMIBC
BCG intravesical therapy entails an induction schedule
of 6 weekly instillations starting a few weeks after tumour
resection, which is followed by a maintenance schedule
of additional BCG instillations every 3–6 months over
1–3 years17. BCG immunotherapy induces initial com-
plete response rates of 55–65% for high-risk papillary
tumours and 70–75% for CIS17–23. Conversely, despite
high success rates, as many as 25–45% of patients will
not benefit from therapy, and an additional 40% of
patients will eventually relapse despite BCG immuno-
therapy showing initial success17,18,23. In addition, BCG
immunotherapy is associated with many adverse effects,
which — although minor — can lead to intolerance
(20%) and shortened or altered treatment schedules24,25.
Maintenance schedules vary considerably between
published studies and institutions and even vary accord-
ing to patient tolerance and compliance with treatment,
and many of these variations have been tested in clinical
studies. Specific variables tested include the number of
instillations and the dose of BCG26. The SWOG 8507
protocol of 6 instillations over 6 weeks followed by a
maintenance schedule of weekly BCG instillations for
3 weeks at 3 months, 6 months, 12 months, 18 months,
24 months, 30 months, and 36 months induced supe-
rior protection against recurrence and progression
compared with induction treatment alone17. An EORTC
study showed that, in patients with high-risk disease, a
full-dose induction course and a 3-year BCG mainte-
nance schedule, according to the SWOG 8507 proto-
col, was associated with reduced recurrence compared
with a full-dose induction course and a 1-year main-
tenance schedule, but no long-term differences were
found between the groups for progression or death27.
Another EORTC study showed that in patients with
intermediate-risk disease, induction therapy followed
by a 3-week maintenance schedule of BCG signifi-
cantly reduced metastasis and cancer-related death
compared with induction and a 3-week maintenance
schedule of intravesical epirubicin28. Efforts are under-
way to determine whether reducing the number of
BCG instillations during the induction phase will also
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
be efficacious (NIMBUS trial (NTR4011; EudraCT
2010-019181-91))29.
Patients who do not respond to BCG therapy can be
classified into three groups: BCG refractory, BCG relaps-
ing, or BCG intolerant30. The distinctions between these
groups are important, as each classification represents
a different response following therapy: BCG refractory
is the persistence of high-grade tumours after induc-
tion and one maintenance course of BCG; BCG relapse
is the reappearance of a tumour after a disease-free
state; and BCG intolerance is the inability to tolerate
at least one full induction course of BCG. These dif-
ferent groups require different follow-up treatment
strategies15,16,30–32. For patients in any of the three non­
responder groups, radical cystectomy is typically recom-
mended for treatment of progressive disease, but several
bladder-preserving strategies have been developed.
These strategies, some of which are still in the experi-
mental phase, include immunotherapy, chemotherapy,
device-assisted therapies, radiotherapy, and treatment
with genetically engineered adenovirus, toxin–protein
conjugates, nanotechnologies, and engineered non-live
BCG30,32,33. Multiple trials are ongoing, with some show-
ing promising early results in selected patients who do
not respond to BCG, but long-term results are still some
way off. Consequently, the conservative approach of
bladder preservation is currently considered oncolog-
ically inferior to radical cystectomy, the gold-standard
treatment for patients who do not respond to BCG15,16.
Finally, owing to the potential contamination of stock,
Sanofi halted BCG production in 2012 and subsequently
left the market entirely in mid-2017. The discontinua-
tion of production, followed by a second BCG shortage
in 2014, profoundly affected patient treatment until
2015 (Ref.34). During the BCG shortage, patients with
bladder cancer received reduced doses of BCG or alter-
native adjuvant chemotherapy, such as mitomycin C34.
Notably, the price of mitomycin C increased dramati-
cally starting in 2014 (Refs34,35), suggesting that the BCG
shortage affected more than just patient standard of care.
Improved understanding of the immunobiology of
BCG-induced tumour immunity is, therefore, necessary,
to improve efficacy, reduce intolerance, anticipate failure,
and ultimately apply this knowledge to the d ­ evelopment
of new efficacious treatments to replace BCG.
BCG-mediated tumour immunity
The mechanisms by which BCG immunotherapy medi-
ates tumour immunity have been widely studied, but
they are still not completely understood. Findings from
preclinical and clinical studies demonstrate that a robust
local inflammatory response to BCG involves several
steps (Fig. 2).
BCG attachment to the urothelium. Animal studies
suggest that BCG binds to the urothelium through the
interaction between molecules expressed in the bacte-
rial wall and fibronectin in the urothelium36–44. Bevers
and colleagues36 described two possible mechanisms:
a physico­chemical interaction following damage of the
glycosaminoglycan layer, allowing BCG closer access to
the bladder wall, and a specific receptor–ligand-mediated
www.nature.com/nrurol
 Reviews

William Coley, the father of does not support this conclusion. Indeed, this concept
immunotherapy, established that will be difficult to test in humans, and ­mechanisms of
microorganisms have therapeutic attachment remain poorly understood.
benefit for cancer patients, 1893
leading to the testing of many Raymond Pearl investigated over
bacteria, including BCG, although 1,600 cadavers, reporting that BCG internalization. Whether BCG is taken up in
Coley’s toxins did not contain 'tuberculous lesions' were more the bladder remains unclear. BCG can be found in the
mycobacterium160. than twice as prevalent in cadavers
1928 urine of both mice and humans in the hours following
without tumours compared with
those with malignancy, leading to intravesical instillation48,49. Notably, the amount of BCG
Lloyd Old and Donald Clarke the idea of tuberculin as a therapy decreases rapidly in mouse urine in the 24 hours after
challenged mice with tumour cells for cancer161.
at specific intervals following BCG 1959 instillation48. In humans, BCG disappeared rapidly with
administration and observed that similar kinetics: 96% of patient urine contained BCG
mice receiving BCG at least 7 days 2 hours after instillation, and only 16% of urine samples
before tumour challenge were Analysis of 287 death certificates
protected from developing cancer162. 1970 revealed that leukaemia incidence were positive 6 days later49. In the same study, the presence
in children aged <15 years was two of mycobacterial DNA also decreased over time, sug-
Guinea pigs, sensitized to BCG or
times higher in nonvaccinated gesting that BCG does not persist in the tissue; however,
individuals than in those vaccinated
not, were challenged intradermally 1971 with BCG163. further study is needed to support this conclusion49.
with a hepatocellular carcinoma cell In the context of uropathogenic Escherichia coli
line; intralesional injection of BCG (UPEC) infection of the urinary tract, tissue-resident
induced tumour regression and
prevented lymph node metastasis A study in a Chicago population macrophages take up the bacteria within hours of
1972
only in sensitized animals164. demonstrated that leukaemia experimental instillation, before neutrophil infiltra-
rates were approximately
2.00/100,000/year in unvaccinated
tion50. UPEC can also be found inside urothelial cells;
Berton Zhar described the ideal children and 0.31/100,000/year in the bacteria mediate invasion and in doing so cause
conditions for BCG therapy for 1974 BCG-vaccinated children165. considerable tissue damage51,52. Extrapolating from the
cancer, in which the bacteria must example of UPEC, BCG probably encounters urothe-
be alive and in close proximity to
the malignancy and the tumour lial cells first, and in the event of tissue damage, BCG
burden must be small166. 1976 Alvaro Morales first described the is exposed to bladder-resident macrophages, which are
use of BCG intravesical instillation,
in which 7/10 patients with postulated to reside in the intermediate urothelial cell
A randomized controlled trial recurrent NMIBC were tumour-free layer53. Internalization of BCG by urothelial cells is con-
confirmed the positive impact of 1980 at follow-up14. troversial, and the pathway by which BCG enters bladder
BCG in preventing bladder tumour cancer cells is not well understood. Redelman-Sidi and
recurrences167.
colleagues54 showed that bladder cancer cell lines differed
The FDA approved the use of in their susceptibility to BCG infection owing to muta-
1990 intravesical BCG for NMIBC.
tions in the PTEN–PI3K, RAS, and CDC42–RAC1–
PAK1 pathways, in which decreased PTEN expression
and oncogenic activation of the RAS and PAK1 pathways
increased BCG uptake by bladder cancer cells through
increased macropinocytosis. Similar to a mechanism
Fig. 1 | A brief history of BCg and cancer. A timeline showing the steps leading to the described for UPEC55, BCG might be internalized via
FDA approval of the use of intravesical BCG for patients with non-muscle-invasive receptor-mediated interactions with integrins56. BCG
bladder cancer (NMIBC) is presented.
attachment and internalization into bladder cancer cells
are dependent on fibronectin opsonization of BCG and
attachment via fibronectin. Antigen 85 and fibronec- are inhibited by anti-β1 integrin and anti-α5 integrin sub-
tin attachment protein (FAP), expressed by BCG, are unit antibodies56. Some studies have suggested that nor-
thought to be crucial for attachment of BCG to the mal urothelial cells cannot internalize BCG, but tumour
urothelium37–41. As fibronectin is found in the basement cells, particularly those from high-grade lesions, can take
membrane and the submucosa of the bladder wall, up bacteria43,57–60. Teppema et al.43 described BCG inter-
fibronectin-mediated attachment of BCG might occur nalization via a phagocytic mechanism into the human
preferentially in the presence of a disrupted urothelium, T24 bladder carcinoma cell line, which was also used by
such as at electrocauterization sites where fibrin clots Redelman-Sidi and co-workers43,54. Importantly, internal-
form40,42–44. Supporting this hypothesis, patients tak- ization studies are performed almost exclusively in vitro
ing the fibrin clot inhibitor warfarin had a higher risk or in animals and should be extended to human stud-
of recurrence and progression after BCG therapy than ies. Notably, BCG immunotherapy is given a few weeks
those not on warfarin45. However, patients from the after transurethral resection, at which time the patient
same study who were taking aspirin were at reduced risk is assumed to have little to no residual tumour burden
of recurrence and progression compared with those not or extensive epithelial damage. Thus, whether an in vivo
taking aspirin, complicating interpretation of the results. role for BCG internalization by tumour cells or urothelial
In addition, two other studies failed to demonstrate a cells exists in the context of therapy remains unknown.
significant impact of fibrin clot inhibitors in patients
treated with BCG compared with those not receiving Induction of innate immune responses. BCG immuno­
these drugs in the course of their therapy46,47. Thus, while therapy induces both local and systemic immune
preclinical mouse studies suggest that fibronectin has a responses. Intravesical BCG instillation induces the
role in bacterial attachment, indirect evidence in humans activation of urothelial cells and antigen-presenting

Nature Reviews | Urology

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews

Prevention of
recurrence and
5 progression

Recurrence or
progression
4b

TH1 cell bias TH2 cell bias

Induction
Attachment of adaptive Combination
1 4a responses immunotherapy?
Repeated
instillations

Failure to
induce adaptive
3b immunity
Robust Poor immune
infiltration infiltration
Induction
Invasion of innate
2 3a responses Combination
immunotherapy?

Fig. 2 | BCg-induced tumour immunity. Following BCG instillation, BCG is thought to attach to (step 1) and invade
(step 2) the urothelium. BCG therapy induces an innate immune response (step 3a), including cytokine expression and
immune cell infiltration, which increases with each instillation (circular arrow). Failure to induce infiltration would lead to
inefficient adaptive immune responses (step 3b). A robust innate response is necessary to support a strong T helper 1 (TH1)
cell-biased adaptive immune response (step 4a). A TH2 cell-biased T cell response does not provide adequate protection
(step 4b). Together, appropriate host responses lead to lasting protection against tumour recurrence and progression
(step 5). Novel immunotherapeutic approaches used in combination with BCG (teal boxes), might help to reverse
ineffective immune responses. Dotted boxes indicate steps supported by in vitro and preclinical studies, although data
from human studies are needed.

cells (APCs), which produce cytokines and chemokines
that attract granulocytes and mononuclear cells to the
bladder61. These events are characterized by the typical
epithelioid and gigantocellular granulomas found in
the bladder wall after BCG instillation, which contain
macrophages, dendritic cells, lymphocytes, neutrophils,
and fibroblasts61–64. In vitro studies using human urothe-
lial carcinoma cell lines demonstrate that BCG induces
upregulation of cytokine production, including IL-6,
IL-8, granulocyte–macrophage colony-stimulating fac-
tor (GM-CSF) and tumour necrosis factor (TNF)59,65,66.
In human studies, cytokines and chemokines found in
the urine following BCG instillation include IL-1β, IL-8,
IL-15, IL-18, CXC-chemokine ligand 10 (CXCL10),
GM-CSF, CC-chemokine ligand 2 (CCL2), and CCL3
(Refs66–69). Urinary levels of these cytokines and chemok-
ines are detectable within the first 24 hours after BCG
instillation, with peak expression after 2–8 hours61,66.
Interestingly, BCG can induce the expression of the
major histocompatibility complex (MHC) class II on
urothelial cells, perhaps indicating that they have a role
as APCs43,60,70–72.
Following uptake by professional APCs and puta-
tive internalization by urothelial cells, BCG-induced
cytokine and chemokine expression leads to immune
cell recruitment. Immune cells found in the bladder and
urine after BCG immunotherapy include neutrophils,
monocytes, macrophages, T cells, B cells, and natural
killer (NK) cells66,73–79. Repeated instillations trigger
a robust immune response that increases in intensity
over the course of therapy66,77. Bisiaux and colleagues66
described this event as a prime–boost mechanism
of the innate immune response, which is compara-
ble to the memory capacity of the adaptive immune
system: the first instillation primes and the following
instillations boost the innate response to BCG.
Neutrophils have multiple roles in the response to
BCG immunotherapy. In a mouse model of bladder
cancer, depletion of neutrophils abolished monocyte
and CD4+ T cell infiltration into the bladder, abrogated
the therapeutic effect of BCG, and reduced survival
to the level of untreated mice73. Neutrophils might also
act directly as antitumour effector cells, as they are
phagocytic, generate reactive oxygen intermediates,
and release lytic enzymes with antimicrobial poten-
tial and proapoptotic factors such as TNF-related
apoptosis-inducing ligand (TRAIL; also known as
TNFSF10)80–83.
In addition to neutrophils, other cytotoxic cells found
in the bladder following BCG immunotherapy include
CD8+ T cells, NK cells, and macrophages75,78,84–86. NK
cells are part of the innate immune system and kill
tumour cells in an antigen-independent manner. The
role of NK cells in BCG-induced antitumour activity is
unclear. In vitro and in vivo studies have demonstrated
that modulating NK cell activity does not significantly
affect bladder cancer cell cytolysis or treatment efficacy
in mice79,87. However, Brandau and colleagues78 reported
reduced efficacy of BCG immunotherapy in NK
cell-depleted mice compared with nondepleted mice,
including both increased tumour growth and reduced
survival. The role of NK cells in BCG-induced cytotox-
icity is supported by additional reports, but their role in
human disease remains to be explored86,88,89.

www.nature.com/nrurol
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Induction of adaptive immunity. BCG antigens are pre-
sented on the cell surface of APCs and urothelial cells via
MHC class II61,70,71. These molecules interact with CD4+
T cell receptors, leading to activation and differentiation
to a primarily T helper 1 (TH1) cell immune response90.
Cytotoxic CD8+ T lymphocytes recognize tumour cells
through antigen presentation via MHC class I. Their
activation is facilitated by a TH1 cell cytokine environ-
ment and is mediated by IFNγ67. The balance between
T H1 cell and T H2 cell responses is consequential,
as a TH1 cell-biased response, characterized by the pro-
duction of IL-2, IL-12, IFNγ, TNF, and TNFβ, is asso-
ciated with successful BCG immunotherapy, whereas
a TH2 cell response, characterized by the production
of IL-4, IL-5, IL-6, and IL-10, has been correlated with
BCG nonresponsiveness67,91–94. The necessity of T cells
in response to BCG immunotherapy is well estab-
lished, as athymic nude mice bearing bladder tumours
show no antitumour response following BCG instilla-
tion95. In addition, depletion of either CD4+ or CD8+
T cells abrogates BCG-induced antitumour activity76.
Using tumour samples from patients who had been
treated with BCG, Pichler et al.96 observed that an
increased tumour-infiltrating CD4+ T cell count and
an increased CD4+:CD8+ T cell ratio were significantly
associated with improved patient response to BCG.
In a mouse model, Biot and colleagues48 reported that
repeated, and not single, intravesical instillations of
BCG induced robust infiltration of CD4+ and CD8+
T cells into the bladder. Mice that received subcutane-
ous vaccination of BCG before BCG instillation showed
a stronger acute inflammatory response after the first
instillation and faster T cell infiltration into the bladder
than unvaccinated mice48. This intravesical inflamma-
tory response was significantly reduced when T cells
were depleted, suggesting that pre-existing BCG-specific
T cells increase the inflammatory response during intra-
vesical BCG instillation48. Retrospective analysis of clini-
cal trial data showed that 5-year recurrence-free survival
(RFS) was significantly improved in patients with
high-risk NMIBC who had a positive purified protein
derivative (PPD) test before intravesical BCG therapy,
which is indicative of a history of exposure to BCG or
tuberculosis, compared with those with a negative PPD
skin test at the onset of treatment (80% versus 45%)48.
These data suggest that BCG vaccination before BCG
immunotherapy might improve the therapeutic response
in clinical practice. Further studies, such as the phase II
PRIME trial to assess the efficacy and safety of BCG vac-
cination before BCG induction therapy in patients with
high-grade NMIBC, are needed to assess the relevance
and the best protocol of BCG immunization before BCG
intravesical treatment97,98.
A systemic immune response also arises following
BCG therapy, as demonstrated by increased lympho­
proliferation, mycobacteria-specific humoral responses,
conversion of the PPD skin test from negative to positive,
and increased serum levels of cytokines and chemokines,
such as IFNγ, IL-1, IL-2, IL-8, TNF, CCL2, and CCL5
(Refs99–102). As a consequence of the immune response
to BCG immunotherapy, moderate-to-severe local and
systemic adverse effects, such as fever or cystitis, can be
Nature Reviews | Urology
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews
observed during the course of treatment and might be
associated with treatment efficacy103,104. The frequent
local adverse effects, such as bacterial or chemical
cystitis or haematuria, reflect a bladder inflammatory
response and mainly occur in the first 48 hours after
instillation103. Systemic adverse events include symptoms
such as mild-to-severe fever, flu-like syndrome, and
asthenia105. Rarely, severe systemic adverse effects occur,
such as a disseminated infection known as BCG-itis,
which can manifest as sepsis, pneumonitis, polyarthri-
tis or reactive arthritis, rash, meningitis, hepatitis, or
mycotic aneurism106–108. Mechanisms underlying this
rare infection are still unknown but might be related to
haematogenous spread of BCG, facilitated by disrup-
tion of the urothelial barrier, immunodeficiency, or a
type IV-hypersensitivity response109,110. Adverse effects
can occur at any time during the course of therapy,
with some reports revealing a delay of several years110.
Severe adverse effects require treatment with antituber-
culosis agents and corticosteroids and contraindicate
the further use of BCG instillations111. The 2014 EORTC
genitourinary cancers group study 30962 compared
efficacy and adverse effects after different maintenance
protocols (one-third dose versus full dose given for
1 year versus 3 years) and found that 62.0% of patients
randomly assigned to receive BCG maintenance for
1 year completed the treatment compared with 36.4% of
patients randomly assigned to receive BCG for 3 years112.
Instillations were delayed or temporarily stopped owing
to adverse effects for 15.7% of all patients. Overall, 49.4%
of patients started but did not complete the protocol.
Inefficacy and other factors such as death, ineligibility,
patient refusal, or being lost to follow-up monitoring
were the main reasons for stopping treatment. Adverse
events (local in 63.0% and systemic in 30.6% of patients)
were the reason for stopping treatment for 7.8% of
patients, including 6.2% within the first year112. Adverse
events mainly occurred during the first year of treatment
and were comparable between all groups, suggesting
that these sequelae arise independently of BCG dose112.
In a previous study, the EORTC attempted to correlate
BCG-related adverse effects with clinical outcome but
did not find any correlation113. As some of these adverse
effects arise in part as a result of the immune response
to BCG, a correlation between adverse effects and treat-
ment efficacy or patient outcomes might help to iden-
tify biomarkers of treatment response early in patients.
However, this concept requires additional study, as no
studies have conclusively demonstrated a concordance
among adverse effects, severity, and treatment efficacy.
Major questions remain. First, how does BCG, a bac-
terium, induce an antitumour response? Second, is the
antitumour response specific to tumour cells, or is it a
BCG-specific immune response, in which a side effect
is antitumour activity? No data are available to provide
a clear answer to these questions. One hint that BCG
induces specific tumour immunity comes from a 2016
study showing that animals rechallenged with tumour
cells following tumour challenge and BCG therapy
mount an adaptive response and effectively eliminate
these tumour cells114. Unravelling this puzzle is crucial to
determining whether new therapies should be aimed at
Reviews
inducing a specific tumour microenvironment, directing
a specific tumour antigen response, or both.
Do BCG strains differ therapeutically?
The antitumour response to and tolerance of BCG
immunotherapy are dependent upon the host’s
immune system. However, BCG itself might also have
a role. Since its worldwide distribution in 1924, the first
Pasteur BCG strain has evolved into many substrains
as a result of serial passage115. Today, commercialized
BCG substrains vary genetically from one another, lead-
ing to phenotypic and immunogenic differences116–120.
Furthermore, pharmaceutical formulation and the num-
ber of colony-forming units (for live BCG) per dose also
vary substantially121. From these observations emerges
the hypothesis that antitumour responses might differ
depending on which BCG substrain is used for treat-
ment. However, as only one or two BCG strains are
used in each country, comparative data on optimal dose,
efficacy, and tolerance are scarce.
By exposing human urothelial tumour cell lines to
various BCG substrains, including BCG Russia, Japan,
Moreau, Danish, Glaxo, Tice, Connaught, and Phipps
substrains, Secanella-Fandos et al.122 reported that BCG
Connaught and Russia substrains induced more IL-6 and
IL-8 production and inhibited tumour growth in vitro
more than other substrains. In an orthotopic murine
model of bladder cancer, the BCG Connaught strain
induced increased BCG-specific CD8+ T cell priming and
more robust inflammatory cell infiltration into the blad-
der compared with the BCG Tice strain123. Sequencing
of the BCG Connaught, Tice, and Pasteur genomes
revealed 35 single-nucleotide polymorphisms and 13
insertions or deletions that differed between the strains,
including a mutation in the gene encoding the Cu-Zn
superoxide dismutase (SOD1) protein in BCG Tice,
which might lead to a loss in enzyme activity123. As SOD1
protects bacilli from oxidative stress, BCG Tice might
be more susceptible to reactive oxygen species and,
therefore, less viable in vivo than other strains124,125.
In the 1990s, BCG Pasteur was withdrawn from the
market and, therefore, was discontinued in Switzerland,
providing the opportunity to directly compare the
remaining two strains in use, BCG Connaught and
BCG Tice 126. In a prospective randomized study,
Rentsch et al.123 compared tolerance and efficacy of the
Connaught and Tice BCG substrains. RFS at 5 years
was significantly higher in the Connaught group than
in the Tice group (74% versus 48%; P = 0.01). However,
progression-free survival (PFS) and overall survival did
not differ between the groups (94.1% versus 87.9% for
PFS (P = 0.34) and 84.9% versus 93.6% for overall sur-
vival (P = 0.26))123. Adverse effects were also similar in
the groups123. This study was limited by the small num-
ber of patients (131 patients analysed), the lack of data on
smoking (a risk factor for bladder cancer), and the lack
of follow-up maintenance treatment. As an additional
complication, all patients were vaccinated for BCG in
childhood without data on the strain used. Despite the
limitations and different elements that could influence
response to specific BCG substrains, the findings do
provide support for the continued investigation of BCG
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
strain differences126. Other studies have compared BCG
Tokyo and BCG Connaught strains and observed no
differences in complete response, 2-year and 5-year
RFS, or frequency of adverse effects127,128. No differences
were observed in two trials comparing the BCG Glaxo
and Pasteur strains, but these studies were limited by the
small numbers of patients and statistical biases owing to
the small number of tumour recurrences129,130. A 2016
retrospective study that compared BCG Connaught
with BCG Tice in 2,099 patients with high-grade T1
bladder tumours showed a lower recurrence rate for the
BCG Connaught strain131. However, when considering
patients who received some form of maintenance ther-
apy, BCG Tice was more effective than BCG Connaught
in terms of time to first recurrence (HR 0.66, 95% CI
0.47–0.93; P = 0.019), with no significant differences in
time to progression or overall survival131.
BCG Connaught is no longer manufactured by
Sanofi, but the strains BCG Tice and BCG RIVM
(Rijksinstituut voor Volkesgezondheid en Milieu, a
Dutch strain) are widely used. Vegt et al.132 compared
5-year RFS between patients receiving intravesical
instillations of mitomycin C, BCG Tice, or BCG RIVM
(induction treatment without maintenance) for primary
or recurrent CIS, stage Ta bladder cancer, or stage T1
bladder cancer. No statistically significant difference was
observed in 5-year RFS between BCG Tice and BCG
RIVM (36% versus 54%)132. Finally, a 2017 network
meta-analysis that compared several BCG substrains
with chemotherapy clearly showed that BCG immuno-
therapy is superior to chemotherapeutic approaches, but
differences in efficacy between BCG substrains were not
apparent121. Thus, BCG strains vary in their genetic pro-
file, cell wall components, titre, and formulation, which
all have a potential effect on efficacy. Further compar-
ative studies are needed to identify whether an optimal
strain exists.
Improving upon the success of BCG
More than 40 years after the first use of BCG in bladder
cancer, many questions remain unanswered. How BCG
stimulates an antitumour response, and whether this
response is specifically targeted or a positive side effect
of a global intravesical inflammatory response, is still
unknown. Although BCG immunotherapy is efficacious,
which patients will have a positive response is difficult
to predict, and the alternative treatment in those who
do not respond is radical cystectomy15,16,133. Given that
cystectomy is an invasive procedure associated with con-
siderable morbidity, bladder-sparing therapies are under
investigation to improve the action of BCG and/or to
treat patients who do not respond to BCG.
Combination therapy using BCG. One way to augment
the response to BCG is to combine it with other thera-
peutic approaches. IFNα has been proposed for use in
combination with BCG owing to its predicted synergy
with BCG-induced innate immune responses in order
to improve tumour-specific immunity in patients with
bladder cancer134. IFNα administered concomitantly
with BCG has not generally contributed to adverse
effects, but in one study, patients receiving IFNα2b
www.nature.com/nrurol

experienced higher frequency of fever and constitu-
tional symptoms than those treated with BCG134–136.
Importantly, none of the studies showed a significant
improvement in outcome in patients receiving BCG
and IFNα combination therapy over those receiving only
BCG134–136. Despite these negative results, more recent
efforts have investigated the use of an IFNα-expressing
adenovirus as therapy to prolong local tumour micro­
environment exposure to IFNα independently of BCG.
In a phase II trial, virus administration was well tolerated,
and durable responses were observed in a small number of
patients (n = 40) after 2 years of follow-up monitoring 137,
suggesting that IFNα-expressing adenovirus improves
patient outcomes.
Additional combinatorial approaches include genet-
ically altering BCG or administering the bacteria simul-
taneously with other cytokines, such as IL-2 or IFNγ33,138.
Many of these agents have been or are currently under
investigation in early-phase clinical trials139. However,
among the approaches that have been tested, few, if any,
have outperformed BCG therapy, which might be caused
by the high response rate observed initially following
therapy136. Research is, therefore, needed to reduce
recurrence and improve PFS in patients with NMIBC as
well as to identify the most efficacious bladder-sparing
Anti-CTLA4
antibodies
CTLA4 B7
TCR
T cell MHC APC
PD-1
PD-L1
• Nivolumab • Atezolizumab
• Avelumab
• Pembrolizumab
• Durvalumab
Fig. 3 | Immune checkpoint molecule inhibitors being tested in NMIBC. T cell
activation is regulated by various co-stimulatory and inhibitory checkpoints. Both
agonistic antibodies to activating receptors and blocking antibodies to inhibitory
receptors can stimulate T cell activity and are being tested in various solid tumours.
Activation of T cells first requires an antigen-presenting cell (APC), such as a dendritic
cell, to present an antigen. Here, an APC presents a tumour antigen complexed to major
histocompatibility complex (MHC) class I to the T cell via the T cell receptor (TCR).
Co-stimulatory signals are also needed at this time. At this point, B7 on an APC can bind
to cytotoxic T lymphocyte antigen 4 (CTLA4), creating an inhibitory signal, but
anti-CTLA4 antibodies can inhibit the inhibitory signal by binding to CTLA4. Once the
activated T cell is in the tumour environment, it can recognize the antigen presented by
an APC cell in the tumour. At this time, the programmed cell death 1 (PD-1) receptor can
also send an inhibitory signal to the T cell when the receptor binds to programmed cell
death 1 ligand 1 (PD-L1), which is often expressed on tumour cells. Inhibition of PD-L1
(for example, with atezolizumab, durvalumab, or avelumab) or PD-1 (for example, with
pembrolizumab or nivolumab) could block that signal. NMIBC, non-muscle-invasive
bladder cancer. Figure adapted from Ref.168, Macmillan Publishers Limited.
Nature Reviews | Urology
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews
strategies. Importantly, as the initial response rate
of BCG can be very high, trials addressing this chal-
lenge must be appropriately powered to compare the
­performance of newer drugs with that of BCG.
Checkpoint blockade inhibition in NMIBC. Enthusiasm
for immunotherapy in bladder cancer has increased dra-
matically following the advent of new targeted immuno-
therapies. Immune checkpoint molecule inhibitors are
leaders in this field, and include anti-cytotoxic T lympho-
cyte antigen 4 (CTLA4), anti-programmed cell death 1
(PD-1), and anti-programmed cell death 1 ligand
1 (PD-L1) antibodies (Fig. 3). CTLA4 is expressed exclu-
sively on T cells and is a competitive receptor of CD28,
which binds with an increased affinity to B7 expressed on
the APC surface140,141. Thus, when B7 binds CTLA4, the
co-signal needed for T cell activation (B7–CD28 inter-
action) following T cell receptor interaction with the
MHC–antigen complex is inhibited, leading to inhibition
of cell cycle progression. Anti-CTLA4 is a monoclonal
antibody that binds to CTLA4, blocking its activity to
restore co-signalling and T cell activation141. PD-1 is a
receptor expressed on activated T cells, and PD-L1 (its
ligand) is expressed on tumour cells, activated T cells, NK
cells, and APCs140. The binding of PD-L1 to PD-1 induces
an inhibitory signal that reduces T cell cytokine produc-
tion and proliferation and limits inflammatory responses
to infection and autoimmunity 140. Anti-CTLA4,
anti-PD-1, and anti-PD-L1 checkpoint inhibitors
improve antitumour immune responses by restoring T
lymphocyte activation and have been tested in multiple
tumour types140. Atezolizumab (anti-PD-L1), pembroli-
zumab (anti-PD-1), nivolumab (anti-PD-1), avelumab
(anti-PD-L1), and durvalumab (anti-PD-L1) have shown
durable objective response rates in patients with locally
advanced or metastatic bladder cancer7,10,142–148.
Whether checkpoint inhibition has a role in BCG
therapy, or more specifically BCG failure, is unknown.
Intriguingly, PD-L1 is highly expressed in BCG-induced
granulomata found in patients who have not responded
to BCG and is associated with bladder cancer stage
progression149. These findings are not conclusive evi-
dence, but a link could conceivably exist between BCG
failure and T cell exhaustion resulting from check-
point inhibition. Currently, several anti-PD-L1 and
anti-PD-1 drugs are undergoing testing in patients with
NMIBC who are refractory to BCG or patients with
very-high-risk NIMBC who have not been exposed to
BCG (NCT02451423 (Ref.150), NCT02625961 (Ref.151),
and NCT02792192 (Ref.152)). Two trials are specifically
testing the efficacy of BCG in synergy with checkpoint
blockade inhibitors in NMIBC and the effect of locally
instilled checkpoint blockade inhibitors in combination
with BCG (NCT02324582 (Ref.153) and NCT02808143
(Ref. 154) , respectively) 114,155. To date, five checkpoint
blockade inhibitors (atezolizumab, pembrolizumab,
durvalumab, avelumab, and nivolumab) have been
approved for frontline or second-line use in patients with
MIBC, and they represent a major treatment advance
in a disease that has seen little innovation in more than
30 years. Whether these molecules will have a similarly
positive effect in the treatment of NMIBC needs to be
Reviews

determined. In addition, limitations to these thera-
pies must not be forgotten: restoring T cell activation
can induce immune-related adverse effects that vary
in frequency and severity from patient to patient, the
long-term effect on immunomodulation is unknown,
and these drugs are expensive. Importantly, much like
BCG immunotherapy, we lack knowledge of factors that
predict response to identify patients who would benefit
from checkpoint blockade inhibition. Additional pre-
clinical and clinical trials are needed to further dissect
the mechanisms of action and response as well as the
efficacy of and tolerance to these drugs in general and
in combination with BCG.
Imminent molecular approaches. Several studies have
made great gains in establishing a molecular classifi-
cation system for bladder cancer. The majority of the
initial studies focused on MIBC3–6, but molecular sub-
types in NMIBC have also been reported. Notably,
most low-risk and intermediate-risk NMIBC tumours
fall into a category called urobasal A subtype, which is
characterized by high or aberrant expression of fibro-
blast growth factor receptor 3 (FGFR3), G1/S-specific
cyclin D1, the transcription factor tumour protein 63
(p63), retinoblastoma-like protein 2 (RB2), and keratin 5
(K5, also known as KRT5), K13 (also known as KRT13),
K15 (also known as KRT15), and K17 (also known as
KRT17)6,156. Molecular subtyping is important, as it ena-
bles more precise tumour classification than traditional
tumour stage and grade determination using histo-
pathological assessment. Indeed, RNA sequencing data
from more than 1,300 non-muscle-invasive tumours
classified NMIBC into three categories that roughly
reflect patient prognosis157,158. Some overlap with CIS
and muscle-invasive tumour signatures was observed,
suggesting that subtyping of NMIBC can be used for
the early identification of patients at increased risk of
disease progression157,158. In addition, identifying the
molecular subtype might predict response to therapy, as
has been suggested for MIBC159, or enable identification
of patients who will or will not respond to BCG therapy.
Conclusions
BCG therapy is one of the great success stories of can-
cer immunotherapy. However, the exact mechanisms
underlying its ability to induce lasting tumour immu-
nity are still incompletely understood. The immune
pathways induced by BCG are complex and result in a
robust immune response that leads to adaptive immu-
nity and antitumour activity. This response reduces
recurrence and infiltrative progression of bladder can-
cer. Many questions surrounding BCG therapy remain
unanswered. What is different in the 30% of patients
who do not respond to BCG therapy? How can we iden-
tify them? How can we prevent treatment intolerance?
How can we augment or even replace BCG therapy with
more targeted therapy? Finally, the most puzzling ques-
tion also remains unanswered: how do bacteria such as
BCG induce a specific antitumour immune response?
The identification of new mechanisms and pathways
involved in BCG immunotherapy might help to answer
these questions and reveal important clues indicating
how newer immunotherapies can be developed to target
those at risk of experiencing recurrence or progression.
Published online xx xx xxxx

1. Ferlay, J. et al. Cancer incidence and mortality
patterns in Europe: estimates for 40 countries in
2012. Eur. J. Cancer 49, 1374–1403 (2013).
2. Antoni, S. et al. Bladder cancer incidence and
mortality: a global overview and recent trends.
Eur. Urol. 71, 96–108 (2017).
3. Robertson, A. G. et al. Comprehensive molecular
characterization of muscle-invasive bladder cancer.
Cell 171, 540–556 (2017).
4. Cancer Genome Atlas Research Network.
Comprehensive molecular characterization of urothelial
bladder carcinoma. Nature 507, 315–322 (2014).
5. Damrauer, J. S. et al. Intrinsic subtypes of high-grade
bladder cancer reflect the hallmarks of breast cancer
biology. Proc. Natl Acad. Sci. USA 111, 3110–3115
(2014).
6. Sjodahl, G. et al. Toward a molecular pathologic
classification of urothelial carcinoma. Am. J. Pathol.
183, 681–691 (2013).
7. Rosenberg, J. E. et al. Atezolizumab in patients with
locally advanced and metastatic urothelial carcinoma
who have progressed following treatment with
platinum-based chemotherapy: a single-arm,
multicentre, phase 2 trial. Lancet 387, 1909–1920
(2016).
8. Flaig, T. W. et al. Proceedings of the 3rd Annual Albert
Institute for Bladder Cancer Research Symposium.
Bladder Cancer 3, 211–223 (2017).
9. Sanli, O. et al. Bladder cancer. Nat. Rev. Dis. Primers
3, 17022 (2017).
10. Plimack, E. R. et al. Safety and activity of
pembrolizumab in patients with locally advanced or
metastatic urothelial cancer (KEYNOTE-012): a
non-randomised, open-label, phase 1b study. Lancet
Oncol. 18, 212–220 (2017).
11. Irani, J. Epidemiology of bladder cancer [French].
Progres Urol. 13, 1207–1208 (2003).
12. Pfister, C. et al. CCAFU Recommendations 2013:
Bladder carcinoma [French]. Progres Urol. 23 (Suppl. 2),
105–125 (2013).
13. Sylvester, R. J. et al. Predicting recurrence and
progression in individual patients with stage Ta T1
bladder cancer using EORTC risk tables: a combined
analysis of 2596 patients from seven EORTC trials.
Eur. Urol. 49, 466–465 (2006).
14. Morales, A., Eidinger, D. & Bruce, A. W. Intracavitary
Bacillus Calmette-Guerin in the treatment of
superficial bladder tumors. J. Urol. 116, 180–183
(1976).
Reference 14 is the first report of the use of BCG
for bladder cancer in humans demonstrating
protective responses.
15. Babjuk, M. et al. EAU guidelines on non-muscle-
invasive urothelial carcinoma of the bladder: update
2016. Eur. Urol. 71, 447–461 (2017).
16. Roupret, M. et al. CCAFU french national guidelines
2016–2018 on bladder cancer [French]. Progres Urol.
27 (Suppl. 1), 67–91 (2016).
17. Lamm, D. L. 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. J. Urol. 163, 1124–1129 (2000).
Reference 17 is a comprehensive study that
demonstrates that maintenance therapy improves
outcomes in patients with NMIBC.
18. Lamm, D. L. et al. A randomized trial of intravesical
doxorubicin and immunotherapy with bacille
Calmette-Guerin for transitional-cell carcinoma
of the bladder. N. Engl. J. Med. 325, 1205–1209
(1991).
19. Sylvester, R. J., van der Meijden, A. P., Witjes, J. A. &
Kurth, K. Bacillus calmette-guerin versus
chemotherapy for the intravesical treatment of
patients with carcinoma in situ of the bladder: a
meta-analysis of the published results of randomized
clinical trials. J. Urol. 174, 86–91 (2005).
Reference 19 is a meta-analysis that establishes
that BCG is the best adjuvant therapy to prevent
recurrence in patients with NMIBC.
20. Sylvester, R. J. Natural history, recurrence, and
progression in superficial bladder cancer.
ScientificWorldJournal 6, 2617–2625 (2006).
21. Sylvester, R. J. Bacillus Calmette-Guerin treatment of
non-muscle invasive bladder cancer. Int. J. Urol. 18,
113–120 (2011).
22. Askeland, E. J., Newton, M. R., O’Donnell, M. A. &
Luo, Y. Bladder cancer immunotherapy: BCG and
beyond. Adv. Urol. 2012, 181987 (2012).
23. Lamm, D. L. Long-term results of intravesical therapy
for superficial bladder cancer. Urol. Clin. North Amer.
19, 573–580 (1992).
24. O’Donnell, M. A. Optimizing BCG therapy. Urol. Oncol.
27, 325–328 (2009).
25. van der Meijden, A. P. et al. Maintenance Bacillus
Calmette-Guerin for Ta T1 bladder tumors is not
associated with increased toxicity: results from a
European Organisation for Research and Treatment of
Cancer Genito-Urinary Group Phase III Trial. Eur. Urol.
44, 429–434 (2003).
26. Yokomizo, A. et al. Randomized controlled study of the
efficacy, safety and quality of life with low dose Bacillus
Calmette-Guerin instillation therapy for nonmuscle
invasive bladder cancer. J. Urol. 195, 41–46 (2016).
27. Oddens, J. et al. Final results of an EORTC-GU cancers
group randomized study of maintenance Bacillus
Calmette-Guerin in intermediate- and 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. 63, 462–472 (2013).
28. Sylvester, R. J. 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. Eur. Urol. 57, 766–773
(2010).
29. EU clinical trials register. Treatment of high grade
non-muscle invasive urothelial carcinoma of the

www.nature.com/nrurol
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

bladder by standard number and dose of intravesical
BCG instillations versus reduced number of
intravesical instillations with standard dose of BCG.
European Medicines Agency https://www.
clinicaltrialsregister.eu/ctr-search/trial/2010-019181-
91/DE (2013).
30. Kamat, A. M. et al. Expert consensus document:
consensus statement on best practice management
regarding the use of intravesical immunotherapy with
BCG for bladder cancer. Nat. Rev. Urol. 12, 225–235
(2015).
31. Kamat, A. M. et al. Definitions, end points, and clinical
trial designs for non-muscle-invasive bladder cancer:
recommendations from the International Bladder
Cancer Group. J. Clin. Oncol. 34, 1935–1944 (2016).
32. Kamat, A. M. et al. BCG-unresponsive non-muscle-
invasive bladder cancer: recommendations from the
IBCG. Nat. Rev. Urol. 14, 244–255 (2017).
33. Grossman, H. B. et al. Innovation in bladder cancer
immunotherapy. J. Immunother. 39, 291–297 (2016).
34. Messing, E. M. The BCG Shortage. Bladder Cancer 3,
227–228 (2017).
35. Davies, B. J., Hwang, T. J. & Kesselheim, A. S. Ensuring
access to injectable generic drugs - the case of
intravesical BCG for bladder cancer. N. Engl. J. Med.
376, 1401–1403 (2017).
36. Bevers, R. F., Kurth, K. H. & Schamhart, D. H. Role of
urothelial cells in BCG immunotherapy for superficial
bladder cancer. Br. J. Cancer 91, 607–612 (2004).
37. Wiker, H. G. & Harboe, M. The antigen 85 complex: a
major secretion product of Mycobacterium
tuberculosis. Microbiol. Rev. 56, 648–661 (1992).
38. Ratliff, T. L., Kavoussi, L. R. & Catalona, W. J. Role of
fibronectin in intravesical BCG therapy for superficial
bladder cancer. J. Urol. 139, 410–414 (1988).
39. Aslanzadeh, J., Brown, E. J., Quillin, S. P., Ritchey, J. K.
& Ratliff, T. L. Characterization of soluble fibronectin
binding to Bacille Calmette-Guerin. J. Gen. Microbiol.
135, 2735–2741 (1989).
40. Zhao, W. et al. Role of a Bacillus Calmette-Guerin
fibronectin attachment protein in BCG-induced
antitumor activity. Int. J. Cancer 86, 83–88 (2000).
41. Abou-Zeid, C. et al. Characterization of
fibronectin-binding antigens released by
Mycobacterium tuberculosis and Mycobacterium
bovis BCG. Infection Immun. 56, 3046–3051 (1988).
42. Ratliff, T. L., Palmer, J. O., McGarr, J. A. & Brown, E. J.
Intravesical Bacillus Calmette-Guerin therapy for
murine bladder tumors: initiation of the response by
fibronectin-mediated attachment of Bacillus
Calmette-Guerin. Cancer Res. 47, 1762–1766
(1987).
43. Teppema, J. S., de Boer, E. C., Steerenberg, P. A. &
van der Meijden, A. P. Morphological aspects of the
interaction of Bacillus Calmette-Guerin with urothelial
bladder cells in vivo and in vitro: relevance for
antitumor activity? Urol. Res. 20, 219–228 (1992).
44. Kavoussi, L. R., Brown, E. J., Ritchey, J. K. & Ratliff, T. L.
Fibronectin-mediated Calmette-Guerin Bacillus
attachment to murine bladder mucosa. Requirement
for the expression of an antitumor response. J. Clin.
Invest. 85, 62–67 (1990).
45. Boorjian, S. A., Berglund, R. K., Maschino, A. C.,
Savage, C. J. & Herr, H. W. Fibrin clot inhibitor
medication and efficacy of Bacillus Calmette-Guerin for
bladder urothelial cancer. J. Urol. 182, 1306–1312
(2009).
46. Witjes, J. A., vd Meijden, A. P., Doesburg, W. &
Debruyne, F. M. Influence of fibrin clot inhibitors on the
efficacy of intravesical Bacillus Calmette-Guerin in
the treatment of superficial bladder cancer. The Dutch
Southeast Cooperative Urological Group. Eur. Urol.
23, 366–370 (1993).
47. Lipsky, M. J., Badalato, G. M., Motamedinia, P.,
Hruby, G. W. & McKiernan, J. M. The effect of fibrin
clot inhibitors on the immunomodulatory efficacy of
Bacillus Calmette-Guerin therapy for non-muscle-
invasive bladder cancer. Urology 81, 1273–1278
(2013).
48. Biot, C. et al. Preexisting BCG-specific T cells improve
intravesical immunotherapy for bladder cancer.
Sci. Transl Med. 4, 137ra172 (2012).
Reference 48 demonstrates that mice with
pre-existing cellular immunity to BCG have a more
rapid immune response to BCG therapy and are
better protected against tumour challenge
following intravesical BCG instillation; patients with
BCG immune memory also demonstrate superior
protection following BCG therapy.
49. Durek, C. et al. The fate of Bacillus Calmette-Guerin
after intravesical instillation. J. Urol. 165, 1765–1768
(2001).
Nature Reviews | Urology
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
50. Mora-Bau, G. et al. Macrophages subvert adaptive
immunity to urinary tract infection. PLOS Pathog. 11,
e1005044 (2015).
51. Mulvey, M. A. et al. Induction and evasion of host
defenses by type 1-piliated uropathogenic Escherichia
coli. Science 282, 1494–1497 (1998).
52. Martinez, J. J., Mulvey, M. A., Schilling, J. D.,
Pinkner, J. S. & Hultgren, S. J. Type 1 pilus-mediated
bacterial invasion of bladder epithelial cells. EMBO J.
19, 2803–2812 (2000).
53. Ingersoll, M. A. & Albert, M. L. From infection to
immunotherapy: host immune responses to bacteria
at the bladder mucosa. Mucosal Immunol. 6,
1041–1053 (2013).
54. Redelman-Sidi, G., Iyer, G., Solit, D. B. & Glickman, M. S.
Oncogenic activation of Pak1-dependent pathway of
macropinocytosis determines BCG entry into bladder
cancer cells. Cancer Res. 73, 1156–1167 (2013).
55. Eto, D. S., Jones, T. A., Sundsbak, J. L. & Mulvey, M. A.
Integrin-mediated host cell invasion by type 1-piliated
uropathogenic. Escherichia coli. PLOS Pathog. 3,
e100 (2007).
56. Kuroda, K., Brown, E. J., Telle, W. B., Russell, D. G. &
Ratliff, T. L. Characterization of the internalization of
Bacillus Calmette-Guerin by human bladder tumor
cells. J. Clin. Invest. 91, 69–76 (1993).
57. Durek, C. et al. Bacillus-Calmette-Guerin (BCG) and 3D
tumors: an in vitro model for the study of adhesion
and invasion. J. Urol. 162, 600–605 (1999).
58. Becich, M. J., Carroll, S. & Ratliff, T. L. Internalization
of bacille Calmette-Guerin by bladder tumor cells.
J. Urol. 145, 1316–1324 (1991).
59. Bevers, R. F., de Boer, E. C., Kurth, K. H. & Schamhart,
D. H. BCG-induced interleukin-6 upregulation and
BCG internalization in well and poorly differentiated
human bladder cancer cell lines. Eur. Cytokine
Network 9, 181–186 (1998).
60. Ikeda, N., Toida, I., Iwasaki, A., Kawai, K. & Akaza, H.
Surface antigen expression on bladder tumor cells
induced by Bacillus Calmette-Guerin (BCG): a role of
BCG internalization into tumor cells. Int. J. Urol. 9,
29–35 (2002).
61. Mitropoulos, D. N. Novel insights into the mechanism
of action of intravesical immunomodulators. In Vivo
19, 611–621 (2005).
62. Cosma, C. L., Sherman, D. R. & Ramakrishnan, L. The
secret lives of the pathogenic mycobacteria. Annu. Rev.
Microbiol. 57, 641–676 (2003).
63. Flynn, J. L., Chan, J. & Lin, P. L. Macrophages and
control of granulomatous inflammation in tuberculosis.
Mucosal Immunol. 4, 271–278 (2011).
64. Lage, J. M., Bauer, W. C., Kelley, D. R., Ratliff, T. L. &
Catalona, W. J. Histological parameters and pitfalls in
the interpretation of bladder biopsies in Bacillus
Calmette-Guerin treatment of superficial bladder
cancer. J. Urol. 135, 916–919 (1986).
65. de Boer, E. C. et al. Role of interleukin-8 in onset of
the immune response in intravesical BCG therapy for
superficial bladder cancer. Urol. Res. 25, 31–34
(1997).
66. Bisiaux, A. et al. Molecular analyte profiling of the
early events and tissue conditioning following
intravesical Bacillus Calmette-Guerin therapy in
patients with superficial bladder cancer. J. Urol. 181,
1571–1580 (2009).
67. Luo, Y., Chen, X. & O’Donnell, M. A. Role of Th1 and
Th2 cytokines in BCG-induced IFN-gamma production:
cytokine promotion and simulation of BCG effect.
Cytokine 21, 17–26 (2003).
68. De Boer, E. C. et al. Induction of urinary interleukin-1
(IL-1), IL-2, IL-6, and tumour necrosis factor during
intravesical immunotherapy with Bacillus
Calmette-Guerin in superficial bladder cancer.
Cancer Immunol. Immunother. 34, 306–312 (1992).
69. Bohle, A. & Brandau, S. Immune mechanisms in
Bacillus Calmette-Guerin immunotherapy for
superficial bladder cancer. J. Urol. 170, 964–969
(2003).
70. Stefanini, G. F. et al. Class I and class II HLA antigen
expression by transitional cell carcinoma of the
bladder: correlation with T cell infiltration and BCG
treatment. J. Urol. 141, 1449–1453 (1989).
71. Prescott, S. et al. HLA-DR expression by high grade
superficial bladder cancer treated with BCG.
Br. J. Urol. 63, 264–269 (1989).
72. Lattime, E. C., Gomella, L. G. & McCue, P. A. Murine
bladder carcinoma cells present antigen to
BCG-specific CD4+ T cells. Cancer Res. 52,
4286–4290 (1992).
73. Suttmann, H. et al. Neutrophil granulocytes are
required for effective Bacillus Calmette-Guerin
immunotherapy of bladder cancer and orchestrate
Reviews
local immune responses. Cancer Res. 66, 8250–8257
(2006).
74. Rosevear, H. M., Lightfoot, A. J., O’Donnell, M. A. &
Griffith, T. S. The role of neutrophils and TNF-related
apoptosis-inducing ligand (TRAIL) in Bacillus
Calmette-Guerin (BCG) immunotherapy for urothelial
carcinoma of the bladder. Cancer Metastasis Rev. 28,
345–353 (2009).
75. Luo, Y. & Knudson, M. J. Mycobacterium bovis
Bacillus Calmette-Guerin-induced macrophage
cytotoxicity against bladder cancer cells. Clin. Dev.
Immunol. 2010, 357591 (2010).
76. Ratliff, T. L., Ritchey, J. K., Yuan, J. J., Andriole, G. L. &
Catalona, W. J. T cell subsets required for intravesical
BCG immunotherapy for bladder cancer. J. Urol. 150,
1018–1023 (1993).
Reference 76 is one of the earliest studies to
establish that an adaptive immune response is
necessary for BCG-mediated tumour immunity.
77. de Boer, E. C. et al. Leukocytes in the urine after
intravesical BCG treatment for superficial bladder
cancer. A flow cytofluorometric analysis. Urol. Res. 19,
45–50 (1991).
78. Brandau, S. et al. NK cells are essential for effective
BCG immunotherapy. International journal of cancer.
J. Int. Cancer 92, 697–702 (2001).
79. Wang, M. H., Flad, H. D., Bohle, A., Chen, Y. Q. &
Ulmer, A. J. Cellular cytotoxicity of human natural
killer cells and lymphokine-activated killer cells
against bladder carcinoma cell lines. Immunol. Lett.
27, 191–197 (1991).
80. Ludwig, A. T. et al. Tumor necrosis factor-related
apoptosis-inducing ligand: a novel mechanism for
Bacillus Calmette-Guerin-induced antitumor activity.
Cancer Res. 64, 3386–3390 (2004).
81. Simons, M. P., O’Donnell, M. A. & Griffith, T. S. Role of
neutrophils in BCG immunotherapy for bladder cancer.
Urol. Oncol. 26, 341–345 (2008).
82. Kresowik, T. P. & Griffith, T. S. Bacillus Calmette-Guerin
immunotherapy for urothelial carcinoma of the
bladder. Immunotherapy 1, 281–288 (2009).
83. Leitch, A. E., Lucas, C. D. & Rossi, A. G. Editorial:
Neutrophil apoptosis: hot on the TRAIL of
inflammatory resolution. J. Leukocyte Biol. 90,
841–843 (2011).
84. Pryor, K. et al. Bacillus Calmette-Guerin (BCG)
enhances monocyte- and lymphocyte-mediated
bladder tumour cell killing. Br. J. Cancer 71, 801–807
(1995).
85. Saint, F. et al. Mechanisms of action of BCG: towards a
new individualized therapeutic approach? [French].
Progres Urol. 10, 1118–1126 (2000).
86. Suttmann, H. et al. Mechanisms of Bacillus
Calmette-Guerin mediated natural killer cell activation.
J. Urol. 172, 1490–1495 (2004).
87. Ratliff, T. L., Shapiro, A. & Catalona, W. J. Inhibition of
murine bladder tumor growth by bacille
Calmette-Guerin: lack of a role of natural killer cells.
Clin. Immunol. Immunopathol. 41, 108–115 (1986).
88. Sonoda, T., Sugimura, K., Ikemoto, S., Kawashima, H.
& Nakatani, T. Significance of target cell infection and
natural killer cells in the anti-tumor effects of Bacillus
Calmette-Guerin in murine bladder cancer. Oncol.
Rep. 17, 1469–1474 (2007).
89. Garcia-Cuesta, E. M. et al. NKG2D is a key receptor
for recognition of bladder cancer cells by IL-2-
activated NK cells and BCG promotes NK cell
activation. Front. Immunol. 6, 284 (2015).
90. Zuiverloon, T. C. et al. Markers predicting response to
Bacillus Calmette-Guerin immunotherapy in high-risk
bladder cancer patients: a systematic review. Eur. Urol.
61, 128–145 (2012).
91. McAveney, K. M., Gomella, L. G. & Lattime, E. C.
Induction of TH1- and TH2-associated cytokine mRNA
in mouse bladder following intravesical growth of the
murine bladder tumor MB49 and BCG
immunotherapy. Cancer Immunol. Immunother. 39,
401–406 (1994).
92. Luo, Y. Blocking IL-10 enhances Bacillus
Calmette-Guerin induced T helper Type 1 immune
responses and anti-bladder cancer immunity.
Oncoimmunology 1, 1183–1185 (2012).
93. Saint, F. et al. Prognostic value of a T helper 1 urinary
cytokine response after intravesical Bacillus
Calmette-Guerin treatment for superficial bladder
cancer. J. Urol. 167, 364–367 (2002).
Reference 93 details one of the first clinical studies
emphasizing the role of TH1 cell immunity in
reducing recurrence rate following BCG
immunotherapy.
94. Riemensberger, J., Bohle, A. & Brandau, S. IFN-γ and
IL-12 but not IL-10 are required for local tumour
Reviews
surveillance in a syngeneic model of orthotopic
bladder cancer. Clin. Exp. Immunol. 127, 20–26
(2002).
95. Ratliff, T. L., Gillen, D. & Catalona, W. J. Requirement
of a thymus dependent immune response for
BCG-mediated antitumor activity. J. Urol. 137,
155–158 (1987).
96. Pichler, R. et al. Tumor-infiltrating immune cell
subpopulations influence the oncologic outcome after
intravesical Bacillus Calmette-Guerin therapy in
bladder cancer. Oncotarget 7, 39916–39930 (2016).
97. US National Library of Medicine. ClinicalTrials.gov
https://ClinicalTrials.gov/show/NCT02326168 (2017).
98. Messing, E. M. Words of wisdom. Re: Preexisting
BCG-specific T cells improve intravesical
immunotherapy for bladder cancer. Eur. Urol. 62,
935–936 (2012).
99. van der Meijden, A. P. et al. Immune reactions in
patients with superficial bladder cancer after
intradermal and intravesical treatment with Bacillus
Calmette-Guerin. Cancer Immunol. Immunother. 28,
287–295 (1989).
100. Winters, W. D. & Lamm, D. L. Antibody responses to
Bacillus Calmette-Guerin during immunotherapy in
bladder cancer patients. Cancer Res. 41, 2672–2676
(1981).
101. Kelley, D. R. et al. Prognostic value of purified protein
derivative skin test and granuloma formation in
patients treated with intravesical Bacillus
Calmette-Guerin. J. Urol. 135, 268–271 (1986).
102. Taniguchi, K. et al. Systemic immune response after
intravesical instillation of Bacille Calmette-Guerin
(BCG) for superficial bladder cancer. Clin. Exp.
Immunol. 115, 131–135 (1999).
103. Saint, F. et al. Urinary leukocytes as a new prognostic
marker of therapeutic response and of adverse effects
associated with the maintenance treatment with
endovesical BCG, for the prophylaxis of superficial
bladder tumors [French]. Progres Urol. 11,
1242–1250 (2001).
104. Jallad, S., Goubet, S., Symes, A., Larner, T. & Thomas, P.
Prognostic value of inflammation or granuloma after
intravesival BCG in non-muscle-invasive bladder
cancer. BJU Int. 113, E22–E27 (2014).
105. Patard, J. J., Chopin, D. K. & Boccon-Gibod, L.
Mechanisms of action of Bacillus Calmette-Guerin in
the treatment of superficial bladder cancer. World J.
Urol. 11, 165–168 (1993).
106. Gonzalez, O. Y. et al. Spectrum of Bacille
Calmette-Guerin (BCG) infection after intravesical BCG
immunotherapy. Clin. Infecti. Dis. 36, 140–148
(2003).
107. Marquez-Batalla, S., Fraile-Villarejo, E.,
Belhassen-Garcia, M., Gutierrez-Zubiaurre, N. &
Cordero-Sanchez, M. Disseminated infection due to
Mycobacterium bovis after intravesical BCG
instillation. World J. Clin. Cases 2, 301–303 (2014).
108. Lamm, D. L. et al. Incidence and treatment of
complications of Bacillus Calmette-Guerin intravesical
therapy in superficial bladder cancer. J. Urol. 147,
596–600 (1992).
109. Elkabani, M., Greene, J. N., Vincent, A. L., VanHook, S.
& Sandin, R. L. Disseminated Mycobacterium bovis
after intravesicular Bacillus calmette-Gu rin treatments
for bladder cancer. Cancer Control 7, 476–481
(2000).
110. Perez-Jacoiste Asin, M. A. et al. Bacillus
Calmette-Guerin (BCG) infection following intravesical
BCG administration as adjunctive therapy for bladder
cancer: incidence, risk factors, and outcome in a
single-institution series and review of the literature.
Medicine 93, 236–254 (2014).
111. Rischmann, P., Desgrandchamps, F., Malavaud, B. &
Chopin, D. K. BCG intravesical instillations:
recommendations for side-effects management.
Eur. Urol. 37 (Suppl. 1), 33–36 (2000).
112. Brausi, M. et al. Side effects of Bacillus
Calmette-Guerin (BCG) in the treatment of
intermediate- and high-risk Ta, T1 papillary carcinoma
of the bladder: results of the EORTC genito-urinary
cancers group randomised phase 3 study comparing
one-third dose with full dose and 1 year with 3 years
of maintenance BCG. Eur. Urol. 65, 69–76 (2014).
113. Sylvester, R. J., van der Meijden, A. P., Oosterlinck, W.,
Hoeltl, W. & Bono, A. V. The side effects of Bacillus
Calmette-Guerin in the treatment of Ta T1 bladder
cancer do not predict its efficacy: results from a
European Organisation for Research and Treatment of
Cancer Genito-Urinary Group Phase III Trial. Eur. Urol.
44, 423–428 (2003).
114. Vandeveer, A. J. et al. Systemic immunotherapy of
non-muscle invasive mouse bladder cancer with
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
avelumab, an anti-PD-L1 immune checkpoint inhibitor.
Cancer Immunol. Res. 4, 452–462 (2016).
115. Herr, H. W. & Morales, A. History of Bacillus
Calmette-Guerin and bladder cancer: an
immunotherapy success story. J. Urol. 179, 53–56
(2008).
116. Behr, M. A. & Small, P. M. A historical and molecular
phylogeny of BCG strains. Vaccine 17, 915–922
(1999).
117. Behr, M. A. BCG—different strains, different vaccines?
Lancet Infect. Dis. 2, 86–92 (2002).
118. Hayashi, D. et al. Biochemical characteristics among
Mycobacterium bovis BCG substrains. FEMS
Microbiol. Lett. 306, 103–109 (2010).
119. Brosch, R. et al. Genome plasticity of BCG and impact
on vaccine efficacy. Proc. Natl Acad. Sci. USA 104,
5596–5601 (2007).
120. Rodriguez-Alvarez, M., Mendoza-Hernandez, G.,
Encarnacion, S., Calva, J. J. & Lopez-Vidal, Y.
Phenotypic differences between BCG vaccines
at the proteome level. Tuberculosis 89, 126–135
(2009).
121. Boehm, B. E. et al. Efficacy of Bacillus Calmette-Guerin
strains for treatment of nonmuscle invasive bladder
cancer: a systematic review and network
meta-analysis. J. Urol. 198, 503–510 (2017).
122. Secanella-Fandos, S., Luquin, M. & Julian, E.
Connaught and Russian strains showed the highest
direct antitumor effects of different Bacillus
Calmette-Guerin substrains. J. Urol. 189, 711–718
(2013).
123. Rentsch, C. A. et al. Bacillus Calmette-Guerin strain
differences have an impact on clinical outcome in
bladder cancer immunotherapy. Eur. Urol. 66,
677–688 (2014).
124. Dussurget, O. et al. Role of Mycobacterium
tuberculosis copper-zinc superoxide dismutase.
Infection Immun. 69, 529–533 (2001).
125. Piddington, D. L. et al. Cu, Zn superoxide dismutase of
Mycobacterium tuberculosis contributes to survival in
activated macrophages that are generating an
oxidative burst. Infection Immun. 69, 4980–4987
(2001).
126. Noon, A. P. & Kulkarni, G. S. All Bacillus
Calmette-Guerin (BCG) strains are equal, but some
BCG strains are more equal than others. Eur. Urol. 66,
689–691 (2014).
127. Sengiku, A. et al. A prospective comparative study
of intravesical Bacillus Calmette-Guerin therapy
with the Tokyo or Connaught strain for nonmuscle
invasive bladder cancer. J. Urol. 190, 50–54
(2013).
128. Inamoto, T. et al. Comparable effect with minimal
morbidity of low-dose Tokyo 172 strain compared with
regular dose Connaught strain as an intravesical
Bacillus Calmette-Guerin prophylaxis in nonmuscle
invasive bladder cancer: results of a randomized
prospective comparison. Urol. Ann. 5, 7–12
(2013).
129. Mukherjee, A., Persad, R. & Smith, P. J. Intravesical
BCG treatment for superficial bladder cancer:
long-term results using two different strains of BCG.
Br. J. Urol. 69, 147–150 (1992).
130. Fellows, G. J. et al. 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).
Br. J. Urol. 73, 639–644 (1994).
131. Witjes, J. A. 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. 34,
484.e19–484.e25 (2016).
132. Vegt, P. D. et al. A randomized study of intravesical
mitomycin C, Bacillus Calmette-Guerin Tice and
Bacillus Calmette-Guerin RIVM treatment in pTa-pT1
papillary carcinoma and carcinoma in situ of the
bladder. J. Urol. 153, 929–933 (1995).
133. Kamat, A. M. et al. Predicting response to intravesical
Bacillus Calmette-Guerin immunotherapy: are we
there yet? a systematic review. Eur. Urol. 75,
738–748 (2017).
134. Lamm, D., Brausi, M., O’Donnell, M. A. & Witjes, J. A.
Interferon alfa in the treatment paradigm for
non-muscle-invasive bladder cancer. Urol. Oncol. 32,
35.e21–30 (2014).
135. Nepple, K. G. et al. Bacillus Calmette-Guerin with or
without interferon α-2b and megadose versus
recommended daily allowance vitamins during
induction and maintenance intravesical treatment of
nonmuscle invasive bladder cancer. J. Urol. 184,
1915–1919 (2010).
136. Shepherd, A. R., Shepherd, E. & Brook, N. R.
Intravesical Bacillus Calmette-Guerin with interferon-α
versus intravesical Bacillus Calmette-Guerin for
treating non-muscle-invasive bladder cancer.
Cochrane Database Syst. Rev. 3, CD012122
(2017).
137. Shore, N. D. et al. Intravesical rAd-IFNα/Syn3 for
patients with high-grade, Bacillus Calmette-Guerin-
refractory or relapsed non-muscle-invasive bladder
cancer: a phase ii randomized study. J. Clin. Oncol.
35, 3410–3416 (2017).
138. Steinberg, R. L., Brooks, N. A., Thomas, L. J., Mott, S. L.
& O’Donnell, M. A. Bacillus Calmette-Guerin strain
may not effect recurrence-free survival when used
intravesically with interferon-α2b for non-muscle-
invasive bladder cancer. Urol. Oncol. 35, 201–207
(2017).
139. Donin, N. M. et al. Immunotherapy for the treatment
of urothelial carcinoma. J. Urol. 197, 14–22
(2017).
140. Carosella, E. D., Ploussard, G., LeMaoult, J. &
Desgrandchamps, F. A. Systematic review of
immunotherapy in urologic cancer: evolving roles
for targeting of CTLA-4, PD-1/PD-L1, and HLA-G.
Eur. Urol. 68, 267–279 (2015).
141. Buchbinder, E. I. & Desai, A. CTLA-4 and PD-1
pathways: similarities, differences, and implications of
their inhibition. Am. J. Clin. Oncol. 39, 98–106
(2016).
142. Balar, A. V. et al. Atezolizumab as first-line treatment
in cisplatin-ineligible patients with locally advanced
and metastatic urothelial carcinoma: a single-arm,
multicentre, phase 2 trial. Lancet 389, 67–76
(2017).
143. Sharma, P. et al. Nivolumab monotherapy in recurrent
metastatic urothelial carcinoma (CheckMate 032):
a multicentre, open-label, two-stage, multi-arm,
phase 1/2 trial. Lancet. Oncol. 17, 1590–1598
(2016).
144. Bellmunt, J. et al. Pembrolizumab as second-line
therapy for advanced urothelial carcinoma. N. Engl.
J. Med. 376, 1015–1026 (2017).
145. Powles, T. et al. MPDL3280A (anti-PD-L1) treatment
leads to clinical activity in metastatic bladder cancer.
Nature 515, 558–562 (2014).
146. Apolo, A. B. et al. Avelumab, an anti-programmed
death-ligand 1 antibody, in patients with refractory
metastatic urothelial carcinoma: results from a
multicenter, phase ib study. J. Clin. Oncol. 35,
2117–2124 (2017).
147. Massard, C. et al. Safety and efficacy of durvalumab
(MEDI4736), an anti-programmed cell death ligand-1
immune checkpoint inhibitor, in patients with
advanced urothelial bladder cancer. J. Clin. Oncol. 34,
3119–3125 (2016).
148. Powles, T. et al. Efficacy and safety of durvalumab in
locally advanced or metastatic urothelial carcinoma:
updated results from a phase 1/2 open-label study.
JAMA Oncol. 3, e172411 (2017).
149. Inman, B. A. et al. PD-L1 (B7-H1) expression by
urothelial carcinoma of the bladder and BCG-induced
granulomata: associations with localized stage
progression. Cancer 109, 1499–1505 (2007).
Several years before the advent of checkpoint
blockade inhibition in bladder cancer, reference
149 reports a link between PD-L1 expression,
response to BCG, and bladder cancer stage
progression.
150. US National Library of Medicine. ClinicalTrials.gov
https://ClinicalTrials.gov/show/NCT03007719 (2018).
151. US National Library of Medicine. ClinicalTrials.gov
https://ClinicalTrials.gov/show/NCT02625961 (2018).
152. US National Library of Medicine. ClinicalTrials.gov
https://ClinicalTrials.gov/show/NCT02792192 (2018).
153. US National Library of Medicine. ClinicalTrials.gov
https://ClinicalTrials.gov/show/NCT02324582 (2018).
154. US National Library of Medicine. ClinicalTrials.gov
https://ClinicalTrials.gov/show/NCT02808143 (2018).
155. Rouanne, M., Loriot, Y., Lebret, T. & Soria, J. C.
Novel therapeutic targets in advanced urothelial
carcinoma. Crit. Rev. Oncol. Hematol. 98, 106–115
(2016).
156. Sjodahl, G. et al. A molecular taxonomy for urothelial
carcinoma. Clin. Cancer Res. 18, 3377–3386 (2012).
157. Lerner, S. P. & Robertson, A. G. Molecular subtypes of
non-muscle invasive bladder cancer. Cancer Cell 30,
1–3 (2016).
158. Hedegaard, J. et al. Comprehensive transcriptional
analysis of early-stage urothelial carcinoma.
Cancer Cell 30, 27–42 (2016).
159. Seiler, R. et al. Impact of molecular subtypes in
muscle-invasive bladder cancer on predicting
www.nature.com/nrurol

response and survival after neoadjuvant
chemotherapy. Eur. Urol. 72, 544–554 (2017).
160. Hoption Cann, S. A., van Netten, J. P. & van Netten, C.
Dr William Coley and tumour regression: a place in
history or in the future. Postgraduate Med. J. 79,
672–680 (2003).
161. Pearl, R. On the pathological relations between cancer
and tuberculosis. Exp. Biol. Med. (Maywood) 26,
73–75 (1928).
162. Old, L. J., Clarke, D. A. & Benacerraf, B. Effect of
Bacillus Calmette-Guerin infection on transplanted
tumours in the mouse. Nature 184 (Suppl. 5),
291–292 (1959).
163. Davignon, L., Robillard, P., Lemonde, P. & Frappier, A.
BCG vaccination and leukemia mortality. Lancet 2,
638 (1970).
164. Zbar, B. & Tanaka, T. Immunotherapy of cancer:
regression of tumors after intralesional injection of
living Mycobacterium bovis. Science 172, 271–273
(1971).
165. Rosenthal, S. R. et al. BCG vaccination and
leukemia mortality. JAMA 222, 1543–1544
(1972).
Nature Reviews | Urology
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
166. Zbar, B. & Rapp, H. J. Immunotherapy of guinea
pig cancer with BCG. Cancer 34, 1532–1540
(1974).
167. Lamm, D. L. et al. Bacillus Calmette-Guerin
immunotherapy of superficial bladder cancer. J. Urol.
124, 38–40 (1980).
168. Carlo, M. I. et al. Checkpoint inhibitors and
other novel immunotherapies for advanced renal
cell carcinoma. Nat. Rev. Urol. 13, 420–431
(2016).
Acknowledgements
The authors thank Y. Neuzillet (Department of Urology,
Hopital Foch, Suresnes, France) for the critical reading of this
manuscript. The authors also thank the French Association of
Urology (AFU) and LabEx ImmunoOnco for funding support
for research on BCG and non-muscle-invasive bladder
cancer.
Author contributions
Both authors researched data for the article, made a substan-
tial contribution to discussion of content, and wrote,
reviewed, and edited the manuscript before submission.
Reviews
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
Review criteria
A systematic search was performed in the PubMed and
Cochrane databases. Manuscripts not available as full text were
excluded. The principal keywords used for publication selection
were “BCG”, “immunotherapy”, “intravesical”, “instillation”,
“NMIBC”, and “mechanism of action”. The following specific key-
words and associations were added using [AND] or [OR]: “attach-
ment”, “internalization”, “immune response”, “innate”,
“adaptive”, “immune cell”, “cytotoxic”, “antitumour”, “side
effects”, “BCG vaccination”, “BCG strain”, and “checkpoint inhib-
itors”. Manuscripts that were accessible in English or French were
selected, including preclinical studies and clinical trials, system-
atic reviews, and meta-analyses, in addition to the latest versions
of the European and French Associations of Urology Guidelines.
To perform an exhaustive search, no publication year limit
was used, and the last search was updated in November 2017.