Bladder Cancer Immunotherapy: BCG and Beyond
Mycobacterium bovis bacillus Calmette-Guérin (BCG) has become the predominant conservative treatment for nonmuscle invasive bladder cancer. Its mechanism of action continues to be defined but has been shown to involve a T helper type 1 (Th1) immunomodulatory response. While BCG treatment is the current standard of care, a significant proportion of patients fails or do not tolerate treatment. Therefore, many efforts have been made to identify other intravesical and immunomodulating therapeutics to use alone or in conjunction with BCG. This paper reviews the progress of basic science and clinical experience with several immunotherapeutic agents including IFN-α, IL-2, IL-12, and IL-10.
Hindawi Publishing Corporation
Advances in Urology
Volume 2012, Article ID 181987, 13 pages
Review A rticle
Bladder Cancer Immunotherapy: BCG and Beyond
Eric J. Askeland, Mark R. New ton, Michael A. O’Donnell, and Yi Luo
Depar tment of Urology, University of Iowa, 375 New ton Road, 3204 MERF, Iowa City, IA 52242, USA
Correspondence should be addressed to Yi Luo, email@example.com
Received 16 March 2012; Accepted 11 May 2012
Academic Editor: Trinity J. Bivalacqua
Copyright © 2012 Eric J. Askeland et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Mycobacterium bovis bacillus Calmette-Gu
erin (BCG) has become the predominant conservative treatment for nonmuscle invasive
bladder cancer. Its mechanism of action continues to be deﬁned but has been shown to involve a T helper type 1 (Th1)
immunomodulatory response. While BCG treatment is the current standard of care, a signiﬁcant proportion of patients fails or do
not tolerate treatment. Therefore, many eﬀorts have been made to identify other intravesical and immunomodulating therapeutics
to use alone or in conjunction with BCG. This paper reviews the progress of basic science and clinical experience with several
immunotherapeutic agents including IFN-α, IL-2, IL-12, and IL-10.
With more than 73,000 estimated cases diagnosed in 2012,
bladder cancer is the ﬁfth most common malignancy in the
United States, responsible for more than 14,000 deaths per
year . Urothelial carcinoma accounts for 90% of bladder
tumors, of which approximately 70% are conﬁned to layers
above the muscularis propria—the so-called nonmuscle
invasive bladder cancer (NMIBC). These tumors (previously
termed “superﬁcial bladder tumors”) include stages Ta, T1,
and Tis, o ccurring in 70%, 20%, and 10% of NMIBC cases,
respectively . Standard primary treatment for NMIBC is
transurethral resection (TUR); however, recurrence rates for
TUR a lone can be as high as 70% with up to 30% progressing
to muscle invasive disease requiring cystectomy .
High rates of recurrence and progression have prompted
investigation into a myriad of treatments attempting to
decrease the burden of this disease. Mycobacterium bovis
erin (BCG) is the most well known
and studied of these adjunctive treatments. Since its ﬁrst
description in 1976 by Morales et al. , intravesical BCG
has become the standard therapy for NMIBC, superior to any
other single chemotherapeutic agent for reducing recurrence
and preventing progression. Typical complete response rates
are 55–65% for papillary tumors and 70–75% for carcinoma
in situ (CIS), which inversely indicates that 30–45% of
patients will be BCG failures [5–7]. Of the complete respon-
ders,upto50%willhavearecurrence. Furthermore, side
eﬀects range from cystitis and irritative voiding symptoms to
much more uncommon life-threatening BCG sepsis. Up to
20% of patients are BCG intolerant due to these side eﬀects
Understanding of BCG, both its mechanisms (which
remain incompletely characterized) and its obvious limita-
tions, is critical to improving the eﬃcacy of therapy. The
initial step after BCG instillation is binding of BCG to
ﬁbronectin expressed on the urothelium, after which the
mycobacterium is internalized by both normal and malig-
nant cells, resulting in urothelial activation and subsequent
inﬂammatory responses in the bladder . BCG antigens
can be presented at the cell surfaces of urothelial and antigen-
presenting cells in the context of major histocompatibility
complex (MHC) class II, stimulating CD4
inducing a primarily T helper type (Th) 1 immune response
. This complex and robust immune reaction evoked
by BCG is evidenced by a massive transient secretion of
cytokines in voided urine, including interleukin (IL)-1, IL-
2, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, interferon-
inducible protein (IP)-10, tumor necrosis factor (TNF)-α,
granulocyte-monocyte colony stimulating fac tor (GMCSF),
and interferon (IFN)-γ . While the role each of these
cytokines plays in urothelial carcinoma treatment is not
2 Advances in Urology
IL-1, IL-6, IL-8,
Activated B cell
Figure 1: Suggested cascade of immune responses in bladder mucosa induced by intravesical BCG instillation. Attachment of BCG to
urothelial cells including carcinoma cells triggers release of cytokines and chemokines from these cells, resulting in recruitment of var ious
types of immune cells into the bladder wall. Activation of phagocytes and the new cytokine environment lead to the diﬀerentiation of na
T cells into TH1 and/or TH2 cells that direct immune responses toward cellular or humoral immunity, respectively. The therapeutic
eﬀect of BCG depends on a proper induction of TH1 immune responses. IL-10 inhibits TH1 immune responses, whereas IFN-γ inhibits T H2
immune responses. Blocking IL-10 or inducing IFN-γ can lead to a TH1-dominated immunity that is essential for BCG-mediated bladder
completely clear, Th1 cytokines (e.g., IFN-γ, IL-2, and IL-
12) have been associated with BCG response, while Th2
cytokines (e.g., IL-10 and IL-6) correlate with BCG failure,
as illustrated in Figure 1 [13–16]. Since the advent of BCG
therapy, a signiﬁcant amount of data has accumulated to
support maintenance treatments, which typically consist
of a series of shorter treatments at 3–6-month intervals,
often based on the time table developed by the Southwest
Oncology Group .
While success has improved with the addition of main-
tenance treatments, the combination of intrave sical therapy,
surveillance, and repeat surgical procedures place enormous
costs on the US healthcare system, approaching $4 billion
annually . Prompted by the burden of patients either with
BCG refractory disease or who are intolerant of treatment,
the search goes on for therapeutic improvements. Given that
the eﬀect of BCG is immune mediated, decades of research
have focused on adjunctive immunotherapies including IFN-
α, IL-2, IL-10, and IL-12. This paper summarizes and
integrates key points for the clinical urologic oncologist.
Interferons (IFNs) are glycoproteins initially isolated in
the 1950s and valued for their antiviral properties. Three
typeshavebeenisolated,IFN-α (which is actually a family
of interferons), IFN-β,andIFN-γ.IFN-α and IFN-β are
grouped as “Type I” interferons, whereas IFN-γ is a “Type
II” interferon. The Type I interferon receptor has 2 compo-
nents, IFNAR-1 and IFNAR-2, which subsequently bind and
phosphorylate Jak molecules initiating a cascade resulting
in gene transcription . The IFN-α family is well known
to stimulate natural killer (NK) cells, induce MHC class
I response, and increase antibody recognition . They
have antineoplastic properties by direct antiproliferative
eﬀects and complex immunomodulatory eﬀects , both of
which could be advantageous for bladder cancer treatment.
Clinically available preparations include IFN-α2a (Roferon-
A, Roche Laboratories, Nutley, NJ) and IFN-α2b (Intron-
A, Schering Plough, Kenilworth, NJ), though to date most
research involves IFN-α2b. There has been interest in IFN-
α2b both alone and in combination with BCG, where
a synergistic response has been described. Conceptually,
combining BCG and IFN makes sense. BCG eﬃcacy depends
on the induction of a robust Th1 cytokine proﬁle, and
IFN-α2b has been shown to potentiate the Th1 immune
response . However, despite theoretical promise, data
after translation to clinical practice has been mixed.
For many years, IFN-α was thought to exert antitumor
activity primarily through direct antiproliferative properties
. At least part of this eﬀect has been shown to be
Advances in Urology 3
mediated by directly inducing tumor cell death. IFN-α has
been documented to independently induce Tumor-Necrosis-
Factor-Related Apoptosis-Inducing Ligand (TRAIL) expres-
sion in UM-UC-12 bladder cancer cells , which subse-
quently triggers apoptosis in cells expressing the appropriate
cell death receptor. Cell death occurs ultimately by Fas-
associated protein with death-domain- (FADD-) dependent
activation of the death inducing signaling complex (DISC)
followed by activation of caspase-8. Furthermore, Tecchio
and colleagues have demonstrated that IFN-α can stimulate
TRAIL mRNA as well as the release of a bioact ive soluble
TRAIL protein from neutrophils and monocytes, which
induces apoptotic activity on TRAIL-sensitive leukemic cell
lines . It also appears that IFN-α apoptotic eﬀects may
not be limited to TRAIL; rather it may trigger caspase-
8 via both cell death receptor-dependent and independent
pathways . Much like IFN-α, BCG has also been
shown to induce TRAIL , which has correlated with
patient response to BCG therapy and has been a source
of overlapping research interest. Other direct IFN-α eﬀects
include enhancing cytotoxicity of CD4
T cells, increasing
antigen detection by upregulating MHC class I expression
[20, 25, 26]. Direct suppression of proliferation by induc-
tion of tumor suppressor genes or inhibition of tumor
oncogenes has also been described . Also contributing
to antiproliferative properties, IFN-α has been documented
to decrease angiogenesis and basic ﬁbroblast growth fac-
tor. Additionally, it downregulates matrix metalloprotease-9
(MMP-9) mRNA as well as the MMP-9 t ranslational protein
in murine bladder tumors . Interestingly, it has also
been demonstrated that an optimal biologic dose with higher
frequency, rather than maximal tolerated dose, produced
the most signiﬁcant decreases in angiogenesis. Signiﬁcantly
decreased angiogenesis has also been documented in human
urothelium during and after IFN-α2b treatment follow ing
transurethral resection of superﬁcial bladder tumors .
In vivo monotherapy with IFN-α2b for bladder cancer
in humans has been explored by multiple groups. In 1990,
Glashan published data from a randomized controlled trial
evaluating high dose (100 million unit) and low dose (10
million unit) IFN-α2b regimens in patients with CIS .
Patients were treated weekly for 12 weeks and monthly
thereafter for 1 year. The high and low dose groups had
complete response rates of 43% and 5%, respectively. Of
the high dose patients achieving a complete response, 90%
remained disease-free at a notably short 6 months of follow-
up. The primary side eﬀects of treatment were ﬂu-like
symptoms (8% low dose, 17% high dose) but without
the ir ritative symptoms seen so often in BCG therapy.
When IFN-α2b was investigated alone to treat BCG failures,
eight of twelve patients had recurrence at initial three-
month evaluation and only one of twelve was disease-free
at 24 months . Another tri al conducted by Portillo and
colleagues randomized 90 pT1 bladder cancer patients to
either intravesical treatment or placebo groups as primary
prophylaxis after complete resection . They utilized a
similar dosing schedule but used 60 million units IFN-α2b.
At 12 months of followup, recurrence rates were signiﬁcantly
lower for IFN-α2b group than placebo, 28.2% versus 35.8%,
respectively. However, after 43 months, rates were similar—
53.8% and 51.2%, respectively, indicating that treatment
beneﬁt of IFN-α2b alone may not be durable.
Given the described antiproliferative and immunomod-
ulatory eﬀects of IFN- α, combination therapy with BCG
has held tantalizing promise. Gan et al. found signiﬁcantly
greater antitumor activity with combination therapy than
BCG alone: 14/15 mice receiving BCG/IFN-α versus 8/15
mice receiving only BCG became tumor-free after 5 weekly
intralesional treatments . In an in vitro study comparing
BCG plus IFN-α to BCG alone, our group demonstrated a
66-fold increase in IFN-γ production in peripheral blood
mononuclear cell (PBMC) cultures . Since IFN-γ is a
major Th1-restricted cytokine found in patients responding
to BCG therapy, it has been used routinely as a surrogate
marker for Th1 immune response in studies examining eﬀect
of IFN-α . It appears that IFN-α by itself generates a
neglig ible Th1 response, as no signiﬁcant levels of IFN-
γ were detected after IFN-α was incubated alone with the
PBMCs. We have also demonstrated that the augmented
IFN-γ production persisted e ven with reduced doses of BCG.
These ﬁndings give credence to the idea that adding Th1-
stimulating cytokines may allow for a decrease in BCG
doses, thereby decreasing side eﬀects thought to be directly
related to BCG. Further augmenting Th1 diﬀerentiation,
IFN-α was found to increase levels of several Th1 cytokines,
including IL-12 and TNF-α as well as decreasing known Th1
inhibitory cytokines IL-10 and IL-6 by 80–90% and 20–30%,
Clinical investigations with the combination of IFN-α2b
and BCG began initially in BCG refractory patients but were
subsequently expanded to BCG na
ıve patients. Stricker et al.
found the combination to be safe, with a similar side eﬀect
proﬁle to BCG alone . In 2001, O’Donnell and colleagues
reported on combination therapy administered to 40 patients
who had failed at least 1 course of BCG alone . At 24
months, 53% of patients were disease-free. Patients with two
or more prior BCG failures faired similarly to patients with
only one. Lam et al. in 2003 repor ted on the treatment of
32 patients, of which 20 (63%) were BCG failures. At 22
months’ median followup, 12 of the 20 BCG failure patients
(60%) remained disease-free . In a smaller trial, Punnen
et al. documented a 50% disease-free rate after combination
therapy at 12 months’ followup in 12 patients with BCG
refractory disease . A subsequent large community-based
phase II clinical trial examined 1106 patients from 125 sites
with NMIBC, which were split into BCG na
ıve and BCG
refractory groups . At median 24 months’ followup,
tumor-free rates were 59% and 45%, respectively. In this
larger trial, patients who had two or more courses of prior
BCG thera py had a worse outcome when compared to
patients who had 1 or less, likely indicating more resistant
disease. A recent study limited to BCG na
demonstrated similar disease-free rate of 62% but with much
longer median followup of 55.8 months . Furthermore,
after evaluating failure patterns and response rates to BCG
plus IFN-α, Gallagher et al. found that patients who recurred
more than 12 months after initial BCG treatments had
similar tumor-free rates at 24 months when compared to
4 Advances in Urology
ıve patients . However, patients who recurred
within a year of receiving their initial BCG treatments did
signiﬁcantly worse, with disease-free rates of 34–43% at
24 months, indicating that additional immunotherapy may
not be appropriate. Overall, while promising, these data
are unable to deﬁne any treatment beneﬁt of combination
therapy over BCG alone.
To date, the only randomized trial comparing BCG alone
to BCG plus IFN-α was a multicenter study of 670 BCG
ıve patients with CIS, Ta, or T1 urothelial carcinoma .
This was a four-arm t rial evaluating eﬃcacy of megadose
vitamins as well as BCG and IFN. Patients were randomized
to 1 of 4 groups: BCG plus recommended daily vitamins,
BCG plus megadose daily vitamins, BCG plus IFN-α2b plus
recommended daily vitamins, and BCG plus IFN-α2b plus
megadose daily vitamins. At 24-month followup, median
recurrence-free survival was similar across all groups, though
the two IFN-α2b groups experienced higher incidence of
constitutional symptoms and fever (P < 0.05).
Lastly, there are multiple areas where additional research
is warranted. A recent evolution in combination therapy has
been the development of an IFN-α2b expressing strain of
recombinant BCG (rBCG-IFN-α) from the Pasteur strain
of BCG. An initial in vitro study documented enhanced
IFN-γ expression in PBMCs after incubation with rBCG-
IFN-α as compared to standard BCG . A subsequent
study reported that rBCG-IFN-α increased cytotoxicity up
to 2-fold over standard BCG in PBMC cultures. Both
NK cells and CD8
T cells were identiﬁed
as primary contributors to the increased cytotoxicity .
Combining IFN-α2b with other antiproliferative agents has
shown in vitro promise. Louie et al. reported that a com-
bination of IFN-α2b and maitake mushroom D-fraction
(PDF) could reduce T24 bladder cancer cell proliferation
by 75%, accompanied by G
cell cycle arrest . Another
combination recently published this year documented that
adding grape seed proanthocyanin signiﬁcantly enhanced
antiproliferative eﬀects of IFN-α2b, with >95% growth
reduction in T24 bladder cancer cells. Cell cycle analysis also
cell cycle arrest, with Western blots conﬁrming
expression of G
cell cycle regulators . Lastly, several
groups have investigated gene therapy with a recombinant
adenovirus delivery system (rAd-IFN/Syn3), which could
potentially result in sustained therapeutic IFN-α2b levels
for long periods of time. Nagabhushan et al. were able to
demonstrate delivery and expression of IFN in the bladder
as wel l as signiﬁcant tumor regression in mice. Phase I
trials with rAd-IFN/Syn3 were ongoing at the time of their
publication in 2007 .
The discovery and characterization of interleukin-2 (IL-
2) was one of the most important breakthroughs in the
ﬁeld of immunology. Prior to its discovery, lymphocytes
were thought to be terminally diﬀerentiated and incapable
of proliferation [47, 48]. In 1975, it was discovered that
the supernatant of murine splenic cell cultures could
stimulate thymocytes, suggesting a native eﬀector protein
was responsible for this mitogenic activity [48, 49]. When
initially examined independently by diﬀerent investigators,
this “eﬀector protein” was given multiple working names
including thymocyte-stimulating factor (TSF), thymocyte
mitogenic factor (TMF), T cell growth factor (TCGF),
costimulator, killer cell helper factor (KHF), and secondary
cytotoxic T-cell-inducing factor (SCIF) . In 1979, it
was recognized that these factors likely represented the
same entity, and the nomenclature was standardized with
the term “interleukin” (between leukocytes). Thus, the
“eﬀector protein” was named IL-2, diﬀerentiating it from
the only other interleukin known at that time, IL-1 .
Regardless of the nomenclature, this protein was recognized
to promote proliferation of primary T cells in vitro,which
revolutionized the experimental armamentarium in the ﬁeld
of immunology [ 47, 49, 51].
Since the discovery of IL-2-mediated control of T-
cell growth in culture, there has been much progress in
elucidating its mechanisms. It was discovered relatively early
that IL-2 enhances the production of cytotoxic lymphocytes
which are capable of lysing tumor cells while leaving normal
cells unharmed [51–53]. These IL-2 activated lymphocytes
became known as “lymphokine-activated killer” (LAK) cells
and were thought to play a large role in antitumor immune
function [51–53]. Additionally, it was noted that IL-2
functions to augment the cytotoxic activity of NK cells and
monocytes [54, 55 ]. It has e ven been discovered that IL-2 is
important for the activation of B cells . As the CD4
and Th2 cell cytokine proﬁles were deﬁned, it became clear
that IL-2 is predominantly a Th1-secreted cytokine .
The cytotoxic antitumor capabilities induced in lympho-
cytes by IL-2 make it a potential cancer immunotherapeutic
agent. To date, multiple studies have demonstrated regres-
sion of metastatic disease following systemic IL-2 treatment
in some cancers . Rosenberg et al. reported on 157
patients with a heterogenous mix of metastatic cancers
refractory to other treatments including renal cell, colon
cancer, breast cancer, and lymphoma. Patients were treated
with either IL-2 and LAK cells or IL-2 alone. Between
the two groups, 9 complete and 20 partial responses were
obtained. Signiﬁcant morbidity has been reported with
systemic IL-2 much of which is secondary to increased
capillary permeability [58, 59] and includes weight gain,
hypotension, oliguria, elevated creatinine, and bilirubin.
These tend to resolve with cessation of IL-2 therapy ;
however, Rosenberg repor ted 4 treatment-related deaths
among their 157 patients. Despite the reports of morbidity,
IL-2 seemed to oﬀer hope to patients with few treatment
With regard to bladder cancer, interest was stimulated
after multiple investigators identiﬁed elevated IL-2 levels (as
well as other cytokines) in urine of patients following BCG,
suggesting an immunomodulatory eﬀect of BCG [ 60–67].
Additionally, an elevation in IL-2 receptor expression has
been documented on T cells in voided urine after BCG
therapy [64, 66]. Increased levels of urinary IL-2 have also
been found to correlate with BCG response, which supports
the concept that a Th1 cytokine proﬁle confers a favorable
response to BCG [15 ]. Furthermore, elevated IL-2 has been
Advances in Urology 5
reported in the ser um of patients following BCG instillation,
which suggests both a local and systemic immune response
to therapy [68, 69]. These ﬁnding s led to the conclusion that
IL-2 may have a therapeutic use in bladder cancer.
One of the ﬁrst clinical trials reported evidence of bladder
tumor regression following intralesional injections of IL-
2, with no adverse events recorded . Multiple murine
studies have demonstrated that systemic administration of
IL-2, w ith or without BCG, can signiﬁcantly decrease tumor
size, suppress tumor growth, and improve mean survival
[71–73]. A small clinical study investigating systemic IL-2
administration eﬀects on low-stage bladder cancer found
a complete and partial response rate in 5 of 12 patients,
though 2 patients discontinued therapy due to toxicity .
The poor side eﬀect proﬁle of systemic IL-2 administration
subsequently prompted a shift to utilize IL-2 as an intrav-
esical therapy. Reports of intravesical use revealed a much
improved side eﬀectproﬁleaswellassomeeﬃcacy alone or
when combined with BCG [75–78]. Den Otter et al. admin-
istered intravesical IL-2 alone after incomplete tr a nsurethral
resection of grade 1-2, T1 papillary urothelial carcinoma, and
documented “marker lesion” regression in 8 of 10 patients
. Additional experiments have focused on de veloping
recombinant-IL-2-secreting strains of BCG [80–85]. Animal
models using this approach have shown that compared
to native BCG, IL-2-secreting BCG strains have increased
IFN-γ production, induced a more favorable IFN-γ to IL-
4 ratio, improved antigen-speciﬁc proliferation, enhanced
antitumor cytotoxicity, and mounted a Th1 cytokine proﬁle
even in immunosuppressed or IL-4 transgenic mice (two
conditions which favor a Th2 response) [80–83, 85]. More
recent animal and in vitro studies have investigated IL-2
transfecting dendritic cells (D Cs), immobilized streptavidin-
tagged bioactive IL-2 on the biotinylated surface of murine
bladder mucosa, and development of a murine IL-2 surface
modiﬁed bladder cancer vaccine [86–89]. Since IL-2 plays
a crucial role in the Th1 response, it will continue to be a
source of interest for immunotherapy of bladder cancer.
Interleukin-12 (IL-12) has been the focus of signiﬁcant
cancer research among cytokines as well. In 1987, it was
discovered through in vitro experiments that there existed a
factor which synergized with IL-2 in promoting a cytotoxic
T lymphocyte (CTL) response . This factor was given
the name cytotoxic lymphocyte maturation factor (CLMF)
. Shortly thereafter a factor was discovered that induced
IFN-γ production, enhanced T cell responses to mitogens,
and augmented NK cell cytotoxicity . This factor was
provisionally called natural killer cell stimulatory factor
(NKSF) . It did not take long to discover that these
factors represented the same entity, thus the nomenclature
converged and this protein was termed IL-12 [91–95].
Although initially discovered in a B cell lymphoma, it was
subsequently found that I L-12 is primarily involved with the
regulation of T cells, causing proliferation of both activated
T cell subsets while causing minimal
proliferation of resting PBMCs [90, 92]. This concept is
supported by studies demonstrating that the IL-12 receptor
is upregulated in activated T and NK cells, but not in
activated B cells . IL-12 potentiates a Th1-speciﬁc
immune response, and it was later discovered that DCs
produce IL-12 and thus direct the development of Th1 cells
T cells [96, 97]. Additionally, IL-12 can, by
itself, stimulate the activation of nonspeciﬁc LAK cells and
facilitate the genera tion of an allogeneic CTL response .
IL-12 has even been found to play a role in the activation
of neutrophils [99, 100]. Mu ltiple studies have demonstrated
that IL-12 strongly inhibits neovascularization, thought to
be mediated through its induction of IFN-γ [101–104].
Furthermore, the mechanism by which IL-12 enhances the
cytolytic eﬀect of NK cells is primarily via the perforin
pathway [105, 106].
Multiple animal studies have shown tumor responsive-
ness to immunomodulation with IL-12. Using systemic or
peritumoral injections, IL-12 showed antitumor proper ties
in murine sarcoma, melanoma, renal cell carcinoma, lung
cancer, colon cancer, breast cancer, and bladder cancer mod-
els [102, 107–111]. Increases in serum IFN-γ were observed
in mice treated with IL-12 . Antitumor eﬃcacy was
lost in CD8
-depleted mice, but not CD4
or NK-deﬁcient mice, suggesting that the primary mediators
of the antitumor IL-12 eﬀect are CD8
T cells [107, 108].
Some of these studies saw eﬀectiveness even with metastatic
disease, including bladder cancer [107, 108, 111]. Multiple
murine studies have also revealed added eﬀectiveness with
IL-12 administered in combination with chemotherapeutic
agents [109, 112–114]. Additionally, IL-12 therapy has shown
synergistic activity when combined with radiation therapy in
mice [110, 115]. Various delivery systems for IL-12 therapy
have been tested in mice using viral and retroviral vectors
to elicit an IL-12 response [116–120]. These constructs have
shown some eﬀectiveness as anticancer therapeutics [116–
119]. IL-12 as an intravesical therapy for bladder cancer has
shown great success in mouse models. BCG was found to b e
a potent stimulus for IL-12 expression, and neutralization of
IL-12 signiﬁcantly dampened the induction of IFN-γ by BCG
. BCG therapy for murine bladder cancer was essentially
found to be ineﬀective in IL-12 knock-out mice, suggesting
a crucial role for IL-12 in the BCG response . When
IL-12 is used as a therapy with BCG, it causes a synergistic
induction of IFN-γ . Intravesical IL-12 treatment alone
was found to be eﬀective for the treatment of orthotopically
placed bladder tumors in mice, and urinary IFN-γ was
subsequently found to be signiﬁcantly elevated [111, 123].
These observations further support the importance of IFN-
γ induction for eﬀective immunotherapy of bladder cancer.
More recently, multiple attempts have been made to improve
the delivery of intravesical IL-12 to the bladder mucosa to
improve eﬃcacy. One method utilized cationic liposome-
mediated IL-12 gene therapy, which showed improved
survival and tumor-speciﬁc immunologic memor y in mice
. Another method utilized chitosan, a mucoadhesive
biopolymer, to increase IL-12 delivery to urothelial surfaces
. This method showed improved eﬃcacy over IL-12
alone in a mouse model .
6 Advances in Urology
The great success of IL-12 in treating various murine can-
cers subsequently led to experiments testing its use on human
cancers, though with mixed success. Initial trials focused
on systemic IL-12 treatment for metastatic cancer, though
progress was initially halted when several patients suﬀered
severe toxic eﬀects from the treatment a nd two patients
died from the therapy . A phase I trial of systemically
administered IL-12 in 40 patients with advanced malignancy
found a dose-dependent increase in circulating IFN-γ with
administration . Experiments on the per ipheral blood
of these patients showed augmented NK cell cytolytic activity
and enhanced T cell proliferation . Unfortunately, of
these 40 patients there was only one partial response and one
transient complete response . Further studies looking
at chronic administration of twice weekly IL-12 in patients
with metastatic cancer found that it is well tolerated and
induces costimulatory cytokines (including IFN-γ).
However, in a cohort of 28 patients, there was only one
patient with a partial response and two with prolonged
disease stabilization, with one of these patients eventually
exhibiting tumor regression . Similar low response
rates have been seen with systemic IL-12 in other studies
of advanced malignancies [130–134 ]. Various combinations
of immunotherapy have been tested with systemic IL-12
in humans. A phase I study examined systemic IL-12 with
low dose IL-2 and showed it was well tolerated, and the
addition of IL-2 signiﬁcantly augmented IFN-γ production
as well as the NK response . Of 28 patients, there was
one partial response and two pathologic responses .
Another phase I study using systemic IL-12 with IFN-α2b
showed acceptable toxicity, but with no response in 41
patients . As discussed previously, intravesical IL-12
showed great promise for the topical treatment of bladder
cancer in a mouse model; however, this success did not
translate clinically in humans. A phase I study of intravesical
IL-12 therapy in patients with superﬁcial bladder cancer
showed minimal toxicity, but disappointing eﬃcacy .
A total of 15 patients were enrolled in this study, of which
12 had no visible pretreatment lesions . Of these 12
patients, 7 remained disease-free and 5 had recurrence within
4 weeks. The remaining 3 patients with pretreatment lesions
had persistent disease at followup . Perhaps the most
disparaging results were that there was negligible IFN-γ-
induced in the urine and serum of these patients post-
treatment, suggesting minimal immunologic eﬀect from
intravesical IL-12 therapy . Despite the disappointing
results from human studies, IL-12 remains an important
target for the treatment of bladder cancer.
Unlike other cytokines previously discussed, interleukin-10
(IL-10) is distinct in that its primary eﬀect is to promote
a Th2 response and thus dampen the immunotherapeutic
eﬀects of BCG for the treatment of bladder cancer [54,
138, 139 ]. As a result, it may have therapeutic value not
by its native funct ion, but by abrogation of its native
function. IL-10 was ﬁrst characterized in 1989. It was
initially termed cytokine synthesis inhibitory factor (CSIF),
a rather ﬁtting name, because it was found to inhibit the
production of several cytokines produced by Th1 clones
. The most important of these cytokines was IFN-γ,
which was recognized as an important player in the Th1
response. As discussed previously, it is a key contributor
in the immunotherapeutic eﬀectiveness of BCG [ 140, 141 ].
Further studies showed that IL-10 prevented a delayed-
type hypersensitivity (DTH) response to BCG and the
neutralization or abrogation of IL-10 prolonged a response,
thus further supporting its role in the Th1/2 response [138,
142]. Several human tumors, including melanoma, non-
small-cell lung carcinoma, renal cell carcinoma, and bladder
cancer, have been found to express ele vated levels of IL-10
[143–147]. It is speculated that production of IL-10 by tumor
cells may represent an “escape mechanism” whereby tumor
cells avoid Th1-immune-mediated tumoricidal eﬀects .
There has been signiﬁcant progress in determining the
regulation and mechanism of IL-10 function since its discov-
ery, particularly with regard to its role in tumor immunology.
It is secreted by multiple cell types including Th2 cells, B cells,
and monocytes/macrophages [140, 148–150]. Like many
other cytokines, IL-10 is known to autoregulate itself by
downregulating its own mRNA synthesis . It has been
shown to block the accumulation of macrophages and DCs
at tumor sites, which are important players in the cellular
immune response [151, 152]. Additionally, it compromises
DCs ability to stimulate T-cells causing induction of antigen-
speciﬁc anergy of T cells . Furthermore, it down-
regulates the expression of MHC class II and costimulatory
molecules, thus preventing a cellular immune response to
tumor cells [154–156]. During activation of CD4
the presence of IL-10 can cause them to diﬀerentiate into
T regulatory cells 1 (Tr1), leading to peripheral tolerance
. IL-10 further reduces cellular tumoricidal activity by
preventing release of reactive nitrogen/oxygen intermediates
by macrophages and NK cells, a key step in their eﬃcacy
during cellular immune defense [139, 158].
Successful treatment of bladder cancer with BCG, as
discussed previously, requires a Th1 cytokine proﬁle. IL-
10 antagonizes the production of a Th1 milieu, thus its
neutralization has been targeted as a potential means to
augment the BCG response. Several murine studies have
demonstrated that after IL-10 knockout mice are inoculated
with bladder cancer, they have an improved BCG and local
immune response, increased bladder mononuclear inﬁltrate,
enhanced DTH responses, greater antitumor activity, and
prolonged survival [54, 138, 143]. Although murine IL-10
knockout studies are conceptually important, studies focused
on IL-10 neutralization hold more promise as clinically
useful therapeutics. Murine bladder cancer studies utilizing
anti-IL-10 neutralizing antibody have shown similar results,
with BCG treatment inducing an enhanced DTH response
and increased bladder mononuclear inﬁltrate [138, 142].
More recent eﬀorts have been placed at targeting the IL-
10 receptor. The IL-10 receptor is composed of two known
subunits (IL-10R1 and IL-10R2), and the IL-10R1 subunit
plays the predominant role in sig nal transduction . In
in vitro studies, we have recently shown that splenocytes
incubated with BCG and anti-IL-10-receptor 1 monoclonal
Advances in Urology 7
antibody (anti-IL-10R1 mAb) produced signiﬁcantly higher
IFN-γ than those incubated with BCG plus anti-IL-10-
neutralizing antibody, suggesting that interference with IL-10
signal transduction may be more eﬀective than neutralizing
IL-10 protein (17). In in vivo studies, mice treated with
BCG and anti-IL-10R1 mAb showed increased urinary IFN-
γ production compared to BCG controls . In a similar
murine experiment, there was improved overall and tumor-
free state in mice treated with BCG plus anti-IL-10R1 mAb
compared to BCG treatment controls, though this diﬀerence
did not reach statistical signiﬁcance . Most recently, in
an experiment designed to follow murine survival after inoc-
ulation with a luciferase-expressing MB49 bladder cancer
cells, we discovered that control mice and BCG-only treated
mice developed histologically conﬁrmed lung metastasis,
whereas mice treated w ith BCG and anti-IL-10R1 mAb
developed no metastasis [unpublished data]. This diﬀerence
was statistically signiﬁcant and raises questions as to anti-IL-
for local bladder cancer control. Conﬁrmatory experiments
and mechanistic studies are necessary, but anti-IL-10R1
mAb shows great potential in not only local bladder cancer
control, but also possibly systemic immunomodulation for
the prevention of metastatic bladder cancer.
Bladder cancer is a disease that places signiﬁcant social and
ﬁnancial burdens both on the patient and on society, costing
nearly $4 billion annually in the U.S. BCG, which stimulates
a robust immune response in most patients and has become
the standard of care after surgical resection of nonmuscle
invasive disease. However, despite treatment, a signiﬁcant
portion of patients still recur or are intolerant of BCG side
eﬀects. Multiple immunotherapies including IFN-α,IL-2,
IL-12, and IL-10 have been investigated, either as adjuncts
with BCG or as a solo replacement therapy. To date, there
are a multitude of encouraging in vitro and murine studies;
however, no clinical data has yet been reported, which is
compelling enough to change the standard of care, yet many
practitioners continue to use adjunctive immunotherapy
based on basic science data and theoretical beneﬁt. At our
institution, for instance, BCG or BCG/IFN-α refractory
disease is often treated with “quadruple therapy”—a com-
bination of BCG, IFN-α, IL-2, and GM-CSF. The widespread
use of immunother a py for bladder cancer highlights the need
for additional basic science and clinical research to further
our understanding of tumor biology, human immunology,
and the treatment of urothelial carcinoma.
E. J. Askeland and M. R. Newton contributed equally to the
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