Hindawi Publishing Corporation
Advances in Urology
Volume 2012, Article ID 181987, 13 pages
EricJ. Askeland,Mark R.Newton, MichaelA.O’Donnell, andYiLuo
Department of Urology, University of Iowa, 375 Newton 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
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, occurring in 70%, 20%, and 10% of NMIBC cases,
respectively . Standard primary treatment for NMIBC is
transurethral resection (TUR); however, recurrence rates for
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
bacillus Calmette-Gu´ 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
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, up to 50% will have a recurrence . 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
presenting cells in the context of major histocompatibility
complex (MHC) class II, stimulating CD4+T cells and
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 factor (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
TH0 CD4+T 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 various
types of immune cells into the bladder wall. Activation of phagocytes and the new cytokine environment lead to the differentiation of na¨ ıve
CD4+T cells into TH1 and/or TH2 cells that direct immune responses toward cellular or humoral immunity, respectively. The therapeutic
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 intravesical 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
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
types have been isolated, IFN-α (which is actually a family
of interferons), IFN-β, and IFN-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
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 Urology3
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 bioactive 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 translational protein
in murine bladder tumors . Interestingly, it has also
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 following
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 irritative 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 trial 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
of IFN-α . It appears that IFN-α by itself generates a
negligible 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 productionpersistedevenwithreduceddoses ofBCG.
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
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 reported 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
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 therapy 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¨ ıve patients
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
4Advances in Urology
BCG na¨ 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
na¨ ıve patients with CIS, Ta, or T1 urothelial carcinoma .
This was a four-arm trial 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
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
CD56+CD8−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 G1cell 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
revealed G1cell cycle arrest, with Western blots confirming
expression of G1 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 well 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 even been discovered that IL-2 is
important for the activation of B cells . As the CD4+Th1
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 reported 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 . Furthermore, elevated IL-2 has been
Advances in Urology5
reported in the serum of patients following BCG instillation,
which suggests both a local and systemic immune response
to therapy [68, 69]. These findings led to the conclusion that
IL-2 may have a therapeutic use in bladder cancer.
tumor regression following intralesional injections of IL-
2, with no adverse events recorded . Multiple murine
studies have demonstrated that systemic administration of
IL-2, with 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 effect profile as well as some efficacy alone or
when combined with BCG [75–78]. Den Otter et al. admin-
istered intravesical IL-2 alone after incomplete transurethral
documented “marker lesion” regression in 8 of 10 patients
. Additional experiments have focused on developing
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 (DCs), 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].
subsequently found that IL-12 is primarily involved with the
regulation of T cells, causing proliferation of both activated
CD4+and CD8+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
from na¨ ıve CD4+T cells [96, 97]. Additionally, IL-12 can, by
itself, stimulate the activation of nonspecific LAK cells and
facilitate the generation of an allogeneic CTL response .
IL-12 has even been found to play a role in the activation
of neutrophils [99, 100]. Multiple 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 properties
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+-depleted mice
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
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 be
a potent stimulus for IL-12 expression, and neutralization of
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 memory 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
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 and 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 peripheral blood
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
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 function, 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 elevated levels of IL-10
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-
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+T cells,
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
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 signal transduction . In
in vitro studies, we have recently shown that splenocytes
incubated with BCG and anti-IL-10-receptor 1 monoclonal
Advances in Urology7
antibody (anti-IL-10R1 mAb) produced significantly higher
IFN-γ than those incubated with BCG plus anti-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 with BCG and anti-IL-10R1 mAb
developed no metastasis [unpublished data]. This difference
was statistically significant and raises questions as to anti-IL-
10R1 mAb’s role as more than just an augmentation to BCG
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
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
production of this paper.
 R. Siegel, D. Naishadham, and A. Jemal, “Cancer statistics,
2012,” CA Cancer Journal for Clinicians, vol. 62, no. 1, pp.
 J. Y. Ro, G. A. Staerkel, and A. G. Ayala, “Cytologic and
histologic features of superficial bladder cancer,” Urologic
Clinics of North America, vol. 19, no. 3, pp. 435–453, 1992.
 T. J. Kemp, A. T. Ludwig, J. K. Earel et al., “Neutrophil
stimulation with Mycobacterium bovis bacillus Calmette-
Gu´ erin (BCG) results in the release of functional soluble
 A. Morales, D. Eidinger, and A. W. Bruce, “Intracavitary
Bacillus Calmette Guerin in the treatment of superficial
bladder tumors,” Journal of Urology, vol. 116, no. 2, pp. 180–
 M. C. Hall, S. S. Chang, G. Dalbagni et al., “Guideline for the
management of nonmuscle invasive bladder cancer (stages
Ta, T1, and Tis): 2007 update,” Journal of Urology, vol. 178,
no. 6, pp. 2314–2330, 2007.
 D. L. Lamm, B. A. Blumenstein, E. D. Crawford et al., “A ran-
domized trial of intravesical doxorubicin and immunother-
apy with bacille Calmette-Guerin for transitional-cell carci-
noma of the bladder,” New England Journal of Medicine, vol.
325, no. 17, pp. 1205–1209, 1991.
 A. Morales, P. Ottenhof, and L. Emerson, “Treatment
of residual, non-infiltrating bladder cancer with bacillus
Calmette-Guerin,” Journal of Urology, vol. 125, no. 5, pp.
 P. U. Malmstr¨ om, H. Wijkstr¨ om, C. Lundholm et al., “5-
Year followup of a randomized prospective study comparing
mitomycin C and bacillus Calmette-Guerin in patients with
superficial bladder carcinoma,” Journal of Urology, vol. 161,
no. 4, pp. 1124–1127, 1999.
 A. P. M. Van der Meijden, R. J. Sylvester, W. Oosterlinck,
W. Hoeltl, and A. V. Bono, “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,” European Urology, vol. 44, no. 4, pp. 429–434, 2003.
 L. R. Kavoussi, E. J. Brown, J. K. Ritchey, and T. L. Ratliff,
to murine bladder mucosa. Requirement for the expression
of an antitumor response,” Journal of Clinical Investigation,
vol. 85, no. 1, pp. 62–67, 1990.
 T. C. M. Zuiverloon, A. J. M. Nieuweboer, H. V´ ekony, W. J.
Kirkels, C. H. Bangma, and E. C. Zwarthoff, “Markers pre-
dicting response to bacillus Calmette-Gu´ erin immunother-
European Urology, vol. 61, no. 1, pp. 128–145, 2012.
 Y. Luo, X. Chen, and M. A. O’Donnell, “Role of Th1 and
Th2 cytokines in BCG-induced IFN-γ production: cytokine
promotion and simulation of BCG effect,” Cytokine, vol. 21,
no. 1, pp. 17–26, 2003.
 R. Kaempfer, L. Gerez, H. Farbstein et al., “Prediction
of response to treatment in superficial bladder carcinoma
through pattern of interleukin-2 gene expression,” Journal of
Clinical Oncology, vol. 14, no. 6, pp. 1778–1786, 1996.
 G. N. Thalmann, A. Sermier, C. Rentsch, K. M¨ ohrle, M.
G. Cecchini, and U. E. Studer, “Urinary interleukin-8 and
18 predict the response of superficial bladder cancer to
intravesical therapy with bacillus Calmette-Guerin,” Journal
of Urology, vol. 164, no. 6, pp. 2129–2133, 2000.
 F. Saint, J. J. Patard, P. Maille et al., “Prognostic value of a T
helper 1 urinary cytokine response after intravesical bacillus
Calmette-Guerin treatment for superficial bladder cancer,”
Journal of Urology, vol. 167, no. 1, pp. 364–367, 2002.
 T. M. De Reijke, E. C. De Boer, K. H. Kurth, and D.
H. J. Schamhart, “Urinary cytokines during intravesical
8 Advances in Urology
bacillus Calmette-Guerin therapy for superficial bladder
cancer: processing, stability and prognostic value,” Journal of
Urology, vol. 155, no. 2, pp. 477–482, 1996.
 D. L. Lamm, B. A. Blumenstein, J. D. Crissman et al.,
“Maintenance bacillus Calmette-Guerin immunotherapy for
recurrent Ta, T1 and carcinoma in situ transitional cell
Group study,” Journal of Urology, vol. 163, no. 4, pp. 1124–
 E. Jonasch and F. G. Haluska, “Interferon in oncological
practice: review of interferon biology, clinical applications,
and toxicities,” Oncologist, vol. 6, no. 1, pp. 34–55, 2001.
 A. M. Kamat and D. L. Lamm, “Immunotherapy for bladder
cancer,” Current urology Reports, vol. 2, no. 1, pp. 62–69,
 F. Belardelli, M. Ferrantini, E. Proietti, and J. M. Kirkwood,
“Interferon-alpha in tumor immunity and immunotherapy,”
Cytokine and Growth Factor Reviews, vol. 13, no. 2, pp. 119–
 A. Papageorgiou, L. Lashinger, R. Millikan et al., “Role of
tumor necrosis factor-related apoptosis-inducing ligand in
Cancer Research, vol. 64, no. 24, pp. 8973–8979, 2004.
 C. Tecchio, V. Huber, P. Scapini et al., “IFNα-stimulated
neutrophils and monocytes release a soluble form of TNF-
related apoptosis-inducing ligand (TRAIL/Apo-2 ligand)
displaying apoptotic activity on leukemic cells,” Blood, vol.
103, no. 10, pp. 3837–3844, 2004.
 A. Papageorgiou, C. P. N. Dinney, and D. J. McConkey,
Cancer Biology and Therapy, vol. 6, no. 6, pp. 872–879, 2007.
 A. T. Ludwig, J. M. Moore, Y. Luo et al., “Tumor necrosis
factor-related apoptosis-inducing ligand: a novel mechanism
for Bacillus Calmette-Gu´ erin-induced antitumor activity,”
Cancer Research, vol. 64, no. 10, pp. 3386–3390, 2004.
 M. J. Droller and D. Gomolka, “Enhancement of natural
cytotoxicity in lymphocytes from animals with carcinogen-
induced bladder cancer,” Journal of Urology, vol. 129, no. 3,
pp. 625–629, 1983.
 P. Parronchi, M. De Carli, R. Manetti et al., “IL-4 and IFN (α
and γ) exert opposite regulatory effects on the development
of cytolytic potential by Th1 or Th2 human T cell clones,”
Journal of Immunology, vol. 149, no. 9, pp. 2977–2983, 1992.
 J. W. Slaton, P. Perrotte, K. Inoue, C. P. N. Dinney,
and I. J. Fidler, “Interferon-α-mediated down-regulation of
angiogenesis-related genes and therapy of bladder cancer are
dependent on optimization of biological dose and schedule,”
Clinical Cancer Research, vol. 5, no. 10, pp. 2726–2734, 1999.
 A. Giannopoulos, I. Adamakis, K. Evangelou et al., “In-
terferon-a2b reduces neo-microvascular density in the ’nor-
mal’ urothelium adjacent to the tumor after transurethral
resection of superficial bladder carcinoma,” Onkologie, vol.
26, no. 2, pp. 147–152, 2003.
 R. W. Glashan, “A randomized controlled study of intrav-
esical α-2b-interferon in carcinoma in situ of the bladder,”
Journal of Urology, vol. 144, no. 3, pp. 658–661, 1990.
 M. A. Hudson and T. L. Ratliff, “Failure of intravesical
interferon-alfa-2b for the treatment of patients with super-
ficial bladder cancer previously failing intravesical BCG
Therapy,” Urologic Oncology, vol. 1, no. 3, pp. 115–118, 1995.
 J. Portillo, B. Martin, R. Hernandez et al., “Results at 43
months’ follow-up of a double-blind, randomized, prospec-
tive clinical trial using intravesical interferon alpha-2b in the
prophylaxis of stage pT1 transitional cell carcinoma of the
bladder,” Urology, vol. 49, no. 2, pp. 187–190, 1997.
 Y. H. Gan, Y. Zhang, H. E. Khoo, and K. Esuvaranathan,
“Antitumour immunity of Bacillus Calmette-Guerin and
interferon alpha in murine bladder cancer,” European Journal
of Cancer, vol. 35, no. 7, pp. 1123–1129, 1999.
 Y. Luo, X. Chen, T. M. Downs, W. C. DeWolf, and M. A.
O’Donnell, “IFN-α 2B enhances Th1 cytokine responses in
bladder cancer patients receiving Mycobacterium bovis bacil-
lus Calmette-Guerin immunotherapy,” Journal of Immunol-
ogy, vol. 162, no. 4, pp. 2399–2405, 1999.
 P. Stricker, K. Pryor, T. Nicholson et al., “Bacillus Calmette-
Guerin plus intravesical interferon alpha-2b in patients with
superficial bladder cancer,” Urology, vol. 48, no. 6, pp. 957–
 M. A. O’Donnell, J. Krohn, and W. C. DeWolf, “Salvage
intravesical therapy with interferon-α2B plus low dose bacil-
lus Calmette-Guerin is effective in patients with superficial
bladder cancer in whom bacillus Calmette-Guerin alone
previously failed,” Journal of Urology, vol. 166, no. 4, pp.
 J. S. Lam, M. C. Benson, M. A. O’Donnell et al., “Bacillus
Calmete-Gu´ erin plus interferon-α2B intravesical therapy
maintains an extended treatment plan for superficial bladder
cancer with minimal toxicity,” Urologic Oncology, vol. 21, no.
5, pp. 354–360, 2003.
 S. P. Punnen, J. L. Chin, and M. A. Jewett, “Management
of bacillus Calmette-Guerin (BCG) refractory superficial
bladder cancer: results with intravesical BCG and Interferon
combination therapy,” The Canadian Journal of Urology, vol.
10, no. 2, pp. 1790–1795, 2003.
 F. N. Joudi, B. J. Smith, and M. A. O’Donnell, “Final results
from a national multicenter phase II trial of combination
bacillus Calmette-Gu´ erin plus interferon α-2B for reducing
recurrence of superficial bladder cancer,” Urologic Oncology,
vol. 24, no. 4, pp. 344–348, 2006.
 S. Bazarbashi, H. Soudy, M. Abdelsalam et al., “Co-
administration of intravesical bacillus Calmette-Gu´ erin and
interferon α-2B as first line in treating superficial transitional
cell carcinoma of the urinary bladder,” British Journal of
Urology International, vol. 108, no. 7, pp. 1115–1118, 2011.
 B. L. Gallagher, F. N. Joudi, J. L. Maym´ ı, and M. A.
O’Donnell, “Impact of previous Bacille Calmette-Gu´ erin
failure pattern on subsequent response to Bacille Calmette-
Gu´ erin plus interferon intravesical therapy,” Urology, vol. 71,
no. 2, pp. 297–301, 2008.
 K. G. Nepple, A. J. Lightfoot, H. M. Rosevear, M. A.
O’Donnell, and D. L. Lamm, “Bacillus Calmette-Gur´ ein
with or without interferon α-2b and megadose versus rec-
ommended daily allowance vitamins during induction and
maintenance intravesical treatment of nonmuscle invasive
bladder cancer,” Journal of Urology, vol. 184, no. 5, pp. 1915–
 Y. Luo, X. Chen, R. Han, and M. A. O’Donnell, “Recom-
binant bacille Calmette-Gu´ erin (BCG) expressing human
interferon-alpha 2B demonstrates enhanced immunogenic-
ity,” Clinical and Experimental Immunology, vol. 123, no. 2,
pp. 264–270, 2001.
 W. Liu, M. A. O’Donnell, X. Chen, R. Han, and Y. Luo,
“Recombinant bacillus Calmette-Gu´ erin (BCG) expressing
interferon-alpha 2B enhances human mononuclear cell
cytotoxicity against bladder cancer cell lines in vitro,” Cancer
Immunology, Immunotherapy, vol. 58, no. 10, pp. 1647–1655,
Advances in Urology9
 B. Louie, S. Rajamahanty, J. Won, M. Choudhury, and S.
Konno, “Synergistic potentiation of interferon activity with
maitake mushroom d-fraction on bladder cancer cells,”
British Journal of Urology International, vol. 105, no. 7, pp.
 A.I. Fishman, B. Johnson, B. Alexander, J. Won, M. Choud-
hury, and S. Konno, “Additively enhanced antiproliferative
effect of interferon combined with proanthocyanidin on
bladder cancer cells,” Journal of Cancer, vol. 3, pp. 107–112,
 T. L. Nagabhushan, D. C. Maneval, W. F. Benedict et al.,
“Enhancement of intravesical delivery with Syn3 potentiates
interferon-α2b gene therapy for superficial bladder cancer,”
 S. Gillis and K. A. Smith, “Long term culture of tumour
 G. Di Sabato, D. M. Chen, and J. W. Erickson, “Production
by murine spleen cells of an activity stimulating the PHA
responsiveness of thymus lymphocytes,” Cellular Immunol-
ogy, vol. 17, no. 2, pp. 495–504, 1975.
 D. M. Chen and G. Di Sabato, “Further studies on the
thymocyte stimulating factor,” Cellular Immunology, vol. 22,
no. 2, pp. 211–224, 1976.
 S. B. Mizel and J. J. Farrar, “Revised nomenclature for
antigen-nonspecific T-cell proliferation and helper factors,”
Cellular Immunology, vol. 48, no. 2, pp. 433–436, 1979.
 J. Shaw, V. Monticone, G. Mills, and V. Paetkau, “Effects
of costimulator on immune responses in vitro,” Journal of
Immunology, vol. 120, no. 6, pp. 1974–1980, 1978.
 I. Yron, T. A. Wood, P. J. Spiess, and S. A. Rosenberg, “In
vitro growth of murine T cells. V. The isolation and growth
of Immunology, vol. 125, no. 1, pp. 238–245, 1980.
 M. T. Lotze, E. A. Grimm, and A. Mazumder, “Lysis of
fresh and cultured autologous tumor by human lymphocytes
11 I, pp. 4420–4425, 1981.
 C. S. Henney, K. Kuribayashi, D. E. Kern, and S. Gillis,
“Interleukin-2 augments natural killer cell activity,” Nature,
vol. 291, no. 5813, pp. 335–338, 1981.
 M. Malkovsky, B. Loveland, and M. North, “Recombinant
interleukin-2 directly augments the cytotoxicity of human
monocytes,” Nature, vol. 325, no. 6101, pp. 262–265, 1987.
of interleukin 2 receptors on activated human B cells,”
Journal of Experimental Medicine, vol. 160, no. 5, pp. 1450–
 T. R. Mosmann, H. Cherwinski, and M. W. Bond, “Two
types of murine helper T cell clone. I. Definition according
to profiles of lymphokine activities and secreted proteins,”
Journal of Immunology, vol. 136, no. 7, pp. 2348–2357, 1986.
 S. A. Rosenberg, M. T. Lotze, and L. M. Muul, “A progress
report on the treatment of 157 patients with advanced cancer
using lymphokine-activated killer cells and interleukin-2
or high-dose interleukin-2 alone,” New England Journal of
Medicine, vol. 316, no. 15, pp. 889–897, 1987.
 D. E. Webb, H. A. Austin, A. Belldegrun, E. Vaughan, W. M.
Linehan, and S. A. Rosenberg, “Metabolic and renal effects of
Nephrology, vol. 30, no. 3, pp. 141–145, 1988.
 T. L. Ratliff, E. O. Haaff, and W. J. Catalona, “Interleukin-
2 production during intravesical bacille Calmette-Guerin
therapy for bladder cancer,” Clinical Immunology and Im-
munopathology, vol. 40, no. 2, pp. 375–379, 1986.
 E. O. Haaff, W. J. Catalona, and T. L. Ratliff, “Detection of
interleukin 2 in the urine of patients with superficial bladder
tumors after treatment with intravesical BCG,” Journal of
Urology, vol. 136, no. 4, pp. 970–974, 1986.
 W. H. De Jong, E. C. De Boer, A. P. M. Van Der Meijden et
al., “Presence of interleukin-2 in urine of superficial bladder
cancer patients after intravesical treatment with bacillus
Calmette-Guerin,” Cancer Immunology Immunotherapy, vol.
31, no. 3, pp. 182–186, 1990.
 A. B¨ ohle, C. Nowe, A. J. Ulmer et al., “Detection of urinary
TNF, IL 1, and IL 2 after local BCG immunotherapy for
bladder carcinoma,” Cytokine, vol. 2, no. 3, pp. 175–181,
 E. C. De Boer, W. H. De Jong, P. A. Steerenberg et
al., “Leukocytes and cytokines in the urine of superficial
bladder cancer patients after intravesical immunotherapy
with Bacillus Calmette-Guerine,” In Vivo, vol. 5, no. 6, pp.
 E. C. De Boer, W. H. De Jong, P. A. Steerenberg 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 Immunology Immunotherapy, vol. 34, no. 5, pp. 306–
 D. Balbay, M. Bakkaloglu, H.¨Ozen et al., “Detection of
urinary interleukin-2, interleukin-2 receptor, and tumor
necrosis factor levels in patients with superficial bladder
tumorsafter intravesical BCGimmunotherapy,” Urology, vol.
43, no. 2, pp. 187–190, 1994.
 T. M. De Reijke, E. C. De Boer, K. H. Kurth, and D.
H. J. Schamhart, “Urinary interleukin-2 monitoring during
the optimal number of instillations?” Journal of Urology, vol.
161, no. 1, pp. 67–71, 1999.
 K.Taniguchi, S.Koga, M.Nishikido etal., “Systemic immune
response after intravesical instillation of bacille Calmette-
Guerin (BCG) for superficial bladder cancer,” Clinical and
 C. Magno, D. Melloni, A. Gal` ı et al., “The anti-tumor activity
of bacillus Calmette-Guerin in bladder cancer is associated
with an increase in the circulating level of interleukin-2,”
Immunology Letters, vol. 81, no. 3, pp. 235–238, 2002.
 G. Pizza, G. Severini, and D. Menniti, “Tumour regression
after intralesional injection of interleukin 2 (IL-2) in bladder
cancer. Preliminary report,” International Journal of Cancer,
vol. 34, no. 3, pp. 359–367, 1984.
 K. E. Lee, G. H. Weiss, R. W. O’Donnell, and A. T. K.
with intravesical Bacillus Calmette Guerin and systemic
Interleukin 2,” Journal of Urology, vol. 137, no. 6, pp. 1270–
 S. Ikemoto, M. Kamizuru, S. Wada, S. Nishio, T. Kishimoto,
and M. Maekawa, “Combined effect of interleukin 2 and
Bacillus Calmette-Guerin in the therapy of mice with
transitional cell carcinoma,” Urologia Internationalis, vol. 47,
no. 4, pp. 250–254, 1991.
 D. R. Riggs, W. F. Tarry, J. I. DeHaven, J. Sosnowski, and D.
L. Lamm, “Immunotherapy of murine transitional cell car-
cinoma of the bladder using alpha and gamma interferon in
combination with other forms of immunotherapy,” Journal
of Urology, vol. 147, no. 1, pp. 212–214, 1992.
10 Advances in Urology
 A. Tubaro, F. Velotti, A. Stoppacciaro et al., “Continuous
low-stage bladder cancer: a phase IB study,” Cancer, vol. 68,
no. 1, pp. 56–61, 1991.
 P. A. Merguerian, L. Donahue, and A. T. K. Cockett, “In-
traluminal interleukin 2 and bacillus Calmette-Guerin for
treatment of bladder cancer: a preliminary report,” Journal
of Urology, vol. 137, no. 2, pp. 216–219, 1987.
 E. Huland and H. Huland, “Local continuous high dose
interleukin 2: a new therapeutic model for the treatment of
advanced bladder carcinoma,” Cancer Research, vol. 49, no.
19, pp. 5469–5474, 1989.
 A. T. K. Cockett, R. S. Davis, L. R. Cos, and L. L. Wheeless,
“Bacillus calmette-guerin and interleukin-2 for treatment of
superficial bladder cancer,” Journal of Urology, vol. 146, no. 3,
pp. 766–770, 1991.
 L. G. Gomella, D. E. McGinnis, E. C. Lattime et al.,
“Treatment of transitional cell carcinoma of the bladder with
intravesical interleukin-2: a pilot study,” Cancer Biotherapy,
vol. 8, no. 3, pp. 223–227, 1993.
interleukin-2 in T1 papillary bladder carcinoma: regression
of marker lesion in 8 of 10 patients,” Journal of Urology, vol.
159, no. 4, pp. 1183–1186, 1998.
 M. A. O’Donnell, A. Aldovini, R. B. Duda et al., “Recom-
binant Mycobacterium bovis BCG secreting functional
interleukin-2 enhances gamma interferon production by
 P. J. Murray, A. Aldovini, and R. A. Young, “Manipula-
tion and potentiation of antimycobacterial immunity using
recombinant bacille Calmette-Gu´ erin strains that secrete
cytokines,” Proceedings of the National Academy of Sciences of
 L. Slobbe, E. Lockhart, M. A. O’Donnell, C. Mackintosh,
G. De Lisle, and G. Buchan, “An in vivo comparison of
bacillus Calmette-Guerin (BCG) and cytokine- secreting
BCG vaccines,” Immunology, vol. 96, no. 4, pp. 517–523,
 H. Yamada, S. Matsumoto, T. Matsumoto, T. Yamada, and
U. Yamashita, “Murine IL-2 secreting recombinant Bacillus
Calmette-Guerin augments macrophage-mediated cytotoxi-
city against murine bladder cancer MBT-2,” Journal of Urol-
ogy, vol. 164, no. 2, pp. 526–531, 2000.
 Y. Luo, X. Chen, A. Szilvasi, and M. A. O’Donnell, “Co-
expression of interleukin-2 and green fluorescent protein
reporter in mycobacteria: in vivo application for monitoring
antimycobacterial immunity,” Molecular Immunology, vol.
37, no. 9, pp. 527–536, 2000.
 S. L. Young, M. A. O’Donnell, and G. S. Buchan, “IL-2-
secreting recombinant bacillus Calmette Guerin can over-
come a Type 2 immune response and corticosteroid-induced
immunosupression to elicit a Type 1 immune response,”
International Immunology, vol. 14, no. 7, pp. 793–800, 2002.
 Y. G. Li, Z. P. Wang, J. Q. Tian et al., “Dendritic cell trans-
fected with secondary lymphoid-tissue chemokine and/or
interleukin-2 gene-enhanced cytotoxicity of t-lymphocyte in
human bladder tumor cell s in vitro,” Cancer Investigation,
vol. 27, no. 9, pp. 909–917, 2009.
 X.Huang,H.S. Yu,Z.Chen,J.L.Li,Z.M.Hu,andJ.M.Gao,
“A novel immunotherapy for superficial bladder cancer by
the immobilization of streptavidin-tagged bioactive IL-2 on
the biotinylated mucosal surface of the bladder wall,” Chinese
Journal of Cancer, vol. 29, no. 6, pp. 611–616, 2010.
 X. Zhang, X. Shi, J. Li et al., “Novel immunotherapy for
metastatic bladder cancer using vaccine of human inter-
leukin-2 surface-modified MB 49 cells,” Urology, vol. 78, no.
3, pp. 722.el–722.e6, 2011.
 H. L. Wong, D. E. Wilson, J. C. Jenson, P. C. Familletti,
D. L. Stremlo, and M. K. Gately, “Characterization of a
factor(s) which synergizes with recombinant interleukin 2 in
promoting allogeneic human cytolytic T-lymphocyte re-
sponses in vitro,” Cellular Immunology, vol. 111, no. 1, pp.
 M. Kobayashi, L. Fitz, M. Ryan et al., “Identification and
purification of natural killer cell stimulatory factor (NKSF),
a cytokine with multiple biologic effects on human lympho-
cytes,” Journal of Experimental Medicine, vol. 170, no. 3, pp.
 A. S. Stern, F. J. Podlaski, J. D. Hulmes et al., “Purification to
homogeneity and partial characterization of cytotoxic lym-
phocyte maturation factor from human B-lymphoblastoid
cells,” Proceedings of the National Academy of Sciences of the
 M. K. Gately, B. B. Desai, A. G. Wolitzky et al., “Regulation
of human lymphocyte proliferation by a heterodimeric
cytokine, IL-12 (cytotoxic lymphocyte maturation factor),”
Journal of Immunology, vol. 147, no. 3, pp. 874–882, 1991.
 U. Gubler, A. O. Chua, D. S. Schoenhaut et al., “Coexpression
cytotoxic lymphocyte maturation factor,” Proceedings of the
National Academy of Sciences of the United States of America,
vol. 88, no. 10, pp. 4143–4147, 1991.
 D. S. Schoenhaut, A. O. Chua, A. G. Wolitzky et al., “Cloning
and expression of murine IL-12,” Journal of Immunology, vol.
148, no. 11, pp. 3433–3440, 1992.
 B. B. Desai, P. M. Quinn, A. G. Wolitzky, P. K. A. Mongini,
R. Chizzonite, and M. K. Gately, “IL-12 receptor. II. Dis-
tribution and regulation of receptor expression,” Journal of
Immunology, vol. 148, no. 10, pp. 3125–3132, 1992.
 R. Manetti, P. Parronchi, M. G. Giudizi et al., “Natural killer
cell stimulatory factor (interleukin 12 [IL-12]) induces T
helper type 1 (Th1)-specific immune responses and inhibits
the development of IL-4-producing Th cells,” Journal of
Experimental Medicine, vol. 177, no. 4, pp. 1199–1204, 1993.
 S. E. Macatonia, N. A. Hosken, M. Litton et al., “Dendritic
cells produce IL-12 and direct the development of Th1 cells
from naive CD4+ T cells,” Journal of Immunology, vol. 154,
no. 10, pp. 5071–5079, 1995.
 M. K. Gately, A. G. Wolitzky, P. M. Quinn, and R. Chizzonite,
“Regulation of human cytolytic lymphocyte responses by
interleukin-12,” Cellular Immunology, vol. 143, no. 1, pp.
 K. Collison, S. Saleh, R. Parhar et al., “Evidence for IL-12-
activated Ca2+and tyrosine signaling pathways in human
neutrophils,” Journal of Immunology, vol. 161, no. 7, pp.
 G. R. Yeaman, J. E. Collins, J. K. Currie, P. M. Guyre, C.
R. Wira, and M. W. Fanger, “IFN-γ is produced by poly-
morphonuclear neutrophils in human uterine endometrium
and by cultured peripheral blood polymorphonuclear neu-
trophils,” Journal of Immunology, vol. 160, no. 10, pp. 5145–
 E. E. Voest, B. M. Kenyon, M. S. O’Reilly, G. Truitt, R. J.
by interleukin 12,” Journal of the National Cancer Institute,
vol. 87, no. 8, pp. 581–586, 1995.
Advances in Urology11
 S. Dias, R. Boyd, and F. Balkwill, “IL-12 regulates VEGF
and MMPs in a murine breast cancer model,” International
Journal of Cancer, vol. 78, no. 3, pp. 361–365, 1998.
 C. M. Coughlin, K. E. Salhany, M. S. Gee et al., “Tumor cell
responses to IFNγ affect tumorigenicity and response to IL-
12 therapy and antiangiogenesis,” Immunity, vol. 9, no. 1, pp.
 C. M. Coughlin, K. E. Salhany, M. Wysocka et al.,
“Interleukin-12, and interleukin-18 synergistically induce
murine tumor regression which involves inhibition of angio-
genesis,” Journal of Clinical Investigation, vol. 101, no. 6, pp.
 W. Hashimoto, T. Osaki, H. Okamura et al., “Differential
antitumor effects of administration of recombinant IL-18 or
of Immunology, vol. 163, no. 2, pp. 583–589, 1999.
 T. Kawamura, K. Takeda, S. K. Mendiratta et al., “Cutting
edge: critical role of NK1 T cells in IL-12-induced immune
responses in vivo,” Journal of Immunology, vol. 160, no. 1, pp.
 M. J. Brunda, L. Luistro, R. R. Warrier et al., “Antitumor
and antimetastatic activity of interleukin 12 against murine
tumors,” Journal of Experimental Medicine, vol. 178, no. 4,
pp. 1223–1230, 1993.
binant IL-12 administration induces tumor regression in
association with IFN-γ production,” Journal of Immunology,
vol. 153, no. 4, pp. 1697–1706, 1994.
“Optimal scheduling of interleukin 12 and chemotherapy in
the murine MB-49 bladder carcinoma and B16 melanoma,”
Clinical Cancer Research, vol. 3, no. 9, pp. 1661–1667, 1997.
“Optimal scheduling of interleukin-12 and fractionated
radiation therapy in the murine lewis lung carcinoma,”
Radiation Oncology Investigations, vol. 6, no. 2, pp. 71–80,
 M. A. O’Donnell, Y. Luo, S. E. Hunter, X. Chen, L. L.
Hayes, and S. K. Clinton, “Interleukin-12 immunotherapy
of murine transitional cell carcinoma of the bladder: dose
dependent tumor eradication and generation of protective
 R. S. Zagozdzon, A. Giermasz, J. Golab, T. Stoklosa, A.
Jalili, and M. Jak´ obisiak, “The potentiated antileukemic
effects of doxorubicin and interleukin-12 combination are
not dependent on nitric oxide production,” Cancer Letters,
vol. 147, no. 1-2, pp. 67–75, 1999.
 R. Zagozdzon, J. Golab, K. Mucha, B. Foroncewicz, and M.
Jakobisiak, “Potentiation of antitumor effects of IL-12 in
combination with paclitaxel in murine melanoma model in
vivo,” International Journal of Molecular Medicine, vol. 4, no.
6, pp. 645–648, 1999.
 J. Golab, R. Zagozdzon, K. Kozar et al., “Potentiatied anti-
tumor effectiveness of combined therapy with interleukin-
12 and mitoxantrone of L1210 leukemia in vivo,” Oncology
Reports, vol. 7, no. 1, pp. 177–181, 2000.
 B. A. Teicher, G. Ara, K. Menon, and R. G. Schaub, “In
vivo studies with interleukin-12 alone and in combination
with monocyte colony-stimulating factor and/or fraction-
ated radiation treatment,” International Journal of Cancer,
vol. 65, no. 1, pp. 80–84, 1996.
 F. H. L. Lieu, T. S. Hawley, A. Z. C. Fong, and R. G. Hawley,
“Transmissibility of murine stem cell virus-based retroviral
vectors carrying both interleukin-12 cDNAs and a third
gene: implications for immune gene therapy,” Cancer Gene
Therapy, vol. 4, no. 3, pp. 167–175, 1997.
and F. L. Graham, “Direct intratumoral injection of an
adenovirus expressing interleukin-12 induces regression and
expression of interleukin-12,” Human Gene Therapy, vol. 7,
no. 16, pp. 1995–2002, 1996.
 C. L. Addison, J. L. Bramson, M. M. Hitt, W. J. Muller,
J. Gauldie, and F. L. Graham, “Intratumoral coinjection
of adenoviral vectors expressing IL-2 and IL-12 results in
enhanced frequency of regression of injected and untreated
distal tumors,” Gene Therapy, vol. 5, no. 10, pp. 1400–1409,
 J. B. Meko, J. H. Yim, K. Tsung, and J. A. Norton, “High
cytokine production and effective antitumor activity of a
recombinant vaccinia virus encoding murine interleukin 12,”
Cancer Research, vol. 55, no. 21, pp. 4765–4770, 1995.
 L. Zitvogel, H. Tahara, Q. Cai et al., “Construction and
characterization of retroviral vectors expressing biologically
active human interleukin-12,” Human Gene Therapy, vol. 5,
no. 12, pp. 1493–1506, 1994.
 M. A. O’Donnell, Y. Luo, X. Chen, A. Szilvasi, S. E. Hunter,
and S. K. Clinton, “Role of IL-12 in the induction and
potentiation of IFN-γ in response to bacillus Calmette-
Guerin,” Journal of Immunology, vol. 163, no. 8, pp. 4246–
 J. Riemensberger, A. B¨ ohle, and S. Brandau, “IFN-gamma
and IL-12 but not IL-10 are required for local tumour
surveillance in a syngeneic model of orthotopic bladder
cancer,” Clinical and Experimental Immunology, vol. 127, no.
1, pp. 20–26, 2002.
 M. A. O’Donnell, Y. Luo, S. E. Hunter, X. Chen, L. L. Hayes,
and S. K. Clinton, “The essential role of interferon-γ during
of the bladder,” Journal of Urology, vol. 171, no. 3, pp. 1336–
 M. Horinaga, K. M. Harsch, R. Fukuyama, W. Heston, and
W. Larchian, “Intravesical interleukin-12 gene therapy in an
orthotopic bladder cancer model,” Urology, vol. 66, no. 2, pp.
 D. A. Zaharoff, B. S. Hoffman, H. B. Hooper et al.,
“Intravesical immunotherapy of superficial bladder cancer
with chitosan/interleukin-12,” Cancer Research, vol. 69, no.
15, pp. 6192–6199, 2009.
 J. Cohen, “IL-12 deaths: explanation and a puzzle,” Science,
vol. 270, no. 5238, p. 908, 1995.
 M. B. Atkins, M. J. Robertson, M. Gordon et al., “Phase I
evaluation of intravenous recombinant human interleukin
12 in patients with advanced malignancies,” Clinical Cancer
Research, vol. 3, no. 3, pp. 409–417, 1997.
 M. J. Robertson, C. Cameron, M. B. Atkins et al., “Immuno-
logical effects of interleukin 12 administered by bolus intra-
venous injection to patients with cancer,” Clinical Cancer
Research, vol. 5, no. 1, pp. 9–16, 1999.
 J. A. Gollob, J. W. Mier, K. Veenstra et al., “Phase I trial
of twice-weekly intravenous interleukin 12 in patients with
metastatic renal cell cancer or malignant melanoma: ability
to maintain IFN-γ induction is associated with clinical
response,” Clinical Cancer Research, vol. 6, no. 5, pp. 1678–
12Advances in Urology
 J. A. Hurteau, J. A. Blessing, S. L. DeCesare, and W. T.
Creasman, “Evaluation of recombinant human interleukin-
12 in patients with recurrent or refractory ovarian cancer:
a gynecologic oncology group study,” Gynecologic Oncology,
vol. 82, no. 1, pp. 7–10, 2001.
 R. J. Motzer, A. Rakhit, J. A. Thompson et al., “Randomized
multicenter phase II trial of subcutaneous recombinant
advanced renal cell carcinoma,” Journal of Interferon and
Cytokine Research, vol. 21, no. 4, pp. 257–263, 2001.
 R. Lenzi, M. Rosenblum, C. Verschraegen et al., “Phase
I study of intraperitoneal recombinant human interleukin
12 in patients with M¨ ullerian carcinoma, gastrointestinal
primary malignancies, and mesothelioma,” Clinical Cancer
Research, vol. 8, no. 12, pp. 3686–3695, 2002.
 R. Lenzi, R. Edwards, C. June et al., “Phase II study
of intraperitoneal recombinant interleukin-12 (rhIL-12) in
patients with peritoneal carcinomatosis (residual disease
<1cm) associated with ovarian cancer or primary peritoneal
carcinoma,” Journal of Translational Medicine, vol. 5, article
 A. Younes, B. Pro, M. J. Robertson et al., “Phase II clinical
non-Hodgkin’s lymphoma and Hodgkin’s disease,” Clinical
Cancer Research, vol. 10, no. 16, pp. 5432–5438, 2004.
 J. A. Gollob, K. G. Veenstra, R. A. Parker et al., “Phase I trial
carcinoma,” Journal of Clinical Oncology, vol. 21, no. 13, pp.
 C. F. Eisenbeis, G. B. Lesinski, M. Anghelina et al., “Phase
I study of the sequential combination of interleukin-12 and
Clinical Oncology, vol. 23, no. 34, pp. 8835–8844, 2005.
 G. R. Weiss, M. A. O’Donnell, K. Loughlin, K. Zonno, R. J.
Laliberte, and M. L. Sherman, “Phase 1 study of the intraves-
ical administration of recombinant human interleukin-12 in
of the bladder,” Journal of Immunotherapy, vol. 26, no. 4, pp.
 R. Nadler, Y. Luo, W. Zhao et al., “Interleukin 10
induced augmentation of delayed-type hypersensitivity
(DTH) enhances Mycobacterium bovis bacillus Calmette-
Gu´ erin (BCG) mediated antitumour activity,” Clinical and
 Y. Luo, R. Han, D. P. Evanoff, and X. Chen, “Interleukin-
10 inhibits Mycobacterium bovis bacillus Calmette-Gu´ erin
(BCG)-induced macrophage cytotoxicity against bladder
no. 3, pp. 359–368, 2010.
 D. F. Fiorentino, M. W. Bond, and T. R. Mosmann, “Two
types of mouse T helper cell. IV. Th2 clones secrete a factor
that inhibits cytokine production by Th1 clones,” Journal of
Experimental Medicine, vol. 170, no. 6, pp. 2081–2095, 1989.
 D. F. Fiorentino, A. Zlotnik, P. Vieira et al., “IL-10 acts on the
 T. A. Ferguson, P. Dube, and T. S. Griffith, “Regulation
of contact hypersensitivity by interleukin 10,” Journal of
Experimental Medicine, vol. 179, no. 5, pp. 1597–1604, 1994.
 B. K. Halak, H. C. Maguire, and E. C. Lattime, “Tumor-
induced interleukin-10 inhibits type 1 immune responses
directed at a tumor antigen as well as a non-tumor antigen
present at the tumor site,” Cancer Research, vol. 59, no. 4, pp.
 E. C. Lattime, M. J. Mastrangelo, O. Bagasra, W. Li, and D.
Berd, “Expression of cytokine mRNA in human melanoma
tissues,” Cancer Immunology Immunotherapy, vol. 41, no. 3,
pp. 151–156, 1995.
sion of interleukin 10 in human melanoma,” British Journal
of Cancer, vol. 70, no. 6, pp. 1182–1185, 1994.
 M. Huang, J. Wang, P. Lee et al., “Human non-small cell
lung cancer cells express a type 2 cytokine pattern,” Cancer
Research, vol. 55, no. 17, pp. 3847–3853, 1995.
 H. Nakagomi, P. Pisa, E. K. Pisa et al., “Lack of interleukin-2
(IL-2) expression and selective expression of IL-10 mRNA in
human renal cell carcinoma,” International Journal of Cancer,
vol. 63, no. 3, pp. 366–371, 1995.
 T. R. Mosmann, J. H. Schumacher, D. F. Fiorentino, J.
Leverah, K. W. Moore, and M. W. Bond, “Isolation of
Th2-specific cytokine (IL-10), cytokine synthesis inhibitory
factor, by using a solid phase radioimmunoadsorbent assay,”
Journal of Immunology, vol. 145, no. 9, pp. 2938–2945, 1990.
 A. O’Garra, G. Stapleton, V. Dhar et al., “Production of
cytokines by mouse B cells: B lymphomas and normal B cells
produce interleukin 10,” International Immunology, vol. 2,
no. 9, pp. 821–832, 1990.
 R. De Waal Malefyt, J. Abrams, B. Bennett, C. G. Figdor,
and J. E. De Vries, “Interleukin 10(IL-10) inhibits cytokine
synthesis by human monocytes: an autoregulatory role of
IL-10 produced by monocytes,” Journal of Experimental
Medicine, vol. 174, no. 5, pp. 1209–1220, 1991.
 Z. Qin, G. Noffz, M. Mohaupt, and T. Blankenstein,
“Interleukin-10 prevents dendritic cell accumulation and
vaccination with Granulocyte-Macrophage Colony-Stim-
ulating Factor Gene-Modified Tumor Cells,” Journal of Im-
munology, vol. 159, no. 2, pp. 770–776, 1997.
 G. Richter, S. Kruger-Krasagakes, G. Hein et al., “Inter-
leukin 10 transfected into chinese hamster ovary cells
prevents tumor growth and macrophage infiltration,” Cancer
Research, vol. 53, no. 18, pp. 4134–4137, 1993.
 K. Steinbrink, M. W¨ olfl, H. Jonuleit, J. Knop, and A. H.
 R. De Waal Malefyt, J. Haanen, H. Spits et al., “Interleukin
10 (IL-10) and viral IL-10 strongly reduce antigen-specific
human T cell proliferation by diminishing the antigen-
II major histocompatibility complex expression,” Journal of
Experimental Medicine, vol. 174, no. 4, pp. 915–924, 1991.
 L. Ding, P. S. Linsley, L. Y. Huang, R. N. Germain, and E. M.
Shevach, “IL-10 inhibits macrophage costimulatory activity
by selectively inhibiting the up-regulation of B7 expression,”
Journal of Immunology, vol. 151, no. 3, pp. 1224–1234, 1993.
 F. Willems, A. Marchant, J. P. Delville et al., “Interleukin-10
inhibits B7 and intercellular adhesion molecule-1 expression
24, no. 4, pp. 1007–1009, 1994.
 H. Groux, A. O’Garra, M. Bigler et al., “A CD4+ T-cell
subset inhibits antigen-specific T-cell responses and prevents
colitis,” Nature, vol. 389, no. 6652, pp. 737–742, 1997.
 E. Cenci, L. Romani, A. Mencacci et al., “Interleukin-4 and
interleukin-10 inhibit nitric oxide-dependent macrophage
Advances in Urology13 Download full-text
vol. 23, no. 5, pp. 1034–1038, 1993.
 K. W. Moore, R. De Waal Malefyt, R. L. Coffman, and A.
O’Garra, “Interleukin-10 and the interleukin-10 receptor,”
Annual Review of Immunology, vol. 19, pp. 683–765, 2001.
 N. A. Bockholt, M. J. Knudson, J. R. Henning et al., “Anti-
IL-10R1 monoclonal antibody enhances BCG-induced TH1
immune responses and antitumor immunity in a mouse
orthotopic model of bladder cancer,” The Journal of Urology,
vol. 187, pp. 2228–2235, 2012.