Bladder Cancer Immunotherapy: BCG and Beyond

Article (PDF Available)inAdvances in Urology 2012(3):181987 · June 2012with42 Reads
DOI: 10.1155/2012/181987 · Source: PubMed
Abstract
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.

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Hindawi Publishing Corporation
Advances in Urology
Volume 2012, Article ID 181987, 13 pages
doi:10.1155/2012/181987
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, yi-luo@uiowa.edu
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
cited.
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 eorts 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.
1. Introduction
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 [1]. 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 [2]. 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 [3].
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. [4], 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 [57]. Of the complete respon-
ders,upto50%willhavearecurrence[8]. Furthermore, side
eects 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 eects
[9].
Understanding of BCG, both its mechanisms (which
remain incompletely characterized) and its obvious limita-
tions, is critical to improving the ecacy 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 [10]. 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
+
Tcellsand
inducing a primarily T helper type (Th) 1 immune response
[11]. 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 [12]. While the role each of these
cytokines plays in urothelial carcinoma treatment is not
2 Advances in Urology
IL-10
IL-1, IL-6, IL-8,
TNF-α, GM-CSF
TH1 CD4
+
T
cell
Dendritic cells
macrophages
neutrophils
BCG
Urothelial carcinoma
No cell-mediated
antitumor activity
Cell-mediated
antitumor activity
Activated
NK cell
Activated
macrophage
Activated B cell
IL-4
IL-5
IL-10
IL-2
IL-12
cell
TH2 CD4
+
T
TH0 CD4
+
T cell
CD8
+
T cell
IFN-γ
IFN-γ
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 dierentiation of na
¨
ıve
CD4
+
T cells into TH1 and/or TH2 cells that direct immune responses toward cellular or humoral immunity, respectively. The therapeutic
eect 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
cancer destruction.
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 [1316]. 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 [17].
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 [3]. 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 eect 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.
2. Interferon-α2b
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 [18]. The IFN-α family is well known
to stimulate natural killer (NK) cells, induce MHC class
I response, and increase antibody recognition [19]. They
have antineoplastic properties by direct antiproliferative
eects and complex immunomodulatory eects [18], 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 ecacy depends
on the induction of a robust Th1 cytokine profile, and
IFN-α2b has been shown to potentiate the Th1 immune
response [12]. 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
[20]. At least part of this eect 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 [21], 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 [22]. It also appears that IFN-α apoptotic eects may
not be limited to TRAIL; rather it may trigger caspase-
8 via both cell death receptor-dependent and independent
pathways [23]. Much like IFN-α, BCG has also been
shown to induce TRAIL [24], which has correlated with
patient response to BCG therapy and has been a source
of overlapping research interest. Other direct IFN-α eects
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 [20]. 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 [27]. 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 [28].
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 [29].
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 eects 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 [30]. 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 [31]. 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 eects 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 [32]. 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 [12]. 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 eect
of IFN-α [12]. 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 eects thought to be directly
related to BCG. Further augmenting Th1 dierentiation,
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%,
respectively [33].
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 eect
profile to BCG alone [34]. In 2001, O’Donnell and colleagues
reported on combination therapy administered to 40 patients
who had failed at least 1 course of BCG alone [35]. 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 [36]. 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 [37]. 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 [38]. 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
¨
ıve patients
demonstrated similar disease-free rate of 62% but with much
longer median followup of 55.8 months [39]. 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
BCG na
¨
ıve patients [40]. 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 [41].
This was a four-arm t rial evaluating ecacy 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 [42]. 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 [43].
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
1
cell cycle arrest [44]. Another
combination recently published this year documented that
adding grape seed proanthocyanin significantly enhanced
antiproliferative eects of IFN-α2b, with >95% growth
reduction in T24 bladder cancer cells. Cell cycle analysis also
revealed G
1
cell cycle arrest, with Western blots confirming
expression of G
1
cell cycle regulators [45]. 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 [46].
3. Interleukin-2
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 dierentiated 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 eector protein
was responsible for this mitogenic activity [48, 49]. When
initially examined independently by dierent investigators,
this “eector 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) [50]. 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
“eector protein was named IL-2, dierentiating it from
the only other interleukin known at that time, IL-1 [50].
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 [5153]. 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 [5153]. 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 [56]. As the CD4
+
Th1
and Th2 cell cytokine profiles were defined, it became clear
that IL-2 is predominantly a Th1-secreted cytokine [57].
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 [58]. 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 [58];
however, Rosenberg repor ted 4 treatment-related deaths
among their 157 patients. Despite the reports of morbidity,
IL-2 seemed to oer hope to patients with few treatment
options.
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 eect of BCG [ 6067].
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 [70]. 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
[7173]. A small clinical study investigating systemic IL-2
administration eects 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 [74].
The poor side eect 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 eectproleaswellassomeecacy alone or
when combined with BCG [7578]. 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
[79]. Additional experiments have focused on de veloping
recombinant-IL-2-secreting strains of BCG [8085]. 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) [8083, 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 [8689]. 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.
4. Interleukin-12
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 [89]. This factor was given
the name cytotoxic lymphocyte maturation factor (CLMF)
[89]. Shortly thereafter a factor was discovered that induced
IFN-γ production, enhanced T cell responses to mitogens,
and augmented NK cell cytotoxicity [90]. This factor was
provisionally called natural killer cell stimulatory factor
(NKSF) [90]. 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 [9195].
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
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 [95]. 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 genera tion of an allogeneic CTL response [98].
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 [101104].
Furthermore, the mechanism by which IL-12 enhances the
cytolytic eect 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, 107111]. Increases in serum IFN-γ were observed
in mice treated with IL-12 [108]. Antitumor ecacy 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 eect are CD8
+
T cells [107, 108].
Some of these studies saw eectiveness even with metastatic
disease, including bladder cancer [107, 108, 111]. Multiple
murine studies have also revealed added eectiveness with
IL-12 administered in combination with chemotherapeutic
agents [109, 112114]. 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 [116120]. These constructs have
shown some eectiveness 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
[121]. BCG therapy for murine bladder cancer was essentially
found to be ineective in IL-12 knock-out mice, suggesting
a crucial role for IL-12 in the BCG response [122]. When
IL-12 is used as a therapy with BCG, it causes a synergistic
induction of IFN-γ [121]. Intravesical IL-12 treatment alone
was found to be eective 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 eective 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 ecacy. One method utilized cationic liposome-
mediated IL-12 gene therapy, which showed improved
survival and tumor-specific immunologic memor y in mice
[124]. Another method utilized chitosan, a mucoadhesive
biopolymer, to increase IL-12 delivery to urothelial surfaces
[125]. This method showed improved ecacy over IL-12
alone in a mouse model [125].
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 suered
severe toxic eects from the treatment a nd two patients
died from the therapy [126]. 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 [127]. Experiments on the per ipheral blood
of these patients showed augmented NK cell cytolytic activity
and enhanced T cell proliferation [128]. Unfortunately, of
these 40 patients there was only one partial response and one
transient complete response [127]. 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)[129].
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 [129]. Similar low response
rates have been seen with systemic IL-12 in other studies
of advanced malignancies [130134 ]. 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 [135]. Of 28 patients, there was
one partial response and two pathologic responses [135].
Another phase I study using systemic IL-12 with IFN-α2b
showed acceptable toxicity, but with no response in 41
patients [136]. 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 ecacy [137].
A total of 15 patients were enrolled in this study, of which
12 had no visible pretreatment lesions [137]. 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 [137]. 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 eect from
intravesical IL-12 therapy [137]. Despite the disappointing
results from human studies, IL-12 remains an important
target for the treatment of bladder cancer.
5. Interleukin-10
Unlike other cytokines previously discussed, interleukin-10
(IL-10) is distinct in that its primary eect is to promote
a Th2 response and thus dampen the immunotherapeutic
eects 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
[140]. 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 eectiveness 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
[143147]. It is speculated that production of IL-10 by tumor
cells may represent an “escape mechanism whereby tumor
cells avoid Th1-immune-mediated tumoricidal eects [143].
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, 148150]. Like many
other cytokines, IL-10 is known to autoregulate itself by
downregulating its own mRNA synthesis [150]. 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 [153]. Furthermore, it down-
regulates the expression of MHC class II and costimulatory
molecules, thus preventing a cellular immune response to
tumor cells [154156]. During activation of CD4
+
T cells,
the presence of IL-10 can cause them to dierentiate into
T regulatory cells 1 (Tr1), leading to peripheral tolerance
[157]. 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 ecacy
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 eorts 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 [159]. 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 eective 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 [160]. 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 dierence
did not reach statistical significance [160]. 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 dierence
was statistically significant and raises questions as to anti-IL-
10R1mAbsroleasmorethanjustanaugmentationtoBCG
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.
6. Conclusions
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
eects. 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.
Authors’ Contribution
E. J. Askeland and M. R. Newton contributed equally to the
production of this paper.
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    • "Currently, interferons are used for the treatment of chronic viral infections, caused—for example—by hepatitis B virus (HBV) and hepatitis C virus (HCV). Numerous studies confirm that the effect of interferon is augmented after the simultaneous addition of other drugs (Askeland et al. 2012; Lei et al. 2013; Mosa et al. 2014; Majewska et al. 2015a Majewska et al. , 2016). Kuczer et al. have shown that alloferon I is not toxic to Vero cell line in concentration of 360 lg/mL. "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract Over the past two decades there has been intense study of compounds from vertebrates, microorganisms, plants, mushrooms, marine sponges, worms, etc. as well as insects in terms of their antiviral activity. Insects produce a variety of biologically active peptides. One of them is alloferon. The in vitro and in vivo experiments demonstrate that synthetic alloferon has an immunomodulatory properties. It was reported that alloferon and its analogues (alloferon I and II) have antimicrobial properties, as well. The aim of this study was to evaluate in vitro the effect of alloferon I and II, either alone or in combination with human lymphocytes, on human herpesvirus type 1 (HHV-1) McIntyre strain replication. On the base of results we can conclude that alloferon I and II inhibit the replication of HHV-1 McIntyre strain in HEp-2 cells. Enhanced antiviral activity was observed when infected cells were treated with alloferons and unstimulated or phytohemagglutinin PHA-stimulated lymphocytes simultaneously. After application of alloferons and PHA-stimulated lymphocytes to the HHV-1 infected HEp-2 culture, the mean HHV-1 titer reduction for alloferon and II, when used at the highest dose—400 µg/mL, were 3.69 and 3.27 log10/TCID50/mL, respectively.
    Full-text · Article · Dec 2015
    • "Morales et al. [144] were pioneer to prove the successful treatment with BCG on CBNMI. Since then, BCG is the treatment of choice for high-risk NMIBC, being superior to chemotherapy in relation to recurrence and tumor progression rates [139, 143, 145]. The ability of intravesically administered BCG to exert an antineoplastic effect on BC is widely accepted and serves as the basis for its clinical use [146, 147] . "
    [Show abstract] [Hide abstract] ABSTRACT: During the last ten years, graphene oxide has been explored in many applications due to its remarkable electro-conductivity, thermal properties and mobility of charge carriers, among other properties. As discussed in this review, the literature suggests that a total characterization of graphene oxide must be conducted because oxidation debris (synthesis impurities) present in the graphene oxides could act as a graphene oxide surfactant, stabilizing aqueous dispersions. It is also important to note that the structure models of graphene oxide need to be revisited because of significant implications for its chemical composition and its direct covalent functionalization. Another aspect that is discussed is the need to consider graphene oxide surface chemistry. The hemolysis assay is recommended as a reliable test for the preliminary assessment of graphene oxide toxicity, biocompatibility and cell membrane interaction. More recently, graphene oxide has been extensively explored for drug delivery applications. An important increase in research efforts in this emerging field is clearly represented by the hundreds of related publications per year, including some reviews. Many studies have been performed to explore the graphene oxide properties that enable it to deliver more than one activity simultaneously and to combine multidrug systems with photothermal therapy, indicating that graphene oxide is an attractive tool to overcome hurdles in cancer therapies. Some strategic aspects of the application of these materials in cancer treatment are also discussed. In vitro studies have indicated that graphene oxide can also promote stem cell adhesion, growth and differentiation, and this review discusses the recent and pertinent findings regarding graphene oxide as a valuable nanomaterial for stem cell research in medicine. The protein corona is a key concept in nanomedicine and nanotoxicology because it provides a biomolecular identity for nanomaterials in a biological environment. Understanding protein corona-nanomaterial interactions and their influence on cellular responses is a challenging task at the nanobiointerface. New aspects and developments in this area are discussed.
    Full-text · Article · Jan 2015
    • "In the present case, the enlarged lymph nodes at bilateral neck level II areas turned out to be chronic granulomatous lymphadenopathy, which might be induced by either BCG lymphadenitis or mycobacterial infection. BCG irrigation after bladder cancer surgery is increasingly employed as a conservative treatment method [18]. BCG lymphadenitis was reported in some cases after BCG irrigation [19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Primary lung adenoid cystic carcinoma (ACC) is extremely rare and accounts for approximately 0.1%-0.2% of all lung cancers. ACC of the head and neck has generally been regarded as a slow-growing, low-grade malignancy which has a tendency for local recurrence and frequent distant metastasis. When ACC of the lung is identified, physicians must determine whether it represents distant metastasis or a primary lung cancer. Thyroid transcription factor-1 staining is one of the most useful methods to differentiate primary from metastatic lesions in lung cancer. Herein we report a case of metachronous, not synchronous, ACC at the peripheral lung followed by ACC presentation at the base of the tongue, and review of relevant literatures.
    Full-text · Article · Sep 2014
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