- Statistical Analysis
Statistical analysis was performed using the SPSS ver. 22.0 (IBM
Corporation, NY, USA). The predictive value of each mast cell mediators
and clinical parameters were assessed by chi-square test. The
differences in the urinary mast cell mediators levels between the
patients with NMIBC and healthy participants were examined using the
Mann–Whitney U test. The relationship between the urinary mast cell
mediator levels and the patients’ response to immunotherapy was also
evaluated using the Mann–Whitney U test. The serial changes in the
urinary mast cell mediators measured at four different visits were
analyzed by the Wilcoxon signed-rank, Friedman, and Post-Hoc tests. A
p-value of less than 0.05 was accepted as statistically significant.
Results
The average age at the time of immunotherapy was 56.1 (37-79) years in
patients diagnosed with NMIBC. There were 13 men and 6 women. Fourteen
patients were diagnosed with Ta high-grade and 5 with T1 high-grade
bladder cancer. There was no patient with carcinoma in situ. During the
follow-up, while 18 patients completed two-year maintenance BCG therapy,
one underwent radical cystectomy. The cystoscopic evaluations undertaken
at three-month intervals revealed that 7 of 19 patients did not respond
to immunotherapy (non-responders), and 12 responded well (responders).
Among the non-responders, recurrence was observed in six patients and
progression in one patient. The average time to recurrence or
progression was 9.4 (3-18) months. The mean age was 52.2 ± 10.5 years in
responders and 61.4 ± 10.6 years in non-responders (p = 0.098). The
remaining baseline clinicopathological findings are shown inTable 1 .
The serial changes in estimated marginal means of urinary
N-methylhistamine, histamine, and tryptase levels in responders and
non-responders are shown in Figure 1 . There was no
statistically significant difference between the immunotherapy
responders and non-responders in terms of the urinary N-methylhistamine,
histamine, and tryptase changes (p > 0.05). However, a
statistically significant increase was observed in the estimated
marginal means of urinary N-methylhistamine (p = 0.027) and histamine (p
= 0.004) levels measured at the second visit compared to the first visit
in patients treated with BCG (Table 2 ). Although there were no
statistically significant differences between the second and third
visits (p = 0.053 and p = 0.26), a statistically significant decrease
was detected in the estimated marginal means of N-methylhistamine levels
measured last visit compared to the third visit (p = 0.013). Concerning
the urinary tryptase levels, no statistically significant differences
were obtained in terms of immunotherapy response and changes of urinary
tryptase levels at different visits in patients treated with BCG (p
> 0.05).
The estimated marginal means of urinary N-methylhistamine, histamine,
and tryptase levels measured at the first visit before immunotherapy in
patients with NMIBC and measured at a single visit in healthy
participants are given in Table 2 . There were no statistically
significant differences in the estimated marginal means of urinary
histamine (p = 0.307) and tryptase (p = 0.816) levels between the
patients diagnosed with NMIBC and healthy participants. However, the
estimated marginal means of urinary N-methylhistamine levels were
significantly higher in patients with NMIBC than healthy participants (p
= 0.005).
The mast cell response to initial intravesical BCG immunotherapy between
BCG responders and non-responders is also evaluated. The increase in the
estimated marginal means of urinary N-methylhistamine after initial BCG
was 27.24 nmol/ml in responders and 41.51 nmol/ml in non-responders (p =
0.340). The increase in the estimated marginal means of urinary
histamine was 19.03 nmol/ml in responders and 27.64 nmol/ml in
non-responders (p = 0.801). The increase in the estimated marginal means
of urinary tryptase was 5.21 nmol/ml in responders and 7.51 nmol/ml in
non-responders (p = 0.108) (Figure 1).
Discussion
In the present study, we observed that urinary N-methylhistamine and
histamine levels were increased significantly with the onset of
immunotherapy, and N-methylhistamine levels were significantly decreased
when immunotherapy was terminated. Although we did not find
statistically significant differences between the responders and
non-responders, the estimated marginal means of Pre-BCG
N-methylhistamine were significantly higher in patients with NMIBC than
healthy participants.
A few studies have shown valuable changes in the BCG-induced urinary
immune microenvironment. In this field, IL-17+ mast cell, interleukins,
TNF-α, IFN-γ, and soluble ICAM-1 levels have been examined [7, 12,
13]. But there is no available data in the literature that can
determine mast cell activation in patients with NMIBC treated with BCG.
Our study determined that the urinary N-methylhistamine and histamine
levels increased with BCG immunotherapy, and N-methylhistamine decreased
with the termination of this BCG immunotherapy. Tryptase is considered
an unstable mast cell mediator, and therefore there was no statistically
significant change in the tryptase levels due to BCG immunotherapy.
Clinically useful tools to predict disease recurrence and progression
are much needed. Studies on predicting immunotherapy response started
with measuring purified protein derivative (PPD)-associated BCG
response. In a study, the median recurrence-free survival was 25 months
in the PPD-negative group and was not available in the PPD positive
group (p < 0.05) [14]. But there was only one study about
the mast cell-related immunotherapy response. [7]. This study has
confirmed the predictive value of IL-17+ mast cells in patients with
NMIBC treated with BCG immunotherapy, and higher numbers of IL-17+ cells
have been found associated with improved event-free survival. However,
there is no available data in the literature to determine urinary
N-methylhistamine, histamine, and tryptase levels in patients with
NMIBC. In our study, the lack of a statistically significant difference
between the mast cell mediators and immunotherapy response can be
explained by the small size of the patient group. The samples’
quantitative differences suggest that statistically significant
differences could be found in further studies designed with larger
patient groups.
There are a few studies on urinary markers in identifying NMIBC
patients. Although some studies have reported promising results in
determining bladder cancer with urinary, immune markers, there was no
mast cell-related markers in the literature [15, 16]. In our study,
increased urinary N-methylhistamine levels were found in patients with
NMIBC compared to healthy participants. These results can be discussed
in several aspects. The evaluation of the samples obtained at the first
visit before BCG instillation in patients with NMIBC excluded the
BCG-related immunological response. However, the samples were taken
after the re-TUR procedure, suggesting that a resection-induced mast
cell activation may have been effective. Therefore, it can be considered
that resection-associated mast cell activation alone can be effective
against tumor cells.
A few studies suggest that a decrease in immune system function in
elderly patients may weaken the BCG response. Kanematsu et al. were the
first to report significantly reduced protection from tumor recurrence
and reduced tuberculin skin test reactivity in patients aged
>80 years treated with BCG [17]. A phase 2 study
revealed that patients older than 80 years had the poorest
recurrence-free survival, and thus being over 80 was an independent
predictor of recurrence (hazard ratio: 1.56) [18]. Furthermore, age
was also an independent predictor of progression by the Club Urologico
Espanol de Tratamiento Oncologico (CUETO) group [19]. Although in
our study, the mean age was reported higher in immunotherapy
non-responders, these differences were not found statistically
significant due to the low number of patients (p = 0.098).
Despite the promising results, our study has certain limitations. First,
it was a single-center study with a relatively low number of cases.
Second, it is considered that increasing the number of visits could
provide more detailed information about the changes in mast cell
mediators. Third, although all patients with NMIBC were high grade,
excluding other clinical and pathological conditions that could affect
immunotherapy response can be considered another limitation. Future
studies with a larger sample size and longer follow-up are needed to
predict BCG response at the beginning of the treatment.
Conclusions
According to our knowledge, this is the first study to determine that
urinary N-methylhistamine and histamine levels were increased
significantly with receiving immunotherapy, and N-methylhistamine levels
were significantly decreased when immunotherapy was terminated. Although
there are no statistically significant differences between the
immunotherapy responders and non-responders, the estimated marginal
means of Pre-BCG N-methylhistamine levels are significantly higher in
patients with NMIBC than healthy participants. These results are
promising for further studies to be conducted on mast cell activation.