- 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.