Original Study
Development of Predictive Value of Urinary Cytokine Profile Induced During Intravesical Bacillus Calmette-Guérin Instillations for Bladder Cancer Jérôme Rigaud,1,2 Alexandra Leger,2 Marie-Claire Devilder,2 Olivier Bouchot,1 Marc Bonneville,2 Emmanuel Scotet2 Abstract We describe a local reaction to intravesical bacillus Calmette-Guérin (BCG) instillations for the treatment of bladder cancer, with different profiles of urinary cytokine production during treatment. This analysis reflects a specific immune response induced in each patient. Their assessment could allow a more reliable selection of the patients eligible for this type of treatment and could help justify maintenance BCG therapy. Background: We sought to demonstrate a correlation between the response to treatment and the profile of urinary cytokine production during bacillus Calmette-Guérin (BCG) therapy. Material and Methods: From December 2008 to February 2011, 23 patients were included in a prospective study. All patients received 6 instillations of BCG weekly. The mean follow-up period of the population was 16.9 8.4 months. Refractory disease or recurrence was observed in 5 patients. Urine samples were collected and stored at 80 C, before and 4 hours after the first, third, and sixth BCG instillations. The cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17, interferon-gamma [IFNg] and tumor necrosis factor-alpha) were quantified within the collected urine samples using cytometric bead array analysis. The quantitative variables were analyzed using Student’s t test, and regression statistical analysis was performed. Results: Urinary cytokine production had increased strongly 4 hours after the sixth instillation but only mildly to moderately after the first and third instillations. IL-2 and IL-6 showed the most dramatic changes after the BCG instillations. Different urinary cytokine production profiles were demonstrated. A trend was observed for the BCG-refractory/recurrence group, with high baseline IL-6 levels, followed by low IL-6 levels before the instillations; low baseline IL-2 levels with only minor changes during treatment; the absence of IFNg and IL-17 production; and a low peak of cytokine production at the end of treatment. Conclusion: Analysis of the urinary cytokine production levels during BCG therapy reflect a specific immune response induced in each patient. Their assessment could allow a more reliable selection of patients eligible for this type of treatment and could help justify the use of maintenance BCG therapy. Clinical Genitourinary Cancer, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: BCG, IL-2, IL-6, NMIBC, Recurrence, Response
Introduction The treatment of nonemuscle-invasive bladder cancer (NMIBC) is essentially conservative, with transurethral endoscopic resection 1
Service d’Urologie, Hôpital Hôtel Dieu, Nantes, France Institut National de la Santé et de la Recherché Médicale U892, Centre de Recherche en Cancérologie, Institute for Therapeutic Research at the University of Nantes, Nantes, France 2
Submitted: Dec 10, 2014; Revised: Jan 9, 2015; Accepted: Jan 16, 2015 Address for correspondence: Jérôme Rigaud, MD, PhD, Clinique Urologique, CHU Hôtel Dieu, 1 Place Alexis Ricordeau, Nantes 44000, France E-mail contact: [email protected]
1558-7673/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clgc.2015.01.005
allowing complete resection of the tumor while preserving the urinary bladder. According to the European Organisation for Research and Treatment of Cancer risk tables1 and to decrease the risk of recurrence or progression of NMIBC, transurethral resection can be completed using intravesical instillations of mitomycin C (chemotherapy) or bacillus Calmette-Guérin (BCG) (immunotherapy).2,3 Intravesical BCG therapy is essentially indicated for high-risk NMIBC. Two meta-analyses have shown that maintenance BCG therapy is associated with a reduction in the risk of progression compared with that in the control groups.2,4 However, the efficacy of intravesical BCG therapy has often been counterbalanced by its
Clinical Genitourinary Cancer Month 2015
-1
Cytokines and BCG in Bladder Cancer adverse effects, with only 16% of patients receiving the complete treatment and the others having to stop maintenance therapy because of adverse effects.5 However, the mechanism of action of BCG against bladder cancer has not yet been fully elucidated. Many studies have been conducted to elucidate the mechanisms of the antitumor action of BCG and have essentially focused on the presence of inflammatory infiltrates in the bladder after treatment, urinary cytokine production, activation of cytotoxic cells, and the mechanisms of cytotoxicity induced by BCG in vitro. Overall, similar kinetics of the cytokines were demonstrated, with a maximum peak at 4 to 6 hours after instillation of BCG and essentially between the fourth and sixth instillations.6-8 Many studies have analyzed the correlation of the production of each cytokine alone and the response to treatment. The originality of the present study is that we sought to analyze the profiles of urinary cytokine production in an attempt to identify the immune markers relevant for patient selection (those more likely to experience a treatment response) and the prediction of intravesical BCG therapy efficacy. The primary aim of the present study was to test whether the local immune response induced by BCG instillation, as assessed by urinary cytokine production (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17, interferon-gamma [IFNg], and tumor necrosis factor-alpha [TNFa]), correlated with the patients’ response to treatment. Our underlying hypothesis was that little or no activation of the local (bladder) immune system after BCG instillations would have a limited affect on the bladder tumor outcome without a decreased risk of recurrence or progression.
Materials and Methods A single-center, prospective, nonrandomized study was conducted. The inclusion criteria for the present study were bladder cancer and an indication for intravesical BCG therapy (high-risk NMIBC). The patients with muscle-invasive bladder cancer (MIBC) were excluded. All patients were BCG naive.
Population A total of 23 consecutive patients were included from December 2008 to February 2011. The study population included 4 women and 19 men, with a mean age of 68.3 8.3 years. All patients had NMIBC at high risk of recurrence at the first transurethral resection of the bladder (Table 1). All patients with a low-grade papillary tumor had associated carcinoma in situ (Cis). A second transurethral resection was performed in 15 patients, with a mean interval between the first and second resections of 54.5 22.4 days. The second resection was performed for a stage pT1 tumor in 14 of these 15 patients (93.3%) and for a stage pTa plus Cis tumor in 1 patient (25%). Histologic examination of the second resection specimen demonstrated the absence of residual tumor in 10 patients (66.7%), the persistence of isolated Cis in 4 (26.7%), and the persistence of a stage pT1a plus Cis tumor in 1 patient (6.6%).
Induction BCG Therapy
2
-
All patients received a course of induction therapy that included 6 intravesical instillations of BCG (ImmuCyst) at a dose of 81 mg
Clinical Genitourinary Cancer Month 2015
Table 1 Histologic Findings From First Bladder Tumor Resection Variable
Patients (n)
Histologic stage Isolated Cis
4 (17.4)
pTa
4 (17.4)
pT1
15 (65.2)
Associated Cis (pTa or pT1) pTa þ Cis
4 (17.4; 100% of pTa)
pT1 þ Cis
7 (30.7; 46.7% of pT1)
Histologic grade Low
3 (13.1)
High
20 (86.9)
Data in parentheses are percentages. Abbreviation: Cis ¼ carcinoma in situ.
once weekly. The mean interval between the last bladder resection and the start of BCG instillations was 58.7 27.2 days.
Post-BCG Follow-up The efficacy of treatment was assessed in all patients by endoscopic examination (cystoscopy) and cytologic examination after the last BCG instillation, with a mean interval between the last BCG instillation and endoscopy of 42.9 15.5 days. The patients without recurrence subsequently underwent regular follow-up examinations with cystoscopy and urine cytology every 3 months during the first year and every 6 months for 5 years. After the 6 induction BCG instillations and follow-up cystoscopy examinations with or without biopsy, treatment failure (BCGrefractory tumor) was observed in 3 patients (13%) owing to the persistence of either Cis (2 patients) or a pT1 plus Cis tumor (1 patient). Of these 3 patients, 2 (with persistent Cis) underwent cystectomy and 1 (with pT1 plus Cis and metastatic disease) underwent systemic chemotherapy. Of the other 20 patients who did not have recurrence detected, 13 received maintenance BCG therapy and 7 underwent regular follow-up examinations without maintenance BCG treatment. The mean follow-up period for the entire population was 16.9 8.4 months after the post-BCG cystoscopy. NMIBC recurrence was observed in 2 patients (high-grade pTa and pT1a plus Cis), but no case of progression to MIBC was observed. No patient died during the follow-up period (Figure 1). We defined 3 groups of patients: those with BCG-refractory disease (persistent tumor at the end of induction BCG), those with recurrence (recurrence developing during the follow-up period after a first response to the BCG instillations), and those without recurrence (no recurrence during the follow-up period).
Urine Sampling The urine samples were collected in a uniform manner during voluntary voiding, before (H0) and 4 hours (H4) after the first (day 1 [D1]), third (D21), and sixth (D42) BCG instillations (ie, a total of 6 urine samples: D1H0, D1H4, D21H0, D21H4, D42H0, and D42H4). The urine samples were stored immediately in nonsterile tubes and frozen at 80 C for urinary cytokine analysis.
Jérôme Rigaud et al Figure 1 Patient Follow-up After Bacillus Calmette-Guérin (BCG) Therapy
Abbreviation: Cis ¼ carcinoma in situ.
Labeling of Cytokines and FACSArray Analysis Urinary assays were performed using the BD CBA Flex Th1/ Th2/Th17 Set to measure the following cytokine panel: IL-2, IL-4, IL-6, IL-10, IL-17, IFNg, and TNFa. The choice of this set was to analyze the T helper 1 (Th1), T helper 2 (Th2), and gammaedelta responses. The cytokine bead array approach is a fluorescent immunoassay, which allows the simultaneous detection and quantification of various selected cytokines, within a same sample using flow cytometry. In brief, the urine tubes, which had been immediately stored at 80 C after collection, were thawed in a water bath, and the cytokine assays were performed according to the manufacturer’s protocol. All samples were analyzed using a BD FACSArray Bioanalyzer device and BD FACSArray System Software. Before the analysis, the cytometer was calibrated using beads, according to the manufacturer’s protocol. Standard samples were used to assess the concentration of the selected cytokines for each urine sample, and the quantitative values are expressed in pg/mL.
Statistical Analysis All clinical and laboratory data were entered into an Excel 2010 database. Descriptive data analysis was performed using Excel and is presented in the form of graphs or tables. The endpoint of the present study was the value of the cytokines during the BCG instillations. Statistical analysis was performed using StatView, version 5 (SAS Institute, Cary, NC). Quantitative variables were analyzed using Student’s t test. The results were considered statistically significant at P < .05. Regression statistical analysis was performed to demonstrate a correlation between cytokine production as a function of time during BCG therapy.
concentrations of IL-2, IL-4, IL-6, and IL-10 showed the most significant changes, with only low levels of production observed for the other cytokines (Figure 2).
Correlation Analysis of Cytokine Production Regression statistical analysis was performed of the IL-2, IL-4, IL-6, and IL-10 urine concentrations to establish a correlation of cytokine production during BCG therapy. IL-6 production during treatment correlated systematically with the time at the different dosages (Table 3). Similarly, the urinary level of IL-2, IL-4, IL-6, and IL-10 at D21H4 correlated strongly with the cytokine levels at D42H4.
Urinary Cytokine Production Profiles Stratified by Each Patient An analysis of all the cytokines stratified by each patient demonstrated quantitatively different cytokine production profiles for some patients during treatment, reflecting the variable immune responses. Globally, the 2 cytokines with significant levels of production were IL-2 and IL-6. Two types of cytokine production profiles were observed: A progressive increase in IL-2 and IL-6 levels during treatment A decrease in IL-6 levels during treatment in association with stable low levels of IL-2 (< 5 pg/mL) The cytokine levels at D42H4 were studied in particular detail. These demonstrated 4 different cytokine production profiles in terms of the levels of cytokine production and the number of cytokines secreted at D42H4:
Results
Production of all cytokines assayed (5 patients)
Urinary Cytokine Values
Production of IL-2 and IL-6, in association with production of IL10 and/or IL-17, and almost no production of IFNg and TNFa (8 patients)
Quantitative values of the various selected cytokines, according to the date and time of sampling, are listed in Table 2. The
Clinical Genitourinary Cancer Month 2015
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Cytokines and BCG in Bladder Cancer Table 2 Urinary Cytokine Assays Variable
IL-2
IL-4
IL-6
IL-10
IL-17
IFNg
TNFa
D1H0 Mean
1.70
0.28
62.77
0.00
0.00
0.00
SD
1.22
0.65
130.40
0.00
0.00
0.00
0.00 0.00
Median
1.66
0.00
16.20
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
3.32
2.11
564.70
0.00
0.00
0.00
0.00
Mean
1.65
0.73
91.62
0.00
0.00
0.00
2.40
SD
1.53
1.34
275.72
0.00
0.00
0.00
10.73
Median
1.88
0.00
25.24
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
5.36
5.35
1255.79
0.00
0.00
0.00
47.99
Mean
1.75
0.76
35.10
0.39
2.42
0.54
0.37
SD
1.82
2.31
109.83
1.83
11.34
2.55
1.71
Median
1.67
0.00
4.19
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
8.06
10.54
517.00
8.58
53.18
11.95
8.03
Mean
10.05
0.16
54.90
1.75
0.00
0.00
0.56
SD
21.51
0.52
143.19
7.36
0.00
0.00
2.00
Median
3.11
0.00
11.38
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
97.77
1.76
653.98
33.83
0.00
0.00
8.85
1.79
0.66
30.84
0.00
1.00
0.06
0.00
D1H4
D21H0
D21H4
D42H0 Mean SD
1.41
1.37
79.44
0.00
4.78
0.29
0.00
Median
2.23
0.00
3.33
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
4.88
5.96
371.70
0.00
22.94
1.38
0.00
Mean
211.15
0.47
465.38
516.78
233.08
165.29
256.77
SD
671.35
1.14
1119.48
1641.52
562.53
755.34
855.42
Median
5.47
0.00
13.81
5.30
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
3160.56
4.99
4525.77
6863.08
2533.22
3627.13
3544.96
D42H4
Abbreviations: D1H0 ¼ day 1, before instillation; D1H4 ¼ day 1, 4 hours after instillation; D21H0 ¼ day 21, before instillation; D21H4 ¼ day 1, 4 hours after instillation; D42H0 ¼ day 42, before instillation; D42H4 ¼ day 42, 4 hours after instillation; IL ¼ interleukin; IFNg ¼ interferon-gamma; SD ¼ standard deviation; TNFa ¼ tumor necrosis factor-alpha.
Production of only IL-2 and IL-6, with almost no production of the other cytokines (8 patients)
Urinary Cytokine Production Profiles According to Response to Treatment
The complete absence of any cytokine production (2 patients)
The cytokine production profiles were analyzed in more detail for the 3 patients with an immediate response refractory to BCG and the 2 patients who developed a relapse during treatment. Although the statistical analysis did not provide any significant results and the sample size was too small to allow for subgroup analysis, the cytokine production profile of these 5 patients tended to be similar:
Analysis of Urinary Cytokines According to Response to BCG
4
-
No significant difference was demonstrated in the levels of production of each cytokine according to the response to BCG therapy (no recurrence vs. refractory/recurrence). However, it was difficult to compare this population in terms of short- and mediumterm recurrence, because some of the patients received maintenance BCG therapy and others did not.
Clinical Genitourinary Cancer Month 2015
IL-6 was high (> 200 pg/mL) at the beginning of treatment but then tended to be decreased on the assays performed before each
Jérôme Rigaud et al Figure 2 Quantitative Values of Urinary Cytokines for Overall Population (Logarithmic Scale). Letters (eg, AJ, AR, BA) Indicate the Different Patients
Abbreviations: D1H0 ¼ Day 1, before instillation; D1H4 ¼ Day 1, 4 Hours after instillation; D21H0 ¼ Day 21, before instillation; D21H4 ¼ Day 21, 4 Hours after instillation; D42H0 ¼ Day 42, before instillation; D42H4 ¼ Day 42, 4 Hours after instillation.
instillation (H0) in association with a weak reaction at H4 after each instillation
No IL-17 production was observed for any of these 5 patients throughout treatment, even at D42H4
IL-2 was low at the beginning of treatment and on the assays performed before each instillation (H0) and tended to decrease or remain stable during treatment
Low levels of production were observed for the other cytokines, in particular, at D42H4
No IFNg production was observed for any of these 5 patients throughout treatment, even at D42H4 Table 3 P Values From Regression Statistical Analysis for IL2, IL-4, IL-6, and IL-10 Urine Concentrations Between Production as a Function of Time During BCG Therapy Variable
IL-2
IL-4
IL-6
IL-10
D1H0 vs. D1H4 D21H0 vs. D21H4 D42H0 vs. D42H4 D1H0 vs. D21H0 D21H0 vs. D42H0 D1H4 vs. D21H4 D21H4 vs. D42H4
.1036 .2466 .9220 .0604 .9494 .1068 .0100
.1904 .6266 .6872 .0005 .7100 .5443 .0046
Development of Predictive Value of Urinary Cytokine Profile Induced During Intravesical Bacillus Calmette-Guérin Instillations for Bladder Cancer Jérôme Rigaud,1,2 Alexandra Leger,2 Marie-Claire Devilder,2 Olivier Bouchot,1 Marc Bonneville,2 Emmanuel Scotet2 Abstract We describe a local reaction to intravesical bacillus Calmette-Guérin (BCG) instillations for the treatment of bladder cancer, with different profiles of urinary cytokine production during treatment. This analysis reflects a specific immune response induced in each patient. Their assessment could allow a more reliable selection of the patients eligible for this type of treatment and could help justify maintenance BCG therapy. Background: We sought to demonstrate a correlation between the response to treatment and the profile of urinary cytokine production during bacillus Calmette-Guérin (BCG) therapy. Material and Methods: From December 2008 to February 2011, 23 patients were included in a prospective study. All patients received 6 instillations of BCG weekly. The mean follow-up period of the population was 16.9 8.4 months. Refractory disease or recurrence was observed in 5 patients. Urine samples were collected and stored at 80 C, before and 4 hours after the first, third, and sixth BCG instillations. The cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17, interferon-gamma [IFNg] and tumor necrosis factor-alpha) were quantified within the collected urine samples using cytometric bead array analysis. The quantitative variables were analyzed using Student’s t test, and regression statistical analysis was performed. Results: Urinary cytokine production had increased strongly 4 hours after the sixth instillation but only mildly to moderately after the first and third instillations. IL-2 and IL-6 showed the most dramatic changes after the BCG instillations. Different urinary cytokine production profiles were demonstrated. A trend was observed for the BCG-refractory/recurrence group, with high baseline IL-6 levels, followed by low IL-6 levels before the instillations; low baseline IL-2 levels with only minor changes during treatment; the absence of IFNg and IL-17 production; and a low peak of cytokine production at the end of treatment. Conclusion: Analysis of the urinary cytokine production levels during BCG therapy reflect a specific immune response induced in each patient. Their assessment could allow a more reliable selection of patients eligible for this type of treatment and could help justify the use of maintenance BCG therapy. Clinical Genitourinary Cancer, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: BCG, IL-2, IL-6, NMIBC, Recurrence, Response
Introduction The treatment of nonemuscle-invasive bladder cancer (NMIBC) is essentially conservative, with transurethral endoscopic resection 1
Service d’Urologie, Hôpital Hôtel Dieu, Nantes, France Institut National de la Santé et de la Recherché Médicale U892, Centre de Recherche en Cancérologie, Institute for Therapeutic Research at the University of Nantes, Nantes, France 2
Submitted: Dec 10, 2014; Revised: Jan 9, 2015; Accepted: Jan 16, 2015 Address for correspondence: Jérôme Rigaud, MD, PhD, Clinique Urologique, CHU Hôtel Dieu, 1 Place Alexis Ricordeau, Nantes 44000, France E-mail contact: [email protected]
1558-7673/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clgc.2015.01.005
allowing complete resection of the tumor while preserving the urinary bladder. According to the European Organisation for Research and Treatment of Cancer risk tables1 and to decrease the risk of recurrence or progression of NMIBC, transurethral resection can be completed using intravesical instillations of mitomycin C (chemotherapy) or bacillus Calmette-Guérin (BCG) (immunotherapy).2,3 Intravesical BCG therapy is essentially indicated for high-risk NMIBC. Two meta-analyses have shown that maintenance BCG therapy is associated with a reduction in the risk of progression compared with that in the control groups.2,4 However, the efficacy of intravesical BCG therapy has often been counterbalanced by its
Clinical Genitourinary Cancer Month 2015
-1
Cytokines and BCG in Bladder Cancer adverse effects, with only 16% of patients receiving the complete treatment and the others having to stop maintenance therapy because of adverse effects.5 However, the mechanism of action of BCG against bladder cancer has not yet been fully elucidated. Many studies have been conducted to elucidate the mechanisms of the antitumor action of BCG and have essentially focused on the presence of inflammatory infiltrates in the bladder after treatment, urinary cytokine production, activation of cytotoxic cells, and the mechanisms of cytotoxicity induced by BCG in vitro. Overall, similar kinetics of the cytokines were demonstrated, with a maximum peak at 4 to 6 hours after instillation of BCG and essentially between the fourth and sixth instillations.6-8 Many studies have analyzed the correlation of the production of each cytokine alone and the response to treatment. The originality of the present study is that we sought to analyze the profiles of urinary cytokine production in an attempt to identify the immune markers relevant for patient selection (those more likely to experience a treatment response) and the prediction of intravesical BCG therapy efficacy. The primary aim of the present study was to test whether the local immune response induced by BCG instillation, as assessed by urinary cytokine production (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17, interferon-gamma [IFNg], and tumor necrosis factor-alpha [TNFa]), correlated with the patients’ response to treatment. Our underlying hypothesis was that little or no activation of the local (bladder) immune system after BCG instillations would have a limited affect on the bladder tumor outcome without a decreased risk of recurrence or progression.
Materials and Methods A single-center, prospective, nonrandomized study was conducted. The inclusion criteria for the present study were bladder cancer and an indication for intravesical BCG therapy (high-risk NMIBC). The patients with muscle-invasive bladder cancer (MIBC) were excluded. All patients were BCG naive.
Population A total of 23 consecutive patients were included from December 2008 to February 2011. The study population included 4 women and 19 men, with a mean age of 68.3 8.3 years. All patients had NMIBC at high risk of recurrence at the first transurethral resection of the bladder (Table 1). All patients with a low-grade papillary tumor had associated carcinoma in situ (Cis). A second transurethral resection was performed in 15 patients, with a mean interval between the first and second resections of 54.5 22.4 days. The second resection was performed for a stage pT1 tumor in 14 of these 15 patients (93.3%) and for a stage pTa plus Cis tumor in 1 patient (25%). Histologic examination of the second resection specimen demonstrated the absence of residual tumor in 10 patients (66.7%), the persistence of isolated Cis in 4 (26.7%), and the persistence of a stage pT1a plus Cis tumor in 1 patient (6.6%).
Induction BCG Therapy
2
-
All patients received a course of induction therapy that included 6 intravesical instillations of BCG (ImmuCyst) at a dose of 81 mg
Clinical Genitourinary Cancer Month 2015
Table 1 Histologic Findings From First Bladder Tumor Resection Variable
Patients (n)
Histologic stage Isolated Cis
4 (17.4)
pTa
4 (17.4)
pT1
15 (65.2)
Associated Cis (pTa or pT1) pTa þ Cis
4 (17.4; 100% of pTa)
pT1 þ Cis
7 (30.7; 46.7% of pT1)
Histologic grade Low
3 (13.1)
High
20 (86.9)
Data in parentheses are percentages. Abbreviation: Cis ¼ carcinoma in situ.
once weekly. The mean interval between the last bladder resection and the start of BCG instillations was 58.7 27.2 days.
Post-BCG Follow-up The efficacy of treatment was assessed in all patients by endoscopic examination (cystoscopy) and cytologic examination after the last BCG instillation, with a mean interval between the last BCG instillation and endoscopy of 42.9 15.5 days. The patients without recurrence subsequently underwent regular follow-up examinations with cystoscopy and urine cytology every 3 months during the first year and every 6 months for 5 years. After the 6 induction BCG instillations and follow-up cystoscopy examinations with or without biopsy, treatment failure (BCGrefractory tumor) was observed in 3 patients (13%) owing to the persistence of either Cis (2 patients) or a pT1 plus Cis tumor (1 patient). Of these 3 patients, 2 (with persistent Cis) underwent cystectomy and 1 (with pT1 plus Cis and metastatic disease) underwent systemic chemotherapy. Of the other 20 patients who did not have recurrence detected, 13 received maintenance BCG therapy and 7 underwent regular follow-up examinations without maintenance BCG treatment. The mean follow-up period for the entire population was 16.9 8.4 months after the post-BCG cystoscopy. NMIBC recurrence was observed in 2 patients (high-grade pTa and pT1a plus Cis), but no case of progression to MIBC was observed. No patient died during the follow-up period (Figure 1). We defined 3 groups of patients: those with BCG-refractory disease (persistent tumor at the end of induction BCG), those with recurrence (recurrence developing during the follow-up period after a first response to the BCG instillations), and those without recurrence (no recurrence during the follow-up period).
Urine Sampling The urine samples were collected in a uniform manner during voluntary voiding, before (H0) and 4 hours (H4) after the first (day 1 [D1]), third (D21), and sixth (D42) BCG instillations (ie, a total of 6 urine samples: D1H0, D1H4, D21H0, D21H4, D42H0, and D42H4). The urine samples were stored immediately in nonsterile tubes and frozen at 80 C for urinary cytokine analysis.
Jérôme Rigaud et al Figure 1 Patient Follow-up After Bacillus Calmette-Guérin (BCG) Therapy
Abbreviation: Cis ¼ carcinoma in situ.
Labeling of Cytokines and FACSArray Analysis Urinary assays were performed using the BD CBA Flex Th1/ Th2/Th17 Set to measure the following cytokine panel: IL-2, IL-4, IL-6, IL-10, IL-17, IFNg, and TNFa. The choice of this set was to analyze the T helper 1 (Th1), T helper 2 (Th2), and gammaedelta responses. The cytokine bead array approach is a fluorescent immunoassay, which allows the simultaneous detection and quantification of various selected cytokines, within a same sample using flow cytometry. In brief, the urine tubes, which had been immediately stored at 80 C after collection, were thawed in a water bath, and the cytokine assays were performed according to the manufacturer’s protocol. All samples were analyzed using a BD FACSArray Bioanalyzer device and BD FACSArray System Software. Before the analysis, the cytometer was calibrated using beads, according to the manufacturer’s protocol. Standard samples were used to assess the concentration of the selected cytokines for each urine sample, and the quantitative values are expressed in pg/mL.
Statistical Analysis All clinical and laboratory data were entered into an Excel 2010 database. Descriptive data analysis was performed using Excel and is presented in the form of graphs or tables. The endpoint of the present study was the value of the cytokines during the BCG instillations. Statistical analysis was performed using StatView, version 5 (SAS Institute, Cary, NC). Quantitative variables were analyzed using Student’s t test. The results were considered statistically significant at P < .05. Regression statistical analysis was performed to demonstrate a correlation between cytokine production as a function of time during BCG therapy.
concentrations of IL-2, IL-4, IL-6, and IL-10 showed the most significant changes, with only low levels of production observed for the other cytokines (Figure 2).
Correlation Analysis of Cytokine Production Regression statistical analysis was performed of the IL-2, IL-4, IL-6, and IL-10 urine concentrations to establish a correlation of cytokine production during BCG therapy. IL-6 production during treatment correlated systematically with the time at the different dosages (Table 3). Similarly, the urinary level of IL-2, IL-4, IL-6, and IL-10 at D21H4 correlated strongly with the cytokine levels at D42H4.
Urinary Cytokine Production Profiles Stratified by Each Patient An analysis of all the cytokines stratified by each patient demonstrated quantitatively different cytokine production profiles for some patients during treatment, reflecting the variable immune responses. Globally, the 2 cytokines with significant levels of production were IL-2 and IL-6. Two types of cytokine production profiles were observed: A progressive increase in IL-2 and IL-6 levels during treatment A decrease in IL-6 levels during treatment in association with stable low levels of IL-2 (< 5 pg/mL) The cytokine levels at D42H4 were studied in particular detail. These demonstrated 4 different cytokine production profiles in terms of the levels of cytokine production and the number of cytokines secreted at D42H4:
Results
Production of all cytokines assayed (5 patients)
Urinary Cytokine Values
Production of IL-2 and IL-6, in association with production of IL10 and/or IL-17, and almost no production of IFNg and TNFa (8 patients)
Quantitative values of the various selected cytokines, according to the date and time of sampling, are listed in Table 2. The
Clinical Genitourinary Cancer Month 2015
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Cytokines and BCG in Bladder Cancer Table 2 Urinary Cytokine Assays Variable
IL-2
IL-4
IL-6
IL-10
IL-17
IFNg
TNFa
D1H0 Mean
1.70
0.28
62.77
0.00
0.00
0.00
SD
1.22
0.65
130.40
0.00
0.00
0.00
0.00 0.00
Median
1.66
0.00
16.20
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
3.32
2.11
564.70
0.00
0.00
0.00
0.00
Mean
1.65
0.73
91.62
0.00
0.00
0.00
2.40
SD
1.53
1.34
275.72
0.00
0.00
0.00
10.73
Median
1.88
0.00
25.24
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
5.36
5.35
1255.79
0.00
0.00
0.00
47.99
Mean
1.75
0.76
35.10
0.39
2.42
0.54
0.37
SD
1.82
2.31
109.83
1.83
11.34
2.55
1.71
Median
1.67
0.00
4.19
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
8.06
10.54
517.00
8.58
53.18
11.95
8.03
Mean
10.05
0.16
54.90
1.75
0.00
0.00
0.56
SD
21.51
0.52
143.19
7.36
0.00
0.00
2.00
Median
3.11
0.00
11.38
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
97.77
1.76
653.98
33.83
0.00
0.00
8.85
1.79
0.66
30.84
0.00
1.00
0.06
0.00
D1H4
D21H0
D21H4
D42H0 Mean SD
1.41
1.37
79.44
0.00
4.78
0.29
0.00
Median
2.23
0.00
3.33
0.00
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
4.88
5.96
371.70
0.00
22.94
1.38
0.00
Mean
211.15
0.47
465.38
516.78
233.08
165.29
256.77
SD
671.35
1.14
1119.48
1641.52
562.53
755.34
855.42
Median
5.47
0.00
13.81
5.30
0.00
0.00
0.00
Minimum
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Maximum
3160.56
4.99
4525.77
6863.08
2533.22
3627.13
3544.96
D42H4
Abbreviations: D1H0 ¼ day 1, before instillation; D1H4 ¼ day 1, 4 hours after instillation; D21H0 ¼ day 21, before instillation; D21H4 ¼ day 1, 4 hours after instillation; D42H0 ¼ day 42, before instillation; D42H4 ¼ day 42, 4 hours after instillation; IL ¼ interleukin; IFNg ¼ interferon-gamma; SD ¼ standard deviation; TNFa ¼ tumor necrosis factor-alpha.
Production of only IL-2 and IL-6, with almost no production of the other cytokines (8 patients)
Urinary Cytokine Production Profiles According to Response to Treatment
The complete absence of any cytokine production (2 patients)
The cytokine production profiles were analyzed in more detail for the 3 patients with an immediate response refractory to BCG and the 2 patients who developed a relapse during treatment. Although the statistical analysis did not provide any significant results and the sample size was too small to allow for subgroup analysis, the cytokine production profile of these 5 patients tended to be similar:
Analysis of Urinary Cytokines According to Response to BCG
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No significant difference was demonstrated in the levels of production of each cytokine according to the response to BCG therapy (no recurrence vs. refractory/recurrence). However, it was difficult to compare this population in terms of short- and mediumterm recurrence, because some of the patients received maintenance BCG therapy and others did not.
Clinical Genitourinary Cancer Month 2015
IL-6 was high (> 200 pg/mL) at the beginning of treatment but then tended to be decreased on the assays performed before each
Jérôme Rigaud et al Figure 2 Quantitative Values of Urinary Cytokines for Overall Population (Logarithmic Scale). Letters (eg, AJ, AR, BA) Indicate the Different Patients
Abbreviations: D1H0 ¼ Day 1, before instillation; D1H4 ¼ Day 1, 4 Hours after instillation; D21H0 ¼ Day 21, before instillation; D21H4 ¼ Day 21, 4 Hours after instillation; D42H0 ¼ Day 42, before instillation; D42H4 ¼ Day 42, 4 Hours after instillation.
instillation (H0) in association with a weak reaction at H4 after each instillation
No IL-17 production was observed for any of these 5 patients throughout treatment, even at D42H4
IL-2 was low at the beginning of treatment and on the assays performed before each instillation (H0) and tended to decrease or remain stable during treatment
Low levels of production were observed for the other cytokines, in particular, at D42H4
No IFNg production was observed for any of these 5 patients throughout treatment, even at D42H4 Table 3 P Values From Regression Statistical Analysis for IL2, IL-4, IL-6, and IL-10 Urine Concentrations Between Production as a Function of Time During BCG Therapy Variable
IL-2
IL-4
IL-6
IL-10
D1H0 vs. D1H4 D21H0 vs. D21H4 D42H0 vs. D42H4 D1H0 vs. D21H0 D21H0 vs. D42H0 D1H4 vs. D21H4 D21H4 vs. D42H4
.1036 .2466 .9220 .0604 .9494 .1068 .0100
.1904 .6266 .6872 .0005 .7100 .5443 .0046
