Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort
Abstract
:1. Introduction
2. Results
2.1. Association of Treg and TAM in the Cancerous Area with Baseline Characteristics
2.2. Correlation among Treg, TAM, and IL6 in the Bladder Tumor Microenvironment
2.3. Prognostic Role of Baseline Treg and TAM in NMIBC Treated with Intravesical BCG
3. Discussion
4. Materials and Methods
4.1. Data Collection of the Patients
4.2. Immunohistochemical Staining and Quantification
4.3. Adjuvant Intravesical Therapy for NMIBC after TURBT
4.4. Statistical Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
BCG | Bacille Calmette–Guérin |
CIS | Carcinoma in situ |
FR | Hazard ratio |
FOXP3 | Forkhead box P3 |
HPF | High-power microscopic fields |
IHC | Immunohistochemistry |
IL6 | Interleukin-6 |
LVI | Lymphovascular invasion |
MIBC | Muscle-invasive bladder cancer |
NF-κB | Nuclear factor-κB |
NMIBC | Non-muscle invasive bladder cancer |
PFS | Progression-free survival |
RC | Radical cystectomy |
RFS | Recurrence-free survival |
TAM | Tumor-associated macrophage |
TURBT | Transurethral resection of bladder tumor |
Treg | Regulatory T cell |
UC | Urothelial carcinoma |
References
- Miyake, M.; Fujimoto, K.; Hirao, Y. Active surveillance for nonmuscle invasive bladder cancer. Investig. Clin. Urol. 2016, 57 (Suppl. S1), S4–S13. [Google Scholar] [CrossRef] [PubMed]
- Fernandez-Gomez, J.; Madero, R.; Solsona, E. Predicting nonmuscle invasive bladder cancer recurrence and progression in patients treated with bacillus Calmette-Guerin: The CUETO scoring model. J. Urol. 2009, 182, 2195–2203. [Google Scholar] [CrossRef] [PubMed]
- Sylvester, R.J.; van der Meijden, A.P.; Oosterlinck, W. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: A combined analysis of 2596 patients from seven EORTC trials. Eur. Urol. 2006, 49, 466–477. [Google Scholar] [CrossRef] [PubMed]
- Witjes, J.A.; Compérat, E.; Cowan, N.C.; De Santis, M.; Gakis, G.; Lebret, T.; Ribal, M.J.; Van der Heijden, A.G.; Sherif, A.; European Association of Urology. EAU guidelines on muscle-invasive and metastatic bladder cancer: Summary of the 2013 guidelines. Eur. Urol. 2014, 65, 778–792. [Google Scholar] [CrossRef] [PubMed]
- Reis, L.O.; Moro, J.C.; Ribeiro, L.F.; Voris, B.R.; Sadi, M.V. Are we following the guidelines on non-muscle invasive bladder cancer? Int. Braz. J. Urol. 2016, 42, 22–28. [Google Scholar] [CrossRef] [PubMed]
- Lamm, D.L.; Blumenstein, B.A.; Crawford, E.D. A randomized trial of intravesical doxorubicin and immunotherapy with bacilli Calmette-Guerin for transitional-cell carcinoma of the bladder. N. Engl. J. Med. 1991, 325, 1205–1209. [Google Scholar] [CrossRef] [PubMed]
- Raj, G.V.; Herr, H.; Serio, A.M.; Donat, S.M.; Bochner, B.H.; Vickers, A.J.; Dalbagni, G. Treatment paradigm shift may improve survival of patients with high risk superficial bladder cancer. J. Urol. 2007, 177, 1283–1286. [Google Scholar] [CrossRef] [PubMed]
- Kitamura, H.; Tsukamoto, T. Immunotherapy for urothelial carcinoma: Current status and perspectives. Cancers 2011, 3, 3055–3071. [Google Scholar] [CrossRef] [PubMed]
- Abebe, F. Is interferon-gamma the right marker for bacilli Calmette-Guérin-induced immune protection? The missing link in our understanding of tuberculosis immunology. Clin. Exp. Immunol. 2012, 169, 213–219. [Google Scholar] [CrossRef] [PubMed]
- Pichler, R.; Fritz, J.; Zavadil, C.; Schäfer, G.; Culig, Z.; Brunner, A. Tumor-infiltrating immune cell subpopulations influence the oncologic outcome after intravesical Bacillus Calmette-Guérin therapy in bladder cancer. Oncotarget 2016, 7, 39916–39930. [Google Scholar] [CrossRef] [PubMed]
- Nunez-Nateras, R.; Castle, E.P.; Protheroe, C.A.; Stanton, M.L.; Ocal, T.I.; Ferrigni, E.N.; Ochkur, S.I.; Jacobsen, E.A.; Hou, Y.X.; Andrews, P.E.; et al. Predicting response to bacillus Calmette-Guérin (BCG) in patients with carcinoma in situ of the bladder. Urol. Oncol. 2014, 32, e23–e30. [Google Scholar] [CrossRef] [PubMed]
- Suriano, F.; Santini, D.; Perrone, G.; Amato, M.; Vincenzi, B.; Tonini, G.; Muda, A.; Boggia, S.; Buscarini, M.; Pantano, F. Tumor associated macrophages polarization dictates the efficacy of BCG instillation in non-muscle invasive urothelial bladder cancer. J. Exp. Clin. Cancer Res. 2013, 32, 87. [Google Scholar] [CrossRef] [PubMed]
- Kumari, N.; Dwarakanath, B.S.; Das, A.; Bhatt, A.N. Role of interleukin-6 in cancer progression and therapeutic resistance. Tumour Biol. 2016, 37, 11553–11572. [Google Scholar] [CrossRef] [PubMed]
- Morales, A.; Eidinger, D.; Bruce, A.W. Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J. Urol. 1976, 116, 180–183. [Google Scholar] [CrossRef]
- Winerdal, M.E.; Marits, P.; Winerdal, M.; Hasan, M.; Rosenblatt, R.; Tolf, A.; Selling, K.; Sherif, A.; Winqvist, O. FOXP3 and survival in urinary bladder cancer. BJU Int. 2011, 108, 1672–1678. [Google Scholar] [CrossRef] [PubMed]
- Horn, T.; Laus, J.; Seitz, A.K.; Maurer, T.; Schmid, S.C.; Wolf, P.; Haller, B.; Winkler, M.; Retz, M.; Nawroth, R.; et al. The prognostic effect of tumour-infiltrating lymphocytic subpopulations in bladder cancer. World J. Urol. 2016, 34, 181–187. [Google Scholar] [CrossRef] [PubMed]
- Boström, M.M.; Irjala, H.; Mirtti, T.; Taimen, P.; Kauko, T.; Ålgars, A.; Jalkanen, S.; Boström, P.J. Tumor-Associated Macrophages Provide Significant Prognostic Information in Urothelial Bladder Cancer. PLoS ONE 2015, 10, e0133552. [Google Scholar] [CrossRef] [PubMed]
- Sjödahl, G.; Lövgren, K.; Lauss, M.; Chebil, G.; Patschan, O.; Gudjonsson, S.; Månsson, W.; Fernö, M.; Leandersson, K.; Lindgren, D.; et al. Infiltration of CD3+ and CD68+ cells in bladder cancer is subtype specific and interacts the outcome of patients with muscle-invasive tumors. Urol. Oncol. 2014, 32, 791–797. [Google Scholar] [CrossRef] [PubMed]
- Mahmoud, S.M.; Paish, E.C.; Powe, D.G.; Macmillan, R.D.; Lee, A.H.; Ellis, I.O.; Green, A.R. An evaluation of the clinical significance of FOXP3+ infiltrating cells in human breast cancer. Breast Cancer Res. Treat. 2011, 127, 99–108. [Google Scholar] [CrossRef] [PubMed]
- Shimizu, K.; Nakata, M.; Hirami, Y.; Yukawa, T.; Maeda, A.; Tanemoto, K. Tumor-infiltrating FOXP3+ regulatory T cells are correlated with cyclooxygenase-2 expression and are associated with recurrence in resected non-small cell lung cancer. J. Thorac. Oncol. 2010, 5, 585–590. [Google Scholar] [CrossRef] [PubMed]
- Ponticiello, A.; Perna, F.; Maione, S.; Stradolini, M.; Testa, G.; Terrazzano, G.; Ruggiero, G.; Malerba, M.; Sanduzzi, A. Analysis of local T lymphocyte subsets upon stimulation with intravesical BCG: A model to study tuberculosis immunity. Respir. Med. 2004, 98, 509–514. [Google Scholar] [CrossRef] [PubMed]
- Bhattacharya, D.; Dwivedi, V.P.; Maiga, M.; Maiga, M.; Van Kaer, L.; Bishai, W.R.; Das, G. Small molecule-directed immunotherapy against recurrent infection by Mycobacterium tuberculosis. J. Biol. Chem. 2014, 289, 16508–16515. [Google Scholar] [CrossRef] [PubMed]
- Bhattacharya, D.; Dwivedi, V.P.; Kumar, S.; Reddy, M.C.; Van Kaer, L.; Moodley, P.; Das, G. Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis. J. Biol. Chem. 2014, 289, 33404–33411. [Google Scholar] [CrossRef] [PubMed]
- Pollard, J.W. Tumour-educated macrophages promote tumour progression and metastasis. Nat. Rev. Cancer 2004, 4, 71–78. [Google Scholar] [CrossRef] [PubMed]
- Takayama, H.; Nishimura, K.; Tsujimura, A.; Nakai, Y.; Nakayama, M.; Aozasa, K.; Okuyama, A.; Nonomura, N. Increased infiltration of tumor associated macrophages is associated with poor prognosis of bladder carcinoma in situ after intravesical bacillus Calmette-Guerin instillation. J. Urol. 2009, 181, 1894–1900. [Google Scholar] [CrossRef] [PubMed]
- Ayari, C.; LaRue, H.; Hovington, H.; Decobert, M.; Harel, F.; Bergeron, A.; Têtu, B.; Lacombe, L.; Fradet, Y. Bladder tumor infiltrating mature dendritic cells and macrophages as predictors of response to bacillus Calmette-Guérin immunotherapy. Eur. Urol. 2009, 55, 1386–1395. [Google Scholar] [CrossRef] [PubMed]
- Hasita, H.; Komohara, Y.; Okabe, H.; Masuda, T.; Ohnishi, K.; Lei, X.F.; Beppu, T.; Baba, H.; Takeya, M. Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma. Cancer Sci. 2010, 101, 1913–1919. [Google Scholar] [CrossRef] [PubMed]
- Hinz, S.; Pagerols-Raluy, L.; Oberg, H.H.; Ammerpohl, O.; Grüssel, S.; Sipos, B.; Grützmann, R.; Pilarsky, C.; Ungefroren, H.; Saeger, H.D.; et al. FOXP3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer. Cancer Res. 2007, 67, 8344–8350. [Google Scholar] [CrossRef] [PubMed]
- Miyake, M.; Gotoh, D.; Shimada, K.; Tatsumi, Y.; Nakai, Y.; Anai, S.; Torimoto, K.; Aoki, K.; Tanaka, N.; Konishi, N.; et al. Exploration of risk factors predicting outcomes for primary T1 high-grade bladder cancer and validation of the Spanish Urological Club for Oncological Treatment scoring model: Long-term follow-up experience at a single institute. Int. J. Urol. 2015, 22, 541–547. [Google Scholar] [CrossRef] [PubMed]
- Miyake, M.; Hori, S.; Morizawa, Y.; Tatsumi, Y.; Nakai, Y.; Anai, S.; Torimoto, K.; Aoki, K.; Tanaka, N.; Shimada, K.; et al. CXCL1-Mediated Interaction of Cancer Cells with Tumor-Associated Macrophages and Cancer-Associated Fibroblasts Promotes Tumor Progression in Human Bladder Cancer. Neoplasia 2016, 18, 636–646. [Google Scholar] [CrossRef] [PubMed]
- Miyake, M.; Hori, S.; Morizawa, Y.; Tatsumi, Y.; Toritsuka, M.; Ohnishi, S.; Shimada, K.; Furuya, H.; Khadka, V.S.; Deng, Y.; et al. Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder. Oncotarget 2017, 8, 36099–36114. [Google Scholar] [CrossRef] [PubMed]
- Schwarz, S.; Butz, M.; Morsczeck, C.; Reichert, T.E.; Driemel, O. Increased number of CD25+ FOXP3+ regulatory T cells in oral squamous cell carcinomas detected by chromogenic immunohistochemical double staining. J. Oral Pathol. Med. 2008, 37, 485–489. [Google Scholar] [CrossRef] [PubMed]
Variables | N | Treg (FOXP3+ Cell) | TAM (CD204+ Cell) | ||||
---|---|---|---|---|---|---|---|
Low | High | p Value | Low | High | p Value | ||
Total | 154 (100%) | 86 (56%) | 68 (44%) | − | 92 (59%) | 62 (41%) | − |
Sex | 0.0012 | 0.45 | |||||
Male | 137 (89%) | 83 (61%) | 54 (39%) | 78 (57%) | 59 (43%) | ||
Female | 17 (11%) | 3 (18%) | 14 (82%) | 8 (47%) | 9 (53%) | ||
Age at initial TURBT | |||||||
categorical | 0.056 | 0.75 | |||||
<60 | 18 (12%) | 15 (83%) | 3 (17%) | 11 (61%) | 7 (39%) | ||
60 to 70 | 55 (36%) | 34 (62%) | 21 (38%) | 32 (58%) | 23 (42%) | ||
>70 | 81 (52%) | 43 (53%) | 38 (47%) | 43 (53%) | 38 (47%) | ||
Continuous | |||||||
median (IQR) | 71 (65−76) | 69 (63−76) | 73 (69−79) | 0.024 | 71 (64−76) | 71 (68−77) | 0.22 |
T category | <0.001 | 0.25 | |||||
Ta | 68 (44%) | 52 (76%) | 16 (24%) | 41 (60%) | 27 (40%) | ||
T1 | 73 (47%) | 30 (41%) | 43 (59%) | 36 (49%) | 37 (51%) | ||
Tis | 13 (9%) | 10 (77%) | 3 (23%) | 9 (69%) | 4 (31%) | ||
Tumor grade | <0.001 | 0.16 | |||||
Low | 71 (46%) | 53 (75%) | 18 (25%) | 44 (62%) | 27 (38%) | ||
High | 83 (54%) | 39 (47%) | 44 (53%) | 42 (51%) | 41 (49%) | ||
Tumor architecture | 0.98 | 0.69 | |||||
Papillary | 134 (87%) | 80 (60%) | 54 (40%) | 74 (55%) | 60 (45%) | ||
Non-papillary | 20 (13%) | 12 (60%) | 8 (40%) | 12 (60%) | 8 (40%) | ||
Multiplicity | 0.64 | 0.71 | |||||
Single | 88 (57%) | 54 (61%) | 34 (39%) | 48 (55%) | 40 (45%) | ||
Multiple | 66 (43%) | 38 (58%) | 28 (42%) | 38 (58%) | 28 (42%) | ||
Tumor size | 0.45 | 0.99 | |||||
Less than 3 cm | 119 (77%) | 73 (61%) | 46 (39%) | 69 (58%) | 50 (42%) | ||
3 cm or more | 35 (23%) | 19 (54%) | 16 (46%) | 17 (49%) | 18 (51%) | ||
CIS | 0.011 | 0.29 | |||||
No | 91 (59%) | 62 (68%) | 29 (32%) | 54 (59%) | 37 (41%) | ||
Yes | 63 (41%) | 30 (48%) | 33 (52%) | 32 (51%) | 31 (49%) | ||
LVI (in T1 tumor, n = 73) | 0.66 | 0.93 | |||||
Negative | 49 (67%) | 21 (43%) | 28 (57%) | 24 (49%) | 25 (51%) | ||
Positive | 24 (33%) | 9 (38%) | 15 (62%) | 12 (50%) | 12 (50%) | ||
Intravesical adjuvant therapy | 0.65 | 0.37 | |||||
No | 64 (42%) | 41 (64%) | 23 (36%) | 40 (62%) | 24 (38%) | ||
BCG | 71 (46%) | 40 (56%) | 31 (44%) | 36 (51%) | 35 (49%) | ||
Chemotherapy | 19 (12%) | 11 (58%) | 8 (42%) | 10 (53%) | 9 (47%) |
Variables | N | Intravesical Recurrence-Free Survival | Progression-Free Survival | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Univariate | Multivariate † | Univariate | Multivariate † | ||||||||||
HR | 95% CI | p Value | HR | 95% CI | p Value | HR | 95% CI | p Value | HR | 95% CI | p Value | ||
Sex | |||||||||||||
Male | 63 (89%) | 1 | 1 | ||||||||||
Female | 8 (11%) | 0.64 | 0.24–1.72 | 0.52 | NA | 1.24 | 0.25–6.21 | 0.98 | NA | ||||
Age | |||||||||||||
≤70 | 37 (52%) | 1 | 1 | ||||||||||
>70 | 34 (48%) | 1.05 | 0.56–1.97 | 0.91 | NA | 1.88 | 0.73–4.86 | 0.22 | NA | ||||
T stage | |||||||||||||
Ta or isolated Tis | 30 (42%) | 1 | 1 | ||||||||||
T1 | 41 (58%) | 1.20 | 0.64–2.26 | 0.69 | NA | 1.29 | 0.49–3.40 | 0.81 | NA | ||||
Tumor grade | |||||||||||||
Low | 13 (18%) | 1 | 1 | 1 | |||||||||
High | 58 (82%) | 0.68 | 0.26–0.96 | 0.04 | 0.81 | 0.24–1.16 | 0.10 | 1.85 | 0.56–6.09 | 0.48 | NA | ||
Multiplicity | |||||||||||||
Single | 38 (54%) | 1 | 1 | ||||||||||
Multiple | 33 (46%) | 0.74 | 0.39–1.39 | 0.78 | NA | 1.20 | 0.46–3.15 | 0.46 | NA | ||||
Tumor size | |||||||||||||
<3 cm | 52 (73%) | 1 | 1 | ||||||||||
≥3 cm | 19 (27%) | 0.76 | 0.38–1.52 | 0.58 | NA | 1.05 | 0.39–2.87 | 0.89 | NA | ||||
Concomitant CIS | |||||||||||||
No | 22 (31%) | 1 | 1 | ||||||||||
Yes | 49 (69%) | 0.48 | 0.24–1.06 | 0.097 | NA | 2.48 | 0.91–6.74 | 0.14 | NA | ||||
Treg | |||||||||||||
Low | 31 (44%) | 1 | 1 | 1 | 1 | ||||||||
High | 40 (56%) | 2.53 | 1.32–4.86 | 0.001 | 3.07 | 1.55–6.07 | 0.001 | 3.38 | 1.29–8.88 | 0.027 | 3.43 | 1.20–9.74 | 0.021 |
TAM | |||||||||||||
Low | 35 (49%) | 1 | 1 | 1 | 1 | ||||||||
High | 36 (51%) | 2.31 | 1.27–4.30 | 0.029 | 1.39 | 0.68–2.84 | 0.37 | 3.35 | 1.29–8.66 | 0.052 | 2.50 | 0.79–8.02 | 0.12 |
IL6+ UC cells | |||||||||||||
Low | 32 (45%) | 1 | 1 | ||||||||||
High | 39 (55%) | 1.36 | 0.73–2.56 | 0.22 | NA | 1.39 | 0.53–3.61 | 0.54 | NA |
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Miyake, M.; Tatsumi, Y.; Gotoh, D.; Ohnishi, S.; Owari, T.; Iida, K.; Ohnishi, K.; Hori, S.; Morizawa, Y.; Itami, Y.; et al. Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort. Int. J. Mol. Sci. 2017, 18, 2186. https://doi.org/10.3390/ijms18102186
Miyake M, Tatsumi Y, Gotoh D, Ohnishi S, Owari T, Iida K, Ohnishi K, Hori S, Morizawa Y, Itami Y, et al. Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort. International Journal of Molecular Sciences. 2017; 18(10):2186. https://doi.org/10.3390/ijms18102186
Chicago/Turabian StyleMiyake, Makito, Yoshihiro Tatsumi, Daisuke Gotoh, Sayuri Ohnishi, Takuya Owari, Kota Iida, Kenta Ohnishi, Shunta Hori, Yosuke Morizawa, Yoshitaka Itami, and et al. 2017. "Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort" International Journal of Molecular Sciences 18, no. 10: 2186. https://doi.org/10.3390/ijms18102186