Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Patients and Ethic Statement
2.2. CTC Blood Sample Processing
2.3. RNA Extraction
2.4. Reverse Transcription
2.5. Digital Droplet PCR (ddPCR)
2.6. CTC Sample Staining
2.7. Statistical Analysis
2.8. Availability of Data
3. Results
3.1. Detection of CTCs in the Blood of NMIBC Patients
3.2. Detection of Different EMT Markers in CTCs from NMIBC Patients
3.3. Gene Expression Profile of the PD-L1 and EMT Genes in Blood CTCs from NMIBC Patients
3.4. Correlation between PD-L1, EMT Genes and Recurrence-Free Survival (RFS) in CTCs from NMIBC Patients
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Antoni, S.; Ferlay, J.; Soerjomataram, I.; Znaor, A.; Jemal, A.; Bray, F. Bladder Cancer Incidence and Mortality: A Global Overview and Recent Trends. Eur. Urol. 2017, 71, 96–108. [Google Scholar] [CrossRef]
- Babjuk, M.; Böhle, A.; Burger, M.; Capoun, O.; Cohen, D.; Compérat, E.M.; Hernández, V.; Kaasinen, E.; Palou, J.; Rouprêt, M.; et al. EAU Guidelines on Non–Muscle-invasive Urothelial Carcinoma of the Bladder: Update 2016. Eur. Urol. 2017, 71, 447–461. [Google Scholar] [CrossRef] [PubMed]
- Singer, S.; Ziegler, C.; Schwalenberg, T.; Hinz, A.; Götze, H.; Schulte, T. Quality of life in patients with muscle invasive and non-muscle invasive bladder cancer. Support. Care Cancer 2013, 21, 1383–1393. [Google Scholar] [CrossRef] [PubMed]
- Witjes, J.A.; Bruins, H.M.; Cathomas, R.; Compérat, E.M.; Cowan, N.C.; Gakis, G.; Hernández, V.; Espinós, E.L.; Lorch, A.; Neuzillet, Y.; et al. European Association of Urology Guidelines on Muscle-invasive and Metastatic Bladder Cancer: Summary of the 2020 Guidelines. Eur. Urol. 2021, 79, 82–104. [Google Scholar] [CrossRef] [PubMed]
- Xylinas, E.; Robinson, B.; Kluth, L.; Volkmer, B.; Hautmann, R.; Küfer, R.; Zerbib, M.; Kwon, E.; Thompson, R.; Boorjian, S.; et al. Association of T-cell co-regulatory protein expression with clinical outcomes following radical cystectomy for urothelial carcinoma of the bladder. Eur. J. Surg. Oncol. 2014, 40, 121–127. [Google Scholar] [CrossRef] [PubMed]
- Powles, T.; Eder, J.P.; Fine, G.D.; Braiteh, F.S.; Loriot, Y.; Cruz, C.; Bellmunt, J.; Burris, H.A.; Petrylak, D.P.; Teng, S.-L.; et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 2014, 515, 558–562. [Google Scholar] [CrossRef] [PubMed]
- Kawahara, T.; Ishiguro, Y.; Ohtake, S.; Kato, I.; Ito, Y.; Ito, H.; Makiyama, K.; Kondo, K.; Miyoshi, Y.; Yumura, Y.; et al. PD-1 and PD-L1 are more highly expressed in high-grade bladder cancer than in low-grade cases: PD-L1 might function as a mediator of stage progression in bladder cancer. BMC Urol. 2018, 18, 97. [Google Scholar] [CrossRef] [PubMed]
- Balar, A.V.; Castellano, D.; O’Donnell, P.H.; Grivas, P.; Vuky, J.; Powles, T.; Plimack, E.R.; Hahn, N.M.; de Wit, R.; Pang, L.; et al. First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): A multicentre, single-arm, phase 2 study. Lancet Oncol. 2017, 18, 1483–1492. [Google Scholar] [CrossRef]
- Rosenberg, J.E.; Hoffman-Censits, J.; Powles, T.; van der Heijden, M.S.; Balar, A.V.; Necchi, A.; Dawson, N.; O’Donnell, P.H.; Balmanoukian, A.; Loriot, Y.; et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: A single-arm, multicentre, phase 2 trial. Lancet 2016, 387, 1909–1920. [Google Scholar] [CrossRef] [Green Version]
- Mahmoudian, R.A.; Mozhgani, S.; Abbaszadegan, M.R.; Mokhlessi, L.; Montazer, M.; Gholamin, M. Correlation between the immune checkpoints and EMT genes proposes potential prognostic and therapeutic targets in ESCC. J. Mol. Histol. 2021, 52, 597–609. [Google Scholar] [CrossRef]
- Tsutsumi, S.; Saeki, H.; Nakashima, Y.; Ito, S.; Oki, E.; Morita, M.; Oda, Y.; Okano, S.; Maehara, Y. Programmed death-ligand 1 expression at tumor invasive front is associated with epithelial-mesenchymal transition and poor prognosis in esophageal squamous cell carcinoma. Cancer Sci. 2017, 108, 1119–1127. [Google Scholar] [CrossRef]
- Lamouille, S.; Xu, J.; Derynck, R. Molecular mechanisms of epithelial–mesenchymal transition. Nat. Rev. Mol. Cell Biol. 2014, 15, 178–196. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Na, L.; Wang, Z.; Bai, Y.; Sun, Y.; Dong, D.; Wang, W.; Zhao, C. WNT7B represses epithelial-mesenchymal transition and stem-like properties in bladder urothelial carcinoma. Biochim. Biophys. Acta Mol. Basis Dis. 2022, 1868, 166271. [Google Scholar] [CrossRef]
- Wang, H.; Mei, Y.; Luo, C.; Huang, Q.; Wang, Z.; Lu, G.-M.; Qin, L.; Sun, Z.; Huang, C.-W.; Yang, Z.-W.; et al. Single-Cell Analyses Reveal Mechanisms of Cancer Stem Cell Maintenance and Epithelial–Mesenchymal Transition in Recurrent Bladder Cancer. Clin. Cancer Res. 2021, 27, 6265–6278. [Google Scholar] [CrossRef] [PubMed]
- Breyer, J.; Gierth, M.; Shalekenov, S.; Aziz, A.; Schäfer, J.; Burger, M.; Denzinger, S.; Hofstädter, F.; Giedl, C.; Otto, W. Epithelial–mesenchymal transformation markers E-cadherin and survivin predict progression of stage pTa urothelial bladder carcinoma. World J. Urol. 2016, 34, 709–716. [Google Scholar] [CrossRef]
- Alsuliman, A.; Colak, D.; Al-Harazi, O.; Fitwi, H.; Tulbah, A.; Al-Tweigeri, T.; Al-Alwan, M.; Ghebeh, H. Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: Significance in claudin-low breast cancer cells. Mol. Cancer 2015, 14, 149. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ock, C.-Y.; Kim, S.; Keam, B.; Kim, M.; Kim, T.M.; Kim, J.-H.; Jeon, Y.K.; Lee, J.-S.; Kwon, S.K.; Hah, J.H.; et al. PD-L1 expression is associated with epithelial-mesenchymal transition in head and neck squamous cell carcinoma. Oncotarget 2016, 7, 15901–15914. [Google Scholar] [CrossRef] [Green Version]
- Santoni, G.; Morelli, M.B.; Amantini, C.; Battelli, N. Urinary Markers in Bladder Cancer: An Update. Front. Oncol. 2018, 8, 362. [Google Scholar] [CrossRef] [Green Version]
- Chalfin, H.J.; Kates, M.; van der Toom, E.E.; Glavaris, S.; Verdone, J.E.; Hahn, N.M.; Pienta, K.J.; Bivalacqua, T.J.; Gorin, M.A. Characterization of Urothelial Cancer Circulating Tumor Cells with a Novel Selection-Free Method. Urology 2018, 115, 82–86. [Google Scholar] [CrossRef]
- Massagué, J.; Obenauf, A.C. Metastatic colonization by circulating tumour cells. Nature 2016, 529, 298–306. [Google Scholar] [CrossRef]
- Jiang, H.; Gu, X.; Zuo, Z.; Tian, G.; Liu, J. Prognostic value of circulating tumor cells in patients with bladder cancer: A meta-analysis. PLoS ONE 2021, 16, e0254433. [Google Scholar] [CrossRef] [PubMed]
- Nicolazzo, C.; Colangelo, L.; Corsi, A.; Carpino, G.; Gradilone, A.; Sonato, C.; Raimondi, C.; Gaudio, E.; Gazzaniga, P.; Gianni, W. Liquid Biopsy in Rare Cancers: Lessons from Hemangiopericytoma. Anal. Cell. Pathol. 2018, 2018, 9718585. [Google Scholar] [CrossRef] [Green Version]
- Msaouel, P.; Koutsilieris, M. Diagnostic value of circulating tumor cell detection in bladder and urothelial cancer: Systematic review and meta-analysis. BMC Cancer 2011, 11, 336. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Soave, A.; Riethdorf, S.; Dahlem, R.; Von Amsberg, G.; Minner, S.; Weisbach, L.; Engel, O.; Fisch, M.; Pantel, K.; Rink, M. A nonrandomized, prospective, clinical study on the impact of circulating tumor cells on outcomes of urothelial carcinoma of the bladder patients treated with radical cystectomy with or without adjuvant chemotherapy. Int. J. Cancer 2017, 140, 381–389. [Google Scholar] [CrossRef] [PubMed]
- Anantharaman, A.; Friedlander, T.; Lu, D.; Krupa, R.; Premasekharan, G.; Hough, J.; Edwards, M.; Paz, R.; Lindquist, K.; Graf, R.; et al. Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients. BMC Cancer 2016, 16, 744. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gazzaniga, P.; De Berardinis, E.; Raimondi, C.; Gradilone, A.; Busetto, G.M.; De Falco, E.; Nicolazzo, C.; Giovannone, R.; Gentile, V.; Cortesi, E.; et al. Circulating tumor cells detection has independent prognostic impact in high-risk non-muscle invasive bladder cancer. Int. J. Cancer 2014, 135, 1978–1982. [Google Scholar] [CrossRef] [Green Version]
- Busetto, G.M.; Ferro, M.; Del Giudice, F.; Antonini, G.; Chung, B.I.; Sperduti, I.; Giannarelli, D.; Lucarelli, G.; Borghesi, M.; Musi, G.; et al. The Prognostic Role of Circulating Tumor Cells (CTC) in High-risk Non–muscle-invasive Bladder Cancer. Clin. Genitourin. Cancer 2017, 15, e661–e666. [Google Scholar] [CrossRef]
- Aydin, A.M.; Baydar, D.E.; Hazir, B.; Babaoglu, B.; Bilen, C.Y. Prognostic significance of pre- and post-treatment PD-L1 expression in patients with primary high-grade non-muscle-invasive bladder cancer treated with BCG immunotherapy. World J. Urol. 2020, 38, 2537–2545. [Google Scholar] [CrossRef]
- Breyer, J.; Wirtz, R.M.; Otto, W.; Erben, P.; Worst, T.; Stoehr, R.; Eckstein, M.; Denzinger, S.; Burger, M.; Hartmann, A. High PDL1 mRNA expression predicts better survival of stage pT1 non-muscle-invasive bladder cancer (NMIBC) patients. Cancer Immunol. Immunother. 2018, 67, 403–412. [Google Scholar] [CrossRef]
- Jang, M.H.; Kim, H.J.; Kim, E.J.; Chung, Y.R.; Park, S.Y. Expression of epithelial-mesenchymal transition–related markers in triple-negative breast cancer: ZEB1 as a potential biomarker for poor clinical outcome. Hum. Pathol. 2015, 46, 1267–1274. [Google Scholar] [CrossRef]
- Zhao, D.; Besser, A.H.; Wander, S.A.; Sun, J.; Zhou, W.; Wang, B.; Ince, T.; Durante, M.; Guo, W.; Mills, G.; et al. Cytoplasmic p27 promotes epithelial–mesenchymal transition and tumor metastasis via STAT3-mediated Twist1 upregulation. Oncogene 2015, 34, 5447–5459. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, B.; Miyake, H.; Nishikawa, M.; Fujisawa, M. Expression profile of epithelial-mesenchymal transition markers in non–muscle-invasive urothelial carcinoma of the bladder: Correlation with intravesical recurrence following transurethral resection. Urol. Oncol. Semin. Orig. Investig. 2014, 33, 110.e11–110.e18. [Google Scholar] [CrossRef] [PubMed]
- Hara, I.; Miyake, H.; Hara, S.; Arakawa, S.; Kamidono, S. Significance of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinase Expression in the Recurrence of Superficial Transitional Cell Carcinoma of the Bladder. J. Urol. 2001, 165, 1769–1772. [Google Scholar] [CrossRef]
Data | Patients |
---|---|
Gender, n (%) | |
Male | 41 (82%) |
Female | 9 (18%) |
Age years | |
Range | (47–93) |
Media | 73.2 |
Median | 74 |
Tumor histology Papillary | 37 (74%) |
Not Papillary | 13 (26%) |
Pathological Tumor Grade | |
Low | 40 (80%) |
High | 10 (20%) |
Pathological Tumor Stage | |
Tx | 1 (2%) |
Tis | 3 (6%) |
Ta | 36 (72%) |
T1 | 10 (20%) |
Recurrence | |
Yes | 20 (40%) |
No | 30 (60%) |
Tissue Infiltration | |
Yes | 5 (10%) |
No | 45 (90%) |
Instillations | |
Yes (BCG or BCG plus MitC) | 2 (4%) |
No | 48 (96%) |
EPCAM | CK | EPCAM | CK | ||
---|---|---|---|---|---|
NMIBC 1 | 0.2458 | 0.31145 | NMIBC 23 | 0.10238 | 0.06905 |
NMIBC 2 | 0.1179 | 0.16667 | NMIBC 24 | 0.34483 | 0.16034 |
NMIBC 3 | 0.4273 | 0.33752 | NMIBC 25 | 0.0416 | 0.00000 |
NMIBC 4 | 0.0315 | 0.36292 | NMIBC 26 | 0.0691 | 0.05647 |
NMIBC 5 | 0.2771 | 0.11547 | NMIBC 27 | 0.1963 | 0.12313 |
NMIBC 6 | 0.344 | 0.13298 | NMIBC 28 | 0.2425 | 0.14434 |
NMIBC 7 | 0.1339 | 0.0324 | NMIBC 29 | 0.0484 | 0.04449 |
NMIBC 8 | 0.4008 | 0.2776 | NMIBC 30 | 0.0516 | 0.01572 |
NMIBC 9 | 0.1803 | 0.03673 | NMIBC 31 | 0.07946 | 0.04948 |
NMIBC 10 | 0.0858 | 0.11489 | NMIBC 32 | 0.02271 | 0.15895 |
NMIBC 11 | 0.2591 | 0.22878 | NMIBC 33 | 0.41441 | 0.20751 |
NMIBC 12 | 0.50918 | 0.28367 | NMIBC 34 | 0.095 | 0.12333 |
NMIBC 13 | 0.05625 | 0.04 | NMIBC 35 | 0.22199 | 0.12474 |
NMIBC 14 | 0.6005 | 0.04381 | NMIBC 36 | 0.01373 | 0.00000 |
NMIBC 15 | 0.09119 | 0.20061 | NMIBC 37 | 0.17112 | 0.24293 |
NMIBC 16 | 0.02533 | 0.06267 | NMIBC 38 | 0.21712 | 0.08065 |
NMIBC 17 | 1.27187 | 0.11111 | NMIBC 39 | 0.07692 | 0.15858 |
NMIBC 18 | 0.1788 | 0.49721 | NMIBC 40 | 0.17618 | 0.10277 |
NMIBC 19 | 0.1521 | 0.07606 | NMIBC 41 | 0.20774 | 0.12194 |
NMIBC 20 | 0.3 | 0.18844 | NMIBC 42 | 0.37625 | 0.27421 |
NMIBC 21 | 0.02426 | 0.05391 | NMIBC 43 | 0.12639 | 0.00000 |
NMIBC 22 | 0.052 | 0.07067 |
Percentage CTCs Positive after Cell Screen Filtration | n (%) |
---|---|
Positive | 43 (89.6%) |
Negative | 5 (10.4%) |
CTC specificity after ddPCR | |
EpCAM + /CK + /CD45- | 40 (93.0%) |
EpCAM + /CK − /CD45- | 3 (7.0%) |
EpCAM − /CK + /CD45- | 0 (0.0%) |
Patients pathological grade and stage | n (%) |
Total | 40 (100%) |
Tis | 3 (7.5%) |
Ta LG | 27 (67.5%) |
Ta HG | 4 (10.0%) |
T1 LG | 6 (15.0%) |
T1 HG | 0 (0.0%) |
EpCAMlow | (n = 14/40, 35.0%) |
---|---|
Tis | 1/14 (7.1%) |
Ta | 9/14 (64.3%) |
T1 | 4/14 (28.6%) |
EpCAMhigh | (n = 26/40, 65.0%) |
Tis | 2/26 (7.7%) |
Ta | 22/26 (84.6%) |
T1 | 2/26 (7.7%) |
VIMlow | (n = 18/40, 45.0%) |
Tis | 3/18 (16.7%) |
Ta | 13/18 (72.2%) |
T1 | 2/18 (11.1%) |
VIMhigh | (n = 22/40, 55.0%) |
Ta | 18/22 (81.8%) |
T1 | 4/22 (18.2%) |
T | E-CTCs | H-CTCs | M-CTCs |
---|---|---|---|
Tis (n = 3, 7.5%) | 0 (0.0%) | 1 (33.3%) | 2 (66.7%) |
Ta (n = 31, 77.5%) | 5 (16.1%) | 17 (54.8%) | 9 (29.1%) |
T1 (n = 6, 15.0%) | 2 (33.3%) | 4 (66.7%) | 0 (0.0%) |
Variables | n | p-Value | HR | 95% CI | Chi-Square |
---|---|---|---|---|---|
Age: <60 vs. >60 years >60 years = 29/40 (72.5%) vs. <60 years = 11/40 (27.5%) | 40 | =0.2355 | 0.4168 | 0.1545–1.5827 | - |
Pathological Grade: Low vs. High Low Grade = 36/40 (90%) vs. High Grade = 4/40 (10%) | 40 | =0.1719 | 0.3344 | 0.1143–1.1234 | - |
T stage: Tis vs. Ta vs. T1 Tis = 3/40 (7.5%) vs. Ta = 31/40 (77.5%) vs. T1 = 6/40 (15.0%) | 40 | =0.1019 | - | 4.5680 | |
Sex: Female vs. Male Female = 7/40 (17.5%) vs. Male = 33/40 (82.5%) | 40 | =0.6378 | 0.6987 | 0.1879–2.7854 | - |
Histology: Papillary vs. Not Papillary Papillary = 29/40 (72.5%) vs. Not Papillary = 11/40 (27.5%) | 40 | =0.0832 | 2.4631 | 0.8657–10.3978 | - |
Variables | n | p-Value | Hazard Ratio (95% CI) |
---|---|---|---|
Age: <60 vs. >60 years | 40 | =0.2594 | 0.5335–10.5083 |
Pathological Grade: Low vs. High | 40 | =0.8370 | 0.2634–5.2006 |
T stage: Tis vs. Ta vs. T1 | 40 | = 0.1679 | 0.8390–2.7830 |
Sex: Female vs. Male | 40 | =0.6511 | 0.3159–6.3487 |
Histology: Papillary vs. Not Papillary | 40 | =0.0900 | 0.1389–1.1474 |
Molecular Variables | Univariate Analysis | Multivariate Analysis | ||
---|---|---|---|---|
Hazard Ratio (95% CI) | p-Value | Hazard Ratio (95% CI) | p-Value | |
PD-L1 | 1.0721–6.8003 | <0.03 * | 1.3267–9.5962 | <0.01 * |
TWIST1 | 0.5286–3238.7085 | <0.05 * | 1.4868–8518.2648 | <0.03 * |
TIMP2 | 1.5390–133.7713 | <0.02 * | 0.6190–128.9769 | =0.11 |
VIM | 0.2926–27.0342 | <0.33 | ns | ns |
ZEB1 | 0.1792–159.9790 | <0.36 | ns | ns |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Morelli, M.B.; Amantini, C.; Rossi de Vermandois, J.A.; Gubbiotti, M.; Giannantoni, A.; Mearini, E.; Maggi, F.; Nabissi, M.; Marinelli, O.; Santoni, M.; et al. Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer. Cancers 2021, 13, 5989. https://doi.org/10.3390/cancers13235989
Morelli MB, Amantini C, Rossi de Vermandois JA, Gubbiotti M, Giannantoni A, Mearini E, Maggi F, Nabissi M, Marinelli O, Santoni M, et al. Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer. Cancers. 2021; 13(23):5989. https://doi.org/10.3390/cancers13235989
Chicago/Turabian StyleMorelli, Maria Beatrice, Consuelo Amantini, Jacopo Adolfo Rossi de Vermandois, Marilena Gubbiotti, Antonella Giannantoni, Ettore Mearini, Federica Maggi, Massimo Nabissi, Oliviero Marinelli, Matteo Santoni, and et al. 2021. "Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer" Cancers 13, no. 23: 5989. https://doi.org/10.3390/cancers13235989
APA StyleMorelli, M. B., Amantini, C., Rossi de Vermandois, J. A., Gubbiotti, M., Giannantoni, A., Mearini, E., Maggi, F., Nabissi, M., Marinelli, O., Santoni, M., Cimadamore, A., Montironi, R., & Santoni, G. (2021). Correlation between High PD-L1 and EMT/Invasive Genes Expression and Reduced Recurrence-Free Survival in Blood-Circulating Tumor Cells from Patients with Non-Muscle-Invasive Bladder Cancer. Cancers, 13(23), 5989. https://doi.org/10.3390/cancers13235989