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Plasticity in muscle-invasive bladder cancer before and after cisplatin-based neoadjuvant chemotherapy
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IBCN Abstracts / Urologic Oncology: Seminars and Original Investigations 35 (2017) 608–621
Plasticity in muscle-invasive bladder cancer before and after cisplatinbased neoadjuvant chemotherapy Roland Seiler, Ewan A. Gibb, Mandeep Takhar, Kim Van Kessel, Bas W. G. van Rhijn, Brian Winters, James Douglas, Qiqi Wang, Voleak Choeurng, Nicholas Erho, Christine Buerki, Elai Davicioni, Gottfrid Sjödahl, George N. Thalmann, Ellen C. Zwarthoff, Joost L. Boormans, Marc Dall’Era, Michiel S. van der Heijden, Jonathan Wright, Peter C. Black. Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia;, Department of Urology, University of Bern, Bern, Switzerland;, GenomeDx Biosciences, Inc., Vancouver, British Columbia, Canada;, Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands;, Department of Surgical Oncology, Division of Urology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands;, Department of Urology, University of Washington School of Medicine, Seattle, Washington, USA;, Department of Urology, University Hospital of Southampton, Hampshire, UK;, Division of Urological Research, Department of Translational Medicine, Lund University, Malmö, Sweden;, UC Davis Comprehensive Cancer Center, Sacramento, California, USA Introduction: Cisplatin-based neoadjuvant chemotherapy (NAC) induces a wide spectrum of responses and outcomes in patients with muscle-invasive bladder cancer (MIBC). Here, we investigate the biological and clinical characteristics of matched pre- and post-NAC tumors. Methods: Radical cystectomy (RC) samples were available for gene expression analysis from 134 patients treated with cisplatin-based NAC who had residual MIBC, of which 116 had matched pre-NAC samples. In addition, the tumor bed (scar) of 21 post-NAC RC specimens with complete pathologic response (ie. pT0N0) was profiled. Unsupervised consensus clustering was performed and the clusters were investigated for their biological and clinical characteristics. H&E staining and immunohistochemistry (IHC) were used to confirm tissue sampling and the gene expression analysis. Results: Unsupervised consensus clustering yielded a robust 4-cluster solution. Consensus clusters (CC) 1 and 2 expressed genes consistent with a basal-like (KRT5/6, KRT14) and a luminal-like (GATA3, PPARG) phenotype, respectively. CC3 expressed a strong T-cell signature, markers for T-cell receptor and chemokine signaling as well as checkpoint molecules (CTLA4, CD80). Conversely, CC4 was associated with wound healing/scarring (MYH11, SHH, CNN1). This ‘scar-like’ character of CC4 was highly consistent with the scar samples (n ¼ 21). IHC for GATA3, PPARG, CD44 and CD8 confirmed the biological characteristics of CC1-4. Pair-wise comparison of samples before and after NAC revealed that pre-NAC samples with basal characteristics were represented in all four post-NAC clusters, but luminal pre-NAC tumors were almost exclusively CC2-4 after NAC. Despite being pathological nonresponders, patients in CC4 had favorable prognosis compared to patients in CC1-3 (p ¼ 0.027). Conclusions: In post-NAC MIBC four biologically and clinically distinct clusters (CC1 basal, CC2 luminal, CC3 infiltrated, CC4 scar-like) were identified. Variability of post-NAC gene expression was greater in the preNAC basal-like tumors compared to luminal-like tumors. This work suggests dynamic gene expression changes occur in response to NAC. Comprehensive immune profiling of patients with urothelial carcinoma of the bladder identifies a novel immune target John P. Sfakianos, Adam D. Farkas, Harry Anastos, Matthey D. Galsky, Nina Bhardwaj. Department of Urology, Icahn School of Medicine at Mount Sinai;, Department of Medicine, Division of Hematology/Oncology, Tisch Cancer Institute Introduction: The introduction of immune checkpoint inhibitors represents a paradigm shift in the clinical management of patients with urothelial carcinoma of the bladder (UCB). However, only a subset of patients will respond to therapy highlighting the importance of identifying novel checkpoint molecules, which may further promote anti-tumor immunity.
Materials and methods: After IRB approval, peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TILs) were analyzed from 20 non-muscle invasive (NMI) and 20 muscle invasive (MI) UCB patients. Cells were stained with a 14 color FACS panel to identify immune subsets and exhaustion markers (PD-1, TIM-3 and TIGIT). Data was analyzed with FlowJo and Prism. Results: A diagnosis of UBC, compared to healthy donors, drove maturation of PBMC NK cells, with increased frequency of CD56dimCD16þ cells, while, in TILs, there was a significant decrease in the frequency of CD56brightCD16- NK cells implying defects in IFNy/TNFa production. The frequency of PD-1 expression in T cells of MI TILs was 15-27% possibly explaining the mixed success of PD-1 blockade, CD4 (16/27% NMI/MI, respectively) and CD8 (15/20% NMI/MI, respectively). Tim-3 expression on TIL NK, CD4, and CD8 T cells increased with stage; 15%, 7%, and 7% (NMI) and 42%, 26% and 29% (MI), respectively (p o0.0005). Tim-3 expression on PBMC T cells also increased with stage (NMI vs MI CD4 (3% and 14%, respectively): p ¼ o.05 CD8 (5% and 15%, respectively): p ¼ o.05). TIGIT expression on NK and T cells did not increase with stage, but was significantly induced in PBMC and TIL in the presence of UBC suggesting it may represent an early marker of immune dysfunction. Conclusion: We report to our knowledge the first comprehensive immune profiling of patients with UCB. We have identified TIM-3 and TIGIT as possible novel targets for patients with UCB.
Inherent and acquired mechanisms of cisplatin resistance in long-term treated urothelial carcinoma cell lines Margaretha A. Skowron, Günter Niegisch, Gommert van Koeveringe, Joep van Roermund, Andrea Romano, Peter Albers, Wolfgang A. Schulz, Michèle J. Hoffmann. Department of Urology, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany;, Department of Urology, Maastricht University Medical Centre, The Netherlands;, Department of Obstetrics and Gynaecology, Maastricht University Medical Centre, The Netherlands Background: Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate the underlying mechanisms and to eventually overcome cisplatin resistance, we generated long-term cisplatin treated (LTT) UC cell lines (UCC) and investigated them for cancer stem cell (CSC) properties conferring inherent resistance and for mechanisms underlying acquired chemoresistance. Material and methods: Cisplatin resistant LTTs were generated from four different UCCs by treatment with escalating cisplatin doses for several months. CD90þ cells were enriched by MACS- or FACS-sorting. Cell growth and proliferation was examined by MTT and Giemsa staining. Cellcycle distribution, apoptosis and ROS levels were quantified by flow cytometry. mRNA and protein expression was measured by qRT-PCR, western blot or immunofluorescence staining. Results: CD90þ UCCs neither exhibited CSC properties nor inherent chemoresistance and were not enriched among LTTs. Phenotypic changes of LTTs could be related to increased expression of EMT- and WNT-target genes. LTTs proliferated more slowly than their parental cell lines, but recovered faster from cisplatin stress. In LTTs, to various extents depending on the cell line, cytoprotective enzymes were induced, NRF2 was activated, glutathione availability was increased and consequently intracellular ROS was decreased, cisplatin exporter molecules were upregulated and cisplatin adduct levels were reduced. Expression of anti-apoptotic factors was generally increased in LTTs. Conclusion: CD90 is not a suitable CSC marker in UC cell lines. LTTs evaded cisplatin stress by phenotypic plasticity via EMT and induction of non-canonical WNT target genes and anti-apoptotic factors. Decreased DNA-damage could largely be attributed to an increased cisplatin inactivation by the NRF2 pathway. The diversity in NRF2 activation and other cytoprotective mechanisms among the four tested LTTs highlights the complexity of cisplatin resistance even within one tumour entity and explains heterogeneity in patient responses to chemotherapy.
IBCN Abstracts / Urologic Oncology: Seminars and Original Investigations 35 (2017) 608–621
Plasticity in muscle-invasive bladder cancer before and after cisplatinbased neoadjuvant chemotherapy Roland Seiler, Ewan A. Gibb, Mandeep Takhar, Kim Van Kessel, Bas W. G. van Rhijn, Brian Winters, James Douglas, Qiqi Wang, Voleak Choeurng, Nicholas Erho, Christine Buerki, Elai Davicioni, Gottfrid Sjödahl, George N. Thalmann, Ellen C. Zwarthoff, Joost L. Boormans, Marc Dall’Era, Michiel S. van der Heijden, Jonathan Wright, Peter C. Black. Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia;, Department of Urology, University of Bern, Bern, Switzerland;, GenomeDx Biosciences, Inc., Vancouver, British Columbia, Canada;, Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands;, Department of Surgical Oncology, Division of Urology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands;, Department of Urology, University of Washington School of Medicine, Seattle, Washington, USA;, Department of Urology, University Hospital of Southampton, Hampshire, UK;, Division of Urological Research, Department of Translational Medicine, Lund University, Malmö, Sweden;, UC Davis Comprehensive Cancer Center, Sacramento, California, USA Introduction: Cisplatin-based neoadjuvant chemotherapy (NAC) induces a wide spectrum of responses and outcomes in patients with muscle-invasive bladder cancer (MIBC). Here, we investigate the biological and clinical characteristics of matched pre- and post-NAC tumors. Methods: Radical cystectomy (RC) samples were available for gene expression analysis from 134 patients treated with cisplatin-based NAC who had residual MIBC, of which 116 had matched pre-NAC samples. In addition, the tumor bed (scar) of 21 post-NAC RC specimens with complete pathologic response (ie. pT0N0) was profiled. Unsupervised consensus clustering was performed and the clusters were investigated for their biological and clinical characteristics. H&E staining and immunohistochemistry (IHC) were used to confirm tissue sampling and the gene expression analysis. Results: Unsupervised consensus clustering yielded a robust 4-cluster solution. Consensus clusters (CC) 1 and 2 expressed genes consistent with a basal-like (KRT5/6, KRT14) and a luminal-like (GATA3, PPARG) phenotype, respectively. CC3 expressed a strong T-cell signature, markers for T-cell receptor and chemokine signaling as well as checkpoint molecules (CTLA4, CD80). Conversely, CC4 was associated with wound healing/scarring (MYH11, SHH, CNN1). This ‘scar-like’ character of CC4 was highly consistent with the scar samples (n ¼ 21). IHC for GATA3, PPARG, CD44 and CD8 confirmed the biological characteristics of CC1-4. Pair-wise comparison of samples before and after NAC revealed that pre-NAC samples with basal characteristics were represented in all four post-NAC clusters, but luminal pre-NAC tumors were almost exclusively CC2-4 after NAC. Despite being pathological nonresponders, patients in CC4 had favorable prognosis compared to patients in CC1-3 (p ¼ 0.027). Conclusions: In post-NAC MIBC four biologically and clinically distinct clusters (CC1 basal, CC2 luminal, CC3 infiltrated, CC4 scar-like) were identified. Variability of post-NAC gene expression was greater in the preNAC basal-like tumors compared to luminal-like tumors. This work suggests dynamic gene expression changes occur in response to NAC. Comprehensive immune profiling of patients with urothelial carcinoma of the bladder identifies a novel immune target John P. Sfakianos, Adam D. Farkas, Harry Anastos, Matthey D. Galsky, Nina Bhardwaj. Department of Urology, Icahn School of Medicine at Mount Sinai;, Department of Medicine, Division of Hematology/Oncology, Tisch Cancer Institute Introduction: The introduction of immune checkpoint inhibitors represents a paradigm shift in the clinical management of patients with urothelial carcinoma of the bladder (UCB). However, only a subset of patients will respond to therapy highlighting the importance of identifying novel checkpoint molecules, which may further promote anti-tumor immunity.
Materials and methods: After IRB approval, peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TILs) were analyzed from 20 non-muscle invasive (NMI) and 20 muscle invasive (MI) UCB patients. Cells were stained with a 14 color FACS panel to identify immune subsets and exhaustion markers (PD-1, TIM-3 and TIGIT). Data was analyzed with FlowJo and Prism. Results: A diagnosis of UBC, compared to healthy donors, drove maturation of PBMC NK cells, with increased frequency of CD56dimCD16þ cells, while, in TILs, there was a significant decrease in the frequency of CD56brightCD16- NK cells implying defects in IFNy/TNFa production. The frequency of PD-1 expression in T cells of MI TILs was 15-27% possibly explaining the mixed success of PD-1 blockade, CD4 (16/27% NMI/MI, respectively) and CD8 (15/20% NMI/MI, respectively). Tim-3 expression on TIL NK, CD4, and CD8 T cells increased with stage; 15%, 7%, and 7% (NMI) and 42%, 26% and 29% (MI), respectively (p o0.0005). Tim-3 expression on PBMC T cells also increased with stage (NMI vs MI CD4 (3% and 14%, respectively): p ¼ o.05 CD8 (5% and 15%, respectively): p ¼ o.05). TIGIT expression on NK and T cells did not increase with stage, but was significantly induced in PBMC and TIL in the presence of UBC suggesting it may represent an early marker of immune dysfunction. Conclusion: We report to our knowledge the first comprehensive immune profiling of patients with UCB. We have identified TIM-3 and TIGIT as possible novel targets for patients with UCB.
Inherent and acquired mechanisms of cisplatin resistance in long-term treated urothelial carcinoma cell lines Margaretha A. Skowron, Günter Niegisch, Gommert van Koeveringe, Joep van Roermund, Andrea Romano, Peter Albers, Wolfgang A. Schulz, Michèle J. Hoffmann. Department of Urology, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany;, Department of Urology, Maastricht University Medical Centre, The Netherlands;, Department of Obstetrics and Gynaecology, Maastricht University Medical Centre, The Netherlands Background: Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate the underlying mechanisms and to eventually overcome cisplatin resistance, we generated long-term cisplatin treated (LTT) UC cell lines (UCC) and investigated them for cancer stem cell (CSC) properties conferring inherent resistance and for mechanisms underlying acquired chemoresistance. Material and methods: Cisplatin resistant LTTs were generated from four different UCCs by treatment with escalating cisplatin doses for several months. CD90þ cells were enriched by MACS- or FACS-sorting. Cell growth and proliferation was examined by MTT and Giemsa staining. Cellcycle distribution, apoptosis and ROS levels were quantified by flow cytometry. mRNA and protein expression was measured by qRT-PCR, western blot or immunofluorescence staining. Results: CD90þ UCCs neither exhibited CSC properties nor inherent chemoresistance and were not enriched among LTTs. Phenotypic changes of LTTs could be related to increased expression of EMT- and WNT-target genes. LTTs proliferated more slowly than their parental cell lines, but recovered faster from cisplatin stress. In LTTs, to various extents depending on the cell line, cytoprotective enzymes were induced, NRF2 was activated, glutathione availability was increased and consequently intracellular ROS was decreased, cisplatin exporter molecules were upregulated and cisplatin adduct levels were reduced. Expression of anti-apoptotic factors was generally increased in LTTs. Conclusion: CD90 is not a suitable CSC marker in UC cell lines. LTTs evaded cisplatin stress by phenotypic plasticity via EMT and induction of non-canonical WNT target genes and anti-apoptotic factors. Decreased DNA-damage could largely be attributed to an increased cisplatin inactivation by the NRF2 pathway. The diversity in NRF2 activation and other cytoprotective mechanisms among the four tested LTTs highlights the complexity of cisplatin resistance even within one tumour entity and explains heterogeneity in patient responses to chemotherapy.