- Email: [email protected]
MP45-07 SYNTHETIC INTRAVESICAL IMMUNOMODULATORY MOLECULES AS POTENTIAL ALTERNATIVES TO BACILLUS CALMETTE-GUERIN (BCG) AGAINST NON-MUSCLE-INVASIVE BLADDER CANCER
THE JOURNAL OF UROLOGYâ
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
MP45-05 TO DISEASE: THE POTENTIAL OF AVOIDING CYSTECTOMIES THROUGH MIRNA PROFILES IN CELL-FREE URINE Chintan Patel*, Burlington, MA; Shiv Patel, Travis Sullivan, Burlington, MA; Alexa Meyer, James McKiernan, New York, NY; John Libertino, Kimberly Christ, Burlington, MA INTRODUCTION AND OBJECTIVES: Neoadjuvant chemotherapy prior to radical cystectomy has been shown to confer a survival advantage in patients with muscle-invasive bladder cancer. When combined with maximal endoscopic resection of visible tumor, 38% of patients can be rendered pathologic stage 0 (pT0) at the time of cystectomy. Previous studies have shown differential expression of microRNA (miRNA) in the cell-free urine of patients with muscle invasive and non-muscle invasive bladder cancer. We aimed to identify miRNA in the urine of patients with a history of invasive bladder cancer and differentiating patients with a complete response to therapy from those with persistent disease. METHODS: Total RNA was isolated from cell-free urine of patients undergoing cystectomy for muscle-invasive cancer (pT0, pT1, and >pT2), cT0 patients on active surveillance with a history of muscle invasive disease, as well as healthy control samples (HC) from patients with no history of cancer. Patient samples were divided into four groups: HC (n¼13), pT0/cT0 (n¼17), pT1 (n¼9) and pT2 (n¼23), consistent with the pathologic tumor stage of the specimen at the time of TURBT or surgery. Pooled RNA isolates from each group were profiled via PCR array of 751 miRNA (Exiqon). MiRNA expression levels were validated on individual samples by qRT-PCR. RESULTS: The number of miRNA detected within each group correlated with the severity of disease where 391 miRNAs were detected in the pT2 group, 137 in the pT1 group, 124 in the pT0/cT0 group, and 73 among the HCs. Hierarchical cluster analysis revealed two main branches; one consisting of the HC and pT0/cT0 pools and the other comprised of the pT1 and pT2 pools. Additionally, 32 miRNA were expressed in the pT1 and pT2 pools, and not detected in the pT0/ cT0 and HC pools. qRT-PCR confirmed differential expression of several miRNA distinguishing the two clustered groups (p<0.05). CONCLUSIONS: This study identified miRNA in cell-free urine differentiating patients with evidence of bladder cancer from those without evidence of disease (pT0/cT0). This miRNA profile may aid in the development of a noninvasive method of confirming response to neoadjuvant therapies, and may provide an adjunct to expectant management and surveillance strategies for the treatment of bladder cancer.
e537
more than 999 neoplastic cells were counted to determine KIs and AIs. The data from the WMTPs and virtual TMAs were analyzed by BlandAltman plot, ordinary least-squares regression and Kendall’s tau. Fixed bias was argued if the 95% confidence interval (CI) for the mean value of difference did not include 0. Proportional bias was argued if the slope of the straight-line regression equation of differences on means differed significantly from 0. RESULTS: Almost all differences between the 2 types of samples were plotted within the lines of limits of agreement for both KI and AI. However, fixed biases were detected in the 1-mm-diameter TMA samples regarding the KI (0.1808; 95% CI: 0.1368 - 0.2248), 0.6mm-diameter TMA samples regarding the KI (0.2220; 95% CI: 0.1763 0.2677), 1-mm-diameter TMA samples regarding the AI (0.05495; 95% CI: 0.03596 - 0.07395), and 0.6-mm-diameter TMA samples regarding the AI (0.06250; 95% CI: 0.04328 - 0.07935). Proportional biases were also detected in the 1-mm-diameter TMA samples regarding the AI (slope: 0.7717, P¼0.0001) and 0.6-mm-diameter TMA samples regarding the AI (slope: 0.7977, P<0.0001). Furthermore, positive correlations between KIs and AIs were observed in WMTPs (r¼0.260, P¼0.044) and the 1-mm-diameter TMA samples (r¼0.375, P¼0.004); however, no correlation was observed in the 0.6-mm-diameter TMA samples (r¼0.200, P¼0.121). CONCLUSIONS: The present study suggests that KIs and AIs obtained from TMA-based samples are susceptible to systematic bias, and the lack of correlation between KIs and AIs cannot be avoided in smaller samples obtained for TMA.
Source of Funding: None
MP45-06 COMPARISON BETWEEN WHOLE MOUNT TISSUE PREPARATIONS AND VIRTUAL TISSUE MICROARRAY SAMPLES FOR MEASUREMENT OF KI-67 AND APOPTOSIS INDICES IN HUMAN BLADDER CANCERS Hisashi Oshiro*, Tokyo, Japan; Bogdan Czerniak, Houston, Armenia; Kentaro Sakamaki, Yokohama, Japan; Koji Tsuta, Tokyo, Japan; Jolanta Bondaruk, Afsaneh Keyhani, Colin Dinney, Houston, Armenia; Takeshi Nagai, Tokyo, Japan; Ashish Kamat, Houston, Armenia INTRODUCTION AND OBJECTIVES: Little is known about the differences in the Ki-67 index (KI) and apoptosis index (AI) determined using tissue microarray (TMA) samples and conventional whole mount tissue preparations (WMTPs) of human bladder cancers. The aim of the present study was to investigate the degree of discrepancy between the 2 different types of samples. METHODS: Using WMTPs of Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling obtained from 30 bladder cancer patients, we created virtual TMA samples consisting of 1-mm and 0.6-mm diameters. In each sample, the most highly reactive areas (hot spots) were digitally photomicrographed and
Source of Funding: This research was supported by Grants-inAid for Specialized Program of Research Excellence for Bladder Cancer Research from the National Cancer Institute of the United States, and for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
MP45-07 SYNTHETIC INTRAVESICAL IMMUNOMODULATORY MOLECULES AS POTENTIAL ALTERNATIVES TO BACILLUS CALMETTEGUERIN (BCG) AGAINST NON-MUSCLE-INVASIVE BLADDER CANCER Gabriela R. Passos, Karen L. Ferrari, Isadora O. Gilli, Juliana A. de Camargo, Mariana A. Davi, Mariana F. G. Fazuoli, Athanase Billis, Leonardo O. Reis*, Campinas, Brazil INTRODUCTION AND OBJECTIVES: Intravesical immunomodulatory molecules mechanisms are unclear; it is thought to involve both immunomodulatory and non-immunomodulatory activities and may be similar to that through which bacillus Calmette-Guerin is thought to
e538
THE JOURNAL OF UROLOGYâ
act. We explored signal transduction cascades of BCG, Imiquimode and Thalidomide against non-muscle-invasive bladder cancer. METHODS: Female Fischer 344 rats, 7 weeks old, received 4 doses of 1.5 mg MNU (N-methyl-n-nitrosourea) intravesically, every other week and were randomized (10/group) to: saline (control MNU); 106 CFU of Connaught BCG; 0.4mg Imiquimod, and 2mg Thalidomide, all intravesically for 6 weeks diluted in 0.2 mL of saline. At week 15 bladders were collected for histopathology and immunoblotting: pAKT, Raptor, p4E-BP1, S6K1, pAMPK, pmTOR, pP53, TLR2 and TLR4. RESULTS: BCG, Imiquimode and Thalidomide showed an intense inflammatory response represented by urothelial hyperplasia, with less tumor compared to control. Overall, BCG and synthetic immunomodulators (Imiquimode and Thalidomide) showed comparable mechanisms of action in most of studied molecules, by ultimately down-regulating the mTOR cascade, potentially through the up-regulation of Toll-like receptors (TLRs), figures. CONCLUSIONS: Synthetic immunomodulatory molecules are potentially important alternative intravesical therapeutics against bladder cancer, with mostly parallel mechanistic effects compared to BCG and apparent lack of prohibitive side effects.
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
MP45-08 GMCSF AND IFNa GENE THERAPY IMPROVED RESPONSE TO BCG IMMUNOTHERAPY IN A MURINE MODEL OF BLADDER CANCER Sin Mun Tham*, Abirami R, Kesavan Esuvaranathan, Ratha Mahendran, Singapore, Singapore INTRODUCTION AND OBJECTIVES: The anti-tumor effects of Bacillus Calmette Guerin (BCG) immunotherapy are dependent on repeated cytokine-driven activation of the cellular immune system. We hypothesize that appropriate cytokine gene therapy can potentiate the response to BCG by directly stimulating the recruitment of immune cells into the bladder. METHODS: The synergistic anti-tumor effects of BCG (OncoTICE) and interferon-alpha (IFNa) and/or granulocyte-macrophage colony stimulating factor (GMCSF) were evaluated in orthotopically implanted MB49 urothelial carcinoma in syngeneic C57BL/6 mice. The cytokines were delivered intravesically using plasmid DNA. Various schedules were analyzed for efficacy. Three days after the last treatment, the bladders were harvested and changes in expression of immune related genes were analysed by real-time polymerase chase reaction. RESULTS: In a schedule where mice received GMCSF gene therapy twice a week for 2 weeks followed by BCG treatment once a week for 4 weeks, there were no improvements in survival and cure rates. When mice received weekly GMCSFþIFNa gene therapy followed by BCG therapy 3 days later for a total of 4 weeks, the cure rate was 88% in mice treated with combined therapy compared to 22-33% of mice treated with single therapy. Mice that received gene therapy and BCG showed an increase in CD40, G-CSF, Pgk1, Smad7, Stat1 and Tgfb and decrease in Ece1, Edn1, Ptgs2, Stat4, Stat6 and Tbx21 compared to gene therapy alone. CONCLUSIONS: It is likely that the second schedule was more effective in curing bladder tumors because gene therapy and BCG immunotherapy were administered early when the tumor was small. Intravesical cytokine gene therapy can prime the bladder and change the immune milieu such that it is more responsive to BCG immunotherapy. Source of Funding: This study (NMRC/CIRG/1335/2012) was funded by the National Medical Research Council, Singapore.
MP45-09 EMETINE DIHYDROCHLORIDE PREFERENTIALLY INHIBITS HIF1a AND HIF2a EXPRESSION IN BLADDER CANCER CELLS Kimberly E. Foreman, Deval Patel, Valerie Davidson, Paul Kuo, Robert Flanigan, Gopal N. Gupta*, Maywood, IL
Source of Funding: “none”
INTRODUCTION AND OBJECTIVES: Hypoxia is a common feature of solid tumors that induces a cascade of tumor glycolysis, angiogenesis, and other cell survival responses by activating transcription through hypoxia inducible factors (HIFs). HIFs are transcription factors that are constitutively expressed and tightly regulated in an oxygen dependent manner. Aberrant hypoxia signaling is considered a significant tumor-promoting event, and previous reports suggest HIFa; is aberrantly overexpressed in bladder cancer, even under physiologic oxygen conditions. We have confirmed expression of functionally active HIF1a and HIF2a in cultured bladder cancer cell lines, but not normal urothelial cells. We recently reported that low, nanomolar concentrations of emetine dihydrochloride (emetine) act synergistically with cisplatin and gemcitabine to inhibit bladder cancer cell proliferation in vitro. Here, we examined the effect of emetine on HIF1a and HIF2a expression in bladder cancer cell lines and begin to elucidate the mechanisms by which emetine inhibits HIFa expression. METHODS: UMUC3, HT1376, and T24 invasive bladder cancer cell lines were cultured under standard conditions. As indicated, cells were treated with emetine, MG132 (a proteasome inhibitor), or cycloheximide (a protein synthesis inhibitor). Western blot was
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
MP45-05 TO DISEASE: THE POTENTIAL OF AVOIDING CYSTECTOMIES THROUGH MIRNA PROFILES IN CELL-FREE URINE Chintan Patel*, Burlington, MA; Shiv Patel, Travis Sullivan, Burlington, MA; Alexa Meyer, James McKiernan, New York, NY; John Libertino, Kimberly Christ, Burlington, MA INTRODUCTION AND OBJECTIVES: Neoadjuvant chemotherapy prior to radical cystectomy has been shown to confer a survival advantage in patients with muscle-invasive bladder cancer. When combined with maximal endoscopic resection of visible tumor, 38% of patients can be rendered pathologic stage 0 (pT0) at the time of cystectomy. Previous studies have shown differential expression of microRNA (miRNA) in the cell-free urine of patients with muscle invasive and non-muscle invasive bladder cancer. We aimed to identify miRNA in the urine of patients with a history of invasive bladder cancer and differentiating patients with a complete response to therapy from those with persistent disease. METHODS: Total RNA was isolated from cell-free urine of patients undergoing cystectomy for muscle-invasive cancer (pT0, pT1, and >pT2), cT0 patients on active surveillance with a history of muscle invasive disease, as well as healthy control samples (HC) from patients with no history of cancer. Patient samples were divided into four groups: HC (n¼13), pT0/cT0 (n¼17), pT1 (n¼9) and pT2 (n¼23), consistent with the pathologic tumor stage of the specimen at the time of TURBT or surgery. Pooled RNA isolates from each group were profiled via PCR array of 751 miRNA (Exiqon). MiRNA expression levels were validated on individual samples by qRT-PCR. RESULTS: The number of miRNA detected within each group correlated with the severity of disease where 391 miRNAs were detected in the pT2 group, 137 in the pT1 group, 124 in the pT0/cT0 group, and 73 among the HCs. Hierarchical cluster analysis revealed two main branches; one consisting of the HC and pT0/cT0 pools and the other comprised of the pT1 and pT2 pools. Additionally, 32 miRNA were expressed in the pT1 and pT2 pools, and not detected in the pT0/ cT0 and HC pools. qRT-PCR confirmed differential expression of several miRNA distinguishing the two clustered groups (p<0.05). CONCLUSIONS: This study identified miRNA in cell-free urine differentiating patients with evidence of bladder cancer from those without evidence of disease (pT0/cT0). This miRNA profile may aid in the development of a noninvasive method of confirming response to neoadjuvant therapies, and may provide an adjunct to expectant management and surveillance strategies for the treatment of bladder cancer.
e537
more than 999 neoplastic cells were counted to determine KIs and AIs. The data from the WMTPs and virtual TMAs were analyzed by BlandAltman plot, ordinary least-squares regression and Kendall’s tau. Fixed bias was argued if the 95% confidence interval (CI) for the mean value of difference did not include 0. Proportional bias was argued if the slope of the straight-line regression equation of differences on means differed significantly from 0. RESULTS: Almost all differences between the 2 types of samples were plotted within the lines of limits of agreement for both KI and AI. However, fixed biases were detected in the 1-mm-diameter TMA samples regarding the KI (0.1808; 95% CI: 0.1368 - 0.2248), 0.6mm-diameter TMA samples regarding the KI (0.2220; 95% CI: 0.1763 0.2677), 1-mm-diameter TMA samples regarding the AI (0.05495; 95% CI: 0.03596 - 0.07395), and 0.6-mm-diameter TMA samples regarding the AI (0.06250; 95% CI: 0.04328 - 0.07935). Proportional biases were also detected in the 1-mm-diameter TMA samples regarding the AI (slope: 0.7717, P¼0.0001) and 0.6-mm-diameter TMA samples regarding the AI (slope: 0.7977, P<0.0001). Furthermore, positive correlations between KIs and AIs were observed in WMTPs (r¼0.260, P¼0.044) and the 1-mm-diameter TMA samples (r¼0.375, P¼0.004); however, no correlation was observed in the 0.6-mm-diameter TMA samples (r¼0.200, P¼0.121). CONCLUSIONS: The present study suggests that KIs and AIs obtained from TMA-based samples are susceptible to systematic bias, and the lack of correlation between KIs and AIs cannot be avoided in smaller samples obtained for TMA.
Source of Funding: None
MP45-06 COMPARISON BETWEEN WHOLE MOUNT TISSUE PREPARATIONS AND VIRTUAL TISSUE MICROARRAY SAMPLES FOR MEASUREMENT OF KI-67 AND APOPTOSIS INDICES IN HUMAN BLADDER CANCERS Hisashi Oshiro*, Tokyo, Japan; Bogdan Czerniak, Houston, Armenia; Kentaro Sakamaki, Yokohama, Japan; Koji Tsuta, Tokyo, Japan; Jolanta Bondaruk, Afsaneh Keyhani, Colin Dinney, Houston, Armenia; Takeshi Nagai, Tokyo, Japan; Ashish Kamat, Houston, Armenia INTRODUCTION AND OBJECTIVES: Little is known about the differences in the Ki-67 index (KI) and apoptosis index (AI) determined using tissue microarray (TMA) samples and conventional whole mount tissue preparations (WMTPs) of human bladder cancers. The aim of the present study was to investigate the degree of discrepancy between the 2 different types of samples. METHODS: Using WMTPs of Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling obtained from 30 bladder cancer patients, we created virtual TMA samples consisting of 1-mm and 0.6-mm diameters. In each sample, the most highly reactive areas (hot spots) were digitally photomicrographed and
Source of Funding: This research was supported by Grants-inAid for Specialized Program of Research Excellence for Bladder Cancer Research from the National Cancer Institute of the United States, and for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
MP45-07 SYNTHETIC INTRAVESICAL IMMUNOMODULATORY MOLECULES AS POTENTIAL ALTERNATIVES TO BACILLUS CALMETTEGUERIN (BCG) AGAINST NON-MUSCLE-INVASIVE BLADDER CANCER Gabriela R. Passos, Karen L. Ferrari, Isadora O. Gilli, Juliana A. de Camargo, Mariana A. Davi, Mariana F. G. Fazuoli, Athanase Billis, Leonardo O. Reis*, Campinas, Brazil INTRODUCTION AND OBJECTIVES: Intravesical immunomodulatory molecules mechanisms are unclear; it is thought to involve both immunomodulatory and non-immunomodulatory activities and may be similar to that through which bacillus Calmette-Guerin is thought to
e538
THE JOURNAL OF UROLOGYâ
act. We explored signal transduction cascades of BCG, Imiquimode and Thalidomide against non-muscle-invasive bladder cancer. METHODS: Female Fischer 344 rats, 7 weeks old, received 4 doses of 1.5 mg MNU (N-methyl-n-nitrosourea) intravesically, every other week and were randomized (10/group) to: saline (control MNU); 106 CFU of Connaught BCG; 0.4mg Imiquimod, and 2mg Thalidomide, all intravesically for 6 weeks diluted in 0.2 mL of saline. At week 15 bladders were collected for histopathology and immunoblotting: pAKT, Raptor, p4E-BP1, S6K1, pAMPK, pmTOR, pP53, TLR2 and TLR4. RESULTS: BCG, Imiquimode and Thalidomide showed an intense inflammatory response represented by urothelial hyperplasia, with less tumor compared to control. Overall, BCG and synthetic immunomodulators (Imiquimode and Thalidomide) showed comparable mechanisms of action in most of studied molecules, by ultimately down-regulating the mTOR cascade, potentially through the up-regulation of Toll-like receptors (TLRs), figures. CONCLUSIONS: Synthetic immunomodulatory molecules are potentially important alternative intravesical therapeutics against bladder cancer, with mostly parallel mechanistic effects compared to BCG and apparent lack of prohibitive side effects.
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
MP45-08 GMCSF AND IFNa GENE THERAPY IMPROVED RESPONSE TO BCG IMMUNOTHERAPY IN A MURINE MODEL OF BLADDER CANCER Sin Mun Tham*, Abirami R, Kesavan Esuvaranathan, Ratha Mahendran, Singapore, Singapore INTRODUCTION AND OBJECTIVES: The anti-tumor effects of Bacillus Calmette Guerin (BCG) immunotherapy are dependent on repeated cytokine-driven activation of the cellular immune system. We hypothesize that appropriate cytokine gene therapy can potentiate the response to BCG by directly stimulating the recruitment of immune cells into the bladder. METHODS: The synergistic anti-tumor effects of BCG (OncoTICE) and interferon-alpha (IFNa) and/or granulocyte-macrophage colony stimulating factor (GMCSF) were evaluated in orthotopically implanted MB49 urothelial carcinoma in syngeneic C57BL/6 mice. The cytokines were delivered intravesically using plasmid DNA. Various schedules were analyzed for efficacy. Three days after the last treatment, the bladders were harvested and changes in expression of immune related genes were analysed by real-time polymerase chase reaction. RESULTS: In a schedule where mice received GMCSF gene therapy twice a week for 2 weeks followed by BCG treatment once a week for 4 weeks, there were no improvements in survival and cure rates. When mice received weekly GMCSFþIFNa gene therapy followed by BCG therapy 3 days later for a total of 4 weeks, the cure rate was 88% in mice treated with combined therapy compared to 22-33% of mice treated with single therapy. Mice that received gene therapy and BCG showed an increase in CD40, G-CSF, Pgk1, Smad7, Stat1 and Tgfb and decrease in Ece1, Edn1, Ptgs2, Stat4, Stat6 and Tbx21 compared to gene therapy alone. CONCLUSIONS: It is likely that the second schedule was more effective in curing bladder tumors because gene therapy and BCG immunotherapy were administered early when the tumor was small. Intravesical cytokine gene therapy can prime the bladder and change the immune milieu such that it is more responsive to BCG immunotherapy. Source of Funding: This study (NMRC/CIRG/1335/2012) was funded by the National Medical Research Council, Singapore.
MP45-09 EMETINE DIHYDROCHLORIDE PREFERENTIALLY INHIBITS HIF1a AND HIF2a EXPRESSION IN BLADDER CANCER CELLS Kimberly E. Foreman, Deval Patel, Valerie Davidson, Paul Kuo, Robert Flanigan, Gopal N. Gupta*, Maywood, IL
Source of Funding: “none”
INTRODUCTION AND OBJECTIVES: Hypoxia is a common feature of solid tumors that induces a cascade of tumor glycolysis, angiogenesis, and other cell survival responses by activating transcription through hypoxia inducible factors (HIFs). HIFs are transcription factors that are constitutively expressed and tightly regulated in an oxygen dependent manner. Aberrant hypoxia signaling is considered a significant tumor-promoting event, and previous reports suggest HIFa; is aberrantly overexpressed in bladder cancer, even under physiologic oxygen conditions. We have confirmed expression of functionally active HIF1a and HIF2a in cultured bladder cancer cell lines, but not normal urothelial cells. We recently reported that low, nanomolar concentrations of emetine dihydrochloride (emetine) act synergistically with cisplatin and gemcitabine to inhibit bladder cancer cell proliferation in vitro. Here, we examined the effect of emetine on HIF1a and HIF2a expression in bladder cancer cell lines and begin to elucidate the mechanisms by which emetine inhibits HIFa expression. METHODS: UMUC3, HT1376, and T24 invasive bladder cancer cell lines were cultured under standard conditions. As indicated, cells were treated with emetine, MG132 (a proteasome inhibitor), or cycloheximide (a protein synthesis inhibitor). Western blot was