MP34-08 CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS CORRELATE WITH RESPONSE TO BCG IMMUNOTHERAPY
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MP34-06 GMCSF GENE THERAPY AND SIRNA TARGETING COX-2 IN A MURINE MODEL OF BLADDER CANCER Sin Mun Tham*, Kesavan Esuvaranathan, Ratha Mahendran, Singapore, Singapore INTRODUCTION AND OBJECTIVES: Bladder cancer is a good model for gene therapy as candidate genes can be transferred directly to the tumor without surgical intervention. Different cytokine genes successfully induced tumor regression in murine models of bladder cancer. In our studies, intravesical gene therapy with either Granulocyte- Macrophage Colony-Stimulating Factor (GMCSF) or siRNA targeting Cox-2 (Cyclooxygenase-2) (siCox2) successfully cured some mice. This study investigates the efficacy of combined therapy in an orthotopic bladder cancer model. METHODS: C57BL/6 mice with bladder tumors received lipo- some-mediated GMCSF (2.5mg) and siCox2 (1mM/mouse) twice a week for 3 weeks for a total of 6 treatments. As controls, mice received each of these therapies singly as well as saline and non-targeting siRNA (siCon). After 28 days, bladders were harvested, weighed and gene expression changes were analyzed by polymerase chase reaction (PCR). RESULTS: 30-33% of mice were cured of bladder tumors with intravesical GMCSF or siCox2 therapy. Local therapy with siCox2 alone resulted in significantly smaller tumors and better survival rates. Mice that were cured after receiving siCox2 therapy showed significant in- crease in activin receptor-like kinase 7 (Alk7), CD4 and angiotensin II receptor 2 (Agtr2). However, GMCSF therapy did not have an additive effect on the therapeutic effects of siCox2. Few gene expression changes were observed between combined GMCSF and siCox2 against GMCSF and siCon, in which significant increase was observed only in CD4. CONCLUSIONS: While local therapy with GMCSF gene ther- apy or silencing of Cox2 with siRNA have anti-tumor activity individually against bladder cancer in mice, combined therapy did not enhance the beneficial effects. More candidate genes have to be studied to find synergistic anti-tumour effects against bladder cancer. Source of Funding: This study (NMRC/CIRG/1335/2012) was funded by the National Medical Research Council, Singapore. MP34-07 DIFFERENTIAL GENE EXPRESSION IN RESPONSIVE VERSUS RECURRENT NON-MUSCLE INVASIVE HIGH-GRADE UROTHELIAL CARCINOMAS AFTER INDUCTION BCG Philip Ho*, Daniel Willis, Houston, TX; Saad Aldousari, Kuwait, Kuwait; Charles Guo, Colin Dinney, Xifeng Wu, Ashish Kamat, Houston, TX INTRODUCTION AND OBJECTIVES: Although BCG remains the intravesical therapy of choice after TURBT for high-grade non- muscle invasive bladder carcinomas (NMIBC), a recurrence and pro- gression rate of up to 40% occurs after induction treatment. BCG failure likely can be attributed, at least partially, to inherent tumoral charac- teristics. In this study, our goal was to seek out differences in gene expression profile between tumors that recurred and those free from recurrence after induction BCG. METHODS: Primary bladder tumors from TURBT specimens were obtained from patients enrolled in a prospective study evaluating predictive markers for response to BCG. The analysis was restricted to high-grade Ta and T1 lesions. Tumors were arrayed using the Illumina Platform e DASL v3 to profile tumors for mRNA differential expression after tumor tissues were identified by a dedicated GU pathologist. RESULTS: mRNA expression profiles of 12 primary tumors that responded to BCG with no subsequent recurrences were com- pared to 5 primary tumors that recurred after BCG. Demographic and tumor stage distribution between the two groups were not statistically different. Median time to recurrence after TURBT was 7 months (range: 4-11 months). Median follow-up for all patients was 50 months. Using Ingenuity Pathway Analysis, we found a significant increase in ex- pression of genes within specific functional pathways by tumors that developed recurrence after BCG: cell cycle progression (p¼9.2e-5), cell death (p¼6.8e-4), necrosis (p¼4.54e-4), apoptosis (p¼7.4e-3), cell proliferation (p¼4.9e-3), migration of antigen presenting cells (p¼0.044), and immunological diseases (p¼0.015). Highly overex- pressed genes include chemokine receptors CXCR2 (fold change (FC) 2.969, p¼7.75e-6) and CXCR4 (FC 2.337, p¼0.022), nuclear receptor NR4A1 (FC 3.776, p¼4.4e-4), and transcription factor SOX2 (FC 2.48, p¼1.02e-3). CONCLUSIONS: Compared to BCG-responsive tumors, high- grade NMIBC that recur after BCG treatment have increased ex- pression of genes implicated in bladder cancer growth and survival, invasion, response to therapy, and metastasis. Source of Funding: Flight Attendant Medical Research Institute (FAMRI) MP34-08 CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS CORRELATE WITH RESPONSE TO BCG IMMUNOTHERAPY Ratha Mahendran*, Sin Mun Tham, Juwita Rahmat, Jen Hwei Sng, Yew Koon Lim, Lata Raman, Ma Zin Mar Wai, Woon Chau Tsang, Edmund Chiong, Yiong Huak Chan, Kesavan Esuvaranathan, Singapore, Singapore INTRODUCTION AND OBJECTIVES: Though BCG is the gold standard for NMIBC there is a significant non responder rate. The reasons for this are not yet established though single nucleotide poly- morphisms (SNPs) in certain genes have been found to correlate with response to therapy. In a clinical study bladder cancer patients who were treated with anti-CTLA4 antibodies had increased CD4 + ICOS hi IFNg expressing T cells over Treg cell numbers in the bladder and peripheral blood indicating a role for CTLA4 in immunosuppression in bladder cancer patients. Further in a mouse model of orthotopic bladder cancer we found that the presence of tumor induced CTLA4 expression in the bladder. Therefore we evaluated seven SNPs in the CTLA4 gene (Rs733618, Rs4553808, Rs5742909, Rs231775, Rs3087243, Rs7565213, Rs960792) that regulate protein expression and function were chosen for analysis. METHODS: We analyzed SNPs in n¼138 bladder cancer pa- tients who had previously been treated with BCG and for whom long term follow-up data was available. We also examined n¼146 healthy controls. Genomic DNA was extracted and subjected to PCR and fol- lowed by High resolution melting (HRM) PCR analysis to identify the SNPs. These were confirmed by sequencing. RESULTS: There were no difference in the incidence of SNPs between healthy controls and patients. By Kaplan Meier analysis GA at Rs4553808 (148 months for GA vs 110 months AA) and CT at Rs5742909 (167 months for CT vs 106 months for CC) correlated with longer time to recurrence. By COX regression analysis, CT at Rs5742909; AA at Rs3087243; AA and AG Rs7565213 and TT at Rs960792 were significantly correlated with reduced risk of recurrence (p<0.05). While GA at Rs3087243 and TT at Rs7565213 correlated with a reduced risk of progression (p<0.05); GA at Rs231775 correlated with an increased risk of progression (p<0.05, HR 39.4, 95% CI 1.97-795.84). Increased survival was observed in patients with CT at Rs5742909 (p<0.05). CONCLUSIONS: It was previously reported that in Asians Rs231775 GG and GA are associated with decreased risk of cancer. Rs231775 GG unlike the AA genotype is associated with significantly higher T cell activation. Here, Rs231775 GA associated with an in- creased risk of disease progression but not AA which is expressed at a lower level in our population. These results indicate that for some bladder cancer patients, CTLA4 blockade may be a beneficial co-therapy. Source of Funding: This study was supported by National University Cancer Institute (NCIS) Pilot Grant (NMRC/CG/ NCIS/2010) e364 THE JOURNAL OF UROLOGY â Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
Transcript of MP34-08 CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS CORRELATE WITH RESPONSE TO BCG IMMUNOTHERAPY
e364 THE JOURNAL OF UROLOGY� Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
MP34-06GMCSF GENE THERAPY AND SIRNA TARGETING COX-2 IN AMURINE MODEL OF BLADDER CANCER
Sin Mun Tham*, Kesavan Esuvaranathan, Ratha Mahendran,Singapore, Singapore
INTRODUCTION AND OBJECTIVES: Bladder cancer is a goodmodel for gene therapy as candidate genes can be transferred directlyto the tumor without surgical intervention. Different cytokine genessuccessfully induced tumor regression in murine models of bladdercancer. In our studies, intravesical gene therapy with either Granulocyte-Macrophage Colony-Stimulating Factor (GMCSF) or siRNA targetingCox-2 (Cyclooxygenase-2) (siCox2) successfully cured some mice. Thisstudy investigates the efficacy of combined therapy in an orthotopicbladder cancer model.
METHODS: C57BL/6 mice with bladder tumors received lipo-some-mediated GMCSF (2.5mg) and siCox2 (1mM/mouse) twice aweek for 3 weeks for a total of 6 treatments. As controls, mice receivedeach of these therapies singly as well as saline and non-targetingsiRNA (siCon). After 28 days, bladders were harvested, weighedand gene expression changes were analyzed by polymerase chasereaction (PCR).
RESULTS: 30-33% of mice were cured of bladder tumors withintravesical GMCSF or siCox2 therapy. Local therapy with siCox2 aloneresulted in significantly smaller tumors and better survival rates. Micethat were cured after receiving siCox2 therapy showed significant in-crease in activin receptor-like kinase 7 (Alk7), CD4 and angiotensin IIreceptor 2 (Agtr2). However, GMCSF therapy did not have an additiveeffect on the therapeutic effects of siCox2. Few gene expressionchanges were observed between combined GMCSF and siCox2against GMCSF and siCon, in which significant increase was observedonly in CD4.
CONCLUSIONS: While local therapy with GMCSF gene ther-apy or silencing of Cox2 with siRNA have anti-tumor activity individuallyagainst bladder cancer in mice, combined therapy did not enhance thebeneficial effects. More candidate genes have to be studied to findsynergistic anti-tumour effects against bladder cancer.
Source of Funding: This study (NMRC/CIRG/1335/2012) wasfunded by the National Medical Research Council, Singapore.
MP34-07DIFFERENTIAL GENE EXPRESSION IN RESPONSIVE VERSUSRECURRENT NON-MUSCLE INVASIVE HIGH-GRADEUROTHELIAL CARCINOMAS AFTER INDUCTION BCG
Philip Ho*, Daniel Willis, Houston, TX; Saad Aldousari, Kuwait, Kuwait;Charles Guo, Colin Dinney, Xifeng Wu, Ashish Kamat, Houston, TX
INTRODUCTION AND OBJECTIVES: Although BCG remainsthe intravesical therapy of choice after TURBT for high-grade non-muscle invasive bladder carcinomas (NMIBC), a recurrence and pro-gression rate of up to 40% occurs after induction treatment. BCG failurelikely can be attributed, at least partially, to inherent tumoral charac-teristics. In this study, our goal was to seek out differences in geneexpression profile between tumors that recurred and those free fromrecurrence after induction BCG.
METHODS: Primary bladder tumors from TURBT specimenswere obtained from patients enrolled in a prospective study evaluatingpredictive markers for response to BCG. The analysis was restricted tohigh-grade Ta and T1 lesions. Tumors were arrayed using the IlluminaPlatform e DASL v3 to profile tumors for mRNA differential expressionafter tumor tissues were identified by a dedicated GU pathologist.
RESULTS: mRNA expression profiles of 12 primary tumorsthat responded to BCG with no subsequent recurrences were com-pared to 5 primary tumors that recurred after BCG. Demographic andtumor stage distribution between the two groups were not statisticallydifferent. Median time to recurrence after TURBT was 7 months (range:4-11 months). Median follow-up for all patients was 50 months. Using
Ingenuity Pathway Analysis, we found a significant increase in ex-pression of genes within specific functional pathways by tumors thatdeveloped recurrence after BCG: cell cycle progression (p¼9.2e-5),cell death (p¼6.8e-4), necrosis (p¼4.54e-4), apoptosis (p¼7.4e-3),cell proliferation (p¼4.9e-3), migration of antigen presenting cells(p¼0.044), and immunological diseases (p¼0.015). Highly overex-pressed genes include chemokine receptors CXCR2 (fold change (FC)2.969, p¼7.75e-6) and CXCR4 (FC 2.337, p¼0.022), nuclear receptorNR4A1 (FC 3.776, p¼4.4e-4), and transcription factor SOX2 (FC 2.48,p¼1.02e-3).
CONCLUSIONS: Compared to BCG-responsive tumors, high-grade NMIBC that recur after BCG treatment have increased ex-pression of genes implicated in bladder cancer growth and survival,invasion, response to therapy, and metastasis.
Source of Funding: Flight Attendant Medical ResearchInstitute (FAMRI)
MP34-08CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS CORRELATEWITH RESPONSE TO BCG IMMUNOTHERAPY
Ratha Mahendran*, Sin Mun Tham, Juwita Rahmat, Jen Hwei Sng,Yew Koon Lim, Lata Raman, Ma Zin Mar Wai, Woon Chau Tsang,Edmund Chiong, Yiong Huak Chan, Kesavan Esuvaranathan,Singapore, Singapore
INTRODUCTION AND OBJECTIVES: Though BCG is the goldstandard for NMIBC there is a significant non responder rate. Thereasons for this are not yet established though single nucleotide poly-morphisms (SNPs) in certain genes have been found to correlate withresponse to therapy. In a clinical study bladder cancer patients whowere treated with anti-CTLA4 antibodies had increased CD4+ICOShi
IFNg expressing T cells over Treg cell numbers in the bladder andperipheral blood indicating a role for CTLA4 in immunosuppressionin bladder cancer patients. Further in a mouse model of orthotopicbladder cancer we found that the presence of tumor induced CTLA4expression in the bladder. Therefore we evaluated seven SNPs inthe CTLA4 gene (Rs733618, Rs4553808, Rs5742909, Rs231775,Rs3087243, Rs7565213, Rs960792) that regulate protein expressionand function were chosen for analysis.
METHODS: We analyzed SNPs in n¼138 bladder cancer pa-tients who had previously been treated with BCG and for whom longterm follow-up data was available. We also examined n¼146 healthycontrols. Genomic DNA was extracted and subjected to PCR and fol-lowed by High resolution melting (HRM) PCR analysis to identify theSNPs. These were confirmed by sequencing.
RESULTS: There were no difference in the incidence of SNPsbetween healthy controls and patients. By Kaplan Meier analysis GAat Rs4553808 (148 months for GA vs 110 months AA) and CT atRs5742909 (167 months for CT vs 106 months for CC) correlated withlonger time to recurrence. By COX regression analysis, CT atRs5742909; AA at Rs3087243; AA and AG Rs7565213 and TT atRs960792 were significantly correlated with reduced risk of recurrence(p<0.05). While GA at Rs3087243 and TT at Rs7565213 correlatedwith a reduced risk of progression (p<0.05); GA at Rs231775 correlatedwith an increased risk of progression (p<0.05, HR 39.4, 95% CI1.97-795.84). Increased survival was observed in patients with CT atRs5742909 (p<0.05).
CONCLUSIONS: It was previously reported that in AsiansRs231775 GG and GA are associated with decreased risk of cancer.Rs231775 GG unlike the AA genotype is associated with significantlyhigher T cell activation. Here, Rs231775 GA associated with an in-creased risk of disease progression but not AA which is expressed at alower level in our population. These results indicate that for some bladdercancer patients, CTLA4 blockade may be a beneficial co-therapy.
Source of Funding: This study was supported by NationalUniversity Cancer Institute (NCIS) Pilot Grant (NMRC/CG/NCIS/2010)
