- Email: [email protected]
REPERFUSION INJURY
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
619 ISOLATION AND ANALYSIS OF CIRCULATING TUMOR CELLS IN RENAL CELL CARCINOMA E. Jason Abel*, Ben P. Casavant, Jacob T. Tokar, Joshua M. Lang, David J. Beebe, Madison, WI INTRODUCTION AND OBJECTIVES: Circulating tumor cells (CTCs) have demonstrated prognostic ability in prostate and breast cancer but similar studies in Renal Cell Carcinoma (RCC) are limited because the current techniques rely on positive selection of cells using cell surface markers rarely expressed in RCC cells. We have designed a novel platform to permit the use of any antibody of interest bound to paramagnetic particles (PMPs) to isolate and purify PMP-bound cells via immiscible oil barriers. The purpose of this study was to demonstrate and validate RCC cell isolation from blood by positive selection of cells expressing carbonic anhydrase nine (CAIX). METHODS: The Vertical Immiscible Filtration Assisted by Surface Tension (VerIFAST) platform was designed (BPC, DJB patent filed) using the relative dominance of surface tension in the microscale to create virtual walls between oil and aqueous phases filtering contaminants in a single step, while maintaining cell viability to permit further analysis. Initial experiments used samples of whole blood spiked with known RCC cell lines (786-0). After optimization of technique and confirmation of capture, samples from patients with locally advanced and metastatic RCC were evaluated after IRB approval and patient consent. Captured cells were stained with cytokeratins and Ki-67. RESULTS: Initial experiments were designed to optimize CTC capture using anti-CAIX antibodies bound to PMP. A high capture efficiency of the spiked 786-0 cells (⬎75% capture efficiency) was able to be demonstrated using commercially available antibodies. Within the device, intracellular staining was demonstrated with successful staining of both cytokeratins and the proliferative marker, Ki-67(figure). Likewise, CTCs have been isolated from RCC patient samples using identical technique and staining for intracellular cytokeratins. CONCLUSIONS: Isolation and purification of CTC cells from peripheral blood of RCC patients using CAIX is possible using this novel platform. We have demonstrated the ability to perform intracellular analyses, which will be important to study the prognostic value of CTC in RCC. Future studies will focus on nucleic acid isolation to investigate tumor heterogeneity and the role of CTC in cancer progression.
THE JOURNAL OF UROLOGY姞
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may prevent RIRI more effectively than Mann. Accordingly, we investigated the protective effects of NAC and Mann on RIRI injury using the in vitro and in vivo models. METHODS: Renal proximal LLC-PK1 cells were employed as our in vitro model while Sprague-Dawley rats were our in vivo model. Effects of NAC and Mann were assessed on chemically-induced ischemia/hypoxia for 24 h using combination of antimycin A (AMA) and 2-deoxyglucose (2DG). Cell viability and malondialdehyde (MDA) assays and biochemical analysis for RIRI markers were also performed. Effects of NAC and Mann were further examined on rats subjected to ischemia through 40 min renal pedicle occlusion, followed by 24 h reperfusion. Either NAC or Mann was given to rats intraperitoneally 30 min prior to ischemia and immediately before the reperfusion period. After 24 h reperfusion, blood samples and kidney specimens were collected for analyses of blood urea nitrogen (BUN) and creatinine (Cr) and histopathological and biochemical evaluations. RESULTS: Although AMA/2DG combination led to a ⬃30% reduction in cell viability and a ⬃44% increase in MDA level (due to oxidative stress), such adverse effects were effectively prevented with NAC, not with Mann. The RIRI markers, heat shock protein 70 (Hsp70) and lipocalin-2 (Lcn2), were also up-regulated under ischemia, but they were diminished to merely the basal levels by NAC (not by Mann). Similarly, the elevated BUN and Cr levels in the ischemic rats declined to nearly the normal level with minimal histological changes in the kidneys by NAC administration. The increased MDA levels and the up-regulated expressions of Hsp70 and Lcn2 in ischemic specimens were also considerably reduced by NAC. CONCLUSIONS: Both in vitro and in vivo studies suggest that oxidative stress may play a critical role in RIRI. However, a potent antioxidant NAC is capable of protecting renal cells from oxidative assault, whereas Mann has little or no such effects. Therefore, it is plausible that NAC should be considered as a more effective perioperative renoprotective agent (compared with Mann). Source of Funding: Departmental
Kidney Cancer: Evaluation & Staging (I) Moderated Poster Session 23 Sunday, May 5, 2013
3:30 PM-5:30 PM
621 PARTIAL AND RADICAL NEPHRECTOMY TRENDS: ANALYSIS OF CASE LOGS FROM CERTIFYING UROLOGISTS Stephen Poon*, Jonathan Silberstein, Ling Chen, Behfar Ehdaie, Philip Kim, Paul Russo, New York, NY Source of Funding: none
620 COMPARISON OF PROTECTIVE EFFECTS OF N-ACETYLCYSTEINE AND MANNITOL ON RENAL ISCHEMIC/ REPERFUSION INJURY Michael Degen*, Andrew Fishman, Bobby Alexander, Muhammad Choudhury, Majid Eshghi, Sensuke Konno, Valhalla, NY INTRODUCTION AND OBJECTIVES: As renal ischemic/reperfusion injury (RIRI) results in significant postoperative morbidity and mortality, pharmaceutical agents have been routinely used as one of the perioperative renoprotective interventions. Among them, mannitol (Mann) has been commonly used but its actual effectiveness has been often debated. As accumulating data suggest that RIRI could be primarily attributed to oxidative stress (generation of oxygen free radicals), it is possible that an antioxidant such as N-acetylcysteine (NAC)
INTRODUCTION AND OBJECTIVES: The concurrent introduction of partial nephrectomy (PN) and laparoscopic radical nephrectomy (LRN) within a short time may be inhibiting diffusion of these techniques, with surgeons opting for one innovation or the other. Using American Board of Urology (ABU) case log data, we investigated contemporary trends in these surgical options and how surgeon and practice characteristics may influence these trends. METHODS: We obtained annualized case log data for nephrectomies from the ABU for all urologists certifying or recertifying from 2002 to 2010, and evaluated trends in nephrectomy use. Logistic regressions were used to evaluate surgeon and practice characteristics as predictors for partial and laparoscopic procedures. RESULTS: From 3,852 case logs submitted by non-pediatric urologists, 48,384 nephrectomies were analyzed. From 2002 to 2010, annual nephrectomies performed as open radical nephrectomies (ORN) decreased from 53% to 27% (figure), and use of LRN moderately increased from 29% to 37%; open PN (OPN) remained stable at 15% and laparoscopic PN (LPN) increased 8-fold, from 2% to 16%. On multivariable analysis, laparoscopy was more likely to be performed by
619 ISOLATION AND ANALYSIS OF CIRCULATING TUMOR CELLS IN RENAL CELL CARCINOMA E. Jason Abel*, Ben P. Casavant, Jacob T. Tokar, Joshua M. Lang, David J. Beebe, Madison, WI INTRODUCTION AND OBJECTIVES: Circulating tumor cells (CTCs) have demonstrated prognostic ability in prostate and breast cancer but similar studies in Renal Cell Carcinoma (RCC) are limited because the current techniques rely on positive selection of cells using cell surface markers rarely expressed in RCC cells. We have designed a novel platform to permit the use of any antibody of interest bound to paramagnetic particles (PMPs) to isolate and purify PMP-bound cells via immiscible oil barriers. The purpose of this study was to demonstrate and validate RCC cell isolation from blood by positive selection of cells expressing carbonic anhydrase nine (CAIX). METHODS: The Vertical Immiscible Filtration Assisted by Surface Tension (VerIFAST) platform was designed (BPC, DJB patent filed) using the relative dominance of surface tension in the microscale to create virtual walls between oil and aqueous phases filtering contaminants in a single step, while maintaining cell viability to permit further analysis. Initial experiments used samples of whole blood spiked with known RCC cell lines (786-0). After optimization of technique and confirmation of capture, samples from patients with locally advanced and metastatic RCC were evaluated after IRB approval and patient consent. Captured cells were stained with cytokeratins and Ki-67. RESULTS: Initial experiments were designed to optimize CTC capture using anti-CAIX antibodies bound to PMP. A high capture efficiency of the spiked 786-0 cells (⬎75% capture efficiency) was able to be demonstrated using commercially available antibodies. Within the device, intracellular staining was demonstrated with successful staining of both cytokeratins and the proliferative marker, Ki-67(figure). Likewise, CTCs have been isolated from RCC patient samples using identical technique and staining for intracellular cytokeratins. CONCLUSIONS: Isolation and purification of CTC cells from peripheral blood of RCC patients using CAIX is possible using this novel platform. We have demonstrated the ability to perform intracellular analyses, which will be important to study the prognostic value of CTC in RCC. Future studies will focus on nucleic acid isolation to investigate tumor heterogeneity and the role of CTC in cancer progression.
THE JOURNAL OF UROLOGY姞
e253
may prevent RIRI more effectively than Mann. Accordingly, we investigated the protective effects of NAC and Mann on RIRI injury using the in vitro and in vivo models. METHODS: Renal proximal LLC-PK1 cells were employed as our in vitro model while Sprague-Dawley rats were our in vivo model. Effects of NAC and Mann were assessed on chemically-induced ischemia/hypoxia for 24 h using combination of antimycin A (AMA) and 2-deoxyglucose (2DG). Cell viability and malondialdehyde (MDA) assays and biochemical analysis for RIRI markers were also performed. Effects of NAC and Mann were further examined on rats subjected to ischemia through 40 min renal pedicle occlusion, followed by 24 h reperfusion. Either NAC or Mann was given to rats intraperitoneally 30 min prior to ischemia and immediately before the reperfusion period. After 24 h reperfusion, blood samples and kidney specimens were collected for analyses of blood urea nitrogen (BUN) and creatinine (Cr) and histopathological and biochemical evaluations. RESULTS: Although AMA/2DG combination led to a ⬃30% reduction in cell viability and a ⬃44% increase in MDA level (due to oxidative stress), such adverse effects were effectively prevented with NAC, not with Mann. The RIRI markers, heat shock protein 70 (Hsp70) and lipocalin-2 (Lcn2), were also up-regulated under ischemia, but they were diminished to merely the basal levels by NAC (not by Mann). Similarly, the elevated BUN and Cr levels in the ischemic rats declined to nearly the normal level with minimal histological changes in the kidneys by NAC administration. The increased MDA levels and the up-regulated expressions of Hsp70 and Lcn2 in ischemic specimens were also considerably reduced by NAC. CONCLUSIONS: Both in vitro and in vivo studies suggest that oxidative stress may play a critical role in RIRI. However, a potent antioxidant NAC is capable of protecting renal cells from oxidative assault, whereas Mann has little or no such effects. Therefore, it is plausible that NAC should be considered as a more effective perioperative renoprotective agent (compared with Mann). Source of Funding: Departmental
Kidney Cancer: Evaluation & Staging (I) Moderated Poster Session 23 Sunday, May 5, 2013
3:30 PM-5:30 PM
621 PARTIAL AND RADICAL NEPHRECTOMY TRENDS: ANALYSIS OF CASE LOGS FROM CERTIFYING UROLOGISTS Stephen Poon*, Jonathan Silberstein, Ling Chen, Behfar Ehdaie, Philip Kim, Paul Russo, New York, NY Source of Funding: none
620 COMPARISON OF PROTECTIVE EFFECTS OF N-ACETYLCYSTEINE AND MANNITOL ON RENAL ISCHEMIC/ REPERFUSION INJURY Michael Degen*, Andrew Fishman, Bobby Alexander, Muhammad Choudhury, Majid Eshghi, Sensuke Konno, Valhalla, NY INTRODUCTION AND OBJECTIVES: As renal ischemic/reperfusion injury (RIRI) results in significant postoperative morbidity and mortality, pharmaceutical agents have been routinely used as one of the perioperative renoprotective interventions. Among them, mannitol (Mann) has been commonly used but its actual effectiveness has been often debated. As accumulating data suggest that RIRI could be primarily attributed to oxidative stress (generation of oxygen free radicals), it is possible that an antioxidant such as N-acetylcysteine (NAC)
INTRODUCTION AND OBJECTIVES: The concurrent introduction of partial nephrectomy (PN) and laparoscopic radical nephrectomy (LRN) within a short time may be inhibiting diffusion of these techniques, with surgeons opting for one innovation or the other. Using American Board of Urology (ABU) case log data, we investigated contemporary trends in these surgical options and how surgeon and practice characteristics may influence these trends. METHODS: We obtained annualized case log data for nephrectomies from the ABU for all urologists certifying or recertifying from 2002 to 2010, and evaluated trends in nephrectomy use. Logistic regressions were used to evaluate surgeon and practice characteristics as predictors for partial and laparoscopic procedures. RESULTS: From 3,852 case logs submitted by non-pediatric urologists, 48,384 nephrectomies were analyzed. From 2002 to 2010, annual nephrectomies performed as open radical nephrectomies (ORN) decreased from 53% to 27% (figure), and use of LRN moderately increased from 29% to 37%; open PN (OPN) remained stable at 15% and laparoscopic PN (LPN) increased 8-fold, from 2% to 16%. On multivariable analysis, laparoscopy was more likely to be performed by