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Purpose: To determine the safety and toxicities of sequential MMC (mitomycin C) + BCG (bacillus Calmette–Guérin) in patients with non–muscle-invasive bladder cancer (NMIBC) and explore evidence for potentiation of BCG activity by MMC.Experimental Design: A 3 + 3 phase I dose-escalation trial of six weekly treatments was conducted in patients with NMIBC. MMC (10, 20, or 40 mg) was instilled intravesically for 30 minutes, followed by a 10-minute washout with gentle saline irrigation and then instillation of BCG (half or full strength) for 2 hours. Urine cytokines were monitored and compared with levels in a control cohort receiving BCG only. Murine experiments were carried out as described previously.Results: Twelve patients completed therapy, including 3 patients receiving full doses. The regimen was well tolerated with no treatment-related dose-limiting toxicities. Urinary frequency and urgency, and fatigue were common. Eleven (91.7%) patients were free of disease at a mean (range) follow-up of 21.4 (8.4–27.0) months. Median posttreatment urine concentrations of IL2, IL8, IL10, and TNFα increased over the 6-week treatment period. A greater increase in posttreatment urinary IL8 during the 6-week period was observed in patients receiving MMC + BCG compared with patients receiving BCG monotherapy. In mice, intravesical MMC + BCG skewed tumor-associated macrophages (TAM) toward a beneficial M1 phenotype.Conclusions: Instillation of sequential MMC + BCG is safe tolerable up to 40-mg MMC plus full-strength BCG. This approach could provide improved antitumor activity over BCG monotherapy by augmenting beneficial M1 TAMs. Clin Cancer Res; 21(2); 303–11. ©2014 AACR.

Supplemental Table. Number (percent) of patients experiencing symptoms by week of therapy

INTRODUCTION AND OBJECTIVES:Despite antibiotic prophylaxis, we have noted an increase in patients presenting with infectious complications after urologic stone procedures despite negative urine cul...

Purpose: To determine the safety and toxicities of sequential MMC (mitomycin C) + BCG (bacillus Calmette–Guérin) in patients with non–muscle-invasive bladder cancer (NMIBC) and explore evidence for potentiation of BCG activity by MMC. Experimental Design: A 3 + 3 phase I dose-escalation trial of six weekly treatments was conducted in patients with NMIBC. MMC (10, 20, or 40 mg) was instilled intravesically for 30 minutes, followed by a 10-minute washout with gentle saline irrigation and then instillation of BCG (half or full strength) for 2 hours. Urine cytokines were monitored and compared with levels in a control cohort receiving BCG only. Murine experiments were carried out as described previously. Results: Twelve patients completed therapy, including 3 patients receiving full doses. The regimen was well tolerated with no treatment-related dose-limiting toxicities. Urinary frequency and urgency, and fatigue were common. Eleven (91.7%) patients were free of disease at a mean (range) follow-up of 21.4 (8.4–27.0) months. Median posttreatment urine concentrations of IL2, IL8, IL10, and TNFα increased over the 6-week treatment period. A greater increase in posttreatment urinary IL8 during the 6-week period was observed in patients receiving MMC + BCG compared with patients receiving BCG monotherapy. In mice, intravesical MMC + BCG skewed tumor-associated macrophages (TAM) toward a beneficial M1 phenotype. Conclusions: Instillation of sequential MMC + BCG is safe tolerable up to 40-mg MMC plus full-strength BCG. This approach could provide improved antitumor activity over BCG monotherapy by augmenting beneficial M1 TAMs. Clin Cancer Res; 21(2); 303–11. ©2014 AACR.

Objectives: With the increasing emphasis on evidence-based medicine, the urology literature has seen a rapid growth in the number of high-quality randomized controlled trials along with increased statistical rigor in the reporting of study results. P-values, CI, and number needed to treat (NNT) are becoming increasingly common in the literature. This paper seeks to familiarize the reader with statistical measures commonly used in the evidence-based literature. Materials and Methods: The meaning and appropriate interpretation of these statistical measures is reviewed through the use of a clinical scenario. Results: The reader will be better able to understand such statistical measures and apply them to the critical appraisal of the literature. Conclusions: P-values, CI, and NNT each provide a slightly different estimate of statistical truth. Together, they provide a more complete picture of the true effect observed in a study. An understanding of these measures is essential to the critical appraisal of study results in evidence-based medicine.

OBJECTIVE To examine the hypothesis that the distribution of nephrocalcinosis in patients with severe hypocitraturia should be symmetric. PATIENTS AND METHODS Patients with profound hypocitraturia defined as a 24-h urine citrate < 50 mg at the time of initial presentation were identified from the metabolic stone clinic database at our academic medical center. Two independent blinded reviewers evaluated all of the abdominal radiographs for the segmental distribution of macroscopic nephrocalcinosis. RESULTS A total of 44 patients met study criteria, with an equal distribution of males and females and a mean age of 55.4 ± 13.7 years. Mean urinary citrate was 28 ± 11 mg/day. Nephrocalcinosis was present in at least one renal segment in 22 patients (50%). Of the 22 patients with nephrocalcinosis, 9 patients (41%) had unilateral nephrocalcinosis and 13 patients (59%) had bilateral nephrocalcinosis. Of the 35 kidneys with nephrocalcinosis, 14 kidneys (40%) had nephrocalcinosis in only one renal segment, 13 kidneys (37%) had nephrocalcinosis in two segments and eight kidneys (23%) had nephrocalcinosis involving all three segments. CONCLUSIONS Despite the systemic nature of severe hypocitraturia, nephrocalcinosis is frequently asymmetric and focal in nature. This suggests that local factors intrinsic to the renal medullary interstitium, such as vascular injury, must play a role in the formation of nephrocalcinosis. Further study to elucidate these intrinsic local factors may further improve the treatment and prevention of urinary stone disease.

Urinary stone disease has historically been viewed as a disorder of mineral metabolism leading to precipitation of minerals out of the urine. Therefore, the medical management of urinary stones has focused on improving the urinary milieu. A growing body of evidence suggests that both cardiovascular risk factors and cardiovascular disease itself are associated with the development of urinary stones. Although disorders of mineral metabolism and cardiovascular diseases are both systemic conditions, patients frequently present with asymmetric renal calculus disease, suggesting that as yet uncharacterized local mechanisms must contribute to the formation of renal calculi. In this review, we examine the links between cardiovascular disease and nephrolithiasis, and discuss the evidence for potential local factors that contribute to stone disease.

Obstructive uropathy is a relatively common condition in which an anatomic or functional problem causes obstruction to normal urinary flow. Obstructive uropathy becomes more prevalent with increasing age and is most frequent as a result of benign prostatic hyperplasia or neurogenic bladder. The clinical manifestations of obstructive uropathy range from little or no symptoms to acute renal failure. Because its prevalence increases with increasing age, the diagnosis and management of obstructive uropathy is particularly relevant to the geriatric population.

OBJECTIVE To develop and apply a standardized evaluation form for assessing the methodological and reporting quality of observational studies of surgical interventions in urology. METHODS An evaluation standard was developed using the Consolidated Standards for Reporting Trials statement and previously reported surgical reporting quality instruments. Consensus scoring among three reviewers was developed using two distinct sets of studies. All comparative observational trials involving therapeutic surgical procedures published in four major urological journals in 1995 and 2005 were randomly assigned to each reviewer. Categories of reporting adequacy included background, intervention, statistical analysis, results and discussion. RESULTS Twenty-seven articles in 1995 and 62 in 2005 met the inclusion criteria; 90% of studies were retrospective. From 1995 to 2005, the overall reporting quality score increased by 3.9 points (95% confidence interval, CI, 2.7–5.9; P = 0.001), from a mean (sd) of 19.1 (3.9) to 23.0 (4.2) on a scale of 0–42. There were significant improvements in the reporting categories of study background (+0.7 points, 95% CI 0.1–1.3, P = 0.043, 0–8-point scale), intervention (+1.6 points, 0.8–2.3, P = 0.001, 0–9-point scale), and statistical analysis (+0.8 points, 0.2–1.4, P = 0.006, 0–9-point scale). There were smaller and statistically insignificant improvements for results (+0.5 points, −0.3 to 1.2, P = 0.217, 0–10-point scale) and discussion reporting (+0.4 points, −0.1 to 0.8, P = 0.106, 0–6-point scale). CONCLUSIONS There have been minor improvements in the reporting of observational studies of surgical intervention between 1995 and 2005. However, reporting quality remains suboptimal. Clinical investigators, reviewers and journal editors should continue to strive for transparent reporting of the observational studies representing the bulk of the clinical evidence for urological procedures.

We investigated the impact of nerve sparing technique on erectile function, urinary continence and health related quality of life after radical perineal prostatectomy using a validated self-assessment questionnaire.The Expanded Prostate Cancer Index Composite questionnaire was administered preoperatively and at defined intervals after surgery to 265 patients who underwent radical perineal prostatectomy at 2 institutions between January 2001 and December 2004. Of these patients 153 (57.7%) and 112 (42.3%) underwent nonnerve sparing and nerve sparing approaches, respectively. Kaplan-Meier analysis was used to determine time to recovery of erectile function (erections firm enough for intercourse) and urinary continence (0 pads per day).Median patient age was 60.6 years. Median followup was 15 months. In multivariate analysis preoperative erectile function (p = 0.005) and preservation of the neurovascular bundle (p = 0.018) were independent predictors of earlier recovery of erectile function, with hazard ratios of 2.3 (95% CI 1.2-4.6) and 4.0 (95% CI 1.5-10.3), respectively. Median time to recovery of urinary continence was 4.8 months in the nerve sparing group and 6.1 months in the nonnerve sparing group (p = 0.001). In multivariate analysis nerve sparing technique (p = 0.001, HR 1.4, 95% CI 1.1-1.9) and age (p = 0.012, HR 1.7, 95% CI 1.3-2.2) were independent predictors of recovery of continence.This analysis suggests that nerve sparing radical perineal prostatectomy is associated with improved recovery of urinary continence and favorable health related quality of life scores and, therefore, should be considered a viable alternative to other nerve sparing approaches.

We determined the potential influence of an early adopter bias in patients undergoing robot assisted laparoscopic prostatectomy.We compared baseline demographic, clinical and health related quality of life characteristics of patients undergoing 3 different surgical procedures for clinically localized prostate cancer following the introduction of robot assisted laparoscopic prostatectomy at our institution. Patients included in this analysis were participating in a prospective health related quality of life study using the SF-12(R) and Expanded Prostate Cancer Index Composite validated questionnaires.Of 402 patients 159 (39%) underwent robot assisted laparoscopic, 144 (36%) underwent radical perineal and 99 (25%) underwent radical retropubic prostatectomy. There were no statistically significant associations between procedure type and patient age (p = 0.267), race (p = 0.725), number of medical comorbidities (p = 0.490), income (p = 0.056) and level of education (p = 0.495). Mean prostate specific antigen was 5.9 +/- 3.3, 7.3 +/- 5.5 and 5.7 +/- 5.0 ng/ml for robot assisted laparoscopic, radical perineal and radical retropubic prostatectomy, respectively (p = 0.030). The proportion of robot assisted laparoscopic, radical perineal and radical retropubic prostatectomy patients with a final Gleason score of 4-6 was 55%, 45% and 39%, respectively (p = 0.037). The proportion of robot assisted laparoscopic, radical perineal and radical retropubic prostatectomy patients with stage T2 disease was 91%, 68% and 80%, respectively (p = 0.001). Statistically significant associations of higher income and education with higher baseline health related quality of life scores were seen in the sexual and physical domains (each p0.01).We failed to find evidence of an early adopter bias for patients undergoing robot assisted laparoscopic prostatectomy. Nevertheless, observational studies comparing robot assisted laparoscopic prostatectomy to radical perineal and radical retropubic prostatectomy should account carefully for patient baseline characteristics to allow meaningful comparisons of surgical outcomes.

Many abstracts presented at scientific meetings never come to full text publication, which is a prerequisite for the critical appraisal of a given study for its validity, impact and generalizability. We determined factors associated with the publication of abstracts presented at the American Urological Association national meeting.All abstracts addressing clinical research accepted for presentation at the 2002 and 2003 meetings of the American Urological Association were reviewed. A comprehensive MEDLINE search was performed for evidence of publication in full manuscript form. Data abstraction and literature searches were done between June 15 and August 30, 2005. Univariate and multivariate analyses were performed to determine the association between abstract characteristics and time to publication.Of the 1,683 abstracts reviewed 740 (44.0%) were published within a median followup of 27.8 months (range 25.9 to 39.7). Time to publication was associated with abstract origin in the United States and the reporting of statistical testing (HR 1.2, 95% CI 1.0-1.4, p=0.040 and HR 1.2, 95% CI 1.1-1.4, p=0.010, respectively). Other variables, such as presentation type, study design, clinical question type and negative outcome, were not predictive.Nonpublication of research findings is a problematic issue that affects more than half of studies 2 years after presentation at the American Urological Association national meeting. Abstracts from the United States and those providing statistical testing were more likely to be published in full text form. Further efforts are warranted to identify and eliminate factors that hinder publication of research to bring it to the scrutiny of a broad audience of urologists.

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.

A 65-year-old man with a history of combined pelvic external beam radiation therapy (EBRT) and brachytherapy for prostatic adenocarcinoma 15 years prior underwent total pelvic exenteration for presumed rectal sarcoma with prostatic invasion. Pathology revealed carcinosarcoma of prostatic origin. This patient exhibited the longest reported interval between initial presentation with prostatic adenocarcinoma and development of carcinosarcoma. This case is also the first reported case of prostatic carcinosarcoma occurring after combined EBRT and brachytherapy. The increasing use of such combination high-dose radiation therapy may potentially lead to an increased incidence of secondary malignancies such as prostatic carcinosarcoma in the future.

Increased fluid intake, and decreased dietary sodium and animal protein intake are thought to reduce the risk of kidney stones but the role of calcium intake is controversial. We evaluated the relationship between dietary factors and incident kidney stone formation.Secondary analysis was done of 78,293 women from the prospective WHI OS (Women's Health Initiative Observational Study) with no history of nephrolithiasis who completed the validated food frequency questionnaire. Multivariate logistic regression was used to determine demographic and dietary factors, and supplement use independently associated with incident kidney stones.Overall 1,952 women (2.5%) reported an incident kidney stone in 573,575 person-years of followup. The risk of incident kidney stones was decreased by 5% to 28% (p = 0.01) with higher dietary calcium intake and by 13% to 31% (p = 0.002) with higher water intake after adjusting for nephrolithiasis risk factors. Conversely higher dietary sodium intake increased the risk of nephrolithiasis by 11% to 61% (p0.001) after adjustment with the most pronounced effect in women with the highest intake. Higher body mass index independently increased the risk of incident nephrolithiasis (adjusted OR 1.19-2.01, p0.001). Animal protein intake was not associated with nephrolithiasis on multivariate analysis.This study adds to the growing evidence underscoring the importance of maintaining adequate fluid and dietary calcium intake. Greater dietary calcium intake significantly decreased the risk of incident kidney stones. In contrast, excess sodium intake increased the risk of incident nephrolithiasis, especially in women with the highest intake. Animal protein intake was not independently associated with nephrolithiasis.

The age of peak incidence for stone disease is 20 to 40 years, although stones are seen in all age groups. There is a male to female ratio of 3:2.We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of interventions for stone removal in people with asymptomatic kidney stones? What are the effects of interventions for the removal of symptomatic renal stones? What are the effects of interventions to remove symptomatic ureteric stones? What are the effects of interventions for the management of acute renal colic? We searched: Medline, Embase, The Cochrane Library, and other important databases up to June 2011 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).We found 21 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.In this systematic review, we present information relating to the effectiveness and safety of the following interventions: antispasmodic drugs, extracorporeal shockwave lithotripsy, intravenous fluids, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, oral fluids, percutaneous nephrolithotomy, and ureteroscopy.

Urinary stone disease is a condition with far-reaching implications. Patients with their initial presentation of acute renal colic generally enter the health care system through 2 routes. Severe cases are seen in the emergency room, whereas more tolerable cases may be seen by primary care physicians. Patients with urinary stone disease are then managed in the long-term by a urologist. This article reviews the epidemiology, pathogenesis, presentation, and short- and long-term management of acute and chronic urinary stone disease. EPIDEMIOLOGY In the United States, the 1988 to 1994 National Health and Nutrition Examination Survey (NHANES III) prospective cohort study found the lifetime prevalence of urinary stone disease to be 5.2%. 1 Stone disease seems to be on the rise, because the 1976 to 1980 NHANES II study recorded a lifetime prevalence of only 3.2%. In NHANES III, lifetime prevalence of stone disease was higher in men at 6.3% compared with 4.1% in women. The risk of stone disease generally seemed to increase as one aged, with the highest prevalence of stone disease in men occurring in the 70- to 74-year-old age group at 13.3%. Women experienced a peak prevalence in their sixth decade of 7.0%. In general, non-Hispanic Caucasian patients had the highest risk of urinary stone disease with a prevalence of 5.9%. Hispanic and African American patients had lower risks of stone disease with prevalences of 2.6% and 1.7%, respectively. Geography

OBJECTIVE To examine the hypothesis that the distribution of nephrocalcinosis in patients with severe hypocitraturia should be symmetric. PATIENTS AND METHODS Patients with profound hypocitraturia defined as a 24-h urine citrate50 mg at the time of initial presentation were identified from the metabolic stone clinic database at our academic medical center. Two independent blinded reviewers evaluated all of the abdominal radiographs for the segmental distribution of macroscopic nephrocalcinosis. RESULTS A total of 44 patients met study criteria, with an equal distribution of males and females and a mean age of 55.4 ± 13.7 years. Mean urinary citrate was 28 ± 11 mg/day. Nephrocalcinosis was present in at least one renal segment in 22 patients (50%). Of the 22 patients with nephrocalcinosis, 9 patients (41%) had unilateral nephrocalcinosis and 13 patients (59%) had bilateral nephrocalcinosis. Of the 35 kidneys with nephrocalcinosis, 14 kidneys (40%) had nephrocalcinosis in only one renal segment, 13 kidneys (37%) had nephrocalcinosis in two segments and eight kidneys (23%) had nephrocalcinosis involving all three segments. CONCLUSIONS Despite the systemic nature of severe hypocitraturia, nephrocalcinosis is frequently asymmetric and focal in nature. This suggests that local factors intrinsic to the renal medullary interstitium, such as vascular injury, must play a role in the formation of nephrocalcinosis. Further study to elucidate these intrinsic local factors may further improve the treatment and prevention of urinary stone disease.

This article introduces practicing urologists to the critical appraisal of systematic reviews and meta-analyses to guide their evidence-based clinical practice.Using a urological clinical case scenario we introduce a 3-step process in evaluating systematic reviews and meta-analyses by considering 1) the validity of the review results, 2) what the results are, and 3) the extent to which the results can and should be applied to patient care.A systematic review seeks to synthesize the medical literature about a specific clinical question using explicit methods to perform a comprehensive literature search, identify and select eligible studies, critically appraise their methods, and judiciously summarize the results considering how they vary with study characteristics. When this summary involves statistical methods, ie a meta-analysis, reviewers can offer a pooled estimate that will have greater precision and will apply more broadly than the individual studies. The quality of the underlying studies, the consistency of results across studies and the precision of the pooled estimate can considerably affect the strength of inference from systematic reviews.Valid systematic reviews of high quality studies can increase the confidence with which urologists and patients make evidence-based decisions. Thus, urologists need to recognize the inherent limitations, understand the results and apply them judiciously to patient care.

The aim of the study was to prospectively assess the role of apical soft tissue biopsies in radical perineal prostatectomy (RPP) patients with documented apical prostate cancer (PCA) involvement. Between June 1998 and May 1999, 77 consecutive men with localized PCA and documented invasion of the prostatic apex underwent RPP by a single surgeon. Soft tissue biopsies were systematically obtained from the prostatic fossa overlying the apex at the time of surgery. Time to biochemical failure was calculated using the Kaplan-Meier method. The rates of positive apical margins and positive apical soft tissue biopsies were 23.4% (18/77) and 15.6% (12/77). The sensitivity, specificity and positive predictive value of positive apical margins for residual apical disease as determined by apical soft tissue biopsy were 41.7, 80, and 28%, respectively. The overall biochemical failure rate was 28.6% (22/77) with a median follow-up of 51 months (range 3-73 months). The 36-month biochemical recurrence-free survival rate was 55.9+/-14.9% for patients with positive apical biopsies and 78.7+/-5.3% for those with negative biopsies (P=0.023). In conclusion, positive apical soft tissue biopsy is an independent predictor of biochemical failure in patients with apical PCA who undergo RPP. Positive apical surgical margins poorly predict residual apical disease that is frequently identifiable by apical soft tissue biopsy. Apical soft tissue biopsies should therefore be obtained in patients with known extensive apical cancer involvement at the time of RPP.

Objectives To prospectively assess the health-related quality-of-life outcomes of patients undergoing robot-assisted laparoscopic prostatectomy using a validated patient self-assessment questionnaire. Methods Patients undergoing robot-assisted laparoscopic prostatectomy between September 2003 and May 2005 were given the Expanded Prostate Cancer Index Composite questionnaire preoperatively and 1, 3, 6, 9, 12, and 18 months postoperatively. Patients with a minimum follow-up of 3 months were included in the analysis. The mean domain-specific health-related quality-of-life scores ± SD and the proportion of patients achieving their baseline scores were calculated. Multivariate proportional hazards regression analysis was used to determine the potential prognostic factors for a return to baseline of the domain scores and continence. Results The median follow-up was 9.5 months. The median time to recovery of the baseline summary scores was 6.6 months (95% confidence interval [CI] 5.9 to 7.2) in the urinary domain, 2.8 months (95% CI 2.0 to 3.7) in the bowel domain, and 3.0 months (95% CI 2.2 to 3.9) in the hormonal domain. The baseline sexual summary score was recovered by 19.2% of patients at 12 months. The median time to return of continence (0 to 1 pads/day) was 4.0 months (95% CI 3.0 to 4.9). The median time to the return of erections firm enough for intercourse was 13.5 months (95% CI 9.9 to 17.1). On multivariate proportional hazards regression analysis, age, body mass index, prostate size, nerve-sparing technique, and number of comorbidities were not significantly associated with the time to recovery of the baseline domain scores or continence. Conclusions Patients undergoing robot-assisted laparoscopic prostatectomy have a favorable health-related quality-of-life recovery profile that appears comparable to those of established surgical approaches.

10042 Background: RCC affects the immunsystem by inhibiting the process of differentiation of antigen-resenting cells from their myeloid precursors by secreting tumor-derived factors such as VEGF. ImC induce a profound state of immune suppression that foster tumor tolerance by inhibition of tumor specific T cells. In this study, we asked whether increased ImC frequencies can be found in peripheral blood from RCC patients and if ImC frequencies correlated positively with increased levels of tumor-derived serum markers. Methods: ImC frequencies from healthy volunteers and RCC patients were determined by FACS. ImC were isolated by magnetic bead separation techniques and their immunosuppressive activity was determined in IFN-γ ELISPOT analyses, CTL and T-cell proliferation assays. The production of reactive oxygen species by ImC was analyzed utilizing specific probes and inhibitors. Tumor-derived serum markers were quantified by ELISA, enzyme immune assays and cytometric bead arrays. Microarray analyses were performed to identify novel, highly specific ImC markers. Results: RCC patients demonstrate higher ImC frequencies (0.8 - 3.2% of total PBMC) compared to healthy donors. The increased ImC frequencies are positively correlated with serum levels of VEGF, PGE-2, IL-13 and M-CSF in RCC patients and the concentration of byproducts of oxidative burst, including iso-prostane and malondialdehyde, was significantly enhanced. Isolated ImC exhibited profound immunosuppressive effects on CTL and CD4+ T cell response in an antigen specific fashion. Immunosuppression by ImC was mediated by release of ROS, including peroxide and superoxide anions and by generation of nitric oxide radicals as evidenced in functional assays. Results from microarray analyses reveal EP-1, EphA5, and PEX-5 as novel ImC markers. Conclusions: These data suggest that RCC may induce the development of immunosuppressive ImC population through secretion of cytokines such as VEGF, IL-13, M-CSF and PGE-2. Inhibition of specific cytokine activity or the use of differentiating agents may represent strategies to decrease immunosuppressive ImC population. Results from a phase I clinical trial investigating the effects of all-trans retinoic acid (ATRA) on differentiation of ImC in RCC patients will be presented. No significant financial relationships to disclose.

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.