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ABSTRACT: OBJECTIVES:: Given the frequent inability to administer a conventional 3-weekly schedule of cisplatin to human subjects with urothelial carcinoma, we evaluated a frequent bolus metronomic schedule in a preclinical system of transitional cell carcinoma (TCC) of the urothelium. We hypothesized that the anti-angiogenic and anti-cell-migratory activity of lower concentrations of cisplatin may confer similar anti-tumor activity and demonstrate less nephrotoxicity than conventional cytotoxic concentrations. MATERIALS AND METHODS:: We evaluated the activity of cisplatin in vitro against human urothelial carcinoma (5637) and endothelial cells (human umbilical vein endothelial cells, HUVECs). The MTT assay was employed to assess viability, and flow cytometry with Annexin-FITC labeling was employed to assess apoptosis. Cell migration in monolayer culture was assessed by scratch assay. Murine xenografts (n = 12 per group) bearing measurable subcutaneous tumors of 5637 cells were administered either no therapy, cisplatin 2 mg/kg/3 days a week (metronomic) or 6 mg/kg/ once a week (standard) intraperitoneal (i.p.) for 4 weeks. Blood was collected from 5 mice per group at baseline and at the end of therapy to measure changes in serum creatinine. RESULTS:: Significant antiproliferative activity, but poor pro-apoptotic activity, was observed against 5637 urothelial carcinoma cells in vitro by MTT assay with cisplatin at concentrations lower than those attainable in vivo. Anti-proliferative activity against HUVECs required higher concentrations than attainable in vivo. Anti-migratory activity was observed against 5637 and HUVECs at earlier time points and with concentrations lower than anti-proliferative concentrations. In murine 5637 xenografts, standard cisplatin was significantly better at inhibiting tumor growth than the no treatment control (P = 0.005), while metronomic therapy was not better than control (P = 0.22). Standard cisplatin was also significantly nephrotoxic (P = 0.016), while metronomic cisplatin (P = 0.374) or no therapy (P = 0.178) did not demonstrate significant nephrotoxicity compared with baseline. CONCLUSIONS:: Cisplatin exhibited anti-cell-migratory activity against urothelial carcinoma and endothelial cells at lower than cytotoxic concentrations. However, a standard preclinical schedule was better than control in vivo for inhibiting tumor growth, while a metronomic schedule was not better. Despite the lower nephrotoxicity of the metronomic schedule, its lower anti-tumor activity suggests that a standard clinical schedule of cisplatin should not be routinely substituted by a split schedule without definitive clinical data.
Urologic Oncology 06/2011; · 3.22 Impact Factor
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ABSTRACT: Dasatinib is an orally administered multitargeted kinase inhibitor that targets Src family tyrosine kinases, Abl, c-Kit, and PDGFR. A preclinical study was conducted to evaluate dasatinib alone or combined with cisplatin for human transitional cell carcinoma (TCC). Expression of Src in a human TCC tissue microarray was evaluated by immunohistochemistry. The activity of dasatinib and/or cisplatin was evaluated in six human TCC cell lines. Western blot was done to assess Src and phosphorylated-Src (p-Src) expression. The activity of dasatinib alone and in combination with cisplatin was determined in murine subcutaneous xenografts. Sixty-two percent to 75% of human TCC expressed Src. Dasatinib displayed significant antiproliferative activity at nanomolar concentrations against two human TCC cell lines (RT4 and Hu456) that exhibited high Src and p-Src expression and were cisplatin-resistant. RT4 cells were the most sensitive and displayed the highest level of Src pathway activation (p-Src/Src ratio). Dasatinib downregulated p-Src in either sensitive or resistant cells. TCC cells that were sensitive to cisplatin (5637 and TCC-SUP) were highly resistant to dasatinib and exhibited low Src expression. Dasatinib showed antitumor activity in RT4 murine xenografts, and the combination of dasatinib and cisplatin was significantly more active than placebo. Combination dasatinib plus cisplatin significantly inhibited proliferation and promoted apoptosis in vivo. In conclusion, dasatinib displayed significant preclinical antitumor activity against Src-overexpressing human TCC with active Src signaling and was highly active in combination with cisplatin in vivo. Further clinical development might be warranted in selected human subjects.
Molecular Cancer Therapeutics 05/2010; 9(5):1128-35. · 5.23 Impact Factor
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ABSTRACT: Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through protein kinase C (PKC)-beta and the phosphatidylinositol 3-kinase/AKT pathways. We preclinically evaluated enzastaurin alone and in combination with gemcitabine for transitional cell cancer (TCC). Immunohistochemistry (IHC) was done on 105 human samples from a microarray to show the expression of PKC-beta. The preclinical antitumor activity of enzastaurin and gemcitabine as single agents and in combination against aggressive human -lines (-SUP and 5637) and murine subcutaneous xenografts bearing 5637 cells was determined. Western Blot was done on tumor cells in vitro to detect signaling through PKC-beta, GSK-3beta, and AKT. The effect on cell migration was determined in vitro. Modulation of proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (CD31) in vivo was determined by IHC. IHC done on human TCC samples from a microarray showed the expression of PKC-beta in 33% of tumors. Enzastaurin induced significant apoptosis and inhibited proliferation in vitro at low micromolar concentrations. The in vitro inhibitory activity of combination enzastaurin and gemcitabine by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay seemed synergistic. Western Blotting revealed down-regulation of Akt, PKC-beta, and GSK-3 beta phosphorylation. Enzastaurin inhibited migration at an earlier time point independent of antiproliferative activity. Combination therapy had significantly superior antitumor activity in murine xenografts compared with untreated controls, whereas single agents did not. IHC showed reduced Ki-67 and CD31 and increased cleaved caspase-3 with combination therapy compared with controls. Enzastaurin showed preclinical antitumor activity against human TCC and enhanced the activity of gemcitabine.
Molecular Cancer Therapeutics 07/2009; 8(7):1772-8. · 5.23 Impact Factor
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ABSTRACT: Sunitinib malate (Pfizer, Inc.) is a multitargeted kinase inhibitor that inhibits vascular endothelial growth factor (VEGF) receptor (R)-1, 2 and 3, platelet-derived growth factor receptors (PDGFR)-alpha and beta, Flt3, RET, and Kit. Angiogenesis and VEGF expression correlate with poor outcomes in human urothelial carcinoma. We designed a preclinical study to examine the efficacy of sunitinib alone and in combination with cisplatin against human urothelial carcinoma.
The in vitro activities of sunitinib and cisplatin alone and in combination were determined against human urothelial carcinoma cell lines, TCC-SUP and 5637. Antitumor activities were also determined in vivo against murine subcutaneous 5637 xenografts. Immunohistochemistry (IHC) was performed to detect VEGFR2 and Kit, and modulation of proliferation, apoptosis, and angiogenesis.
Both cell lines expressed VEGFR2, but did not express Kit. Sunitinib displayed activity against both cell lines in vitro at low micromolar concentrations, which are not attainable in vivo, and was synergistic with cisplatin. Activity was observed for sunitinib at 20 and 40 mg/kg orally once daily for 4 weeks, which attains low nanomolar concentrations in vivo against murine 5637 xenografts. Sunitinib 20 mg/kg/d in combination with cisplatin 4 mg/kg/wk intraperitoneally induced tumor regression compared to no therapy (P < 0.0001) or cisplatin alone (P = 0.06). Cisplatin, sunitinib, and combination treated tumors displayed significantly reduced ki-67 expression compared with control untreated tumors, and the difference was also statistically significant for the combination compared with cisplatin. Cleaved caspase-3 expression was significantly higher for sunitinib single agent and combination therapy compared with untreated controls, and for combination therapy compared with cisplatin alone. CD31 expression was diminished for both single agents and combination therapy compared with untreated tumors.
Sunitinib is preclinically active against urothelial carcinoma, and enhances the activity of cisplatin probably by targeting the stroma.
Urologic Oncology 07/2008; 27(4):391-9. · 3.22 Impact Factor
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ABSTRACT: To evaluate estrogen receptors as a therapeutic target for human bladder cancer.
The ability of the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene to inhibit 5637 human transitional cell carcinoma cell proliferation was determined in vitro and in xenograft studies using 5637 cells in female athymic BALB/c nu/nu mice.
Treatment with tamoxifen, raloxifene, or the pure antiestrogen ICI 182,780 inhibited proliferation of 5637 cells in vitro. In the first xenograft study, raloxifene (10, 100, or 1000 microg/day) administered by oral gavage inhibited the growth of tumors compared with placebo or untreated controls (P <0.05). In a second experiment, tamoxifen (8.3, 125, or 1250 microg/day) delivered by time-release pellet inhibited tumor growth compared with placebo-treated controls (P <0.01). A comparison study in which tamoxifen (8.3 or 125 microg/day) or raloxifene (100 microg/day) was administered by slow-release pellet demonstrated that both SERMs reduced growth compared to placebo-treated controls (P <0.05), with comparable effectiveness. There was no detectable tumor in 17 of 30 treated mice. In all studies, average tumor volumes in SERM-treated animals declined over the course of treatment.
Selective estrogen receptor modulators inhibit the growth of 5637 transitional cell carcinoma cell xenografts, supporting the rationale to evaluate these agents as targeted therapeutics for patients with urothelial carcinoma.
Urology 07/2007; 69(6):1221-6. · 2.43 Impact Factor
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ABSTRACT: Estrogen receptors (ERs) are known to mediate important physiologic responses as well as the growth of some tumors in response to estradiol stimulation. In a previous study the selective ER modulator raloxifene was shown to induce apoptosis in an ERbeta-positive bladder cancer cell line. However, the expression of ERbeta in human bladder cancer has not been thoroughly investigated.
ERalpha and ERbeta expression in 224 bladder tumor samples was evaluated using tissue microarray and immunohistochemistry. Levels of ERalpha and ERbeta protein and mRNA expression were determined in several bladder cancer cell lines using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of estradiol and antiestrogen treatments on RT4 bladder cancer cell growth was determined by cell proliferation assays.
Analyses revealed that only 2 human bladder cancers weakly expressed ERalpha. In contrast, the expression of ERbeta was detected in 141 tumors (63%). ERbeta was expressed in 58% of WHO Grade 1 and 2 tumors, whereas 70% of Grade 3 tumors demonstrated expression (P = .085). Importantly, although only 53% and 55% of Ta and T1 tumors demonstrated ERbeta expression, 80% of T2, 81% of T3, and 75% of T4 tumors showed ERbeta expression. The differences in ERbeta expression between Ta/T1 and T2/T3/T4 tumors were found to be highly significant (P < .001). Metastatic transitional cell carcinomas had ERbeta expression (80%) comparable to that of muscle invasive bladder cancers. Western blot analysis detected ERbeta protein expression in each of the 5 bladder cancer cell lines tested, whereas no or very low levels of ERalpha were found. Quantitative RT-PCR revealed that higher levels of ERbeta than ERalpha mRNA were present in 5637, T-24, TSU-Pr1, and TCC-Sup bladder cancer cells, whereas ER-alpha mRNA levels were greater than ERbeta in RT4 cells. Treatment with 17beta-estradiol modestly increased RT4 cell growth, whereas the antiestrogens, 4-hydroxtamoxifen, raloxifene, or ICI 182,780 inhibited the growth of RT4 cells.
ERbeta is the dominant receptor expressed in bladder cancer cell lines and in the majority of human bladder tumors. Moreover, the degree of ERbeta expression increases with increasing stage and grade of differentiation. Antiestrogens have an inhibitory effect on the growth of bladder cancer cells in vitro.
Cancer 07/2006; 106(12):2610-6. · 4.77 Impact Factor
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ABSTRACT: To test the hypothesis that elevated urinary levels of soluble E-cadherin (sE-cadherin) would aid in the detection of transitional cell carcinoma (TCC) of the urinary bladder.
We performed sE-cadherin staining of one murine (MBT2) and four human (RT4, 5637, T24, and TCCSUP) bladder cancer cell lines. sE-cadherin levels were also determined in voided urine of 188 consecutive subjects at risk for TCC recurrence, 31 patients with other uro-pathologic conditions, and 10 healthy subjects using a commercially-available ELISA kit. sE-cadherin was analyzed continuously and categorically on the basis of its median distribution.
Moderately and poorly differentiated bladder cancer cell lines had decreased cellular E-cadherin expression, whereas RT4, a well differentiated cell line, had preserved expression. All cell lines had measurable sE-cadherin levels in their conditioned media. The area under the ROC curve of sE-cadherin for the detection of TCC was 0.719 (95%CI, 0.637-0.801; p<0.001). Higher levels of sE-cadherin were associated with positive cytology results (p=0.012) and muscle invasive tumor stage (p=0.009). Urinary sE-cadherin was more sensitive, but less specific than urinary cytology for the detection of bladder TCC. In a multivariable logistic regression analysis, higher sE-cadherin and positive cytology were both associated with an increased risk of bladder TCC (p=0.048 and p<0.001, respectively). Combination of cytology and sE-cadherin allowed categorization of patients into three significantly different risk groups for bladder cancer. Adjustment of sE-cadherin for urinary creatinine levels did not affect any of the outcomes.
Urinary level of sE-cadherin may add information to cytology in the detection of bladder TCC.
European Urology 08/2005; 48(1):69-76. · 8.49 Impact Factor
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ABSTRACT: The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.
Cancer Research 04/2005; 65(5):1961-72. · 7.86 Impact Factor
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ABSTRACT: The serine-threonine kinase mammalian target of rapamycin has emerged as a potential target for cancer therapy. Rapamycin and rapamycin analogs are undergoing clinical trials and have induced clinical responses in a subgroup of patients. Rapamycin has also been reported to enhance the efficacy of several cytotoxic agents. The aim of this study was to determine the nature of the interactions between rapamycin and chemotherapeutic agents used as first- and second-line agents against breast cancer.
We performed a multiple drug effect/combination index isobologram analysis in cells sensitive and resistant to rapamycin alone in vitro, and we evaluated the in vivo efficacy of combination therapy in a rapamycin-sensitive model.
In vitro, synergistic interactions were observed in combinations with paclitaxel, carboplatin, and vinorelbine. Additive effects were observed in combinations with doxorubicin and gemcitabine. Rapamycin dramatically enhanced paclitaxel- and carboplatin-induced apoptosis. This effect was sequence dependent and mediated at least partly through caspase activation. Furthermore, rapamycin enhanced chemosensitivity to paclitaxel and carboplatin in HER2/neu-overexpressing cells, suggesting a potential approach to these poorly behaving tumors. Cell lines that are resistant to the growth-inhibitory effect of rapamycin were also resistant to rapamycin-mediated chemosensitization. In vivo, rapamycin combined with paclitaxel resulted in a significant reduction in tumor volume compared with either agent alone in rapamycin-sensitive tumors.
Rapamycin potentiates the cytotoxicity of selected chemotherapeutic agents in cell lines sensitive to the effects of rapamycin due to aberrations in the phosphatidylinositol 3'-kinase/Akt pathway, suggesting that combination therapy may be effective in patients selected for aberrations in this pathway.
Clinical Cancer Research 11/2004; 10(20):7031-42. · 7.74 Impact Factor
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ABSTRACT: Molecular Therapy (2004) 9, S297–S297; doi: 10.1016/j.ymthe.2004.06.875
785. Synergism of Combined Radiation and Suicide Gene Therapy Using a Chimeric Adenovirus Vector for Bladder Cancer
Kazumasa Matsumoto1, Christian T. F. Freund1, Weiguo Jian1, Jiang Yu1, Patricia Yotnda2, Alan R. Davis2 and Seth P. Lerner11Scott Department of Urology, Baylor College of Medicine, Houston, TX2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX
Molecular Therapy 04/2004; · 6.87 Impact Factor
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ABSTRACT: Rapamycin inhibits the serine-threonine kinase mammalian target of rapamycin (mTOR), blocking phosphorylation of p70 S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) and inhibiting protein translation and cell cycle progression. Rapamycin and its analogues are currently being tested in clinical trials as novel-targeted anticancer agents. Although rapamycin analogues show activity in clinical trials, only some of the treated patients respond. The purpose of this study is to identify determinants of rapamycin sensitivity that may assist the selection of appropriate patients for therapy.
Breast cancer cell lines representing a spectrum of aberrations in the mTOR signaling pathway were tested for rapamycin sensitivity. The expression and phosphorylation state of multiple components of the pathway were tested by Western blot analysis, in the presence and absence of rapamycin.
Cell proliferation was significantly inhibited in response to rapamycin in 12 of 15 breast cancer cell lines. The ratio of total protein levels of 4E-BP1 to its binding partner eukaryotic initiation factor 4E did not predict rapamycin sensitivity. In contrast, overexpression of S6K1, and phosphorylated Akt independent of phosphatase and tensin homologue deleted from chromosome 10 status, were associated with rapamycin sensitivity. Targeting S6K1 and Akt with small interfering RNA and dominant-negative constructs, respectively, decreased rapamycin sensitivity. Rapamycin inhibited the phosphorylation of S6K1, ribosomal S6 protein, and 4E-BP1 in rapamycin-resistant as well as -sensitive cells, indicating that its ability to inhibit the mTOR pathway is not sufficient to confer sensitivity to rapamycin. In contrast, rapamycin treatment was associated with decreased cyclin D1 levels in the rapamycin-sensitive cells but not in rapamycin-resistant cells.
Overexpression of S6K1 and expression of phosphorylated Akt should be evaluated as predictors of rapamycin sensitivity in breast cancer patients. Furthermore, changes in cyclin D1 levels provide a potential pharmacodynamic marker of response to rapamycin.
Clinical Cancer Research 03/2004; 10(3):1013-23. · 7.74 Impact Factor
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ABSTRACT: We investigated the relationship between cyclooxygenase-2 (COX-2) expression and molecular alterations commonly found in transitional cell carcinoma (TCC) of the bladder and determined whether COX-2 immunoreactivity is associated with cancer stage, progression and survival in patients undergoing radical cystectomy.
Immunohistochemical staining for COX-2 was done in archival tumor specimens from 80 patients who underwent radical cystectomy. Immunoreactivity was categorized as positive (reactivity in greater than 10% tumor cells) or negative. Microvessel density, E-cadherin, pRB, p16, p21, p53 and transforming growth factor (TGF)-beta1 and its receptors (types I and II) were also studied because evidence suggests a biological association between COX-2 and alteration of these molecules.
COX-2 was over expressed in 62 patients (78%). COX-2 over expression was associated with muscle invasive pathological stage (p = 0.022), TGF-beta1 over expression (p = 0.004), decreased E-cadherin expression (p < 0.001), and altered expression of pRB (p = 0.003) and p16 (p = 0.006). At a median followup of 101 months COX-2 over expression was associated with disease progression (p = 0.038) and bladder cancer specific survival (p = 0.042). However, when adjusted for the effects of standard pathological features, only lymph node metastasis was associated with bladder cancer progression (p = 0.027) and mortality (p = 0.042).
COX-2 is commonly expressed in patients with bladder TCC. Using the cutoff of 10% abnormal COX-2 expression is associated with the degree of invasiveness, alterations in TGF-beta1 and pRB/p16 pathways, and loss of cell adhesion. While COX-2 expression has limited prognostic value in patients with bladder TCC, it may serve as a target for therapy with selective COX-2 inhibitors.
The Journal of Urology 10/2003; 170(3):985-9. · 3.75 Impact Factor
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ABSTRACT: PurposeWe investigated the relationship between cyclooxygenase-2 (COX-2) expression and molecular alterations commonly found in transitional cell carcinoma (TCC) of the bladder and determined whether COX-2 immunoreactivity is associated with cancer stage, progression and survival in patients undergoing radical cystectomy.Materials and MethodsImmunohistochemical staining for COX-2 was done in archival tumor specimens from 80 patients who underwent radical cystectomy. Immunoreactivity was categorized as positive (reactivity in greater than 10% tumor cells) or negative. Microvessel density, E-cadherin, pRB, p16, p21, p53 and transforming growth factor (TGF)-β1 and its receptors (types I and II) were also studied because evidence suggests a biological association between COX-2 and alteration of these molecules.ResultsCOX-2 was over expressed in 62 patients (78%). COX-2 over expression was associated with muscle invasive pathological stage (p = 0.022), TGF-β1 over expression (p = 0.004), decreased E-cadherin expression (p < 0.001), and altered expression of pRB (p = 0.003) and p16 (p = 0.006). At a median followup of 101 months COX-2 over expression was associated with disease progression (p = 0.038) and bladder cancer specific survival (p = 0.042). However, when adjusted for the effects of standard pathological features, only lymph node metastasis was associated with bladder cancer progression (p = 0.027) and mortality (p = 0.042).ConclusionsCOX-2 is commonly expressed in patients with bladder TCC. Using the cutoff of 10% abnormal COX-2 expression is associated with the degree of invasiveness, alterations in TGF-β1 and pRB/p16 pathways, and loss of cell adhesion. While COX-2 expression has limited prognostic value in patients with bladder TCC, it may serve as a target for therapy with selective COX-2 inhibitors.
The Journal of Urology.
