Ronald B. Moore

Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model.

An animal tumour model that mimics the human counterpart is essential for preclinical evaluation of new treatment modalities. The objective of this study was to develop and characterize such a model. To accomplish this, the established AY-27 rat bladder transitional cell carcinoma (TCC) cell line was transplanted orthotopically into Fischer CDF344 female rats. AY-27 TCC cells were grown in monolayer cell culture and instilled intravesically as single cell suspensions into bladders that had been conditioned with mild acid washing. Tumour growth was assessed weekly by subjecting the rats to magnetic resonance imaging (MRI). At intervals following implantation and MRI tumour detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies. Flow cytometry was also performed for detection of Fas or Fas-ligand expression on AY-27 cells. The overall tumour establishment was 95% (97/102 rats) at 12–50 days, while in a subgroup of animals sacrificed at 16 days, 80 out of 82 animals (97%) developed TCC, the majority of which was superficial. Tumour stage was assessed by gross pathology and light microscopy. Histological examination of the tumour specimens confirmed the presence of grade II–III TCC. Immunocytochemistry confirmed that the tumour model maintained the features of TCC. The changes seen on MRI correlated well with the extent of tumour invasion identified histologically. Patchy carcinoma in situ could be detected histologically 12–13 days post-inoculation, and progressed to papillary tumour or invasive disease thereafter. Neither Fas nor Fas-ligand was expressed on AY-27 cells. The orthotopic AY-27 TCC model is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.

Bladder outlet obstruction: progression from inflammation to fibrosis

Study Type – Aetiology (case control)u2028Level of Evidenceu20033b

Associations Among Age, Comorbidity and Clinical Outcomes After Radical Cystectomy: Results From the Alberta Urology Institute Radical Cystectomy Database

PURPOSEnWe determined the associations among age, comorbidity and clinical outcomes after radical cystectomy.nnnMATERIALS AND METHODSnThe study was a retrospective cohort analysis of 314 consecutive patients with primary bladder cancer treated with radical cystectomy between January 2000 and December 2006 in Edmonton, Canada. Comorbidity was obtained through a medical record review using the Adult Comorbidity Evaluation-27 instrument. The main clinical outcomes were 90-day mortality, early postoperative complications, and major and minor early postoperative complications. Logistic regression analyses were used to determine predictors of clinical outcomes.nnnRESULTSnThe 90-day mortality, any early postoperative complications, and major and minor early postoperative complications occurred in 18 (5.7%), 148 (47.1%), 78 (24.8%) and 92 (29.3%) patients, respectively. In univariate and multivariate logistic regression analysis age was not associated with 90-day mortality or early postoperative complications. In contrast, compared to patients with no or mild comorbidity, multivariate logistic regression analysis adjusted for age and surgeon procedure volume showed that severe comorbidity was associated with an increased risk of 90-day mortality (OR 6.4, p = 0.03). In addition, compared to patients with no or mild comorbidity, multivariate logistic regression analysis adjusted for age, sex, surgeon procedure volume, type of urinary tract reconstruction and American Joint Committee on Cancer stage showed that moderate and severe comorbidity were associated with any early postoperative complications (moderate OR 5.2, p <0.001; severe OR 7.0, p <0.001), major early postoperative complications (moderate OR 11.4, p <0.001; severe OR 15.2, p <0.001) and minor early postoperative complications (moderate OR 2.1, p = 0.019; severe OR 2.2, p = 0.038).nnnCONCLUSIONSnIncreasing comorbidity was independently associated with an increased risk of 90-day mortality and early postoperative complications after radical cystectomy.

Adverse Renal Outcomes in Subjects Undergoing Nephrectomy for Renal Tumors: A Population-Based Analysis

BACKGROUNDnThere has been increasing interest in determining renal outcomes after nephrectomy for renal tumors. Previous studies have not assessed all relevant risk factors, including proteinuria.nnnOBJECTIVEnWe sought to determine the risk and predictors for the development of adverse renal outcomes in a population-based cohort of subjects undergoing partial or complete nephrectomy.nnnDESIGN, SETTING, AND PARTICIPANTSnA large population-based data set was used to identify all subjects undergoing nephrectomy in Alberta, Canada, from 2002 to 2007 using administrative codes. Comorbid conditions were determined using validated algorithms, and baseline estimated glomerular filtration rate (eGFR) and proteinuria status were determined.nnnMEASUREMENTSnPostsurgical outcomes of end-stage renal disease, acute dialysis, chronic kidney disease (CKD) (eGFR <30 ml/min per 1.73 m(2)), and rapidly progressive CKD (eGFR <60 ml/min per 1.73 m(2) and eGFR loss ≥4 ml/min per 1.73 m(2) per year) were assessed. The risk and risk factors for developing the composite renal outcome were determined using a multivariable Cox proportional hazards model.nnnRESULTS AND LIMITATIONSnOf 1151 subjects, 10.5% developed an adverse renal outcome over a mean of 32 mo. Complete (vs partial) nephrectomy was associated with a hazard ratio (HR) of 1.75 (95% confidence interval [CI], 1.02-2.99) for the primary outcome, as was lower baseline eGFR. Subjects with proteinuria were more likely to experience the primary outcome (42% vs 9%), conferring an adjusted HR of 2.40 (95% CI, 1.47-3.88).nnnCONCLUSIONSnClinically important adverse renal outcomes are common in patients undergoing nephrectomy for renal tumors. In addition to baseline eGFR and the extent of the renal mass removed, proteinuria is a strong independent risk factor. Assessment of proteinuria, in addition to other risk factors, should be performed to inform prognosis and the optimal treatment strategy.

Associations Between Comorbidity, and Overall Survival and Bladder Cancer Specific Survival After Radical Cystectomy: Results From the Alberta Urology Institute Radical Cystectomy Database

PURPOSEnWe determined the associations between comorbidity, and overall survival and bladder cancer specific survival after radical cystectomy.nnnMATERIALS AND METHODSnThe Alberta Urology Institute Radical Cystectomy database is an ongoing multi-institutional computerized database containing data on all adult patients with a diagnosis of primary bladder cancer treated with radical cystectomy in Edmonton, Canada from April 1994 forward. The current study is an analysis of consecutive database patients treated between April 1994 and September 2007. Comorbidity information was obtained through a medical record review using the Adult Comorbidity Evaluation 27 instrument. The outcome measures were overall survival and bladder cancer specific survival. Cox proportional regression analysis was used to determine the associations between comorbidity, and overall survival and bladder cancer specific survival.nnnRESULTSnOf the database patients 160 (34%), 225 (48%) and 83 (18%) had no/mild comorbidity, moderate comorbidity and severe comorbidity, respectively. Compared to patients with no or mild comorbidity, multivariate Cox proportional regression analyses that included age, adjuvant chemotherapy, surgeon procedure volume, pathological T stage, pathological lymph node status, total number of lymph nodes removed, surgical margin status and lymphovascular invasion showed that increased comorbidity was independently associated with overall survival (moderate HR 1.59, 95% CI 1.16-2.18, p = 0.004; severe HR 1.83, 95% CI 1.22-2.72, p = 0.003) and bladder cancer specific survival (moderate HR 1.50, 95% CI 1.04-2.15, p = 0.028; severe HR 1.65, 95% CI 1.04-2.62, p = 0.034).nnnCONCLUSIONSnIncreased comorbidity was independently associated with an increased risk of overall mortality and bladder cancer specific mortality after radical cystectomy.

Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis

Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 versus Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.

Metabolic Modulation of Clear-cell Renal Cell Carcinoma with Dichloroacetate, an Inhibitor of Pyruvate Dehydrogenase Kinase.

BACKGROUNDnClear-cell renal cell carcinoma (ccRCC) exhibits suppressed mitochondrial function and preferential use of glycolysis even in normoxia, promoting proliferation and suppressing apoptosis. ccRCC resistance to therapy is driven by constitutive hypoxia-inducible factor (HIF) expression due to genetic loss of von Hippel-Lindau factor. In addition to promoting angiogenesis, HIF suppresses mitochondrial function by inducing pyruvate dehydrogenase kinase (PDK), a gatekeeping enzyme for mitochondrial glucose oxidation.nnnOBJECTIVEnTo reverse mitochondrial suppression of ccRCC using the PDK inhibitor dichloroacetate (DCA).nnnDESIGN, SETTING, AND PARTICIPANTSnRadical nephrectomy specimens from patients with ccRCC were assessed for PDK expression. The 786-O ccRCC line and two animal models (chicken in ovo and murine xenografts) were used for mechanistic studies.nnnOUTCOME MEASUREMENTS AND STATISTICAL ANALYSISnMitochondrial function, proliferation, apoptosis, HIF transcriptional activity, angiogenesis, and tumor size were measured in vitro and in vivo. Independent-sample t-tests and analysis of variance were used for statistical analyses.nnnRESULTSnPDK was elevated in 786-O cells and in ccRCC compared to normal kidney tissue from the same patient. DCA reactivated mitochondrial function (increased respiration, Krebs cycle metabolites such as α-ketoglutarate [cofactor of factor inhibiting HIF], and mitochondrial reactive oxygen species), increased p53 activity and apoptosis, and decreased proliferation in 786-O cells. DCA reduced HIF transcriptional activity in an FIH-dependent manner, inhibiting angiogenesis in vitro. DCA reduced tumor size and angiogenesis in vivo in both animal models.nnnCONCLUSIONSnDCA can reverse the mitochondrial suppression of ccRCC and decrease HIF transcriptional activity, bypassing its constitutive expression. Its previous clinical use in humans makes it an attractive candidate for translation to ccRCC patients.nnnPATIENT SUMMARYnWe show that an energy-boosting drug decreases tumor growth and tumor blood vessels in animals carrying human kidney cancer cells. This generic drug has been used in patients for other conditions and thus could be tested in kidney cancer that remains incurable.

Disease progression and kidney function after partial vs. radical nephrectomy for T1 renal cancer

PURPOSEnPartial nephrectomy (PN) for early stage renal cancer preserves renal function better than radical nephrectomy (RN) and is generally considered oncologically similar. The Intergroup European Organisation for Research and Treatment of Cancer trial comparing outcomes after PN vs. RN, however, showed reduced overall survival in the PN group. Our aim was to evaluate recurrence, death, and renal function after PN vs. RN for T1 tumors in a Canadian population.nnnMATERIALS AND METHODSnFrom 2000 to 2015, 2,358 patients with a first occurrence of a clinical T1 renal cancer who underwent PN or RN were identified from the Canadian Kidney Cancer Information System. Clinical, surgical, and pathologic parameters were analyzed. Time to progression was compared after PN vs. RN using a Cox proportional hazards model, adjusted for pertinent variables.nnnRESULTSnInclusion criteria were met in 1,615 PN and 743 RN. Preoperative characteristics appeared similar in both groups. Time to progression was not different after PN vs. RN, adjusted for potential confounders (hazard ratio = 1.17 [95% CI: 0.8-1.72, P = 0.42]). Postoperative estimated glomerular filtration rate at 1 and 3 years was significantly greater for PN vs. RN in a linear regression model, accounting for preoperative estimated glomerular filtration rate.nnnCONCLUSIONSnThese results suggest that progression-free survival after PN and RN in patients with T1 renal cancer was similar, but that there was better preservation of renal function after PN. This suggests that both PN and RN have similar oncological efficiency, and that selection of surgical approach should be based on other factors such as technical feasibility, potential complications, and preservation of renal function.

The effect of photodynamic therapy on rat urinary bladder with orthotopic urothelial carcinoma

To assess the effect of whole‐bladder photodynamic therapy (PDT) on a rat model with orthotopic superficial bladder cancer, as PDT is an alternative intravesical therapy for treating superficial bladder cancer, based on an interaction between a photosensitizer and light energy to induce oxygen radicals that destroy tissue by lipid peroxidation.

A molecular complex of bovine milk protein and oleic acid selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic rat bladder tumour model.

Novel intravesical therapies are needed for superficial bladder cancer that reduce the risk of infection associated with Bacillus Calmette–Guérin (BCG) and further destabilization of the urothelium associated with cytotoxic chemotherapy. Experimental therapies to date have included photodynamic therapy, oncolytic viruses, gene therapy (antisense oligonucleotides and silencing RNA), cytokine therapy, death receptor agonists (tumour‐necrosis‐factor‐related apoptosis‐inducing ligand and anti‐DR5 monoclonal antibody), naturally occurring substances (curcumin and deguelin) and human α‐lactalbumin made lethal to tumour cells (HAMLET). HAMLET, a natural occurring product in milk, induces apoptosis in urothelial cancer cells but has limitations in clinical application because of its human source. A previous study in patients with bladder cancer has demonstrated that intravesical HAMLET (daily for 5 days before tumour resection) caused selective apoptotic tumour cell death. BAMLET, the bovine equivalent of HAMLET, is a complex of bovine α‐lactalbumin and oleic acid (bLAC) that has been shown in vitro to accumulate in the endolysosomal compartment of tumour cells and induce leakage of lysosomal cathepsins into the cytosol followed by activation of the pro‐apoptotic protein Bax. This is the first in vivo study to show that BAMLET (bLAC) induces apoptosis in urothelial cancer cells and controls the growth of high risk urothelial cancer in a syngeneic rat orthotopic model. This same bladder cancer model system has been used to test other novel therapies, including BCG, and therefore provides a relative comparison of its effectiveness with other intravesical therapies.