Matthew J. Knudson

Mycobacterium bovis Bacillus Calmette-Guérin-Induced Macrophage Cytotoxicity against Bladder Cancer Cells

Many details of the molecular and cellular mechanisms involved in Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer have been discovered in the past decades. However, information on a potential role for macrophage cytotoxicity as an effector mechanism is limited. Macrophages play pivotal roles in the host innate immunity and serve as a first line of defense in mycobacterial infection. In addition to their function as professional antigen-presenting cells, the tumoricidal activity of macrophages has also been studied with considerable interest. Studies have shown that activated macrophages are potent in killing malignant cells of various tissue origins. This review summarizes the current understanding of the BCG-induced macrophage cytotoxicity toward bladder cancer cells with an intention to inspire investigation on this important but underdeveloped research field.

Bladder Volume at Onset of Reflux on Initial Cystogram Predicts Spontaneous Resolution

PURPOSE Reflux grade is the factor most commonly used to predict spontaneous reflux resolution. We evaluated other potential predictive factors aside from reflux grade relative to spontaneous resolution. MATERIALS AND METHODS We reviewed the records of 20 males and 98 females who were diagnosed with primary vesicoureteral reflux between ages 0 and 7 years between 1990 and 2000. Age, sex, height, weight, reflux grade, bladder volume at onset of reflux and laterality were recorded for the first, second and most recent voiding cystourethrogram or nuclear cystogram before spontaneous resolution or operative intervention. RESULTS Of 118 patients 75 (64%) had spontaneous resolution, 27 (23%) underwent corrective surgery and 16 (13%) are still being followed. Average age at diagnosis was 2.3 years (range 1 day to 7.7 years) and average followup was 4.3 years (range 0.2 to 14). Average time to spontaneous resolution was 2.2 years (range 0.5 to 10.3) vs a time to operative treatment of 3.6 years (range 0.2 to 11.2). There was a significantly higher spontaneous resolution rate for lower reflux grades (p = 0.0004). Reflux occurring at greater than 75% of predicted bladder capacity had a significantly higher resolution rate (p = 0.0005). The initial height and weight percentile was not significant for predicting spontaneous resolution. Breakthrough urinary tract infections were negative predictors of spontaneous resolution (p <0.0001). CONCLUSIONS In addition to grade, bladder volume relative to predicted bladder capacity at the onset of reflux appears to provide additional prognostic information regarding the likelihood of spontaneous resolution of primary vesicoureteral reflux.

Abnormal Renal Scans and Decreased Early Resolution of Low Grade Vesicoureteral Reflux

PURPOSE Limited studies suggest a relationship between scarring on renal scan and failure to resolve vesicoureteral reflux. We evaluated the impact of abnormal renal scans on early vesicoureteral reflux resolution. MATERIALS AND METHODS The medical records and renal scans were reviewed of children diagnosed with primary reflux between 1988 and 2004. We defined an abnormal renal scan as renal scarring or relative renal function 40% or less. Reflux resolution was noted 1 and 2 years after diagnosis. RESULTS Renal scan data were available on 161 children with vesicoureteral reflux, including 127 girls and 34 boys. Relative renal function was 15% or less in 7 children, 16% to 35% in 14, 36% to 40% in 18 and greater than 40% in 122. Of the 161 patients 79 (43%) had an abnormal renal scan, including 37% with grades 1 to 3 reflux. The rate of 2-year reflux resolution in the abnormal and normal renal scan groups was 13% vs 53%. Of children with grades II and III reflux those with an abnormal renal scan were less likely to have reflux resolution compared to those with normal renal scans (23% vs 55% and 4% vs 41, respectively, p <0.05). The same relationship was present at 1 year for grades 2 and 3 (18% vs 49% and 4% vs 30, respectively, p <0.05). CONCLUSIONS Abnormal renal scans are an important independent predictor of early failure to resolve vesicoureteral reflux. An abnormal renal scan should be considered when counseling families about the likelihood of early reflux resolution. Performing a renal scan may be indicated in select patients.

Anti-Interleukin-10R1 Monoclonal Antibody Enhances Bacillus Calmette-Guérin Induced T-Helper Type 1 Immune Responses and Antitumor Immunity in a Mouse Orthotopic Model of Bladder Cancer

PURPOSE Proper induction of the T-helper type 1 immune response is required for effective bacillus Calmette-Guérin immunotherapy for bladder cancer. Interleukin-10 down-regulates the T-helper 1 response and is associated with bacillus Calmette-Guérin failure. We investigated whether blocking interleukin-10 receptor 1 would enhance the bacillus Calmette-Guérin induced T-helper type 1 immune response and anti-bladder cancer immunity in a mouse model. MATERIALS AND METHODS Splenocytes were incubated with bacillus Calmette-Guérin or bacillus Calmette-Guérin plus control IgG1, anti-interleukin-10 receptor 1 mAb or anti-interleukin-10 neutralizing mAb, followed by enzyme-linked immunosorbent assay of interferon-γ production. Bladder RNA was extracted after intravesical bacillus Calmette-Guérin plus intraperitoneal IgG1 or anti-interleukin-10 receptor 1 mAb and analyzed by reverse transcriptase and/or quantitative polymerase chain reaction. Urine was collected and analyzed by enzyme-linked immunosorbent assay. Mice bearing a luciferase expressing MB49 orthotopic tumor were treated with intravesical bacillus Calmette-Guérin plus intraperitoneal IgG1 or anti-interleukin-10 receptor 1 mAb. Tumor response was assessed by bioluminescent imaging and bladder weight measurement. RESULTS Bacillus Calmette-Guérin plus anti-interleukin-10R1 mAb induced significantly higher interferon-γ production by splenocytes than bacillus Calmette-Guérin plus anti-interleukin-10 mAb. Bacillus Calmette-Guérin plus anti-interleukin-10 receptor 1 mAb also induced significantly higher interferon-γ mRNA and protein in bladder and urine, respectively, in a dose dependent manner. Treatment with phosphate buffered saline, bacillus Calmette-Guérin plus control IgG1 and bacillus Calmette-Guérin plus anti-interleukin-10 receptor 1 mAb showed a 0% tumor-free rate with a 20% death rate, a 20% tumor-free rate with a 20% death rate and a 40% tumor-free rate with a 0% death rate, respectively. Bladder weight also revealed the effect of anti-interleukin-10 receptor 1 mAb on the bacillus Calmette-Guérin induced bladder tumor response. CONCLUSIONS Anti-interleukin-10 receptor 1 mAb enhanced the bacillus Calmette-Guérin induced T-helper type 1 immune response and anti-bladder cancer immunity. A humanized form of this mAb warrants future investigation for bacillus Calmette-Guérin treatment of bladder cancer.

Adding Renal Scan Data Improves the Accuracy of a Computational Model to Predict Vesicoureteral Reflux Resolution

PURPOSE We previously developed a computational model to predict vesicoureteral reflux resolution 1 and 2 years after diagnosis. Previous studies suggest that an abnormal renal scan may be a predictor of the failure of vesicoureteral reflux to resolve. We investigated whether the addition of renal scan data would improve the accuracy of our computational model. MATERIALS AND METHODS Medical records and renal scans were reviewed on 161 children, including 127 girls and 34 boys, with primary reflux between 1988 and 2004. In addition to the 9 input variables from our prior model, we added renal scan data on decreased relative renal function (40% or less in the refluxing kidney) and renal scars. Resolution outcome was evaluated 1 and 2 years after diagnosis. Data sets were prepared for 1 and 2-year outcomes, and randomized into a modeling set of 111 and a cross-validation set of 50. The model was constructed using neUROn++. RESULTS A logistic regression model had the best fit with an ROC area of 0.945 for predicting reflux resolution in the 2-year model. This was improved compared to our previous model without renal scan data. A prognostic calculator using this model can be deployed for availability on the Internet, allowing input variables to be entered and calculating the odds of resolution. CONCLUSIONS This computational model uses multiple variables, including renal scan data, to improve individualized prediction of early reflux resolution with almost 95% accuracy. The prognostic calculator is a useful tool for predicting individualized vesicoureteral reflux resolution.

Distal ureteral diameter measurement objectively predicts vesicoureteral reflux outcome

OBJECTIVE Vesicoureteral reflux (VUR) grading may be difficult when discrepancies exist between the degree of dilation of the pyelocalyceal system and the ureter. Resolution may be more accurately predicted by the appearance of the distal ureter. We analyzed a novel, objective method of evaluating VUR based on the diameter of the distal ureter. METHODS Seventy-nine voiding cystourethrograms were reviewed (18 boys; 61 girls; aged 1 month to 7.5 years). The largest ureteral diameter within the false pelvis was measured and normalized by dividing by the distance from the L1-L3 vertebral body to give the distal ureteral diameter: L1-L3 ratio (UDR). Clinical outcome was defined as spontaneous resolution or surgical correction. RESULTS A significant association between grade and UDR existed (p < 0.0001). Mean UDR was significantly greater in those who underwent surgical correction (0.34 ± 0.02 vs 0.18 ± 0.02; p < 0.0001). Logistic regression analysis demonstrated a significant association of UDR with outcome controlling for grade (p = 0.001). Grade effect on outcome when controlling for UDR was not significant (p = 0.76). Odds ratio for surgical correction corresponding to a 0.1 increase in UDR equaled 2.25 (95% CI: 1.39, 3.64). CONCLUSION UDR provides an objective measurement of VUR and appears more predictive of clinical outcome than grade in this series.

877 ANTI-IL10-R1 MONOCLONAL ANTIBODY ENHANCES BACILLUS CALMETTE-GUERIN (BCG) INDUCED TH1 AND ANTI-BLADDER CANCER IMMUNE RESPONSES IN VITRO AND IN VIVO

nodal stage, they are associated with inferior disease outcomes. Detection of even 1 CTC/7.5 mL blood in UCB patients prior to RC is an independent predictor for disease recurrence and cancer-related death. These findings are very important for future investigations, as they are in contradiction to theories supporting a cut-off value of 5 or more CTC needed for accurate outcome prediction. Therefore, CTC may represent a feasible biomarker for monitoring response to neoadjuvant and adjuvant chemotherapy.

Abstract 5390: Anti-IL10-R1 monoclonal antibody with concomitant bacillus calmette-Guérin (BCG) prevents metastatic bladder cancer in an in vivo mouse model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Induction of Th1 immunity is required for effective intravesical BCG immunotherapy of bladder cancer. IL-10 down-regulates the Th1 response and is associated with BCG failure. We previously demonstrated that blocking IL-10 receptor (IL-10R) by systemic administration of anti-IL-10R1 mAb enhanced intravesical BCG (living Pasteur strain) treatment of bladder cancer in an orthotopic mouse model. Here we investigated the effect of anti-IL-10R1 mAb on clinically used BCG (lyophilized TICE strain) for induction of anti-bladder cancer immunity. Methods: Splenocytes were incubated with BCG alone or plus control IgG or ant-IL-10R1 mAb for 24 hours, followed by ELISA analysis of IFN-γ production. Bladder RNA was extracted after 6 treatments (twice weekly) with intravesical (i.b.) BCG plus intraperitoneal (i.p.) control IgG or anti-IL-10R1 mAb, followed by qPCR analysis of IFN-γ mRNA expression. Three groups of 20 mice were inoculated with luciferase-expressing MB49 bladder cancer cells and treated with i.b. PBS plus i.p. PBS (Group 1), i.b. BCG plus i.p. control IgG (Group 2), or i.b. BCG plus i.p. anti-IL-10R1 mAb (Group 3) 2x/wk for 6 total treatments. Mice were monitored weekly using IVIS luminescence and followed for 76 days. A bioluminesence cutoff of 5X104 p/s was used to establish the presence of bladder cancer. At animal death bladders were collected, weighed and processed for histological analysis. In suspected cases of metastatic disease the affected organs were also collected for histological analysis. Results: BCG plus anti-IL-10R1 mAb induced increased IFN-γ production by splenocytes in a dose-dependent manner. BCG plus anti-IL-10R1 mAb substantially increased IFN-γ mRNA. Eleven, 17 and 9 mice from Groups 1, 2 and 3, respectively, developed bladder cancer. One mouse in Group 2 and 2 mice in Group 3 showed cancer regression. 36%, 53% and 0% of Groups 1, 2 and 3 developed metastatic bladder cancer, respectively (p=0.020). While bladder weights for the groups were different, the differences did not reach statistical significance. Conclusions: Anti-IL-10R1 mAb enhances BCG-induced Th1 immune responses both in vitro and in vivo. Intravesical BCG plus anti-IL-10R1 mAb shows statistical significance in preventing metastasis of bladder cancer in an orthotopic bladder tumor mouse model. Anti-IL-10R1 mAb may prove useful in clinical practice for the prevention of metastatic bladder cancer in high-risk patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5390. doi:1538-7445.AM2012-5390

492 RECOMBINANT B7-DCIG ENHANCES BACILLUS CALMETTE-GURIN (BCG)-INDUCED TH1 AND ANTI-BLADDER CANCER IMMUNE RESPONSES IN VITRO AND IN VIVO

INTRODUCTION AND OBJECTIVES: B7-DCIg, AMP-224, is a recombinant Fc fusion protein composed of the extracellular domain of B7-DC fused to the hinge and Fc domain of IgG. B7-DCIg interacts with PD-1, reduces the dysfunctional CD8 T cells, and promotes an effective antitumor response. In vitro M. tuberculosis stimulation of human PBMC upregulates PD-1 expression in T cells. We proposed to test if B7-DCIg could enhance BCG-induced anti-bladder cancer immune responses. METHODS: For in vitro evaluation of B7-DCIg on BCG induction of immune responses, mouse splenocytes were prepared and cultured in the absence or presence of Pasteur strain BCG and/or different doses of B7-DCIg for 3 days, followed by ELISA analysis of interferon gamma (IFN) in culture supernatants. For in vivo evaluation of B7-DCIg on BCG induction of immune responses, mice were treated twice weekly with intravesical (i.b.) PBS plus intraperitoneal (i.p.) PBS, i.b. BCG (0.1 OD/dose) plus i.p. IgG2a (400 g/dose), or i.b. BCG (0.1 OD/dose) plus i.p. B7-DCIg (400 g/dose). The bladders were collected after treatments 2, 4 and 6, and analyzed for IFNas well as other mRNAs by RT-PCR. ImageJ software was used to quantify PCR products on agarose gels. To evaluate the effect of B7-DCIg on BCG treatment of bladder cancer, C57BL/6 mice were implanted i.b. with luciferase-expressing MB49 (MB49-Luc) bladder cancer cells at day 0 and treated with i.b. BCG (0.1 OD/dose) plus i.p. B7-DCIg (400 g/dose) twice weekly starting at day 1 for a total of 6 treatments. Control mice were treated with i.b. PBS plus i.p. PBS or i.b. BCG plus i.p. IgG2a. Bioluminescence was measured with Xenogen IVIS imaging at days 7, 14, and 21. At day 23 mice were euthanized and bladder weights measured. RESULTS: B7-DCIg enhanced BCG-induced splenocyte IFNproduction in a dose-dependent manner in vitro. B7-DCIg also enhanced BCG-induced bladder expression of IFNand tumor necrosis factor related apoptosis-inducing ligand (TRAIL) mRNAs in vivo. In the orthotopic tumor model, BCG treatment reduced bladder weight by 53% (p 0.0503 vs. PBS-treated group). Combination of BCG with B7-DCIg further reduced bladder weight by 5% (p 0.0257 vs. PBStreated group). In addition, the combination treatment also led to a delay in tumor growth by bioluminescence compared to BCG-treated group. CONCLUSIONS: B7-DCIg enhanced BCG-induced Th1 and anti-bladder cancer immune responses in mice. B7-DCIg may serve as a potential agent in combination with BCG for the treatment of bladder cancer in humans.

Abstract 1782: Recombinant B7-DCIg enhances Bacillus Calmette-Guérin (BCG)-induced Th1 and anti-bladder cancer immune responses in vitro and in vivo

Introduction and Objective: B7-DCIg, AMP-224, is a recombinant Fc fusion protein composed of the extracellular domain of B7-DC fused to the hinge and Fc domain of IgG. B7-DCIg interacts with PD-1, reduces the dysfunctional CD8 + T cells, and promotes an effective antitumor response. In vitro M. tuberculosis stimulation of human PBMC upregulates PD-1 expression in T cells. We proposed to test if B7-DCIg could enhance BCG-induced anti-bladder cancer immune responses. Methods: For in vitro evaluation of B7-DCIg on BCG induction of immune responses, mouse splenocytes were prepared and cultured in the absence or presence of Pasteur strain BCG and/or different doses of B7-DCIg for 3 days, followed by ELISA analysis of interferon gamma (IFN-γ) in culture supernatants. For in vivo evaluation of B7-DCIg on BCG induction of immune responses, mice were treated twice weekly with intravesical (i.b.) PBS plus intraperitoneal (i.p.) PBS, i.b. BCG (0.1 OD/dose) plus i.p. IgG2a (400 μg/dose), or i.b. BCG (0.1 OD/dose) plus i.p. B7-DCIg (400 μg/dose). The bladders were collected after treatments 2, 4 and 6, and analyzed for IFN- γ as well as other mRNAs by RT-PCR. ImageJ software was used to quantify PCR products on agarose gels. To evaluate the effect of B7-DCIg on BCG treatment of bladder cancer, C57BL/6 mice were implanted i.b. with luciferase-expressing MB49 (MB49-Luc) bladder cancer cells at day 0 and treated with i.b. BCG (0.1 OD/dose) plus i.p. B7-DCIg (400 μg/dose) twice weekly starting at day 1 for a total of 6 treatments. Control mice were treated with i.b. PBS plus i.p. PBS or i.b. BCG plus i.p. IgG2a. Bioluminescence was measured with Xenogen IVIS imaging at days 7, 14, and 21. At day 23 mice were euthanized and bladder weights measured. Results: B7-DCIg enhanced BCG-induced splenocyte IFN- γ production in a dose-dependent manner in vitro. B7-DCIg also enhanced BCG-induced bladder expression of IFN- γ and tumor necrosis factor related apoptosis-inducing ligand (TRAIL) mRNAs in vivo. In the orthotopic tumor model, BCG treatment reduced bladder weight by 53% (p = 0.0503 vs. PBS-treated group). Combination of BCG with B7-DCIg further reduced bladder weight by 5% (p = 0.0257 vs. PBS-treated group). In addition, the combination treatment also led to a delay in tumor growth by bioluminescence compared to BCG-treated group. Conclusions: B7-DCIg enhanced BCG-induced Th1 and anti-bladder cancer immune responses in mice. B7-DCIg may serve as a potential agent in combination with BCG for the treatment of bladder cancer in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2011-1782