Eric J. Askeland

Bladder Cancer Immunotherapy: BCG and Beyond

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has become the predominant conservative treatment for nonmuscle invasive bladder cancer. Its mechanism of action continues to be defined but has been shown to involve a T helper type 1 (Th1) immunomodulatory response. While BCG treatment is the current standard of care, a significant proportion of patients fails or do not tolerate treatment. Therefore, many efforts have been made to identify other intravesical and immunomodulating therapeutics to use alone or in conjunction with BCG. This paper reviews the progress of basic science and clinical experience with several immunotherapeutic agents including IFN-α, IL-2, IL-12, and IL-10.

Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette–Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity

Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)‐10 down‐regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL‐10 signalling could enhance the BCG‐induced Th1 response and anti‐tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti‐IL‐10 receptor 1 monoclonal antibody (anti‐IL‐10R1 mAb) increased the interferon (IFN)‐γ to IL‐10 ratio in both splenocyte cultures and urine. Mice bearing luciferase‐expressing MB49 (MB49‐Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate‐buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti‐IL‐10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K‐ras oncogene) known to be expressed in MB49‐Luc cells. Induction of ras mutation‐specific IFN‐γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti‐IL‐10R1 mAb, induces enhanced anti‐tumour immunity that is protective against lung metastasis. Anti‐IL‐10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high‐risk patients.

Urological Manifestations of Duchenne Muscular Dystrophy

PURPOSE Duchenne muscular dystrophy is a dystrophinopathy affecting males that is associated with multiple organ system complications. To our knowledge urological complications of Duchenne muscular dystrophy have been described only anecdotally to date. MATERIALS AND METHODS We reviewed the medical charts of 135 patients with Duchenne or Duchenne-Becker muscular dystrophy for demographics and disease progression, urological diagnoses, intervention and followup. RESULTS Of 135 patients 67 (50%) had at least 1 documented urological diagnosis and 38 (28%) had multiple manifestations. Lower urinary tract symptoms were the most common urological diagnosis (32% of patients). Survival analysis revealed a median age at onset of lower urinary tract symptoms of 23 years (95% CI 17.7-23.9). Intervention was required in 12 patients (9%), most commonly due to nephrolithiasis. Urological morbidity increased with Duchenne muscular dystrophy progression when stratified by clinical progression. Lower urinary tract symptoms were more common in nonambulatory patients (40.7% vs 19%, p = 0.007), those with a diagnosis of scoliosis (44% vs 19.7%, p = 0.003) and/or scoliosis spine surgery (60% vs 22%, p <0.001), and those on invasive respiratory support (53% vs 29%, p = 0.046). Likewise, nephrolithiasis was more common in nonambulatory patients (10% vs 0%, p = 0.017), those with scoliosis (12% vs 0%, p = 0.004) and/or scoliosis spine surgery (20% vs 1%, p <0.001), and those on invasive respiratory support (29% vs 3%, p <0.001). Only 28% of patients with a urological manifestation were referred to urology. CONCLUSIONS As these patients transition into adolescence and adulthood, the increased prevalence of urological manifestations warrants increased awareness and referral to urologists.

Immunotherapy of Urinary Bladder Carcinoma: BCG and Beyond

Urothelial carcinoma of the bladder is the second most common urologic neoplasm after prostate carcinoma in the United States, with an estimated 70,510 new cases and 14,880 deaths in 2012 [1]. Global prevalence of bladder cancer is estimated at >1 million and is steadily increasing. This disease places enormous economic burden on the U.S. health care system due to its requirements of surgical resection, repeated intravesical therapies, and lifelong medical follow-up. Urothelial carcinoma accounts for 90% of bladder tumors. At the time of diagnosis, 20-25% of cases are muscle invasive (stage T2 or higher) and are typically treated with surgical resection (radical cystectomy) [2]. The remainders are confined to layers above the muscularis propria – so-called nonmuscle invasive bladder cancer (NMIBC). These cancers (also termed “superficial bladder cancer“) include tumors confined to the urothelium (Ta), tumors invading the lamina propria (T1), and carcinoma in situ (Tis, a flat erythematous lesion), occurring in 70%, 20% and 10% of NMIBC cases, respectively [2]. Transurethral resection of bladder tumor (TURBT) is the standard primary treatment for Ta and T1 lesions; however, recurrence rates for TURBT alone can be as high as 70% with up to 30% progressing to muscle invasive disease requiring cystectomy [3]. The high rates of recurrence and significant risk of progression in higher grade tumors mandate additional therapy with intravesical agents. While limiting the systemic exposure, intravesical therapy allows the destruction of residual microscopic tumor and circulating tumor cells after TURBT by exposure to therapeutic agents, thereby preventing reimplantation. To date, intravesical therapy has been used as an adjuvant treatment after TURBT to prevent recurrence and progression of the disese and is also the treatment of choice for Tis that is not feasible for TURBT.

Cell cycle progression score predicts metastatic progression of clear cell renal cell carcinoma after resection.

BACKGROUND The outcome of surgically resected, apparently localized, clear cell renal carcinoma (ccRCC) is uncertain. OBJECTIVE To evaluate if cell cycle progression (CCP) gene expression can predict future metastasis. METHODS Pathologic T2a-T3b tumors at University of Iowa were reviewed. Patients with known or suspected metastasis, lymph node involvement or who received neoadjuvant or adjuvant radiation, chemotherapy or immunotherapy were excluded. Case and control cohorts were defined as those who did or did not develop metastatic disease within 5 years. Measured levels of 31 cell cycle genes and 15 control genes from the tumor were calculated as a CCP score. Additionally, gene expression data for a separate ccRCC cohort was downloaded from The Cancer Genome Atlas (TCGA). RESULTS Univariate analysis of 26 cases and 38 controls revealed that the CCP score predicted progression to metastasis (OR 2.65, p = 0.0091). In multivariate logistic regression modeling, CCP expression remained a significant independent predictor for progression (p = 0.026). The CCP score was also significantly associated with distant metastasis in the TCGA renal cancer cohort in both univariate (p = 1.0 × 10-9) and multivariate (p = 5.6 × 10-3) analysis. CONCLUSION The CCP score has prognostic value in predicting metastatic progression after resection of organ-confined ccRCC.

Abstract 4742: BCG in combination with anti-IL-10R1 monoclonal antibody induces specific antitumor immunity in an orthotopic bladder cancer model.

Introduction and Objective: We previously demonstrated that combination therapy with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) effectively prevented bladder cancer metastasis in a murine MB49 orthotopic tumor model. MB49 cells express the male minor histocompatibility (HY) antigen that, although is immunogenic, induces no tumor rejection in female mice. Here we investigated whether the combination therapy could enhance HY-specific immune responses and whether this HY-specific immunity contributed to the prevention of bladder cancer metastasis in this model. Methods: Four groups of female mice (n = 5) were inoculated intravesically (i.b.) with 1 X 106 luciferase–expressing MB49 (MB49-Luc) cells and treated intraperitoneally (i.p.) with PBS, control IgG (200μg), or anti-IL-10R1 mAb (200μg) at day 0. Starting at day 1, mice were treated twice weekly with i.b. PBS plus i.p. PBS (Group 1), i.b. PBS plus i.p. anti-IL-10R1 mAb (Group 2), i.b. BCG plus i.p. control IgG (Group 3), or i.b. BCG plus i.p. anti-IL-10R1 mAb (Group 4) for a total of 6 treatments. Tumor growth was monitored using the IVIS luminescence system. Mice were sacrificed for analysis at day 21. Splenocytes were prepared, pooled and cultured in the presence of a HY antigen peptide or a control ovalbumin (OVA) peptide. After 3 days culture supernatants were collected and analyzed for IFN-γ production by ELISA. After 5 days splenocytes were used as effector cells in a cytotoxicity assay. Results: Tumor implantation was confirmed for all groups. Splenocytes produced no IFNγ in response to OVA peptide stimulation. Splenocytes from Groups 1 and 3 (treated with no anti-IL-10R1 mAb) produced no IFN-γ in response to HY peptide stimulation. In contrast, splenocytes from Groups 2 and 4 (treated with anti-IL-10R1 mAb) produced significantly increased IFN-γ in a dose-dependent manner in response to HY peptide stimulation, with Group 4 (combination therapy) splenocytes being more potent. Accordingly, Group 4 splenocytes exhibited profound cytotoxicity against MB49-Luc cells with 69.8%, 51.2% and 25.6% killing at an E:T ratio of 50:1, 25:1 and 12.5:1, respectively. However, splenocytes from Group 2 (anti-IL-10R1 mAb monotherapy) showed marginal killing of MB49-Luc cells. Conclusions: BCG in combination with anti-IL-10R1 mAb induced HY-specific effector cells cytotoxic to MB49-Luc bladder cancer cells. Anti-IL-10R1 mAb may prove useful in clinical practice for the prevention of metastatic bladder cancer in high-risk patients. Citation Format: Eric J. Askeland, Mark R. Newton, Xu Wang, Xiaohong Chen, Ryan W. Askeland, Michael A. O9Donnell, Yi Luo. BCG in combination with anti-IL-10R1 monoclonal antibody induces specific antitumor immunity in an orthotopic bladder cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4742. doi:10.1158/1538-7445.AM2013-4742

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