Sam J. Brancato

Targeted therapies in urothelial carcinoma.

Purpose of review Greater understanding of the biology and genetics of urothelial carcinoma is helping to identify and define the role of molecules and pathways appropriate for novel-targeted therapies. Here, we review the targeted therapies that have been reported or are in ongoing urothelial carcinoma clinical trials, and highlight molecular targets characterized in preclinical and clinical studies. Recent findings Trials in nonmuscle-invasive bladder cancer are evaluating the role of immunotherapy and agents targeting vascular endothelial growth factor (VEGF) or fibroblast growth factor receptor-3. In muscle-invasive bladder cancer, neoadjuvant studies have focused on combining VEGF agents with chemotherapy; adjuvant studies are testing vaccines and agents targeting the human epidermal growth factor receptor 2, p53, and Hsp27. In the first-line treatment of metastatic urothelial carcinoma, tubulin, cytotoxic T-lymphocyte antigen 4, Hsp27, and p53 are novel targets in clinical trials. The majority of targeted agents studied in urothelial carcinoma are in the second-line setting; new targets include CD105, polo-like kinase-1, phosphatidylinositide 3-kinases (PI3K), transforming growth factor &bgr; receptor/activin receptor-like kinase &bgr;, estrogen receptor, and the hepatocyte growth factor receptor (HGFR or MET). Summary Development of targeted therapies for urothelial carcinoma is still in early stages, consequently there have been no major therapeutic advances to date. However, greater understanding of urothelial carcinoma and solid tumor biology has resulted in a proliferation of clinical trials that could lead to significant advances in treatment strategies.

Epidermal Growth Factor Receptor (EGFR)-targeted Photoimmunotherapy (PIT) for the Treatment of EGFR-expressing Bladder Cancer

The use of light as a means of therapy for bladder cancer has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT), which targets EGFR-expressing bladder cancer. Anti-EGFR antibody panitumumab was labeled with the photoabsorber (PA), IRDye 700Dx (IR700), to create a panitumumab-IR700 antibody–PA conjugate that is activated by near-infrared radiation (NIR). Bladder cancer tissue microarray (TMA) and bladder cancer cell lines were analyzed for expression of EGFR. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most bladder cancers have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Panitumumab-IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Panitumumab alone, panitumumab-IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed toward cell death. NIR-PIT with panitumumab-IR700 reduced growth compared with only panitumumab-IR700–treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Panitumumab-IR700–induced PIT selectively kills EGFR-expressing bladder cancer cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of bladder cancer and ultimately in patients. Mol Cancer Ther; 16(10); 2201–14. ©2017 AACR.

Nonmuscle invasive bladder cancer： a primer on immunotherapy

Intravesical Bacillus Calmette-Guérin (BCG) has long been the gold standard treatment of nonmuscle invasive bladder cancer. Recently, there has been an emergence of novel immunotherapeutic agents, which have shown promise in the treatment of urothelial cell carcinoma. These agents aim to augment, modify, or enhance the immune response. Such strategies include recombinant BCG, monoclonal antibodies, vaccines, gene therapy, and adoptive T-cell therapy. Here, we review the emerging immunotherapeutics in the treatment of nonmuscle invasive bladder cancer.

Chronic Colovesical Fistula Leading to Chronic Urinary Tract Infection Resulting in End-Stage Renal Disease in a Chronic Granulomatous Disease Patient

A 46-year old man with X-linked chronic granulomatous disease (CGD) being followed at the National Institute of Health with uncontrolled CGD colitis who developed chronic colovesical fistula, and end-stage renal disease (ESRD). Despite aggressive medical management of symptoms with immunomodulators and antibiotic prophylaxis, the chronic colovesical fistula led to chronic pyelonephritis, recurrent urinary tract infections, persistent air in the collecting system and bladder, and post-renal obstruction resulting in renal failure. Patient is now hemodialysis dependent and required diverting loop ileostomy placement. This report highlights multiple potential etiologies of rising serum creatinine in patients with CGD.

Evolving immunotherapy strategies in urothelial cancer.

The treatment of nonmuscle-invasive urothelial carcinoma with bacillus Calmette-Guérin (BCG) represents the importance of immunotherapy in the treatment of cancer. Despite its clinical efficacy, up to 30% of patients will ultimately experience progression to muscle-invasive disease. This, along with an improved understanding of the biologic pathways involved, has led to efforts to improve, enhance, or alter the immune response in the treatment of urothelial carcinoma. A number of novel therapeutic approaches currently are being pursued, including recombinant BCG to induce T helper type 1 (Th1) immune responses, nonlive Mycobacterium agents, targeted agents toward cancer-associated antigens, immune-modulating vaccines, and adoptive T-cell therapies. Here, we review the current and future immunotherapy treatment options for patients with urothelial cancer.

Robot-Assisted Partial Cystectomy

Radical cystectomy is the standard of care for localized muscle-invasive bladder cancer as well as refractory high grade, nonmuscle invasive urothelial cell carcinoma or carcinoma in situ. However, it is a highly morbid procedure that adversely impacts both urinary and sexual functions. To minimize these side effects without compromising oncological efficacy, bladder sparing strategies such as trimodal therapy and partial cystectomy have evolved. In carefully selected patients, many contemporary series have reported comparable oncologic outcomes and decreased morbidity compared to radical cystectomy. In this chapter, we present the indications and techniques of robot-assisted laparoscopic partial cystectomy for bladder cancer.

Abstract 3311: Molecular targeted photoimmunotherapy as a treatment for bladder cancer

Introduction: Bladder cancer (BCa) is a common cancer and is the most expensive malignancy to treat from diagnosis to death. Although no new therapies have been introduced in the past two decades, data from ‘The Cancer Genome Atlas (TCGA)’ confirms that Epidermal Growth Factor Receptor (EGFR) family and its downstream signaling molecules are altered in almost 72% of muscle invasive bladder cancer (MIBC) cases. However, anti-EGFRs to date have failed to demonstrate any benefit over standard of care chemotherapy. We have applied a technique of employing monoclonal antibodies (mAbs) conjugated with photo-activatable compounds (IRDye 700Dx, a phthalocyanine dye) that are activated by near-infrared light (NIR). When incubated with the conjugate, exposure to NIR destroys only targeted cells. We explored photoimmunotherapy (PIT) in a panel of BCa cell lines. The purpose of this study was to study the efficacy and mechanism of action of PIT using the anti-EGFR panitumumab (Pan)-IR700 immunoconjugate as a selective therapeutic strategy for bladder cancer. Methods: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines. The cytotoxicity of Pan-IR700 was analyzed using LIVE/DEAD and IC50 was measured using the MTS assay. The type of cell death was examined by fluorometric caspase assay, Annexin V-PI staining, and TEM. The mechanism of cell death was elucidated using fluorometric measurement of reactive and singlet oxygen species. Results: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines and cell lines with increasing amounts of surface EGFR (RT4, TCCSUP, 5637 and UMUC-5) were selected for further studies. Pan-IR700 rapidly killed UMUC-5 cells (high EGFR expressions) with an IC50 of 4 nM at 4 J/cm2 NIR. In TCCSUP cells (low surface EGFR), the same IC50 of Pan-IR700 could be achieved at 64 J/cm2 of NIR. No significant cytotoxicity was observed in the presence of IR700 or NIR alone, or in cell lines without any EGFR expression. Absence of any caspases and presence of most cells in the late apoptosis/necrosis quadrant of Annexin V-PI staining suggested that PIT kills cells by necrosis. In TEM, these cells showed classic features of necrotic cell death such as swollen organelles, disintegrated plasma membrane and depleted cytoplasm and nucleoplasm. Cells undergoing PIT showed production of ROS and singlet oxygen species in large amounts immediately after exposure to NIR. However, only the singlet oxygen quencher (NaN3) and not the ROS quencher (Trolox) was able to protect the cells from PIT induced cell death indicating partial involvement of singlet oxygen in PIT-induced necrosis of cells. Conclusions: PIT is a new targeted treatment for bladder cancer. Our data demonstrate that Pan-IR700-induced PIT selectively and efficiently kills EGFR-expressing bladder cancer cells in vitro and therefore warrants further preclinical therapeutic studies in in vivo bladder cancer models. Citation Format: Reema Railkar, Quentin Li, Srinivas Vourganti, Sam J. Brancato, Peter L. Choyke, Hisataka Kobayashi, Piyush K. Agarwal. Molecular targeted photoimmunotherapy as a treatment for bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2015-3311

MP34-14 TARGETING EPIDERMAL GROWTH FACTOR RECEPTOR USING PHOTOIMMUNOTHERAPY IN THE TREATMENT OF BLADDER CANCER

INTRODUCTION AND OBJECTIVES: More than 67,000 new cases of bladder cancer (BCa) are diagnosed yearly in the US and w70-80% of these patients present with nonmuscle invasive (NMI) tumors and 70% of these tumors recur despite optimal treatment. More effective targeted agents are needed to better manage this disease. To this end, we developed a novel platform using photoimmunotherapy (PIT) to treat BCa. This technique involves conjugation of a targeted monoclonal antibody to a phthalocyanine dye (IR700), which is activated by near-infrared light (NIR) and destroys the targeted cells though the production of reactive oxygen species. The monoclonal antibody binds to the tumor cell surface receptor of interest. The objective was to explore the efficacy of PIT using anti-epidermal growth factor receptor (EGFR) panitumumab (Pan)-IR700 immunoconjugate as a selective therapeutic strategy for NMI BCa. METHODS: Using flow cytometry surface staining, the surface expression of EGFR was profiled in several BCa cell lines and particular cells were chosen for further study. In vitro, cells of interest were incubated with no drug, 5-25 ug/ml panitumumab alone (Pan), a blocking condition, or Pan-IR700 conjugate for one hour followed by conditions of 0 or 4 J/cm NIR irradiation. Cytotoxicity was evaluated with flow cytometric detection of amine reactive viability dye and propidium iodide as well as utilizing raw hemocytometer counts of trypan blue exclusion. RESULTS: We successfully created the Pan-IR700 immunoconjugate as demonstrated by gel electrophoresis. We found that high levels of EGFR were expressed on the cell surface of human bladder cancer UMUC5 cells by flow cytometry and used this as a model for targeted therapy. We also demonstrated that EGFR was expressed in TCCSUP cells but at levels lower than UMUC5. Raw counts of cell viability by trypan exclusion after treatment revealed near complete cell death (98% of cells nonviable) in the Pan-IR700 plus NIR group in UMUC5 cells but minimal cell death in the same group in TCCSUP cells. However, increasing NIR irradiation to 64 J induced a more robust effect in TCCSUP cells. In all groups, light exposure alone resulted in minimal toxicity. CONCLUSIONS: PIT represents a targeted, cytotoxic approach that can be tailored to specific BCa cell surface receptors. Our observations indicate that Pan-IR700-induced PIT selectively and effectively triggers cell death in bladder cancer cells that express EGFR in vitro. We are currently developing in vivo rodent models of this platform in the hopes to fast track this technology into Phase I clinical trials in human.