Oncology Resident Ohio State College of Veterinary Medicine Columbus, Ohio
Transitional cell carcinoma (TCC) accounts for >90% of canine malignant tumors occurring in the urinary bladder. TCC tumors are generally inoperable and unresponsive to traditional chemotherapy, indicating a need for more effective therapies. Toceranib phosphate (Palladia) is a multi-target receptor tyrosine kinase (RTK) inhibitor that exhibits potent activity against members of the split kinase family of RTKs, including vascular endothelial growth factor receptor, platelet-derived growth factor receptor, Kit, and Flt-3, resulting in both direct antitumor and antiangiogenic activity. Toceranib (TOC) demonstrated single agent activity against a variety of tumor types in a phase 1 study in dogs with cancer, including several carcinomas. In this clinical trial, 3 of 4 dogs with bladder TCC treated with TOC alone had stable disease for 10 weeks or greater. Preliminary retrospective studies suggested approximately 86.7% of dogs with TCC experienced clinical benefit (partial response or clinically meaningful disease stabilization) following toceranib (TOC) treatment; however, the basis for the observed responses to TOC is not known. The purpose of this study was to evaluate normal canine bladder tissues, primary bladder TCC tumors, and established TCC cell lines for the expression and activation of VEGFR1, VEGFR2, PDGFRα, PDGFRβ, and KIT to assess whether dysregulation of these RTKs may contribute to the biological activity of TOC. To provide an initial assessment of RTK expression, Real Time PCR was performed on primary TCC tissue samples (N=10) and TCC cell lines (N=5) to detect VEGFR2, PDGFRα, PDGFRβ, and KIT mRNA. Transcript for VEGFR2, PDGFRα, and PDGFRβ was detected in all TCC tissue samples and TCC cell lines; however, mRNA for KIT was not detectable in any samples. The Proteome Profiler™ Human Phospho-RTK Array Kit (R & D Systems) provided a platform to assess phosphorylation of 42 different RTKs in primary TCC tissue specimens using the available flash frozen tumor specimens and TCC cell lines. In concordance with these data, PDGFRα, and PDGFRβ were found to be phosphorylated in all tumor samples and cell lines and KIT activation was not observed on the arrays. While message for VEGFR2 was identified in all tumor samples and cell lines, all samples exhibited low basal phosphorylation levels of this RTK. Core samples from all tumor samples and normal bladder tissues were available for evaluation and a tissue microarray was constructed to evaluate expression of receptors of interest and determine VEGFR, PDGFRα, and PDGFRβ immunoreactivity and localization in tumor cells and supporting stroma. Studies are ongoing to evaluate the in vitro activity of TOC on cell viability, apoptosis, and VEGFR, PDGFRα, and PDGFRβ phosphorylation in TCC cell lines. Taken together, our findings demonstrate that known targets of TOC are expressed/activated in primary TCC tumors and TCC cell lines. Given the observed phosphorylation of PDGFRα and PDGFRβ, these RTKs merit further investigation as to their role in mediating the biology of TCC and their contribution to TOC’s activity.