Primary urothelial carcinoma tumor samples

Primary canine UC tumor samples (N = 9) were collected from clinical cases treated at The Ohio State University Veterinary Medical Center (OSU-VMC). Owner consent for tissue collection was obtained in all cases in accordance with established hospital protocols and approved Institutional Animal Care and Use Committee protocols (IACUC #2010A0015). Tissue collections were performed by the OSU-VMC Blue Buffalo Clinical Trials Office and Veterinary Clinical Research Shared Resource. Fresh tumors collected at the time of surgery or euthanasia were flash frozen in liquid nitrogen and placed in formalin and processed for routine paraffin embedding for histopathology. Briefly, sections were routinely sectioned at 4–5 µm thickness and stained with hematoxylin and eosin (H&E) on a Leica ST5020 autostainer (Leica Biosystems, Buffalo Grove, IL) using a routine and quality-controlled protocol. All tumor samples were confirmed to be urothelial carcinoma by board certified veterinary anatomic pathologists at OSU-VMC.

Sections were evaluated using a semi-quantitative assessment of the approximate percentage of total tumor area comprised of necrotic debris using a digital grid with a total size of 3.2 × 2.3 mm and each square of the grid measuring 0.2 × 0.2 mm applied via Olympus CellSens Imaging Software. Semi-quantitative methods were developed in consultation with a board-certified veterinary comparative pathologist to assess the approximate percentage of total tumor area comprised of necrotic debris with subcategories < 5%, 5–10%, > 10%, and > 50% of total tumor. This was a pilot study with the intent to characterize the expression and phosphorylation of receptor tyrosine kinases in primary canine UC tumors and UC cell lines, so no power calculation was made.