Immunohistochemical assessment of COX-2 enzyme in canine renal cell carcinomas COX-2 is an inducible enzyme involved in the production of prostaglandins, modulating pathologic events such as inflammation, wound healing and carcinogenesis. In the normal kidney COX-2 is only expressed in cells of the macula densa. Recent studies demonstrate that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX enzymes, can reduce the incidence of cancer in humans and animals and may be potential targets for therapeutic and preventive strategies. Renal cell carcinomas (RCCs) are the most common canine primary renal tumours with a reported prevalence of 0.3% to 1.5% of all canine neoplasms (Edmonson et al., 2015). Information regarding the molecular mechanisms that contribute to tumorigenesis in RCCs is scant, although the expression and distribution of COX-2 has been demonstrated to be prognostically significant in human renal carcinomas, the presence of COX- 2 has been analysed in only two cases of canine RCC (Khan et al., 2001). Histological Analysis Objectives A. Suárez-Bonnet 1 , A. Stoll 2 , S. Carvalho 3 , A. Lara 3 & S.L. Priestnall 2 1 Unidad de Histología y Patología Animal, Instituto Universitario de Sanidad Animal, Universidad de Las Palmas de Gran Canaria, Arucas, Gran Canaria, Spain. 2 Department of Pathology and Pathogen Biology and 3 Oncology Service, The Royal Veterinary College, Hawkshead Lane, Hatfield, Hertfordshire, United Kingdom. In a large case series of canine renal cell carcinomas: 1. Characterise the histological types and mitotic index. 2. Analyse the expression and distribution of COX-2 enzyme. Materials & Methods 31 formalin-fixed paraffin embedded tissue blocks of canine RCC from the archives of veterinary pathology diagnostic laboratories within Europe and North America. 1. Histological tumour types were classified independently by 3 veterinary pathologists according to WHO criteria (Meuten et al., 2004). Hale's colloidal iron stain was used to identify Solid Chromophobe cell type tumours. 2. Immunohistochemical staining was performed using a monoclonal anti-COX-2 antibody (Thermo) on a BondMax autostainer (Leica). COX-2 was semiquantitatively scored based on the intensity (0-3) and percentage (0-3) of tumour expressing the enzyme cells to give a total COX-2 score, 0-9 (Pires et al., 2010). The cellular localisation of COX-2 was also recorded. Immunohistochemical Analysis Tubular type. Total score = 0 Papillary type. Total score = 3 , Cytoplasmic and perinuclear Tubular type. Total score = 6, Apical Tubular type. Total score = 6, Cytoplasmic and apical Tubular type. Total score = 9, Cytoplasmic Discussion & Conclusions Results Acknowledgements Alejandro Suárez-Bonnet was awarded a bursary by the Journal of Comparative Pathology Educational Trust to visit the Royal Veterinary College and contribute to this work. Tubular type RCC (H&E, x10) Papillary type RCC (H&E, x10) Solid Chromophobe cell-type RCC (H&E, x20) Sarcomatoid type RCC (H&E, x20) Tubular type RCC (H&E, x40) Solid Chromophobe cell-type RCC (Colloidal iron, x20) References • Edmonson EF, Hess AM, Powers BE. Prognostic significance of Histologic Features in Canine Renal Cells Carcinomas: 70 Nephrectomies. 2015. Veterinary Pathology 52:260-268. • Khan KNM, Stanfield KM, Trajkovic D, Knapp DW. Expression of Cyclooxygenase-2 in canine renal carcinoma. 2001, Veterinary Pathology 38:116-119. • Meuten DJ, Everitt J, Inskeep W, Jacobs RM, Peleteiro M, Thompson KG. Histological classification of tumors of the urinary system of domestic animal. Washington, DC: Armed Forces Institute of Pathology and the World Health Organization, 2004. • Pires I, Garcia A, Prada J, Queiroga FL. COX-1 and COX-2 expression in canine cutaneous, oral and ocular melanocytic tumours. 2010. Journal of Comparative Pathology 143:142-149. • Schwandt A, Garcia JA, Elson P, Wyckhouse J, Finke JH, Ireland J, Triozzi P, Zhou M, Dreicer R, Rini BI.Clinical and immunomodulatory effects of celecoxib plus interferon-alpha in metastatic renal cell carcinoma patients with COX-2 tumor immunostaining. 2011. Journal of Clinical Immunology 31:690-698. • Guimarães MJ, Carvalho MI, Pires I, Prada J, Gil AG, Lopes C, Queiroga FL. Concurrent expression of cyclo-oxygenase-2 and epidermal growth factor receptor in canine malignant mammary tumours. 2014. Journal of Comparative Pathology 150:27-34. Papillary-type RCC (Colloidal iron, x20) Solid Clear cell-type RCC (H&E, x40) Papillary type RCC (H&E, x40) Solid Clear cell-type RCC (H&E, x10) Canine renal carcinoma: A large, moderately well-demarcated, necrotic, cystic and haemorrhagic mass effaces and replaces half of the kidney. Post fixed nephrectomy specimen. Normal kidney: Positive immunostaining for COX-2 in cells of the macula densa (MD) only. Solid clear cell-type. Total score = 9, Cytoplasmic 0 10 20 30 40 50 60 70 80 90 100 Tubular Papillary Solid 58.1 16.1 25.8 Percentage of total cases Predominant tumour type • Tubular-type carcinomas were the most frequent histological type (58.1%). • Mitotic index ranged from 0-75. Mean MI for tubular was 24.9, solid 8.1 and papillary 14.2. • COX-2 expression: 83.9% tumours were positive. Mean total COX-2 scores by histological type, tubular; 3.1, solid; 2.9 and papillary; 2.6. These differences were not statistically significantly different. • Localisation: COX-2 was detected apically in the cell membrane (46.2% cases), perinuclear zone (15.4%) and in the cytoplasm (26.9%) of neoplastic cells. Three cases (11.5%) had apical-perinuclear and/or apical- cytoplasmic expression. • To the author´s knowledge this is the first comprehensive study to analyse the expression of COX-2 in a large series of canine RCC. • In contrast to normal canine kidney, in which COX-2 expression is restricted to the macula densa, in RCC most of the cases (83.9%) exhibited COX-2 overexpression in concordance with studies in humans (Schwandt et al., 2011). • COX-2 distribution in different cell compartments (cytoplasmic, perinuclear and cytoplasmic) has also been demonstrated in other carcinomas, suggesting a dynamic role during carcinogenesis (Guimaraes et al., 2014). • Finally we suggest that COX-2 overexpression in canine RCC could represent cellular reprograming during the neoplastic transformation process or may indicate a major role of the macula densa in the histiogenesis of a large proportion of canine RCC. View publication stats View publication stats