PATHOLOGY OF THE UROGENITAL TRACT IN DOMESTIC ANIMALS

Even though pathology of the urogenital tract in domestic animals is a wide field of research several topics are still poorly investigated, tumors in particular. Moving from this observation, my research is organized into two main topics: canine urothelial carcinoma and tumours of the genital system (ovarian and testicular tumours). My research into urothelial carcinoma began with an analysis of the grading systems proposed in veterinary medicine. The relationship between tumour morphology and clinical behaviour is a key point in oncology. In this scenario, pathologists and clinicians play a pivotal role in the identification and testing of reliable grading systems based on standardized parameters to predict patient prognosis. Dogs with bladder urothelial carcinoma (BUC) were recently proposed as a “large animal” model for the study of human BUCs due to their similar morphology and metastasis locations. BUC grading systems are consolidated in human medicine, while in veterinary medicine, the BUC grading systems that have been proposed for canine tumours are not yet applied in routine diagnostics. These latter systems have been proposed, decade by decade, over the last thirty years, and the reason for their scarce applications is mainly related to a lack of specific cutoff values and studies assessing their prognostic relevance. However, for any prognostic study, reliable grading is necessary. During the beginning of my PhD, I published a review on grading systems for urothelial carcinomas to give an overview of the BUC grading systems available in both human and veterinary pathology and provide an extensive description and a critical evaluation to support veterinary researchers in the choice of possible grading systems to apply in future studies on canine BUCs. After analysing grading systems, I further studied dog urothelial carcinomas by analysing the microenvironment of these tumours, specifically, I studied the expression of an extracellular matrix protein called periostin in normal bladder and dog urothelial carcinomas. The tumour microenvironment is considered one of the main players in cancer development and progression and may influence the behaviour of cancer cells. Periostin (POSTN) is an extracellular matrix protein, and its main functions are induction of fibrillogenesis, fibroblastic cell proliferation and migration, enhancing regeneration in normal tissue, and promoting metastasis in case of neoplasia. POSTN has already been studied in humans in several normal tissues, inflammatory processes, and neoplasms, revealing an important role in tumour progression in various types of cancer, such as colon, lung, head and neck, breast, ovarian, and prostate. In these latter, high levels of POSTN are usually associated with a more aggressive tumour behaviour, tumour advanced stages, and poor prognosis, while in human bladder urothelial carcinoma (BUC), unlike in most tumours, POSTN expression seems to be downregulated. The expression of this marker has been poorly investigated in veterinary medicine; thus, the present Thesis aims to immunohistochemically investigate the presence and the intensity of POSTN expression in canine BUCs and to determine a possible relationship between POSTN expression and histopathological features such as mitotic count and muscular and vascular invasions. For the present retrospective study, archived samples from 45 canine BUCs and 6 non-neoplastic canine bladders were considered for histological evaluation and immunohistochemical examination for the expression of POSTN. POSTN expression was semi-quantitatively assessed considering both the percentage of the neoplastic stroma positive for POSTN and the intensity of the immunohistochemical labelling. The fact that POSTN protein is less expressed in urothelial carcinomas than in the normal bladder supports what was reported in human BUCs and, together with the negative correlation between mitotic count and protein expression that emerged in the present retrospective study, encourages further prospective follow-up studies to verify the possible role of POSTN in canine BUCs as a prognostic marker, and also as a possible target for the development of future anticancer therapies. My research during this PhD also involved the genital system, in particular, I described two rare cases of testicular sarcoma. Testicular tumours are common in dogs and, among them, interstitial cell tumours, seminomas and subletacular cell tumours are the most reported. Mesenchymal testicular tumours are rarely reported in humans as in veterinary medicine where only three cases of sarcomas (leiomyomas and leiomyosarcomas) have been described in two stallions and a ram. The present cases regarded a 12-year-old mixed-breed dog and a 10-year-old American Staffordshire Terrier who underwent bilateral orchiectomy. Formalin-fixed testes were referred for histopathological diagnosis. At gross examination, in one of the testes of both dogs, a white, firm and variably cystic testicular mass, effacing and replacing the testicular parenchyma was detected. Samples were collected from both neoplastic and contralateral testes, routinely processed for histology and serial sections were also examined immunohistochemically with primary antibodies against cytokeratins, vimentin, Von Willebrand factor, inhibin-α, α-smooth muscle actin, smooth muscle myosin and desmin. Histopathological features as well as the immunohistochemical results, positive for vimentin, actin, myosin and desmin, confirmed the mesenchymal origin and the myoid phenotype of both testicular tumours supporting the diagnoses of leiomyosarcoma. To the author’s knowledge, these are the first cases of primary testicular sarcoma reported in the canine species. However, even rare, these tumours deserve to be considered in routine diagnosis when a testicular spindle cell tumour is observed. The immunohistochemical panel applied was useful to distinguish the present tumours from differentiated Sertoli cell tumours confirming the diagnosis of leiomyosarcoma. Studies on the genital apparatus then led to a retrospective immunohistochemical study on ovarian tumours in the bitch. In canine species, ovarian epithelial tumours may derive from 3 structures: surface cells (SE), subsurface epithelial structures (SES) and rete ovarii (RO). Since no data are available concerning the possibility of individuating the precise structure of origin of canine ovarian epithelial cancer, and since encouraging results were obtained last year, the study continued on a larger number of cancers. Five normal canine ovaries and 33 tumours were tested immunohistochemically for CK7, CKAE1/AE3, CK19, HBME and Desmin. In normal ovaries, CK AE1/AE3 was expressed by all epithelial structures, CK7, HBME and Desmin were highly expressed by SE and less or not expressed in SES and RO. Within the normal ovary, CK7 is markedly expressed by the superficial epithelium while it is weakly and inconstantly expressed by the SES. In contrast, CK19 is diffusely and intensely positive also in SES and, therefore, we hypothesise that tumours that are inhibin negative, that develop beneath a preserved epithelium and are positive for CK19, but with little or no signal for CK7, may originate from SES. The data of the present Thesis confirm that is sometimes difficult to draw a line between benign and malignant forms but, unfortunately discriminating cut-offs are not individuated yet. Although considering the difficulty of distinguishing between benign and malignant forms, the results of this Thesis have made it possible to confirm previous studies and to suggest immunohistochemical markers that can contribute to delineate the structure of origin of canine ovarian epithelial neoplasms. We recommend continuing the work by incorporating an immunohistochemical panel including inhibin-α, HBME (with antigenic unmasking), CK7, CK19 and desmin into diagnostic practice.