Exploring the prognostic significance of new vessel formation and its relationship with CD44 and hypoxic factors immunoexpression in canine appendicular osteosarcoma.

Introduction: Angiogenesis imbalance is essential for tumour growth, with hypoxia being the most important driver. Moreover, tumour cells can contribute to forming blood vessels by vasculogenic mimicry (VM) or recalling the endothelial-progenitor-cells through vasculogenesis. Microvessel density (MVD) increase and VM are associated with poor prognosis in human osteosarcoma (OS), while their significance in canine OS is unclear. The cancer-stem-cell marker CD44 can represent a key factor due to its involvement in MVD and VM increase. The aim of this work is the investigation of MVD, VM and CD44 relationship among them and with hypoxic factors, including VEGF!, clinical-pathological features and prognosis in canine OS. Materials and Methods: MVD and VM were assessed in 23 canine appendicular OS samples through PAS-CD31 staining. MVD was quantified with Weidner’s method and VM identified as CD31-negative-PAS-positive cavities containing erythrocytes. CD44 and hypoxic factors were evaluated by immunohistochemistry. Analyses of survival and univariate Log-Rank association were used to examine all variables. Variable relationships (excluding survival data) were estimated by Student’s or Mann-Whitney test. Results: Results showed MVD increase in male dogs and high-grade tumours (p"0.05). VM was present in 7/23 cases, in association with higher CD44 immunoexpression (p"0.05) and reduced overall survival (p"0.05). A positive tendency between CD44 and VEGF! immunostaining was also observed (p<0.1). Conclusions: For the first time, VM and CD44 and their positive relation were observed in canine OS tissues, together with VM prognostic value. A larger number of cases is necessary to confirm these preliminary results and understand the mechanisms involved in hypoxia-related cancer progression.