Abstract. Vasculogenic mimicry (VM) is a key developmental program, frequently activated during cancer invasion and metastasis. The aim of the present study was to evaluate the role of VM in orbital rhabdomyosarcoma (RMS), the correlation between VM and tumor differentiation, recurrence and survival duration, as well as the contribution of epithelial cell kinase (EphA2) and matrix metalloproteinase-2 (MMP-2) in VM initiation. A total of 32 patients were enrolled to investigate the associations between VM in orbital RMS tumors and clinical characteristics, as well as its impact on overall survival. VM was identified and confirmed by CD31/periodic acid-Schiff double staining, while the presence of EphA2 and MMP-2 were examined by immunohistochemical analysis. VM was identified in eleven patients, particularly those with poorly differentiated orbital RMS (P=0.001). Patients with VM exhibited significantly worse survival rates (P= 0.001, log-rank test), a significantly increased risk of mortality (P=0.008) and EphA2 and MMP-2 expression levels were enhanced (P=0.005 and 0.001, respectively). The VM and mitotic rate were independent predictors of poor prognosis (P=0.001 and 0.004, respectively), indicated by multivariate Cox proportional hazards models. These results demonstrated that VM is present in orbital RMS and represents an independent prognostic factor for overall survival. In addition, overexpression of EphA2 and MMP-2 may promote VM formation in orbital RMS. IntroductionOrbital rhabdomyosarcoma (RMS) as a prevalent malignancy, and is the most common malignant tumor of the orbit amongst children (1,2). Malignant tumors require a sufficient blood supply in order to support their growth. It was previously believed that blood vessels were only able to be formed by endothelial cells. However, the rigid cellular identity of the blood vessel has since been questioned, following the observation that tumor cells are also capable of forming extravascular networks in high-grade malignancies (3). The process of vasculogenic mimicry (VM), describes the mechanism by which highly aggressive tumor cells are able to form vessel-like structures, as a result of their high plasticity. It has therefore been suggested that tumors may develop vascularization via angiogenesis, and that this process is associated with poor prognosis (4-6).Orbital RMS is a solid tumor with the morphological characteristic of bidirectional differentiation, which is the cellular basis of the formation of VM (7). However, to the best of our knowledge, no specific study regarding the novel blood-supplying pattern in orbital RMS has previously been published. Furthermore, the precise molecular mechanism of VM formation remains to be elucidated. Epithelial cell kinase (EphA2) and matrix metalloproteinase-2 (MMP-2) have been found to be a crucial inducers of VM (8,9), therefore it is of significant interest to examine the role of EphA2 and MMP-2 in VM formation in orbital RMS. The present pilot study was designed to investigate whether VM occurs in orbital...