Investigation of Prognostic Value of Claudin-5, PSMA, and Ki67 Expression in Canine Splenic Hemangiosarcoma

Abstract

Splenic hemangiosarcoma (HSA) is a malignant tumor of endothelial cells that affects middle-aged and elderly dogs and is characterized by the formation of new blood vessels, commonly associated with necrotic and hemorrhagic areas. Despite its importance in veterinary medicine, few studies have identified markers with prognostic value for canine HSA. Thus, this study aimed to associate the clinicopathological findings (prostate-specific membrane antigen [PSMA], Claudin-5, and Ki67 gene and protein expression) with overall survival in HSA-affected patients. Fifty-three formalin-fixed and paraffin-embedded canine splenic HSA samples, previously diagnosed by histopathological examination, were used in this study. Claudin-5, PSMA, and Ki67 protein expression levels were evaluated by immunohistochemistry, and gene expression was evaluated by quantitative polymerase chain reaction. Claudin-5 protein overexpression was observed in patients with metastasis (p = 0.0078) and with stage III tumors compared to those with stage I and II tumors (p = 0.0451). In patients treated with surgery alone, low PSMA gene and protein expression (p = 0.05 and p = 0.0355, respectively) were associated with longer survival time. Longer survival time was observed in patients with a low Ki67 index (p = 0.0488). Our results indicate that Claudin-5 protein expression is associated with metastatic status, and PSMA gene and protein expression, and Ki67 index are associated with survival time.

Keywords: Ki67; canine spleen neoplasia; endothelial splenic neoplasia; proliferative index; prostate-specific membrane antigen.

Figure 1

(A) Overall survival in HSA-affected dogs according to clinical pathological criteria. (B) Patients with metastatic splenic HSA experienced a shorter survival time (p < 0.0001). (C) Patients at stage III show shorter survival time than stage I and II patients (p = 0.0444). (D) Patients that received surgical or surgery associated with adjuvant chemotherapy (CT) as treatment show a longer survival time than those that did not receive treatment or only surgery treatment (p < 0.0001).

Figure 2

Immunoexpression of Claudin-5, PSMA, and Ki67 in canine hemangiosarcoma (HSA) samples. (A) Low Claudin-5 immunoexpression in a capillary HSA sample. (B) Canine HSA showing high Claudin-5 expression in neoplastic endothelial cells. (C) Low Ki67 expression in a solid HSA sample. (D) High Ki67 expression in a canine HSA sample. (E) Low PSMA expression in a solid HSA sample. (F) High PSMA expression in neoplastic endothelial cells (arrows) in a capillary HSA sample. Note the negative expression in red cells (asterisk), confirming satisfactory endogenous peroxidase blocking. Harris hematoxylin counterstain, 40×.

Figure 3

(A) Metastatic samples have higher Claudin-5 expression compared with non-metastatic samples (p = 0.0078). (B) Association of Claudin-5 expression with tumor stage, with stage III tumors showing higher expression than stage I and II tumors (p = 0.0451).

Figure 4

Representative image of each antibody in capillary, cavernous and solid patterns. It is possible to observe the negative control for each antibody and PSMA, Ki67, and Clauding-5 expression in the different histological subtypes. Harris hematoxylin counterstaining, DAB, 40.

Figure 5

(A) Patients that underwent surgery as single therapy show low PSMA protein expression associated with increased survive time (p = 0.0355). (B) Patients treated with surgery associated with chemotherapy show the opposite relationship between PSMA and overall survival. Patients with low PSMA protein expression experience a shorter survival time (p = 0.0113). (C) A longer survival time is observed in patients treated with surgery with a lower Ki67 index (p = 0.0488) and (D) a longer survival time is observed for patients with low Ki67 index in those treated with chemotherapy (p = 0.0143).

Figure 6

Matrix of multiple correlations for all studied markers. Positive correlations are shown in blue and negative correlations are shown in red. The strength of correlations is depicted by color intensity.