COMPARATIVE EVALUATION OF PROGNOSTIC MARKERS IN CANINE AND FELINE MELANOMAS

The present PhD project investigates animal spontaneous models of non-UV induced melanomas, namely canine oral melanoma and feline iris diffuse melanoma (FDIM), which shares unique similarities in biological behavior with human mucosal melanoma and human iris melanoma, respectively. The project investigates selected markers related to the pathogenesis and prognosis of these tumors, i.e. gene and proteins that have been implicated in the progression and metastasis in human, canine and feline melanomas, such as Leukotriene A4 Hydrolase (LTA4H), Fragile X mental retardation-related protein 1 (FXR1) and matrix-metalloproteinases (MMPs). LTA4H is an enzyme of the arachidonic acid cascade, FXR1 is a RNA binding protein, MMPs a family of proteolytic enzymes of the extracellular matrix. The specific aims of the project are: 1) the validation of anti-FXR1 antibodies in the canine species; 2) the investigation of the expression of LTA4H and FXR1 in canine oral melanoma; 3) the study of FXR1-induced modulation of MMPs in canine oral melanoma; 4) the study of MMPs and tumor-matrix interaction in feline diffuse iris melanoma. 1) Two different commercially available polyclonal anti-human FXR1 antibodies were validated for use in dogs. Western blot experiments highlighted the specificity of cross-reaction. Immunohistochemistry described for the first time the specific distribution of FXR1 protein in canine normal tissues, and then the expression of FXR1 in a pool of canine melanocytic tumors. 2) LTA4H and FXR1 genes and proteins expression was investigated in FFPE canine oral melanomas (histology and immunohistochemistry, n=36, from 32 dogs; RT-PCR, subset n=23; clinical follow-up, subset n=13). ΔCt expression values ranged 0.76-5.11 for LTA4H and 0.22-6.24 range for FXR1 (out of range in 3 cases). The immunohistochemical expression of the proteins was evaluated as IRS-score (percentage of positive cells combined with intensity of the staining). IRS-score of LTA4H and FXR1 proteins did not correlate with the expression of the codifying genes. LTA4H and FXR1 seemed not correlated with the known criteria of malignancy or with the clinical outcome, when available. 3) Since FXR1 belongs to a family of RNA binding protein able to modulate the mRNA coding for the proteolytic enzyme MMP-9, MMP-9 and its inhibitor TIMP-2 were investigated by immunohistochemistry in canine oral melanomas to assess the association of FXR1 with MMP-9 and the association of MMPs activity with the clinical outcome. MMP-9 expression seemed not associated with FXR1 in canine oral melanomas. Anyway, intense levels of MMP-9/TIMP-2 were observed in cases with high expression of FXR1 and with unfavorable clinical outcome in canine oral melanoma. 4) The expression of MMPs in FDIM was investigated. Immunohistochemical expression of MMP-9/TIMP-2 was investigated in 62 FDIM and results were compared with the histological grade and mitotic index. MMP-9 and TIMP-2 were expressed in 77.4% and 71.0% FDIM, respectively. Increasing MMP-9 and TIMP-2 paralleled with high histological grades and high mitotic index.