Lymphangiogenesis does not influence progression and outcome of canine mast cell tumours

Introduction: Mast cell tumours often metastatize to the skin, regional lymph nodes and internal organs. Prodromic for metastasis is the increase in the haematic and lymphatic network, driven by VEGF receptors and ligands, in particular VEGFR3 influencing lymphangiogenesis. In malignant mastocytoma an increase is known in the number of blood vessels (angiogenesis) that acquires prognostic value. The aim of this study was to evaluate whether tumoral lymphangiogenesis develops and if it has prognostic value. Material and methods: Thirty-one primary cutaneous mast cell tumours, single or multiple and with one year follow-up, have been classified according to the Patnaik's grading system. An immunohistochemical (IHC) anti-laminin/anti-VEGFR3 double stain has been used to identify lymphatics when negative for laminin. IHC stained sections have been evaluated for blood vessels (showing positive laminin stain), lymphatic vessels (negative laminin stain), then further subgrouped for presence/absence of VEGFR3 positivity. The count has been carried out on 5 intratumoral (fields randomly chosen amid the tumour stroma) and extratumoral areas (between the external border of the neoplasm and surrounding derma/subcutaneous tissue). The randomly chosen areas were of 0.789 square millimeters each. The data did not have a normal distribution, therefore they were processed for statistical analysis with the Spearman rank correlation test. Results: According to Patnaik's grading system 15 out of 31 were graded I, 11 were graded II and 5 of 31 were graded III. Four were multiple and 27 single tumours. Twelve dogs had died and 19 were still alive at one year post surgery. IHC double stain evidenced laminin in the basement membrane stained brown, and the blue positivity to VEGFR3 in the cytoplasm/cell membrane of endothelial cells. In all the cases, the quantity of micro vessels of the four typologies (haematics: laminin+/VEGFR3+ or laminin+/VEGFR3-; lymphatics: laminin-/VEGFR3+ or laminin-/VEGFR3-) have been compared. Spearman's test has pointed out that the number of lymphatics with or without VEGFR3 expression did not vary between intratumoral vs extratumoral fields, Patnaik grade progression, single vs multiple tumours, dead vs alive animals at one year follow-up. The only significant changes included an increase in the haematic vessels without VEGFR3 expression in extratumoral (median 1.6) vs intratumoral (median 0.4) stroma (P=0.041), in multiple (median 3.2) vs single (median 0.6) neoplasms (P=0.013) and in dead (median 1.7) vs alive (median 0.4) animals at one year follow-up (P=0.005). Discussion: The expression of VEGFR3 indicates a response to angiogenetic factors produced during tumour growth in vessel endothelium (mainly of lymphatics). On the basis of the obtained results, no objective data support a true increase in lymphatics at the tumour borders, where the functionality of lymphatic vessels is better preserved than in intratumoral areas, nor the number of lymphatics was enhanced in multiple than single tumours or in dead compared with alive animals. It seems that lymph node involvement in mastocytomas is due to the spread through the preexisting lymphatics and true lymphangiogenesis does not happen. This study confirms the role of haemangiogenesis, but not of lymphangiogensis, as prognostic factor in canine mast cell tumours.