Lymphangiogenesis in mammary tumours of the cat assessed by VEGF-3 expression

Introduction. Lymphatics play a crucial role in the formation of metastasis, and only recently were found specific markers, such as VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) that has been employed in this study. It is found in both lymphatic and blood vessels during embryogenesis, and only in lymphatics after birth. This study is aimed to verify if lymphangiogenesis develops (de novo or from pre-existing lymphatic vessels) in the intra(IT)/extratumoral(ET)stroma of a series of feline mammary tumours. Materials and Methods. The samples were 6 cases of normal mammary gland (NMG), 10 benign (BN) and 32 malignant (MN) neoplasms, all formalin-fixed and paraffin-wax embedded, and diagnosed on hematoxylin-eosin slides. Malignancies were graded into non-infiltrating (stage 0) and infiltrating with stromal invasion (stage I) or lymphatic or blood emboli and/or regional lymph node metastases (stage II). More 4 μm sections from the same samples were immunohistochemically (IH) stained with a laminin/VEGFR-3 double stain. Lymphatics expressing and non-expressing VEGFR-3 were counted in 10 intratumoral/intramammary(IT/IM) and 20 extratumoral /extramammary (ET/EM) fields, and were negative for laminin and positive or negative for VEGFR-3. Counts included VEGFR-3 positive, VEGFR-3 negative and total (the sum of both) lymph vessels. Results. A red intracytoplasmic staining is characteristic of anti-VEGFR-3 IH positivity, and a sub-endothelial or sub-epithelial (periductular and perialveolar) brown staining of anti-laminin positivity. In NMG, BT and MT, the positive or negative or total VEGFR-3 lymphatic vessels had a significantly higher number in the ET(EM) vs IT(IM) fields (Spearman test, P<0.01 for all comparisons). Comparing IM with IT fields no difference was detected in the number of lymphatic vessels, whereas there was a significantly higher number of total and VEGFR-3 negative (Spearman test, P<0.05), but not of VEGFR-3 positive lymphatics (Spearman test, P=0.26) in ET vs EM fields. No difference emerged comparing IT or ET counts among the 3 histological grades in MT, except for VEGFR-3 positive lymphatic vessels in stage II carcinomas vs stage 0 and I (Spearman test, P<0.05), which were more numerous. Discussion: The significantly higher number of lymphatic vessels in the ET vs IT stroma is likely to reflect the expansive growth of the tumour and the concentration of vessels in the extra(peri)tumoral areas. This is enforced by the significant increase in VEGFR-3 negative and total but not VEGFR-3 positive lymphatics in ET vs EM fields. In spite of an extremely limited lymphangiogenesis, early metastases of mammary carcinomas are located in the regional lymph node. This indicates that the neoplastic cells leave the primary tumour through pre-existing but not de novo lymphatics, and chemical mediators may attract neoplastic cells to the lymphatics. Conclusions: In the comparison between malignancies and benign tumours and normal glands, the number of lymphatics is not increased. The known ability of carcinomas to an early spread via the lymphatics is not consequent on a real increase of lymphatics in the intra/extratumoral stroma, but it may depend on the ligand/receptor interaction between neoplastic cells and lymphatic endothelium.