Malignant melanoma (MM) metastasises to distant organs probably via the lymphatic system. Tumours stimulate new lymphatic vessel formation (lymphangiogenesis) via secretion of Vascular Endothelial Growth Factor C (VEGF-C) or D and this facilitates metastasis in animal models (Mandriota et al. 2001, Skobe et al. 2001). Lymphatic vessel density (LD) can be used to predict MM metastasis (Shields et al. 2003, Dadras et al. 2003) and that VEGF-C and D expression have prognostic potential. We have determined VEGF-C and D expression in melanoma and assessed their association with lymphatic density, lymphatic endothelial cell proliferation and metastatic potential.
Normal skin (n = 11) and 17 archival samples of MM (11 metastatic, 6 non metastatic, obtained with Local Ethical Committee approval, North Bristol NHS Trust). Lymphatics were identified by immunohistochemical staining with rabbit anti-human LYVE-1 (4.2 µg/ml, gift of Dr Jackson), VEGF-C with goat anti-human VEGF-C (1.14 µ/ml, Santa Cruz,), VEGF-D with goat anti-human VEGF-D (10 µg/ml) and Ki67 with mouse anti-human ki67 (4 µg/ml, Oncogene). LD was then calculated for the whole sample.
LD was significantly greater in metastatic MM (12.8 ± 1.6 mm2) than non metastatic MM (5.4 ± 1.1 mm2, P < 0.01 Mann Whitney), clearly discriminating between tumours that had metastasised with those that had not. 13/17 (76 %) tumours expressed VEGF-C and 9/17 (53 %) expressed VEGF-D. Intensity of staining was assessed blindly by subjective scoring. There was no significant difference in the intensity or frequency of staining in metastatic compared to non-metastatic samples for either VEGF-C or VEGF-D. Furthermore, no evidence of Ki67 expression in lymphatic vessels was seen in the non metastatic or the metastatic group indicating that lymphatic endothelial cell proliferation was not occurring at the time of biopsy. From this small study, we can conclude that although a significantly increased LD is seen in metastatic MM, and lymphangiogenic growth factors VEGF-C and D are expressed, lymphatic endothelial cell proliferation was not actively occurring at the time of melanoma excision. Since LD was increased this suggests that lymphangiogenesis had occurred prior to excision and maybe an early feature of melanomas that have a high probability of forming distant metastasis.