Lymphatic density and metastatic spread in human malignant melanoma

Abstract

Malignant melanoma (MM), the most common cause of skin cancer deaths, metastasises to regional lymph nodes. In animal models of other cancers, lymphatic growth is associated with metastasis. To assess if lymphatic density (LD) was increased in human MM, and its association with metastasis, we measured LD inside and around archival MM samples (MM, n=21), and compared them with normal dermis (n=11), basal cell carcinoma (BCC, n=6) and Merkel cell carcinoma (MCC), a skin tumour thought to metastasise through a vascular route (MCC, n=6). Lymphatic capillary density (mm(-2)), as determined by immunohistochemical staining with the lymphatic specific marker LYVE-1, was significantly increased around MM (10.0+/-2.5 mm(-2)) compared with normal dermis (2.4+/-0.9 mm(-2)), BCC (3.0+/-0.9 mm(-2)) and MCC (2.4+/-1.4 mm(-2)) (P<0.0001). There was a small decrease in LD inside MM (1.1+/-0.7 mm(-2)) compared with normal dermis, but a highly significant decrease in BCC (0.14+/-0.13) and MCC (0.12+/-2.4) (P<0.01 Kruskal-Wallis). Astonishingly, LD discriminated between melanomas that subsequently metastasised (12.8+/-1.6 mm(-2)) and those that did not (5.4+/-1.1 mm(-2), P<0.01, Mann-Whitney). Lymphatic invasion by tumour cells was seen mainly in MM that metastasised (70% compared with 12% not metastasising, P<0.05 Fisher's Exact test). The results show that LD was increased around MMs, and that LD and tumour cell invasion of lymphatics may help to predict metastasis. To this end, a prognostic index was calculated using LD, lymphatic invasion and thickness that clearly discriminated metastatic from nonmetastatic tumours.

Figure 1

Lymphatic density in normal dermis, intra-tumoural LD and dermis immediately around three types of skin cancer. (A) Lymphatic density inside the tumour (green) was always lower than that outside the tumour (red). There was a significant increase in LD around the tumour in MM compared with normal dermis, BCC and MCC. See Results for values and detailed statistical analysis. Lymphatic capillaries in dermis stained with LYVE-1 antibody (arrows) in normal (B), BCC (C), MM (D) and MCC (E). Lymphatic vessels stained inside tumours in BCC (F), melanoma (G) and MCC (H). Inset is negative control staining. Bar: (B–G), 50 μm; (H), 100 μm.

Figure 2

Relation between LD and metastasis in MM. (A) Lymphatic density outside (solid bars, mean±s.e.m.) and inside (stippled bars) MM classified according to whether the tumour had subsequently metastasised (P<0.001, two-way ANOVA). Post hoc tests showed significant difference between metastatic and nonmetastatic epitumoral LD (P<0.01, Mann–Whitney U), and between epi- and intratumoral LD for both nonmetastatic (P<0.05) and metastatic (P<0.001, Bonferroni), but not between metastatic and nonmetastatic intratumoral LD. (B) Plot of LD against thickness for nonmetastatic (open squares) and metastatic (filled diamonds) melanoma. There was no significant correlation for the pooled group (r=−0.2, P>0.1).

Figure 3

Lymphatic and vascular invasion in MM. Tumour cells were seen inside lymphatic capillaries (positive for LYVE-1) in both MM (A) and MCC (B). Vascular invasion (tumour cells inside PECAM positive, LYVE-1 negative vessels) was also seen in MM (C) and MCC (D). (E) Frequency of vascular (black) and lymphatic (stippled) invasion in MM and MCC. Although lymphatic and vascular invasion were equally common in MM, vascular invasion was significantly more common than lymphatic invasion in MCC P<0.05, Fisher's exact test. (F) Invasion frequency observed in metastatic (red) and nonmetastatic melanomas (blue). Lymphatic invasion alone, vascular invasion alone and both lymphatic and vascular invasion were significantly more common in metastatic than nonmetastatic melanomas. Combined vascular and lymphatic invasion was a particularly strong prognostic sign for metastasis (although not significantly different from either alone). Invasion of neither lymphatic nor vascular microvessels indicated a favourable prognosis, that is, no metastasis. Bar (A, C, D), 50 μm; (B), 100 μm.

Figure 4

Prognostic index PI for MM plotted on a logarithmic scale. PI is the product of LD squared, thickness and a lymphatic invasion factor (1 for no invasion, 2 for lymphatic invasion). The inclusion of vascular invasion made no significant difference to the separation of the results from the two groups. All nonmetastatic tumours had a PI of <119 (log. value 2.08). All metastatic tumours were above this value.