Tumour Angiogenesis in Uveal Melanoma Is Related to Genetic Evolution

Abstract

Increased angiogenesis is associated with a higher metastasis- and mortality rate in uveal melanoma (UM). Recently, it was demonstrated that genetic events, such as 8q-gain and BAP1-loss, influence the level of immune infiltrate. We aimed to determine whether genetic events, and specific cytokines, relate to angiogenesis in UM. Data from UM patients who underwent enucleation between 1999 and 2008 were analysed. Microvascular density (MVD) and the presence of infiltrating immune cells were determined with immunohistochemistry (IHC) and immunofluorescence in 43 cases. Chromosome status, BAP1 IHC and mRNA expression of angiogenesis-related genes were known in 54 cases. Tumours with monosomy 3/BAP1-loss showed a higher MVD compared to tumours with disomy 3/normal BAP1 expression (p = 0.008 and p = 0.004, respectively). Within BAP1-positive lesions (n = 20), 8q-gain did not relate to MVD (p = 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (p = 0.041), Von Willebrand Factor (VWF) (p = 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (p = 0.024), and increased numbers of tumour-infiltrating macrophages (CD68+, p = 0.017; CD68+CD163+, p = 0.031) and lymphocytes (CD4+, p = 0.027). Concluding, vascular density of UM relates to its genetic profile: Monosomy 3 and BAP1-loss are associated with an increased MVD, while an early event (gain of 8q) is not independently related to MVD, but may initiate a preparation phase towards development of vessels. Interestingly, VEGF-B expression is decreased in UM with a high MVD.

Keywords: BAP1; VEGF-B; angiogenesis; chromosomes; macrophages; oncology; uveal melanoma.

Figure 1

Patient survival in relation to MVD and mRNA gene expression. Groups (high vs. low) were based on the median vessel counts and mRNA gene expression values. (a) Immunohistochemistry (IHC) counts of MVD (n = 43), (b) mRNA gene expression of CD68 macrophages (n = 54), (c) mRNA gene expression of VEGF-A (n = 54), and (d) mRNA gene expression of VEGF-B (n = 54).

Figure 2

Tumour genetics in relation to MVD and mRNA gene expression. Gain of chromosome 8q is an early event in UM development, while loss of BAP1 is a later event. (a) IHC counts of MVD (n = 43), (b) mRNA gene expression of CD68 macrophages (n = 54), (c) mRNA gene expression of VEGF-A (n = 54), (d) mRNA gene expression of VEGF-B (n = 54), (e) mRNA gene expression of HIF1a (n = 54), and (f) mRNA gene expression of VHL (n = 54). (p-values were obtained using Mann–Whitney U tests, comparing BAP1+ and n8q with BAP1+ and 8qgain patients, and all BAP1+ with all BAP1− patients. Abbreviations: BAP1+, BAP1-positive; BAP1−, BAP1-negative; n8q, normal chromosome 8q; 8qgain, gain of chromosome 8q).

Figure 2

Tumour genetics in relation to MVD and mRNA gene expression. Gain of chromosome 8q is an early event in UM development, while loss of BAP1 is a later event. (a) IHC counts of MVD (n = 43), (b) mRNA gene expression of CD68 macrophages (n = 54), (c) mRNA gene expression of VEGF-A (n = 54), (d) mRNA gene expression of VEGF-B (n = 54), (e) mRNA gene expression of HIF1a (n = 54), and (f) mRNA gene expression of VHL (n = 54). (p-values were obtained using Mann–Whitney U tests, comparing BAP1+ and n8q with BAP1+ and 8qgain patients, and all BAP1+ with all BAP1− patients. Abbreviations: BAP1+, BAP1-positive; BAP1−, BAP1-negative; n8q, normal chromosome 8q; 8qgain, gain of chromosome 8q).

Figure 3

Tumour genetics in relation to MVD. (a) Within all 43 patients, patients were compared based on status of BAP1, chromosome 3 or chromosome 8q. (b) Within either disomy 3 (n = 22) or monosomy 3 (n = 21) patients, patients were compared based on status of BAP1. (p-values were obtained using Mann–Whitney U tests. Abbreviations: BAP1+, BAP1-positive; BAP1−, BAP1-negative; D3, Disomy 3; M3, Monosomy 3; 8q normal, normal chromosome 8q; 8q gain, gain of chromosome 8q).