Aims Several anti-tumour properties have been ascribed to the tissue inhibitor of matrix metalloproteinases-3 (TIMP3) gene, including inhibition of neovascularisation in tumour xenografts. Reduced protein expression has been linked to promoter hypermethylation and allelic loss of heterozygosity in various human malignancies. In melanoma-positive lymph nodes from patients, we evaluated the association between TIMP3 expression, vessel density, macrophage infiltration and potential correlations with disease-free survival (DFS) and overall survival (OS). Patients and methods TIMP3 expression was analysed by immunohistochemistry (IHC) in melanoma lymph node biopsies of stage III melanoma patients (n = 43). Blood vessel density and macrophage infiltration were quantitatively assessed and correlation with TIMP3 expression was investigated. Methylation status of the gene promoter was determined using methylation-specific polymerase chain reaction (MSP). Protein expression and promoter methylation status were investigated for associations with DFS and OS. Results Reduced expression of TIMP3, as determined by IHC, was observed in 74% of the cases (32 in 43). A significant inverse correlation was observed between TIMP3 expression and vessel density (p = 0.031). Correlation between TIMP3 expression and macrophage infiltration was not statistically significant (p = 0.369). MSP analysis revealed methylation of the gene promoter in 18% (7 in 38) of the analysed cases. No differences in OS and DFS were observed between cases with high and low TIMP3 expression. Gene promoter methylation was significantly associated with both poor 5-year DFS (p = 0.024) and OS (p = 0.034). Conclusions Our data indicate that TIMP3 is a dominant negative regulator of angiogenesis in cutaneous melanoma and gene silencing by promoter methylation is associated with poor outcome.

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Keywords Angiogenesis, Macrophage infiltration, Melanoma, Promoter methylation, TIMP3
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Journal European Journal of Cancer
Mooppilmadham Das, A, Koljenović, S, Oude Ophuis, C.M.C, Van Der Klok, T. (Thom), Galjart, B. (Boris), Nigg, A.L, … ten Hagen, T.L.M. (2016). Association of TIMP3 expression with vessel density, macrophage infiltration and prognosis in human malignant melanoma. European Journal of Cancer, 53, 135–143. doi:10.1016/j.ejca.2015.09.014