title: Association of TIMP3 expression with vessel density, macrophage infiltration and prognosis in human malignant melanoma.
abstract: OBJECTIVE
  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).
  
  
  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.
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Asha M
authors.initials: AM
authors.lastname: Das
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Senada
authors.initials: S
authors.lastname: Koljenović
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Charlotte M C
authors.initials: CM
authors.lastname: Oude Ophuis
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Thom
authors.initials: T
authors.lastname: van der Klok
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Boris
authors.initials: B
authors.lastname: Galjart
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands; Erasmus Optical Imaging Center, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Alex L
authors.initials: AL
authors.lastname: Nigg
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands; Erasmus Optical Imaging Center, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Wiggert A
authors.initials: WA
authors.lastname: van Cappellen
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Vincent
authors.initials: V
authors.lastname: Noordhoek Hegt
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Winand N M
authors.initials: WN
authors.lastname: Dinjens
authors.affiliation: Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Peggy N
authors.initials: PN
authors.lastname: Atmodimedjo
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Cindy E
authors.initials: CE
authors.lastname: Vermeulen
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Cornelis
authors.initials: C
authors.lastname: Verhoef
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands; Gustave Roussy Cancer Campus Grand Paris, Villejuif, France.
authors.forename: Alexander M M
authors.initials: AM
authors.lastname: Eggermont
authors.affiliation: Department of Surgical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands. Electronic address: t.l.m.tenhagen@erasmusmc.nl.
authors.forename: Timo L M
authors.initials: TL
authors.lastname: ten Hagen
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015331:Cohort Studies; D019175:DNA Methylation / Q000502:physiology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D008207:Lymphatic Metastasis; D008264:Macrophages / Q000502:physiology*; D008297:Male; D008545:Melanoma / Q000098:blood supply* / Q000378:metabolism / Q000401:mortality; D008875:Middle Aged; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology; D011379:Prognosis; D012878:Skin Neoplasms / Q000098:blood supply* / Q000378:metabolism / Q000401:mortality; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3
keywords: Angiogenesis; Macrophage infiltration; Melanoma; Promoter methylation; TIMP3