The prognostic value of metalloproteinases and angiogenic factors in ovarian carcinoma

Abstract

The objective of this study was to analyze the correlation between matrix metalloproteinases (MMPs) and angiogenic genes and survival in advanced-stage ovarian carcinomas. Primary and metastatic ovarian carcinomas from patients diagnosed with FIGO stage III–IV disease and followed up to 20 years were studied using mRNA in situ hybridization (ISH). Expression of MMP-2, MMP-9, membrane-type 1-MMP (MT1-MMP), the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and basic fibroblast growth factor (bFGF) was studied. MMP-2, MMP-9 and TIMP-2 mRNA was detected in both tumor and stromal cells, while MT1-MMP was largely confined to tumor cells. In univariate analysis of primary tumors, TIMP-2 and MMP-9 mRNA expression correlated with poor outcome. In metastatic lesions, mRNA expression of TIMP-2, MMP-2, and MT1-MMP correlated with poor survival. In a multivariate analysis of primary tumors, TIMP-2 expression in stromal cells (P=0.006) and MMP-9 expression in tumor cells (P=0.011) retained their predictive value. Intense expression of bFGF mRNA and weak expression of IL-8 mRNA was detected in both stromal and tumor cells in most cases, while VEGF mRNA expression was limited to a few cases. Angiogenic mRNA expression showed no correlation with disease outcome in survival analysis (P>0.05). We conclude that bFGF is the major angiogenic factor expressed in ovarian carcinoma at the mRNA level. MMP-2, MMP-9, MT1-MMP and TIMP-2 are valid markers of poor survival in advanced-stage ovarian carcinoma.

Introduction

Local invasion, as well as distant spread, of malignant neoplasms, involves degradation of subepithelial and -endothelial basement membranes and modification of the extracellular matrix (ECM) (Liotta et al., 1983). This process is now known to depend on complex interactions between tumor cells, stromal cells and the endothelium, mediated by a large number of adhesion molecules, proteolytic enzymes, ECM components and transcription factors (Werb, 1997). Matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent enzymes, mediate the invasive and metastatic properties of most malignant tumors, in addition to their ubiquitous presence in benign reparative and inflammatory processes, through their ability to degrade most ECM components (Crawford and Matrisian, 1994–5, Aznavoorian et al., 1993, Liotta and Stetler-Stevenson, 1991). MMP-2 (Gelatinase A, 72 kD type IV collagenase) and MMP-9 (Gelatinase B, 92 kD type IV collagenase) are able to degrade type IV collagen, a component of all basement membranes, thereby facilitating stromal and vascular invasion by tumor cells (Aznavoorian et al., 1993). The enzymatic activity of MMPs is traditionally postulated to be regulated by tissue inhibitors of metalloproteinases (TIMP), a family presently comprised of four proteins with a molecular size of 21–28 kD. However, recent reports have demonstrated a role for TIMP and membrane-type MMPs (MT-MMP) in the cell-surface mediated activation of MMP-2 and MMP-9 (Chambers and Matrisian, 1997).

The ability of solid tumors to grow locally, and subsequently disseminate to distant organs, is dependent upon the formation of new blood vessels (angiogenesis), reflecting both the need for nutrient supply and the facilitation of vascular invasion by tumor cells (Folkman, 1985, Folkman, 1990, Fidler and Ellis, 1994). Several molecules have been recognized as angiogenic factors (Fidler and Ellis, 1994, Folkman and Klagsbrun, 1987). Vascular endothelial growth factor (VEGF) is a 32–34 kDa protein, expressed in four forms of 121–206 amino-acid residues, as a result of alternative splicing, exhibiting a mitogenic effect on endothelial cells (Tischer et al., 1991, Keck et al., 1989). Basic fibroblast growth factor (bFGF), a 146 amino-acid polypeptide, is part of a family of molecules encoded by at least seven genes (Goldfarb, 1990), involved in embyogenesis, angiogenesis and wound healing (Gospodarowicz et al., 1986). Interleukin-8 (IL-8) is a cytokine with a chemotactic effect on neutrophils, as well as angiogenic activity (Koch et al., 1992).

Carcinoma of the ovary is the leading cause of death from gynecological cancer in the western countries, with an overall median 5-year survival rate of 35% (Parkin et al., 1999). Two-third of the patients are diagnosed with stage III or IV disease, reflecting the tendency of these tumors to spread widely within the peritoneal cavity and occasionally to distant organs. However, data is lacking regarding the prognostic significance of MMP and angiogenic gene expression in ovarian tumors. A prognostic role was demonstrated for MMP-2 in one report using immunohistochemistry (Westerlund et al., 1999). The clinical significance of VEGF expression in ovarian carcinoma remains inconclusive (Paley et al., 1997, Hartenbach et al., 1997). Cytoplasmic levels of bFGF, as measured by immunoassay, correlated with better survival in the only study published to date (Obermair et al., 1998). The prognostic role of MMP-9, MT1-MMP and TIMP-2 and IL-8 in ovarian carcinoma has not been reoprted to date.

The objective of the present study was to evaluate the role of MMP-2, MMP-9, MT1-MMP, TIMP-2, VEGF, IL-8 and bFGF as prognostic markers in advanced-stage ovarian carcinomas.