Matrix Metalloproteinases -2 and -9, Vascular Endothelial Growth Factor, Basic Fibroblast Growth Factor and CD105- Micro-Vessel Density are Predictive Markers of Non-Muscle Invasive Bladder Cancer and Muscle Invasive Bladder Cancer Subtypes

Abstract

Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) remodeling during embryogenesis, wound healing and tumor development. Aberrant expressions and activation of MMP-2 and MMP-9 are examined as one of the major attributes acquired by tumor neoangiogenic markers including vascular endothelial growth factor (VEGF), basic growth factor (bFGF) and CD105-microvessel density (CD105-MVD) during bladder tumorigenesis. The present study examined the levels of MMP-2, MMP-9, VEGF, bFGF and CD105 to elucidate the relationship among them and associated clinical features in the given cohort of non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) patients. Real time-quantitative PCR was done to examine the gene expressions of MMP-2, MMP-9, VEGF, bFGF and CD105 in 70 NMIBC and 40 MIBC patients. Western blotting and immunohistochemical staining were done to check their immunolevels followed by statistical analyses of their expressions with patients' demographic variables. Scanning electron microscopic (SEM) studies were done in representative non-muscle and muscle invasive tumor specimens to elucidate the tumor vasculature and extent of neoangiogenesis. The study reported an increase in gene expression and immunolevels of MMP-2, MMP-9, VEGF, bFGF and CD105-MVD with tumor stage and tumor grade. Statistical studies examined the relevant associations of their expressions with tumor stage, tumor grade, tumor size, tumor type, and tobacco chewing/smoking history of patients. SEM studies revealed marked differences in the vascular architecture and their spatial distribution indicated by increase in vascular density, vascular sprout proliferation and new blood vessel formation with tumor stage and tumor grade. The discriminatory ability of MMP-2, MMP-9, VEGF, bFGF and CD105-MVD in the diagnosis of NMIBC and MIBC was confirmed by ROC curve analysis which revealed the high sensitivity and low specificity of these markers in a given cohort of patients. Observed positive correlations of angiogenic markers with MMP-2 and MMP-9 in the given cohorts of NMIBC and MIBC patients explain their possible effects on bladder tumorigenesis via vascular angiogenesis. Cox regression (univariate and multivariate) and Kaplan-Meier along with log-rank survival analysis examined the strong expressions of these markers as the predictive indicators of poor survival probability (OS, RFS, PFS, and CSS) in 52 NMIBC and 36 MIBC patients. Significant associations of expressions of MMP-2, MMP-9, VEGF, bFGF and CD105-MVD with clinical variables emphasized their significance in diagnosis of NMIBC and MIBC patients. Survival analysis identified these markers as the independent prognosticators of poor survival of NMIBC and MIBC patients. Nevertheless, multi-center analysis is required to validate their importance in the clinical management of NMIBC and MIBC patients.

Keywords: Angiogenesis; Basic fibroblast growth factor; Matrix metalloproteinases; Microvessel density; NMIBC and MIBC patients; Vascular endothelial growth factor.