Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma

Abstract

We examined the expression levels of a number of metastasis-related genes to determine the relationship of these levels to the development of metastasis in renal cell carcinoma. Gene expression was examined in 46 formalin-fixed, paraffin-embedded, archival specimens of primary organ-confined, clear-cell, renal cell carcinoma from patients who had undergone radical nephrectomy. Twenty samples were from patients who did not have metastasis after a median of 48 months; 26 were from patients with either synchronous or metachronous metastases. Microvessel density was assessed by anti-CD-34 immunohistochemical analysis. The expression levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), matrix metalloproteinases (MMP)-2 and -9, and E-cadherin were examined at the periphery of the tumor by a colorimetric in situ mRNA. The expression levels of bFGF, VEGF, IL-8, MMP-2, and MMP-9 were significantly higher in primary renal tumors from patients with either synchronous or metachronous metastases than those who were disease-free at a median of 48 months of follow-up. Multivariate analysis of disease-free survival showed that the ratio of MMP-9 to E-cadherin (P = 0.012) and the expression level of bFGF expression (P = 0.045), were independent predictors for the development of metastases. The expression levels of bFGF, VEGF, and IL-8 did not correlate with microvessel density, which in itself was not a significant predictor of progression (P = 0.21). In summary, expression levels of genes that regulate metastasis angiogenesis can predict the metastatic potential in individual patients with organ-confined clear-cell renal carcinoma.

Figure 1.

In situ hybridization analysis of angiogenic factors in nonmetastatic and metastatic RCC (original magnification, ×200). Hybridization with a hyperbiotinylated poly(dT)₂₀ probe confirmed the integrity and lack of mRNA degradation. A positive reaction in this assay stains red. The expression intensity of each factor was determined in both cells at the periphery of the tumor and divided by the expression in normal proximal tubular epithelium. This value was normalized to the expression of poly(dT)₂₀ in neoplastic and normal tissue. Note higher expression of bFGF and VEGF relative to poly(dT) in metastatic primary tumor. In addition, an example of anti-CD-34 immunohistochemistry in organ-confined renal primary tumors from both nonmetastatic and metastatic groups. Note similar vascular density in both tumors.

Figure 2.

Correlation of MVD and the expression of angiogenic factors. The absolute value of MVD in each tumor specimen did not correlate with the relative intensity of bFGF, VEGF, or IL-8.

Figure 3.

Scattergram of angiogenic factor expression in primary tumors from nonmetastatic and metastatic groups. The optimal cutoff values for relative gene expression as a predictor for recurrence were 150%, 130%, and 120% for bFGF, VEGF, and IL-8, respectively.