Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients

Abstract

Purpose: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Pre-clinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a) evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b) compare the prognostic significance of VEGF isoforms; and (c) analyze the combined effects of HER-2/neu and VEGF on clinical outcome. Experimental Design: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF121-206 ELISA uses antibodies that bind to VEGF121 and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF165-206 ELISA uses antibodies that bind to VEGF165 and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF121-206 and VEGF165-206 were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Results: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF121-206 and VEGF165-206 expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (Χ2 test: P < 0.001 for both VEGF121-206 and VEGF165-206). VEGF121-206 and VEGF165-206 demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF121-206, P = 0.0068; VEGF165-206, P = 0.0046; multivariate analysis: VEGF 121-206, P = 0.1475; VEGF165-206, P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF121-206 and VEGF165-206 were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF121-206, P = 0.0003; VEGF165-206, P = 0.0038; multivariate analysis: VEGF121-206, P = 0.0103; VEGF 165-206, P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF121-206, P = 0.0280; VEGF165-206, P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF121-206/WEGF 165-206 results in additional prognostic information for survival (VEGF121-206, P = 0.0133; VEGF165-206 P = 0.0092). Conclusion: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.