Clinical significance of the quantitative assessment of the cytosolic concentration of HER-2/neu protein in breast cancer by immunoenzymatic assay (ELISA)

Abstract

Purpose: Retrospective analysis to assess the prognostic and predictive value of HER-2/ neu expression in breast tumors, quantified by enzyme immunoassay (ELISA).

Methods: Quantification of HER-2/neu was performed on cytosolic extracts from 914 cases of primary invasive breast carcinomas. Relapse-free and overall survival data were available from 889 patients. The prognostic value of HER-2/neu levels was assessed considering them as a continuous, dichotomic or quartile variable.

Results: Cytosolic HER-2/neu levels ranged widely in breast carcinomas (median: 746.5 NHU/mg; range: 2.8-80,000 NHU/mg protein). HER-2/neu protein levels were significantly higher in either moderately or poorly differentiated tumors, as well as in those showing a ductal histological type, aneuploidy or a high S-phase fraction. There was a significant and positive association between cytosolic and membranous HER-2/neu levels (n=162, r sub S=0.53; P<0.0001). In addition, cytosolic HER-2/neu level correlated weakly with progesterone receptors but not with estrogen receptors. Elevated cytosolic HER-2/neu levels (> or =1,400 NHU/mg protein) were associated with a high probability of both shortened relapse-free survival and overall survival. This same cut-off value was obtained when we divided the overall group of patients in a training set. However, this HER-2/neu value did not achieve any statistical significance in a validation set used to make sure that the cut-off was correct. Nevertheless, when we divided the obtained data into three different groups with respect to the quartile values (Q) of the intratumoral oncoprotein levels (< or = Q1 vs Q1-Q2 vs > Q3), we observed that patients with either low HER-2/ neu levels (< or = Q1) or high HER-2/neu levels (> Q3) had shorter both relapse-free survival and overall survival curves than those patients with intermediate HER-2/neu levels. On the other hand, high HER-2/neu levels predicted a poor response to adjuvant chemotherapy but not to adjuvant hormonal therapy with tamoxifen.

Conclusions: The results of the present investigation indicate that by quantitatively determining the content of HER-2/neu oncoprotein, groups of high-risk breast cancer patients could be identified, for a more effective clinical management.

Fig. 1

Distribution of HER-2/neu cytosolic levels in 914 patients with breast carcinoma

Fig. 2

Correlation between HER2-/ neu levels in membranous and in cytosolic tumoral samples

Fig. 3

Maximum likelihood determination of the cutoff value for the cytosolic HER-2/neu tumoral content (NHU/mg protein) to predict relapse-free survival in 889 patients with breast cancer. P-values obtained for each cutoff value are plotted against the value itself. Statistical significance is indicated by the horizontal line at the 0.05 level. Analyses lead to the definition of a HER-2/neu content of 1,400 NHU/mg protein as the optimal cutoff (χ² =6.3; P=0.012)

Fig. 4

Relapse-free survival (a) and overall survival (b) as a function of the cut-off value of HER-2/neu cytosolic levels in 889 patients with breast carcinoma

Fig. 5

Relapse-free survival (a) and overall survival (b) as a function of the quartile values of the HER-2/neu cytosolic levels in 889 patients with breast carcinoma

Fig. 6

Relapse-free survival (a) and overall survival (b) as function of the HER-2/neu cytosolic levels in 265 patients with breast carcinoma and chemotherapy as the single systemic adjuvant therapy

Fig. 7

Relapse-free survival (a) and overall survival (b) as function of the HER-2/neu cytosolic levels in 125 patients with breast carcinoma and treated with chemotherapy plus tamoxifen as systemic adjuvant therapy