Determination of Cytosol c-erbB-2 Protein in Breast Cancer by Sandwich Enzyme Immunoassay

Introduction

Many investigators have indicated that overexpression and amplification of human proto-oncogene c-erbB-2 (HER-2/neu) is an independent prognostic factor of primary breast cancer (1-5). Overexpression of c-erbB-2 protein and the gene amplification in breast cancer tissues have been determined by immunohistochemical staining (IHC) or Western or Southern blotting. IHC of c-erbB-2 protein in breast cancer tissues has been especially widely used, since this method is easily performed. However, the evaluation of staining intensity and localization of c-erbB-2 protein in cancer cells has not been sufficiently standardized to predict a poor prognosis of breast cancer (6-8). Sugano et al. (9) presented a new technique to determine the levels of cytosol c-erbB-2 protein in breast tissues using a sandwich enzyme immunoassay (EIA) and demonstrated a strong positive correlation between overexpression of cytosol c-erbB-2 protein and the gene amplification in breast cancer. In the present study, we measured the levels of cytosol c-erbB-2 protein in benign and malignant breast tissues using this sandwich EIA and evaluated the relationship between overexpression of c-erbB-2 protein and clinicopathological factors.

Materials and Methods

Patients

One hundred and sixty nine cases that had been treated at the National Cancer Center Hospital  between September 1994 and December 1995 were analyzed. Patients' characteristics are shown in Table 1. Patients with stages I-IIIB breast cancer underwent radical mastectomy or quadrantectomy with axillary dissection, patients with benign breast disease had an excisional biopsy and patients with stage IV or recurrent breast cancer had an incisional biopsy of the primary sites or tumorectomy of the recurrent sites. From the resected specimens, pathological diagnosis was made on paraffin-embedded hematoxylin-eosin-stained sections and biological markers (hormonal receptor status and c-erbB-2 protein level) were examined.

Table1. Patients' characteristics

Histological grade was determined based on the number of mitoses and architectural and cytological atypia determined by a modified Bloom and Richardson grading system (10). Estrogen receptor (ER) and progesterone receptor (PR) in the cytosol fraction were determined by a dextran-coated charcoal assay . The higher limit cut-off value of each assay is 5 fmol/mg protein. Serum tumor markers (CEA, CA15-3) were examined before surgical treatment.

Measurement of Cytosol c-erbB-2 Protein

Breast tissues and recurrent tumors were kept below -80°C until used. They were weighed, minced with scissors and homogenized with a sevenfold volume of TED buffer (pH 7.4) containing 10 mM Tris-HCl, 3 mM NaN₃, 12 mM thioglycerol, 10% glycerol and 1 mM Na₂EDTA. The homogenate was centrifuged at ×105 000 g for 60 min and the cytosol fraction was obtained from the supernatant. Protein concentration was determined by Lowry's method using bovine serum albumin as a standard.

Two monoclonal antibodies that react with the extracellular domains of c-erbB-2 protein, 6G10 and SV-2-61 [gamma] (Nichirei, Tokyo, Japan), were used for the sandwich EIA (11,12). The cytosol fraction was diluted twofold with TED buffer before use as a sample for measurement. The 6G10-coated polystyrene bead was incubated with a mixture of 50 µl of the sample or standard and 200 µl of PBS-based reaction buffer for 2 h at room temperature. The bead was washed in 2 ml of saline and then incubated with 200 µl of horseradish peroxidase-labeled SV-2-61 [gamma] (Fab)₂ (0.95 ng/µl) for 2 h at room temperature. The bead was washed in saline again and incubated with 300 µl of a mixture of o-phenylenediamine dihydrochloride (OPD) (2 mg/ml) and sodium perborate for 30 min at room temperature. After the reaction had been stopped by adding 2 ml of phosphoric acid, the absorbance of the reaction mixture was measured at 492 nm. The level of c-erbB-2 protein was calculated from a calibration graph constructed with the use of recombinant c-erbB-2 (Nichirei). The cut-off level of c-erbB-2 protein was set at 18 ng/mg protein, since this level was correlated with c-erbB-2 gene amplification detected by dot blot hybridization or differential polymerase chain reaction (9).

Statistical significance was determined by the Kruskal-Wallis test for differences between c-erbB-2 protein levels and clinical stages and by the chi-squared test or Fisher's exact probability test for differences between c-erbB-2 protein expression and clinicopathological factors in breast cancer.

Results

The distribution of c-erbB-2 protein levels examined in this study is illustrated in Fig. 1. The levels of c-erbB-2 protein in benign breast disease were all <18 ng/mg protein. The mean and median levels of c-erbB-2 protein in stages I-IIIB breast cancer, stage IV breast cancer and recurrent breast cancer were 18.7; 7.1, 60.6; 7.9, and 82.5; 21.1 ng/mg protein, respectively. The distribution of c-erbB-2 protein levels was significantly different between the three groups of breast cancer (p < 0.05 for the Kruskal-Wallis test). In addition, c-erbB-2 overexpression ([ge]18 ng/mg protein) in these groups was shown in 26 of the 139 cases of stages I-IIIB breast cancer (18.7%), four of the 12 cases of stage IV breast cancer (33.3%) and seven of the 13 recurrent cases (53.8%) (Table 2). The positive rates of c-erbB-2 overexpression were also statistically different between the groups (p = 0.0097 for the chi-squared test).
 

Figure1. Levels of cytosol c-erbB-2 protein in benign breast disease and breast cancer. The levels of the range and mean ± standard deviation in benign breast disease, stages I-IIIB breast cancer, stage IV breast cancer and recurrent breast cancer were 0 to 11.6 and 4.0 ± 4.7; 0 to 327.9 and 18.7 ± 42.2; 0 to 315 and 60.6 ± 107.6; and 3.4 to 368.8 and 82.5 ± 11.3 ng/mg protein, respectively. The distribution of c-erbB-2 protein levels was significantly different between stages I-IIIB breast cancer, stage IV breast cancer and recurrent breast cancer (p < 0.05, Kruskal-Wallis test).

Table2. Overexpression of c-erbB-2 protein in breast cancer

NIDC, non-invasive ductal carcinoma; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma.
*Positive cases had cytosol c-erbB-2 protein levels of [ge]18 ng/mg protein. ^[dagger]Analysis of significance by the chi-squared test. ^[Dagger]Three of the four positive cases had invasive ductal carcinoma with a predominantly intraductal component.

Table3. Correlation between c-erbB-2 expression and ER or PR status

*Positive cases had cytosol c-erbB-2 protein levels of [ge]18 ng/mg protein. ^[dagger]Analysis of significance by the chi-squared test or Fisher's exact probability test.

In stages I-IIIB breast cancer, overexpression of c-erbB-2 protein was associated with histological grade and serum CEA level, but not with other clinicopathological factors: menopausal status, tumor size, clinical stage, histological subtype, lympho-vascular invasion, nodal metastasis, intraductal component in cancer lesion and serum CA15-3 level (Table 2).

The correlation between c-erbB-2 protein expression and hormonal receptor status is summarized in Table 3. There was no correlation between c-erbB-2 expression and ER or PR status in stages I-IIIB breast cancer, stage IV breast cancer or recurrent breast cancer. However, there was an inverse correlation between c-erbB-2 overexpression and ER in the group of stages I-IIIB plus IV breast cancer (p = 0.0369 for the chi-squared test).

Discussion

We determined the levels of cytosol c-erbB-2 protein in breast cancer by sandwich EIA. This method is a standard method and therefore c-erbB-2 overexpression can be easily determined in the cytosol fraction together with hormonal receptors. Overexpression of c-erbB-2 protein determined by sandwich EIA was detected in 12.3-18.5% of primary breast cancer (9,13-15) and we also found it in 18.7% of stages I-IIIB breast cancer cases. In the present study, four of the 10 cases with HG 1 breast cancer overexpressed c-erbB-2 protein and three of them were invasive ductal carcinoma with a predominantly intraductal component including comedo carcinoma. A similar result was found in comedo carcinoma in situ (6). However, histological grades 2 and 3 were marginally associated with overexpression of c-erbB-2 protein (p = 0.066 for the chi-squared test).

The positive rate of c-erbB-2 protein expression was higher in advanced breast cancer than in operable breast cancer. These results suggest that c-erbB-2 overexpression of breast cancer is associated with aggressive behavior and that targeting therapies to neutralize c-erbB-2 gene product may be more indicated for advanced breast cancer patients than for early breast cancer patients.

Overexpression of c-erbB-2 protein in breast cancer is reported to be inversely related to ER and PR status in many reports (1-5,7,9,13-15). The present study, however, showed an inverse correlation between c-erbB-2 protein levels and ER status in the group of stages I-IIIB plus IV breast cancer, but not in stages I-IIIB breast cancer. This discrepancy may be explained in two ways. First, c-erbB-2 protein levels in the cytosol fraction by EIA may not be completely parallel to the membrane staining intensity by IHC on paraffin-embedded tissues (16). Second, advanced breast cancer, in which c-erbB-2 overexpression frequently occurs, can have the phenotype of hormone-independent cell growth and it tends to develop into ER-negative and PR-negative breast cancer (Table 3). A recent report indicates that overexpression of HER-2 (c-erbB-2) receptor in ER-positive tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcript (17).

The prognostic importance of the overexpression of c-erbB-2 protein in this study remains to be determined because of the short follow-up period. In addition to the cytosol c-erbB-2 protein levels, serum c-erbB-2 protein levels in the same breast cancer patients are being examined periodically and will be analyzed for their predictive recurrence value. In a recent randomized trial, overexpression of c-erbB-2 protein was associated with response to doxorubicin-based chemotherapy for operable breast cancer patients and resulted in survival benefits for patients receiving high-dose chemotherapy (18). Whether overexpression of c-erbB-2 protein in primary and metastatic breast cancer has an influence on the responsiveness to chemohormonal therapy or not is under investigation (19-21). We have ongoing clinical trials with Amyloxine in breast cancer patients, who are randomized by determination of cytosol c-erbB-2 protein levels using this sandwich EIA. This issue also will be evaluated in the future.

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Abbreviations: IHC, immunohistochemical staining; EIA, enzyme immunoassay; ER, estrogen receptor; PR, progesterone receptor; CEA, carcinoembryonic antigen; CA15-3, carbohydrate antigen 15-3