Simultaneous analysis of c‐erb B‐2 expression and DNA content in breast cancer using flow cytometry

Abstract

The c-erbB-2 oncogene is frequently amplified and overexpressed in human breast cancer. We have studied the c-erbB-2 protein in conjunction with DNA content in frozen samples from breast cancers using flow cytometry. The cell suspensions were obtained by mechanical disaggregation followed by a short fixation in 1% paraformaldehyde. The level of c-erbB-2 expression was calculated as a fluorescence index, taking into account the relative amount of total cellular fluorescence compared to non-specific fluorescence. The flow cytometric value correlated with immunohistochemical results obtained with the same monoclonal antibody (c-neu, clone 9G6). Overexpression of c-erbB-2 was significantly more frequent in DNA aneuploid tumors than in DNA diploid ones and correlated with increasing S-phase fraction and estrogen receptor negativity. In 10 DNA multiploid tumors, the different aneuploid stemlines uniformly expressed c-erbB-2, supporting the hypothesis that overexpression is an early event in breast cancer. Of the 172 tumors, the 37 (22%) judged as positive with immunohistochemistry showed a somewhat higher rate of distant recurrence than others (P = 0.14). The fluorescence index was significantly associated with prognosis (P = 0.0012), as it was also among the immunohistochemically positive cases. If the degree of overexpression is important, then flow cytometry could be a feasible technique for classification.