ERBB-2 (HER2/NEU) GENE COPY NUMBER, P185(HER-2) OVEREXPRESSION, AND INTRATUMOR HETEROGENEITY IN HUMAN BREAST-CANCER

Amplification of the ERBB-2 (HER-2/neu) gene is accompanied by overexpression of its cell surface receptor product, p185(HER-2), Heterogeneity has been observed for both the gene copy number and the level of overexpression of its protein product. To better understand their relationship, correlation between the level of cellular expression of p185(HER-2) and ERBB-2 gene amplification was studied in four human breast cancer cell lines (BT-474, SK-BR-3, MDA-453, and MCF-7) and in a primary human breast tumor sample. The relative expression of p185(HER-2) was measured by immunofluorescence by using flow and/or image cytometry while correlated DNA analysis was performed on the same cells by fluorescence in situ hybridization to determine ERBB-2 gene and chromosome 17 copy numbers. Marked heterogeneity was observed in both protein expression and ERBB-2 copy number. Despite this heterogeneity, and in accordance with previous studies, the average levels of p185(HER-2) expression correlated well with average ERBB-2 gene copy numbers in the four lines examined (r = 0.99), When the relationship between copy number and protein expression was studied on a cell-by-cell basis, p185(HER-2) expression correlated with both the absolute number of ERBB-2 gene copies/cell (r = 0.59-0.63) and chromosome 17 copy number (r = 0.45-0.61). It is of interest that there was weak or no correlation between p185(HER-2) protein expression and the ERBB-2 copy number:chromosome 17 copy number ratio (r = 0.0-0.25). In more than one-half of cells expressing a high level of p185(HER-2), the chromosome 17 copy number was high (two or three times the average copy number), whereas <2% of an unselected population had a high chromosome 17 copy number. Bromodeoxyuridine incorporation indicated that the S-phase-labeling index was homogeneous across various p185(HER-2)-expressing subpopulations in the SK-BR-3 cell line. Analysis of the primary breast tumor sample showed results similar to the cell lines, supporting the strong possibility of a mechanistic link among p185(HER-2) overexpression, ERBB-2 amplification, and high chromosome 17 copy number.