Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

Abstract

HER-2/neu oncogene status and total cellular p185(HER-2) content were simultaneously analyzed in 415 invasive breast-cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut-off value of 1.7 for the ratio between the intensity of the HER-2/neu gene band and the reference gene band, to consider the HER-2/neu gene amplified, and of 260 fmol/mg protein, to consider p 185(HER-2) over-expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER-2/neu gene amplification. Of these tumors, 87% showed over-expression of the p185(HER-2). Of the remaining 352 specimens that did not display HER-2/neu gene amplification, 97% showed no p185(HER-2) over-expression (P < 0.0001). In 40 selected samples with a p185(HER-2) level lower than 260 fmol/mg protein, the degree of p185(HER-2) phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185(HER-2) level higher than 260 fmol/mg protein exhibited a considerable degree of p185(HER-2) phosphorylation (P < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER-2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER-2/neu oncogene is amplified or p185(HER-2) is over-expressed.