HER-2lneu oncogene status and total cellular p I 8SHER-' content were simultaneously analyzed in 4 I 5 invasive breast-cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut-off value of I .7 for the ratio between the intensity of the HER-2lneu gene band and the reference gene band, to consider the HER-2lneu gene amplified, and of 260fmollmg protein, to consider p I 8SHER-' over-expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER-2lneu gene amplification. Of these tumors, 87% showed over-expression of the p18SHER-'. Of the remaining 352 specimens that did not display HER-2lneu gene amplification, 97% showed no p I 8SHER-' over-expression ( p < 0.OOOi). In 40 selected samples with a p18SHER-' level lower than 260 fmollmg protein, the degree of p185HER-2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p18SHER-' level higher than 260 fmollmg protein exhibited a considerable degree of p I 8SHER-' phosphorylation ( p < 0.0001). Our data suggest that: (i) differential PCR and ELISA. which are relatively simple procedures, give similar information on HER-2lneu 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-Zlneu oncogene is amplified or p I 8SHER-' is over-expressed.o 1996 Wiley-Liss, Inc.The HER-21neu proto-oncogene encodes a 185-kDa transmembrane glycoprotein which is a growth-factor receptor with intracellular tyrosine-kinase activity. Overexpression of p185HER-2 leads to receptor dimerization, tyrosinekinase activation, receptor autophosphorylation, and the subsequent activation of kinase substrates involved in the cellular signal-transduction mechanisms, which eventually affects the nuclear transcription of genes regulating cell-cycle progression (Hynes and Stern, 1994). Several studies have shown a strong correlation between HER-2Ineu gene amplification and/or p185HER-2 over-expression, and poor prognosis in breast cancer (Slamon et al., 1987(Slamon et al., , 1989Tandon et al., 1989;Gullick et al., 1991;McCann et al., 1991;Press et al., 1993), although divergent results have also been reported (van de Vijver et al., 1988;Zhou et al., 1989;Borg et al,, 1991;Clark and McGuire, 1991;Berns et al., 1992). HER-2Ineu gene amplification has been studied mainly through the Southern-blotting procedure (Zhou et al., 1989; Slamon et al., 1987Slamon et al., , 1989. A polymerase chain reaction procedure (differential PCR) has been developed to study HER-21neu amplification in cultured cell lines and in solid tumors (Frye et al., 1989;Liu et al., 1992;Neubauer et al., 1992).Studies of p185HER-2 expression have been carried out mainly by immunohistochemistry (Van de Vijver et al., 1988;Gullick et al., 1991;Press et al., 1993) and exceptionally by Western blot (Slamon et al., 1989;Tandon et al., 1989). Both procedures provide a semi-quantitative estimation of oncoprotein...