Approximately 50% of patients presenting with breast cancer have lymph node-negative tumors. Although the overall prognosis for such patients is substantially better than for those with positive nodes, 30% will still succumb to metastatic disease. Experience with node-positive disease suggests that this 'false-negative' patient cohort may derive significant survival benefit from adjuvant systemic therapy. Identification of high- risk node-negative patients is therefore an area of intense clinical interest. Expression of the c-erbB-2 (neu, HER-2) receptor has been proposed as one criterion for predicting the natural history of human breast cancer, but the predictivity of this receptor in early-stage disease is an issue of much debate. Recent observations by my colleagues and I may help establish the true pathogenetic and prognostic significance of the c-erbB-2 phenotype. We have shown that c-erbB-2 receptors may exist in two interconvertible forms - an activated 175kDa tyrosine-phosphorylated form (p175) and an inactive 185kDa tyrosine-dephosphorylated form (p185). The existence of these two forms (which are indistinguishable by conventional c-erbB-2 detection techniques) provides a possible explanation for the clinical polemic mentioned above. I propose to clarify the clinical significance of c-erbB-2 expression in human breast cancer by identifying and quantifying receptor activation in vivo using a novel experimental approach. The project has three objectives: The first objective is to prepare polyclonal antisera against p175 and p185 using c-erbB-2 peptides containing the prime activation and inactivation sites respectively; The second objective is to generate monoclonal antibodies to the intact p175 and p185 molecules obtained by expressing mutant full-length human c- erbB-2 cDNAs in baculovirus; The third objective is to determine the relative expression of p175 and p185 in normal, preinvasive and invasive breast tissue, and thus to establish the biologic and prognostic significance of these c-erbB-2 receptor isoforms in human breast cancer. These objectives are particularly feasible given my current clinical liaison with surgical and pathology services within the Institute, the laboratory availability of an ultrasensitive PhosphorImager for isotope studies, an unlimited supply of high-affinity phosphotyrosine antibody for receptor activity controls, and the experience of myself and my colleagues in the growth factor/tyrosine kinase field. Achievement of these objectives will prepare the way for a more ambitious grant application in which I propose assessing the clinical value of anti-p175 and/or anti-p185 in a prospective study of patients undergoing adjuvant systemic treatment for node-negative breast cancer.