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Activation State-specific Monoclonal Antibody Detects Tyrosine Phosphorylated p185^neu/erbB-2 in a Subset of Human Breast Tumors Overexpressing This Receptor¹

Departments of Pathology [M. P. D., D. F. S.] and Internal Medicine [M. P. D.], Yale University School of Medicine, New Haven, Connecticut 06510

Amplification of the neu/erbB-2/HER-2 gene and/or overexpression of its receptor tyrosine kinase product, p185^neu/erbB-2 (p185), occurs in 15–40% of primary human breast tumors and is variably correlated with poor patient prognosis. Variability in predictive accuracy likely results from activation of p185 by agonist(s) in only a subset of tumors in which it is overexpressed, which may greatly affect outcomes. As a first step toward evaluating this hypothesis, we previously produced a polyclonal antibody that specifically recognizes the activated, tyrosine-phosphorylated forms of p185 and the closely related epidermal growth factor receptor (L. Bangalore et al., Proc. Natl. Acad. Sci. USA, 89: 11637–11641, 1992). We now describe the production of a mAb, PN2A, that specifically recognizes tyrosine-phosphorylated p185 and bears no cross-reactivity with closely related receptors. Furthermore, we demonstrate its reactivity in immunohistochemical staining of paraffin-embedded, formalin-fixed tumor samples. In a series of five p185-overexpressing human tumors examined thus far, PN2A reactivity was detected in two, indicating that p185 is phosphorylated and hence actively signaling in a subset. This reagent will facilitate both clinical and research analyses of p185 activity. Furthermore, this work serves as a prototype for similar analyses of other tyrosine phosphoproteins.

¹ Supported in part by grants from the Mathers Foundation and USPHS Grants CA45708 and SPORE 5-P20-CA58202. M. P. D. has a postdoctoral fellowship research award from the United States Army Medical Research and Development Command (Grant DAMD17-94-J-4135). The content of the information contained herein does not necessarily reflect the position or the policy of the United States Government, and no official endorsement should be inferred.

² To whom requests for reprints should be addressed.

Received 11/ 2/94. Accepted 3/ 6/95.

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