Immunohistochemical analysis of the topographical relationship between the estrogen- and progesterone receptor in five human breast cancers. A simultaneous demonstration of both nuclear receptors in the same section by using a computer-assisted image processing.

A technique is described which allows precise assessment of the topographical relationship between the estrogen receptor (ER) and the progesterone receptor (PR) in the same histological section. It is based on the analysis of the results of immunohistochemical double staining by computer-assisted image processing. Five human ductal breast cancers were examined. The simultaneous demonstration of both receptors consists in the following principal steps: The primary antisera against the ER (monoclonal rat antibody) and the PR (monoclonal mouse antibody) are incubated simultaneously, but only the anti-ER antibody is demonstrated in the first staining step by using a goat anti-rat antibody as the linking antibody and the PAP complex from the rat, both antisera from the ER-ICA kit. The result is stored as a digitized grey image ("1. object image"). Then the colored end product and the residual peroxidase activity of the PAP complex are removed. In the second staining sequence the anti-PR-antibody is demonstrated by using a rabbit anti-mouse antibody as the linking antibody and the PAP complex from the mouse. The result is exactly positioned and also stored as a digitized grey image ("2. object image"). Though antibodies raised in different species were used, cross-reactivity could not be avoided. Grey values generated by cross-reactivity between the different antibody systems are evaluated in negative controls and are eliminated in the object images. The remaining (specifically stained) structures of both object images are copied into a final image so that the topographical relationship of the ER and the PR becomes obvious. The results show that in the five carcinomas investigated three types of receptor-positive tumour cells can be distinguished: Cells which coexpress the ER and the PR (1), cells which express either the ER (2) or the PR (3). The number of tumour cells showing one of these expression patterns varies from tumour to tumour.