No abstract

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.

"Receptogram Analysis" has been developed as a pattern-oriented approach for predicting endocrine response in breast cancer based upon quantification of the estrogen receptor immunocytochemical assay (ERICA), using a Quantimet Imaging System. Response prediction was evaluated in 58 stage I11 and IV patients receiving endocrine therapy (primarily Tamoxifen). The Receptogram is a composite of the univariate distributions of nuclear receptor content, IOD(S), and concentration (MOD), and their bivariate contour plot; where (S) is the calculated nuclear radius in section. MOD distributions were classified into four types based upon peak modality and kurtosis (I-IV), and contour plots were classified into four subtypes (A-D) based upon contour slope. Patients failing therapy were ERICA-or their receptogram revealed co-existent ER+ and ER-tumor cells (type 11), highly skewed MOD distributions lacking defined peaks (type IV), or contours with nearly horizontal slope (type C). Response was realized in 9/16 type I patients, with a single positive MOD peak, and in 9/15 type I11 patients, with discrete, multimodal MOD peaks. In contrast, 0/8 type 11,0112 type IV, and 0/10 type C patients were responders. Receptogram analysis was superior to cytosol assay (DCC) as a response discriminant: positive predictive value, 53% vs. 33%; negative predictive value, 100% vs. 75%; sensitivity, 100% vs. 83%; specificity, 68% vs. 23%; and accuracy, 78% vs. 41%, respectively. Alternately, patients were assigned to potentially responsive or nonresponsive groups based upon thresholded mean receptor parameters: field MOD, mean nuclear MOD (NMOD), and mean NMOD(PF) where PF is the ER+ nuclear fraction. While these parameters correlated with DCC (r = .72, 0.69, and 0.69), they were only marginally better in predictive value.Key terms: Steroid receptor heterogeneity, densitometric imaging, monoclonal antibodies, human breast cancer prognosis, peroxidase-antiperoxidase, tamoxifenThe evaluation of estrogen receptor status in breast cancer is a paramount consideration that influences the prognosis and the selection of subsequent anti-estrogen therapy. The recent availability of monoclonal antibodies to estrogen receptor protein (ER) (13)(14)(15)17,18,24) has led to the development of an immunocytochemical assay (ERICA) (24) for detecting ER based upon the indirect immunoperoxidase technique (21,311. This approach provides the specificity required for sensitive detection of high-affinity type I ER (18,34) and the reaction stoichiometry for densitometric evaluation of concentration differences on a true ratio scale (32). Since the ER monoclonal antibodies bind to epitopes distal to the steroid binding site, masking of receptors by steroid or anti-steroid does not lower assay values as in the traditional dextran-coated charcoal method (DCC) (17,23,34). The methodology has revealed heterogeneity of receptor staining in breast cancer cells in many patients with ER+ cytosol assays 'This work was supported by American Cancer Society grant PDT317A (R.J.S.)...