Comparison of microscopic imaging strategies for evaluating immunocytochemical (PAP) steroid receptor heterogeneity.

Receptogram analysis was compared with three other imaging strategies for immunocytochemical assay of estrogen receptors. These included nuclear-specific methods for analysis of nuclear integrated optical density (IOD) or mean optical density (MOD) histograms, and field-specific methods, where the pixel optical density (POD) histogram was evaluated for the composite nuclear phase. Measurements in culture and in breast cancer cryosections were treated separately to isolate geometric considerations. In culture receptograms the modality of IOD and MOD histograms and their bivariate contour maps revealed one, two, or more subpopulations with discrete receptor content and concentration. However, when the field of nuclei was imaged as a whole, regardless of the number of subpopulations, POD histograms showed two minima, defining three intranuclear phases. This was due to mottling and variegation of intranuclear chromatin and nucleolar immunostaining and not to differences between nuclei. These limitations were also revealed in breast cancer sections. In POD histograms, % unstained pixels did not provide a reliable estimate of % receptor negative nuclei, as determined by their enumeration. In sections, correction of IOD for nuclear volume variability was essential to suppress artifactual peaks not representing differences in receptor content. This was achieved by multiplying nuclear IOD by the spherical nuclear radius (S) of individual slab sections. Peaks of IOD(S) then reflected receptor content on a true ratio scale. Only receptogram analysis, which incorporates these strategies, permitted objective evaluation of receptor heterogeneity at the level of tumor subpopulations.

Quantitative imaging of immunocytochemical (PAP) estrogen receptor staining patterns in breast cancer sections.

"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 III 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 II), 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 III patients, with discrete, multimodal MOD peaks. In contrast, 0/8 type II, 0/12 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 non-responsive 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.