Physical basis of the 'magnification rule' for standardized Immunohistochemical scoring of HER2 in breast and gastric cancer

Background: Detection of HER2/neu receptor overexpression and/or amplification is a prerequisite for efficient anti-HER2 treatment of breast and gastric carcinomas. Immunohistochemistry (IHC) of the HER2 protein is the most common screening test, thus precise and reproducible IHC-scoring is of utmost importance. Interobserver variance still is a problem; in particular in gastric carcinomas the reliable differentiation of IHC scores 2+ and 1+ is challenging. Herein we describe the physical basis of what we called the 'magnification rule': Different microscope objectives are employed to reproducibly subdivide the continuous spectrum of IHC staining intensities into distinct categories (1+, 2+, 3+). Methods: HER2-IHC was performed on 120 breast cancer biopsy specimens (n = 40 per category). Width and color-intensity of membranous DAB chromogen precipitates were measured by whole-slide scanning and digital morphometry. Image-analysis data were related to semi-quantitative manual scoring according to the magnification rule and to the optical properties of the employed microscope objectives. Results: The semi-quantitative manual HER2-IHC scores are correlated to color-intensity measured by image-analysis and to the width of DAB-precipitates. The mean widths +/- standard deviations of precipitates were: IHC-score 1+, 0.64 +/- 0.1 mu m; score 2+, 1.0 +/- 0.23 mu m; score 3+, 2.14 +/- 0.4 mu m. The width of precipitates per category matched the optical resolution of the employed microscope objective lenses: Approximately 0.4 mu m (40x), 1.0 mu m (10x) and 2.0 mu m (5x). Conclusions: Perceived intensity, width of the DAB chromogen precipitate, and absolute color-intensity determined by image-analysis are linked. These interrelations form the physical basis of the 'magnification rule': 2+ precipitates are too narrow to be observed with 5x microscope objectives, 1+ precipitates are too narrow for 10x objectives. Thus, the rule uses the optical resolution windows of standard diagnostic microscope objectives to derive the width of the DAB-precipitates. The width is in turn correlated with color-intensity. Hereby, the more or less subjective estimation of IHC scores based only on the staining-intensity is replaced by a quasi-morphometric measurement. The principle seems universally applicable to immunohistochemical stainings of membrane-bound biomarkers that require an intensity-dependent scoring.