Abstract
BackgroundDetection 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+).MethodsHER2-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.ResultsThe 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 μm; score 2+, 1.0 ± 0.23 μm; score 3+, 2.14 ± 0.4 μm. The width of precipitates per category matched the optical resolution of the employed microscope objective lenses: Approximately 0.4 μm (40×), 1.0 μm (10×) and 2.0 μm (5×).ConclusionsPerceived 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 5× microscope objectives, 1+ precipitates are too narrow for 10× 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.
Highlights
Detection of The HER2 protein (HER2)/neu receptor overexpression and/or amplification is a prerequisite for efficient anti-HER2 treatment of breast and gastric carcinomas
Carcinomas classified as IHC 2+ were subsequently tested with dualcolor chromogenic in situ hybridization (ISH) for amplification of the HER2/Neu Gene (INFORM HER2 Dual ISH DNA Probe Cocktail Assay, Ventana Medical Systems Inc., Tucson, USA)
HER2-IHC scoring categories reflect the width of DABprecipitates In total, n = 120 cases of invasive breast carcinoma were analyzed which yielded 4800 individual measurements
Summary
Detection of HER2/neu receptor overexpression and/or amplification is a prerequisite for efficient anti-HER2 treatment of breast and gastric carcinomas. A prerequisite for specific treatment is the demonstration of HER2 receptor overexpression by immunohistochemistry (IHC) and/or HER2/neu gene amplification by in-situ hybridization (ISH) [4,5,6]. A central review of 394 HER2 stained specimens from 19 French pathological institutions revealed a false positive rate of 5% but a false negative rate of 27.4% [10]. This problem has recently been addressed by the panelists of the new HER2 testing guideline for gastric and gastroesophageal cancer [5]. It remains unclear to the reader how this particular scoring problem can be resolved in clinical practice
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