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

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.Electronic supplementary materialThe online version of this article (10.1186/s13000-018-0696-x) contains supplementary material, which is available to authorized users.

Hyperinsulinemia Promotes Esophageal Cancer Development in a Surgically-Induced Duodeno-Esophageal Reflux Murine Model

“…On the basis of our previous retrospective study [ 18 ], we hypothesized the involvement of other growth factor receptors and their cooperation with the insulin/insulin-like growth factor-1 axis in the evolution of these tumors. HER2 overexpression plays a critical role in the development, progression and metastasis of many malignancies such as breast cancer [ 12 , 13 , 19 ], gastric cancer [ 20 , 21 ] and esophageal cancer [ 18 , 22 ]. To evaluate whether in our carcinogenesis model (related exclusively to duodenal content chronic reflux) that HER2 could play a role, HER2 total protein expression was evaluated in esophageal tissue on both non-operated and operated mice.…”

Comparison of HercepTest™ mAb pharmDx (Dako Omnis, GE001) with Ventana PATHWAY anti-HER-2/neu (4B5) in breast cancer: correlation with HER2 amplification and HER2 low status

“…For cases of IHC 1 + staining intensity (i.e., faint/barely perceptible membrane staining), the percentage of stained cells ≤ 10% or > 10% was recorded separately according to Ventana Instructions for Use (IFU) [ 22 ]. Intensity scoring was performed by applying the magnification rule as published previously by our group [ 24 , 25 ].…”

Unusual Patterns of HER2 Expression in Breast Cancer: Insights and Perspectives

“…Especially when it comes to distinguish between scores 1+ and 2+, the subjectivity of the interpretation of the scoring may lead to discordant results among pathologists and/or institutions. Given the central role of IHC HER2 evaluation in guiding therapy, some authors have developed the so-called magnification rule [4]. The latter uses the different magnifications of the objectives to perform a sort of triage across scores.…”