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

ABSTRACT: 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+).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.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 ?m; score 2+, 1.0?±?0.23 ?m; score 3+, 2.14?±?0.4 ?m. The width of precipitates per category matched the optical resolution of the employed microscope objective lenses: Approximately 0.4 ?m (40×), 1.0 ?m (10×) and 2.0 ?m (5×).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 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.

SUBMITTER: Scheel AH

PROVIDER: S-EPMC5848460 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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

Scheel Andreas H AH Penault-Llorca Frédérique F Hanna Wedad W Baretton Gustavo G Middel Peter P Burchhardt Judith J Hofmann Manfred M Jasani Bharat B Rüschoff Josef J

Diagnostic pathology 20180312 1

<h4>Background</h4>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 ...[more]

PMID: 29530054

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Project description:Hormones and growth factors accelerate cell proliferation of breast cancer cells, and these molecules are well investigated targets for drug development and application. The mechanisms of cell proliferation of breast cancers lacking estrogen receptor (ER) and HER2 have not been fully understood. The purpose of the present study is to find genes that are differentially expressed in breast cancers and that might significantly contribute to cell proliferation in these cancers. Forty tumor samples, consisting of ten each of immunohistochemically ER(+)/HER2(-), ER(+)/HER2(+), ER(-)/HER2(+), and ER(-)/HER2(-) cancer were analyzed using oligonucleotide microarrays. Both genes and tumor samples were subjected to hierarchical clustering. ER(+)/HER2(-) breast cancers and ER(-)/HER2(-) cancers tended to form a tumor cluster, but HER2 positive breast cancers were split into different tumor clusters. Significant differential expression between IHC-ER(-)/HER2(-) and other tumors was defined as having an expression level at least 2-fold higher or 2-fold lower, and analyzed by multi-step two-way ANOVA. Genes overexpressed differently in IHC-ER(-)/HER2(-) breast cancers compared to other all three types were 8 genes (FABP7, GABRP, GAL, CXCL13, CDC42EP4, C2F, FOXM1, CSDA), and underexpressed genes were nine including ITGB5, KIAA0310, MAGED2, PRSS11, SORL1, TGFB3, KRT18, CPE, BCAS1. No gene was directly related to cell proliferation such as cyclins, cyclin-dependent kinase, p53, p16, and the pRb and p21 families. We had a particular focus on a transcriptional factor E2F-5 from a list of genes overexpressed in ER negative breast cancers compared to ER positive breast cancers, and further examined. Gene amplification of E2F-5 was detected in 5/57 (8.8%) in breast cancers by FISH. No point mutation was found at the binding domain with DNA or dimerization partner of E2F-5. Immunohistochemically E2F-5 positive cancers were more frequent in ER(-)/HER2(-) cancer (14/27, 51.9%) than in other types of cancer (5/30, 16.7%) (p=0.05). E2F-5 positive cancers had higher Ki-67 labeling index (59.5%) than E2F-5 negative cancers (36.3%). E2F-5 positive cancers showed higher histological grade including metaplastic carcinoma, and worse clinical outcome with shorter disease free survival in node negative patients. In conclusion, we demonstrated that there is a population of breast cancer with overexpression of a cell cycle related transcriptional factor E2F-5. E2F-5 positive breast cancers were frequent in ER(-)/HER2(-) group with high Ki-67 labeling index, high histological grade and worse clinical outcome. Keywords: immunohistochemical phenotype

Dataset Information

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

Publications

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

Similar Datasets

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