Automated Quantitative RNA in Situ Hybridization for Resolution of Equivocal and Heterogeneous ERBB2 (HER2) Status in Invasive Breast Carcinoma Zhen.

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Automated Quantitative RNA in Situ Hybridization for Resolution of Equivocal and Heterogeneous ERBB2 (HER2) Status in Invasive Breast Carcinoma Zhen Wang, Bryce P. Portier, Aaron M. Gruver, Son Bui, Hongwei Wang, Nan Su, Hong-Thuy Vo, Xiao-Jun Ma, Yuling Luo, G. Thomas Budd, Raymond R. Tubbs The Journal of Molecular Diagnostics Volume 15, Issue 2, Pages 210-219 (March 2013) DOI: 10.1016/j.jmoldx.2012.10.003 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Automated quantitative RNA ISH. A: Consecutive sections of an HER2-positive breast cancer FFPE block were stained for HER2 mRNA by fully automated and manual RNAscope assays. dapB, bacterial gene as negative control. Brown granules are HER2 mRNA signals detected by horseradish peroxidase–DAB. Blue, nuclei counterstained with hematoxylin. Original magnification, ×10. B: Quantification of RNAscope-stained slides. Left panel, image of a scanned RNAscope slide stained for POLR2A. Brown punctate dots are DAB signals. Nuclei are counterstained blue. Right panel, single-cell level quantification of mRNA. Green squares denote individual signal dots identified by TargetQuant software. Black lines outline cell boundaries. Original magnification, ×40. The Journal of Molecular Diagnostics 2013 15, 210-219DOI: (10.1016/j.jmoldx.2012.10.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Analysis of HER2 mRNA in training and validation sets by automated quantitative RNAscope HER2 assay and qPCR analysis. A and B: Examples of RNAscope staining of HER2-positive (A) and HER2-negative (B) cases. Original magnification, ×20. C: Distribution of RNAscope HER2 scores in the training set according to HER2 status by FISH and IHC. The horizontal line at 0.375 denotes the cutoff separating HER2-positive and HER2-negative cases. D: Distribution of qPCR HER2 values in the training set, according to HER2 status by FISH and IHC. The horizontal line at 7.0 denotes the cutoff for separating HER2-negatives cases from HER2-positive cases. E: Distribution of RNAscope HER2 scores in the validation set according to HER2 status by FISH and IHC. The horizontal line at 0.375 denotes the cutoff. F: Distribution of qPCR HER2 values in the validation set according to HER2 status by FISH and IHC. The horizontal line at 7.0 denotes the cutoff. G and I: Pairwise scatterplot between RNAscope and FISH ratio (G), between qPCR and FISH ratio (H), and between RNAscope and qPCR (I) in the validation set. The Journal of Molecular Diagnostics 2013 15, 210-219DOI: (10.1016/j.jmoldx.2012.10.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Resolution of FISH-equivocal cases by automated quantitative RNAscope HER2 assay and qPCR analysis. A: Distribution of RNAscope HER2 scores according to reflex HER2 IHC testing results. B: Distribution of qPCR HER2 values according to reflex HER2 IHC testing results. The Journal of Molecular Diagnostics 2013 15, 210-219DOI: (10.1016/j.jmoldx.2012.10.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Representative images of breast carcinomas showing heterogeneous amplification/expression of HER2. A: H&E staining. B: HER2 IHC staining (brown). C: RNAscope HER2 mRNA staining (brown). D: HER2 Dual ISH staining. Black, HER2. Red, CEP17. Original magnification, ×20. The Journal of Molecular Diagnostics 2013 15, 210-219DOI: (10.1016/j.jmoldx.2012.10.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions