Identification of HER2 Immunohistochemistry-Negative, FISH-Amplified Breast Cancers and Their Response to Anti-HER2 Neoadjuvant Chemotherapy

I-sanna Gibbons-Fideler, MD; Hiroaki Nitta, PhD; Adrian Murillo, PhD; Gary Tozbikian, MD; Peter Banks, MD; Anil V. Parwani, MD, PhD; Zaibo Li, MD, PhD

Disclosures

Am J Clin Pathol. 2019;151(2):176-184. 

In This Article

Materials and Methods

Patient Cohort and Specimens

This study was approved by the Ohio State University Institutional Review Board. A total of 1,107 consecutive invasive BCs with both HER2 IHC and FISH results on core biopsy specimens during a 3-year period (November 2013 to December 2016) were included. HER2 status was determined by HER2 IHC (clone 4B5; Ventana) and HER2 FISH using the dual-color Vysis FDA-approved PathVysion HER2 DNA Probe Kit (Abbott Molecular). One hundred tumor cells were scored using computer assistance (BioView; Abbott) to enumerate SpectrumOrange (O) HER2 and SpectrumGreen (G) centromeric chromosome 17 (CEP17) signals. Mean HER2/CEP17 ratio was calculated by BioView from all selected cells, with results confirmed by fluorescence microscopy. Cells with no signals or signals of only one color (ie, 1G and 1O) were not scored. The HER2 IHC results were interpreted by subspecialized breast pathologists, and HER2 FISH results were interpreted by subspecialized molecular pathologists based on the 2013 ASCO/CAP HER2 testing guideline.[9]

HER2 Gene Protein Assay

To confirm the HER2 IHC and FISH results, the recently developed gene protein assay (GPA), combining HER2 IHC and dual in situ hybridization (ISH), was performed on fresh-cut sections from original blocks according to a previously described method.[11–13] The GPA combines PATHWAY anti-HER2/neu (4B5) rabbit monoclonal primary antibody (Ventana) for IHC and the INFORM HER2 Dual ISH DNA probe cocktail (Ventana) for ISH. Briefly, HER2 IHC was performed first using the iVIEW DAB IHC Detection Kit (Ventana), then followed by hybridization using a cocktail of the 2,4 dinitrophenyl (DNP)–labeled HER2 probe and digoxigenin (DIG)–labeled chromosome 17 centromere (CEN17) probe. The HER2 gene and CEN17 signals were detected using the ultraView Silver ISH DNP Detection Kit and ultraView Red ISH DIG Detection Kit (Ventana), respectively. HER2 IHC and ISH signals were visualized with conventional light microscopy in which HER2 protein showed brown staining in the cell membranes, while the HER2 gene appeared as discrete black signals and CEN17 as red signals in the nuclei. GPA results were interpreted according to the 2013 HER2 ASCO/CAP guidelines for both HER2 IHC and HER2 ISH.[9] HER2 gene and CEN17 signals were enumerated by counting at least 50 nuclei, and the areas with stronger IHC staining were selected for counting in cases with intratumoral heterogeneity.

HER2 RNA ISH

HER2 RNA ISH was performed to evaluate HER2 RNA levels. The slides were dewaxed and pretreated using the RNAScope Sample Prep kit (Advanced Cell Diagnostics) according to the manufacturer's recommendations. The wax solvent was applied to the slide twice and incubated on the slide for 8 minutes each time at 60°C. Target retrieval solution was applied to the slide and incubated for 30 minutes at 97°C. A protease solution was also applied to the slide for 20 minutes at 37°C. RNAScope HER2 target probe and RNAScope VS Universal HRP Amp kits (Advanced Cell Diagnostics) were applied to the slide according to the manufacturer's instructions. The hybridized messenger RNA (mRNA) target probes were detected using the Discovery mRNA Purple HRP kit (Ventana). All mRNA ISH assays were conducted on the Discovery Ultra automated staining platform (Ventana).

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