The FDA-Approved Breast Cancer HER2 Evaluation kit (HercepTest; Dako) May Miss Some HER2-Positive Breast Cancers

Frank Schneider, MD; Yulan Jin, MD, PhD; Kevin Van Smaalen, MD; Evin H. Gulbahce, MD; Rachel E. Factor, MD; Xiaoxian Li, MD, PhD


Am J Clin Pathol. 2019;151(5):504-510. 

In This Article

Materials and Methods

Case Selection

This project was initially designed for validation of an FDA-approved kit for HER2 IHC in our laboratory. We randomly selected 52 specimens from 52 patients to have approximately 20 HER2+ cases (22 in this study) from 2014 to 2017. The patient demographic data and tumor characteristics are summarized in Table 1. The institutional review board at Emory University approved this study.

IHC Analysis and Interpretation

IHC assays were performed and interpreted based on the 2013 ASCO/CAP guidelines. All tissues were initially fixed either in 10% neutral buffered formalin (core biopsy specimens or excisions) or methanol-based Cytolyt (Hologic) (fluids and transbronchial aspirate) within 1 hour. Cytology specimens initially fixed in Cytolyt were processed into cell block preparation and transferred to 10% neutral buffered formalin. Formalin-fixed, paraffin-embedded (FFPE) blocks were cut at a 4-μm thickness and put on a positively charged glass slide. For the LDT IHC assay, slides were stained with the HercepTest Her2 antibody (Dako) using an automated Leica Bond III stainer (Leica Biosystems) following on-board antigen retrieval in citrate buffer for 20 minutes at pH 6.0. For the FDA-approved assay, the Dako HercepTest kit was used with the Dako Autostainer Link 48 per the manufacturer's instructions. Appropriate positive controls were used in all cases. Results were evaluated and interpreted by two independent pathologists (F.S. and X.L.) blinded to the FISH results.

FISH Analysis

FISH was performed on FFPE tissue using the PathVysion HER2 DNA probe kit (Abbott) to detect HER2 and CEP17 copy number. After scanning the entire tumor on the slide, at least 60 tumor cells were evaluated at the area with the most HER2 copy number per cell. HER2 FISH results were interpreted based on the 2013 ASCO/CAP guidelines.

Both FISH and IHC tests (FDA kit and LDT) were performed using the same block for all cases.

IHC and FISH Concordance Definition

IHC and FISH results were considered concordant if the HER2 gene was amplified by FISH analysis and the IHC result was positive (3+) or equivocal (2+) or the HER2 gene was not amplified by FISH and IHC was negative or equivocal.