Abstract
BackgroundThe eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section.MethodsThe HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays.ResultsHER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively.ConclusionsWe have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297
Highlights
The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively
When the brightfield ISH (BISH) step was carried out after the DAB-based IHC step, the DNP hapten on the HER2 DNA probe bound non- to nuclei and to some of the DAB staining, producing silver background staining during the BISH detection step for DNP-labeled DNA
The optimized HER2 gene-protein assay appropriately stains clinical breast-cancer tissue samples Using a previously Food and Drug Administration (FDA)-approved HER2 IHC assay, we identified clinical breast cancer tissue samples with HER2 expression scores of 3+ (Figure 3A, B, C), 2+ (Figure 3D, E, F), 1+ (Figure 3G, H, I), and 0 (Figure 3J, K, L)
Summary
The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. The oncogene HER2, which encodes human epidermal growth factor receptor 2 (HER2) protein, is amplified in 20–30% of breast cancer cases [1] and is the target of HER2-directed anti-cancer therapies. The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) have introduced guidelines for HER2 status assessments based on the level of HER2 protein overexpression determined by immunohistochemistry (IHC) and on the level of HER2 gene amplification determined by in situ hybridization (ISH) on formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue sections [2]. Which of the two methods is superior for assessing the HER2 status of breast cancer patients is unclear
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