A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the <it>HER2</it> gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

• Main Authors: Nitta Hiroaki (Author), Kelly Brian D (Author), Padilla Mary (Author), Wick Nikolaus (Author), Brunhoeber Patrick (Author), Bai Isaac (Author), Singh Shalini (Author), Ranger-Moore Jim (Author), Bieniarz Chris (Author), Tsuda Hitoshi (Author), Grogan Thomas M (Author)
• Format: Book
• Published: BMC, 2012-05-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

<p>Abstract</p> <p>Background</p> <p>The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the <it>HER2</it> gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by <it>in situ</it> hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved <it>HER2</it> & 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.</p> <p>Methods</p> <p>The 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 <it>HER2</it> & 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 <it>HER2</it> 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 <it>HER2</it> & CEN17 BISH assays.</p> <p>Results</p> <p>HER2 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 <it>HER2</it> & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, <it>HER2</it> 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 <it>HER2</it> gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and <it>HER2</it> & CEN17 BISH assays, respectively.</p> <p>Conclusions</p> <p>We have developed a protocol that allows simultaneous visualization of the HER2 IHC and <it>HER2</it> & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and <it>HER2</it> 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 <it>HER2</it> & CEN17 BISH assays.</p> <p>Virtual slides</p> <p>The virtual slides for this article can be found here: <url>http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297</url></p>

Item Description

10.1186/1746-1596-7-60
1746-1596