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

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

A novel gene–protein assay for evaluating HER2 status in gastric cancer: simultaneous analyses of HER2 protein overexpression and gene amplification reveal intratumoral heterogeneity

“…Hirschmann et al [18] reported the simultaneous analysis of HER2 gene and HER2 protein on a single slide in a small study with 25 gastric cancers, in which the same antibody and DISH probes from the current study were used but without naphthol phosphate. Recently, Nitta et al [12] described the diagnostic utility of the HER2 GPA technology in breast cancer, especially in equivocal cases or cases showing intratumoral heterogeneity of HER2. The present study is the first to evaluate the concordance of the HER2 statuses obtained using conventional methods (single IHC and DISH assays) and GPA in a large number of gastric cancer cases.…”

“…HER2 immunohistochemistry, dual-color in situ hybridization, and gene-protein assay HER2 IHC, HER2 and chromosome 17 centromere (CEN17) DISH, and HER2 GPA assays were performed as previously described by Nitta et al [12]. Briefly, for HER2 IHC, HER2 protein expression was detected using the PATHWAY HER-2/neu rabbit monoclonal antibody (clone 4B5; Ventana Medical Systems, Inc., Tucson, AZ, USA) and the iVIEW DAB detection kit (Ventana) on a BenchMark XT automated slide staining system (Ventana).…”

“…The utility of GPA technology has been demonstrated in breast cancer, especially in equivocal cases or cases showing intratumoral heterogeneity [12].…”

Signatures of post-zygotic structural genetic aberrations in the cells of histologically normal breast tissue that can predispose to sporadic breast cancer

“…PTs from all these subjects were characterized as either HER2-positive or Luminal B HER2-positive, as part of routine clinical assessment. From large-format paraffin blocks, several tissue cores were removed, sectioned, and stained using the HER2 tricolor Dual ISH DNA Probe Cocktail Assay, according to the standard protocol (Roche Diagnostics Scandinavia AB) (Nitta et al 2012). These tissue cores were selected based on their proximity to the original UM samples that were taken prior to fixation of the whole breast tissue (Figs.…”

“…New tissue samples were taken from the immediate vicinity of these biopsy sites for morphological ERBB2/HER2 assessment. We applied this strategy in the combined analysis of ERBB2 expression and copy number analysis using the HER2 tricolor Dual ISH DNA Probe Cocktail Assay, allowing visualization of expression of the HER2 protein as well as the copy number variation of ERBB2 and the centromere of Chromosome 17 (Nitta et al 2012). Eleven cases from the Falun cohort were selected for this analysis, based on the results of ERBB2 analysis in UM samples from the Illumina platform.…”