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

摘要: 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 protein overexpression status tumor as in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine US Food and Drug Administration (FDA)-approved & chromosome 17 centromere (CEN17) brightfield ISH (BISH) IHC assays into a single automated gene-protein assay allowing simultaneous detection all three targets tissue section. optimized using formalin-fixed, paraffin-embedded (FFPE) samples xenograft tumors MCF7 [HER2 negative (non-amplified gene, negative)] Calu-3 positive (amplified positive)]. performed rabbit monoclonal anti-HER2 antibody (clone 4B5) conventional 3,3'-diaminobenzidine detection. CEN17 BISH signals were visualized horseradish peroxidase-based silver alkaline phosphatase-based red systems, respectively with cocktail 2,4-dinitrophenyl-labeled digoxigenin-labeled probes. performance on microarray slides containing 189 randomly selected FFPE clinical cores compared that separate assays. optimal when protocol used before (rather than after) protocol. sequential use steps sections appropriately co-localized protein, after mitigating background staining naphthol phosphate-containing buffer step. obtained multiplex demonstrated high concordance those assays, We have developed allows visualization targets. This facilitated determination samples, particularly cases equivocal exhibited heterogeneity. produced results virtually equivalent FDA-approved virtual this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297