Manual DISH was performed using a DNA‐specific dual‐color probes ZytoDot2C SPEC HER2/CEN 17 Probe Kit (ZytoVision, Bremerhaven, Germany), or INFORM HER2 Dual ISH DNA Probe Assays were performed on a BenchMark XT Staining Platform (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's instructions.
Benchmark xt staining platform
Manufactured by Roche
The BenchMark XT Staining Platform is a lab equipment product designed for automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining procedures. It provides consistent and reproducible staining results by automating the staining process.
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6 protocols using benchmark xt staining platform
1
HER2 gene copy detection protocol
2
Quantitative HER2 Amplification Assessment
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The INFORM HER2 Dual ISH DNA Probe Assay was used on 5-μm sections using the BenchMark XT Staining Platform (Ventana Medical Systems). All samples were processed following the FDA-approved protocol. Samples with >70% of the cells with a DM (small dispersed dots distributed throughout the nucleus) or HSR (tightly clustered dots in discrete regions of the nucleus) patterns were classified accordingly. Cases not falling into these categories because of the presence of both HSR and DM patterns in the same sample were classified as mixed; for example, samples with 40% and 60% of cells with DM and HSR patterns, respectively, were classified as mixed while samples with 20% and 80% of cells with DM and HSR patterns, respectively, were classified as HSR (see text for details). Two teams including certified pathologists led by V. P. and R. M. blindly assessed the pattern of HER2 amplification in the series of samples from patients treated with neoadjuvant trastuzumab. The pattern of amplification in the series of samples from patients treated with adjuvant trastuzumab was assessed by V. P. The HER2/centromere 17 probe signal ratio was determined based on the quantification of 20 cells in at least two different fields, as recommended in ASCO/CAP 2013 guideline [11 (link)].
3
Standardized HER2 IHC Staining Protocol
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IHC was performed using a BenchMark XT Staining Platform (Ventana Medical Systems, Tucson, AZ) with an automatic staining protocol. Standard IHC for HER2 was performed to make a decision whether to treat with Trastuzumab. An anti-HER2/neu (4B5) rabbit monoclonal antibody (Roche Diagnostics, Penzberg, Germany) was used as the primary antibody with the appropriate dilution. Reacted antibodies were visualized enzymatically using brown diaminobenzidene (DAB) as a substrate.
4
Automated HER2 Dual ISH Assay
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INFORM HER2 Dual ISH DNA Probe Assays were performed on a BenchMark XT Staining Platform (Ventana Medical Systems, Tucson, AZ). The run protocol was established so that the entire assay procedure, consisting of baking, deparaffinization, pretreatment, hybridization, stringency wash, signal detection and counterstaining, was completed as a one-step fully automated assay. Each assay was finished within 13.5 h on this system.
5
HER2 Immunohistochemistry Protocol
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HER2‐IHC with a rabbit anti‐HER2/neu (4B5) monoclonal antibody (Roche Diagnostics, Penzberg, Germany) as the primary antibody was performed using a BenchMark XT Staining Platform (Ventana Medical Systems, Tucson, AZ) with an automatic staining protocol. HER2‐IHC were performed on all samples.
6
HER2 Gene Amplification Evaluation by DISH
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DISH was performed with a BenchMark XT Staining Platform (Ventana Medical Systems, Tucson, AZ) using an INFORM HER2 Dual ISH DNA Probe Assay and 4-μm tissue sections. The kit contains two DNA probes, the Inform® ErbB2 DNA Probe (labeled with black) and the Inform® Chromosome 17 Probe (CEP17) (labeled with red). DISH signals were observed using a conventional light microscope. The HER2/CEP17 ratio was assessed using the scoring criteria of fluorescence in situ hybridization (FISH) for breast cancer [9 (link)]. The HER2 and CEP17 signals were counted in 20 tumor cell nuclei in two different areas. HER2 gene amplification was quantitatively assessed by evaluating the HER2/CEP17 ratio and the average number of HER2 signals in each cell. The designation “amplified” was assigned if the HER2/CEP17 ratio was ≥2.0 or if the HER2/CEP17 ratio was < 2.0 but the average number of HER2 signals in each cell was ≥6.0. Conversely, the designation “not amplified” was assigned if the HER2/CEP17 ratio was < 2.0 and the average number of HER2 signals was < 4.0. When a case had a HER2/CEP17 signal count ratio of < 2.0 and an average number of HER2 signals per cell of ≥4.0 and < 6.0, signals were counted in another 20 nuclei, and the result was determined in a total of 40 tumor cell nuclei.
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