We performed HER2 SISH on the Ventana Benchmark automated instrument (Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturer’s protocols for INFORM HER2 DNA and chromosome 17 probes. We performed testing for the HER2 gene and chromosome 17 on sequential sections. Two sections were baked at 60 °C for 20 min. The HER2 DNA probe was denatured at 95 °C for 12 min, and hybridization was performed at 52 °C for 2 h. The chromosome 17 probe was denatured at 95 °C for 12 min, and hybridization was performed at 44 °C for 2 h. After hybridization, appropriate stringency washes were performed three times at 72 °C for the HER2 probe and three times at 59 °C for the chromosome 17 probe. Both DNP-labeled probes were visualized using a rabbit anti-DNP primary antibody and the ultraView SISH Detection Kit (Ventana). The slides were counterstained with hematoxylin for examination by light microscopy. Evaluation of HER2 gene amplification status was performed in a blinded manner using the ASCO/CAP guidelines [20 (link)].
Ultraview sish detection kit
Manufactured by Roche
Sourced in United States
The UltraView SISH Detection Kit is a laboratory equipment used for the detection and visualization of target DNA sequences in tissue samples. It utilizes a silver-enhanced in situ hybridization (SISH) technology to amplify and detect specific nucleic acid targets. The kit provides the necessary reagents and components to perform this analysis.
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Lab products found in correlation
Benchmark xt, by Roche (6 mentions)
Inform her2 dna and chromosome 17 probes, by Roche (4 mentions)
Ventana benchmark, by Roche (2 mentions)
Pr clone 1e2, by Roche (2 mentions)
Her2 pathway clone 4b5, by Roche (2 mentions)
Egfr dna probe, by Roche (2 mentions)
Ultraview universal dab detection kit, by Roche (2 mentions)
Inform her2 dna probe, by Roche (2 mentions)
Benchmark xt staining system, by Roche (1 mentions)
Clone sp1, by Roche (1 mentions)
Optiview dab detection kit, by Roche (1 mentions)
Ki 67 clone 30 9, by Roche (1 mentions)
Inform her2 dual ish assay, by Roche (1 mentions)
Benchmark autostainer, by Roche (1 mentions)
Clone pgr636, by Agilent Technologies (1 mentions)
Herceptest, by Agilent Technologies (1 mentions)
Ultraview red ish dig detection kit, by Roche (1 mentions)
Inform her2 dual ish dna probe cocktail, by Roche (1 mentions)
Her2 dna probe, by Roche (1 mentions)
Benchmark ultra stainer, by Roche (1 mentions)
19 protocols using ultraview sish detection kit
1
HER2 SISH Evaluation Protocol
2
Immunohistochemical Evaluation of Breast Cancer
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The immunohistochemistry analysis was performed using specific antibodies against ER, Clone SP1 (Ventana Medical Systems Inc., Tucson, AZ, USA); PR Clone 1E2 (Ventana Medical Systems); Ki67, Clone 30-9 (Roche Diagnostics K.K., Tokyo, Japan); AR Clone SP107 (Cell-MarqueTM, Rocklin, CA, USA); HER2 PATHWAY Clone 4B5 (Ventana Medical Systems). Immunostaining was performed using the Ventana Benchmark XT staining system with Optiview DAB detection kit. In cases of HER2 with equivocal immunohistochemical score (2+), we performed HER2 gene amplification by ultra-View SISH Detection Kit (Ventana Medical Systems). Evaluation of immunostaining and SISH for HER2 was based on American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations [6 (link)]. ER, PR and AR expression was considered positive if at least 10% immunostained tumor nuclei were detected in the sample [17 (link)]. Ki67 was scored low if <14% of tumor nuclei were positive and high if ≥14% of tumor nuclei were positive [35 (link)].
3
KRAS and EGFR Analyses in Tumors
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KRAS mutation analysis was performed with the DxS KRAS mutation kit (DxS Ltd, Manchester, UK). Detailed methods for EGFR IHC and EGFR GCN have been described [8] (link). In brief, three µm sections were first stained with EGFR (clone 5B7) mAb (Ventana Medical Systems/Roche Diagnostics, Tucson AZ, USA). Stainings were performed with BenchMark XT (Ventana/Roche) using ultraVIEW Universal DAB Detection Kit (Ventana/Roche). EGFR gene was detected from subsequent five µm sections with EGFR DNA Probe (Ventana/Roche) and ultraVIEW SISH Detection Kit (Ventana/Roche). In each tumor, EGFR GCN of forty tumor cells was analyzed using a 40x objective by two observers (ML, JS) from areas of highest IHC reactivity. The investigators were blinded of the clinical information. To evaluate the average EGFR GCN within each tumor, five tumor areas were arbitrarily chosen. From each of these areas, EGFR GCN of 20 randomly selected cancer cells was counted by two observers (TA, JS). The results were reported as mean and range within each area.
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KRAS mutation analysis was performed with the DxS KRAS mutation kit (DxS Ltd, Manchester, UK). Detailed methods for EGFR IHC and EGFR GCN have been described [8] (link). In brief, three µm sections were first stained with EGFR (clone 5B7) mAb (Ventana Medical Systems/Roche Diagnostics, Tucson AZ, USA). Stainings were performed with BenchMark XT (Ventana/Roche) using ultraVIEW Universal DAB Detection Kit (Ventana/Roche). EGFR gene was detected from subsequent five µm sections with EGFR DNA Probe (Ventana/Roche) and ultraVIEW SISH Detection Kit (Ventana/Roche). In each tumor, EGFR GCN of forty tumor cells was analyzed using a 40x objective by two observers (ML, JS) from areas of highest IHC reactivity. The investigators were blinded of the clinical information. To evaluate the average EGFR GCN within each tumor, five tumor areas were arbitrarily chosen. From each of these areas, EGFR GCN of 20 randomly selected cancer cells was counted by two observers (TA, JS). The results were reported as mean and range within each area.
4
HER2 Copy Number Assessment by SISH
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HER2 copy number status determined with SISH was performed using a dual probe (INFORM HER2 Dual ISH assay, Ventana Medical Systems, Tucson, AZ, USA, catalog Nr: 780-4332) containing the HER2 region (17q11.2–q12, directly labelled with black) and the centromeric region of chromosome 17 (17p11.1–q11.1, directly labelled with red). The signals were detected with the ultraVIEW SISH Detection Kit and the UltraView red ISH detection kit as per instructions by the manufacturer. The in situ hybridisation procedure was carried out using the Ventana Benchmark autostainer according to the manufacturer recommendation.
At the NCI, ISH Protease 3 (cat. #5273331001, Ventana Medical Systems), INFORM HER2 DNA Probe (cat. #5273439001, Ventana Medical Systems), and Rabbit anti-DNP (cat. #5273447001, Ventana Medical Systems) were used. Bound probe was visualised using silver deposition (ultraVIEW SISH Detection Kit, Ventana Medical Systems) followed by haematoxylin counterstain.
Scoring of SISH signals followed the guidelines established in the ToGA trial (Bang et al, 2010 (link)).
At the NCI, ISH Protease 3 (cat. #5273331001, Ventana Medical Systems), INFORM HER2 DNA Probe (cat. #5273439001, Ventana Medical Systems), and Rabbit anti-DNP (cat. #5273447001, Ventana Medical Systems) were used. Bound probe was visualised using silver deposition (ultraVIEW SISH Detection Kit, Ventana Medical Systems) followed by haematoxylin counterstain.
Scoring of SISH signals followed the guidelines established in the ToGA trial (Bang et al, 2010 (link)).
5
Standardized TNBC Subtyping by IHC
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TNBC status was evaluated by immunohistochemistry using specific antibodies against monoclonal rabbit ER antibody, Clone SP1 (Neomarker) and monoclonal mouse anti-human PR antibody, Clone PgR 636 (Dako). ER and PgR expression was interpreted as positive if at least 1% immunostained tumor nuclei were detected in the sample, according with ASCO/CAP recommendations for immunohistochemical testing of hormone receptors in breast cancer [17 (link)]. HER2 protein expression was determined using FDA approved HercepTest™ (K5206 DAKO) and evaluated according to the manufacturer’s instructions. HER2 gene amplification was determined by ultra-View SISH Detection Kit (Ventana Medical Systems, Tucson, USA). Tumors were classified according to the 2013 ASCO/CAP recommendations [18 (link)]. Given that the study included patients diagnosed over almost 20 years in different hospital centers, all surgical specimens of TNBC patients were reviewed independently by three experienced pathologists to achieve a consensus on morphologic criteria and to standardize the results at the current guidelines recommendations for ER, PgR and HER2 immunohistochemistry [17 (link)].
6
HER-2 Gene Amplification Assessment by Dual-ISH
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Dual-ISH was performed in HER-2 cases with equivocal membrane staining (if scored IHC 2+) according to manufactures recommendations by using the Ventana INFORM HER-2 Dual ISH/DNA Probe Cocktail and visualized utilizing two-color chromogenic in situ hybridization (ultraVIEW SISH Detection KIT and ultraVIEW Red ISH DIG Detection Kit, Ventana Medical Systems, Mannheim, Germany). HER-2 gene amplification was determined by the count of visualized copies of the HER-2 gene and visualized copies of chromosome 17. Ratios above 2.0 were considered amplified. IHC 3+ or IHC 2+/Dual-ISH positive (amplified) were classified HER-2 positive; IHC 0, IHC 1+ or IHC 2+/Dual-ISH negative (not amplified) were defined HER-2 negative [23 (link)]. IHC and Dual-ISH analyses were performed independently by two different observers (HS and IN).
7
HER2 Status Determination Protocol
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HER2 status had been determined by HER2 fluorescence in situ hybridization or silver in situ hybridization (SISH) for cases that were equivocal by HER2 immunohistochemistry. In cases whose HER2 status was not determined, HER2 SISH assays were performed with INFORM HER2 DNA and chromosome 17 probes (Ventana Medical Systems) using an ultraView SISH Detection Kit (Ventana Medical Systems) as previously described [19 (link)]. At least 50 cells were evaluated for each case and HER2 status was determined according to the updated 2013 ASCO/CAP guidelines [18 (link)]. A HER2 copy number of 6.0 or higher per cell, or a HER2:CEP17 ratio of 2 or higher was defined as amplified. Cases with HER2/CEP17 ratios < 2 and HER2 copy numbers of 4 to 6 signals per cell were considered equivocal. HER2 copy numbers of < 4 signals per cell and HER2/CEP17 ratios < 2 were defined as non-amplified. In this study, HER2-equivocal cases were regarded as HER2–non-amplified for statistical analysis.
8
Dual-Color SISH for Gene Status
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The genetic status of EGFR, HER2, c-MYC, and MET was evaluated by the dual-colour SISH technique. Briefly, consecutive unstained TMA slides were stained following the manufacturer’s protocol using the target gene DNA and corresponding CEP probes. The following probes were used: EGFR DNA and Chromosome 7 probes, HER2 DNA and Chromosome 17 probes, c-MYC DNA and Chromosome 8 probes, and MET DNA and Chromosome 7 probes (Ventana Medical System, Tucson, AZ, USA). The target gene DNA and CEP probes were allowed to co-hybridize on the same slides and were visualized by the Ventana ultraView SISH detection kit on the Ventana BenchMark XT automated slide stainer. The target gene and corresponding CEP signals were detected as black and red signals, respectively.
9
HER2 Status Determination Protocols
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HER2 status was determined via HER2 fluorescence in situ hybridization or silver in situ hybridization (SISH) for cases with equivocal HER2 IHC. HER2 SISH was also performed in cases that showed discrepant results in HER2 status between primary and metastatic tumors. HER2 SISH assays were performed with INFORM HER2 DNA and Chromosome 17 probes (Ventana Medical Systems) using an ultraView SISH Detection Kit (Ventana Medical Systems) as previously described [17 (link)]. After scanning the whole section, at least 50 cells were evaluated in each case, and HER2 status was determined according to the 2013 ASCO/CAP guidelines [14 (link)]. In this study, HER2-equivocal cases were regarded as HER2-non-amplified for statistical analysis.
10
Immunohistochemical and SISH Analysis of EGFR and HER2 in Tumors
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For each tumour, the most representative formalin-fixed paraffin-embedded (FFPE) tissue block was chosen and new sections were cut for both IHC staining and SISH. The methods for EGFR IHC and EGFR SISH have been described previously [7 (link)], and HER2 IHC was performed similarly with monoclonal HER2 antibody (clone 4B5, Ventana Medical Systems/Roche Diagnostics, Tucson, AZ, USA). HER2/Chr17 double-SISH was detected with HER2 DNA Probe and INFORM Chromosome 17 Probe (Ventana/Roche) and performed with ultraView SISH Detection Kit and ultraView Alkaline Phosphatase (AP) Red ISH Detection Kit (Ventana/Roche).
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