Inform her2 dual ish dna probe cocktail

To detect both WTHER2 and d16HER2 isoform expression, we used the anti-human c-erbB-2 A0485 polyclonal antibody (dilution 1:500; Dako) on 3-μm FFPE sections. IHC was performed using a BenchMark Ultra Platform (Ventana Medical Systems, Tucson, AZ) with the Optiview DAB Detection Kit (Ventana Medical Systems). HER2 immunoreactivity in breast and gastric carcinomas was scored as 0, 1+, 2+, 3+ according to international guidelines systems^{34 ,37 (link),38 (link)}. HER2 immunoreactivity in colon carcinomas was evaluated according to guidelines for gastric carcinomas.
Dual color silver in situ hybridization (DC-SISH) was performed on a BenchMark Ultra Platform (Ventana Medical Systems) according to the manufacturer’s protocol. The gene status was assessed on 3-μm FFPE sections using the INFORM HER2 Dual ISH DNA Probe Cocktail (Ventana Medical Systems). HER2 gene amplification was evaluated according to previously described scoring systems^{35 (link),37 (link),38 (link)}. Briefly, HER2 gene amplification was defined as positive when HER2/CEP17 ratio was >2 or when HER2 gene copy number was of >6 for breast carcinomas or of >6 for gastric and colon carcinomas.
Scoring systems for HER2 protein expression and gene amplification evaluations were applied for clinical samples, cell lines and PDX.

FISH was used for evaluation of the EGFR status (Vysis LSI EGFR SpectrumOrange/CEP7 Spectrum Green Probe, Abbott) [n = 5] while HER2 [n = 11] and c-MET [n = 9] genes were evaluated using CISH (dual EGFR DNP/CEP 7 DIG probes; INFORM HER2 Dual ISH DNA Probe Cocktail; commercially available c-MET and chromosome 7 DIG probe; Ventana, Tucson, AZ) as previously described [19 (link),21 (link)]. The tumors were considered amplified for HER2 when HER2/CEP17 ratio >2 [22 (link)]; EGFR was amplified when EGFR/CEP7 ratio >2 or >15 EGFR gene copies per cell were observed in >10% of analyzed cells [20 (link)]. cMET was amplified if >5 cMET copies on average were observed [19 (link)].

Tissue HER2 status was analyzed by immunohistochemistry (IHC) (clone Ventana pathway HER2 4B5) in all tumors and by in situ hybridization (ISH) for score 2+ tumors by IHC (INFORM HER2 dual ISH DNA probe cocktail, Ventana) according to the ASCO and to experts recommendations [1 (link), 16 (link), 17 ]. The laboratory regularly contributes to the National Breast Pathology External Quality Assessment scheme.

Dual colour SISH was performed according to the INFORM HER2 Dual ISH DNAProbe cocktail and ultraVIEW SISH DNP Detection Kit/ultraVIEW RED ISH DIG Detection Kit for HER2 and Chr 17 quantitation and procedure (Ventana/Roche). Both probes were labeled with 2,4-dinitrophenol (DNP) and digoxigenin (DIG). The tissue slides were deparaffinized with EZ Prep at 65°C-76°C and heat pretreated with EZ Prep-diluted Cell Conditioner 2 (CC2) at 90°C followed by protease digestion with ISH Protease 3 for 16 min at 37°C. The genomic DNA tissue sections and the nick-translated HER2 and Chr 17 probes were co-denatured by heat treatment for 20 min at 80°C followed by a hybridization step for 6 h at 44°C. The HER2 signals were detected using rabbit anti-DNP antibody for 20 min at 37°C and incubated with a HRP-conjugated goat anti-rabbit antibody for 16 min at 37°C after three 8 min stringency washes. The signal was detected as silver deposits with silver acetate, hydroquinone and hydrogen peroxide. For Chr 17 detection, the slides were incubated with mouse anti-DIG antibody for 20 min at 37°C and with an AP-conjugated goat anti-mouse antibody for 24 min at 37°C. The Chr 17 signal was developed as red dot staining with fast red and naphthol phosphate. The slides were finally counterstained with hematoxylin II and bluing reagent before mounting.

CISH was performed by using Ventana Medical Systems, Inc.’s (Ventana) INFORM HER2 Dual ISH DNA Probe Cocktail as intended to determine HER2 gene status by enumeration of the ratio of the HER2 gene to chromosome 17. The HER2 and chromosome 17 probes are detected using two color ISH in formalin-fixed, paraffin-embedded human cancer tissue specimens following staining on VENTANA BenchMark XT automated slide stainer, and visualized by light microscopy. The INFORM HER2 Dual ISH DNA Probe Cocktail has been approved by the US Food and Drug Administration for selection of patients to HER2 targeted therapies in breast cancer.