Inform her2 dual ish assay

Eligible patients were age 18 years or older with clinical stage cT2 to cT4 (> 2 cm)/cN0 to cN3/cM0 (> 2 cm) disease confirmed by a central laboratory (Targos Molecular Pathology, Kassel, Germany), to be HER2-positive breast cancer (immunohistochemistry 3+ [PATHWAY anti-HER2/neu {4B5} assay; Ventana Medical Systems, Santa Clara, CA]), or in situ hybridization–positive, defined as a HER2/CEP17 ratio of 2 or greater (INFORM HER2 Dual ISH assay; Ventana Medical Systems).^{15 (link)} Both tests were conducted in parallel for all patients; however, only one test needed to be positive to establish HER2-positive status.

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)).