Her2 iqfish pharmdx

The primary breast cancer tissue sample for each patient was retested for HER2 status to ensure proper classification as HER2-negative. HER2 protein overexpression was evaluated by immunohistochemistry (IHC) using an FDA-approved monoclonal antibody (clone 4B5, Ventana) directed against the internal domain of the c-erbB-2 oncoprotein (HER2). The IHC results were categorized as follows: 0, 1+ = negative result; 2+ = equivocal result; and 3+ = positive result, according to the published American Society of Clinical Oncology (ASCO) guidelines [8 ] (Table 1). Tissues with 2+ staining (equivocal) were assessed for HER2 amplification with fluorescence in situ hybridization (FISH) as per ASCO guidelines [8 ] using an FDA-approved probe set (HER2 IQFISH pharmDx™, Dako), and defined as HER2/CEP17 ratio ≥ 2.0. Carcinoma with 0 or 1+ IHC staining or 2+ IHC staining and concurrent negative HER2 FISH (HER2/CEP17 ration < 2.0) were classified as HER2-negative primary breast malignancies.

Following written consent, patients’ archived primary breast cancer tissue samples were retested for HER2 status, to ensure proper classification of the pathology samples as HER2-negative (See Fig. 1 for protocol schema). HER2 protein overexpression was evaluated by immunohistochemistry using an FDA-approved monoclonal antibody (clone 4B5, Ventana) directed against the internal domain of the c-erbB-2 oncoprotein (HER2). The immunohistochemical results were categorized as follows: 0, 1+ = negative result; 2+ = equivocal result; and 3+ = positive result, according to published American Society of Clinical Oncology (ASCO) guidelines (11 (link)) (Table 1). Tissues with 2+ staining (equivocal) were assessed for HER2 amplification with fluorescence in situ hybridization (FISH) as per ASCO guidelines (11 (link)) using an FDA-approved probe set (HER2 IQFISH pharmDx™, Dako), and defined as HER2/CEP17 ratio ≥ 2.0. Carcinoma with 0 or 1+ immunohistochemical staining or 2+ immunohistochemical staining and concurrent negative HER2 FISH (HER2/CEP17 ration < 2.0) were classified as “HER2-negative” primary breast malignancies.

HER2 testing was performed at Clinical Laboratory Improvement Amendments (CLIA) laboratories at MSK and UC. HER2 amplification was assessed by fluorescence in-situ hybridization (FISH) using FDA approved probe sets (PathVysion, Abbott and HER2 IQFISH pharmDx™, Dako), and defined as HER2/CEP17 ratio ≥2.0 as used in clinical trials.^{8 , 9 (link)}(Figure 1)HER2 mutation was assessed by fragment analysis, mass spectrometry genotyping and Sanger sequencing for indels in exon 20. Where tissue was available, HER2 protein overexpression was assessed by immunohistochemistry (IHC) using the 4B5 Ventana antibody, and defined as 2+ or 3+ by published methods identical to those used for breast cancers.^{4 (link), 9 (link)} In both laboratories, each test was reviewed independently by two experts.