Human epidermal growth factor receptor (HER)-2 testing. (A): Immunohistochemistry (IHC). This panel depicts the four categories of HER-2 IHC staining including 0 and 1+ (negative), 2+ (equivocal), and 3+ (positive) using the American Society of Clinical Oncology–College of American Pathologists guidelines for HER-2 IHC scoring. (B): Fluorescence in situ hybridization (FISH). This panel demonstrates a case of invasive duct carcinoma, on the left, negative for HER-2 gene amplification (gene copy number <4) and a case of HER-2 gene–amplified breast cancer (gene copy number >6), on the right, using the Ventana Inform single probe system (Ventana Medical Systems, Inc., Tucson, AZ). (C): True negative HER-2 IHC. In this image, the patient’s tumor is negative (0+) for HER-2 by IHC (Ventana Pathway™, Ventana Medical Systems, Inc., Tucson, AZ). Note the 3+ positive control section from another patient with known HER-2 3+ positive disease in the red control box to the left confirming that the staining procedure for the current patient was performed properly. (D): Pitfall in HER-2 gene amplification testing by FISH. In this panel, the central portion of the mixed ductal carcinoma in situ (DCIS) and invasive breast cancer is HER-2 gene amplified (inset to the left). However, this area is the in situ carcinoma component and should not be scored or reported for HER-2 gene copy number. The invasive portion of the tumor seen to the right, where the HER-2 gene copy number should be calculated, is HER-2 unamplified (inset to the right). Cases such as this one can, on occasion, lead to a false conclusion that HER-2–amplified breast cancer may frequently metastasize as HER-2–unamplified disease when, in fact, the invasive carcinoma was not HER-2 amplified to begin with. (E): Chromogenic in situ hybridization (CISH). This image depicts an invasive duct carcinoma with significant HER-2 gene amplification determined by the Invitrogen SpotLight® CISH assay (Invitrogen, Inc., Carlsbad, CA).