Immunohistochemistry

The IHC staining protocol using the HercepTest (mAb) was performed as described by the manufacturer [20]. Freshly cut tissue was processed on the Dako Omnis platform (Agilent Technologies, Santa Clara, CA) together with kit control slides for every staining run, using an automated staining protocol validated for HER2 detection [20].

IHC staining using the PATHWAY® HER-2/neu rabbit monoclonal antibody 4B5 was performed according to the recommendations of the manufacturer [22]. Freshly cut tissue was processed on the Ventana BenchMark ULTRA (Ventana Medical Systems, Roche Diagnostics, Tucson, AZ) together with kit control slides for every staining run, using an automated staining protocol validated for HER2 detection [22].

IHC staining for HER2 was independently evaluated by three trained pathologists (IN, MK, JR), followed by a consensus session for discordantly scored samples to define a consensus score for each case. IHC stains of the two assays were read after a 2-week wash-out period, and all the pathologists were blinded to the FISH results. In addition to a pre-study training provided by Dako/Agilent, all investigators had extensive experience in HER2 evaluation, having served over the past 20 years as readers in most of the trastuzumab, pertuzumab, and T-DM1 approval BC studies by Targos GmbH (Kassel, Germany) (for a review of studies screened by first-generation HercepTest (poly), see [23]).

IHC scoring was performed according to the 2018 ASCO/CAP guidelines [18]. Accordingly, cases with complete intense staining in ≤ 10% of tumor cells, as well as cases with intense and lateral or basolateral (“U-type”) staining, were included in the IHC 2 + category. For cases of IHC 1 + staining intensity (i.e., faint/barely perceptible membrane staining), the percentage of stained cells ≤ 10% or > 10% was recorded separately according to Ventana Instructions for Use (IFU) [22]. Intensity scoring was performed by applying the magnification rule as published previously by our group [24, 25].