Sp384

Immunohistochemistry (IHC) staining for ROS1 was performed on 2 μm thick formalin‐fixed paraffin‐embedded (FFPE) tumor tissue sections using two distinct antibodies for ROS1: the rabbit monoclonal antibody clone D4D6 (Cell Signaling Technology) at 1:100 dilution, and the rabbit monoclonal antibody clone SP384 (ready to use antibody, Ventana ). The lung adenocarcinoma cell line HCC‐78 with known SLC34A2‐ROS1 rearrangement was used as positive control. Two experienced board‐certified pathologists assessed ROS1 IHC expression. The IHC cytoplasmic staining was qualified as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong). As there are no standardized cut off values, Score 1+ in any percentage of tumor cells was considered positive (requiring further testing).
Fluorescence in situ hybridization (FISH) assays were performed using a ROS1 break‐apart probe set (Abbott, Illinois), which hybridizes with the 5′ telomeric (labeled with Spectrum Orange) a 3′ centromeric (labeled with Spectrum Green) sequence of the ROS1 gene. A minimum of 50 tumor nuclei were evaluated with a Zeiss Axio Imager Z2 fluorescent microscope. The assay was considered positive when ≥15% of the tumor cells exhibited break‐apart or separate green signals.

For both primary antibodies, ROS1 IHC expression was performed on a BenchMark ULTRA staining automated IHC system (Ventana Medical Systems, Tucson, Arizona). IHC staining was performed on 4-µm-thickness FFPE tissue samples using D4D6 (Cell Signaling Technology, Danvers, Massachusetts) and SP384 (Ventana Medical Systems, Tucson, Arizona) clones. The protocols of both antibodies are briefly shown in Table 1. For both clones, the staining protocols of the antibodies were made according to the datasheet, and the positive control staining was obtained before the study.
IHC scores were semiquantitatively graded according to their staining intensities as follows: strong staining (3 +) visible using x 4 objective with > 5% tumor cells, moderate staining (2 +) visible using x 20 objective with > 5% tumor cells, weak staining (1 +) visible using x 40 objective with > 5% tumor cells or any staining intensity with ≤ 5% tumor cells, and negative staining (0) or absence of expression.^{28 (link)} For all samples, IHC was also interpreted as homogeneously or heterogeneously stained.