Clone d5f3

We performed immunohistochemical staining using antibodies against ALK (clone D5F3; Roche) and p53 (clone DO‐7; Roche) in the TMAs. The immunohistochemical results were evaluated as follows: strong granular cytoplasmic staining of ALK in tumor cells (any percentage) was defined as ALK positivity; staining of p53 in greater than or equal to 10% of tumor cells was defined as p53 positivity. For PD‐L1, TMAs were stained for PD‐L1 using PD‐L1 IHC 22C3 pharmDx assays (Agilent Technologies, Inc.) and an Autostainer Link 48 using an automated staining protocol. If the membrane of the tumor cells was stained, the cells were considered positive for PD‐L1 protein expression. The tumor proportion score (TPS) of PD‐L1 in tumor cells was defined as the percentage of PD‐L1‐positive tumor cells in the TMA tumor sections. If the TPS was greater than or equal to 1%, the section was defined as having positive expression.

ALK and ROS1 gene rearrangements and MET amplification were evaluated by the standard FISH method. Briefly, unstained sections obtained from FFPE blocks or cytoblocks were incubated with an ALK and ROS1 dual-color probe (IQFISH Break Apart Probe Agilent Technologies, Santa Clara, CA, USA). In each case, at least 100 tumor nuclei were evaluated. Cells were considered positive if a break-apart pattern of orange and green signals, at least one single orange signal, or a combination of both patterns were seen. Tumors with at least 15% of cells with ALK or ROS1 rearrangements were defined as positive. In ambiguous or equivocal cases, ALK or ROS1 immunohistochemical stains (clone D5F3, Ventana, Tucson, AZ, USA and clone D4D6, Cell Signaling, Danvers, MA, USA, respectively) were performed. The presence of MET gene amplification was evaluated using the MET IQFISH Probe with CEP7 (Agilent Technologies, Santa Clara, CA, USA). Amplification was reported in cases with a MET Probe/CEP7 Ratio ≥2 and/or gene copy number ≥5.