Ros1 probe

IHC was performed using tissue microarray slides and antibodies against anaplastic lymphoma kinase (ALK; D5F3, 1:50; Cell Signaling Technology, Danvers, MA, USA), ROS1 (D4D6, 1:50; Cell Signaling), and RET (C-19, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The IHC results were obtained using the OptiView Detection Kit (Ventana Medical System, Tucson, AZ, USA). All cases were reviewed by two experienced pathologists (YJC and HSS). All cases were further analyzed to confirm rearrangement of ALK, ROS1, and/or RET using FISH and a break-apart probe for ALK (Abbott Molecular, Abbot Park, IL, USA), a ROS1 probe (Abbott Molecular), and a RET probe (ZytoVision, Bremerhaven, Germany) [29 (link)].

To identify ALK and ROS1 rearrangements, IHC was performed using the ALK (rabbit monoclonal, clone D5F3, Cell Signaling Technology, Danvers, MA, USA) and ROS1 (rabbit monoclonal, clone D4D6, Cell Signaling Technology, Danvers, MA, USA) antibodies. In IHC positive cases, fluorescence in situ hybridization (FISH) was performed using a break-apart ALK probe (Vysis LSI Dual Color, Break Apart Rearrangement Probe kit, Abbott Molecular, Abbott Park, IL, USA) or ROS1 probe (Abbott Molecular). ALK or ROS1 rearrangements were scored as positive when more than 15% of tumor cells displayed split signals or isolated signals containing a kinase domain (red for ALK and green for ROS1).