Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) analysis was performed on 4 μM formalin-fixed and paraffin-embedded tissue sections following a standardized protocol that included pretreatment (dewax/proteolysis), denaturation, probe application, and hybridization; application of 4′,6-diamidino-2-phenylindole (DAPI)/antifade solution; and analysis of slides using Leica fluorescent microscopy (DM6000B). ROS1 (ROS proto-oncogene 1, receptor tyrosine kinase) rearrangement was tested by FISH using a dual-color break-apart probe (ZytoLight Spec ROS1, ZytoVision, Germany) as previously described [20].

One case (case No. 1) was subjected to a targeted panel for multigene profiling using the Oncomine focus assay on the Ion Torrent Personal Genome Machine (Ion PGM, Thermo Fisher Scientific). Reaffirmation of the next-generation sequencing findings for copy number gain used FISH for c-MYC. The XL MYC BA spectral orange-labeled probe hybridizing proximal to the MYC gene region at 8q24.21 and a green-labeled probe hybridizing distal to the MYC gene region at 8q24.21 were applied (Metasystems Probes GmbH, Altlussheim, Germany). No centromeric probe was used. The number of c-MYC signals per cell was counted in 100 tumor cells and averaged. c-MYC copy number gain was defined as average copy number ≥ 3.0.