DNA was extracted from paraffin‐embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany). Ten nanograms of DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the Ion AmpliSeq Cancer Hotspot Panel version 2 (Thermo Fisher Scientific, Waltham, MA), which covers mutation hotspots of 50 genes. The panel includes driver mutations, oncogenes, and tumor suppressor genes. By mid‐2018, the gene panel was expanded using the 161‐gene next‐generation sequencing panel of Oncomine Comprehensive Assay version 3 (Thermo Fisher Scientific), which covers genetic alterations and gene fusions. The complete list of the gene panel is provided in the supplemental online Appendix . The Ampliseq cancer hotspot panel was sequenced with an Ion PGM (Thermo Fisher Scientific) and the Oncomine Comprehensive Assay version 3 on an Ion S5 sequencer (Thermo Fisher Scientific).
The identified genetic variants were classified according to a five‐tier system comprising the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, and benign [12 ]. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.
The identified genetic variants were classified according to a five‐tier system comprising the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, and benign [