MET and EGFR gene copy numbers were evaluated by FISH; a CEP7 centromere probe (Abbott Molecular) was used as a control. High-level MET amplification was defined as tight gene clusters of ≥ 15 copies in ≥ 10% of tumor cells, or a MET: CEP7 ratio of ≥2. A cutoff of ≥5 copies of MET/ cell was predefined as the criterion for FISH-positive status (FISH+), based on prior prognostic data supporting this cutoff in NSCLC (4 (link)). Tumors were considered EGFR FISH+ based on a scoring system used in multiple clinical studies (19 (link)). Further details are included in the Supplementary Methods section .
DNA and RNA were isolated from macrodissected tissue to enrich for tumor content. EGFR and KRAS mutations were evaluated using the DxS Genotyping Kit. MET exon 14 variants were evaluated by Surveyor nuclease digestion and detection by WAVE analysis (Transgenomics, Inc.). A polymorphism at position N375S of MET was evaluated by pyrosequencing on the Pyromark Q24 (11 (link)). Expression of MET, HGF, EGFR, amphiregulin (AREG), and epiregulin (EREG) mRNA transcripts was evaluated by qRT-PCR on the Biomark platform (Fluidigm). The primer/probes used for profiling are shown inSupplementary Table S2 and further details are included in the Supplementary Methods section .
DNA and RNA were isolated from macrodissected tissue to enrich for tumor content. EGFR and KRAS mutations were evaluated using the DxS Genotyping Kit. MET exon 14 variants were evaluated by Surveyor nuclease digestion and detection by WAVE analysis (Transgenomics, Inc.). A polymorphism at position N375S of MET was evaluated by pyrosequencing on the Pyromark Q24 (11 (link)). Expression of MET, HGF, EGFR, amphiregulin (AREG), and epiregulin (EREG) mRNA transcripts was evaluated by qRT-PCR on the Biomark platform (Fluidigm). The primer/probes used for profiling are shown in
