FGFR3 Gene Copy Number Evaluation

FGFR3 IHC positive, but FGFR3::TACC3 fusion-negative samples were analyzed with FISH for potential FGFR3 amplification. FFPE slides were hybridized with an IGH/FGFR3 (IGH, immunoglobulin heavy locus) translocation dual fusion FISH probe (Cytocell, Cambridge, UK). FFPE slides were prepared for FISH using protocol as described by Richardson et al.^{22 (link)} To determine FGFR3 gene copy numbers, 50 tumor cell nuclei per tumor were assessed on FGFR3 and IGH gene copy numbers at 100× magnification using a Leica DM5500 B microscope system with Leica application suite advanced fluorescence software (Leica Microsystems, Rijswijk, The Netherlands). An FGFR3-IGH ratio was calculated and defined as <1.5: normal copy numbers, 1.5–2.0: copy number gain, >2: gene amplification.^{23 (link)}