MYCN amplification status
MYCN oncogene copy number was assayed by FISH on the TMAs, using a direct commercial probe (Surefish 2p24 MYCN 277kb p5; Agilent Technologies Inc., Santa Clara, CA, USA). Briefly, the TMA sections were deparaffinized, dehydrated, and treated with HCl (0.2N), followed by proteolytic digestion with pepsin (750 U/ml) and serial ethanol dehydration. Hybridization was performed overnight at 37 °C in a humidified chamber. The slides were counterstained with 0.2 μmol DAPI in an antifade solution. Samples were blind analyzed by manual counting by two independent investigators. Digital images were obtained using a confocal microscope (NIKON Instruments Inc., Tokyo, Japan). MYCNwas considered non-amplified in samples with 2–9 copies and amplified in samples with ≥ 10 positive signals.