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.