Op 87765

FISH analysis of the TMAs was performed using a two‐color KDM5D/X chromosome (CenX) probe mix, which was provided by the Molecular Cytogenesis Core at Memorial Sloan Kettering Cancer Center (New York, USA). From the 194 male patients with SCCs, TMAs of 173 tumors were available for FISH analysis. The probe mix consisted of a bacterial artificial chromosome clone containing full‐length KDM5D (Yq11) (RP11‐188C1 and RP11‐204P21; labeled with red dUTP) and a centromeric repeat plasmid specific to CenX (pSV2x5; labeled with green dUTP). Probe labeling, tissue processing, hybridization, post‐hybridization washing, and fluorescence detection were performed according to standard laboratory procedures [15 (link)]. Images were obtained with an all‐in‐one fluorescence microscope (BZ‐X800; KEYENCE, Osaka, Japan) equipped with a Plan Apochromat ×40 objective (NA0.95, BZ PA40; KEYENCE). Red fluorescence was detected using a TexasRed filter (ex: 560/40 nm, em: 630/75 nm, dichroic: 585 nm, OP‐87765; KEYENCE) and green fluorescence was detected using a GFP filter (ex: 470/40 nm, em: 525/50 nm, dichroic: 495 nm, OP‐87763; KEYENCE). A minimum of 50–300 nuclei per case were evaluated. Copy number loss of KDM5D was defined as cases in which more than 90% of tumor cells showed absence of a red (KDM5D) signal, as previously described [15 (link)].