Expression and mutation analyses of FOXO3 and PRDM1. (A) FISH analysis of NKL. The red signal indicates the BAC probe 65306RP11-118H13 encompassing FOXO3, and the green signal indicates the centromere probe for chromosome 6 (CEP6, D6Z1; Abbott). The FOXO3 region shows heterogeneous loss. (B) PRDM1 and FOXO3 expression in NK-cell neoplasms (cell line and clinical samples). Semiquantitative RT-PCR was performed to validate the results of oligo-microarray analysis in 7 NK cell lines including NKL (from left; NKYS, SNK6, SNK10, HANK1, NK92, NKL, and KHYG1) and 7 clinical samples (from left; sample nos. 25, 27, 28, 30, 26, 31, and 32). Normal NK cells from one representative donor were also analyzed. Each band was converted to a numerical value using ImageJ software, and the ratio to β-actin was calculated. The ratio of normal NK cells and each neoplastic sample is shown below each band. *Samples with a 6q21 deletion; **HANK1 accompanied the deletion of PRDM1, but not FOXO3. (C) Mutation analysis of FOXO3 and PRDM1. The schema outlines the functional domain and location of the mutation. The mutation is shown as triangle on the map. The electrofluorogram shows that the mutation is located on exon 5 in NK92 and is aligned with the case without mutation. Missense mutations of FOXO3 were found in 3 cases and nonsense mutations of PRDM1 were found in 2 cases.