Interphase FISH to confirm CNA status in cells from twins clinically discordant for ETV6-RUNX1 positive ALL. In each case the ETV6-RUNX1 ES (Vysis extra signal) probe was cohybridized with a fosmid probe (W12-3139G6) for the PAX5 gene and a BAC probe (RP-675D23) for the region on 10p shown to be gained in the cells of the twin with leukemia by SNP array. The PAX5 probe is labeled with biotin-16-dUTP (pseudocolored pink), and the 10p probe is labeled with digoxigenin-11-dUTP (pseudocolored brown). In panel A, a normal bone marrow cell from the healthy twin (6b) shows 2 red (RUNX1 gene), 2 green (ETV6 gene), 2 pink (PAX5 gene), and 2 brown (10p) signals, respectively. In panel B, a leukemic cell from twin 6a shows 1 yellow (ETV6-RUNX1 gene fusion), 2 red (normal RUNX1 and remnant from rearranged RUNX1 gene, respectively), 1 pink (PAX5), and 3 brown (10p) signals, respectively. This confirms the findings by SNP array of a deletion of ETV6 and PAX5 and gain of 10p in this twin. In panel C, an ETV6-RUNX1 fusion gene positive cell in the unaffected twin (6b) has the following signal pattern: 1 yellow (ETV6-RUNX1 gene fusion), 2 red (normal and remnant from rearranged RUNX1), 1 green (normal ETV6), 2 pink (PAX5), and 2 brown (10p) signals, indicating that the “driver” mutations seen in the leukemic twin (loss of second ETV6, loss of PAX5, gain of 10p) are not present in the preleukemic, fusion gene positive cells of the unaffected twin. Five cells with the ETV6-RUNX1 fusion were identified in the unaffected twin (twin 6b), of a total of 4251 scored.