G-band and FISH analysis of an ins(22;3) in Ph⁻ cells of a CML patient undergoing dasatinib treatment (patient 3). (A) Partial G-band karyotype showing the products of the ins(22;3)(q11;q26q21) (right) beside the normal chromosome homologues (left). Part of the q-arm of chromosome 3 is seen, inverted, within the structure of the abnormal chromosome 22. A simplified diagrammatic representation of the structural rearrangement resulting in this abnormality is shown (inset). (B) Structure of the EVI1 locus together with the composition and relative coverage of the dual-color EVI1 FISH probe (Kreatech Biotechnology, Amsterdam, The Netherlands). The probe system employs 2 separate components specific for the 5′ and 3′ portions of the locus, labeled in red and green, respectively. In cells without a 3q26 rearrangement, 2 red/green fusion signals are produced. In contrast, physical separation of 5′ and 3′ hybridization signals, or splitting of 1 of these component signals, is visible in cells with rearrangement of this region. (C) Analysis of an ins(22;3)–positive bone marrow metaphase cell from patient 3 using the dual-color EVI1 probe. One intact red/green fusion signal is observed on the normal chromosome 3 homologue, marking the unrearranged EVI1 locus. In contrast, the second EVI1 hybridization signal reveals relocation and rearrangement of this region, with part of the red 5′ signal hybridized toward the distal end of the der(22), whereas the remains of the red 5′ signal together with the green 3′ signal are present toward the centromere of this marker. This finding is consistent with an inversion of chromosome 3 involving a break distal to EVI1 and a subsequent insertion of 3q material, including the inverted region, into chromosome 22.