Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of Acute Myeloid Leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes generating a 5′RUNX1/3′CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8. In 1 to 8.5% of AML cases insertions events generating a 5′RUNX1/3′CBFA2T1 fusion gene have been reported, whereas the occurrence of inversions accompanying the t(8;21) has never been observed. We report a screening of 82 AML cases bearing the RUNX1/CBFA2T1 rearrangement detected by RT-PCR; all cases were tested by Fluorescence In Situ Hybridization (FISH) with BAC and PAC clones specific for CBFA2T1 and RUNX1 genes. This analysis allowed us to reveal five cases with ins(21;8), one with ins(8;21), and two with a pericentric chromosome 8 inversion followed by a t(8;21) translocation. A detailed molecular cytogenetic characterization of breakpoints has been performed in all cases.

FISH co-hybridization experiments with CBFA2T1 and RUNX1 probes revealed the presence of a functional fusion gene on the der(21) instead of the der(8) chromosome in five cases with ins(21;8); a single fusion signal on the der(8) chromosome was detected in the case with ins(8;21). The use of the same clones in FISH studies showed the presence of a single unexpected fusion signal on the 8p derivative chromosome in addition to faint CBFA2T1 and RUNX1 signals on the long arm of der(8) and der(21) chromosomes, respectively. These results suggested that a pericentric chromosome 8 inversion involving CBFA2T1 gene occurred and that the chromosome 21 was rearranged with the 8p derivative chromosome.

Appropriate chromosome 21 and 8 BAC clones were employed to precisely define the size of inserted regions in cases with insertions and the breakpoint on the 8p derivative chromosome in cases showing pericentric chromosome 8 inversion. The insertion size turned out to be very heterogeneous, ranging from a minimum of 2.4 Mb to a maximum of 44 Mb. In both cases with chromosome 8 inversion, the CBFA2T1 gene represents the breakpoint at the chromosome 8 long arm whereas the 8p breakpoint showed different mapping positions in 8p21.3 and 8p21.1, respectively.

Our results illustrate that (1) heterogeneous mechanisms can lead to the generation of the 5′RUNX1/3′CBFA2T1 chimeric gene; (2) molecular cytogenetic techniques may identify cryptic chromosomal changes, not detected by conventional cytogenetic analysis; (3) the crucial role of the 5′RUNX1/3′CBFA2T1 fusion gene in leukemogenesis does not depend on its location.

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