Philadelphia Negative BCR/ABL1 Positive CML: FISH Mapping and aCGH Reveal Breakpoint Hotspots.

Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder defined by expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase. The fusion gene is a result of t(9;22)(q34;q11) or related variant rearrangements leading to formation of the Philadelphia chromosome (Ph). In less than 2% of the CML patients the bone marrow cells appear to have a normal karyogram although the fusion gene can be identified by molecular means and located by FISH on 22q11 and less frequently on 9q34 or on a third chromosome. The biology and clinical significance of the genetic rearrangements in Ph negative BCR/ABL1 positive disease are controversial and widely discussed. Two alternative mechanisms have been postulated but not confirmed: simple insertion or multiple step model. Although sequencing analysis as early as 1990 (Morris and Heisterkamp) showed that a large part of the 9q34 region is present in the masked Ph, little has been done to clarify the underlying rearrangements. Aided by genome array CGH and FISH, we constructed a comprehensive map of the regions flanking ABL1 and BCR genes in 9 patients and one cell line with this form of CML. The main results show:

1. the formation of the BCR/ABL1 fusion is associated with relocation of sequences well in excess of the ABL1 and BCR gene in all 10 samples,

2. the involvement of downstream 3′ ABL1 is diversely associated with distal breakpoint clusters in the regions of the RAP-GEF1 and RXRA genes,

3. the disease progression can be accompanied by sequential breakages/reunions within the regions flanking the BCR/ABL1 fusion and

4. the presence of multiple cryptic rearrangements of both 9q34 and 22q11 segments identifies a sub-group of patients with shorter survival thus demonstrating the clinical significance of genome instability in Ph negative, BCR/ABL1 positive CML.