EVI1 activation in blast crisis CML due to juxtaposition to the rare 17q22 partner region as part of a 4-way variant translocation t(9;22)

Abstract

Background: Variant translocations t(9;22) occur in 5 to 10% of newly diagnosed CMLs and additional genetic changes are present in 60-80% of patients in blast crisis (BC). Here, we report on a CML patient in blast crisis presenting with a four-way variant t(9;22) rearrangement involving the EVI1 locus.

Methods: Dual-colour Fluorescence In Situ Hybridisation was performed to unravel the different cytogenetic aberrations. Expression levels of EVI1 and BCR/ABL1 were investigated using real-time quantitative RT-PCR.

Results: In this paper we identified a patient with a complex 4-way t(3;9;17;22) which, in addition to BCR/ABL1 gene fusion, also resulted in EVI1 rearrangement and overexpression.

Conclusion: This report illustrates how a variant t(9;22) translocation can specifically target a second oncogene most likely contributing to the more aggressive phenotype of the disease. Molecular analysis of such variants is thus warranted to understand the phenotypic consequences and to open the way for combined molecular therapies in order to tackle the secondary oncogenic effect which is unresponsive to imatinib treatment.

Figure 1

Representative karyotype from bone marrow of the patient. Arrows indicate a derivative chromosome 3 with an addition, a marker chromosome, two derivative chromosomes 9 (der(9)), del(16)(q23), the loss of a chromosome 17 and a Ph-chromosome.

Figure 2

FISH analysis on metaphases from bone marrow slides. (A) FISH with the dual color, dual fusion BCR/ABL1 probe generates two fusion signals (ABL1 red and BCR green): one on a marker chromosome [der(17)] and one on the Ph-chromosome, one red signal on a normal chromosome 9 and one green signal on a normal chromosome 22. (B) EVI1 specific probes RP11-362K14 (red, distal from EVI1) and RP11-82C9 (green, EVI1 overlapping) showed a co-localized red-green signal on the normal chromosome 3, a green signal on the derivative chromosome 3 [der(3)] and two red signals translocated onto identical but unidentified chromosomes [der(9)]. (C) FISH with WCPs for chromosomes 3 (purple), 9 (red) and 22 (green), revealed one normal chromosome 9, two derivative chromosomes 9 [der(9)], a der(17) containing a piece of chromosome 22 and chromosome 9, a normal chromosome 3 (norm 3), a derivative chromosome 3 [der(3)] and a Ph-chromosome. (D) WCP of chromosomes 17 (green) and 22 (red), combined with a purple centromeric probe for chromosome 3, indicated that a translocation between the long arm of chromosome 3 and the long arm of chromosome 17 had occurred as well as a translocation between chromosome 17 and 22.

Figure 3

EVI1 expression measured by qRT-PCR. (A) qRT-PCR for EVI1 with primers located 5' of the transcript revealed ectopic EVI1 expression in the patient after normalization with several household genes, the cell line K562 was used as a positive control, the K562 expression was set to 1, Y-axis in log-scale (B) EVI1 qRT-PCR with primers located 3' of the transcript (cEVI1) indicated an ectopic expression of a 5' variant EVI1 transcript, the cell line K562 was used as a positive control, the K562 EVI1 expression was set to 1, Y-axis in log-scale.

Figure 4

Schematic representation of possible generation of the 4-way t(3;9;17;22). (A) Formation of the t(9;22)(q34, q11). (B) Three-way translocation involving the EVI1 locus on 3q26, part of the distal long arm of chromosome 17 and the der(9)t(9;22). (C) Representation of the derivative chromosomes 3, 9, 17 and 22 in our patient.