High-throughput fish screening reveals the accurate incidence of IGH@ translocations in childhood acute lymphoblastic leukaemia

Chromosomal translocations lead to oncogene activation or gene fusion in hematological malignancies, where they play an important role both in diagnosis and as an indicator of prognosis. Translocations involving the immunoglobulin heavy chain locus (IGH@) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) have recently been characterised and found to be different from those reported in the mature B-cell malignancies, although their expression is always deregulated due to the juxtaposition of transcriptional enhancers within the locus. The IGH@ locus rearranges with multiple genes including: the CEBP family of transcription factors, cytokine receptors: EPOR and CRLF2, and the inhibitory transcription factor, ID4. We have developed a high-throughput screening approach to ascertain the true incidence of IGH@ translocations in BCP-ALL. We have screened an entire childhood clinical treatment trial (ALL2003) using the Vysis LSI IGH break-apart rearrangement probe (Abbott Molecular) with automated scanning using the CytoVision 7.2 SPOT counting system (Leica Microsystems). We optimised slide set up, hybridisation and scanning templates for capture and automated scoring of 200 cells in over 2000 patients. We have identified IGH@ translocations in 4% (115/2580) of childhood ALL with the translocation positive population ranging from 5% (cut off value of 3%) to 100% of nuclei. A median age of 10 years and white cell count of 11x109/L was observed in patients with IGH@ translocations. The median age was significantly higher than all patients entered to this trial (5 years). This is consistent with our previous findings of IGH@ translocations being frequently observed in adolescents and young adults. We identified the involvement of CRLF2 in 19%, the CEBP gene family in 5%, and ID4 in 6% of the IGH@ translocations. DNA was available for Multiplex Ligation-dependent Probe Amplification (MLPA) from 43 IGH@ translocation patients. Copy number alterations were investigated using the MLPA kit P335-A1 (MRC Holland), which includes probes for IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and the PAR1 region: CRLF2, CSF2RA, IL3RA. We identified deletions in 40%, 30%, 19%, 7%, 9%, 12% and 9%, respectively. The number of gene deletions ranged from zero (37%), one (28%), two (26%), three (7%) and five (2%) deleted in the same patient. We have also screened an entire adult trail (MRC UKALLXII) for the presence of IGH@ translocations, identifying 8% (58/690) of patients whose outcome was inferior to other BCP-ALL subtypes. Interestingly, in 70% and 57% of IGH@ positive children and adults, respectively, the translocation partner remains to be elucidated, suggesting the presence of as yet unidentified cryptic rearrangements. Characterisation of these partners is necessary to identify additional oncogenes that may be involved in leukaemogenesis or provide potential therapeutic targets.