MULTIPLEX PCR TO RAPIDLY IDENTIFY IKZF1 (IKAROS) GENE BREAKPOINTS IN BCRABL1-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Background: Deletions of IKZF1 gene, encoding for a regulator of lymphocyte differentiation, occur in more than 80% of adult patients (pts) with Ph+ ALL and significantly affect the prognosis. The deletions either involve the entire IKZF1 locus, resulting in loss of function, or delete an internal subset of exons, resulting in the expression of dominant negative isoforms. To better stratify Ph+ ALL pts according to IKZF1 status, we set up, validate and assess the routine applicability of a IKZF1 deletion screening strategy based on a multiplex-PCR. Patients and methods: We studied 87 adult BCR-ABL1+ ALL pts. For each type of common IKZF1 deletion (D4-7, D2-7, D4-8) an appropriate pair of primers will be designed using Primer 3 (http://frodo.wi.mit.edu/primer3/). Their ability to efficiently amplify the deletion was tested. PCR amplifications was performed using 50 ng genomic DNA as template for each reaction and a FastStart Taq DNA Polymerase (Roche Diagnostics). For each patient 3 amplicons were generated and sequenced (amplicon A for D4-7, amplicon B for D2-7 and amplicon C for D4-8). Moreover, a multiplex amplification strategy was assessed and a forth amplicon was generated and sequenced (forward primers were localized on intron 1 and 3, the reverse ones in the intron 7 and at the end of the gene, after the exon 8). The size of amplicons depends on the positions of the breakpoints in the IKZF1 gene and on the number of nucleotides added at the conjunction. It was in the range of 450-600 nucleotides. A positive control was used for each run. Results: On 87 pts previously analyzed by SNPs array, we identify IKZF1 deletions in 69/89 (78%) samples. Deletions were: 51 D4-7, 12 D2-7 and 4 D4-8; in 2 cases the deletion was extended to all gene, and 2 pts presented two breakpoints simultaneously. Multiplex PCR and subsequent sequencing were performed for most common deletions on 40 pts confirming previous results and indentifying the precise genomic positions of breakpoints and the nucleotides added at the conjunction. This rearrangement has been used to strictly monitor Minimal Residual Disease (MDR) during the follow-up and at relapse to confirm the clonal fidelity. Conclusions: We set-up a method rapid sensitive and with less amount of DNA sample, to screen Ph+ ALL pts at diagnosis and to monitor MRD during the treatment. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus