Expression of different isoforms of the B-cell mutator activation-induced cytidine deaminase (AID) in BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL) Patients

Background: BCR-ABL1-positive ALL is the most frequent and prognostically unfavorable subtype of adult ALL, mainly because of genetic instability. Activation-induced cytidine deaminase (AID) introduces single-strand breaks into target DNA and produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and Methods: We analyzed 61 adult de novo BCR-ABL1-positive ALL patients (pts) and 60 CML (chronic phase and myeloid/lymphoid blast crisis) pts. AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5#8217; end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. AID expression was also found in lymphoid blast crisis CML (50%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform; 19/61 (31%) co-expressed the wild-type and different AID splice variants with deaminase activity (AID#916;E4a, with a 30 bp deletion of exon 4; AID#916;E4, with the exon 4 deletion; AIDins3, with the retention of intron 3-4); 4/61 (7%) expressed the AID#916;E3-E4 isoform without deaminase activity (deletion of exons 2 and 3). To investigate whether AID introduces DNA-single strand breaks, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA). Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative (median copy number alteration of 14 versus 4. respectively, p<0.03). Recurring copy number abnormalities were identified in genes with an established role in leukemogenesis, such as IKZF1, CDKN2A, CDKN2B, PAX5, MELK, BTG1 and MDS1. Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as mutator outside the Ig gene loci in promoting genetic instability in leukemia cells