PAX5 GENE IS FREQUENTLY REARRANGED IN A LARGE COHORT OF BCR-ABL1-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA: ON BEHALF OF GIMEMA AL & MDS WP

Background. Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, a high frequency of genetic alterations of regulators of B-lymphoid development (Mullighan et al., Nature 2008) was described in B-progenitor ALL. Genetic alterations targeting the B lymphoid transcription factor PAX5 were identified in over 30% of cases. Aim. To characterize, by high resolution SNP arrays, the rearrangements on 9p involving the PAX5 locus. Patients and Methods. Affymetrix Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 arrays and FISH assay using fosmid probes were used to profile the genomic of 98 adult BCR-ABL1-positive ALL patients. Results. The most frequent somatic copy number alterations affecting B-lymphoid development were deletions on 7p12 involving the IKZF1 gene (76/98, 78%), which encodes the transcription factor Ikaros, and mono-allelic copy number changes on 9p chromosome involving the PAX5 gene (31/98, 32%). Overall mono-allelic loss of PAX5 was identified in 28 patients (29%), whereas internal amplification in only 3 cases (3%). Four major PAX5 losses occurred: 1) focal deletions involving only the PAX5 gene in 3 cases (3%) with the minimal overlapping region of 101 kb from 37021876 to 36920536; 2) deletions involving only a portion of PAX5 and flanking genes in 8 (8%) with a median size of 364 kb (range: 154 kb-16395 kb) and ranging from 36603003 on 9p13.3 to 20553365 on 9p21.3; 3) broader deletions involving PAX5 and a variable number of flanking genes in 11 patients (11%) with a median size of 947 kb (range: 567 kb-18208 kb) and ranging from 36948016 on 9p13.3 to 20553365 on 9p21.3; 4) deletion of all chromosome 9 or 9p in 6 patients (6%). In 23 patients (23%) we identified the deletions of both IKZF1 and PAX5; in 51 patients (52%) only the deletion of IKZF1 was found, while the presence of deletion or rearrangements of only PAX5 gene was found in 5 patients (5%). In two cases we identified the loss of IKZF1 and the gain of an internal region of PAX5. According to the type of deletion we could have PAX5 haploinsufficiency or PAX5 mutants with impaired DNA-binding or transactivating activitiy. All these alterations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor. FISH analysis with three overlapping BAC probes encompassing the whole PAX5 gene was performed confirming what obtained by SNP-array analysis. To investigate the consequences of genomic PAX5 alteration in BCR-ABL1-positive ALL patients, quantitative PCR (q-PCR) was used to assess the expression of PAX5 in cases with copy number changes on 9p13.2. Q-PCR confirmed the SNP results demonstrating that genomic alterations on 9p13.2 leads to a significant down-modulation at the transcript level of the Pax5. Conclusions. PAX5 rearrangements occurred at an incidence of about 30% in adult BCR-ABL1 ALL and its impairment may be associated with the development of this for this poor prognosis subtype of leukemia. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus, Fondazione del Monte di Bologna e Ravenna, PIO project 2007.