LOSS OF THE TUMOR SUPPRESSOR GENE CDKN2A/ARF BY GENOMIC DELETIONS IS A FREQUENT EVENT IN ADULT BCR-ABL1 POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) PATIENTS AND CONTRIBUTES TO DISEASE PROGRESSION

Background: The chromosome 9p21 locus contains the 40-kb region encoding the p16/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. This locus is a major target in the pathogenesis of a number of human tumors and its inactivation has been also documented in childhood ALL. Patients and Methods: In order to assess whether and how it is inactivated in adult BCR-ABL1-positive ALL, we studied 112 adult patients: 78 (70%) were de novo ALL, 15 (13%) were unpaired relapsed cases and 19 (17%) were paired relapsed cases. Their median age was 53 years (range: 18-76) and their median blast percentage was 90% (range, 18-99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of all exons by cloning and subsequent sequencing were also performed. Results: SNP array analysis revealed CDKN2A/ARF and CDKN2B genomic alterations in 33% and 24% of diagnosed patients, respectively. Deletions were in the majority of cases bi-allelic (73% vs 27%) and had a mean size of 100.8 kb (range, 27 kb-300 kb), ranging from 21.82 Mb to 22.12 Mb. In 70% of cases, deletions were limited to CDKN2A/CDKN2B genes, whereas in 30% they also affected neighbour genes and/or the entire chromosome 9. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether CDKN2A loss is responsible for progression, 34 patients were analyzed at the time of relapse and a significant increase in the detection rate of CDKN2A/ARF loss (53%) compared to diagnosis (p = 0.04) was found. In contrast, CDKN2B deletions were found to be not significantly different between diagnosis and relapse (41% vs 24%, p= 0.07). To assess whether deletions affected CDKN2A/ARF transcript levels, we used the Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. This analysis showed that deletions in the 9p21 locus led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005). Finally, the mutation screening of all exons showed that the 9p21 locus is rarely affected by point mutations, since we only identified the D146N and the R128 in the exon 2 of CDKN2A/ARF and the P83 silent mutation in the exon 2 of CDKN2B gene. These mutations were mutually exclusive and were found in only single cases. Conclusions: Loss of the tumor suppressor gene CDKN2A/ARF by genomic deletions is a frequent event in adult Ph+ ALL and it is involved in disease progression. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.