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Loss of CDKN2A, encoding p16/INK4A and p14/ARF tumor suppressors, has been associated with poorer survival and higher leukemia-initiating- cell-frequency in xenograft models (Notta F et al. Nature 2011). Recently, using genome-wide single nucleotide polymorphisms arrays and gene candidate deep exon sequencing, we identified CDKN2A deletions in 29% (29/101) adult newly diagnosed Ph+ ALL patients and in 47% relapsed cases. Accordingly, here we aimed to investigate the functional consequences of genomic deletions and their prognostic value. By quantitative real-time PCR a significant difference in the expression of CDKN2A was observed among cases with heterozygous (p = 0.04) and homozygous (p = 0.01) loss compared to normal cases, suggesting that deletions lead to CDKN2A haploinsufficiency. In order to investigate their clinical implications, data were collected from 81 patients (median follow-up: 25.2 months- range 2.1-148.1; median age at diagnosis: 54 years- range, 18-76; CDKN2A loss in 29, 36%, cases). 72 patients (89%) were enrolled in the GIMEMA clinical trials (12 patients in GIMEMA LAL0201-B protocol, 13 in LAL2000 and 47 in LAL1205 protocols), while 9 patients (11%) were enrolled into institutional protocols. By univariate analysis a shorter overall survival (OS) and disease-free survival (DFS) were found in patients with CDKN2A deletion compared with those wild-type (OS: 27.7 v 38.2 months, respectively, p = 0.0206; DFS: 10.1 vs 56.1 months, respectively, p = 0.0010). Moreover, a higher cumulative incidence of relapse for patients with CDKN2A deletion (73.3 vs 38.1; p = 0.0014) was also recognized. The multivariate analysis confirmed the negative prognostic impact of CDKN2A deletion on DFS (p = 0051). These results show that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation, cell growth and tumor formation by the action of MDM2 and CDK4/6, attractive drugs to prevent disease recurrence could be represented by the inhibitors of MDM2 and CDK4/ CDK6. These drugs are currently under investigation in in vitro studies by our group. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program, Programma di Ricerca Regione – Università 2007 – 2009.

Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the 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 each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. 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 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis.

Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1