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

HIGH-RESOLUTION PHARMACOGENETIC PROFILES OF GENES INVOLVED IN DRUG ABSORPTION, DISTRIBUTION, METABOLISM AND ELIMINATION IN ADULT PHILADELPHIA-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS

Background: Inter-individual variations in genes encoding drug metabolizing enzymes and transporters have been demonstrated to influence the response to therapy. However so far, how these genetic variations interact to produce specific drug related phenotypes in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) has not yet been investigated. Aim: In order to investigate potential genetic structure and related pharmacogenetic profiles, around 2000 variants in more than 200 genes involved in drug absorption, distribution, metabolism and elimination were genotyped and studied with a population genetics approach in 45 Ph+ ALL patients. Methods: The Drug Metabolizing Enzymes and Transporters (DMET™, Affymetrix) platform, covering more than 90% of the most biologically relevant drug absorption, distribution, metabolism and excretion (ADME) markers was used for successfully genotyping 1931 variants in Ph+ ALL patients treated with the tyrosine kinase inhibitor Dasatinib. A model-based clustering method for inferring population structure using genotype data was applied by means of the Structure software assuming a model in which there are K populations - each one of them being characterized by a set of allele frequencies at each locus - to which individuals are probabilistically assigned according to their genotypes. Distribution of the genetic variance observed among the identified leukemia sub-groups was investigated with a locus by locus Analysis of the Molecular Variance (AMOVA) by means of the Arlequin 3.01 package, exploiting information on genotypes allelic content and frequencies. Results: Three different sub-groups (G1, G2, G3), made up of 2, 12 and 31 patients respectively, were identified in the examined ALL sample, according to their different patterns of allele frequency. A statistical support for this finding was provided by AMOVA results which pointed out a substantial level of genetic differentiation among G1 and the other two sub-groups (Fst = 0.099, p0.3) and significant Fst values; whereas a total of 50 loci, located on the NAT2, VKORC1, CYP4F2, CYP2B6, UGT2B7 and CYP2D6 genes, showed moderate to high (>0.08) significant Fst values in the G2/G3 comparison. Conclusions: Differences of allele frequencies observed among the identified ALL sub-groups prove that an evident genetic structure is detectable in our sample by genotyping loci involved in drug metabolism. Supported by: Fondazione GIMEMA Onlus, 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

NEXT GENERATION TRANSCRIPTOME RESEQUENCING IDENTIFIES NOVEL POINT MUTATIONS, GENE EXPRESSION AND ALTERNATIVE SPLICING PROFILES IN BCR-ABL1 POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. The selected cases had previously been profiled by high-resolution SNP and gene expression arrays and candidate gene resequencing. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, most of which successfully mapped to the reference sequence of the human genome. With the exclusion of the T315I mutation in the BCR-ABL kinase domain, seven novel missense mutations were detected after applying stringent criteria to reduce the SNV discovery false positive rate and validating novel substitutions with genomic DNA Sanger sequencing: 4 were exclusively found in the primary ALL sample and affected genes involved in metabolic processes (DPEP1, ZC3H12D, TMEM46) or transport (MVP); 3 missense mutations specific to the relapse sample affected genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21). Differences in mutational patterns suggest that the leukemia clone from which relapsed cells have been developed was not the predominant one at diagnosis and that relapse specific variants were mutations probably acquired during Ph+ ALL progression. Moreover, 4,334 and 3,651 primary ALL and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. The well-known alternatively spliced IKZF1 gene was also detected. Finally, a detailed gene expression profile was obtained indicating that more than 60% of annotated human genes were transcribed in leukemia cells in both diagnosis and relapse phases. Approximately 23% of genes were up-regulated at relapse with respect to diagnosis. Many of these genes affect cell cycle progression (AURORA A, SURVIVIN, PLK1, CDK1, Cyclin A, Cyclin B), suggesting that the loss of cell cycle control and the subsequent increased proliferation play a role in disease progression. Conversely, only 9% of active genes in both samples were down-regulated at relapse with respect to diagnosis. Conclusions: Discovery of novel missense mutations, as well as exhaustive alternative splicing and gene expression profiles were achieved for the first time for a BCR-ABL1+ positive ALL demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations. Supported by AIL, AIRC, FIRB 2006, European LeukemiaNet, GIMEMA ONLUS