High-Resolution Genomic Profiling of Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukaemia (ALL) Identified Novel Recurrent Copy Number Variations Involved in Both Pathogenesis and Resistance to Tyrosine Kinase Inhibitors.

The Ph chromosome is the most frequent cytogenetic aberration associated with ALL and it represents the single most significant adverse prognostic marker. Despite the encouraging results achieved with imatinib, resistance develops rapidly and is quickly followed by disease progression. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors driving both the disease and resistance remains to be defined. In order to identify at submicroscopic level genetic lesions driving leukemogenesis and resistance, we profiled until now the genomes of 18 patients, out of 55 Ph+ ALL patients treated in our institute, at diagnosis (n=11) or at the time of haematological relapse (n=7) during therapy with imatinib or dasatinib. 250 ng of genomic DNA were processed on 500K single nucleotide polymorphism (SNP) array according to protocols provided by the manufacturer (Affymetrix Inc., Santa Clara, CA, USA). The median SNP call rate of analysed samples was 96%. Raw signal data were analyzed by BRLMM algorithm and copy number state was calculated with respect to a set of 48 Hapmap normal individuals and a diploid reference set of samples obtained from acute leukaemia cases in remission. Regions of amplification and deletion were visualized by Integrated Genome Browser and mapped to RefSeq to identify the specific genes involved in the lesion. Our analysis identified multiple copy number alterations per case, with deletions outnumbering amplification almost 3:1. Lesions varied from loss or gain of complete chromosome arms (trisomy 4, monosomy 7, loss of 9p, 10q, 14q, 16q and gain of 1q and 17q) to microdeletions and microduplications targeting genomic intervals. The recurring microdeletions that we detected in at least 50% of patients (both at diagnosis and at relapse) included 1p36.21 (PRAMEF), 3q29 (TFCR), 7p14.1 (AMPH), 8p23 (DEFB105A), 14q11.2 (DAD1), 16p13.11 (PDXDC1, NTAN1, RRN3), 16p11.2 (SNP) and 19p13.2 (CARM1, SMARCA4). A common microamplification was 4q13.2 (TMPRSS11E) and 17q21.31. Some genomic alterations were identified in genes regulating B-lymphocyte differentiation, such as PAX5 (n=3), BLNK (n=1) and VPREB1 (n=6) and in genes with an established role in leukemogenesis, such as MDS, BTG1, MLLT3 and RUNX1. Furthermore, many of the deletions detected included genes encoded for phosphatase proteins (e.g. PTPRD, PPP1R9B, PTPN18) and for zinc-finger proteins without any difference between diagnosis and resistance. It is noteworthy that some lesions felt in regions lacking annotated genes (loss: 2p11.2, 3p12.3, 7q11.21 and 14q32.33; gain: 8q23.3 and 13q21.1). Using high-resolution genome wide approach we showed that Ph+ ALL is a more complex disease characterized by multiple genomic anomalies which may provide new insights into the mechanisms underlying leukemogenesis and may be used as targets for existing or novel drugs. Supported by: European LeukemiaNet, COFIN 2003, Novartis Oncology Clinical Development, AIL.