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Background. Recently, using single nucleotide polymorphism (SNP) microarrays, a high frequency of genetic alterations of regulators of Blymphoid development (Mullighan et al., Nature 2008) was described in paediatric B- ALL, but a comprehensive analysis in adults is still lacking. 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 SNP6.0 arrays and FISH assay were used to profile the genome 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 monoallelic 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 (3%) with the minimal overlapping region of 101 kb; 2) deletions involving only a portion of PAX5 and flanking genes (8%) with a median size of 364 kb; 3) broader deletions involving PAX5 and a variable number of flanking genes (11%) with a median size of 947 kb; 4) deletion of all chromosome 9 or 9p (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 activity. FISH analysis with three overlapping BAC probes encompassing the whole PAX5 gene confirmed what obtained by SNParray analysis. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead 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 poor prognosis subtype of leukemia. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

Recent genome-wide analyses in B-ALL have identified a high frequency of DNA copy-number abnormalities, but the biological and prognostic implications of these abnormalities have not been defined. In order to address this issue, a cohort of 149 adult ALL patients (106 BCR-ABL1-postive, 3 ALL1/AF4, 1 EA/PBX, 39 negative for known molecular rearrangements) were analyzed with the use of singlenucleotide– polymorphism (SNP) microarrays (Affymetrix 250K NspI and SNP6.0), FISH, transcriptional profiling, and resequencing of samples obtained at diagnosis. Lesions varied from loss or gain of complete chromosome arms (trisomy 4, monosomy 7, loss of 9p, gain of 1q) to micro-alterations targeting genomic intervals in which the minimal common region of change involved one or few genes. Lesions in genes involved in early B-cell differentiation (60%) and cell cycle regulation (40%) were observed with relatively high frequency (60%). The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1 (75% in BCR-ABL1 positive and 58% in BCR-ABL1-negative ALL), which encodes the transcription factor Ikaros required for the earliest stages of lymphoid lineage commitment. FISH analysis using a pool of fosmid probes for IKZF1 and genomic quantitative PCR confirmed SNP results. Among the 149 patients, the entire IKZF1 locus was deleted in 19 (13%); in 84 (56%) additional patients, a subgroup of exons or the genomic region immediately upstream of IKZF1 was deleted. In 48 of them, there was a deletion of coding exons 4 through 7, which results in expression of a dominant-negative isoform, Ik6, which cytoplasmatic localization. Using gene-set enrichment analysis to compare the geneexpression signatures of patients with IKZF1 deletion versus not-deleted patients, we identified a unique signatures independent by BCRABL1 and characterized by down-regulation of B-cell lineage genes (e.g. VPREB1, VPREB3, IGLL3, BLK) and up-regulation of genes involved in cell-cycle progression (STK17B, SERPINB9, CDKN1A). We next investigated whether the IKZF1 deletions associated with a poor outcome. Univariate analysis showed that the IKZF1 deletion negatively influenced the cumulative incidence of relapse (p=0.0103) and disease-free survival (p=0.0229). Conclusion. Deletion of IKZF1 is an important event in the development of B-progenitor ALL which significantly influences clinical outcome. European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

Introduction. Acute myeloid leukemia (AML) is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome. However, using conventional chromosome banding techniques alone, karyotype abnormalities are detected in only half of all cases. Aims. To identify novel genomic regions of interest in normal karyotype AML and to identify novel candidate regions and disease-related genes in patients with complex karyotypes using genome-wide SNP-array. Patients and methods. Samples from 70 AML patients with FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype were examined by SNP arrays (Human Mapping 250K NspI and SNP6.0, Affymetrix). Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different genetic lesions (gains/losses) involving complete chromosome arms (del 16q, i(13q10), del 3p, del 7p, monosomy 9) or submicroscopic genomic intervals were identified in a substantial proportion of cases (55%) with a prevalence of gains respect to losses. Focal genetic alterations were detected at the breakpoints of previously cytogenetically identified chromosomal translocations, such as t(2;3)(p22-23)(q26-27) and t(1;11)(p32;q23). Hemizygous deletions were identified at 2q33.3-q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 9p21.3- p21.2 (CDKN2A-2B), at 12p13 (ETV6), at 17q12 (NF1) and 21q21.2 (RUNX1). Most frequent gains affected the oncogene MYC at 8q24 (4.33 Mb), the ABC transporters genes at 17q24, PTPRM at 18p11 and ERG at 21q22. Other recurring genetic lesions were uncommon and were identified only in single cases. Some lesions affected regions with a single gene, such as: ETAA1, FIGN, STK32B, PRAGMIN, PCM1, GLIS3, MRGPRX1, SESN3, BCL2L14 or lacking annotated genes. Marked differences in the combination of copy number anomalies were identified across the different genetic subtypes of AML. Conclusion. These data demonstrated that, in contrast to adult acute lymphoblastic leukaemia (ALL), AML is characterized by relatively few recurring copy number alterations, and that spectrum of genetic anomalies is significantly associated with AML disease subtype. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.

Background. BCR-ABL1-positive ALL is the most frequent and prognostically unfavorable subtype of adult ALL, mainly because of genetic instability. Activation-induced cytidine deaminase (AID) introduces single-strand breaks into target DNA and produces immunediversity by inducing somatic hypermutations and class-switch recombinations (CSR) in human immunoglobulin genes (Ig). Aim. We investigated the expression of the AID in BCR-ABL1-positive ALL. Patients and Methods. 61 adult de novo BCR-ABL1-positive ALL patients (pts) were analyzed. AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5’ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results. On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform (AID-FL); 19/61 (31%) co-expressed the wild-type and different splice variants. In particular, we found an isoform with a 30 bp deletion of exon 4 (AID-deltaE4a); an isoform characterized by the deletion of the entire exon 4 (AID-deltaE4) which led to a C-terminal truncation due a frameshift. In 2 patients, AIDins3, characterized by retention of intron 3, was expressed. This variant maintained the cytidine domain but lacked the class switch recombination (CSR) domain due a frameshift. Four out 61 Ph+ ALL patients (7%) expressed the AID deltaE3-E4 isoform without deaminase activity but retaining intact the CSR domain. Since splicing induced alterations in the nuclear export signal (NES) at the Cterminus we found that. AID-FL exhibited predominant cytoplasmic localization, as did the AID-deltaE4a and AID-deltaE3E4 variants whereas the C-terminal truncated AID-deltaE4 showed a slightly increased nuclear localization. Conclusions. Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutator outside the Ig gene loci in promoting genetic instability in leukemia cells. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus.

The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 kinase and defines a subgroup of ALL with a particularly unfavorable prognosis. ALL cells derived from B cell precursors typically carry rearranged immunoglobulin heavy chain (IGH) variable (V) region genes devoid of somatic mutations. Somatic hypermutation is restricted to mature germinal center B cells and depends on expression of activationinduced cytidine deaminase (AID), which introduces single-strand breaks into target DNA. Since, at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in Ph+ ALL and blast crisis chronic myeloid leukemia (CML) patients. On 35 adult Ph+ ALL patients, we detected AID mRNA and protein in 16 (46%), moreover AID expression was also found in blast crisis B-lymphoid lineage CML patients (n=2) but not in myeloid lineage (n=5) or in chronic phase CML (n=30). AID expression correlated with the BCR-ABL1 transcript levels and with the frequency of mutations within the BCR-ABL1 kinase domain that confer resistance to the BCR-ABL1 kinase inhibitors and disappeared after treatment with imatinib at the time of remission. To investigate whether AID introduces DNA-SSB in Ph+ ALL, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA) which is able to detect at high resolution and throughout the genome copy number abnormalities. We identified region of high level amplification and homozygous deletion in all patients. Overall, deletions outnumbered amplifications 3:1 and on average, we found 10 lesions per case, with a median of 6 losses and 2 gains. Patients who expressed AID had a higher number of alterations respect to patients who were AID negative (median copy number alteration was 8.5, range 4-22, versus 4, range 1-10, respectively, p

Introduction. Acute myeloid leukemia (AML) is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome of this disease. However, using conventional chromosome banding techniques alone, karyotype abnormalities are detected in only half of all AML cases. Aims.We sought to identify novel genomic regions of interest in normal karyotype AML and to identify novel candidate regions and disease-related genes in patients with complex karyotypes using genome-wide high resolution SNP-array. Patients and Methods. Samples from 57 AML patients with FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype were examined. Genomic DNA was isolated from mononuclear AML cells and applied to GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 array microarrays (Affymetrix, Santa Clara, CA) following the manufacturer’s instructions. Copy number aberrations were scored using the Hidden Markov Model and the segmentation approach available within the Partek software package as well as by visual inspection. All aberrations were calculated with respect to a set of 270 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission in order to reduce the noise of raw copy number data. When available, in order to exclude inherited copy number variants a comparison to paired constitutional DNA and to paired remission DNA was performed. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results. A wide spectrum of different genetic lesions (gains/losses) involving complete chromosome arms (del 16q, i(13q10), del 3p, del 7p, monosomy 9) or submicroscopic genomic intervals were identified in a substantial proportion of cases (55%) without differences in the frequency of losses or gains. Focal genetic alterations were detected at the breakpoints of previously cytogenetically identified chromosomal translocations, such as t(2;3)(p22-23)(q26-27) and t(1;11)(p32;q23). Hemizygous deletions were identified at 2q33.3-q34 involving ERBB4 (verb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 9p21.3- p21.2 (CDKN2A-2B), at 12p13 (ETV6), at 17q12 (NF1) and 21q21.2 (RUNX1). Most frequent alterations affected the oncogene MYC at 8q24.13 - q24.21 (4.33 Mb), the ABC transporters genes ABCA8, ABCA9, ABCA6, ABCA5, ABCA10 at 17q24, PTPRM at 18p11.31-p11.23 and ERG at 21q22. Other recurring genetic lesions were uncommon and were identified only in single cases. Some lesions affected regions with a single gene, such as: ETAA1, FIGN, STK32B, PRAGMIN, PCM1, GLIS3, MRGPRX1, SESN3, BCL2L14 or lacking annotated genes. Marked differences in the combination of copy number anomalies were identified across the different genetic subtypes of AML. Patients with normal karyotype showed no relevant genetic alterations. Conclusions. These data demonstrated that, in contrast to adult acute lymphoblastic leukaemia (ALL), AML is characterized by relatively few recurring copy number alterations, and that spectrum of genetic anomalies is significantly associated with AML disease subtype. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.