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Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases regulated by Ataxia-Telangiectasia and Rad3-related (ATR) kinases and involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Deregulation of these pathways has been previously described in BCR-ABL positive cells and involved in chemoresistance. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, in this study we aimed to investigate the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in Ph+ ALL and to determine potential biomarkers of functional inhibition. We first examined Chk1 and Chk2 mRNA expression levels in 45 newly diagnosed Ph+ ALL patients, in their paired remission samples, in 14 relapsed cases and in 3 Ph+ cell lines (BV-173, SUPB-15 and K562) by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation). Higher transcript levels of Chk1 but not Chk2 were found in newly diagnosed patients compared to remission samples (p = 0.0009 for Chk1 and p= 0.8183 for Chk2). Chk1 transcript levels were comparable between diagnosis and relapsed cases (p = 0.5728), suggesting that the ATR-Chk1 pathway is strongly activated in Bcr-Abl-positive cells. This was confirmed by phosphorylation of Chk1 Ser317 by western blotting analysis in Ph+ cell lines. We then evaluated the effect of PF-0477736 as single agent on cell viability using the Cell Proliferation Reagent WST-1 (Roche). BV-173, SUPB-15 and K562 cell lines were incubated with increasing concentration of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1–0.5 μM, with BV-173 being the most sensitive, while K562 the most resistant. These results were confirmed in primary blasts cells from a Ph+ ALL patient with wild-type Bcr-Abl and from 3 cases harboring the T315I Bcr-Abl mutation found to be insensitive to the available TKIs (IC50 ranged from 0.1–0.5 μM at 48 hours). Consistent with the WST-1 results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in both cell lines and primary cells. To test whether increased apoptosis resulted from Chk1 inhibition, we assessed the changes in phosphorylation of Cdc25c phosphatase, which is inactivated by Chk1 to prevent mitotic entry, and of {gamma}H2AX, which is increased in response to DNA damage. Western blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 and increases levels of {gamma}H2AX. Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening by amplification and subsequent sequencing of all coding exons of p53. All cell lines except for K562 and primary leukemia cells lacked mutations in the p53 gene, demonstrating that in Ph+ ALL PF-0477736 is highly effective also on p53 wild-type tumor cells. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on 3 treated Ph+ cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p < 0.05) of 211 genes including those involved in apoptosis and cell cycle (CEBPB, CUL1, Histone H1-H2A, 2B family clusters, Histone H4, DHX15, SNCB, FOS) and DNA damage, such as DNA-damage-inducible transcript 3 (DDIT3) and growth-arrest and DNA damage-inducible proteins GADD34 and GADD45a, suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in Ph+ ALL cells. In conclusion, for the first time we demonstrate in a large cohort of Ph+ ALL patients an over-expression of Chk1, providing a strong rational for its inhibition. In vitro treatment of Ph+ ALL cells with PF-0477736 alone resulted in reduction of inhibitory phosphorylation of Cdc25c, inhibition of proliferation and induction of biomarkers of DNA damage and apoptosis, suggesting that single-agent Chk1/2 inhibition may be an effective treatment strategy for Ph+ ALL.

Ninety-six depressed outpatients from 5 centres were given viloxazine 200–400 mg/day. The treatment produced significant clinical improvement as evaluated by the Hamilton Rating Scale for Depression. Thirteen patients dropped-out because of possible side effects. Many untoward symptoms were described by all the patients. To distinguish between illness-related symptoms (IRSs) and potential side effect symptoms (SESs) a new approach was taken, using an algorithm that provides a decision strategy based on the time course both of the symptom and the illness. By this procedure, 90 of the 187 claimed untoward symptoms were identified as IRSs. Of the 97 potential SESs, only 36 were spontaneously volunteered, and the remaining 61 symptoms were elicited on specific questioning. Whenever possible, volunteered potential SESs were assessed to determine the relationship between the drug treatment and the adverse reaction. It was found that only a few instances of gastric disturbance and exacerbation of anxiety were probably violxazine-related.

Mother's own milk represents the optimal source for preterm infant nutrition, as it promotes immune defenses and gastrointestinal function, protects against necrotizing enterocolitis, improves long-term clinical outcome and is hypothesized to drive gut microbiota assembly. Preterm infants at birth usually do not receive their mother's milk directly from the breast, because active suckling and coordination between suckling, swallowing and breathing do not develop until 32-34 weeks gestational age, but actual breastfeeding is usually possible as they grow older. Here, we enrolled moderately preterm infants (gestational age 32-34 weeks) to longitudinally characterize mothers' milk and infants' gut and oral microbiomes, up to more than 200 days after birth, through 16S rRNA sequencing. This peculiar population offers the chance to disentangle the differential contribution of human milk feeding per se vs. actual breastfeeding in the development of infant microbiomes, that have both been acknowledged as crucial contributors to short and long-term infant health status. In this cohort, the milk microbiome composition seemed to change following the infant's latching to the mother's breast, shifting toward a more diverse microbial community dominated by typical oral microbes, i.e., Streptococcus and Rothia. Even if all infants in the present study were fed human milk, features typical of healthy, full term, exclusively breastfed infants, i.e., high percentages of Bifidobacterium and low abundances of Pseudomonas in fecal and oral samples, respectively, were detected in samples taken after actual breastfeeding started. These findings underline the importance of encouraging not only human milk feeding, but also an early start of actual breastfeeding in preterm infants, since the infant's latching to the mother's breast might constitute an independent factor helping the health-promoting assembly of the infant gut microbiome.

Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. ©2011 AACR.

Abstract 2574 Recently, using genome-wide single nucleotide polymorphism arrays and gene candidate deep exon sequencing, we identified lesions in CDKN2A gene, encoding p16/INK4A and p14/ARF tumor suppressors, in 27% (32/117) adult newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients and in 47% (14/30) relapsed cases. Clinically, in our cohort CDKN2A deletions were associated by univariate analysis to a worse outcome in terms of overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) (OS: 27.7 vs 38.2 months, p = 0.0206; DFS: 10.1 vs. 56.1 months, p = 0.0010; CIR: 73.3 vs 38.1, p = 0.0014). Noteworthy, the negative prognostic impact of CDKN2A deletion on DFS was also confirmed by the multivariate analysis (p = 0.0051). These results showed 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 and tumor cell growth by the action of MDM2, a negative regulator of p53, we investigated the preclinical activity of the MDM2 antagonist RG7112 in primary B-ALL patient samples and leukemic cell line models. BV-173, SUPB-15 and K562 Ph+ cell lines were incubated with increasing concentration of RG7112 (0.5–10 μM) and its inactive enantiomer for 24, 48 and 72 hours (hrs). MDM2 inhibition by RG7112 resulted in a dose and time-dependent cytotoxicity with IC50 (24 hrs) of 2 μM for BV-173 and SUPB-15 which harbor homozygous deletion of CDKN2A but wild-type p53. No significant changes in cell viability were observed in K562 p53-null cell line after incubation with RG7112. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/Propidium Iodide analysis showed a significant increase in apoptosis after 24 hrs in BV-173, SUPB-15 and in primary leukemia blasts, whereas no apoptosis was observed in K562 cells. To examine the possible mechanisms underlying RG7112-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased upon treatment with RG7112 after 24 hrs of incubation with concentrations equal to the IC50. These data demonstrate the ability of RG7112 to activate the intrinsic apoptotic pathway by a p53-dependent mechanism. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was next performed, comparing sensitive cell lines (BV-173 and SUPB-15) after 24 hrs exposure to 2 μM RG7112 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). They include genes involved in cell cycle and apoptosis control (e.g. Histone H1, TOP2, GAS41, H2AFZ) and in the down-regulation of the Hedgehog signaling (e.g. BMI1, BMP7, CDKN1C, POU3F1, CTNNB1, PTCH2) with a strong repression of stemness genes and re-activation of INK4/ARF as illustrated in Figure 1. Actually, both GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. In our data they were strongly down-regulated (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells, suggesting that these genes have a potential as new biomarkers of activity. In conclusion, inhibition of the p53–MDM2 interaction by RG7112 can activate the p53 pathway, resulting in apoptosis and inhibition of stemness genes in B-ALL with wild-type p53. Our findings provide a strong rational for further clinical investigation of RG7112 in Ph+ ALL. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione–Università 2007–2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Introduction: Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods: 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) ALL and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results: A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus (exon 2) that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; p= 7.1 x 10-3). Conclusions: Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Recently, the rs564398 has been demonstrated by Cunnington M.S. et al. (2010) to alter ANRIL expression leading in turn to a deregulation of CDKN2A/B genes. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program (PIO), PRIN 2008, Programma di Ricerca Regione – Università 2007 – 2009.

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.

Background: Approximately 30% of adult ALL patients are characterized by the presence of the Philadelphia (Ph) chromosome, which derives from a reciprocal translocation t(9;22)(q34;q11) and results in a chimeric BCR-ABL oncogene. The prognosis for this subset of patients treated with standard therapies, including multi-agent chemotherapy, imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and nilotinib are second generation TKIs developed to overcome resistance to imatinib in relapsed Ph+ leukemias. Here, we sought to determine the efficacy of these agents in patients with relapsed Ph+ ALL. Methods: We retrospectively evaluated the single-center experience of dasatinib, nilotinib, and experimental third-generation TKIs administered as second or subsequent lines of therapy. We evaluated the efficacy of these agents in 25 adult patients with relapsed Ph+ ALL. All patients were previously treated with imatinib.Results: The median age at time of diagnosis was 50 years (range, 18-74 years). Seventeen patients were male and 8 were female. Ten patients presented with a BCR-ABL P190 fusion protein and corresponding fusion transcript, and the remaining patients exhibited BCR-ABL P210. Nineteen patients received dasatinib, 2 patients nilotinib, and the remaining 4 patients were treated with third-generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third, and fourth relapse, respectively. A mutational analysis was performed twice in all the patients: before administering TKIs (9 patients with wild type BCR-ABL, 16 with mutated BCR-ABL, including T315I) and at the time of subsequent relapse. Gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Thirteen out of 25 patients (52%) achieved a hematologic response (HR) (11 patients treated with dasatinib, 1 patient with nilotinib, and 1 patient with a third-generation experimental TKI). Ten patients also achieved a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range, 2-29 months), median duration of HR, CyR, and MolR was 117 days (range, 14-385 days); progression free survival was 162 days with dasatinib and 91 days with nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 patients with wild-type BCR-ABL treated with dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusions: Second- and third-generation TKIs represent a valid approach in treating patients with relapsed Ph+ adult ALL. The subsequent relapse in Ph+ ALL patients is often associated with the emergence of mutations conferring resistance to TKIs.Acknowledgments: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

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.

Abstract 12 Background: Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim: To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. 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 Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion: PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures: No relevant conflicts of interest to declare.

Background. Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes by deaminating deoxycytidine residues in mature germinal center B cells. AID can also induce mutations in non-Ig genes. In addition to the full-length isoform, five splice variants have been reported in normal B cells and in mature B-cell disorders. Furthermore, aberrant expression of AID has been detected in B-lineage acute lymphoblastic leukemia (B-ALL) and a strong correlation between AID expression and BCR/ABL-positivity has been found; intriguingly, BCR/ABL+ ALL, the most frequent genetically-defined ALL in adults, is characterized by a high genetic instability. Aims. To deepen the role of AID in BCR/ABL+ ALL, AID expression was studied in these patients and then its association with gene expression profiling was evaluated. Methods. We analyzed 16 adult BCR/ABL+ ALL patients at the onset of disease for AID expression: after cDNA amplification, PCR products were loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Subsequently, gene expression profiling experiments were performed on the same set of patients using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analysis was carried out using the dChip software. Unsupervised and supervised analyses (Analysis of Variance, ANOVA) were used to evaluate the presence of potential subsets and to compare the BCR/ABL+ ALL subgroup based on AID results. Results. AID mRNA was detected in 7 patients: 4 expressed only the full-lenght isoform, 2 displayed the splice variant that retains exons 1, 2 and 5, while 1 patient co-expressed the full-lenght isoform and two splice variants. The remaining cases proved negative for AID expression. By unsupervised clustering, gene expression profiling data analysis revealed that AID-fulllenght and AID-splice variants patients display a similar profile. This finding was confirmed by supervised analysis: in fact, comparison on the 3 AID subsets (i.e. AID-full-lenght, AID-splice variants and AID-negative) grouped AID-full-lenght and AID-splice variants in the same cluster, as opposed to AID-negative cases. Among the 1151 differentially expressed genes, 328 resulted specifically and homogeneously upregulated in AIDfull- lenght patients. Of interest, this group of patients was characterized by the overexpression of a large set of genes involved in i) DNA repair (PCNA, RPA3, RAD50, XRCC4, TP53), ii) DNA replication (ORC5L, MCM10, CETN3, ANAPC7), iii) cell cycle regulation (CDK6, MKI67, MYC), iv) nucleotide metabolism (NT5C3, PPAT, PRPS2). Functional annotation analysis, performed using the DAVID software, corroborated these findings. Conclusions. These results indicate that AID-full-lenght and splice variants exert a similar genomic profile, distinct from AIDnegative patients, suggesting that gene expression profiling is mainly affected by AID positivity or negativity, rather than by the presence of AID-splice variants. Furthermore, AID positive patients are characterized by a peculiar signature in which genes involved in DNA repair and replication are overrepresented, indicating a possible deregulation of these mechanisms: it is intriguing to speculate that this phenomenon might be implicated in the genetic instability observed in these patients. Functional studies are ongoing to confirm this hypothesis.

Background. Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, a high frequency of genetic alterations of regulators of B-lymphoid development (Mullighan et al., Nature 2008) was described in B-progenitor ALL. Genetic alterations targeting the B lymphoid transcription factor PAX5 were identified in over 30% of cases. 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 Genome-Wide Human SNP 6.0 arrays and FISH assay using fosmid probes were used to profile the genomic 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 mono-allelic 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 in 3 cases (3%) with the minimal overlapping region of 101 kb from 37021876 to 36920536; 2) deletions involving only a portion of PAX5 and flanking genes in 8 (8%) with a median size of 364 kb (range: 154 kb-16395 kb) and ranging from 36603003 on 9p13.3 to 20553365 on 9p21.3; 3) broader deletions involving PAX5 and a variable number of flanking genes in 11 patients (11%) with a median size of 947 kb (range: 567 kb-18208 kb) and ranging from 36948016 on 9p13.3 to 20553365 on 9p21.3; 4) deletion of all chromosome 9 or 9p in 6 patients (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 activitiy. All these alterations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor. FISH analysis with three overlapping BAC probes encompassing the whole PAX5 gene was performed confirming what obtained by SNP-array analysis. To investigate the consequences of genomic PAX5 alteration in BCR-ABL1-positive ALL patients, quantitative PCR (q-PCR) was used to assess the expression of PAX5 in cases with copy number changes on 9p13.2. Q-PCR confirmed the SNP results demonstrating that genomic alterations on 9p13.2 leads 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 for this poor prognosis subtype of leukemia. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus, Fondazione del Monte di Bologna e Ravenna, PIO project 2007.

Introduction. The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukaemia (ALL) with the worst clinical prognosis. Aim and Methods. To identify oncogenic lesions that combine with BCRABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0) and genomic PCR to study 106 cases of adult BCR-ABL1-positive ALL. Results. The most frequent somatic copy number alteration was a deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 of 106 patients (75%). Two major deletions occurred: the first one (D4-7) was characterised by a loss of exons 4 through 7 (44/106, 42%), and the second one (D2-7) that we cloned was characterised by removal of exons 2 through 7 (20/106, 19%). A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the D4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localisation and oncogenic activity, while the D2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion was also identified in the progression of chronic myeloid leukaemia (CML) to lymphoid blast crisis (66%), but never in myeloid blast crisis or chronic phase CML, or in acute myeloid leukaemia patients. Known DNA sequence and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences (RSS) recognised by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination. Using gene-set enrichment analysis to compare the gene-expression signatures of patients with IKZF1 deletion versus not-deleted patients, we identified a unique signatures characterized by down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3, BLK) and upregulation of genes involved in cell-cycle progression (STK17B, SERPINB9, CDKN1A). These findings suggest that genetic alterations of IKZF1 influence the transcriptome and contribute to an impaired B-cell differentiation. We next investigated whether the IKZF1 deletions associated with a poor outcome. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). Conclusions. Deletion of IKZF1 is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome. It is likely that Ikaros loss combines with BCRABL1 to induce lymphoblastic leukaemia, arresting B-lymphoid maturation. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus, Fondazione del Monte di Bologna e Ravenna, PIO project 2007.

Background. The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. The constitutively active tyrosine kinase encoded by BCR-ABL blocks the B-cell differentiation, prevents apoptosis and also causes genetic instability. Recently, the introduction of array-based analysis of single nucleotide polymorphism (SNP) has allowed the rapid determination of genome-wide allelic information at high density for a DNA sample. Aims. To identify, at submicroscopic level, genetic lesions which can escape standard cytogenetic observations and may provide new insights into the alternative process underlining leukemogenesis and resistance in Ph+ ALL. Methods.We profiled, until now, the genomes of 36 out of 55 Ph+ ALL patients. The median age was 55 years (range, 18-76) and the median blast percentage was 90% (range, 76-100%). 250 ng of genomic DNA were processed on 500K SNP array according to protocols provided by the manufacturer (Affymetrix Inc., USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH), real-time quantitative PCR and western-blot analyses were performed to validate our results. Results. We identified region of high level amplification and homozygous deletion in all patients, with deletions outnumbering amplification almost 3:1. Copy number alterations most frequently involved chromosome 7 and 8. 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. Sub-microscopic lesions encompassing single or only few genes were observed with relative high frequency. Many of these copy number alterations were associated with cellular proliferation and/or apoptosis [e.g. CDKN2A and CDKN2B (n=8), GADD45A (n=2), FAS (n=2), BTG1(n=2)]. We also frequently observed anomalies in genes involved in early B-cell differentiation. These include the paired box gene PAX5 (9p13, n=5) which is a critical determinant of B-lineage commitment; the B-cell adapter containing a SH2 domain protein BLNK (10q23.2-q23.33, n=1); the pre-B lymphocyte gene VPREB1 (22q11.22, n=8) and the transcription factor IKZF1 (7p13-p11.1, n=9), which is required for normal lymphoid development. Reverse transcriptase-PCR and western-blot analysis for IKZF1 showed that in some cases this deletion was associated with the expression of the dominant-negative isoform Ik6 with cytoplasmatic localization. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, TOX, MDS, PBX1, RUNX1, ETV1 and PTEN. It is noteworthy that some lesions felt in regions lacking annotated genes. Conclusions. High-resolution SNP-based genomic profiling is a reliable and valid method for the identification of genomic anomalies and genes relevant for the development of leukaemia. Multiple independent copy number anomalies were frequently observed in genes involved in cell cycle regulation and B-cell differentiation, suggesting that these processes could contribute to the poor prognosis of Ph+ ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.

Among all cytogenetic subtypes of ALL, the BCR-ABL1 fusion gene, which is causative of chronic myeloid leukemia (CML), defines the subgroup of ALL with the worst prognosis. The reasons of the aggressive nature of BCR-ABL1-positive ALL have not yet been completely understood. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL and blastic transformation of CML, we used an integrated molecular approach including high resolution genomic analysis of copy number alterations by single nucleotide polymorphism (SNP) arrays (500K, Affymetrix Inc., Santa Clara, CA, USA), genomic amplification, cloning, deep sequencing, gene expression profiling (GEP) and analysis of epigenetic changes to study 97 de novo BCR-ABL1- positive ALL adult patients (pts). High level amplification and homozygous deletions were identified in all pts, with deletions outnumbering amplification almost 3:1. The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros identified in 59/97 pts (61%). Ikaros is required for the earliest stages of lymphoid lineage commitment and acts as tumor suppressor in mice. The IKZF1 deletions were predominantly mono-allelic (64% vs 36%) and were limited to the gene in all cases, identifying IKZF1 as the genetic target. Real-time quantitative PCR and FISH analysis confirmed SNP results. We characterized and mapped all breakpoints to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4–7 (37/97, 38%) and due to breakpoints occurring in the region spanning from 50380371 to 50380388 and from 50431123 to 50431167 in introns 3 and 7, respectively; type B characterized by removal of exons 2–7 (18/97, 19%) and due to a variable pattern of breakpoints in introns 1 (from 50317112 to 50317936) and 7. A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of both deletions correlated with the expression of a dominant-negative isoform Ik6 with cytoplasmic localization and oncogenic activity, and of an aberrant transcript containing only exons 1 and 8, respectively. Interestingly, 2 pts with a homozygosis deletion showed simultaneously both type A and B deletions. In other 2 pts, the deletion involved the whole IKZF1 gene. The IKZF1 alteration was not a frequent event across different leukemias, since it was identified also at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML (n=10), chronic phase CML (n=30) and acute myeloid leukemia (n=28). Known DNA sequences and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH), suggesting that IKZF1 deletions could arise from aberrant RAG and/or AID-mediated recombination. GEP analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to a block of B-cell differentiation. For 83 out of 97 BCR-ABL1 ALL pts correlation between molecular data and clinical outcome was available. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 resulting in either haploinsuffciency and in the expression of the dominant negative isoforms is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

No abstract available.

Abstract 2795 In the era of molecular target therapy, whereas Imatinib has shown an overall survival rate of 85% and an estimated event-free survival of 55% in a 8 year result update of IRIS trial, several in vitro data have confirmed that Ph+ stem cells housed within BM niches are resistant to TKI treatments. Second generation TKI Nilotinib has been designed with improved target specificity over Imatinib. Its efficacy in the treatment of patients with CML who are resistant to or intolerant to Imatinib led to the registration of a clinical trial CAMN107EIC01, a phase IIIb, multicentre, open-label study applying Nilotinib in the treatment of newly diagnosed CML. The aim of the current study (CAMN107EIC01 sub-study N10) is to define BM microenvironment markers that nurture and determine leukemic stem cell fate in the BM niches of Nilotinib treated patients. We enrolled 37 patients involving 21 Italian centers, from whom written informed consent has been obtained for sub-study N10. Patients have been monitored by Real Time RT-PCR (RT-qPCR) for the expression of the fusion mRNA BCR-ABL, as specified by the core protocol. Major Molecular Response (MMR) is defined as detectable disease ≤0.1% BCR-ABL according to the international scale (IS). Plasma and mononuclear cells have been collected from BM and PB samples of the enrolled patients at the screening visit (V0) and after 3 months of treatment (V4). We purified total RNA from BM and PB mononuclear cells of the enrolled patients to screen by RT-qPCR the expression of 10 genes (ARF, cKIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the regulation of the stemness and survival signaling of hematopoietic stem cells. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN). Moreover, we evaluated by multiplex ELISA assay BioPlex, the BM plasma concentration level of 20 cytokines (IL1a, IL1b, IL3, IL6, IL7, IL8, IL10, IL12, IL15, G-CSF, M-CSF, SCF, SDF1, TRAIL, HGF, PDGFbb, GM-CSF, MIP-1a, TNFa and VEGF), known to be key factors in the interaction of Ph+ stem cells to BM microenvironment. The interim analysis of MMR until the 12th month is available in 26 out of 37 enrolled patients. We observed that MMR is achieved during the first 12 months of treatments in 17 out of 26 (65%) patients. Molecular analysis showed that the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) and cytokine signaling (SET) were significantly down-regulated at V0 in PB mononuclear cells of patients achieving MMR in comparison to cells from non-responding patients (4vs21 NANOg NCN, p=0.05; 8vs32 SET NCN, p=0.05). These data were also confirmed on BM patient's sample. Moreover, we investigated the concentration level of the above-mentioned soluble factors in BM plasma samples of all 37 enrolled patients at both V0 and V4. We observed that the BM plasma level of several cytokines produced by leukemic cells significantly decreased during the first 3 months of Nilotinib treatment: IL3 (54vs3ng/ml; p=0.02), M-CSF (56vs12 ng/ml; p=0.005), SCF (170vs104 ng/ml; p=0.007), HGF (7565vs705 ng/ml; p Disclosures: Paratore: Novartsi: Employment. Galimberti:Novartis: Employment. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 575 Incorporation of the tyrosine kinase inhibitor (TKI) imatinib in the frontline treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) has significantly improved the anti-leukemic efficacy of induction therapy. In contrast to chronic myeloid leukemia (CML), however, responses are short-lived and relapse is frequently associated with the selection of Bcr-Abl kinase domain (KD) mutations, fostered by the high genetic instability of Ph+ ALL cells. The advent of the 2nd-generation TKIs dasatinib and nilotinib has brought additional treatment options both for newly diagnosed and for imatinib-resistant pts. To analyze the changes they have brought in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL KD mutation analyses done in our laboratory from January 2004 to June 2011. Overall, 781 tests on 258 Ph+ ALL pts (number of tests per pt, range: 1–15) were performed by denaturing high-performance liquid chromatography (D-HPLC) followed by direct sequencing of D-HPLC-positive cases. One hundred and fourty-three pts were analyzed because of imatinib resistance. One hundred and one out of 143 (71%) pts scored positive for one or more KD mutations. Similarly to what is know to occur in CML, hematologic and cytogenetic resistance were by far more frequently associated with mutations than molecular resistance (Bcr-Abl transcript increase as assessed by RT-Q-PCR). Overall, mutations at thirteen residues were detected. In contrast to what can be observed in CML, three mutations were by far the most frequent, accounting for almost 75% of the mutated cases: T315I (n=38 pts, 37%), E255K/V (n=19 pts, 18%) and Y253H (n=19 pts, 18%). The other mutations were, in order of frequency: F359V/I, M244V, M351T, F317L, G250E, Q252H, L387M, D276G, L248R, E279K. Nine out of 103 (9%) pts had two mutations, in the same (2 pts) or in different (7 pts) subclones. In 84 pts who were analyzed because they were reported to have developed resistance to dasatinib (n=72) or nilotinib (n=12) as 2nd- or 3rd-line TKIs, 65 (77%) had newly acquired mutations (57/72 dasatinib-resistant pts and 8/12 nilotinib-resistant pts). The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib and in 2/8 cases acquiring mutations on nilotinib. Other recurrent newly acquired mutations were F317L, V299L, T315A in dasatinib-resistant pts and Y253H and E255K in nilotinib-resistant pts. Thirty out of 65 pts (46%) were positive for multiple mutations (2 to 4 mutations, in the same or in different subclones or both) that emerged under the same TKI in 11 cases (37%) and accumulated as a consequence of multiple lines of TKI therapy in the remaining 19 (63%) cases. Mutation analysis was also performed in 15 resistant pts enrolled in a clinical trial of dasatinib as first-line treatment for Ph+ ALL. Twelve pts were positive for mutations; 11/12 had a T315I. Sixty-one pts were analyzed at the time of diagnosis in order to assess whether TKI-resistant mutations could already be detectable. Only two pts (3%) were positive for mutations: one patient had an F311L that disappeared after one month of nilotinib treatment; an additional patient was positive only by D-HPLC, but not by the less sensitive direct sequencing – most likely for the T315I mutation that shortly after the start of dasatinib treatment outgrew and led to resistance. Taking advantage of the recent availability of a next-generation sequencing platform (Roche 454), allowing high sensitivity (0.01%) mutation scanning of the KD, samples collected at the time of diagnosis and during follow-up from selected Ph+ ALL cases who developed mutations and resistance to TKI therapy were retrospectively analyzed – but the mutations were not always already detectable at diagnosis. In conclusion: a) although 2nd generation TKIs may ensure a more rapid debulking of the neoplastic clone and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy; b) the clinical usefulness of mutation screening of Ph+ ALL pts at diagnosis before TKI start, even with highly sensitive approaches is low – not all mutations pre-exist since genetic instability remains high and fosters mutational events anytime during treatment. Supported by PRIN, FIRB, AIL and AIRC. Disclosures: Soverini: ARIAD: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Foà:Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baccarani:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 3580 Introduction: Although treatment with tyrosine kinase inhibitors (TKIs) has revolutionized the management of adult patients with BCR-ABL1 -positive acute lymphoblastic leukemia (ALL) and significantly improved response rates, relapse is still an expected and early event in the majority of them. It is usually attributed to the emergence of resistant clones with mutations in BCR-ABL1 kinase domain or to BCR-ABL1 -independent pathways but many questions remain unresolved about the genetic abnormalities responsible for relapse after TKI and chemotherapy-based regimens. Patients and methods: In an attempt to better understand the genetic mechanisms responsible for this phenomenon, we have analyzed matched diagnosis-relapse samples from 30 adult BCR-ABL1 -positive ALL patients using high resolution Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI, n=15 pairs and Genome-Wide Human SNP 6.0, n= 15 pairs). Genetic differences were analyzed in terms of copy number changes and loss of heterozygosity (LOH) events. 20 patients were enrolled in clinical trials of GIMEMA AL Working Party and treated with imatinib alone or in combination with conventional chemotherapy (40%) or dasatinib as frontline therapy (60%). The median age at diagnosis was 54 years (range 23–74) and the median blast cell count was 97% (range 60–99). The median time to relapse was 27 months (range, 9–104). 10 patients were treated according to the GMALL trials, a high-dose chemotherapy based protocol in combination with imatinib. The median age at diagnosis was 65 years (range 19–79) and the median leucocyte count was 37300/μl (range 5000 – 220000/μl). The median time to relapse was 9.8 months (range, 3 – 25). Results: First, we compared diagnosis and relapse samples for the presence of macroscopic (> 1.5 MB) copy number alterations (CNA). Novel acquired macroscopic CNAs were detected in 7/20 (35%) TKI relapse cases and included losses of 3p12-p14, 5q34, 9q34, 10q24 and 12p13-p12 and gains of 1q, 9q34-q33 and 22q and in 4/10 (40%) chemotherapy-relapse cases and included losses of 9p21 and 12q21–22 and gains of all chromosome 8 or part of it in 2 patients. Since no common patterns of acquired alterations were observed, it is likely that relapse may be due to a more generalized genetic instability rather than to specific mechanisms. Moreover, chemotherapy did not select resistant clones with higher number of alterations. 8/20 (40%) TKI resistant cases and 4/10 chemotherapy resistant patients harbored the same CNAs present in the matched diagnosis sample (losses of 9p21 in 7 cases, 7p and 22q11 in single cases and gains of chromosomes 1q, 4, 8q, 17q and 21), indicating a common clonal origin. In contrast, in 5/20 (25%) TKI resistant cases and 4/10 (40%) chemotherapy resistant patients macroscopic CNAs present at diagnosis were lost at relapse (losses of chromosomes 7, 11q, 14q, 15q, 16q and 19p and gains of 5q, 8q, 9q34 and 22q11). Thereafter, we compared diagnosis and relapse samples for microscopic CNAs (< 1.5 MB). The alteration most frequently acquired at relapse was loss of the tumor suppressor CDKN2A (53% vs 33 % of diagnosis). Other common acquired CNAs at relapse included gains of ABC transporter genes, such as ABCC1, ABCC6 (1q41) and BCL8 (15q11); losses affected EBF1 (5q33) and IGLL3 (22q11) genes involved in B-cell development, BTG1 (12q21) involved in cell cycle regulation and CHEK2 (22q12) involved in DNA repair. Interestingly, for all relapse cases analysis of IKZF1 deletions, identified in 80% of patients, demonstrated a clonal relationship between diagnostic and relapse samples, suggesting that this alteration is not acquired with relapse but it is maintained with fidelity from diagnosis working as a marker of disease. The majority (92%) of relapse samples harbored at least some of the CNAs present in the matched diagnosis sample, indicating a common clonal origin. Conclusions: Genomic copy number changes evolving from diagnosis to relapse have been identified demonstrating that a diversity of alterations contributes to relapse and with the most common alterations targeting key regulators of tumor suppression, cell cycle control, and lymphoid/B cell development. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, Ateneo RFO grants, PIO program, Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Ottmann:Novartis Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 1677 The BCR-ABL1 positive ALL is the most frequent and prognostically most unfavorable subtype of ALL in adults. Treatment strategies based on tyrosine kinase inhibitors of first and second generation have improved overall treatment results, with a rapid and complete remission (CR) rate ranging 90%. Nevertheless most patients experienced hematological relapse in a short time, also after hematopoietic stem cell transplantation. Therefore, the detection of residual leukemic cells by measuring BCR-ABL1 transcript level is absolutely required to monitor minimal residual disease and to detect early relapse. Different assays based on quantitative polymerase chain reaction (qPCR) are available and they vary in respect to the type of instrument used, the primer and probe location, the real-time chemistry and the control gene used. Differences among methods can lead to a variation in the sensitivity and measurement reliability. In order, to investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared the molecular results obtained by conventional qPCR with those obtained by nanofluidic Fluidigm approach. At the time of writing, 60 BCR-ABL1-positive ALL samples (42 positive for the p190 BCR-ABL1 isoform and 18 for the p210) were analyzed. First, we performed a TaqMan absolute quantitative PCR (Applied Biosystems 7900HT Fast Real-Time PCR System); RNA integrity was evaluated using the control gene ABL1. Both BCR-ABL1 or ABL1 copy number copies were derived by the interpolation of cycle threshold (Ct) values to the appropriate standard curve obtained using different plasmid dilutions (Ipsogen), each containing known BCR-ABL1 or ABL1 gene copies. Samples resulted in complete (38.3%) or major (61.7%) molecular response (BCR-ABL1/ABL1 ratio ≤ 0.001 or < 0.1, respectively and according to the International Scale). Subsequently, we quantified again the samples using the 12.765 Digital array (Fluidigm). This is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Samples are portioned prior to qPCR into the single chambers of the panel; as fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Following amplification, digital raw data are processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per chamber using the Poisson probabilistic distribution. First, we assessed the sensitivity and reproducibility of the assay using six serial dilutions of plasmids (Ipsogen) expressing known copy number of BCR-ABL1 p190 transcript (10000; 1000; 100; 50; 10; 1 copies). A 2 μl volume of input leukemia cDNA was loaded and two panels for each dilution were used. Analysis parameters chosen for digital raw data processing were automated set threshold and target Ct range 20–40. Results showed a detection rate until a copy of target sequence (Fig. 1) and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 positive ALL samples with a positive control for each chip: digital array resulted positive in 22 major molecular response samples (59.46%; 3.25 median number of copies detected, range 0.5–33.5), and in 1 complete molecular response sample (4.34%). Moreover, the comparison between TaqMan and Fluidigm methods resulted in the detection of the same BCR-ABL1 copies in 53.34% (group I); in 35.0% the quantity detected by Fluidigm was lower than that detected by TaqMan (group II); finally, in 11.66% the Fluidigm approach resulted more sensitive than TaqMan (group III). In conclusion, we demonstrated that the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, as demonstrated with plasmid serial dilution detection rate and with samples in molecular remission and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Abstract 1670 Among genes that are supposed to be involved in altered molecular pathways leading to leukemogenesis, the CDKN2A/B locus is particularly noteworthy since it encodes for the INK4-class cyclin dependent kinase inhibitors p15 INK4B, p16 INK4A and p14 ARF, which act as tumor suppressors. The CDKN2A/B locus has been found to be inactivated in several haeamatologic malignancies mainly due to deletion, aberrant repression or epigenetic silencing, whereas little is known about the potential role of its single nucleotide polymorphisms (SNPs) in leukemia susceptibility. To investigate whether polymorphisms within this broad genomic region can correlate with increased susceptibility to haeamatologic malignancies, an association study was performed by genotyping 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions, in a case-control cohort made up of 332 samples: 149 leukemia patients, including Philadelphia positive (Ph+) ALL (n=92) and acute myeloid leukemia (AML) samples (n=57), and 183 unrelated healthy controls. Ph+ ALL patients showed a median age of 52 years (18-78 years); 24 cases (26%) expressed the p210 oncoprotein, 62 (67%) the p190 and 6 (7%) both the proteins. AML patients showed a median age of 53 years (21-71 years) and they included FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype subtypes. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected among those included in the Affymetrix Genome-Wide Human SNP Array 6.0 to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Furthermore, all leukemia samples were also previously genotyped using the GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix). A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with Minor Allele Frequency (MAF) 20% missing call rates, were instead excluded from the association analysis. Potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and an Odd Ratio (OR) of 2 (95% CI, 1.20 to 3.33; p= 7.1 × 10-3). Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

In Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) treated with tyrosine kinase inhibitors (TKIs), responses are generally short-lived and relapse is most often accompanied by selection of point mutations in the Bcr-Abl kinase domain (KD). In order to assess whether mutations are already present at the time of diagnosis, we analyzed RNA samples from 13 pts with newly diagnosed Ph+ ALL enrolled on the GIMEMA LAL1205 protocol – designed to assess activity, safety and tolerability of dasatinib monotherapy as first-line treatment for adult Ph+ ALL. The pts investigated (males/ females: 6/7; median age: 56 years, range 30–73) included 6 pts who subsequently relapsed with dasatinib-resistant mutations, 2 pts who relapsed without evidence of mutations as assessed by direct sequencing and 5 pts in persistent remission. Screening for low level mutations was performed by cloning the entire Bcr-Abl KD (amino acid 206 through 524) in a bacterial vector and sequencing 150 to 200 independent clones for each pt. All pts, irrespective of their subsequent response to dasatinib, had evidence of aberrant KD sequences due to point mutations (13/13 pts), small insertions (2/13 pts) or small deletions (1/13 pts). Two to five point mutations were detectable for each pt. In some cases, multiple mutations could be found to co-exist in the same cloned fragment. A total of 45 different point mutations (including 18 silent mutations, 4 nonsense mutations and 23 missense mutations) were observed. Base substitutions were scattered all over the KD and there were no mutation hotspots. Thirty-nine out of 45 (87%) mutations were transitions: G>A (n=14), A>G (n=10), C>T (n=9), T>C (n=6). Such a high prevalence of transitions (which normally occur 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is predominant. Activation-induced cytidine deaminase (AID) has been recently shown to be aberrantly expressed in a proportion of Ph+ ALLs and has been implicated in the development of at least some Bcr-Abl mutations; correlation with AID expression will be presented. Mutations were detected in no more than three independent clones, suggesting that they were present at low levels. Mutations detected in less than two clones were considered only if independently validated by a specifically designed ASO-PCR assay run on the original cDNA sample. The majority of point mutations detected have never been reported in association with TKI resistance. However, three pts were found to harbor known imatinib- or dasatinib-resistant mutations, including two T315I mutations that were later on selected and led to relapse. This supports the theory that mutations randomly arise as a consequence of a high genetic instability and very few of them will confer a growth advantage under the selective pressure of TKIs. Six samples from newly diagnosed chronic phase CML (CP-CML) pts were screened for comparison with the same cloning approach, including 2 pts who later progressed with evidence of KD mutations after 3 and 6 months from the start of imatinib, respectively, and 4 pts who achieved a stable molecular response on imatinib. Both pts who experienced early relapse on imatinib had low level mutations detectable at the time of diagnosis, whereas none of the clones from CP-CML imatinib responders had evidence of KD mutations. The results herein presented support the concept that Ph+ ALL cells have a particularly high genomic instability that allows early escape from TKI inhibitor therapy. Only a fraction of CML cases share this feature. Our data highlight the importance of understanding the mechanism(s) responsible for this ‘mutator’ phenotype, as well as how to interfere therapeutically with it.

Abstract 1024 Although treatment with tyrosine kinase inhibitors has revolutionized the management of adult patients with BCR-ABL1-positive ALL and significantly improved response rates, relapse is still an expected and early event in the majority of them. It is usually attributed to the emergence of resistant clones with mutations in BCR-ABL1 kinase domain or to BCR-ABL1-independent pathways but many questions remain unresolved about the totality of genetic abnormalities and the knowledge of which alterations really matter for relapse. In an attempt to better understand the genetic mechanisms responsible for this phenomenon, we have analyzed matched diagnosis-relapse samples by three high resolution approaches: genome wide single nucleotide polymorphisms (SNPs) array (Affymetrix Genome-Wide Human SNP Array 6.0), gene expression profiling (Affymetrix Human Exon 1ST Array) and whole transcriptome deep sequencing by Illumina/Solexa technology. (RNA-Seq) approach. An additional subset of 30 adult BCR-ABL1+ positive ALL cases were analyzed by SNP and gene-expression arrays and by candidate gene sequencing in order to validate specific alterations. For whole transcriptome deep sequencing (RNA-seq) 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 alternative splicing (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. 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. At diagnosis, 6 novel missense single nucleotide variations (SNVs) were detected after applying stringent criteria to reduce the SNV discovery false positive rate and validating novel substitutions with genomic DNA Sanger sequencing: 5 affected genes involved in metabolic processes (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46), one transport (MVP); these mutations disappeared at relapse and in this phase 3 novel missense mutations affecting genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21) were found. Furthermore, the T315I mutation in the Bcr-Abl kinase domain was also identified. These 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 acquired only during leukemia progression. Only the R20Q DPEP1 mutation was found in 1/30 additional BCR-ABL1 positive adult cases. By RNA-seq, 4,334 and 3,651 primary ALL and relapse isoforms with at least one AS event were identified. 240 of these genes were known cancer-related alternatively spliced genes, of which kinases and transcription regulators were the most represented functional classes. An average of 1.5 and 1.3 AS per isoform was estimated. The well-known alternatively spliced IKZF1 isoform was also detected both at the diagnosis and relapse. 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. In conclusion, this study provided, for the first time, a quite comprehensive overview of a BCR.-ABL1-positive ALL transcriptome, identifying novel mutations, changes in gene expression levels and AS events potentially involved in ALL 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. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Background: BCR-ABL1-positive ALL is the most frequent and prognostically most unfavorable subtype of adult ALL. The main reason for the poor clinical outcome of BCR-ABL1-positive ALL is genetic instability. However, how normal B-cell precursor cells acquire the genetic changes that lead to transformation and progression has not been completely defined. Activation-induced cytidine deaminase (AID) produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: 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 BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and methods: We analyzed 61 adult de novo Ph+ ALL patients (pts) and 60 CML pts (chronic phase and myeloid/lymphoid blast crisis). 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 for automated capillary gel electrophoresis 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 41 (67%). AID expression correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. Moreover, AID expression was also found in lymphoid blast crisis CML (60%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were expressed. In 10/61 (16%) BCR-ABL1-positive ALL pts the full-length isoform (GeneBank accession number NM_020661) was identified, in 16/61 (26%) the co-expression of the wild-type isoform and of different AID splice variants was found and in 15/61 (25%) only the expression of splice variants was found. These can result from retention of intron 4 (Variant A), omission of exon 4 (Variant B) and 3 (Variant C), and from a deletion of 30 bp in the initial portion of exon 4 (Variant D). In the wild-type mRNA, codon 148 spans exons 3 and 4. In both variants A and B, mRNA splicing disrupts this codon and causes a frameshift, which results in a premature stop codon. If translated, the splice variants produce truncated proteins of 187 and 145 amino acids, respectively. However, the putative deaminase active site, encoded by exon 3, is preserved in both splice variants, but is lacking in the variant C. Since enforced expression of Pax5 induces endogenous AID gene expression, we analyzed the expression levels of Pax5 in all pts (ΔΔCt method, GAPDH as control gene). As expected, we found a very strong difference (p Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as a mutator outside the Ig gene loci in promoting genetic instability in leukemia cells.

Abstract 3074 Poster Board III-11 Background BCR-ABL1-positve Acute Lymphoblastic Leukemia (ALL) is the most common ALL subtype in adults and is associated with poor prognosis. The pathogenesis of this leukemia is related to the expression of the BCR-ABL1 fusion transcript, but additional recurrent genetic lesions are suspected to be involved in its development and progression. Aim A Next-Generation Sequencing Technology was used to sequence the whole transcriptome of leukemia cells from a BCR-ABL1-positive ALL patient at diagnosis and at relapse following tyrosine kinase inhibitor (TKI) therapy with the aim to detect acquired mutations cooperating with BCR-ABL1 in leukemia manifestation and drug-resistance. Methods Poly(A) RNA was extracted from leukemia cells and used to prepare double-stranded cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained 36 base-pair (bp) sequence reads were mapped to the reference sequence of the human genome (UCSC hg18, NCBI build 36.1) to identify single nucleotide variants (SNVs) and to estimate reads density corresponding to RNA from each known exon, canonical splice event or new candidate gene. This approach allowed us to define a detailed Digital Gene Expression (DGE) profile. Reads that showed no match to the reference genome were subsequently mapped to a dataset of all possible splice junctions created by in silico pairwise combination of the exons of all annotated genes (UCSC knownGene file) to identify alternative splicing (AS) events. Results Whole Transcriptome Shotgun Sequencing (RNA-seq) analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples respectively, achieving approximately 90% diploid coverage and detecting transcripts from 62% and 64% of human annotated genes. The great majority of these active genes (78% at diagnosis and 73% at relapse) showed very low expression levels, with a number of reads per kilobase of exon model per million mapped reads (RPKM value) from 0.01 to 10, whereas 20% and 24% showed moderate expression levels (RPKM 10-100), as well as only 2% and 3% resulted highly expressed (RPKM 100-8000). Moreover, 6,390 and 4,671 AS events were also identified within 4,334 diagnosis and 3,651 relapse annotated transcripts, with the already described ALL-related Ik6 Ikaros isoform observed in both samples. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. Of greater interest as potential ALL-related mutations, 124 and 115 non annotated SNVs were also found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 72 resulted diagnosis and relapse private variants. In particular, the analysis was focused to the coding sequences of annotated genes, finding that non-synonymous changes were one out of the 19 shared between the two samples and affected a transmembrane receptor gene (PLXNB2). Six out of the 12 diagnosis private variants, affecting genes involved in metabolic process (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) or transport (MVP) and 5 out of the 30 relapse private variants, affecting genes involved in cell cycle regulation (ABL1, CDC2L1), catalytic activity (CTSZ, CXorf21) or with unknown function (FAM116B). Most of these diagnosis and relapse non-synonymous private mutations resulted highly expressed, showing frequencies of mutated unique reads higher than 50%. According to this pattern, diagnosis private mutations may be carried by primary leukemic clones that did not develop again at relapse, whereas relapse private mutations have greater probability to be variants acquired during the disease progression. Interestingly, the T315I point mutation in the Abl kinase domain, that confers resistance to many TKIs, was found at relapse but not at diagnosis. Conclusions An accurate expression profile was obtained for the leukemia cells of the examined ALL patient, as well as the discovery of several new non-synonymous mutations affecting genes from different pathways and for which no correlation was previously found with ALL pathogenesis. These findings demonstrate that RNA-Seq represents a suitable and cost-efficient approach for identifying new genes potentially involved in ALL development and progression. Acknowledgments AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo 60% grants, European LeukemiaNet. Disclosures No relevant conflicts of interest to declare.