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Introduction. The proteasome inhibitor bortezomib is a new treatment option for patients with refractory or relapsed ALL, particularly when used in combination with conventional chemotherapy or other targeted agents. Indeed, a limited efficacy of bortezomib alone in ALL patients has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. CX-4945, a potent CK2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. Here, we have explored the cytotoxic effects of a bortezomib/CX-4945 combination in a panel of B- and T-ALL cell lines. Methods. B- (KOPN-8, NALM-6, RS4;11) and T- (MOLT-4, Jurkat, CEM-R) ALL cell lines were pretreated with bortezomib (Selleck Chemicals, Houston TX, USA), for six hours and then with CX-4945 (Selleck Chemicals, Houston TX, USA), for 16/24 hours. MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining and flow cytometric analysis. Protein expression was studied by western blot. Results. Cells were cultured in the presence of bortezomib or CX-4945, either alone or in combination at a fixed ratio. The combined treatment was more effective in reducing cell viability in all B-ALL cell lines and in MOLT-4 cells. Annexin V/PI staining was performed; interestingly, no synergism was detected if the two drugs were administered together since the beginning of treatment. In response to treatment with 2.5 nM bortezomib followed by 5 μM CX-4945, we detected an increase in apoptosis in B-ALL cell lines and in MOLT-4 cells after 24 h. Western blot analysis for the cleaved forms of caspase-3 and PARP, confirmed a higher apoptosis induction by the combined treatment. A reduction in anti-apoptotic Bcl2 concomitant with an increase in proapoptotic Bax, suggested that bortezomib/CX-4945 treatment caused a mitochondrial apoptosis. IRE1a and CHOP (established markers of ER stress/UPR-mediated apoptosis) levels increased in response to the combined treatment, in contrast the expression of GRP78/BIP (a marker of UPR activation) decreased, suggesting that a potential mechanism by which the drug combination induced cell death, involved ER stress induction by bortezomib and the inability to respond by adequate activation of the UPR signaling which was blocked by CX-4945. Conclusions. Here, we demonstrated that the proteasome inhibitor bortezomib and the CK2 inhibitor CX-4945 interact in a synergistic manner to induce apoptosis both in B- and in T-ALL cell lines. Drug cytotoxicity was associated with modulation of the ER stress/UPR signaling pathway. Importantly, the synergism was observed only when bortezomib treatment preceded CX-4945 administration. Therefore, our findings support clinical application of bortezomib in combination with CX- 4945 in B- and T-ALL treatment.

Bcl2 expression in T-ALL cell lines. Western blotting analyses for the basal expression of Bcl2 in untreated T-ALL cell lines. Twenty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. Densitometric analysis was performed using a Chemidoc 810 Imager with the appropriate software (UVP, Upland, CA, USA), and for each cell line, Bcl2 protein expression is indicated relatively to β-actin expression.

Background: About 20% of the childhood AML present MLL gene translocations, that are associated with a very poor prognosis. That is why there is a strong interest in developing novel therapeutic strategies for these patients. As DOT1L is responsible for H3K79 methylation and is associated with MLL-r leukemogenesis, DOT1L inhibitors entered in clinical trials as promising treatments. MLL-r AML also showed an overexpression of FLT3 receptor tyrosine kinase. Therefore, FLT3 inhibitors, such as the multi-tyrosine kinase inhibitor Sorafenib, demonstrated encouraging efficacy in AML. Aims: To investigate the efficacy of a combination treatment using DOT1L inhibitor EPZ-5676 and Sorafenib to treat MLL-r AML. Methods: MLL-r (MOLM13, NOMO1, THP1) and non MLL-r (OCI-AML3, HL60 and U937 harboring CALM-AF10 translocation) AML cell lines, and MLL-r primary samples from pediatric AML patients were used. Flow cytometry analyses were performed to assess absolute cell counting and apoptosis (AnnexinV FITC/PI staining). Protein expression and H3K79 methylation were quantified by Western blot. mRNA expression was studied by quantitative Real-Time PCR (Q-PCR). Results: Firstly, the specific effects of DOT1L inhibition were examined in AML cell lines treated with increasing concentrations of EPZ-5676, up to 18 days. Cell growth was significantly inhibited after at least 8 days of treatment in MOLM13 and NOMO1 cells, but time-dependent apoptosis occurred only in MOLM13, thus suggesting that DOT1L inhibition alone might not be able to induce cytotoxicity. Whereas no effects were observed in HL60, U937 (non MLL-r), or THP1 (MLL-r) cells, both cell growth impairment and apoptosis were detected in non MLL-r OCI-AML3 cells, so that the impact of DOT1L inhibition could not exclusively rely on MLL rearrangements. Repression of DOT1L occurred since the 4th day of treatment, as demonstrated by the complete loss of H3K79me2. To further explore the consequence of this phenomenon, both MLL targets and key component of signaling pathways involved in AML survival (i.e. PI3K, FLT3 and MAPK) were investigated in cells treated with 1 uM EPZ-5676, up to 28 days. Gene expression of HOXA9, MEIS1, FLT3 and CDK6 protein (MLL target) decreased in all cell lines, whereas STAT5 and c-Myc mRNAs, along with STAT5 protein expression, were downregulated only in MLL-r cells. Furthermore, in MOLM13 and NOMO1 cells p-Erk was strongly reduced. Finally, p-Akt slightly decreased in nearly every case, whereas a strong induction in p-S6RP was observed only in U937 cells after prolonged treatment, suggesting the possible involvement of PI3K pathway in drug resistance mechanisms. To increase the benefit of DOT1L inhibition, both AML cell lines and MLL-r primary samples were pretreated with increasing concentration of EPZ- 5676 for 4/8 days, following 24/48h treatment with Sorafenib. This combination resulted in a synergistic effect in nearly all cases. Importantly, EPZ-5676 pretreatment increased Sorafenib-induced cell growth inhibition in EPZ-5676 refractory cells HL60, U937 and THP1. Moreover, in primary AML samples both EPZ-5676 and Sorafenib showed a limited effect as single agent, whereas their combination induced a drastic increase of apoptosis. Summary/Conclusions: These results demonstrated that the single administration of EPZ-5676 has a limited antileukemic activity, which is not restricted to MLL-r AMLs. However, the combination of EPZ-5676 with Sorafenib revealed a synergistic effect in both MLL-r and non MLL-r AMLs, paving the way to innovative and more effective treatments for pediatric AML patients.

Background: Detection of residual leukemic cells by measuring BCR-ABL1 transcript level with assays based on quantitative polymerase chain reaction (qPCR) is necessary in the monitoring of minimal residual disease in patients with BCR-ABL1 positive ALL. Aim: 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 results obtained by conventional qPCR with those obtained by the nanofluidic approach. Patients and methods: We analyzed 60 BCR-ABL1 positive ALL samples expressing p190 (42) or p210 (18) isoform, first by TaqMan absolute qPCR (Applied Biosystems 7900HT Fast Real-Time PCR System). BCR-ABL1 target gene and ABL1 control gene copies were derived by the interpolation of cycle threshold values to the appropriate standard curve obtained using serial plasmid dilutions containing known BCR-ABL1 or ABL1 gene copies. Results: Samples resulted in complete (22) or major (38) molecular response (BCR-ABL1/ABL1 ratio ≪ 0.001 or < 0.1, respectively; 3,8 BCR-ABL1 mean copy number copies). Subsequently we reanalyzed the samples using the 12.765 Digital array (Fluidigm) and the integrated BioMark Digital PCR Analysis software (Fluidigm). The nanofluidic biochip consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR. 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. Sensitivity and reproducibility of the assay were assessed using six serial dilutions of plasmids (Ipsogen) expressing known BCR-ABL1 p190 transcript copy number (10000; 1000; 100; 50; 10; 1 copies). A 2 µl volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 ALL samples: digital array resulted positive in 21 of major molecular response samples (2,13 BCR-ABL1 mean copy number copies) and in any complete molecular response samples. In conclusion, the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL. Aim: To characterize mutations and rearrangements on 9p involving PAX5 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: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); 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); 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); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of 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: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions. Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

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 of 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 Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a 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 in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also 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 were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs.

Mutations in the Bcr-Abl kinase domain (KD) are often detected at the time of resistance to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). It is still unclear whether and in how many pts low level mutations may already be detectable at the time of diagnosis. We therefore analyzed cDNA samples from 22 newly diagnosed pts with Ph+ ALL (n=13) or chronic phase (CP)-CML (n=9) who subsequently received TKI therapy (imatinib, dasatinib or nilotinib). Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a. 206-524) in a bacterial vector and sequencing 200 independent clones for each pt. All pts had evidence of aberrant KD sequences. Three to twelve different mutations were detected in each pt. Each mutation was present in two to five independent clones. A total of 105 mutations (including 35 silent, 5 nonsense, and 65 missense mutations) were observed. The vast majority (98/105, 93%) of them have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 93/105 (89%) mutations were transitions: G>A (n=28), A>G (n=23), C>T (n=22), T>C (n=20). Such a high prevalence of transitions (normally occurring 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is active in Ph+ cells. One of the nine CP-CML pt received hydroxyurea for 6 months before starting imatinib therapy. In this pt, high-sensitivity mutation screening was performed again immediately before imatinib start and showed further accumulation of mutations. Eight Ph+ ALL pts and three CML pts subsequently relapsed with evidence of mutations, but only one with a mutation (T315I) that was already detectable at diagnosis. The remaining eleven pts are in persistent remission after a follow up ranging from 12 to 36 months, although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. Our observations suggest that: a) Bcr-Abl KD mutations can probably be found at diagnosis in all CP-CML and Ph+ ALL pts; b) mutations seem to arise randomly and most of them are silent or not conferring any growth advantage under the selective pressure of TKIs; c) generation of mutations seems to be linked to Bcr-Abl-driven genetic instability; d) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity. This warns against high-sensitivity mutation screening of all CML and Ph+ ALL pts before the start of TKI therapy.

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 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 BCR-ABL1-positive ALL patients (pts) and 60 CML (chronic phase and myeloid/lymphoid blast crisis) pts. 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#8217; 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 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. AID expression was also found in lymphoid blast crisis CML (50%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform; 19/61 (31%) co-expressed the wild-type and different AID splice variants with deaminase activity (AID#916;E4a, with a 30 bp deletion of exon 4; AID#916;E4, with the exon 4 deletion; AIDins3, with the retention of intron 3-4); 4/61 (7%) expressed the AID#916;E3-E4 isoform without deaminase activity (deletion of exons 2 and 3). To investigate whether AID introduces DNA-single strand breaks, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA). Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative (median copy number alteration of 14 versus 4. respectively, p

The activation of Chk1 has been demonstrated to influence response to chemotherapy in patients with BCR-ABL-positive chronic myeloid leukemia patients (Nieborowska-Skorska et al, Cell Cycle 2006). Chk1 and Chk2 are serine threonine kinase activated by different insults leading to DNA damage and involved in DNA repair and cell cycle control. Since, their role has not been established in BCR-ABL+ ALL, we investigated the expression of Chk1/Chk2 in blast cells from adult BCR-ABLpositive ALL patients (n=48) compared with normal bone marrow precursor cells (n=9) using the BioMark instrument (Fluidigm) and Fluidigm Dynamic Array 48 x 48. The expression of Chk1 but not Chk2 was higher (p=0,0005) in BCR-ABL+ leukemia patients (median value 0,69) compared with normal samples (median value 0,27). In order to better understand the biological role of Chk1 in ALL, we included in the analysis different B (BCR-ABL-positive: BV-173 and SUP-B15; BCR-ABL-negative: REH, NALM-6 and NALM-19) and T (MOLT-4, RPMI-8402 and CCRFCEM) leukemia cell lines. The expression of Chk1 was very homogeneous among the cell lines, with no difference between BCR-ABL-positive or negative cells. Since Chk1/2 inhibitors are currently available, we investigated the efficacy of Chk1/2 inhibition by PF-0477736 (Sigma) both as single agent and in combination with tyrosine kinase inhibitors (TKIs), imatinib and nilotinib, in BCR-ABL+ leukemia cell lines. Results showed that the combination (TKIs + Chk1/2 inhibitor) is more effective than the single inhibitors in inducing cell death. In conclusion, adult ALL patients show an higher expression of Chk1 compared with normal bone marrow precursor cells. This abnormal expression could be found also in different B-/T-ALL cell lines, suggesting that the inhibition of Chk1 could be a target for new therapeutic strategies in ALL.

Definite spectra of point mutations in the Bcr-Abl kinase domain (KD) are known to confer resistance to the tyrosine kinase inhibitors (TKIs) imatinib (IM), dasatinib (DAS) and nilotinib (NIL) in Philadelphia-positive (Ph+) leukemias. Until the recent advent of next-generation sequencing (NGS) technologies, no method was available that could conjugate high sensitivity, possibility to screen for any known or unknown sequence variation and accurate quantitation of mutated subclones. Because of these key technical limitations, the clinical relevance of early detection of small mutated subclones could never be fully elucidated. We have set up and optimized a Bcr-Abl KD mutation screening assay taking advantage of the Roche 454 NGS technology on a GS Junior instrument, that allows parallel pyrosequencing of a hundred thousand clonally amplified DNA molecules of 400 bp average length. We have thus designed 4 partially overlapping amplicons covering the whole KD of the Bcr-Abl transcript (a.a. 240–520) to be generated by nested RT-PCR using sequence-specific primers conjugated with multiplex identifiers – allowing to pool and sequence different samples from one or multiple patients (pts) in a single run. The assay proved capable to identify, characterize and quantitate sequence variations in samples from pts already known to harbour mutations as assessed by conventional Sanger sequencing with 100% concordance. Given that the number of sequence reads generated in each run is relatively constant, the lower detection limit is inversely correlated with the number of samples that can simultaneously be analyzed. Sensitivity could thus be easily modulated – analyzing a single sample per run allowed to routinely achieve lower detection limits ranging between 0.02 and 0.05% (that is, to detect as little as 2 to 5 mutated Bcr-Abl transcripts in a total of 10,000 Bcr-Abl transcripts). With this target sensitivity, we first retrospectively analyzed samples collected at the time of diagnosis from selected chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) cases known to have developed a T315I mutation 3 to 9 months after TKI start. We found that the T315I could not always be traced back to the time of diagnosis. We also found that, both in CML and in Ph+ ALL pts, several low-level mutations (in the range of 0.05 to 1%) with apparently no clinical relevance (e.g., never reported in association with TKI resistance) were routinely detectable – and appeared not to be predictors of a higher level of genetic instability. More interestingly, when used for mutation monitoring of de novo Ph+ ALL pts enrolled in clinical trials with DAS or NIL, NGS could highlight emerging resistant clones far earlier than D-HPLC or conventional sequencing. Also, some IM-resistant CML and Ph+ ALL pts who had been scored as unmutated by conventional sequencing could be shown by NGS to harbour DAS- or NIL-resistant mutations at 1–10% level – a phenomenon that is known to happen because of the clonal deselection resulting from the temporary lack of any TKI selective pressure in the interval between IM discontinuation and DAS or NIL start. Analyses will be presented in detail. We can conclude that: 1) the 454 NGS technology on the GS Junior instrument is a reliable and cost-effective method to perform Bcr-Abl KD mutation screening of Ph+ leukemia pts – with a target sensitivity of 0,1%, 4 samples can simultaneously be analyzed with costs comparable to those of conventional Sanger sequencing and the advantage to quantitatively follow the dynamics of mutated subclones over time; 2) in line with a recent report, small mutated subclones with apparently no clinical relevance can be detected both in newly diagnosed CML and Ph+ ALL pts before TKI start – which further underlines that mutation screening of pts at diagnosis is uninformative, if not misleading; 3) during TKI therapy, the high sensitivity of NGS allows to detect emerging Bcr-Abl mutant subclones earlier than D-HPLC or conventional sequencing; this is particularly relevant: i) for mutation monitoring of Ph+ ALL pts, that are highly prone to develop resistance and mutations while on TKI therapy, and ii) in IM-resistant CML and Ph+ ALL pts, where NGS may uncover the presence of DAS- or NIL-insensitive mutations, and may thus help choosing the second-line therapeutic strategy most likely to be successful.

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. 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.

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.

Bcr-Abl kinase domain (KD) mutations may cause resistance to tyrosine kinase inhibitor (TKI) therapy in Ph+ acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). Mutations in rare Ph+ cells have been detected in some imatinib-naïve advanced-phase CML patients (pts), but it is unclear whether low level mutations may already be present at diagnosis in chronic-phase (CP) CML as well as in Ph+ ALL, and whether their detection predicts for subsequent treatment failure. We analyzed cDNA samples from 24 newly diagnosed pts with Ph+ ALL (n=13) or CP-CML (n=11) who subsequently received TKI therapy. Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a. 206-524) in a bacterial vector and sequencing 200 clones for each pt. All pts had evidence of aberrant KD sequences. Three to twelve different mutations were detected in each pt. A total of 115 mutations (41 silent, 5 nonsense, 69 missense mutations) were observed. The majority (107/115, 93%) have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 103/115 (90%) mutations were transitions: G>A (n=30), A>G (n=25), C>T (n=25), T>C (n=23). One of the eleven CP-CML pt received hydroxyurea for 6 months before starting imatinib therapy. In this pt, high-sensitivity mutation screening was performed again immediately before imatinib start and showed further accumulation of mutations. Eight Ph+ ALL pts and three CML pts subsequently relapsed with mutations, but only two with a mutation (T315I) already detectable at diagnosis. The remaining thirteen pts are in persistent remission (follow-up, 12-52 months), although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. We can conclude that: a) mutations can probably be found at diagnosis in all CP-CML and Ph+ ALL pts; b) mutations seem to arise randomly and most of them are silent/not conferring any growth advantage; c) generation of mutations seems to be linked to Bcr-Abl-driven genetic instability; d) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity. This warns against high-sensitivity mutation screening of all pts before the start of therapy.

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.

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. Despite treatment results have improved in the past decade, prognosis of ALL in adult age is still dismal. The identification of genetic alterations through gene expression profile (GEP), and deep molecular cytogenetic analysis by SNPs Array, is leading to a more defined characterization of this heterogeneous disease, in order to obtain an optimized risk stratification and develop targeted therapies in different subgroups of ALL. Ph positive, hypodiploidy, and the involvement of MLL gene on chromosome 11, are associated to an unfavourable outcome. Recently, in pediatric ALL, two newly recognized subsets have been identified: 1) the “BCR-ABL like”, a group of cases characterized by very high incidence of relapse, genetic alteration of IKZF1, of VPREB- 1 and a gene expression profile similar to BCR-ABL1 ALL (Mullighan et al.; N Engl J Med 2009); 2) the “ALL with prednisone induced monocytic differentiation”, a novel subtype of childhood B cell precursor recently described (Mejstrikova et al.; personal communication at BFM meeting Bergamo, 2009). However, it is unknown if these two subtypes of ALL are present also in the adult subset. Methods and result. A 43 years woman was hospitalized due to fever and severe asthenia. Peripheral blood count revealed a moderate leucopenia and a severe anemia. A bone marrow biopsy and the immunophenotype signature led to the diagnosis of pre-B ALL. The conventional cytogenetic analysis showed the trisomy of chromosome 8 in 14 out of 20 metaphases. Molecularly, the search for E2A-PBX, BCR-ABL and TEL-AML1 fusion transcripts was negative. After informed consent was obtained, the patient received a prednisone pre-treatment for 8 days, according to our institutional experimental clinical trial based on AIEOP ALL 2000 Protocol. A progressive increasing of white blood cells count was observed, reaching a severe hyperleucocytosis (WBC 140000/mm3) at the end of steroid therapy. A morphological and immunophenotype analysis of peripheral WBC revealed a wide population constituted by CD14 positive mature monocyte cells. Detailed GEP and SNPs Array analysis were performed both before and after steroid treatment. Experiments for cloning the genomic alterations are ongoing and results will be presented. Conclusion. This report supports the presence of a new subset of adult patients with “ALL with prednisone induced monocytic differentiation”, previously described only in the pediatric context. Funding. Work supported by European LeukemiaNet, FIRB 2008, AIRC, AIL, COFIN, University of Bologna and BolognAIL.

Acute myeloid leukemia (AML) is a hematopoietic disorder characterized by numerous cytogenetic and molecular aberrations that accounts for ~25% of childhood leukemia diagnoses. The outcome of children with AML has increased remarkably over the past 30 years, with current survival rates up to 70%, mainly due to intensification of standard chemotherapy and improvements in risk classification, supportive care, and minimal residual disease monitoring. However, childhood AML prognosis remains unfavorable and relapse rates are still around 30%. Therefore, novel therapeutic approaches are needed to increase the cure rate. In AML, the presence of gene mutations and rearrangements prompted the identification of effective targeted molecular strategies, including kinase inhibitors, cell pathway inhibitors, and epigenetic modulators. This review will discuss several new drugs that recently received US Food and Drug Administration approval for AML treatment and promising strategies to treat childhood AML, including FLT3 inhibitors, epigenetic modulators, and Hedgehog pathway inhibitors.

L-Asparaginase (L-Asp) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid, and its depletion induces leukemic cell death. L-Asp is an important component of treatment regimens for Acute Lymphoblastic Leukemia (ALL). Sensitivity to L-Asp is due to the absence of L-Asparagine synthetase (ASNS), the enzyme that catalyzes the biosynthesis of L-asparagine. ASNS gene is located on 7q21.3, and its increased expression in ALLs correlates with L-Asp resistance. Chromosome 7 monosomy (-7) is a recurrent aberration in myeloid disorders, particularly in adverse-risk Acute Myeloid Leukemias (AMLs) and therapy-related myeloid neoplasms (t-MN), that leads to a significant downregulation of the deleted genes, including ASNS. Therefore, we hypothesized that -7 could affect L-Asp sensitivity in AMLs. By treating AML cell lines and primary cells from pediatric patients with L-Asp, we showed that -7 cells were more sensitive than AML cells without -7. Importantly, both ASNS gene and protein expression were significantly lower in -7 AML cell lines, suggesting that haploinsufficiency of ASNS might induce sensitivity to L-Asp in AMLs. To prove the role of ASNS haploinsufficiency in sensitizing AML cells to L-Asp treatment, we performed siRNA-knockdown of ASNS in AML cell lines lacking -7, and observed that ASNS knockdown significantly increased L-Asp cytotoxicity. In conclusion, -7 AMLs showed high sensitivity to L-Asp treatment due to low expression of ASNS. Thus, L-Asp may be considered for treatment of AML pediatric patients carrying -7, in order to improve the outcome of adverse-risk AMLs and t-MN patients.

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

Abstract 2027 Poster Board II-4 Background. The prognosis of Ph+ adult ALL 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 Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a 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 in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also 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 were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs. Since TKIs are different and have different efficacy, pharmacokinetic, pharmacodynamic and toxicity profiles, and since are all effective, an alternative experimental option for the treatment of Ph+ ALL could be a combination or a rotation of two or more than two TKIs. Moreover, the addition of new promising targeted molecules, such as Aurora kinase or T315I inhibitors may contribute to overcome the problem of resistance to TKIs. Acknowledgments. Work supported by European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: No relevant conflicts of interest to declare.

Introduction High-resolution genome-wide profiling analysis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples has identified many novel somatic genetic alterations, several of which have clear implications for risk stratification or future therapeutic targeting. However, most of the studies focused on children and therefore a deep molecular characterization of adults is still challenging, especially for those cases lacking recurrent fusion genes. Subjects and Methods In order to shed light on the molecular features of this ALL subgroup, we retrospectively analyzed 28 newly diagnosed BCR-ABL1-negative BCP-ALL subjects (19 males/9 females; median age 41.5 years; negative for known fusion genes) and 28 BCR-ABL1-positive BCP-ALL subjects as a comparison group, since it represents the most frequent genetic subgroup in adults with ALL. In BCR-ABL1-negative ALL karyotype was normal in 10/28 (36%), showed abnormalities in 5/28 (18%) and failed or was not available in 13/28 (46%) cases. The overall survival rate was very poor with a median of 14 months (range, 1-75). We analyzed copy number alterations (CNA) of IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and genes within PAR1: CRLF2, CSF2RA, IL3RA by the SALSA MLPA kit P335 IKZF1 (MRC Holland). In addition, mutation status was assessed for TP53, CRLF2, JAK2, LEF1, PAX5 and IL7R by next-generation deep-sequencing (NGS) (Roche Applied Science; IRON-II study oligonucleotide primer plates). Positivity for newly described BCR-JAK2, PAX5-JAK2, ETV6-ABL1, EBF1-PDGFRB, NUP-ABL1 gene fusions occurring in BCR-ABL1-like ALL (Roberts KG et al., Cancer Cell. 2012) was assessed by PCR amplification and sequencing. Finally, SNP arrays (SNP 6.0, Affymetrix) and gene expression profile analyses (GeneChip® Human Transcriptome Array 2.0) were performed to more fully assess genomic complexity. Results Overall, 76% of BCR-ABL1-negative subjects showed an abnormality of at least one of the analyzed genes: 7 (25%) had one, 4 (14%) had two, 6 (21%) had three, and 6 (21%) had four or more alterations. In subjects showing no abnormalities, SNP arrays analysis revealed amplifications of chromosome 1q in 2/6 cases (33%). Deletions of CDKN2A/B were the most frequent (39%) and in 73%, they occurred together with other abnormalities, suggesting that multiple events are needed to induce the full leukemia phenotype. Other common CNA included: deletions of IKZF1 (25%), ETV6 (25%), PAX5 (14%), EBF1 (11%), PAR1 region (11%) and RB1 (7%). NGS showed mutations of TP53 in 18% of cases (W147*, V172L/G, G245C, Del244-246, D259Y), while JAK2 and CRLF2 were mutated in 7% (R683S/G) and 4% (F232C), respectively. No positivity for newly described fusion genes activating tyrosine kinase was confirmed. Importantly, subjects with no abnormalities showed better survival rates compared to those with one or more molecular alterations (p < 0.01). The BCR-ABL1-positive subgroup shared the same CNA of BCR-ABL1-negative cases, such as deletions of IKZF1 (71%), CDKN2A/B (21%), PAX5 (14%), BTG1 (11%), EBF1 (11%), and ETV6 (4%), but they did not show mutations in the analyzed genes. Conclusions BCP-ALL lacking recurrent fusion genes is a highly heterogeneous and complex disease. Current diagnostic procedures need to be revised to improve risk assessment and to guide therapeutic decisions. Supported by AIL, AIRC, PRIN 2010-2011, Programma Ricerca Regione-Università 2010-2012, FP7 “NGS-PTL” project. Disclosures: Soverini: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; ARIAD: Consultancy. Chiaretti:Roche Diagnostics: Research Support Other. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Martinelli:Novartis: Consultancy, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Speaker fees, Speaker fees Other; Ariad: Consultancy, Speaker fees, Speaker fees Other.