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The main reason for the unfavorable clinical outcome of BCR-ABL1-positive acute lymphoblastic leukemia (ALL) is genetic instability. However, how normal B-cell precursors acquire the genetic changes that lead to transformation has not yet been completely defined. We investigated the expression of the activation-induced cytidine deaminase (AID) and its role in clinical outcome in 61 adult BCR-ABL1-positive ALL patients. AID expression was detected in 36 patients (59%); it correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different AID splice variants were identified: full-length isoform; AIDDeltaE4a, with a 30-bp deletion of exon 4; AIDDeltaE4, with the exon 4 deletion; AIDins3, with the retention of intron 3; AIDDeltaE3-E4 isoform without deaminase activity. AID-FL predominantly showed cytoplasmic localization, as did the AID-DeltaE4a and AID-DeltaE3E4 variants, whereas the C-terminal-truncated AID-DeltaE4 showed a slightly increased nuclear localization pattern. AID expression correlated with a higher number of copy number alterations identified in genome-wide analysis using a single-nucleotide polymorphism array. However, the expression of AID at diagnosis was not associated with a worse prognosis. In conclusion, BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutators outside the immunoglobulin (Ig) gene loci in promoting genetic instability.

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1–positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Δ4-7) and by removal of exons 2 through 7 (Δ2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Δ4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Δ2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

The outcome for adults with Philadelphia chromosome (Ph+) leukaemias (chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL)) has been dramatically improved with the use of tyrosine kinase inhibitors (TKIs), but progression and/or relapse are still present in the majority of patients. We reviewed recent findings obtained from analysis of BCR-ABL point mutations, gene expression profiling (GEP) analysis single nucleotide polymorphism (SNP) arrays and characterised by the identification of multiple novel genetic alterations targeting key cellular pathways, including lymphoid differentiation, cell cycle, tumour suppression, apoptosis and drug responsiveness. By GEP analysis, several down/up-expressed genes have been identified. Furthermore, by SNP array analysis, deletions of genes such as IKAROS, PAX5 and CDKN2A-CDKN2B were frequently identified. New therapeutic approaches with novel TKIs are now available. Dasatinib, nilotinib and bosutinib are now in clinical development. Some emerging aurora kinase inhibitors, such as VX-680, PHA-739358, MK-0457 and AS703569, and Smo1 and Hedgehog (Hh) inhibitors promise clinical efficacy against the Bcr-Ab T315I mutant form and leukaemia stem cells, respectively. In this review, we highlight the most promising drugs for the treatment of adult BCR-ABL-positive leukaemias.

Summary Chronic myeloid leukemia (CML) is induced by BCR-ABL1 and can be effectively treated for many years with Imatinib until leukemia cells acquire drug resistance through BCR-ABL1 mutations and progress into fatal B lymphoid blast crisis (LBC). Despite its clinical significance, the mechanism of progression into LBC is unknown. Here, we show that LBC but not CML cells express the B cell-specific mutator enzyme AID. We demonstrate that AID expression in CML cells promotes overall genetic instability by hypermutation of tumor suppressor and DNA repair genes. Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression.

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Genetic variations in patients have strong impact on their drug therapies and responses because the variations may contribute to the efficacy and/or produce undesirable side effects for any given drug. The Drug Metabolizing Enzymes and Transporters (DMET) assay is a high-throughput technology by Affymetrix that is able to simultaneously genotype variants in multiple genes involved in absorption, distribution, metabolism, and excretion of drugs for subsequent clinical applications, i.e., the assay allows for a precise genetic map that can guide therapeutic interventions and avoid side effects.

BMI1 is a key component of the PRC1 (polycomb repressive complex-1) complex required for maintenance of normal and cancer stem cells. Its aberrant expression is detected in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (ALL), but no data exist on BMI1 requirement in ALL cells. We show here that BMI1 expression is important for proliferation and survival of Ph+ ALL cells and for leukemogenesis of Ph+ cells in vivo. Levels of BIM, interferon-α (IFNα)-regulated genes and E2F7 were upregulated in BMI1-silenced cells, suggesting that repressing their expression is important for BMI1 biological effects. Consistent with this hypothesis, we found that: (i) downregulation of BIM or E2F7 abrogated apoptosis or rescued, in part, the reduced proliferation and colony formation of BMI1 silenced BV173 cells; (ii) BIM/E2F7 double silencing further enhanced colony formation and in vivo leukemogenesis of BMI1-silenced cells; (iii) overexpression of BIM and E2F7 mimicked the effect of BMI1 silencing in BV173 and SUP-B15 cells; and (iv) treatment with IFNα suppressed proliferation and colony formation of Ph+ ALL cells. These studies indicate that the growth-promoting effects of BMI1 in Ph+ ALL cells depend on suppression of multiple pathways and support the use of IFNα in the therapy of Ph+ ALL.

We report a case of a 65-year old patient affected by neurofibromatosis 1, documented by the presence of germ-line mutation on the NF1 gene, who developed various hyperproliferative malignant and benign diseases. He was brought to our attention for the diagnosis of acute myeloid leukemia revealed by major fatigue and dyspnea. The disease characteristics at diagnosis were hyperleukocytosis and complex karyotype with the inversion of the chromosome 16, classifying as a high-risk leukemia. The association between leukemia and neurofibromatosis 1 is controversial and needs to be further investigated. Nevertheless, such patients present a wide number of comorbidities that make therapeutic strategies most difficult.

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Background According to the 2013 European LeukemiaNet recommendations, BCR-ABL kinase domain (KD) mutation screening by conventional Sanger sequencing (SS) is recommended in all patients (pts) with progression, failure or warning, since detection of second-generation (2G) TKI-resistant mutations (T315I, F317L/V/I/C, V299L, Y253F/H, E255K/V, F359V/I/C) helps in the selection of the most suitable alternative therapy. However, SS has limited sensitivity. A recent study (Parker et al, J Clin Oncol 2011) has shown that low level mutations (i.e., undetectable by SS) may be identified by mass spectrometry and this may aid in more appropriate selection of the second-line treatment strategy for imatinib-resistant pts. Aims We sought to determine i) whether an UDS-based approach for BCR-ABL KD mutation screening might allow more sensitive detection of 2GTKI-resistant mutations at the time of switchover to second or third-line therapy and ii) whether low level mutations identified by UDS undergo clonal expansion, thus predicting for subsequent failure, if the 2GTKI to which they are insensitive to happens to be chosen. Methods To this purpose, we used UDS to scan the BCR-ABL KD in a cohort of 75 chronic myeloid leukemia (n=50) or Philadelphia chromosome-positive acute lymphoblastic leukemia (n=25) pts who were treated with a 2GTKI (dasatinib or nilotinib) after having failed first-line (n=62) or second-line (n=13) TKI therapy. A total of 235 samples collected at the time of switchover and at subsequent timepoints during second-or third-line treatment were analyzed. All the samples had already been evaluated by SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study. Runs were designed to achieve high sequencing depth. However, raw data analysis with Amplicon Variant Analyzer software (Roche Applied Science) was performed filtering out variants Results Forty-three imatinib-resistant pts failed subsequent second- (n=37) or third-line (n=6) treatment with 2GTKIs (dasatinib, n=28; nilotinib, n=15). By routine SS analysis, 32 BCR-ABL KD mutations had been identified in 29/43 (67%) pts at the time of switchover - informing subsequent-line treatment selection in 12 pts who were found to harbor 2GTKI-resistant mutations. At the time of treatment failure (after a median of 9 months; range, 2-30), SS had detected newly acquired 2GTKI-resistant mutations in all 43 pts. We thus wondered how many of these mutations could have been detected at the time of switchover using a more sensitive approach. By UDS re-analysis, 73 mutations were identified in 35/43 (81%) pts at switchover – UDS detected all the 32 mutations previously identified by SS plus 41 low level mutations (i.e., with an abundance between 1 and 15%). Twenty-four of the 41 low level mutations were resistant to the 2GTKI that happened to be selected (T315I=10; E255K=2; E255V=1; Y253H/F=4; F359V=1; F317L=4; V299L=1) and all invariably expanded becoming detectable by SS at relapse, alone or in combination with pre-existing mutations. Thus, mutations that would have influenced therapeutic decision after first or second TKI failure could have been detected in 17 more pts by UDS (p Thirty pts who achieved a stable response to second- (n=26) or third-line (n=4) treatment with 2GTKIs after having failed first- or second-line treatment, respectively, were also analyzed for comparison. By UDS, a total of 13 mutations were detected – including 6 mutations (6 pts) that had already been identified by SS plus 7 low level mutations (7 pts). No low level mutation resistant to the 2GTKI the pts actually received was detected. Conclusions In comparison to SS, UDS could identify 2GTKI-resistant mutations at the time of switchover in a significantly greater proportion of pts. Low level mutations (down to 1% abundance) detected by UDS were consistently found to expand when the 2GTKI they are insensitive to happened to be chosen. UDS-based screening of the BCR-ABL KD at switchover might thus have enabled more effective therapeutic tailoring. Further evaluation of whether this technology may be implemented in a diagnostic setting is highly warranted. Supported by PRIN 2009 (prot.2009JSMKY), Fondazione CARISBO, AIL, AIRC, FP7 ‘NGS-PTL’, IGA NT 11555 and 13899. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Castagnetti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Baccarani:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Teva: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees Other. 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.

The analyses carried out using 2 different bioinformatics pipelines (SomaticSniper and MuTect) on the same set of genomic data from 133 acute myeloid leukemia (AML) patients, sequenced inside the Cancer Genome Atlas project, gave discrepant results. We subsequently tested these 2 variant-calling pipelines on 20 leukemia samples from our series (19 primary AMLs and 1 secondary AML). By validating many of the predicted somatic variants (variant allele frequencies ranging from 100% to 5%), we observed significantly different calling efficiencies. In particular, despite relatively high specificity, sensitivity was poor in both pipelines resulting in a high rate of false negatives. Our findings raise the possibility that landscapes of AML genomes might be more complex than previously reported and characterized by the presence of hundreds of genes mutated at low variant allele frequency, suggesting that the application of genome sequencing to the clinic requires a careful and critical evaluation. We think that improvements in technology and workflow standardization, through the generation of clear experimental and bioinformatics guidelines, are fundamental to translate the use of next-generation sequencing from research to the clinic and to transform genomic information into better diagnosis and outcomes for the patient.

Background Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Methods The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent n-ethyl-n-nitrosourea. Results Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia. Conclusions In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0206-5) contains supplementary material, which is available to authorized users.

PAX5 encodes for a transcription factor essential for B-lymphoid cell commitment and is rearranged in 30% of B-cell precursor acute lymphoblastic leukemia pediatric patients (BCP-ALL). Since the first characterization of PAX5/ETV6, an increasing number of PAX5 fusion genes have been described in both adult and pediatric BCP-ALL patients. As reported in our recent review, PAX5 fusion partners encode a variety of proteins involved in cell structure (ELN, POM121), transcriptional regulation (ETV6, PML, FOXP1, MLLT3), phosphorylation (JAK2), and unknown functions (C20orf112,7AUTS2). Here we report the characterization of three new PAX5 partner genes in BCP-ALL: CHFR (a novel transcript isoform), SOX5 and POM121C. Moreover, we report cases displaying PAX5/AUTS2 and PAX5/MLLT3 fusions.

Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and a complex network of events contribute to its pathogenesis. Chromosomal rearrangements and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. A recent RNA sequencing (RNAseq) study on 179 AML revealed that fusion events occur in 45% of patients. However, the leukemogenic potential of these fusions and their prognostic role are still unknown. To identify novel rare gene fusions having a causative role in leukemogenesis and to identify potential targets for personalized therapies, transcriptome profiling was performed on AML cases with rare and poorly described chromosomal translocations. Bone marrow samples were collected from 5 AML patients (#59810, #20 and #84 at diagnosis and #21 and #32 at relapse). RNAseq was performed using the Illumina Hiseq2000 platform. The presence of gene fusions was assessed with deFuse and Chimerascan. Putative fusion genes were prioritized using Pegasus and Oncofuse, in order to select biologically relevant fusions. Chimeras not supported by split reads, occurring in reactive samples, involving not annotated or conjoined genes were removed. The remaining fusions were prioritized according to mapping of partner genes to chromosomes involved in the translocation or to Chimerascan and deFuse concordance. The CBFβ-MYH11 chimera was identified in sample #84, carrying inv(16) aberration, thus confirming the reliability of our analysis. Sample #59810 carried the fusion transcript ZEB2-BCL11B (Driver Score, DS=0.7), which is an in-frame fusion and a rare event in AML associated with t(2;14)(q21;q32). The breakpoint of the fusion mapped in exon 2 of ZEB2 (ENST00000558170) and exon 2 of BCL11B (ENST00000357195). Differently from previous data, this fusion transcript showed 3 splicing isoforms. Type 1 isoform is the full-length chimera and it retains all exons of both genes involved in the translocation. Type 2 isoform was characterized by the junction of exon 2 of ZEB2 and exon 3 of BCL11B. In type 3 isoform, exon 2 and 3 of BCL11B were removed, resulting in an mRNA composed by exon 2 of ZEB2 and exon 4 of BCL11B. Gene expression profiling showed an upregulation of ZEB2 and BCL11B transcripts in the patient's blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region. The same samples showed the WT1-CNOT2 chimera, which is a novel out-of-frame fusion (DS= 0.008) related to t(11;12) translocation, identified by cytogenetic analysis. Two new in-frame fusion genes were identified in sample #20: CPD-PXT1 (DS=0.07), which appeared as the reciprocal fusion product of t(6;17) translocation, and SAV1-GYPB, which remained cryptic at cytogenetic analysis (DS=0.8, alternative splicing events are being investigated). SAV1 was downregulated in sample #20 compared to our AML cohort, suggesting the putative loss of a tumour-suppressor gene. Sample #21 carried a t(3;12) translocation and RNAseq identified a novel fusion event between chromosomes 19 and 7, involving the genes OAZ and MAFK (DS=0.9). Finally, no chimeras were confirmed in sample #32 having a t(12;18) translocation. Our data suggest that fusion events are frequent in AML and a number of them cannot be detected by current cytogenetic analyses. Gene fusions cooperate to AML pathogenesis and heterogeneity and we are further investigating the oncogenic potential of the identified translocations. Moreover, the results firmly indicate that different approaches, including G-banding, molecular biology, bioinformatics and statistics, need to be integrated in order to better understand AML pathogenesis and improve patients' stratification, High-resolution sequencing analysis currently represent the most informative strategy to tailor personalized therapies. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

BACKGROUND: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. PATIENTS AND METHODS: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CML WP over a 40-year period. RESULTS: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P < 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P < 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P < 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). CONCLUSIONS: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome. CLINICAL TRIAL NUMBERS: NCT00510926, NCT00514488, NCT00769327, NCT00481052. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

We describe a new AML entity, occurring in 30% of de novo acute myeloid leukemia, due to structural and epigenetic deregulation of the UNCX homeobox (HB) gene. By molecular approaches, we identified a M5 AML patient with a t(7;10)(p22;p14) translocation as the sole cytogenetic anomaly and showing ectopic expression of UNCX (7p22.3), which encode for a transcription factor involved in somitogenesis and neurogenesis. Since UNCX was never reported in association with cancer but only with common myeloid cell proliferation and regulation of cell differentiation, we decided to investigate its contribution to leukemogenesis. We observed UNCX ectopic expression in 32.3% (20/62) and in 8% (6/75) of acute myeloid leukemia (AML) patients and cell lines, respectively. Notably, retroviral-mediated UNCX transfer in CD34+ HSCs induced a slow-down in their proliferation and differentiation and transduced cells showed a lower growth rate but a higher percentage of CD34+ stem cells in liquid culture than controls. Additionally, UNCX infected cells displayed a decrease of MAP2K1 proliferation marker but increase of KLF4, HOXA10, and CCNA1, associated with impaired differentiation and pluripotency. Similarly, UNCX-positive patients revealed alteration of gene pathways involved in proliferation, cell cycle control and hematopoiesis. Since HB genes encode for transcription factors showing a crucial role in normal hematopoiesis and in leukemogenesis, we focused our attention on the role of altered UNCX expression level. Of note, its murine ortholog, (Uncx) was previously described as embedded within a low-methylated regions (≤ 10%) called "canyon" and dysregulated in murine hematopoietic stem cells (HSCs) as a consequence of altered methylation at canyons edges (borders) due to Dnmt3a inactivation. In our hands, UNCX activation was accompanied by methylation changes at both its canyon borders, clearly indicating an epigenetic regulation of this gene, although not induced by DNMT3A mutations. Clinical parameters and correlation with response to therapy will be presented. Taken together, our results indicate that more than 30% of de novo AML have a novel entity with a putative leukemogenic role of UNCX, whose activation may be ascribed to epigenetic regulators. Acknowledgments: MG, CP, GS, and AP(2) and this work was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. CTS, GD and AL are supported by Associazione Italiana Ricerca sul Cancro (AIRC) funding.

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p0.01). We then genotyped these candidate SNPs in a validation series of 748 patients from Italy that resulted genetically compatible with the UK series based on principal component analysis. In a Cox proportional hazard model adjusted for age, sex and clinical stage, four SNPs were confirmed on the basis of their having a hazard ratio (HR) indicating the same direction of effect in the two series and p0.05. The strongest association was provided by rs2107561, an intronic SNP of PTPRG, protein tyrosine phosphatase, receptor type, G; the C allele was associated with poorer survival in both patient series (pooled analysis loge HR = 0.31; 95% CI: 0.15-0.46, p = 8.5 × 10(-5) ). PTPRG mRNA levels in 43 samples of lung adenocarcinoma were 40% of those observed in noninvolved lung tissue from the same patients. PTPRG overexpression significantly inhibited the clonogenicity of A549 lung carcinoma cells and the anchorage-independent growth of the NCI-H460 large cell lung cancer line. These four germline variants represent promising candidates that, with further study, may help predict clinical outcome. In addition, the PTPRG locus may have a role in tumor progression.

Imatinib mesylate (STI571), a specific Bcr-Abl inhibitor, has shown a potent antileukemic activity in clinical studies of chronic myeloid leukemia (CML) patients. Early prediction of response to imatinib cannot be anticipated. We used a standardized quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR) for BCR-ABL transcripts on 191 out of 200 late-chronic phase CML patients enrolled in a phase II clinical trial with imatinib 400 mg/day. Bone marrow samples were collected before treatment, after 12, 20 and at the end of study treatment (52 weeks) while peripheral blood samples were obtained after 2, 3, 6, 10, 14, 20 and 52 weeks of therapy. The amount of BCR-ABL transcript was expressed as the ratio of BCR-ABL to beta2-microglobulin (beta2M). We show that, following initiation of imatinib, the early BCR-ABL level trends in both bone marrow and peripheral blood samples made it possible to predict the subsequent cytogenetic outcome and response. We propose this method as the method of choice for monitoring patients on imatinib therapy. QRT-PCR studies may be able to identify degrees of molecular response that predict both complete cytogenetic response and long term stability, as well as patterns of response that provide an early indication of relapse and imatinib resistance.

The loss-of-function of teneleven-translocation (TET) 2, a Fe2+-oxoglutarate-dependent dioxygenase catalyzing 5 methyl cytosine (5mC) conversion into 5-hydroxymethylcytosine (5hmC), contributes to the hematopoietic transformation in vivo. The aim of our study was to elucidate its role in the phenotype of chronic myeloid leukemia (CML), a myeloproliferative disease caused by the Bcr-Abl rearranged gene. We first confirmed TET2 interaction with the Bcr-Abl protein predicted by a Fourier-based bioinformatic method. Such interaction led to TET2 cytoplasmatic compartmentalization in a complex tethered by the fusion protein tyrosine kinase (TK) and encompassing the Forkhead box O3a (FoxO3a) transcription factor. We then focused the impact of TET2 loss-of-function on epigenetic transcriptional regulation of Bcl2-interacting mediator (BIM), a pro-apoptotic protein transcriptionally regulated by FoxO3a. BIM downregulation is a critical component of CML progenitor extended survival and is also involved in the disease resistance to imatinib (IM). Here we reported that TET2 release from Bcr-Abl protein following TK inhibition in response to IM triggers a chain of events including TET2 nuclear translocation, re-activation of its enzymatic function at 5mC and recruitment at the BIM promoter followed by BIM transcriptional induction. 5hmC increment following TET2 re-activation was associated with the reduction of histone H3 tri-methylation at lysine 9 (H3K9me3), which may contribute with DNA de-methylation reported elsewhere to recast a permissive epigenetic landscape for FoxO3a transcriptional activity. J. Cell. Biochem. 113: 27652774, 2012. (c) 2012 Wiley Periodicals, Inc.

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.

Abstract 2601 Background: Tyrosine Kinase Inhibitors (TKI) have been shown to be very effective for the treatment of Acute Lymphoblastic Leukemia (ALL), with a Complete Hematologic Remission (CHR) rate close to 100%, and a high rate of Complete Cytogenetic and Molecular responses (CCgR and CMR). However, when they are used alone, as single agents, most patients relapse, so that they are currently used in combination with chemotherapy and as a preparation to allogeneic stem cell transplantation (SCT). Since Ph+ ALL is more frequent in the elderly, many patients cannot tolerate intensive chemotherapy and are not eligible for SCT. We have explored if the administration of two TKIs, Nilotinib (NIL) and Imatinib (IM) can improve the results without increasing the toxicity. Aims: To evaluate the response and the outcome of Ph+ ALL patients treated with the sequential administration of NIL and IM, to investigate the type and number of BCR-ABL kinase domain mutations developing during and after the study. Methods: We have designed a study (ClinicalTrials.gov. NCT01025505) in which patients more than 60 years old or unfit for intensive chemotherapy and SCT where treated with two TKIs, NIL 400 mg twice daily, and IM 300 mg twice daily, alternating for 6 weeks for a minimum of 24 weeks (study core) and indefinitely in case of response. The 6-weeks rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the rate of Disease Free Survival (DFS) at 24 weeks (4 courses of treatment); the secondary end points included the evaluation of CHR, CCgR and CMR rates. Mutation analysis was performed by nested RT-PCR amplification of the ABL kinase domain of the BCR-ABL transcript (codons 206 through 421). Amplified products were screened by denaturing-high performance liquid chromatography (D-HPLC). Samples scored positive for the presence of sequence variations were then subjected to direct automatic sequencing to characterize the mutation. Results: 39 patients have been enrolled in 15 Italian hematologic Centers (median age 66 years, range 28–84). Among these, 8 patients were unfit for standard chemotherapy or SCT (median age 50 years, range 28–59). 27 patients were p190, 5 were p210 and 7 were p190/p210. After 6 weeks of treatment, 36 patients were evaluable for response: 34 were in CHR (94%) and 2 in PHR (6%). 23 patients have already completed the study core (24 weeks), 87% were in CHR and 17 are currently continuing therapy in the protocol extension phase. Thus, the OS at 1 year is 79%, and 64% at 2 years. Overall, 1 patient was primarily resistant and 13 patients have relapsed, with a median time to relapse of 7.6 months (range 0.8–16.1 months), for a DFS of 51.3% at 12 months (Figure 1). Mutations detected were T315I in 2 cases, Y253H in 3 cases, T315I and Y253H in 1 case, E255K in 1 case, T315I and E255K in 1 case, E255V and Y253H in 1 case. Two patients were WT. A detailed kinetics of Molecular responses is shown in Table 1. Data on mutational analysis are reported in Table 2. Further details about Cytogenetic and Molecular responses, and about Adverse Events will be provided on site. Conclusions: In this small cohort of Ph+ ALL elderly/unfit patients, the rates of relapse and progression were not likely to be different from the rates observed with Imatinib alone (Vignetti et al, Blood 2007, May 1;109(9):3676-8) and Dasatinib alone (Foà, Blood 2011, Dec 15;118(25):6521-8). It's important to notice that the mutations that occurred at the time of relapse were sensitive to other TKIs (Dasatinib and Ponatinib). Acknowledgments: COFIN, Bologna University, BolognAIL, PRIN, Fondazione del Monte di Bologna e Ravenna, INPDAP. Disclosures: Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau.

Abstract 1749 Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs, mostly bone marrow, skin, liver and gastrointestinal tract. In most adult patients, the systemic form of mastocytosis (SM) is diagnosed, which includes an indolent, an aggressive and a leukemic subvariant. The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 11 patients (male/female=3/8) affected by Mastocytosis, have been referred to our Institution. The median age was 53 years. All the patients underwent a bone marrow biopsy, with flow citometry and molecular biology analysis, in order to identify the presence of D816V mutation of c-Kit gene. Serum tryptase level was tested, resulting elevated in all cases. According to 2008 WHO diagnostic criteria, 9 patients presented with SM, whereas in the other cases a skin isolated involvement was detected. Systemic symptoms were characterized by nausea, diarrhoea, asthenia, weight loss, pruritus and serotine fever, identifying an aggressive form of the disease in 5/11 patients, due to skeletal involvement in three cases, ascitis and liver function impairment in another one and bone marrow disfunction in the fifth one. Therefore, since a first line therapy with supportive care and histamine receptor antagonists wasn't followed by a significant benefit, a personalized use of PKC412 was asked and obtained for all these five patients. Results. From March 2011 three out of the five patients with aggressive SM have received a prolonged period of treatment with PKC412, which was administered orally, at the dosage of 100 mg twice daily, without rest periods. The drug was well tolerated. No serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. In one case we observed, after a first good response, a disease progression, characterized by the sudden reoccurrence of the same symptoms detected at diagnosis, confirmed by a relevant expansion of a pathologic mast cell population in the bone marrow. After a rest period, the drug was readministered, and a second remission was obtained. Conclusions. PKC412 is safe and effective in patients with SM, being able to significantly improve not only the gastrointestinal and systemic symptoms, but also the haematological profile. The persistence of the D816V c-kit mutation, despite a morphologic remission, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, Fondazione del Monte di Bologna e Ravenna, University of Bologna. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy, Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 284 Background and Aims: Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come. We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations. Methods: 72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS. Results: 39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with >20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct. Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant. Conclusions: Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with >20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Nowadays the effectiveness of the treatments for adult Acute Lymphoblastic Leukemia (ALL) patients is still inadequate and frequently many patients after years of response to treatments develop relapses. Thus there is a need to find novel targets for specific therapies and to maximize the effect of the actual treatments. Recently different Checkpoint Kinase (Chk)1/Chk2 inhibitors has been assessed for the treatment of different type of cancers but only few studies have been performed on hematological diseases. We evaluated the effectiveness of the Chk1 inhibitor, LY2606368, as single agent and in combination with tyrosine kinase inhibitors (imatinib and dasatinib) or with the purine nucleoside antimetabolite clofarabine in B-/T- acute lymphoblastic leukemia (ALL) cell lines and in primary blasts. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were incubated with increasing concentrations of drug (1-100 nM) for 24 and 48 hours and the reduction of the cell viability was evaluated using WST-1 reagent. LY2606368 deeply reduced the cell viability in a dose and time dependent manner in all the cell lines, with the BV-173 (6.33 nM IC50 24hrs) and RPMI-8402 (8.07 nM IC50 24hrs) being the most sensitive while SUP-B15 (61.4 nM IC50 24hrs) and REH (96.7 nM IC50 24hrs) being the less sensitive cell lines. Moreover the sensitivity to the compound was no correlated with the different sub-type of ALL or with the mutational status of p53, which is a marker of the functionality of the G1/S checkpoint. The cytotoxic activity was confirmed by the significant increment of apoptosis cells (Annexin V/Propidium Iodide), by the increment of gH2AX foci and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). To understand the relationship between the activation of apoptosis and the effect on cell cycle and to identify hypothetical mechanisms of death, different cell cycle analyses were performed (Propidium Iodide staining). The inhibition of Chk1, deeply changed the cell cycle profile. Indeed in all the cell lines the percentage of cells in S phase and in G2/M phase were reduced by the treatment while the numbers of cells in sub-G1 and G1 phase were increased. The hypothetical function of LY2606368 as a chemosensitizer agent was evaluated combining the compound with different drugs normally used in clinical trials. For each drugs the combination strongly reduced the cell viability when compared to the cytotoxic effect of the single drugs. Moreover the combination showed an additive efficacy in term of induction of DNA damages as showed by the increase number of gH2AX foci and the activation of pChk1 (ser 317). The results found on the cell lines were confirmed also using primary leukemic blast isolated from adult Philadelphia-positive ALL patients. Indeed LY2606368 as single agent or in combination with the Tki, imatinib, was able to deeply reduce the cell viability and to induce DNA damages (gH2AX foci). In conclusion LY2606368 showed a strong cytotoxic activity on B-/T-All cell lines and primary blasts as single agent and in combination with other drugs. In our opinion this data are the basis for a future clinical evaluation of this compound in the treatment of leukemia. Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Martinelli:ROCHE: Consultancy; Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

Abstract 1484 MDM2, a p53-inducible phosphoprotein, binds to the N-terminus of the p53 and negatively regulates its transcriptional activity. New MDM2 antagonists, such as RO5045337 (Roche) and NSC-66811 (Merck), are now available for Phase I/II clinical development, but their activity is dependent on TP53 mutation status. Therefore, in order to efficiently treat B-progenitor acute lymphoblastic leukemia (ALL) patients with an MDM2 antagonist, we set up a sensitive assay to identify TP53 lesions. Deletions and uniparental disomy (UPD) involving TP53 were assessed on 146 DNA samples from Philadelphia-positive (Ph+)(n = 126) and Ph-negative (n = 20) ALL patients by Genome-Wide Human SNP 6.0 array (Affymetrix). No 17p UPD events were detected whereas losses were identified in 2% of cases. Mutations of TP53 were thereafter investigated in 67 samples including 60 Ph+ and 7 Ph-negative cases. Since the majority of the studies in leukemia were focused on genomic alterations and resulted in low rate of TP53 mutations, we aimed to identify RNA mutations and aberrant isoforms due to other mechanisms, such as RNA editing. To this purpose three overlapping shorter amplicons covering the entire coding cDNA sequence (GenBank accession number NM_000546.4) and the untranslated exon 1 [amplicon 1 (491 bp): exons 1–5; amplicon 2 (482 bp): exons 5–8; amplicon 3 (498 bp): exons 8–11)] and a longer amplicon (1,317 bp) starting from exon 1 and ending to exon 11 were sequenced by Sanger method. TP53 mutations were detected in only 6 cases (8.9%), suggesting that these alterations are apparently rare events in B-ALL. They included 4 missense point mutations in the DNA binding domain and in the carboxyl-terminal tetramerization and regulatory domain: C135Y (ex 5), A234T (ex 7), R290C (ex 8) and A347T (ex 10). Interestingly, in two cases we identified aberrant transcripts: 1) a TP53 isoform characterized by retention of introns 5–6–7 and predicted to encode for a truncated protein due a premature stop codon; 2) a TP53 isoform in which the DNA binding domain is lost due to an exon conjunction between the exon 4 and the 3' untraslated region (UTR)(ex4-3'UTR: 7579533–7572842, according to GRCh37/hg19). Next, in order to investigate if low rate of mutations were detectable, we also analyzed our whole transcriptome data obtained using next generation sequencing technology (Illumina/Solexa Genome Analyzer) on 3 Ph+ ALL patients. Curiously, all patients harbored clones ranging from 45% to 94% with TP53 mutations in the DNA binding and tetramerization domains: C182W (ex 5), T231A (ex 7), L330R (ex 9) in the first patient and Stop394S, D393V/H and G389Y/V (ex 11) in the second one. Moreover, in the first and third patient we detected 10 and 13 base exchanges, respectively, located in intron 6 within 7578166–7578142 region, suggesting a retention of this intron in the primary transcript and the dysfunction of the DNA-binding domain. The mechanism of intron retention (with or without mutations) was particularly intrigued since it could be a new mechanism of functional inactivation of TP53. To address this hypothesis we performed amplification of TP53 cDNA followed by single cell cloning and subsequent direct sequencing in 4 patients previously resulted wild-type by Sanger sequencing for TP53. By this approach, all patients showed cDNA alterations. In one case we identified the missense mutation S90P (ex 4) and an aberrant isoform lacking the DNA binding domain and caused by an exon-junction between exons 2 and 7 (ex2-7: 7579866–7577510). In a second patient the P190S (ex 6) and N235S (ex 7) missense mutations were detected. Moreover, an aberrant isoform lacking the DNA binding domain and characterized by an exon-junction between the first part of exon 4 and the last part of exon 7 (ex4-7: 7579581–7577532) was also identified. In the third patient the E285G (ex 8) was found associated with a 3'-UTR base exchange, which was also detected in the remaining fourth patient. In conclusion, we demonstrate for the first time that TP53 alterations at the RNA level, including missense mutations, aberrant exon junctions and internal intron retentions are highly frequent in B-ALL patients and that testing for TP53 mutations with sensitive assay based on RNA analysis is absolutely required. 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. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Background. Fludarabine plus Cytarabine and Idarubicine (FLAI) was proved to be an effective and well tollerated induction regimen for treatment of acute myeloid leukaemia (AML). The trial objective was to assess the efficacy of Etoposide when used in combination with FLAI schedule. Design and Methods. We retrospectively report clinical outcome results of 101 newly diagnosed and younger than 60 years AML patients (median age 46 years, range 18-60 years) treated in a Phase III clinical trial, with FLAIE induction chemotherapy, including Etoposide to the FLAI schedule. Induction consisted of Fludarabine 25mg/m2/day on days 1-5, Idarubicin 6 mg/m2/day on days 1, 3 and 5, Cytarabine 2 g/m2 infused in 4 h, daily on days1-5 and Etoposide 100 mg/m2/day on day 1-5. After induction, all the patients underwent consolidation with Cytarabine (2 g [[Unsupported Character – /]]sqm i.v. infusion on days 1-5) and Idarubicin (12 mg[[Unsupported Character – /]] sqm i.v. infusion on days 1, 3 and 5). All the patients shared the same strategy for intensification, that was allogenic or autologous stem cell transplantation. After consolidation, maintenance treatment with Cytarabine was given to patients who obtained a complete remission but who could not undergo allogenic or autologous stem cell transplantation. More than half of the patients had abnormal karyotypes. Molecular analysis at diagnosis for the more frequent abnormalities was performed. Duration of CR and overall survival was estimated according to the Kaplan-Meier method. The CR duration was dated from start of CR to first evidence of recurrence. Results. After a single induction course of FLAIE, 73 pts obtained a CR (72.2%) and 8 pts a CRp (7.9%) for an overall response rate of 80.2%. Fifteen patients (14.9%) had resistant disease, and 5 (4.9%) died during induction. After a median follow-up of 33 months, 75 patients (76%) are in continuous CR. The median CR duration and OS were 45 and 55 months, respectively. 11 pts underwent ABMT and 44 a BMT. Relapses were more frequent in patients who were not submitted to allogenic stem cell transplantation. Of the 55 transplanted patients, 28 (51.9%; 1 with chromosome 7 abnormality), were alive in CR after a median follow up of 10 months (range, 2 to 45 months) after transplantation, 14 (25.9%) relapsed (median DFS 4 months), and 13 (24%) died in CR or CRp of transplant related complications. The most common grade 3 adverse events included gastro-intestinal toxicities(i.e. nausea, vomiting, mucositis and diarrhoea), liver dysfunction, and skin rash. Conclusion. The combination of etoposide to FLAI is safe and active. Further studies exploring different dosing and scheduling are warranted, particularly in patients with poor-risk AML.

Background: The CDKN2A/B locus is inactivated in several haeamatologic malignancies mainly due to deletion, aberrant repression or epigenetic silencing, but little is known about the potential role of its single nucleotide polymorphisms (SNPs) in leukemia susceptibility. Patients and methods: To investigate whether polymorphisms within this locus 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. The median age was 52 years (18-78 years) and 53 years (21-71 years) in Ph+ ALL and AML patients, respectively. AML cases included FAB M0-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, type 2 diabetes mellitus, frailty. 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). Results: 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. Potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. The 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, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009.

Abstract 1429 The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. The Sonic Hedgehog (Hh) pathway is an essential regulator of multidrug resistance (MDR) in leukemia by exerting it's effect on leukaemia stem cells (LSC). PF-04449913 is a selective and potent small molecule inhibitor of the Hh pathway and leukemia self-renewal and is currently being evaluated in Phase I clinical trials. In order to evaluate the activity of PF-0444913 in overcoming the drug resistance of leukemia stem cells, we studied leukemia stem cell population (CD34+ subpopulation) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies including Myelofibrosis (MF), MDS, Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic leukemia (CMML) and Acute Myeloid Leukemia (AML) patients. We were able to collect and separate highly purified (98%) bone marrow hematopoietic progenitor cells (CD34+ populations) in 5 AML, 1 MF and 2 CML patients, by immunomagnetic separation, and analyze them for gene expression profile (GEP) using Affimetrix HG-U133 Plus 2.0 platform. We have observed that 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Among these genes, we demonstrated a down regulation of Bcl2 (fold change −1.03004; p value= 0.01), ABCA2 (fold change −1.08966; p value=0.03), Bcl2l13 (fold change −1,04259; p-value=0,027642), Bcl2l2 (fold change −1,17214; p-value=0,000768), Casp4 (fold change −1,06551; p-value=0,032428), Casp7 (fold change −1,01569; p-value=0,006688), Casp10 (fold-change −1,3076; p-value=0,050431), ABCF1 (fold change −1,04999; p-value=0,07213). On the contrary, ABCB1 (fold change 1,46592) and ABCG2 (fold change −1,16103) are respectively up and down regulated, with a not statistically significant p-value (0,35375 and 0,288194 respectively). Bcl2 (B-cell lymphoma 2), Bcl2l2 (Bcl2-like protein 2) and Bcl2l13 (Bcl2-like 13) are the founding members of the Bcl-2 family of apoptosis regulator proteins. Recent studies showed that Hh signals upregulate Bcl2 to promote cellular survival. Casp 4,7,10 (Caspases, or c ysteine-asp artic proteases) are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation. ABCA2 (ATP-binding cassette sub-family A member 2), ABCF1 (ATP-binding cassette sub-family F member 1), ABCB1 (ATP-binding cassette sub-family B member 1, MDR1), ABCG2 (ATP-binding cassette sub-family G member 2) belong to the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranes. One mechanism of MDR is the increased expression of ABC drug transporters that mediate energy-dependent transport of drugs out of the cells against a concentration gradient, resulting in low intracellular drug concentrations. This is a common finding in LSC, and represents an important clinical problem for disease eradication. Furthermore, we evaluated Gli1, Gli2 and Smo expression by GEP, comparing data before and after 28 days of treatment with PF-04449913 and, as expected, we observed a down regulation of Gli1 (fold change −1.0775), Smo (fold change −1.07702), and an up regulation of Gli2 (fold change 1.08191). Our results suggest that PF-04449913 is able to revert MRD mechanisms of LSC by a strong down regulation of genes (Bcl-2, Bcl-2l13, Bcl-2l2, ABCA2, and ABCF1), which are critical for chemoresistance in acute and chronic leukemia patients. Therefore, the combination of PF-04449913 with Tyrosine Kinase inhibitors or conventional chemotherapy could represent a valid new therapeutic approach in these haematological malignancies. Acknowledgments. Work supported by Pfizer, European LeukemiaNet, FIRB 2008, PRIN 2009, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jamieson:Wintherix: Equity Ownership; Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria. Cortes:Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. McLachlan:Pfizer: Employment. Van Arsdale:Pfizer: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

Abstract 4261 Background Acute lymphoblastic leukemia (ALL) shows different outcome in children and adults, with event-free-survival (EFS) rates of 70–80% and 30–40% at 5 years, respectively. Results improved in young adults/adolescents with de novo ALL, when treated with pediatric intensive regimens rather than with adult regimens. Clinical studies are ongoing in older patients, toxicity related-therapy seeming the limiting issue. Aims We report a single centre experience on adult ALL patients treated with an intensive pediatric-inspired schedule, aiming to assess its tolerability and efficacy. Methods From 11/07 to 03/11 we treated 24 ALL patients (M/F=17/7) according to modified AIEOP-LAL2000 regimen. Treatment consisted of 7 days steroid pre-treatment, and four drugs 78-days induction (phase IA and IB) after which high risk (HR) patients were treated with three polychemotherapy blocks, while intermediate (IR) and standard risk (SR) patients went on 8-weeks consolidation and subsequent intensification. A 2 cycle consolidation therapy with nelarabine was planned for T-ALL patients. Patients with HLA-matched donor underwent allo-SCT; 2-years maintenance therapy was given to the others. Median age was 30 years (17–47). Results 22/24 patients completed the phase IA, 2 being out for grade IV toxicity (intestinal occlusion and sepsis). 17 (70.8%) obtained a complete remission (CR), 5 (21%) were refractory. One of the resistant patients achieved CR after polychemotherapy blocks, two after phase IB. Median induction duration (IA+IB) was 95 days (82–136); delays were mostly experienced after phase IB, hematologic toxicity being an important cause of delay, with median time to neutrophil count recovery of 28 days (18–34) and 39 days (16–62) for phase IA and IB respectively. A higher absolute number of adverse events during phase IA than during IB was registered (infections and gastrointestinal), without a significant prolongation of phase IA. After induction, 3 of the 19 CR patients received consolidation therapy, then 2/3 underwent allo-SCT. 6 patients received blocks: 3/6 undewent allo-SCT, 2/6 dropped out after the first and the second block for reversible grade II-III renal toxicity and one relapsed. 5 patients were treated with nelarabine, then 2/5 underwent allo-SCT. 3/19 directly underwent allo-SCT, while 1 patient completed the whole therapeutic program because no suitable donors for allo-SCT were found. One patient died after phase IB for pulmonary infection. With a median follow up of 12 months (3–50), 14/24 (58,3%) patients are alive, 8 in CR (5 underwent allo-SCT). 10 patients died, 5 for relapsed/refractory disease, 5 in CR (3 after allo-SCT). Conclusions A pediatric-inspired therapeutic regimen demonstrated a significant hematologic and a subsequent infective toxicity in adult ALL patients, this causing a lack in dose-intensity maintenance. The overall outcome seemed to be comparable to the one reached in other studies conducted on larger population but a longer follow-up and a larger population are needed to draw definitive conclusions. Acknowledgments. BolognAIL, European LeukemiaNet, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO, Project of Integrated Program (PIO), Programma di Ricerca Regione - Università 2007–2009. Disclosures: No relevant conflicts of interest to declare.

Abstract 76 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 γH2AX, 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 γH2AX. 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. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 4029 Background: Azacitidine (AZA) at a dose of 75 mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates and prolongation of survival in high-risk MDS patients (pts) (Fenaux, 2009). However few data are hitherto available concerning the efficacy and safety of Aza in lower risk MDS. A lower dose regimen, AZA 5 (75 mg/mq daily, subcutaneously, for 5 consecutive days every 4 weeks) have shown to induce response rates consistent with the currently approved schedule (Lyons, 2009), however in this study pts were not classified according to IPSS risk. Aim: The use of AZA in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial, AZA 5 regimen was administered to IPSS low-or-intermediate-1 risk pts, for a total of 8 courses, in order to evaluate its efficacy and safety. Furthermore pharmacogenomic studies (GEP, SNP) cytokine network and PI-PLC-beta1 methylation and gene expression, before and at the end of 4th and 8th course of Aza treatment, were planned to identify new biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients (24 males, 10 females), with a median age of 71 (56-84) yrs, with symptomatic transfusion-dependent anemia, previously unresponsive to erythropoietin (EPO) or not expected to respond to EPO, or with severe neutropenia or thrombocytopenia, were enrolled into the study. According to WHO classification, 15 pts had RA, 6 RARS, 7 RCMD and 6 RAEB-1. Results: At present time 30/34 pts are evaluable: 23/30 pts (77%) completed the treatment plan (8 courses), 3/30 pts (10%) are ongoing and 4/30 (13%) died during the treatment period. According to the 2006 International Working Group criteria, overall response rate (ORR) was 60,9 % (14/23 pts): 5 pts (21,7%) achieved complete remission (CR), while 9 pts (39,1%) showed an hematologic improvement (HI) (7 erythroid responses, 1 erythroid/platelet response and 1 neutrophil/platelet response). 9/23 pts (39%) maintained a stable disease (SD). Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%). 4 pts (11,7%) died during treatment (2 pts after the 1th cycle and 2 pts after the 4th course) because of septic shock, gastrointestinal hemorrage, pneumonia, and respiratory distress, respectively. The median duration of response was 3,5 months (range 1–14 months). Surprisingly, 3/14 patients (2 CR and 1 HI erythroid) showed a long duration of response (11, 13 and 14 months, respectively), still ongoing, after discontinuation of AZA. Preliminary data on the lipid signalling pathways suggested a direct correlation between the demethylating effect on PI-PLC-beta1 and responsiveness to treatment. Conclusion: Our study shows that AZA low-dose schedule may be a feasible and effective treatment for low-risk MDS pts and may induce durable responses. Despite AZA safety, extreme caution is needed in pts with age-related comorbidities and/or with severe neutropenia or thrombocytopenia, especially in low-risk MDS. Furthermore, PI-PLC–β1 demethylation and gene expression could represent a new biological marker to predict the clinical response to AZA Disclosures: Off Label Use: In Italy the use of Azicitidine for Low-Risk Myelodysplastic patients is off-label. The use of azacitidine in our study is part of a Phase II clinical trial. Finelli:Celgene: Consultancy.

SummaryAlterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

BACKGROUND Philadelphia chromosome–like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR–ABL1–positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6–NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.)

BACKGROUND: The INTERIM study (ClinicalTrials.gov NCT 00858806) showed that in elderly (> 65 years) Ph+ CML patients selected for a stable complete cytogenetic response (CCgR) lasting > 2 years, the policy of intermittent imatinib treatment (one month on/one month off) may affect the markers of residual disease (CCgR and major molecular response, MMR or MR3.0), but not the clinical outcomes (overall survival and progression-free survival) (Russo D et al, Blood 2013; 121(26):5138-44). AIMS: To update the results of the INTERIM Study, with a follow up ≥ 5 years. METHODS: After 4 years of follow up, patients continouing INTERIM treatment were monitored with peripheral blood RT-Q-PCR every 3 months according to the ELN-2013 guidelines. RESULTS: At 48thmonth, out of 76 patients enrolled in the INTERIM study, 13 (17%) had lost CCgR and MMR, 14 (18%) had lost MMR only and 50 patients (75%) continued INTERIM. The patients who had lost CCgR and/or MMR resumed imatinib continuously and all of them regained the CCgR and the MMR, within 3 to 12 months. No patient progressed to accelerated or blastic phase, or developed clonal chromosomal abnormalities in Ph+ cells, or BCR-ABL mutations. No patient complained of new or more severe side effects during the months “on”. After a follow up ≥ 5 years, 45/76 (59%) enrolled patients are on INTERIM, with a probability of maintaining intermittent administration of 59% (95% CI: 46-69). No patient lost the CCgR and only 9 additional patients lost the MMR while on intermittent treatment. All these patients resumed continuous imatinib treatment and regained the MMR. Thus, at ≥ 5 years, the probability of maintaing CCgR is 80% (95% CI 68-87) and the probability of maintaining the MMR is 61% (95% CI: 48-71). From start of INTERIM, 6 patients died but no deaths were related to CML progression (3 cases of other non haematological neoplasms, 1 stroke, 1 myocardial infarction, 1 chronic obstructive pulmonary disease).The PFS at ≥ 5 years is 94% (95% CI: 89-100) CONCLUSIONS: In summary, with a follow up ≥ 5 years, intermittent imatinib administration (INTERIM) confirmed to be safe, to produce a reversible increase of residual molecular disease in about one third of patients, but not to affect the long-term outcome. Aknowledgments: This work was supported in part by EuropeanLeukemiaNet (contract LSHC-CT-2004-503216) through the European Treatment and Outcome Study (EUTOS), supported by Novartis Oncology Europe, and COFIN 2009 Disclosures Russo: Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy. Martinelli:Novartis: Speakers Bureau; Bristol-Meyers and Squibb: Speakers Bureau; Pfizer: Speakers Bureau. Soverini:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau. Turri:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria. Breccia:novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Abruzzese:Novartis: Consultancy. Tiribelli:Novartis: Consultancy, Honoraria; Bristol-Meyers and Squibb: Consultancy, Honoraria. Rosti:Consultant: Consultancy, Speakers Bureau; Bristol-Meiers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

Myelodisplastic syndromes (MDS) and Acute Myeloid Leukemia (AML) are a group of diseases of the elderly that initiates in a hematopoietic stem cell and are characterized by clonal hematopoiesis and uncertain prognosis, mostly due to cytogenetic background. In both diseases, 5-Azacitidine (5-Aza) has been successful, inducing prolonged survival and delayed AML evolution. To identify the genes mostly predictive of treatment response, we use high-throughput genomic analysis (SNP arrays and/or NGS-RNA-seq and/or NGS-WES and/or GEP) in azacitidine-sensitive and resistant MDS/AML patients. NGS-WES or RNA seq HiSeq 2000 (Illumina) was positively done in 35/214 AML samples (16%), GEP (GeneChip Human Transcriptome Array 2.0, Affymetrix Inc.) was performed in 65/214 AML samples (30%). SNPs arrays (CytoScan HD Array, Affymetrix Inc.) was done in 125/214 AML samples (58%) and 18/32 MDS samples (56%) at diagnosis, then analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.), Nexus Copy Number™ v7.5 (BioDiscovery) and GeneGo MetaCore™ software. We treated 246 adult patients (pts) with MDS or AML: 214 pts were AML and 32 were MDS with a median age of 59 and 70 years, respectively. Forty-five pts were treated with 5-Aza (32 MDS / 13 AML), while 201 AML were treated with conventional chemotherapy. Forty-five MDS/AML pts were treated with at least one complete cycle of 5-Aza (75 mg/sqm/daily). SNP arrays was done in 22/45 (49%), 13 pts were defined “insensitive/resistant”, ie. never achieving clinical complete remission (CCR) and 9 were defined “sensitive”, ie. all of them obtaining CCR. Copy Number Alterations (CNAs) ranged from loss or gain of complete chromosome (chr) arms to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Macroscopic CNAs affecting a complete chromosome or its arms were detected in 5 of 22 pts (23%), while classical cytogenetic was able to detect only two cases of trisomy 8 (9%), suggesting superiority of SNPs array for CNAs identifications. Microscopic CNAs abnormalities were detected in all of the patients affecting all the chromosomes. Of interest, some of them were located on chr 2, 3, 4, 5, 7, 8, 11, 12, 15, 17, 20, X, and were involved genes such as: NPM1, CTNNA1, IRF1, RPS14, SPARC, CBL, ETV6, EZH2, CUX1, CDC25C, EGR1, RUNX1, BRAF, ASXL1, ZRSR2, PHF6, BCOR, CDC25C, EGR1, IRAK1 in loss; RAD21, JAK2, KIT, ZRSR2, PHF6, BCOR, IRAK1 in gain; SIRPB1 both in loss and gain. Moreover we found in LOH these genes: NF1, GATA2, FADD, IDH2, SF3B1, BCOR, PHF6, ZRSR2, STAG2, KDM6A, ATRX, IRF1, NPM1, CBL, CTNNA1, EGR1, IRAK1. Chromosomic aberrations disease-related are more statistically frequent on pts “insensitive” versus pts. “sensitive” (64% vs. 35%) (p≤0.01). Moreover we found that from the median of chromosomic alterations lenghts (in kbp) the group of “insensitive” MDS/AML patients to 5-Aza therapy present more gains and losses than “sensitive” ones. By Nexus Copy Number software, we identify 137 genes highly differentially gain (SIRPB1 and KIT with p ≤ 0.05) or loss (SIRPB1, LCE1C, BCAS1, EXD3 with p ≤ 0.05) or LOH between “insensitive” versus “sensitive” to 5-Aza (p ≤ 0.05). Among these genes, we focused on SIRPB1 (cytoband 20p13, 56Kbps), since it was loss on 14/22 (64%) “insensitive” pts (p=0,023) and gain on 7/22 (70%) “sensitive” ones with a significantly (p=0,0324), respectively. SIRPB1 common deletion region goes from 1571 to 1598 (27 kbps) and common amplified region goes from 1561 to 1591 (30 kbps). By NGS-WES we analyzed 35/214 (16%) AML samples at diagnosis and we searched for point mutations, insertion/deletion or other abnormalities, involved in biomarkers of sensitivity/refractory to 5-Aza or chemoteraphy. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR and again in SIRPB1 an heterozigosity frameshift deletion (c. 388delC; p. H130fs) in exone 2 in a AML pts with normal karyotype. By GEP we further analyzed 48/214 (22%) AML samples for SIRPB1 in order to correlate and confirm the expression or loss of expression of these genes, in correlation with 5-Aza response. We conclude that SIRPB1 is a promising marker of response to 5-Aza treatment in MDS and AML. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7NGS-PTL project. Celgene ITA–VZ–MDS–PI-0298 GRANT-ITA-002 Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Background: AML is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome, and patients (pts) with complex karyotype (CK) have generally a poor outcome. TP53 is the most frequently mutated gene in human tumors. The reported TP53 mutation rate in AML is low (2.1%). In contrast, the incidence of TP53 mutations in AML with a complex aberrant karyotype is higher (69-78%). Aims: To investigate the frequency, the types of mutations, the associated cytogenetic abnormalities and the prognostic role of TP53 mutations in adult AML pts, we focused the screening on subgroups of AML with chromosome abnormalities. Patients and Methods: 886 AML patients were analysed at the Seràgnoli Institute of Bologna between 2002 and 2013 for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, CBF fusion transcripts, DNMT3A, IDH1, IDH2, etc). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform (35/172 pts). 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Of the 886 AML patients beforehand analysed, 172 pts were screened for TP53 mutations and were correlated with cytogenetic analysis (excluding 15 pts where the karyotype was not available). 1. Fifty-two pts (30,2%) have 3 or more chromosome abnormalities, i.e. complex karyotype; 2. 71 (41,3%) presented one or two cytogenetic abnormalities (other-AML) and 3. 34 pts (19,8%) have normal karyotype. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations; seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had complex karyotype while only 6/29 mutated pts have “no CK” (21% and 3% of the entire screened population). Overall, between pts with complex karyotype, TP53 frequency is 44.2%. Regarding the types of the TP53 alterations, 32 were deleterious point mutations (http://p53.iarc.fr/TP53GeneVariations.aspx) and 4 deletions. Forty pts were also analysed for Copy Number Alterations (CNAs) by Affymetrix SNP arrays: several CNAs were found ranged from loss or gain of complete chromosome (chr) arms to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). Seven genes are included in these regions (RGS20, TCEA1, LINC01299, ARMC1, MTFR1, RAD54B, KIAA1429). In addition to the trisomy of the chr 8, others CNAs, located on other chromosomes are significantly associated (p = 0.05) with TP53 mutations: loss of chr 5q, chr 3 (p22.3), chr 12 (p12.3) and the gain of chr 17 (p11.2), chr 16 (p11.2-11.3) and chr 14 (q32.33). The zinc finger gene ZNF705B, implicated in the regulation of transcription was the most differentially associated gene (gain). WES analysis was done in 37 pts, 32 TP53 were wt while 5 pts were TP53 mutated: of importance, CDC27, PLIN4 and MUC4 were found also mutated in 3 out of 5 TP53 mutated (60%). Clinical outcome: as previously reported, alterations of TP53 were significantly associated with poor outcome in terms of both overall survival and disease free-survival (P < 0.0001). Conclusions: Our data demonstrated that TP53 mutations occur in 16.86% of AML with a higher frequency in the subgroup of complex karyotype AML (p< 0.0001–Fischer’s exact test). Since TP53 mutations have predicted to be deleterious and significantly correlated with prognosis, TP53 mutation screening should be recommended at least in complex karyotype AML pts. Furthermore, although further studies in larger numbers of patients are needed, the gain of chromosome 8 was observed to be significantly associated to TP53 mutations pts. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Speakers Bureau; Bristol Mayers Squibb: Speakers Bureau; Pfizer: Speakers Bureau.

Whole exome and transcriptome sequencing (WES and RNAseq) technologies are able to provide a comprehensive analysis of the genomic aberrations acquired by malignant cells, of their synergistic effects and functional consequences. In particular, RNAseq enables the detection of gene fusions originating from rare chromosomal translocations that have been involved in the pathogenesis of Acute Myeloid Leukemia (AML). We performed WES and RNAseq of AML patients to identify novel genetic abnormalities playing a causative role in leukemia development. We collected bone marrow or peripheral blood samples of 31 patients. Sequencing was performed using the Illumina Hiseq2000 platform. WES raw data were analysed with Whole-Exome sequencing Pipeline web tool for variants detection (WEP). The presence of gene fusions was assessed in RNAseq data with deFuse and Chimerascan. Selected genes fusions and variants were validated by Sanger sequencing. By RNAseq we identified a rare gene fusion transcript involving the BCL11B gene, which been previously suggested to play an oncogenic role in AML. The gene encodes for a zinc-finger protein participating to chromatin remodelling and regulating the differentiation and apoptosis of hematopoietic cells. The fusion was identified in a patient with poorly differentiated leukemia phenotype and unfavourable karyotypic abnormalities: 46,XX, t(2;14)(q21;q32), t(11;12)(p15;q22), who received standard chemotherapy, underwent allogeneic bone marrow transplantation and is currently in complete remission. Differently from previous data, the BCL11B translocation was associated neither with FLT3-ITD nor DNMT3A mutations. WES analysis revealed mutations in the TET2 and WTAP genes, which are known to act as co-players in the leukemic transformation. The exome data of our AML cohort identified neither INDELs nor nonsynonymous mutations in the BCL11Bgene, suggesting that the oncogenic function of BCL11B is activated by chromosomal translocations. Gene expression profiling showed a 4-fold upregulation of BCL11B transcript in the patient’s blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region, according to cytogenetic analysis. The increased expression of BCL11B was associated with an upregulation of potential targets including the antiapoptotic protein SPP1. Our data suggest that chromosomal translocations involving the BCL11B gene are rare events in AML and associate with somatic mutations in the malignant transformation of myeloid lineage cells, potentially by altering the differentiation and apoptotic processes. Future studies will investigate putative fusion partners of BCL11Band elucidate the biological consequences of its upregulation in AML pathogenesis. The results highlight the molecular heterogeneity of AML and the need for high-resolution sequencing analysis of leukemic samples at diagnosis in order to tailor personalized therapies. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

BACKGROUND Patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) frequently relapse on imatinib with acquisition of BCR-ABL kinase domain (KD) mutations. To analyze the changes that second-generation tyrosine kinase inhibitors (TKIs) have brought in mutation frequency and type, a database review was undertaken of the results of all the BCR-ABL KD mutation analyses performed in the authors' laboratory from January 2004 to January 2013. METHODS Interrogation of the database retrieved 450 mutation analyses in 272 patients with Ph+ ALL. Prescreening of samples was performed with denaturing high-performance liquid chromatography (D-HPLC), followed by direct sequencing of D-HPLC–positive cases. RESULTS BCR-ABL KD mutations were detected in 70% of imatinib-resistant patients, with T315I, E255K, and Y253H mutations accounting for 75% of cases. Seventy-eight percent of the patients reported to be resistant to second-generation TKIs after imatinib failure were positive for mutations, and 58% of them had multiple mutations. Analysis of patients relapsing on dasatinib revealed a newly acquired T315I mutation in almost two-thirds of the cases. Direct sequencing detected no mutations at diagnosis, even in patients who relapsed after a few months. CONCLUSIONS Second-generation TKIs ensure a more rapid debulking of the leukemic clone and have much fewer insensitive mutations, but long-term disease control remains a problem, and the T315I mutation is revealed to be an even more frequent enemy. BCR-ABL KD mutation screening of patients with Ph+ ALL who are receiving imatinib or second-generation TKIs would be a precious ally for timely treatment optimization. In contrast, the clinical usefulness of conventional direct sequencing at diagnosis seems to be very low. Cancer 2014;120:1002–1009. © 2013 American Cancer Society.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders of the myeloid lineage characterized by peripheral cytopenias and frequent leukemic evolution. MDS differ for clinical presentation, disease behavior and progression and this is the reflection of remarkable variability at molecular level. To this moment disease diagnosis is still dependent on bone marrow morphology that, although high concordance rates among experts are reported, remains subjective. Karyotype analysis is mandatory but diagnosis may be difficult in presence of normal karyotype or non-informative cytogenetics. Standardized molecular markers are needed to better define diagnosis, prediction of disease progression and prognosis. Furthermore, a molecular biology analysis could provide an important therapeutic tool towards tailored therapy and new insights in the disease's biology.

Introduction: The genetic basis of several acute myeloid leukemia (AML) subtypes remains poorly characterized, such as that of acute erythroid leukemia (AEL, AML M6) which is currently subclassified by morphology alone, and is associated with poor outcome. Here we sought to perform a definitive genomic analysis of AEL and translate these findings into faithful experimental models and novel therapeutic approaches. Methods: We studied 151 AEL cases (19.2% pediatric, 4.6% young adult, 21.9% adult and 54.3% older adult). Diagnosis of AEL was centrally confirmed and subclassified according to WHO 2008 and revised 2016 criteria. Whole exome and/or genome sequencing, RNA-sequencing and SNP array analysis were performed on all cases and in 2 AEL cell lines (TF-1 and Hel). Genomic data were compared to those from non-M6 childhood and adult AML from TARGET (n=192) and TCGA (n=197) studies. The functional effects of fusion transcripts and mutated genes were examined in IL-3 dependent Ba/F3 cells, NIH3T3 cells for focus formation assays and/or mouse lineage negative hematopoietic stem cells (lin- HSC) for colony forming and transplantation assays. Avatars of human AEL were established in immunocompromised NSGS and MISTRG mice for preclinical studies. Results: a) Genomic landscape of AEL. We identified 2,250 non-synonymous clonal and subclonal somatic mutations in 1,723 genes with a mean of 16.4 per case (range 2-88) and with missense and frameshift mutations accounting for 47.1% and 22.5% of all mutations, respectively. 78 genes were recurrently mutated in at least 3 cases. In frame fusions were detected in 31% of childhood and 27.5% of adult cases, and were more frequent in cases with complex karyotype. 124 potential driver genes were identified by statistical analysis or known pathogenic role in cancer, 9 of which were recurrent novel targets of mutation, most commonly involving chromatin modification (60.3%), cell cycle/tumor suppression (TP53, 33.8%), DNA methylation (28.5%), transcription regulation (26.5%), splicing (15.9%), NPM1 (11.9%), Ras (11.3%), JAK-STAT signaling (9.9%), the cohesin complex (8.6%), ALK/NTRK1 (4.6%) and PI3K signaling (3.3%). Overall, 33% of cases harbored a mutation in signaling genes amenable to inhibition by tyrosine kinase/Ras inhibitors. Mutations in TP53 and DNA methylation genes were significantly more frequent in adults while mutations in transcription regulators and Ras pathway were more frequent in children. Splicing mutations correlated with MDS phenotype and PI3K alterations with therapy-related AEL. Based on the co-occurrence and exclusivity of mutations 7 main distinct AEL genetic subtypes were defined: 1) pediatric AEL with NUP98-rearrangements (3.3% of all cases); 2) adult complex karyotype AEL with TP53 mutations (33.8%); 3) AEL with MLL-rearrangements (12.6%); 4) NPM1-mutated AEL (11.9%); 5) DNA-methylation/splicing mutated AEL (17.8%); 6) splicing/Ras/transcription regulation mutated AEL (21.2%) and 7) Other (8.6%) (Fig.1A). Mutations of chromatin modifiers occurred independently of karyotype, age and subtype (Fig.1B). NUP98-fusions and mutations in PTPN11, UBTF and GATA1 were more frequent in pediatric AEL compared to non-M6 AML. Among adults, mutations in TP53 and MLL were more frequent in AEL while FLT3, NPM1, DNMT3A and IDH1 were more in frequent in non-M6 subtypes. A complex karyotype, therapy-related AEL, TP53 mutations and NUP98-rearrangements were associated with poor outcome. b) Functional AEL modeling and therapeutic translations. Expression of NUP98-JARID1A in lin- HSC resulted in sustained self-renewal and development of an aggressive transplantable leukemia. At least three classes of signaling pathway mutations are targetable in AEL. ALK mutations in the extracellular MAM domain transformed Ba/F3 cells which were sensitive to crizotinib in vitro. Mutations in the tyrosine kinase domain of NTRK1 transformed NIH3T3 cells and were sensitive to entrectinib in vitro. Targeting of JAK-STAT, mTOR and PI3K pathways were examined in xenografts and sensitivity to JAK2 inhibitor ruxolitinib was confirmed in vivo. Conclusions: We provided the first comprehensive landscape of genomic alterations in AEL and defined distinct genomic groups with unique patterns of mutation occurrence compared to non-M6 AML. Finally, we showed that several pathogenic pathways are amenable to inhibition by approved targeted compounds. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Wei:Novartis: Honoraria, Research Funding. Loh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding.

Survival for childhood acute lymphoblastic leukemia (ALL) now approaches 90% with risk adapted therapy based on National Cancer Institute risk group (NCI RG) at diagnosis, somatic lymphoblast genetics, and early response to therapy as measured by minimal residual disease (MRD). Recent studies have identified multiple somatic genetic mutations in ALL, some of which confer increased risk of relapse or identify opportunities for additional targeted therapies. However, there are few genome sequencing analyses of representative cohorts of childhood ALL treated on contemporary regimens. To define the mutational landscape of childhood B-ALL, we performed whole exome sequencing (WES) on diagnostic tumor and remission samples of 192 patients with B-ALL consecutively enrolled between 4/1/06-9/8/06 on the Children's Oncology Group AALL03B1 classification trial that enrolled 11,145 patients up to age 30, which included patients enrolled on clinical trials for standard-risk (SR) B-ALL (N=5226, age 1-10 years and white blood cell count (WBC) < 50,000/uL) and high-risk (HR) B-ALL (N=2907; age ≥10 years or WBC ≥50,000/uL). An 8-gene expression low density array card identified Ph-like patients, and single nucleotide polymorphism arrays and multiplex ligation assays were used to determine DNA copy number alterations. Approximately 2/3 NCI SR and 1/3 NCI HR patients with sufficient banked samples were selected to reflect a population-based cohort (Table 1). Comparison of those selected vs. those not revealed significantly more NCI SR patients with a higher WBC and MRD, and fewer with double trisomy 4 and 10. Selected NCI HR patients were younger, had a higher WBC, and had more ETV6/RUNX1. There were a total of 3576 non-silent mutations with a median of 15 mutations/case (range 1-134). One case had 102 non-silent mutations and a germline mutation in MSH3. The additional mutations clustered in 11 pathways (Table 2), several of which were novel including cell-matrix interaction (e.g. SSPO, FAT1) and intracellular trafficking/cytoplasmic transport (e.g. DYNC2H1, ANK3, UNC13C). Most commonly altered were B-cell development (49.4%), transcription factors (45.8%), tumor suppressor genes (32.7%), cytoplasmic transport (27.3%), and Ras signaling (26.7%). Several pathways Ras signaling, Jak/STAT, and transcription factor mutations/deletions were associated with genetic lesions commonly used for risk stratification. Other pathways (epigenetic, B-cell development, or tumor suppressor) occurred in all subtypes. The most commonly identified genetic mutations were NRAS (n=33), KRAS (n=26), FLT3 (n=13), PAX5 (n=10), CREBBP (n=10), XBP1 (n=9), WHSC1 (n=7), and UBA2 (n=7). XBP1 and UBA2 mutations were novel. XBP1 (X-Box binding Protein 1) encodes a transcription factor that regulates the unfolded protein response. UBA2 (ubiquitin like modifier activating enzyme 2) encodes a protein involved in sumoylation to regulate protein structure and intracellular localization. Univariable analysis revealed no significant associations with any of these pathways or mutations with an increased risk of relapse with the exception of IKZF1 mutations or deletions (n=36; p=0.0087). Multivariable analysis modeling including IKZF1, age, presenting WBC, gender, NCI RG, ETV6/RUNX1, BCR/ABL1, Ph-like, white race, and MRD revealed only BCR/ABL1 and MRD positivity being significantly predictive of relapse. However, the risk of relapse was significantly increased based on the number of mutations identified in any one single patient (HR 1.02, 95% CI 1.01-1.023, p < 0.0004). Of note, only 18 patients (NCI SR, n=8, NCI HR, n=10) were Ph-like in this cohort, likely explaining the lack of significance between Ph-like status and an increased risk of relapse in this analysis. In summary, WES of a consecutively enrolled cohort of NCI SR and HR patients revealed a large number of novel genetic mutations that could be broadly assigned to 11 classes. Outcomes for patients overall were not influenced by any one of these classes, demonstrating that sentinel genetic alterations currently used in risk stratification are of paramount importance in directing therapy intensification for ALL. These data provide important information about pathways commonly mutated in childhood ALL, identifying classes of drugs that can be considered for clinical testing to further improve outcome. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Hunger:Amgen: Equity Ownership; Pfizer: Equity Ownership; Merck: Equity Ownership; Jazz Pharmaceuticals: Honoraria; Sigma Tau Pharmaceuticals: Honoraria; Erytech: Honoraria; Patent: Patents & Royalties: Dr. Hunger is a co-inventor of a patent (#8658,964) for the identification of novel subgroups in high risk B-ALL and outcome correlations and diagnostic methods related to the same; Spectrum Pharmaceuticals: Honoraria.

Although impressive developments have been made in the treatment of Acute Lymphoblastic Leukemia (ALL) patients, the overall survival is still very poor. With the exception of novel therapeutic strategies based on monoclonal antibodies (Bi-specific T-cell engagers, BiTEs) or immunogenic cells (CART cells), the therapeutic approaches for adult ALL patients are still base on non-selective chemotherapy or on tyrosine kinase inhibitors (TKIs) for the patients harboring the BCR-ABL1 fusion transcript. In addition a large percentage of initial successfully treated patients frequently develop relapses. Thus there is a need to improve the efficacy of conventional therapies, in particular those related to TKIs and to DNA damaging agents, in order to reduce the off-target toxicity and avoid relapses. In the present study we evaluated the in vitro, ex vivo and in vivo efficacy of MK-1775, a specific Wee1 inhibitor, in single agent and in combination with different therapeutic agents normally used for the treatment of B-/T-ALL. We firstly started by evaluated the efficacy of the compound in single agent on a panel of human B and T ALL cell lines (n=8) and on primary cells isolated from the bone marrow of adult B-ALL patients (n=8). The inhibition of Wee1 deeply reduced the cell viability and the proliferation rate, induced the apoptosis and increased the DNA damages of both leukemic cell lines and primary cells. Further cell-cycle analysis showed that in leukemic cell lines the treatment increased the number of cell in late S and G2/M phase. Light microscopy analyses, looking for nuclei morphology, confirmed that MK-1775 increased the number of mitotic cells but it interfered with normal mitotic division (induction of aberrant mitosis as showed by the increment of DNA bridges and micro-nuclei). The effects of the compound on the cell cycle profile and on the G2/M checkpoint were confirmed also in immunoblotting analyses, by the increment of phospho-HH3(ser10) and of Myt1 (mitotic isoform), and by gene expression analysis looking to specific genes involved in the G2/M checkpoints (PrimePcr DNA damage assay, Biorad). In particular genes like GADD45A and CCNB1/CCNB2 were significantly up-regulated between treated and untreated samples. Finally using a T-ALL mouse model we evaluated the effect of MK-1775 in single agent. Although no significative differences were seen between treated and un-treated samples, due to a very aggressive phenotype of the disease (all animal died after only 18 days from the engraftment), molecular analyses confirmed that the treatment induced DNA damages (increase of H2A.X and p-Chk1 ser317) and inhibited Wee1 functionality (reduction of pCDC2) on leukemic blasts isolated from both spleens and bone marrows. To evaluate if the inhibition of the G2/M checkpoint could sensitize leukemic cells to the toxicity of antineoplastic drugs, Philadelphia-negative ALL cell lines and primary leukemic cells (n=9) where treated with increasing concentration of MK-1775 and increasing concentration of the nucleotide analogue, clofarabine. Statistical analyses (Combination index value) confirmed the synergy of the combination in the reduction of the cell viability, in the inhibition of the proliferation and in the induction of the apoptosis. Similar results were seen on Philadelphia-positive ALL cell lines and primary cells (n=3) combining the MK-1775 with the TKI, bosutinib. The simultaneously inhibition of the Wee1 and the BCR-ABL downstream pathway resulted in a synergic inhibition of the cell viability, reduction of the proliferation and induction of apoptosis. In our opinion the pre-clinical results of this study are the basis for a future clinical evaluation of MK-1775 for the treatment of ALL patients. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Speakers Bureau; BMS: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau.

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph(+) acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph(+) ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph(+) ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph(+) ALL patients. (Blood. 2008; 112:3847-3855)

none 24 si BACKGROUND: Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. DESIGN AND METHODS: In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. RESULTS: We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. CONCLUSIONS: Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia. Iacobucci I; Lonetti A; Cilloni D; Messa F; Ferrari A; Zuntini R; Ferrari S; Ottaviani E; Arruga F; Paolini S; Papayannidis C; Piccaluga PP; Soverini S; Saglio G; Pane F; Baruzzi A; Vignetti M; Berton G; Vitale A; Chiaretti S; Müschen M; Foà R; Baccarani M; Martinelli G. Iacobucci I; Lonetti A; Cilloni D; Messa F; Ferrari A; Zuntini R; Ferrari S; Ottaviani E; Arruga F; Paolini S; Papayannidis C; Piccaluga PP; Soverini S; Saglio G; Pane F; Baruzzi A; Vignetti M; Berton G; Vitale A; Chiaretti S; Müschen M; Foà R; Baccarani M; Martinelli G.

We report a case of a patient affected by juvenile polyposis and hereditary hemorrhagic telangiectasia linked to a SMAD4 mutation who developed acute lymphoblastic leukemia positive for the Philadelphia chromosome translocation and with a complex karyotype. During the treatment with the tyrosine kinase inhibitor dasatinib the patient presented recurrent severe gastrointestinal hemorrhages linked to the genetic background and aggravated by thrombocytopenia.

Abstract 692 Background and Aims: In chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant Bcr-Abl mutants. Mutation status of resistant patients is usually investigated by Sanger sequencing (SS) of the Bcr-Abl kinase domain (KD). Novel ultra-deep sequencing (UDS) technologies allow to conjugate higher sensitivity with the unprecedented possibility to perform instant cloning of thousands of DNA molecules. We thus decided to take advantage of an UDS-based approach in order to: 1. resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs; 2. investigate their clonal structure and evolution in relation to time and treatment. Methods: We retrospectively performed a longitudinal analysis of a total of 111 samples from 35 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs (two to four TKIs among imatinib, dasatinib, nilotinib, ponatinib) and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS; 74/111 (67%) were positive for one (n=33) or multiple (n=41) mutations. UDS of the Bcr-Abl KD was done using Roche 454 technology. UDS allowed to achieve a lower detection limit of at least 0.1% – as compared to 20% of SS. Results: Bcr-Abl KD mutation status was found to be more complex than SS had previously shown in 85/111 (77%) samples (representative examples are detailed in Table 1 ). In 33/74 (44%) samples known to harbour one or more mutations by SS, UDS revealed that up to four ‘minor' mutations with 1–20% abundance were present in addition to the ‘dominant' one(s). The type of mutations could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the current or to the previous TKI received. The higher degree of complexity was evident also when the clonal relationships of multiple mutations were reconstructed ( Table 1 ). This revealed that identical mutations may be acquired in parallel by independent populations (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). In addition, longitudinal quantitative follow-up of mutated populations revealed that: 1. complexity generally increases with increasing lines of TKI therapy; 2. with a few exceptions, double compound mutants have higher selective advantage over single mutants but also over triple; 3. however, whether a compound mutant will ultimately take over depends on TKI, treatment duration, competition with other coexisting populations - the same compound mutants behaved differently in different patients receiving the same TKI. Conclusions: 1. sequential changes in the selective pressure exerted by TKIs may result in a heterogeneous mosaic of subclones harbouring different mutations or mutation combinations; 2. the evolution of each subclone is shaped not only by its inherent sensitivity to the specific TKI administered (‘absolute' fitness) but also by the competition with all other coexisting subclones (‘relative' fitness); (nonlinear) acquisition of additional mutations dictates further dynamics of shrinkage/expansion over time; 3. information provided by SS may not always be sufficient to predict responsiveness to a TKI; 4. sensitivity of a single or a compound mutant to a TKI in vivo is dictated by more complex factors than the mere in vitro IC50 value. Table 1 . Sample TKI (line) Mutations by SS (%) Mutations by UDS (%) Mutated populations by UDS (%) CML-04 DAS (3rd, after IM and NIL) T315I (∼60) E255K (∼30) T315I (54.89) E255K (26.15) E255V (1.37) T315I (47.02) E255K (18.63) T315I+E255K (7.52) E255V (1.02) T315I+E255V (0.35) ALL-09 PON (3rd, after IM and DAS) E255K (∼70) T315I (∼50) E255K (76.25) T315I (52.19) Q252H (cag>cac) (14.20) Q252H (cag>cat) (7.49) G250E (0.95) E255K+T315I (51.60) E255K+Q252H (cag>cac) (13.94) E255K+Q252H (cag>cat) (7.34) T315I (5.75) E255K (2.52) E255K+ G250E (0.70) Q252H (cag>cac) (0.25) G250E (0.25) Q252H (cag>cat) (0.15) CML-21 DAS (2nd, after IM) Y253H (∼60) F317L (∼60) Y253H (54.90) F317L (54.40) Y253H+F317L (43.00) Y253H (11.90) F317L (11.40) Disclosures: Soverini: ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Castagnetti: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Luppi: CELGENE CORPORATION: Research Funding. Rosti: Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli: NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Purpose: This phase II prospective study aimed to evaluate the efficacy and safety of 5-days azacytidine (5d-AZA) in patients with low-risk myelodysplastic syndromes (MDS). Second, single-nucleotide polymorphism (SNP) genetic profile and phosphoinositide-phospholipase C (PI-PLC) β1 levels were studied to evaluate possible biologic markers able to predict the hematologic response. Experimental Design: The study tested a lower intensity schedule of azacytidine. The treatment plan consisted of 75 mg/sqm/d subcutaneous administered for 5 days every 28 days, for a total of 8 cycles. Results: Thirty-two patients were enrolled in the study. The overall response rate was 47% (15 of 32) on intention-to-treat and 58% (15 of 26) for patients completing the treatment program. In this latter group, 5 (19%) achieved complete remission (CR) and 10 (38%) had hematologic improvement, according to the International Working Group (IWG) criteria. Three patients have maintained their hematologic improvement after 37, 34, and 33 months without other treatments. Moreover, 21 and 2 of 26 cases completing 8 cycles were transfusion-dependent for red blood cells and platelets at baseline, respectively. Of these, 7 (33%) and 2 (100%) became transfusion-independent at the end of the treatment program, respectively. Grade 3–4 neutropenia occurred in 28% of patients and 4 patients died early due to infections or hemorrhage. SNP results were not significantly correlated to the clinical outcome, whereas PI-PLCβ1 level anticipated either positive or negative clinical responses. Conclusions: 5d-AZA is safe and effective in a proportion of patients with low-risk MDS. PI-PLCβ1 gene expression is a reliable and dynamic marker of response that can be useful to optimize azacytidine therapy. Clin Cancer Res; 19(12); 3297–308. ©2013 AACR.