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Introduction. Chk1 and Chk2 are serine/threonine kinases that play a critical role in determining cellular responses to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. We explored the cellular effects of single-agent inhibition of Chk1/2 by PF-0477736 and its potential use as a therapeutic strategy for the treatment of B-ALL. Methods. 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). Results. BCR-ABL1-positive (BV-173, SUPB-15) and negative cell lines (NALM6, NALM19, REH) were incubated with increasing concentrations of PF-0477736 (0.005-2 M) for 24, 48 and 72 hours. Inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1-1.5 μM, with BV-173 being the most sensitive, while NALM6 the most resistant. All cell lines were TP53 wild-type, CDKN2A deleted. Consistent with the viability results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in all cell lines. Functionally, PF-0477736 decreased the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry and increased the number of H2AX foci, a markers of DNA damage, that culminated in a proportion of cells developing intense staining for H2AX together with nuclear morphological characteristics of apoptosis as demonstrated by immunofluorescence analysis. The efficacy of PF-0477736 was thereafter confirmed in primary blasts from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50: 0.1-0.5 μM at 24 hours); good responders, 36% (IC50: 0.6-1 μM at 24 hours); poor responders, 18% (IC50 > 1 μM at 24 hours). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis was performed on treated cell lines and their untreated counterparts (DMSO 0.1%) after 24 hours. Consistent with a specific Chk1- mechanism of action, treatment resulted in differential expression (p < 0.05) of genes involved in apoptosis and cell cycle (e.g. CEBPB, CUL1, Histone H1-H2A, 2B family clusters) and DNA damage (DDIT3, GADD34 and GADD45a), suggesting that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in B-ALL cells. Conclusions. For the first time we demonstrated the efficacy of PF-0477736 in vitro models of B- ALL, suggesting that single-agent Chk1/2 inhibition may be further evaluated in clinical trials. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, FIRB2006, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer.

Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis.

Background: Although recent high-resolution genome-wide profiling analysis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples has contributed to the identification of many novel somatic genetic alterations, a deep molecular characterization of adult ALL is still challenging, especially for those cases lacking recurrent fusion genes. Aims: In order to better molecularly dissect this ALL subgroup, we performed an integrative molecular approach including gene-candidate high-resolution screening and genome-wide profiling analyses. Methods: We retrospectively analyzed 28 newly diagnosed BCR-ABL1- negative BCP-ALL subjects (19 males/9 females; median age 41.5 years; negative for known fusion genes) and 28 BCR-ABL1-positive BCP-ALL subjects as a comparison group. In BCR-ABL1-negative ALL karyotype was normal in 10/28 (36%). Copy number alterations (CNA) of know leukemiarelated genes, such as IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and genes within PAR1: CRLF2, CSF2RA, IL3RA have been assessed by the SALSA MLPA kit P335 IKZF1 (MRC Holland). In addition, sequence mutations have been investigated in TP53, CRLF2, JAK2, LEF1, PAX5 and IL7R by nextgeneration deep-sequencing (NGS) (Roche Applied Science; IRON-II study oligonucleotide primer plates). Positivity for newly described BCR-JAK2, PAX5- JAK2, ETV6-ABL1, EBF1-PDGFRB, NUP-ABL1 gene fusions occurring in BCR-ABL1-like ALL (Roberts KG et al., Cancer Cell. 2012) was assessed by PCR amplification and sequencing. Finally, SNP arrays (SNP 6.0, Affymetrix), gene expression profile analyses (GeneChip® Human Transcriptome Array 2.0) and whole exome sequencing (Illumina) were performed to more fully assess genomic complexity. Results: Overall, 76% of BCR-ABL1-negative subjects showed an abnormality of at least one of the analyzed known leukemia genes: 7 (25%) had one, 4 (14%) had two, 6 (21%) had three, and 6 (21%) had four or more alterations. In subjects showing no abnormalities, SNP arrays analysis revealed amplifications of chromosome 1q in 2/6 cases (33%). Deletions of CDKN2A/B were the most frequent (39%) and in 73%, they occurred together with other abnormalities, suggesting that multiple events are needed to induce the full leukemia phenotype. Other common CNA included: deletions of IKZF1 (25%), ETV6 (25%), PAX5 (14%), EBF1 (11%), PAR1 region (11%) and RB1 (7%). NGS showed mutations of JAK2 and CRLF2 in 7% (R683S/G) and 4% (F232C), respectively. No positivity for newly described fusion genes activating tyrosine kinase was confirmed. Importantly, subjects with no abnormalities showed better survival rates compared to those with one or more molecular alterations (p

Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). 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, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and 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. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). 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, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and 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. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project.

Monica Bocchia,1 Angelo Michele Carella,2 Antonino Mulè,3 Lorenzo Rizzo,4 Mauro Turrini,5 Maria Chiara Abbenante,2 Roberto Cairoli,4 Valeria Calafiore,3 Marzia Defina,1 Angelo Gardellini,5 Giovanni Luzi,6 Caterina Patti,3 Maria Beatrice Pinazzi,6 Marta Riva,4 Giovanni Rossi,2 Vincenzo Sammartano,1 Luigi Rigacci6 1Hematology Unit, Azienda Ospedaliera Universitaria Senese, University of Siena, Siena, Italy; 2Division of Hematology with Hematologic Intensive Care Unit and Cellular Therapies, Department of Medical Science, Fondazione IRCCS Casa Sollievo Della Sofferenza, Foggia, Italy; 3UOC Hematology and Oncology, Ospedali Riuniti Villa Sofia-Cervello, Palermo, Italy; 4Department of Haematology, Niguarda Cancer Center, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy; 5Division of Hematology, Valduce Hospital, Como, Italy; 6UOC Hematology and Stem Cell Transplant Unit, Ospedale S, Camillo, Rome, ItalyCorrespondence: Angelo Michele Carella, Division of Hematology with Hematologic Intensive Care Unit and Cellular Therapies, Department of Medical Science, Fondazione IRCCS Casa Sollievo della Sofferenza, Viale Cappuccini, San Giovanni Rotondo, Foggia, 71013, Italy, Tel +390882410054, Fax +390882410322, Email am.carella@operapadrepio.itAbstract: Acute myeloid leukemia is a malignant disorder of the bone marrow, characterized by differentiation, clonal expansion, and uncontrolled proliferation of malignant myeloid progenitor cells and by several molecular and genetic abnormalities. A mutation of FMS-like tyrosine kinase 3 gene can be observed in about one-third of cases of acute myeloid leukemia. Two FLT3 inhibitors are actually approved for FLT3 mutated acute myeloid leukemia: midostaurin, a multikinase first generation inhibitor with lower affinity for FLT3 binding, and gilteritinib fumarate, a potent second-generation inhibitor of both FLT3-ITD and TKD. Gilteritinib is a new effective and well-tolerated drug for patients with relapsing or refractory FLT3-positive acute myeloid leukemia. Thanks to its efficacy, low toxicity, its good manageability (oral formulation), this drug is suitable for all the patients, including elderly frail patient with concomitant therapies or pre-existing or underlying diseases, and can be used also in the outpatient setting, reducing risks and costs related to the hospitalization. We report and discuss seven cases of different patients with FLT3 positive acute myeloid leukemia successfully managed with gilteritinib in the real clinical practice.Keywords: acute myeloid leukemia, FLT3, midostaurin, gilteritinib

Background: Precursor T cell ALL/LBL occurs most frequently in late childhood, adolescence, and young adulthood, with a 2:1 male predominance; it comprises 15 and 25 percent of childhood and adult ALL, respectively, and 2 percent of adult non-Hodgkin lymphoma. In adult patients prognosis is poor, due to the high incidence of relapse even after allogenic stem cell transplantation. Aims: To identify new targets, to optimize therapy, to reduce toxicity and to improve clinical outcome. Methods: We retrospectively analysed and currently report clinical outcome results about 52 newly diagnosed and younger than 60 years T-ALL patients (median age 30 years, range 14-73 years) treated, from 2006 to 2012 according to standard chemotherapy regimen, including adapted pediatric-like schedule, BFM protocol, and adult schedules. After induction, all the patients underwent consolidation for at least one course. All the patients shared the same strategy for intensification, that consisted, when available, in allogenic or autologous stem cell transplantation. Detailed data about cytogenetics and molecular biology will be provided on site. Durations of complete remission (CR) and overall survival (OS) were estimated according to the Kaplan-Meier method. The CR duration was calculated from start of CR to first evidence of disease recurrence. Results: Informed consent was obtained; after a single induction course 41/52 patients obtained a CR (78.8%) and 2 patients a partial remission (PR) (3.8%) for an overall response rate (ORR) of 82.6%. Seven patients (13.4%) had resistant disease, and 2 (3.8%) died during induction. After a median follow-up of 22.7 months, 19 patients (36.5%) are still in CR. The median CR duration and OS were 12.3 and 23.15 months, respectively. The most common grade 3 adverse events included gastro-intestinal toxicities (i.e. nausea, vomiting, mucositis and diarrhoea) and liver dysfunction. Summary and Conclusions:Our analysis confirms, in line with literature data, that, despite intensive chemotherapeutic treatments and stem cell transplantation, T-ALL and T-LBL adult patients still show a bad prognosis. The relatively satisfying induction remission rate is followed, in most cases, by a high relapse rate. Therefore, a molecular stratification approach, based on gene-expression profile analysis (Ferrando et al., Cancer Cell 2002) should be routinely performed, in order to identify new targets, to optimize therapy, to reduce toxicity and to improve clinical outcome.

Introduction. Mutations, chromosomal rearrangements and associated gene fusions resulting from inversions, interstitial deletion or translocations represent recurrent findings in leukemia. Nowadays high-throughput “Next Generation Sequencing” technologies and novel developed algorithmic methods allow a unified deep analysis of such alterations with a single base resolution. Taking advantage from these tools, we performed an RNA-Seq (whole transcriptome shotgun sequencing) approach using the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations and fusion genes in 3 adult BCR-ABL1+ ALL cases treated with tyrosine kinase inhibitors. Methods. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case with del(9)(p13p22). All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. Results. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. Thereafter, we analyzed RNA-seq data by deFuse (McPherson A et al. PLoS Computational Biology May 2011), a novel computational method for fusion discovery between a gene and an intergenic region. This software uses clusters of discordant paired-end alignments to inform a split read alignment analysis for finding fusion boundaries. First of all, we evaluated the ability of deFuse to rediscovery the BCR-ABL1 gene fusion, then we focused on the most intriguing fusions such as the PBXIP1- PMVK (pre-B-cell leukemia homeobox interacting protein 1- phosphomevalonate kinase) gene fusion. All new gene fusions are under evaluation in a bigger ALL cohort. Conclusions. This study provided a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations and gene fusions involved in Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna- Ravenna, FIRB 2006, Ateneo RFO grants, PIO project, Programma di Ricerca Regione–Università 2007–2009.

Background: Deletions of IKZF1 gene, encoding for a regulator of lymphocyte differentiation, occur in more than 80% of adult patients (pts) with Ph+ ALL and significantly affect the prognosis. The deletions either involve the entire IKZF1 locus, resulting in loss of function, or delete an internal subset of exons, resulting in the expression of dominant negative isoforms. To better stratify Ph+ ALL pts according to IKZF1 status, we set up, validate and assess the routine applicability of a IKZF1 deletion screening strategy based on a multiplex-PCR. Patients and methods: We studied 87 adult BCR-ABL1+ ALL pts. For each type of common IKZF1 deletion (D4-7, D2-7, D4-8) an appropriate pair of primers will be designed using Primer 3 (http://frodo.wi.mit.edu/primer3/). Their ability to efficiently amplify the deletion was tested. PCR amplifications was performed using 50 ng genomic DNA as template for each reaction and a FastStart Taq DNA Polymerase (Roche Diagnostics). For each patient 3 amplicons were generated and sequenced (amplicon A for D4-7, amplicon B for D2-7 and amplicon C for D4-8). Moreover, a multiplex amplification strategy was assessed and a forth amplicon was generated and sequenced (forward primers were localized on intron 1 and 3, the reverse ones in the intron 7 and at the end of the gene, after the exon 8). The size of amplicons depends on the positions of the breakpoints in the IKZF1 gene and on the number of nucleotides added at the conjunction. It was in the range of 450-600 nucleotides. A positive control was used for each run. Results: On 87 pts previously analyzed by SNPs array, we identify IKZF1 deletions in 69/89 (78%) samples. Deletions were: 51 D4-7, 12 D2-7 and 4 D4-8; in 2 cases the deletion was extended to all gene, and 2 pts presented two breakpoints simultaneously. Multiplex PCR and subsequent sequencing were performed for most common deletions on 40 pts confirming previous results and indentifying the precise genomic positions of breakpoints and the nucleotides added at the conjunction. This rearrangement has been used to strictly monitor Minimal Residual Disease (MDR) during the follow-up and at relapse to confirm the clonal fidelity. Conclusions: We set-up a method rapid sensitive and with less amount of DNA sample, to screen Ph+ ALL pts at diagnosis and to monitor MRD during the treatment. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus.

Loss of CDKN2A, encoding p16/INK4A and p14/ARF tumor suppressors, has been associated with poorer survival and higher leukemia-initiating- cell-frequency in xenograft models (Notta F et al. Nature 2011). Recently, using genome-wide single nucleotide polymorphisms arrays and gene candidate deep exon sequencing, we identified CDKN2A deletions in 29% (29/101) adult newly diagnosed Ph+ ALL patients and in 47% relapsed cases. Accordingly, here we aimed to investigate the functional consequences of genomic deletions and their prognostic value. By quantitative real-time PCR a significant difference in the expression of CDKN2A was observed among cases with heterozygous (p = 0.04) and homozygous (p = 0.01) loss compared to normal cases, suggesting that deletions lead to CDKN2A haploinsufficiency. In order to investigate their clinical implications, data were collected from 81 patients (median follow-up: 25.2 months- range 2.1-148.1; median age at diagnosis: 54 years- range, 18-76; CDKN2A loss in 29, 36%, cases). 72 patients (89%) were enrolled in the GIMEMA clinical trials (12 patients in GIMEMA LAL0201-B protocol, 13 in LAL2000 and 47 in LAL1205 protocols), while 9 patients (11%) were enrolled into institutional protocols. By univariate analysis a shorter overall survival (OS) and disease-free survival (DFS) were found in patients with CDKN2A deletion compared with those wild-type (OS: 27.7 v 38.2 months, respectively, p = 0.0206; DFS: 10.1 vs 56.1 months, respectively, p = 0.0010). Moreover, a higher cumulative incidence of relapse for patients with CDKN2A deletion (73.3 vs 38.1; p = 0.0014) was also recognized. The multivariate analysis confirmed the negative prognostic impact of CDKN2A deletion on DFS (p = 0051). These results show that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation, cell growth and tumor formation by the action of MDM2 and CDK4/6, attractive drugs to prevent disease recurrence could be represented by the inhibitors of MDM2 and CDK4/ CDK6. These drugs are currently under investigation in in vitro studies by our group. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program, Programma di Ricerca Regione – Università 2007 – 2009.

Background: Loss of CDKN2A/B has been associated with poorer survival and higher leukemia-initiating-cell-frequency in xenograft models (Notta F et al. Nature 2011). Here, we aimed to: a) explore the frequency and size of deletions in CDKN2A/B genes in adult BCR-ABL1-positive ALL patients; b) determine the main mechanism of inactivation; c) investigate their prognostic role. Methods: 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes; gene-expression profiling (Affymetrix Human Exon 1ST Array) was performed to draw a specific signature associated with deletions. Results: At diagnosis, CDKN2A and CDKN2B deletions were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in 72% of cases and in 43% of them the minimal overlapping region of the lost area spanned only the CDKN2A/ B genes. In contrast, in 57% of cases the loss was considerable larger and extended sometimes over the entire short arm. Analysis of the genomic status at the relapse showed a marked increase in the detection rate of CDKN2A loss (47%) compared to diagnosis (p = 0.06). The mutation screening, performed by PCR amplification and subsequent sequencing on each exon of ARF/CDKN2A/B, showed that missense point mutations are rare with only the identification of D146N in the CDKN2A exon 2 in one case. Correlation with clinical features demonstrated that deletions of CDKN2A/B are significantly associated with poor outcome both in terms of disease free-survival (p=0.001) and cumulative incidence of relapse (p=0.001). Conclusions: Novel treatment strategies targeting the ARF-MDM2-p53 pathway or CDK4/6 may be effective in this subset of patients to overcome aggressive growth properties and in vitro studies are ongoing to confirm this hypothesis. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program, Programma di Ricerca Regione – Università 2007 – 2009.

Background: Genetic heterogeneity in drug-metabolizing enzyme and transporter genes affects specific drug-related phenotypes in cancer. In order to investigate a relationship between genetic variation and response in AML, we comprehensively assessed the allele frequencies of 1936 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 94 CD33-positive AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform. Patients and methods: All patients were enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy (ClinicalTrials.gov NCT00909168). Cytogenetics, multidrug-resistance phenotype, FLT3 and NPM mutation status, as well as WT1 quantitative expression analyses were performed at diagnosis. Furthermore, high-resolution single nucleotide polymorphism (SNP) array and DMET genotyping analyses (Affymetrix) were also performed. Results: Of the 94 patients, genotype results were evaluable for 91 cases. The median call rate was 99.48 (range, 96.32-100). Firstly, we tested the association among SNPs and response to the induction cycle (FLAI + GO) comparing the genotyping profile of 80 patients in complete (85%) and partial (3%) remission with that of patients (12%) with no response. A highly significant difference (p < 0.001) in the allele frequency of 2 variants, in complete linkage disequilibrium, in the alcohol dehydrogenase enzyme (ADH1A) was found. ADH1A metabolizes the conversion of ethanol to acetaldehyde which is thereafter converted to acetate by ALDH1, which is well known as stem cell marker. These variants were not associated with high risk AML, FLT3 and NPM1 mutations, but strongly influenced response to the induction phase also in a multivariate analysis. Secondly, SNPs were stratified according to liver toxicity and a significant difference in the allele frequency in CYP2E1, involved in the alcohol metabolism was found to be associated with a grade I/II liver toxicity. Conclusions: Genetic variations in ADH1A and CYP2E1 genes were for the first time identified and associated with response and toxicity in AML patients treated with a combination of GO and FLAI regimen. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007–2009.

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.