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Background. Outcome of elderly acute myeloid leukemia (AML) patients is dismal. Tipifarnib (Zarnestra, Z) and Bortezomib (Velcade, V) are new, targeted-treatments that might improve current results. In particular, Z is a farnesyltransferase inhibitor effective in AML, allowing complete remission (CR) rates ranging from 8 to 22%. Notably, the response rate appeared superior among patients with higher RASGRP1/APTX gene expression ratio. V is a proteasome inhibitor, possibly effective in AML as for phase I studies. Interestingly, Z and V appeared synergistic in AML cell lines. Aim. We designed a phase I/II study aiming to assess the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of V in association with standard doses of Z and the efficacy and toxicity of the combination, in AML patients >18 years, unfit for conventional therapy, or >60 years, in relapse. Furthermore, we aimed to assess the correlation between RASGRP1/APTX ratio and treatment response. Methods. V was administered as weekly infusion for three consecutive weeks (days 1, 8, 15), starting from 0.7 mg/m2 and increasing by 0.3 mg/m2 until the DLT was reached. Z was administered at the daily dose of 600 mg BID for 21 consecutive days. Response was assessed at the end of each cycle (28 days). Patients’ withdrawn was planned in case of progression or stable disease after six cycles. Real-time quantitative PCR (q-PCR) was used for RASGRP1/APTX genes evaluation. Results. 45 patients were enrolled: 12 in the phase I and 33 in the phase II. 11/12 patients were evaluable in the phase I. Five patients received V at the dose of 0.7 mg/m2 without reporting DLT. Conversely, DLT was reached at the dose of 1.0 mg/m2 due to grade III SNC toxicity, recorded in 1/6 patients. 22/45 patients were evaluable for treatment response; transient reduction of peripheral blasts (> 50% to baseline) was observed in 20/22 cases. 2 patients achieved CR and 1 obtained a partial response (PR). 1 patient had a haematological improvement (HI), 12/22 had a stable disease (SD) and 5/22 showed progressive disease (PD). Interestingly, the 4 responders (CR+PR+HI) had a significant higher RASGRP1/APTX ratio respect to non responder (p=0.017). Conclusion. We conclude that the MTD of V in association with Z is 1.0 mg/m2. The association seemed to be safe, with response rate (18%) similar to what reported for Z alone. Finally, though in few cases, RASGPR1/APTX ratio was confirmed to be associated to treatment response.

Background . Conventional induction treatment in young Acute Myeloid Leukemia (AML) patients ( Aim of the study . We evaluated the safety profile and the efficacy of two four-drugs induction schedules, adding either fludarabine (25 mg/sqm days 1–5) or mylotarg (3 mg/sqm day 6) to idarubicin (6 mg/sqm days 1, 3, 5), cytarabine (1 g/sqm days 1–5), etoposide (100 mg/sqm days 1–5) (FLAIE and MyAIE, respectively). Methods . Sixty-six consecutive AML patients were enrolled either in the FLAIE (N=44, from 2002 to 2005) or in the MyAIE (N=22, from 2005 to April 2007) schedule, with similar clinical and biological characteristics. The median age was 45 and 48 years, respectively. According to kariotype, WBC count and FLT3 status, seventy and sixty-four percent of cases, respectively, were considered at high risk. Consolidation therapy consisted of 2 cycles of ID-AraC and Ida. Results . The complete remission rate was 75% and 59% for FLAIE and MyAIE, respectively (p=n.s.). Death during treatment rates were 5% and 0. After 1 consolidation course the overall CR rate was 80% and 73%. After a similar median follow up, 27 months (1–62) and 21 months (5–42) respectively, 41% of patients are alive in CR in the FLAIE group (12 SCT and 3 ASCT) and 64% in the MyAIE group (7 SCT and 4 ASCT) (p=n.s.; Chi-square, Fisher’s exact test). Toxicity was comparable in the two regimens. The median time to ANC recovery (>1.0 x 10^9/L) was 31 and 23 days for FLAIE and MyAIE, respectively. The median time to PLT recovery (>100 x 10^9/L) was 28 and 24 days, respectively. The median time of neutropenic fever episodes for patients was 1 and 1.4 in the 2 groups, respectively. Grade III/IV GI toxicities occurred in 11% and 22% of cases, respectively. Conclusions . These data showed that four-drugs intensified induction therapy is a feasible approach in young AML patients. Recent published trials have demonstrated that fludarabine-based induction chemotherapy in high risk AML patients is able to increase the CR rate offered by conventional treatment. Therefore, the limited number of patients involved in this study, the low administration dosage of idarubicin, and the relevant role of fludarabine in induction therapy, can reasonably justify the lower CR rate obtained in patients treated in the MyAIE schedule, if compared with FLAIE regimen and, above all, with standard chemotherapy. In the light of their efficacy and safety profile, fludarabine and mylotarg, in combination with conventional chemotherapy, represent a promising induction regimen in young AML patients; further analyses and randomized pilot trials will define their definite role.

2176 Poster Board II-153 Progression from chronic phase to blast crisis (BC) remains a major hurdle on the road to effective treatment of chronic myeloid leukemia (CML). BC is known to be associated with accumulation of additional genetic alterations, but these alterations have so far been only partially characterized. The development of SNP-arrays as a tool for high-resolution karyotyping now allows to perform high-throughput genome-wide screens for submicroscopic genomic alterations with unprecedented informativity and resolution and to precisely map all the genes involved in these alterations. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution molecular allelokaryotyping of 25 DNA samples from BC (myeloid, n=16; lymphoid, n=9) CML patients (pts). The 6.0 SNP Array technology relies on 1.8 million markers evenly spaced across the genome, with a median inter-marker distance 2 pts are listed in the [Table][1] below. Gains/losses mapping to known regions of copy number variation (CNV) were excluded. | Gene Symbol | Type of CNA | Exons involved | Chromosomal location | No of pts harboring CNAs | |:-----------:| ----------- | -------------- | -------------------- | ------------------------ | | MDM4 | a | All | 7q22.11 | 5 pts | | HGF | a | All | 1q32.1 | 4 pts | | NRAS* | a | All | 1p13.2 | 3 pts | | HRAS* | a | All | 11p15.5 | 3 pts | | KRAS* | a | All | 12p12.1 | 5 pts | | AKT3* | md | One or more | 1q43 | 13 pts | | CDC73 | md | One or more | 1q31.2 | 8 pts | | RB1 | md+a | One or more | 13q14.2 | 8 pts | | ERG | md/bd | One or more | 21q22.3 | 8 pts | | ETS1 | a | One or more | 11q24.3 | 8 pts | | ETV1 | a | One or more | 7p21.2 | 7 pts | | SMAD2 | ma+d | One or more | 18q21.1 | 7 pts | | PIK3CA* | ma+d | One or more | 3p26.32 | 6 pts | | SKAP2 | a | One or more | 7p15.2 | 5 pts | | EPHA3 | a | One or more | 3p11.2 | 5 pts | | KALRN* | md | One or more | 3q21.2 | 5 pts | | FHIT | md | One or more | 3p14.2 | 5 pts | | JAK1 | d | One or more | 1p32.3 | 5 pts | | JAK2 | ma+d | One or more | 9p24.1 | 4 pts | | AKT2* | md | One or more | 19p13.2 | 4 pts | | SOS1* | md | One or more | 2p22.1 | 4 pts | | PTEN* | ma+d | One or more | 10q23.31 | 4 pts | | FGFR2 | a | One or more | 10q26.3 | 4 pts | | ETV5 | a | One or more | 3q27.2 | 4 pts | | MET | a | One or more | 7q31.2 | 4 pts | | PBX4 | md | One or more | 19p13.11 | 4 pts | | NRG1 | md | One or more | 8p12 | 4 pts | | FAF1 | md | One or more | 1p33 | 4 pts | | GAS2 | md | One or more | 11p14.3 | 3 pts | | TEC | a | One or more | 4p12 | 3 pts | | PTCH1 | ma+d | One or more | 9q22.32 | 3 pts | | PIK3R1* | a | One or more | 5q13.1 | 3 pts | | GRB2* | a | One or more | 17q24 | 3 pts | | PAK2* | md | One or more | 3q29 | 3 pts | | RUNX1T1 | a | One or more | 8q21.3 | 3 pts | * Abbreviations: md, monoallelic deletion; bd, biallelic deletion; a, amplification; ma+d, complex pattern of monoallelic deletion of some exons and amplification of other exons All the genes found to harbor CNAs were transcription factors, adaptor proteins, receptor and non-receptor kinases involved in cell proliferation and apoptosis - with a known role as oncogenes or tumor suppressors or oncogene/tumor suppressor interactors. Although these results confirm a high degrees of heterogeneity in the alterations detectable in BC CML pts, members of the RAS pathway (indicated with an asterisk) were the most frequently altered genes. Further characterization by polymerase chain reaction and sequencing is ongoing. In conclusion, the power of 6.0 SNP Array technology allowed us to detect previously unidentified alterations targeting whole or part of key oncogenes or tumor suppressors whose deregulation may play a role in determining the aggressive phenotype of BC CML, and which may represent potential therapeutic targets. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare. [1]: #T1

Introduction Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8). Aims In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany). Results We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies. Conclusion Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far. Disclosures: Kohlmann: Roche Diagnostics: Honoraria; MLL Munich Leukemia Laboratory: Employment. Martinelli:Roche Diagnostics: Research Support Other. Alikian:Roche Diagnostics: Research Support Other. Auber:Roche Diagnostics: Research Support Other. Belickova:Roche Diagnostics: Research Support Other. Bronzini:Roche Diagnostics: Research Support Other. Cazzaniga:Roche Diagnostics: Research Support Other. Chiaretti:Roche Diagnostics: Research Support Other. Ernst:Roche Diagnostics: Research Support Other. Fuellgrabe:Roche Diagnostics: Research Support Other. Gabriel:Roche Diagnostics: Research Support Other. Hernandez:Roche Diagnostics: Research Support Other. Jansen:Roche Diagnostics: Research Support Other. Iacobucci:Roche Diagnostics: Research Support Other. Lion:Roche Diagnostics: Research Support Other. Lode:Roche Diagnostics: Research Support Other. Martinez-Lopez:Roche Diagnostics: Research Support Other. Mills:Roche Diagnostics: Research Support Other. Mossner:Roche Diagnostics: Research Support Other. Machova Polakova:Roche Diagnostics: Research Support Other. Porret:Roche Diagnostics: Research Support Other. Pospisilova:Roche Diagnostics: Research Support Other. Preudhomme:Roche Diagnostics: Research Support Other. Sayitoglu:Roche Diagnostics: Research Support Other. Soverini:Roche Diagnostics: Research Support Other. Spinelli:Roche Diagnostics: Research Support Other. Thiede:Roche Diagnostics: Research Support Other. Vandenberghe:Roche Diagnostics: Research Support Other. Yeoh:Roche Diagnostics: Research Support Other. Hochhaus:Roche Diagnostics: Research Support Other. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Roche Diagnostics: Research Funding.

Point mutations in the kinase domain (KD) of the Bcr-Abl gene are generally regarded as the most frequent mechanism of resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) in patients (pts) with chronic myeloid leukemia (CML). Nearly all studies, however, have focused mainly on pts with advanced disease, where resistance is most often observed. Nowadays, the great majority of pts on IM are early chronic phase (ECP) pts receiving IM as front-line treatment. If, on one hand, the IRIS study demonstrated that response rates are high and relapse is infrequent in ECP, on the other hand we still know very little on the contribution of KD mutations to resistance in this subset of pts. Between January 2005 and July 2007 we analyzed for the presence of Abl KD mutations one hundred and two ECP pts on IM who were referred to our laboratory because their response was defined either as ‘failure’ (n=70 pts) or as ‘suboptimal’ (n=32 pts) according to recently published recommendations (Baccarani et al, Blood 2006). Twenty mutations were detected in 17/70 (24%) pts who failed IM. In particular, mutations were observed in 1/2 pts who showed no hematologic response (HR) at 3 months, 1/10 (10%) pts who showed less than partial cytogenetic response (PCgR) at 12 months, 4/25 (16%) pts who showed less than complete cytogenetic response (CCgR) at 18 months, 6/23 (26%) pts who lost CCgR, 5/10 (50%) pts who lost HR. Mutations were M244V (n=2), G250E (n=1), Y253H (n=4), E255K (n=1), T277A (n=1), E279K (n=1), F311I (n=1), T315I (n=1), M351T (n=3), E355D (n=1), F359V (n=1), H396R (n=3). In 7 pts who progressed to accelerated or blastic phase shortly after, four had mutations: Y253H (n=2 pts), E255K (n=1 pt) and T315I (n=1 pt). Four mutations were detected in 4/32 (13%) pts who had a suboptimal response to IM. In particular, a mutation was observed in 1/11 (9%) pts who showed less than PCgR at 6 months and in 3/21 (14%) pts who showed less than CCgR at 12 months. Mutations were E255K, F317L, M351T, F359V. In both groups no correlation was observed between likelihood of mutation selection and Sokal risk score. We conclude that in ECP pts who receive IM as front-line treatment Abl KD mutations are not the major mechanism of drug-resistance, probably because mutations tend to accumulate during the natural course of the disease as a result of a progressively increasing genetic instability and are therefore a feature of CML clinical deterioration rather than a phenomenon observed only against a background of IM exposure. Our data highlight the need to find out which is the actual predominant mechanism(s) of resistance acting in the setting of ECP - which now gathers the overwhelming majority of CML pts on IM therapy - as a mandatory step towards the development of effective second-line treatment strategies.

Background: BCR-ABL1-positive ALL is the most frequent and prognostically most unfavorable subtype of adult ALL. The main reason for the poor clinical outcome of BCR-ABL1-positive ALL is genetic instability. However, how normal B-cell precursor cells acquire the genetic changes that lead to transformation and progression has not been completely defined. Activation-induced cytidine deaminase (AID) produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and methods: We analyzed 61 adult de novo Ph+ ALL patients (pts) and 60 CML pts (chronic phase and myeloid/lymphoid blast crisis). AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5′ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 41 (67%). AID expression correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. Moreover, AID expression was also found in lymphoid blast crisis CML (60%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were expressed. In 10/61 (16%) BCR-ABL1-positive ALL pts the full-length isoform (GeneBank accession number NM_020661) was identified, in 16/61 (26%) the co-expression of the wild-type isoform and of different AID splice variants was found and in 15/61 (25%) only the expression of splice variants was found. These can result from retention of intron 4 (Variant A), omission of exon 4 (Variant B) and 3 (Variant C), and from a deletion of 30 bp in the initial portion of exon 4 (Variant D). In the wild-type mRNA, codon 148 spans exons 3 and 4. In both variants A and B, mRNA splicing disrupts this codon and causes a frameshift, which results in a premature stop codon. If translated, the splice variants produce truncated proteins of 187 and 145 amino acids, respectively. However, the putative deaminase active site, encoded by exon 3, is preserved in both splice variants, but is lacking in the variant C. Since enforced expression of Pax5 induces endogenous AID gene expression, we analyzed the expression levels of Pax5 in all pts (ΔΔCt method, GAPDH as control gene). As expected, we found a very strong difference (p Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as a mutator outside the Ig gene loci in promoting genetic instability in leukemia cells.

Abstract 1027 Poster Board I-49 Background: Current therapeutic strategies for the treatment of elderly acute myeloid leukemia (AML) patients are still unsatisfactory. Induction regimens relying on anthracycline and cytarabine combination (3+7) still represent the standard reference therapy, with complete remission (CR) rate of 35-60% but no more than 10-20% of patients living more than three years from diagnosis. The addition of other drugs to overcome resistance and prevent subsequent relapse, has often shown greater toxicity without improving results. Aim We designed a phase II study aiming to assess toxicity and efficacy of My-FLAI regimen in patients with newly diagnosed AML aged more than 60 years. Fifty-one patients were enrolled with a median age of 68 years (range 60-76). The median leukocyte count was 10.2 ×109/L (range 0.7-222.7). Twenty-five patients had a secondary AML and 31% had a complex kariotype. Fludarabine, cytarabine and idarubicin were administered for three consecutive days at the daily dose of 25 mg/m2, 1 g/ m2 and 5 mg/ m2 respectively. Gemtuzumab ozogamycin (Mylotarg, My) was infused at day four at the dose of 5 mg. Patients achieving a CR after the induction course were planned to receive a second identical course of My-FLAI. Granulocyte colony-stimulating factor (G-CSF) was administered at physician's discretion to promote granulocytic recovery if clinically indicated. Results: Twenty-seven patients achieved a CR (one of these with an incomplete platelet recovery) and 4 obtained a partial response (marrow blast 5-19%) for an overall response rate (ORR) of 61%. Seventeen patients (33%) showed a resistant disease and three (6%) died during induction (DDI). Seventeen patients received an identical consolidation course. Seven patients were treated with cytarabine-based consolidation and the remaining 27 patients were not treated furthermore. The disease-free survival (DFS) and overall survival (OS) were 6 months (range 2.4-48.0) and 13 months (range 1.1-61.5) respectively. DFS appear to be more favorable in patients receiving additional therapy after two My-FLAI cycles (IDAC n=3; ASCT n= 4; experimental drugs n= 1; My n=4) in comparison to patients no further treated, being 12.8 (range 4.7-18.0) and 5.9 (range 2.4-48.0) months respectively. The median duration of G-CSF administration was 6 days (range 0-35) in induction and 8 days (range 0-15) in consolidation cycle. All patients developed a grade IV hematological toxicity. Median time to ANC recovery (>1 ×109/L) was 21 days (range 10-52) after induction course and 18 days (range 12-20) after consolidation course. Median time to PLT recovery (PLT>20 ×109/L and PLT>100 ×109/L) was 18 days (range 9-54) and 23 days (range 15-78) after induction and 18 days (range 12-20) and 32 days (range 21-110) after the consolidation course. Thirty-six patients had infectious complication; 33 experienced a microbiologically documented infection (n=20 gram+, n=12 gram- n=1 candidemia). Eighteen patients had a fever of unknown origin (FUO) and 12 patients suffered from pulmonary infection (n=2 bacterial; n=8 probable fungal; n=2 aspergillosis). One patient developed a maxillary sinus aspergillosis. Grade III/IV haemorrhagic complications were observed in 5 patients. No severe gastro-intestinal adverse events were observed. With a median follow-up of 8.8 months (range 0.5-61.5) 41 patients died. Five patients died in first CR for cardiac failure (n=2) and for infective complication (n=3). The remaining died for disease. Nine patients are alive, four of them in first CR. Conclusions: The four drug regimen My-FLAI is overall well tolerated in an elderly AML population. However, the efficacy did not appear to be superior to that of standard “3+7” regimen. Acknowledgments: Supported by BolognAIL, AIRC, European LeukemiaNET, COFIN, FIRB 2006, Fondazione del Monte di Bologna e Ravenna Disclosures: No relevant conflicts of interest to declare.

7113 Background: Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and ta...

Abstract 967 Human genome sequence variation, in the form of single nucleotide polymorphisms (SNPs) as well as more complex structural variation (such as insertions, duplications and deletions) in drug-metabolizing enzyme and transporter genes (DMETs), affects each individual's response to anticancer agents and thus the likelihood of experiencing an adverse drug reaction. Therefore, understanding relationships between genetic variation and differential drug responses is a challenge to improve both the safety and efficacy of drugs. Accordingly, in order to investigate a potential association between SNPs and clinical response and toxicity, we comprehensively assessed the allele frequencies of 1931 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 59 AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform (Affymetrix, Inc. Santa Clara, CA, USA). 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 i (eudract: 2007–005248-26; ClinicalTrials.gov NCT00909168). The induction regimen (GO-FLAI) included fludarabine (25 mg/sqm) and Ara-C (2 g/sqm) on days 1–5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (ID-AC and IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 and NPM mutation status, WT1 quantitative expression analyses, were performed at diagnosis in all patients. Furthermore, high-resolution SNP array analysis by GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix) was also performed. DNA processing and genotype identifications were performed according to the Affymetrix DMET platform's instructions. All statistical analyses were performed using the R package 2.11.1. Allele frequencies estimation and principal component analyses (PCAs) were performed using the adegenet library, whereas an ad hoc R function was developed to compute two-side Fisher's exact test p-values for each SNP in order to test their association with clinical response and toxicity.Results of potential interest were limited to those in which the p-value was 6). In conclusion, we performed an exploratory pharmacogenomic study that related SNPs in multidrug enzymes and transporters genes with the efficacy and toxicity of a combination of Gemtuzumab-ozogamicin (GO) with FLAI regimen used in a clinical trial of AML patients younger than 65 years. We identified for the first time a pharmacogenomic panel made up of 1 gene (ADH1A) associated with clinical outcome and 4 genes associated with toxicity. Findings obtained from this exploratory study are being tested in a larger clinical sample sets to confirm the predictive value of the SNP panel. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Martinelli: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 167 Introduction: Acute promyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15;17), resulting in the fusion of the promyelocytic leukemia (PML) gene and retinoic acid receptor α (RARA) gene (PML-RARA). Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for the development of APL. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and be used to better classify the disease by dissection of genomic subsets. Methods: At the time of writing, DNA from the bone marrow of 21 cases of t(15;17) APL at diagnosis were examined. Genomic DNA was isolated from mononuclear APL cells and applied to Genome-Wide Human SNP 6.0 array microarrays (Affymetrix, Santa Clara, CA) following the manufacturer's instructions. Copy number aberrations were scored using the Hidden Markov Model and the segmentation approach available within the Partek software package as well as by visual inspection. All aberrations were calculated with respect to a set of 270 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission in order to reduce the noise of raw copy number data. When available, in order to exclude inherited copy number variants a comparison to paired constitutional DNA and to paired remission DNA was performed. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: In all patients we identified one or more genomic abnormalities ranging from loss or gain of complete chromosome arms (trisomy 8, loss of 20q and loss of 6q) to submicroscopic genomic intervals. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes. Hemizygous deletions were identified at 1p13.3 affecting the glutathione S-transferase mu 1 (GSTM1), at 2q33.3–q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 3p11.2 (EPHA3), at 12p13 (ETV6), and at 17q12 (NF1). Deletions also affected genes involved in cell regulations as CDKN2A (9p21) and RB1 (13q14.2). Most frequent gains affected the TP73 gene at 1p36.3, the oncogene MYC at 8q24 (4.33 Mb), the oncogene ETS1 (11q23.3), the RAS p21 protein activator 3 (RASA3) at 13q34 and PRAME at the 22q11.22. Interestingly, in 2 cases we observed a gain of the region (5p15.33) containing the telomerase reverse transcriptase (TERT) which is important for maintenance of telomere ends. Other recurring genetic lesions were uncommon and were identified only in single cases. Some lesions affected regions lacking annotated genes and they are under investigation for miRNAs. For each alteration we interrogated a collated library of copy-number variants (CNVs, Database of Genomics Variants and USCS Genome Browser) to assure that these regions were not known as CNVs. Conclusion: These data demonstrate that different cooperating events may be involved in the generation of APL. These novel findings may be used to stratify patients according to genomic changes and to facilitate the screening for novel therapeutic targets. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo. Disclosures: No relevant conflicts of interest to declare.

CML is a clonal myeloproliferative disease which typically presents in chronic phase (CP), in which malignant progenitor cells proliferate rapidly but retain much of their ability to differentiate, with the disease later evolving to accelerated phase/blast crisis. Even after the introduction of imatinib, the calculation of the Sokal and the Euro prognostic scores has remained essential in clinical practice, since allow to stratify CML patients at different evolutive risk at diagnosis, guiding therapeutic decisions. More recently, numerous research efforts are ongoing to gain a better understanding about the intrinsic heterogeneity of CML, in order to identify a novel molecular signature which might characterize patients (pts) with different prognosis, and propensity to respond to treatment In the present study we adopted a GEP strategy in an attempt to identify genes and pathways, able to predict and/or elucidate the disease course of CP-CML pts at the time of diagnosis. To this aim, highly enriched CD34+ cells from peripheral blood obtained at diagnosis from pts with untreated Ph+ CML in CP were used throughout the study. Overall, 28 pts were included in the present analysis. They were diagnosed from August 2006 to June 2008; front-line treatment was either imatinib (12 pts) or nilotinib (16 pts). GEP was performed using the Affymetrix HG133 Plus microarray platform as per manufacturer’s recommendation. Raw data was normalized using the RMA algorithm and filtered. Unsupervised analysis was performed by Multidimensional Scaling and hierarchical clustering. Top differential genes were selected by significance analysis of microarrays method (SAM), setting the FDR threshold 0.01. Genes associated with Sokal risk score were searched by various methods (Limma, ANOVA, SAM, ROC analysis, EB-arrays). Analyses were performed using R and Bioconductor, BRB array tools and MeV. To analyse the data, we have first stratified our cohort of pts according to their Sokal risk, into 3 groups (high, intermediate and low risk) of 10, 13 and 5 pts, respectively. We did not identify any significant gene signature uniquely associated with Sokal risk score, even by various supervised techniques. Neither was it possible to obtain a signature from the most extreme phenotypes, i.e. by excluding intermediate-risk pts. Interestingly, however, unsupervised analysis of the whole gene expression data set clearly identified two subgroups of pts (we have named them A and B), which included 15 and 13 pts, respectively. Those two subgroups of pts show a differential expression of a list of at least 461 probe sets, which represents the most significantly up and down regulated probes, without any false positive. Both a hierarchical clustering of these probe sets, as well as a multidimensional scaling plot showed a clear demarcation between the two subgroups of pts, and the Sokal risk score did not co-vary along with their gene expression profile. In the group A of pts, of the 461 probe sets, 317 resulted up-regulated and 144 down-regulated. Genes up-regulated are mainly involved in regulation of transcription and/or gene expression (40/317 probe sets code for zinc finger protein), whereas down-modulated genes are involved in cell differentiation, cell death and cell cycle regulation. Of interest, several genes and functional classes showed the same deregulated expression as previously described during progression to blast crisis of CML (Radich et al. PNAS 2006). Moreover, 6 probe sets (EREG, FOS, IL8, WT1, HSPA1A and DKFZp761P0423) that are part of the Radich “top ten” list of genes most significantly associated with progression in CML, are differentially expressed in our two subgroups. Overall, our data suggests the existence of two biologically distinct subgroups of CML, irrespective of Sokal score, which could be identifiable at diagnosis. Those findings further support a hypothesis that different clinical behaviours of the disease and response to treatment could be associated with different gene expression profiles of individual pts. Our plan is to follow up the pts which we have analyzed in this study and to investigate if those molecular signatures, identified through GEP could be correlated with treatment responses.

Abstract 3262 Poster Board III-1 CML pts display a certain degree of clinical heterogeneity that is documented by the varying levels of response to tyrosine kinase inhibitor therapy and is best reflected by the Sokal risk score. Clinical differences must be a sign of some biological heterogeneity the basis of which, however, are still poorly understood. Today many high-throughput assays are available that allow to unravel the complexity of cancer cells in a genome-wide fashion. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution ( The pathways and genes most frequently affected by CNAs or UPD are listed in the Table below: Base Excision Repair (BER) MUTYH DNA glycosilase 1p34.1 loss/upd 10 pts PNKP Polynucleotide kinase 19q13.33 loss 10 pts NEIL1 DNA glycosilase 15q24.2 loss 7 pts POLB DNA polymerase beta 8p11.21 loss/upd 6 pts PCNA Sliding clamp for DNA polymerases 20p12.3 loss 6 pts Mismatch Repair (MMR) PMS2L5 Mut L homolog - mismatch and loop recognition 7q11.23 loss/upd 11 pts MSH2 Mut S homolog - mismatch and loop recognition 2p21 loss 8 pts POLD1 DNA polymerase delta 19q13.33 loss 8 pts POLA2 DNA polymerase alpha, subunit 2 2p16.3 loss/upd 6 pts POLE2 DNA polymerase epsilon, subunit 2 14q21–22 loss 5 pts Nucleotide Excision Repair (NER) ERCC1 5’ incision subunit of TFIIH complex 19q13.32 loss 11 pts ERCC2 5’ to 3’DNA helicase of TFIIH complex 19q13.32 loss 10 pts XAB2 Transcription-coupled NER factor 19p13.2 loss/upd 9 pts CDK7 Kinase subunit of TFIIH complex 5q13.2 loss 7 pts RPA4 Binds damaged DNA in preincision complexes Xp21.33 loss/upd 7 pts RPA2 Binds damaged DNA in preincision complexes 12q24.31 loss/upd 6 pts Homologous Recombination (HR) RAD51C Homologous pairing 17q23.2 loss/upd 7 pts RAD52 Accessory factor for recombination 12p13.33 loss 7 pts XRCC2 DNA break and crosslink repair 7q36.1 loss 5 pts Non-Homologous End Joining (NHEJ) PRKDC DNA-dependent protein kinase, catalytic subunit 8q11.21 loss/upd 5 pts DCLRE1C Artemis nuclease 10p13 loss 5 pts REV7 DNA polymerase zeta, subunit 1p36.22 gain 5 pts Other genes involved in DNA replication/repair/modification or chromatin remodeling CHAF1A Chromatin assembly factor 19p13.3 loss 15 pts RECQL5 DNA helicase 17q25.1 loss/upd 11 pts RAD9B PCNA-like DNA damage sensor 12q24.11 loss/upd 10 pts CHEK2 DNA-damage checkpoint 22q12.1 loss/upd 8 pts RAD17 RFC-like DNA damage sensor 5q13.2 loss 6 pts TREX1 DNAase III exonuclease 3p12.31 loss/upd 6 pts For some genes (e.g., RAD52), the monoallelic deletion we detected was found to translate into reduced mRNA expression, observation that was also independently confirmed in an additional group of high/intermediate versus low Sokal risk pts. In all the 44 pts, multiple pathways and multiple genes within the same pathway were affected, supporting the hypothesis that the lesions we detected might actually have consequences on DNA integrity despite the known partial functional redundancy of pathways and effectors. For many of the genes identified in this screen, activating or inactivating mutations are known to occur, and together with overexpression or haploinsufficiency, have been linked to a mutator phenotype in several malignant conditions. We are currently investigating whether this may be the case also in CML. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: Baccarani: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau.

none 12 no Therapeutic implications of intra-tumor heterogeneity are still undefined. In this study we report a genetic and functional analysis aimed at defining the mechanisms of chemoresistance in a 43-year old woman affected by stage IVB Burkitt lymphoma with bulky abdominal masses and peritoneal effusion. The patient, despite a transient initial response to chemotherapy with reduction of the bulky masses, rapidly progressed and died of her disease. Targeted TP53 sequencing found that the bulky mass was wild-type whereas peritoneal fluid cells harbored a R282W mutation. Functional studies on TP53 mutant cells demonstrated an impaired p53-mediated response, resistance to ex vivo doxorubicin administration, overexpression of DNA damage response (DDR) activation markers and high sensitivity to pharmacologic DDR inhibition. These findings suggest that intra-tumor heterogeneity for TP53 mutational status may occur in MYC-driven cancers, and that DDR inhibitors could be effective in targeting hidden TP53 mutant clones in tumors characterized by genomic instability and prone to intra-tumor heterogeneity. none Derenzini, Enrico; Iacobucci, Ilaria; Agostinelli, Claudio; Imbrogno, Enrica; Storlazzi, Clelia Tiziana; L`abbate, Alberto; Casadei, Beatrice; Ferrari, Anna; Ghelli Luserna Di Rorà, Andrea; Martinelli, Giovanni; Pileri, Stefano; Zinzani, Pier Luigi Derenzini, Enrico; Iacobucci, Ilaria; Agostinelli, Claudio; Imbrogno, Enrica; Storlazzi, Clelia Tiziana; L`abbate, Alberto; Casadei, Beatrice; Ferrari, Anna; Ghelli Luserna Di Rorà, Andrea; Martinelli, Giovanni; Pileri, Stefano; Zinzani, Pier Luigi

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

Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated. Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of TOT/886 AML pts treated at the Serˆgnoli Institute of Bologna between 2002 and 2013. Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). 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. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Numberª v7.5 (BioDiscovery). Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc). Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype. Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious.. Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm 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). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively). Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p Figure 1: Overall Survival curve of 172 AML patients with (red) or without (blue) TP53 mutations (p< 0.0001). Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher's exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universitˆ 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures No relevant conflicts of interest to declare.

Background: Imatinib (IM) is the drug of choice for the treatment of CML, where it was shown that a complete cytogenetic response (CCgR) and a major molecular response (MMolR) at 12 months (mos) were the best surrogate markers of progression-free survival (PFS) (O’Brien et al, NEJM, 2003; Hughes et al, NEJM 2003). The issue of the MolR is particularly sensitive, because the degree of the MolR may fluctuate over time and among different labs, also for methodological reasons (Hughes et al, Blood 2006). Aim: To evaluate the prognostic value of the MolR based on a retrospective analysis of the patients (pts) who achieved a CCgR with IM 400 mg daily. Patients and Methods: 130 pts who achieved a CCgR for more than 1 year (confirmed CCgR) and were tested for MolR at least 3 times after achieving the CCgR, were analyzed. Cytogenetic response was assessed every 6 mos by conventional analysis of at least 20 marrow cell metaphases. MolR was assessed every 3 mos by RTQ-PCR (TaqMan) on peripheral blood buffy coat cells and was defined as MMolR when BCR-ABL:ABL Results: In 71/130 pts (55%) MolR was always major (stable MMolR). In 19/130 (15%) pts MolR was sometimes major and sometimes less than major (unstable MMolR). In 40/130 pts (30%) MolR was never major (never MMolR). The proportion of pts remaining in CCgR after 5 years was calculated by the Kaplan-Meyer method (Fig. 1) and was 95% for pts in stable MMolR, 77% for pts with unstable MMolR and 62% for pts without a MMolR during follow-up (p Conclusions: These data confirm that achieving a MMolR is prognostically important but point out that the prognostic value of achieving a MMolR is greater if the response is confirmed, and introduce the concept of sustained Mol response, alerting from relying on single determinations. Figure Figure

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.

Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a ‘failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS. Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a ‘warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an ‘optimal response’ in one case; a ‘failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with ‘warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected. Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of ‘failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that: 1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that 2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy. A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis. Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Soverini: Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Metabolic remodeling of cancer is controlled by metabolic enzymes having oncogenic or tumor suppressor functions, along with oncogenes and tumor suppressors, which cooperate with the tissue environment to define specific metabolic profiles (Yuneva et al. Cell met 2012). Dysregulated metabolic pathways contribute to the pathogenesis of Acute Myeloid Leukemia (AML), as demonstrated for IDH1/2 mutations, which force the production of the oncometabolite 2-Hydroxyglutarate (Ward et al. Cancer Cell 2010) and can be selectively targeted (Wang et al. Science 2013). However, the genetic determinants of leukemia metabolic plasticity are largely unexplored. To identify metabolism-related pathogenic mechanisms in AML, we screened 886 AML cases for targeted genomic alterations and performed Whole Exome Sequencing of 143 leukemia samples (100 bp paired-end, HiSeq2000, Illumina), focusing our analysis on 37 AML cases (34 at diagnosis and 3 at relapse). Mutations were called by MuTect and GATK. Moreover, transcriptional analysis was performed on bone marrow cells from 59 AML cases (≥80% blasts) and 7 healthy controls (HTA2.0, Affymetrix). By mapping the mutated genes into functional categories, we identified a previously undescribed class of mutations targeting metabolism-related genes, that we define metabolic acute leukemia genes (MALGs). MALG was the most represented category after signaling pathways (76/915 genomic alterations) and 29/37 patients carried at least one MALG mutation. MALG mutations mostly targeted biosynthesis and catabolism of lipids and of CoA (ACP2, PANK2), bioenergetic pathways, metabolism of amino acids and nucleotides (NUDT18, IMPDH2). Notably, IMPDH2 is a target of MYC, a known regulator of cancer cell metabolism, and balances the nucleotide pool required for DNA replication (Liu et al, Plos one 2008). IMPDH2 was not only mutated but also upregulated at mRNA level in AML compared with controls (p=0.0001), suggesting an oncogenic function of the gene in AML, which is under investigation. Moreover, MYC transcriptional network was affected by additional mutations targeting genes regulating MYC activity (HUWE1, ZBTB17, TRRAP) and degradation (HEPACAM). Mutations in amino acid metabolism affected the synthesis/degradation of serine (PHGDH), glycine (SHMT2), proline (PRODH), tryptophan(CYP1B1) and glutamate (OPLAH), with a glutamate-related metabolic signature being also enriched in AML. These results may be highly relevant to AML therapy, since they may identify patients suitable to glutaminase inhibitor treatment, which is under development by pharmaceutical companies. An additional subset of patients displayed mutations in glucose-dependent bioenergetic pathways: glycolysis (GPI), oxidative phosphorylation (ND1, ND4, ND5, CYTB) and pentose phosphate pathway (H6PD, PGLS). These mutations were mutually exclusive with KRAS/NRAS alterations, which were detected in 8/37 samples. Indeed, oncogenic KRAS stimulates glucose uptake and channeling of glucose intermediates into pentose phosphate pathway (Ying et al. Cell 2012). Mutations in the bioenergetic pathways occurred across different cytogenetic groups and were associated with a poor outcome in terms of overall survival (p=0.016 Fig.1) in our AML cohort. Along with mutations in KRAS- and MYC-oncogenic pathways, which are known to control metabolic processes, we identified a novel functional category of mutated genes involved in metabolism (MALG) in AML. Our results may suggest different types of metabolic remodeling across leukemia subgroups. Mutations targeting a common downstream metabolic pathway are mutually exclusive in our cohort, as shown by KRAS and genes involved in glucose-dependent bioenergetic processes. Glucose metabolism predicts clinical outcome and chemotherapy response in AML (Chen et al. Blood 2014). Our data further suggest that the mutational screening of glucose-related MALGs may define a new subgroup of patients, which could not be identified by cytogenetic analysis. These findings may have implication for AML treatment, since metabolic alterations and genomic determinants of metabolic remodeling are promising targets for tailored therapies, as recently shown for glutaminase and IDH1/2 inhibitors. Acknowledgments: EHA Research Fellowship award, FP7 NGS-PTL project, ELN, AIL, AIRC, progetto Regione-Università 2010-12 Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Martinelli:Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; MSD: Consultancy; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy.

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. Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Martinelli:MSD: Consultancy; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy.

// Francesca Chiarini 1,2 , Annalisa Lonetti 3 , Gabriella Teti 3 , Ester Orsini 3 , Daniela Bressanin 3 , Alessandra Cappellini 4 , Francesca Ricci 5 , Pier Luigi Tazzari 5 , Andrea Ognibene 2 , Mirella Falconi 3 , Pasqualepaolo Pagliaro 5 , Ilaria Iacobucci 6 , Giovanni Martinelli 6 , Sergio Amadori 7 , James A. McCubrey 8 and Alberto M. Martelli 1,3 1 Institute of Molecular Genetics, National Research Council, Bologna, Italy 2 Muscoloskeletal Cell Biology Laboratory, IOR, Bologna, Italy 3 Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 4 Department of Human, Social and Health Sciences, University of Cassino, Cassino, Italy 5 Immunohematology and Transfusion Center, S. Orsola-Malpighi Hospital, Bologna, Italy 6 Department of Specialist, Diagnostic and Experimental Medicine, University of Bologna, Bologna, Italy 7 Department of Hematology, Tor Vergata University Hospital, Rome, Italy 8 Department of Microbiology & Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA. Correspondence: Alberto M. Martelli, email: // Keywords : AML, PI3K/Akt/mTOR signaling, apoptosis, autophagy, combination therapy, leukemia initiating cells Received : November 30, 2012, Accepted : December 17, 2012, Published : December 18, 2012 Abstract Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC 1 ) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G 0 /G 1 -phase cell cycle arrest, apoptosis, and autophagy. CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34 + /CD38 - /CD123 + ), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs.

In 2004, we reported the short-term results of a multicentric, phase 2 study of imatinib 400 mg daily and pegylated interferon-alpha in the treatment of 76 early chronic phase Philadelphia-positive chronic myeloid leukemia patients. In this report, we update the results with an observation time of five years. After two years of treatment, all but 10 patients (13%) had discontinued pegylated interferon-alpha. The complete cytogenetic response rate at five years was 87%, and 94% of complete cytogenetic responders maintained the complete cytogenetic response after five years. All but one complete cytogenetic response also achieved a major molecular response. These data confirm the excellent response to imatinib front-line and the stability of the complete cytogenetic response. Any possible additional benefit of the combination with interferon-alpha remains uncertain, due to low patient compliance.

Background: ALL is a disease characterised by lack of differentiation and uncontrolled proliferation of lymphocyte precursor cells, driven by activation of oncogenes and inactivation of tumour suppressor genes. About 30% of B-cell ALL carries the Philadelphia chromosome (Ph), originated by the translocation t(9;22)(q34;q11), and thus express the fusion gene BCR-ABL. The constitutively active tyrosine kinase encoded by BCR-ABL blocks the differentiation of B precursor cells, prevents apoptosis and also causes genetic instability. This ultimately leads to the acquisition of new mutations, resistance to BCR-ABL inhibitors and disease progression. In particular BCR-ABL stimulates DNA repair by non-homologous end-joining and homologous recombination repair mechanisms after various genotoxic stimuli, but a lot of deletions and abnormalities are also generated. Methods and patients: In order to identify whether some genetic lesions could contribute to genetic instability in Ph+ ALL, we analyzed the genome of 18 Ph+ ALL patients using high-resolution single nucleotide polymorphism (SNP) array. 250 ng of genomic DNA were processed on 250K NspI array according to protocols provided by the manufacturer (Affymetrix Inc., Santa Clara, CA, USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH) and real-time quantitative polymerase chain reaction (PCR) were performed to validate our results. Results: We found a significant deletion on chromosome 22, cytoband q11.22, close to immunoglobulin lambda light chain genes, in the region 20913571-20931814, corresponding to the VpreB1 (CD179a, Pre-B-Lymphocyte gene 1) gene in about 30% of newly diagnosed Ph+ ALL patients. It is highly conserved among mammals and selectively expressed during the pre-B stage of B cells ontogenesis and together with IGLL1 encodes for the surrogate light chain of the pre-B cell receptor. In mice its monoallelic deletion is associated with an increase of lymphocytes in pre-B stage in bone marrow, while its complete absence causes the impossibility to generate mature B cells. Since Ph+ ALL cells are immunophenotypically blocked at the pre-B stage, we are investigating whether VpreB1 deletion could interfere with normal B cell development and thus could have a role in leukeamogenesis and disease progression. Conclusion: This study demonstrated that the use of high-resolution SNP array is a powerful technology to detect genetic anomalies not revealed by classical cytogenetics which may provide new insights into leukemogenesis.

Background: The Bcr-Abl kinase expressed in ALL drives malignant transformation of human pre-B cells and promote aberrant alternative splicing of genes involved in B-cell signaling and differentiation. The fact that Ikaros (Ik) transcription factor functions as a critical regulator of normal lymphocyte development and the observation of rapid development of leukaemia in mice expressing non-DNA binding isoforms, prompted our study to investigate whether normal Ik expression and function might be altered in Ph+ ALL. Patients and methods: Bone marrow and peripheral blood samples from 47 adult patients (median age 55 yrs, 18-76) with Ph+ ALL were collected after informed consent. Reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for exon 1 and exon 7 of Ik, cloning, nucleotide sequencing and western blot analyses were performed to identify the specific Ik isoforms. Results: In 43/47 (90%) patients the Ik6 isoform, lacking all N-terminal zinc-fingers responsible for DNA-binding, was detected and in 23 of them (54%) it was the predominant isoform. Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patients expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In the patients who seemed to express by RT-PCR only full-length Ik isoforms cloning and subsequent sequencing revealed clones harboring different transcripts. We detected an in frame deletion of 10 aa (ΔKSSMPQKFLG) at the end of ex6 due to the selection of an alternative donor splice site. The consequences were impaired activation of transcription and impaired ability to form dimers. We also observed isoforms with a 60-bp insertion (TYGADDFRDFHAIIPKSFSR) immediately upstream of exon 4 at the exon 2/exon 4 junction either alone or together with the in-frame deletion. Also in this in case the aberration is due to the selection of alternative splice site which determines the skipping of exon 3 and the insertion of a sequence inside the intron 3-4 which starts with an alternative acceptor splice site. Aberrant Ik isoforms had a cytoplasmatic localization as revealed by immunofluorescence and confocal laser scanning microscopy analyses. Conclusions: Our data demonstrated that aberrant splicing of Ikaros represents a frequent feature in Ph+ ALL patients and that dominant negative Ik6 splice variant correlates with BCR-ABL mRNA levels, disease progression and relapse.

Purpose Imatinib mesylate (IM) has rapidly become the front-line treatment of Philadelphia-positive (Ph-pos) chronic myeloid leukemia, but the number of patients who were treated and are being treated with IM second-line is still substantial. Patients and Methods We have monitored and analyzed the cytogenetic and molecular response to IM 400 mg/d in a cohort of 277 late chronic phase (LCP) patients who were resistant or intolerant to interferon-α and were observed for 48 to 79 months (median, 72 months). Results One hundred fifty-three patients (55%) achieved a complete cytogenetic response (CCgR). Seventy-seven percent of them were still in CCgR after 5 years. The rate of response loss did not increase over time. The 6-year progression-free survival and overall survival of these 153 complete cytogenetic responders were 90% and 91%, respectively. Molecular response was less than major in 21%, major in 78%, and complete in one patient only. Conclusion These data confirm that, in LCP the CCgR rate to IM is 50% to 60%, and show that CCgR is stable and is associated with a prolonged survival, even if leukemia continues to be molecularly detectable.