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Additional chromosomal abnormalities (ACAs) in Philadelphia-positive cells have been reported in ∼ 5% of patients with newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP). Few studies addressing the prognostic significance of baseline ACAs in patients treated with imatinib have been published previously. The European LeukemiaNet recommendations suggest that the presence of ACAs at diagnosis is a “warning” for patients in early CP, but there is not much information about their outcome after therapy with tyrosine kinase inhibitors. To investigate the role of ACAs in early CP CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective trials of the Gruppo Italiano Malattie Ematologiche dell'Adulto Working Party on CML: 378 patients were evaluable and ACAs occurred in 21 patients (5.6%). The overall cytogenetic and molecular response rates were significantly lower and the time to response was significantly longer in patients with ACAs. The long-term outcome of patients with ACAs was inferior, but the differences were not significant. The prognostic significance of each specific cytogenetic abnormality was not assessable. Therefore, we confirm that ACAs constitute an adverse prognostic factor in CML patients treated with imatinib as frontline therapy. This study was registered with clinicaltrials.gov as NCT00514488 and NCT00510926.

Variant Philadelphia (Ph) chromosome translocations have been reported in 5%-10% of patients with newly diagnosed chronic myeloid leukemia (CML). Variant translocations may involve one or more chromosomes in addition to 9 and 22, and can be generated by 2 different mechanisms, 1-step and 2-step rearrangements, as revealed by fluorescence in situ hybridization. The prognostic significance of the occurrence of variant translocations has been discussed in previous studies. The European LeukemiaNet recommendations do not provide a “warning” for patients with variant translocations, but there is limited information about their outcome after therapy with tyrosine kinase inhibitors. To identify the role of variant translocations in early chronic phase (CP) CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective imatinib trials of the Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) Working Party on CML. Variant translocations occurred in 30 patients (5%). Our data show that the presence of variant translocations has no impact on the cytogenetic and molecular response or on outcome, regardless of the involvement of different mechanisms, the number of involved chromosomes, or the presence of deletions. Therefore, we suggest that patients with variant translocations do not constitute a “warning” category in the imatinib era. This study is registered at www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Background: Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting. Aim: To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. Design: From March 2015 to July 2016, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. Patients: 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. Results: 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Conclusions: Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Acknowledgments: Work supported by ALN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

// Simona Luatti 1 , Carmen Baldazzi 1 , Giulia Marzocchi 1 , Gaia Ameli 1 , Maria Teresa Bochicchio 1 , Simona Soverini 1 , Fausto Castagnetti 1 , Mario Tiribelli 2 , Gabriele Gugliotta 1 , Giovanni Martinelli 1 , Michele Baccarani 1 , Michele Cavo 1 , Gianantonio Rosti 1 , Nicoletta Testoni 1 1 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. and A. Seragnoli”, University of Bologna, “S Orsola-Malpighi” University Hospital, Bologna, Italy 2 Division of Hematology and Bone Marrow Transplantation, Azienda Ospedaliero-Universitaria di Udine, Udine, Italy Correspondence to: Simona Luatti, email: simonaluatti@gmail.com Keywords: CML, BCR/ABL, Philadelphia chromosome, FISH, TKI Received: September 23, 2016 Accepted: January 16, 2017 Published: February 16, 2017 ABSTRACT At diagnosis, about 5% of Chronic Myeloid Leukemia (CML) patients lacks Philadelphia chromosome (Ph), despite the presence of the BCR/ABL rearrangement. Two mechanisms have been proposed about the occurrence of this rearrangement: the first one is a cryptic insertion between chromosomes 9 and 22; the second one involves two sequential translocations: a classic t(9;22) followed by a reverse translocation, which reconstitutes the normal morphology of the partner chromosomes. Out of 398 newly diagnosed CML patients, we selected 12 Ph-negative cases. Six Ph-negative patients treated with tyrosine kinase inhibitors (TKIs) were characterized, in order to study the mechanisms leading to the rearrangement and the eventual correlation with prognosis in treatment with TKIs. FISH analysis revealed cryptic insertion in 5 patients and classic translocation in the last one. In more detail, we observed 4 different patterns of rearrangement, suggesting high genetic heterogeneity of these patients. In our cases, the BCR/ABL rearrangement mapped more frequently on 9q34 region than on 22q11 region, in contrast to previous reports. Four patients, with low Sokal risk, achieved Complete Cytogenetic Response and/or Major Molecular Response after TKIs therapy. Therapy resistance was observed in one patient with duplication of BCR/ABL rearrangement and in another one with high risk. Even if the number patient is inevitably low, we can confirm that the rare Ph-negative CML patients do not constitute a “warning” category, meanwhile the presence of further cytogenetic abnormalities remains an adverse prognostic factor even in TKI era.

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1. We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.

Chromosome number alterations, aneuploidy, is a hallmark of cancer. It occurs in about 15% of acute myeloid leukemia (AML) cases, is generally preserved throughout disease progression (Bochtler et al. Leukemia 2015) and correlates with adverse prognosis (Breems et al. JCO 2008, Papaemmanuil et al. NEJM 2016). This evidence highlights the need of understanding the molecular mechanisms that promote and sustain aneuploidy in AML, in order to define novel potential therapeutic targets. In the NGS-PTL project we profiled the genomic landscape of 536 hematological samples by whole exome sequencing (WES, Illumina). Among them, we analyzed 88 and 68 samples from aneuploid (A-) FLT3-wildtype AML (isolated trisomy and monosomy, complex and monosomal karyotype) and euploid (E-) AML (normal and complex karyotype, A-AML showed an increased genomic instability, as confirmed by a higher mutation load compared with E-AML (median number of variants: 22 (range: 2-95) and 11 (range: 3-45), respectively, pA substitutions, compared with the C>T transition-related signature, which is prevalent in AML. A-AML was associated with mutations and/or heterozygous deletion of TP53 (p We show here for the first time the molecular mechanisms promoting and maintaining aneuploidy in AML. Our results indicate that p53 deficiency, either caused by somatic mutations, copy number loss, impaired DNA damage response and enhanced PLK1 signaling synergize with APCgain, RAD50 structural or functional loss and forced progression through mitosis, to override cell cycle and mitotic checkpoints and allow the formation of daughter cells with an aberrant chromosome number. These mechanisms cooperate with recurrent mutations of genes involved in protein ubiquitination and proteasome-mediated protein catabolic process in A-AML, indicative of the attempt of aneuploid cells to override the proteotoxic stress due to the unbalanced protein load generated by the aneuploid condition. This evidence provides the rationale for exploiting proteasome inhibition (Velcade), p53 reactivation (MDM2/4 inhibitor) and targeting of the cell cycle (CHK1/2 inhibitor) downstream to p53 (WEE1 inhibitor) as strategies for novel combination therapies against aggressive aneuploid AML, which are under clinical investigation in our Institution and may serve as a model for aneuploid tumors. GS and AP: equal contribution. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Ariad: Consultancy, Speakers Bureau; MSD: Consultancy; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Novartis: Speakers Bureau; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

BRCA1 is one of the most important gene associated with familial breast and ovarian cancer susceptibility and is involved in the DNA damage repair and cell cycle arrest. Alterations in BRCA1 and their consequences are well characterized in breast and ovarian tumors, while little is known about its role in Acute Myeloid Leukemia (AML). We aimed to investigate the frequency and the interplay of BRCA1 alterations and patterns of somatic mutations (SNVs) and copy number variations (CNVs) of in AML patients. We genotyped 118 AML samples at either diagnosis or relapse by Single Nucleotide Polymorphism (SNP) array (SNP6.0 and Cytoscan HD, Affymetrix) to detect CNVs. In addition, we performed Whole-Exome Sequencing (WES, 100 bp paired-end, Illumina) of 56 genotyped AML patients to detect SNVs and small indels (MuTect and Varscan 2.0). Differences in survival were assessed using Kaplan-Meier survival analysis and Long-Rank test, or Breslow when indicated. We detected BRCA1 loss in 14 out of 118 patients (12%), ranging from 1,6 Kb to the loss of the entire chromosome 17, with none of the BRCA1 loss patients having a normal karyotype. Moreover, BRCA1 losses significantly co-occurred with PALB2 and RAD50 losses. Notably, the PALB2 loss (chromosome 16) mostly involved exon 12 and was detected also in patients without the BRCA1 loss, with a frequency of 11 out of 118 cases (9%), suggesting a pivotal and previously undescribed role in AML pathogenesis. BRCA1 loss patients were enriched for copy number alterations in genes involved in the regulation of mitotic recombination, DNA repair and positive regulation of kinase activity compared with BRCA1 wild-type cases (p Finally, BRCA1 and PALB2 alterations defined group of patients with poor overall survival (OS). In particular, patients with alterations of either BRCA1 or PALB2 had a worst prognosis compared to patients without alterations in the two genes (p=0.009 and p=0.001, respectively). Patients harboring PALB2 loss had a poorer prognosis compared with patients with complex karyotype (p=0.021, Breslow test). In addition, double BRCA1 and PALB2 loss, single BRAC1 or PALB2 hits and lack of alterations in BRCA1 and PALB2 defined three different classes of risks in our AML cohort (p In summary, we define for the first time a novel subgroup of AML patients characterized by alterations targeting the DDR pathway, and in particular the BRCA1 and PALB2 genes. Our data suggest that a signature of genes involved in the DDR and cell cycle regulation synergize and led to the uncontrolled proliferation, independently of DNA damage accumulation. Our results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Guadagnuolo: CellPly S.r.l.: Employment. Martinelli:Novartis: Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

// Elisa Zuffa 1 , Eugenia Franchini 1 , Cristina Papayannidis 1 , Carmen Baldazzi 1 , Giorgia Simonetti 1 , Nicoletta Testoni 1 , Maria Chiara Abbenante 1 , Stefania Paolini 1 , Chiara Sartor 1 , Sarah Parisi 1 , Giovanni Marconi 1 , Federica Cattina 2 , Maria Teresa Bochicchio 1 , Claudia Venturi 1 , Emanuela Ottaviani 1 , Michele Cavo 1 , Giovanni Martinelli 1 1 “Seragnoli” Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy 2 Bone Marrow Transplant Unit, University of Brescia, Brescia, Italy Correspondence to: Cristina Papayannidis, e-mail: cristina.papayannidis@unibo.it Keywords: AML, FLT3, ultra-deep sequencing, clonal evolution, minimal residual disease Received: April 21, 2015 Accepted: August 24, 2015 Published: September 05, 2015 ABSTRACT FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2–2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.

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

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

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

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

Aneuploidy causes a proliferative disadvantage, mitotic and proteotoxic stress in non-malignant cells ( Torres et al. Science 2007). Chromosome gain or loss, which is the hallmark of aneuploidy, is a relatively common event in Acute Myeloid Leukemia (AML). About 10% of adult AML display isolated trisomy 8, 11, 13, 21 (Farag et al. IJO 2002), or either an isolated autosomal monosomy or monosomal karyotype (Breems et al. JCO 2008). This evidence suggests that tumor-specific mechanisms cooperate to overcome the unfitness barrier and maintain aneuploidy. However, the molecular bases of aneuploid AML are incompletely understood. We analyzed a cohort of 166 cytogenetically-characterized AML patients (80 aneuploid (A-) and 86 euploid (E-)) treated at Seràgnoli Institute (Bologna). Aneuploidy was significantly associated with poor overall survival (median survival: 13 and 26 months in A-AML and E-AML respectively; p=.006, Fig.1). To identify AML-specific alterations having a causative and/or tolerogenic role towards aneuploidy, we integrated high-throughput genomic and transcriptomic analyses. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 70 samples from our A-AML and E-AML cohort of 166 patients. Variants where called with MuTect or GATK for single nucleotide variant and indels detection, respectively. AML samples were genotyped by CytoScan HD Array (Affymetrix). Gene expression profiling (GEP) was also conducted on bone marrow cells from 24 A-AML, 33 E-AML (≥80% blasts) and 7 healthy controls (HTA 2.0, Affymetrix). We detected a significantly higher mutation load in A-AML compared with E-AML (median number of variants: 31 and 15, p=.04) which was interestingly unrelated to patients' age (median age: 63.5 years in A-AML and 62 years in E-AML, Xie et al, Nat. Med. 2014). C>A and C>T substitutions, which are likely mediated by endogenous 5mCdeamination, were the most frequent alterations (Alexandrov et al. Nat. 2013). However, aneuploidy associated with an increased variability in terms of mutational signatures, with the majority of A-AML displaying 3 or more signatures compared to few E-AML cases (p=.04). WES analysis also revealed a specific pattern of somatic mutations in A-AML. A-AML had a lower number of mutations in signaling genes (p=.04), while being enriched for alterations in cell cycle genes (p=.01) compared with E-AML. The mutated genes were involved in different cell cycle phases, including DNA replication (MCM6, PURB, SSRP1), centrosome dynamics (CEP250, SAC3D1, HEPACAM2, CCP110), chromosome segregation (NUSAP1, ESPL1, TRIOBP), mitotic checkpoint (ANAPC7, FAM64A) and regulation (CDK9, MELK, ZBTB17, FOXN3, PPM1D, USP2). Moreover, genomic deletion of cell cycle-related genes was frequently detected in A-AML. Notably, ESPL1 which associated with aneuploidy, chromosome instability and DNA damage in mammary tumors (Mukherjee et al. Oncogene 2014) was mutated and also upregulated in A-AML compared with E-AML (p=.01), the latter showing expression levels comparable to controls. Among the top-ranked genes differentially expressed between A-AML and E-AML, we identified a specific signature characterized by increased CDC20 and UBE2C and reduced RAD50 and ATR in A-AML (p Our data show a link between aneuploidy and genomic instability in AML. Deregulation of the cell cycle machinery, DNA damage and repair checkpoints either through mutations, copy number and transcriptomic alterations is a hallmark of A-AML. The results define specific genomic and transcriptomic signatures that cooperate with leukemogenic pathways, as KRAS signaling, to the development of the aggressive phenotype of A-AML and suggest that a number of A-AML patients may benefit frompharmacological reactivation of TP53pathway (e.g. MDM2 inhibitor, clinical trial NP28679). Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 GS & AP: equal contribution Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Introduction. Autologous Bone Marrow Transplantation (Auto-BMT) is currently rarely used in the treatment of Acute Myeloid Leukemia (AML). However, it may represent a good therapeutic option in a specific subset of patients, mainly in consolidation of both low risk (LR) and MRD negative AML without an available HLA matched donor. Aims. To review our database of AML patients who received Auto-BMT from 2005 to 2014 and who were referred to Bologna Institution, in order to assess the efficacy of the procedure in terms of Overall Survival (OS) and Disease Free Survival (DFS). Patients and methods: From 2005 to 2014, 98 AML patients underwent Auto-BMT in several Italian Institutions. 89/98 patients are evaluable for survival and outcome data. The 89 patients considered (42 female, 47 male), had a median age of 49 years (range 15-70). Cytogenetics was performed in all patients by conventional karyotype (22 patients were also analyzed by Single Nucleotide Polymorphisms Array); molecular analysis (FLT3 TKD and ITD, and NPM1 mutational analysis) was available for 51/89 patients. Molecular monitoring by specific fusion transcripts (CBF-MYH11 and AML1-ETO) was performed in CBF positive leukemias (inv(16) and t(8;21)) at the time of diagnosis, after induction, consolidation courses, and every 3 months in the first 2 years of follow-up. Based on this data, and according to ELN guidelines, a risk stratification identified 41 patients with a LR AML (t(8:21), inv(16) or NPM1+/FLT3- with normal karyotype), 4 patients with a high risk (HR) AML (complex karyotype or FLT3 ITD mutated or inv(3) or t(6;9)) and 44 patients with a standard risk (SR) AML (normal karyotype, other alterations). Results. All the patients received an induction chemotherapy treatment, as follows: a "3+7-like" course in 48 cases, a Fludarabine-based regimen in 20 patients and a Gemtuzumab-ozogamicin (GO)-based regimen in 21. 83/89 (93.3%) patients received a median of 2 consolidation courses of chemotherapy (range 1-4) before proceeding to Auto-BMT, performed in 1st CR. 6/89 (6.7%) patients received Auto-BMT in first relapse. 41 patients relapsed after auto-BMT and were treated with a re-induction chemotherapy, or were enrolled in clinical trials. 24 patients reached a 2nd complete remission, and 12 patients underwent an allogeneic BMT in 2nd CR. With a median follow up of 6 years, the median Overall Survival (OS) of the entire population was 64.3 months (range 5.8-294.2 months); the 1 year OS and the 5 years OS were, 97.1%, and 67.9%, respectively. The median Disease Free Survival (DFS) of the 83 patients treated with Auto-BMT in 1st CR was 36 months (range 1.3-293 months). The 1-year DFS and the 5-years DFS were 85% and 56.7%, respectively. Transplant related mortality (TRM, death in 100 days after BMT) was 1.2% for auto-BMT and 6.5% for allogeneic BMT. First, to assess the role of the number of consolidation courses we compared patients who received none or 1 consolidation course with patients who received 2 or more cycles, who showed a better OS (p= 0.0061, Figure 1). There was no statistical difference in terms of OS between young and elderly patients (cut off=65 years). Second, we compared patients who achieved a negative minimal residual disease status before auto-BMT (n=37) with patients who did not (n=9). MRD negativity offered a significantly better outcome in terms of 5-years OS (83.4% and 50% respectively); the median OS of MRD neg was not yet reached; the median OS of MRD pos was 27 months (p= 0.0130) (Figure 2). Conclusions: Auto-BMT offers a chance to achieve long-term DFS and OS if used as a consolidation therapy both in patients with LR and SR AML. The major role could be played in MRD negative patients, offering the best chances to achieve a long-term OS. Auto-BMT can be also a good choice as consolidation therapy for elderly patients, in which allo-BMT could induce high morbidity and mortality rates. The small patients cohort and the retrospective analysis don't allow us to define the best induction therapy to be used before auto-BMT. However, based on our findings we suggest a therapy schedule including two or more consolidation courses in patients who obtain a first CR, and to proceed then to auto-BMT. Acknowledgments: work supported by ELN, AIL, AIRC, Progetto Regione-Università 2010-12 (L.Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Rodeghiero:Celgene Corporation: Honoraria, Research Funding. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:AMGEN: Consultancy; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; MSD: Consultancy.

Abstract 2027 Poster Board II-4 Background. The prognosis of Ph+ adult ALL patients treated with standard therapies, including multi-agent chemotherapy, Imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a BCR-ABL P210. Nineteen patients received Dasatinib, 2 patients Nilotinib and the remaining 4 patients were treated with third generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third and fourth relapse, respectively. A mutational analysis was performed in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range 2-29 months), median duration of HR, CyR and MolR were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs. Since TKIs are different and have different efficacy, pharmacokinetic, pharmacodynamic and toxicity profiles, and since are all effective, an alternative experimental option for the treatment of Ph+ ALL could be a combination or a rotation of two or more than two TKIs. Moreover, the addition of new promising targeted molecules, such as Aurora kinase or T315I inhibitors may contribute to overcome the problem of resistance to TKIs. Acknowledgments. Work supported by European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: No relevant conflicts of interest to declare.

Background: FLT3 internal tandem duplication (ITD) is frequently detected in AML patients and is an independent predictor of unfavourable outcome, while secondary point mutations in the FLT3 tyrosine kinase domain (KD) are common causes of acquired clinical resistance to FLT3 inhibitors, such as AC220 and Sorafenib. Technologies allowing massively parallel, ultra-deep sequencing (UDS) are currently being evaluated in diagnostic settings since they may conjugate throughputness, sensitivity and accurate quantification of mutated clones. Aim: Since recent whole genome sequencing studies have suggested that FLT3 ITD may evolve from small subclones undetectable at diagnosis by routine PCR, we tested the ability of an UDS strategy for FLT3 mutation screening to highlight small clones harbouring ITD mutations. Furthermore we evaluated if an UDS strategy could highlight in AML patients treated with FLT3 inhibitors emerging clones harbouring critical mutations, anticipating the development of drug-resistance. Methods: 886 AML patients were analyzed in Seràgnoli Institute of Bologna between 2002 and 2013 for a panel of genetic alterations, including FLT3. For our purpose, we retrospectively analyzed five AML (four CN-AML and one t(3;3) AML) who were found negative for FLT3 ITD- at diagnosis by conventional PCR and Sanger Sequencing, but were then found FLT3 ITD+ during follow-up at relapse or disease progression and ten AML (five FLT3+ and five FLT3-) treated with the FLT3 inhibitor AC220. In order to reconstruct the dynamic of mutation emergence, we performed a longitudinal re-analysis of RNA samples with UDS on a Roche GS Junior. UDS achieved a lower detection limit between 0,1% and 1%, depending on the relative number of sequence reads per sample obtained in each run. Results: 886 AML patients were analyzed for a panel of genetic alterations including FLT3 ITD and TKD and 239 of 886 (27%) were FLT3+. In particular 256 were cytogenetically normal (CN) AML and of these 66 (25,8%) were FLT3+ and 192 were FLT3-.UDS strategy revealed that all the CN-AML analyzed already carried at diagnosis a small clone FLT3 ITD+ (allelic ratio 0.2-2%), that increased over time during follow-up, while in the t(3;3) AML the ITD clone emerged only at disease progression. In the three CN patients treated with chemotherapy the ITD+ was a minor clone until complete remission (CR), while after relapse the ITD+ clone expanded; one of these patients carried two rare ITD clones at diagnosis and only one became dominant at relapse, likely through loss of heterozygosity (LOH) of the mutated allele. In one CN patient who was treated only with FLT3 inhibitor AC220 the allelic load of the mutated clone increased over time before treatment and then followed the dynamic of the disease (regression at complete remission). For the five AML FLT3- treated with AC220 we didn’t find any novel FLT3 mutation after treatment, while for the five AML FLT3-ITD+ analyzed, we were able to follow the allelic load of the FLT3-ITD+ clone during treatment and the appearance in two patients of novel TKD mutations after treatment that are able to confer drug resistance (allelic ratio 2-55%). Conclusions: The high sensitivity of UDS technology allows detection of emergence of mutated clones earlier than conventional methods: this is a precious tool to find small clones FLT3 ITD+ that may evolve over time and worsen the prognosis of otherwise good prognosis CN-AML patients and to better calibrate therapy for these patients. Furthermore the prognostic value of determining the presence of FLT3-ITD by UDS is stronger than conventional methods because of the possibility to determine the ratio of mutated versus wild-type allele, the length and the size of insertion within a single analysis. In the setting of FLT3 inhibitors, UDS gives advantage in monitoring MRD by determining exactly the allelic load of mutated FLT3 ITD clones before and after treatment and high sensitivity in highlighting emerging mutated clones during treatment that may confer resistance, giving possibility to switch eventually to other inhibitors before coming out of overt clinical resistance to therapy. For monitoring patients treated with FLT3 inhibitors with UDS we will go on by screening AML treated with Sorafenib, to follow the emergence of any novel critical mutation during treatment. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

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

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.

Introduction PI3P is a key regulator of cell growth, and mediates cell proliferation via PI3K/AKT/mTOR in response to various growth signals. Abnormal activation of genes in its pathway is associated to oncogenic activity and poor Overall Survival (OS). PI3P is also a core activator of autophagy. Which role autophagy plays in cancer is not well established; it can function as a pro-apoptotic mechanism, or it can improve survive to stresses clearing damaged mitochondria and proteins accumulation, preventing apoptosis. Levels and activity of pro-apoptotic and anti-apoptotic proteins, particularly bcl-2 and p53, membrane signaling via mTOR, high levels of cAMP, a complex made by pink/park, promote a switch from apoptotic autophagy toward a mechanism that augment cell resiliency. Our study aims to define the role of PI3P pathways in AML, and to establish if autophagy could reduce the patients' chance to respond to induction, and to worsen OS. Methods We analyzed 208 consecutive newly diagnosed non M3 AML patients, screened for TP53, FLT3, NMP1, IDH1, IDH2, and DNMT3A mutations. Remission status was assessed with bone marrow biopsy. In all the patients, we perform Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD; we perform Whole Exome Sequencing (WES)in 80/208 patients. Survival data were collected prospectively, with a median follow-up of 18 months. Survival analysis was performed with Kaplan Meyer method using log rank test. Univariate and multivariable regression and Cox Hazard Ratio(HR) model was performed. Correlation between variables was assessed with Fisher's exact test. Results We analyzed 4 pathways (Table 1); we selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). PI3K/AKT/mTOR pathway alterations (both gains or losses) were shown to confer worst OS (p = .035, Figure 1a) when compared with unaltered patients; events in these pathways did not affect therapy response. Autophagy pathway alterations were shown to confer worst OS (p AMPK pathway alterations were shown to confer worst OS (p Autophagy switch pathway confer worst OS to patients(p Having at least an altered pathway is associated with worst prognosis (p WES in a sub-cohort of patients did not found any significant mutation in genes we analyzed. This data is consistent with literature. Conclusions Our work investigates for the first time the role of PI3P pathways and autophagy in AML. Surprisingly, it showed that both positive and negative alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP and autophagy pathways were associated with therapy resistance. These results point out that both positive and negative regulation of autophagy could worsen patients OS; a diminished autophagy could be linked to a hyper-proliferative state via activation of AKT/mTOR but an augmented autophagy could give cell resiliency, favoring cytoplasm turnover, damaged mitochondria elimination, and neutralizing oxidative damages to proteins. A pan-PI3K inhibitor could target these mechanisms and improve chemo-sensitivity in high risk AML. Acknowledgment: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Guadagnuolo: CellPly S.r.l.: Employment. Soverini:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Martinelli:Novartis: Speakers Bureau; MSD: Consultancy; Ariad: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Introduction. Conventional induction treatment in young non APL-Acute Myeloid Leukemia (AML) patients is still represented by the association of an antracycline and Cytarabine, which offers a complete remission (CR) rate not inferior to 60%. The addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, already demonstrated to improve clinical outcome, in terms of CR rates. Aims of the study. We retrospectively evaluated and compared the efficacy of different induction schedules, in terms of CR rates and Overall Survival (OS), administered to two groups of AML patients. Group 1 (n=139) was treated with a GO containing course (MyFLAI or MyAIE schedules); Group 2 (n=270) received a non-GO based regimen including or not Fludarabine (FLAI, FLAN, FLAG, 3+7 or DAE). Patients and Methods. From 1997 to 2014,409 newly diagnosed AML patients were treated in 3 Italian Institutions. Their median age was 53 (range 19-74) and 52 (range 17-75) years, respectively. According to karyotype (performed in 392/409 patients), FLT3 (available for 244/409 patients), and NPM1 mutational status (available for 157/409 patients), based on the NCCN-2013 risk stratification criteria, 35.2% of the patients were considered at High Risk (HR) (31.6% and 36.4% in the two groups, respectively) and 7.6% at low risk (LR) (7.8% and 7.0%, respectively). Results. The complete remission (CR) rate after induction was 81.4% and 70.4% for Group 1 and 2, respectively (p=0.01). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 4/139 (2.9%) in Group 1 and 22/270 (8.1%) in Group 2 (p=0.003). Patients treated with GO showed a better OS than patients of Group 2 (Figure 1); the 5-years OS in the two groups was 54.01% and 34.9%, respectively, and different according to age (54.0% and 34.9% respectively (p=0.0003) in patients Then, with a logistic univariate regression analysis.,we explored the impact of GO therapy in terms of CR rate. The use of GO was identified as a strong predictor of the achievement of a 1st CR (p=0.008). Moreover, a logistic multivariate regression analysis (risk and age) confirmed the role of the compound as a predictor of higher CR rate (p=0.013). We also performed a Cox Regression analysis, to investigate the impact of GO therapy, age and risk on OS: the use of GO was confirmed to be an independent predictor of better OS (p In a sub-analysis performed on HR patients, we observed a significantly better outcome in Group 1 than in Group 2 in terms of OS (p=0.0074, 5-year OS 47.7%; in group 2 OS 21.0% respectively, Figure 3) and EFS (p=0.001). However, there was no difference in terms of CR rate (p=ns). In particular, HR patients with adverse karyotype, may benefit from GO induction therapy in terms of OS (5-years OS 42.9% and 12.8% in Group 1 and 2, respectively, p=0.02); nevertheless FLT3 mutations negative impact canÕt be overcome by the drug administration (5-years OS 66.6% and 61.3% in Group 1 and 2, respectively). In SR AML, GO offered a better OS (p=0.036, 5-years OS 51.4% and 41.9% respectively). Comparing with Fisher's exact test the rate of increment in 5-years OS between Group1 and Group2 in HR and SR AML, we demonstrate that treatment with GO gave the higher benefit in HR AML (p=0.0005). Conclusions. These data showed that a four-drugs intensified induction therapy is a feasible approach in AML patients: adding GO at any induction regimen is an independent and strong predictor of better OS and higher CR rates. In our population, GO was not associated with a higher incidence of DDI. This approach, could be strongly recommended in SR and HR AML patients, due to karyotype abnormalities, in which showed an advantage in term of OS if compared with other standard regimens. On the contrary, we donÕt suggest the same schedule in FLT3 mutated patients, in which no benefits have been observed. Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universitˆ 2010-12 (L.Bolondi), FP7 NGS-PTL project. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Fanin:Novartis Farma: Speakers Bureau. Cavo:BMS: Honoraria; Millenium Pharmaceuticals: Honoraria; Jansenn: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; MSD: Consultancy; AMGEN: Consultancy; Ariad: Consultancy.