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Simple Summary IDH1/2 mutations are a common event in acute myeloid leukemia (AML) and represent a therapeutic target. We designed the GIMEMA AML1516 observational protocol to examine the prevalence of IDH1/2 mutations and the associations between IDH mutations and clinico-biological parameters in a cohort of Italian patients affected by AML. By analyzing 284 consecutive adult AML patients, we confirmed that IDH1 and IDH2 mutations are frequently detected-14% and 18%, respectively-at diagnosis. IDH1/2 mutations were significantly associated with an inferior performance status and non-complex karyotype when compared to IDH1/2-WT. With regards to the outcome, in the subset of IDH1/2-mutated patients the rate of complete remission achievement was 60.5% and overall survival at 2 years was 44.5%: these percentages did not significantly differ from IDH1/2-WT patients. However, given the availability of IDH1/2 inhibitors, it is important to recognize IDH1/2-mutated cases up-front to offer patients the most appropriate therapeutic strategy. IDH1/2 mutations are common in acute myeloid leukemia (AML) and represent a therapeutic target. The GIMEMA AML1516 observational protocol was designed to study the prevalence of IDH1/2 mutations and associations with clinico-biological parameters in a cohort of Italian AML patients. We analyzed a cohort of 284 AML consecutive patients at diagnosis, 139 females and 145 males, of a median age of 65 years (range: 19-86). Of these, 38 (14%) harbored IDH1 and 51 (18%) IDH2 mutations. IDH1/2 mutations were significantly associated with WHO PS >2 (p < 0.001) and non-complex karyotype (p = 0.021) when compared to IDH1/2-WT. Furthermore, patients with IDH1 mutations were more frequently NPM1-mutated (p = 0.007) and had a higher platelet count (p = 0.036). At relapse, IDH1/2 mutations were detected in 6 (25%) patients. As per the outcome, 60.5% of IDH1/2-mutated patients achieved complete remission; overall survival and event-free survival at 2 years were 44.5% and 36.1%, respectively: these rates were similar to IDH1/2-WT. In IDH1/2-mutated patients, high WBC proved to be an independent prognostic factor for survival. In conclusion, the GIMEMA AML1516 confirms that IDH1/2 mutations are frequently detected at diagnosis and underlines the importance of recognizing IDH1/2-mutated cases up-front to offer the most appropriate therapeutic strategy, given the availability of IDH1/2 inhibitors.

Background Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. Methods The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. Results We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. Conclusions Our data suggest that WEE1 plays a role in ALL blast’s survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL. Electronic supplementary material The online version of this article (10.1186/s13045-018-0641-1) contains supplementary material, which is available to authorized users.

Background Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. Methods To understand the molecular bases of aneuploid acute myeloid leukemia (A‐AML), this study examined the genomic profile in 42 A‐AML cases and 35 euploid acute myeloid leukemia (E‐AML) cases. Results A‐AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E‐AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A‐AML, which was associated with a 3‐gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A‐AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E‐AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. Conclusions These findings indicate that aneuploidy‐related and leukemia‐specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets., Aneuploid acute myeloid leukemia (A‐AML) is associated with genomic and transcriptional alterations in the cell cycle and protein degradation pathways. The upregulation of PLK1 and CDC20 and the downregulation of RAD50 and of a p53‐related signature are hallmarks of A‐AML.

Myeloid sarcomas (MS) are tumors composed by myeloid elements and developing outside bone marrow. The prognosis is overall poor, only stem cell transplantation being consistently reposted as a potentially curative approach. In this study we explored whether microvessel density, a biomarker of angiogenesis, might be relevant in MS. We studied 60 MS, 24 acute myeloid leukemia, 5 normal bone marrow samples and 2 cases of extramedullary hemopoiesis in patients without evidence of hematological malignancy. We used immunohistochemistry (anti-CD34) to identify and quantify micro-vessel density (MVD) and micro-vessel grading (MVG). We found that MS had significantly higher MVD and MVG than normal bone marrow (p = 0.0002 and p < 0.001, respectively). We then found that cases with monocytic morphology had significantly higher MVD than myelo-monocytic and blastic ones (p = 0.005), while no differences were recorded based on extramedullary site. Finally, we found that higher MVD and higher MVG were associated with inferior outcome in terms of overall survival in multivariate analysis (p = 0.05 and p = 0.02, respectively), when censoring for stem cell transplantation was undertaken. In conclusion, we documented for the first time that increased angiogenesis is characteristic of MS and correlates with survival, suggesting that anti-angiogenic approaches might deserve a clinical evaluation in this setting.

Background Extramedullary involvement of B-cell Acute Lymphoblastic Leukemia (EM-ALL) is a rare occurrence, characterized by dismal outcome and the absence of a defined and shared therapeutic approach. In the landscape of innovative compounds, inotuzumab ozogamicin (IO) is a promising drug, whose mechanism of action relies on the killing of CD22 positive leukemic cells, through the delivery, after cell binding, of a molecule of calicheamicin. Case presentation We report two cases of CD22 positive relapsed EM-ALL treated with IO, obtained as compassionate use. Case 1, a 66 years old woman, affected by Philadelphia (Ph) negative B-ALL, relapsed with extramedullary involvement after 6 standard chemotherapy courses, who reached a complete metabolic response with IO treatment. Case 2, a 67 years old man with Ph positive B-ALL, initially treated with ponatinib, a third generation tyrosine-kinase inhibitor (TKI), obtaining a prolonged deep molecular remission. Nevertheless, for skin relapse during TKI treatment, the patient received local radiotherapy and, shortly after, standard chemotherapy, as multiple abdominal sites of relapse were detected too, with no response. The patient then received IO, obtained as compassionate use, with a good metabolic response. Conclusions These two cases suggest a possible key role of IO in the setting of advanced CD22 positive ALL, and underline its potential activity also in patients with EM involvement, relapsed after or refractory to conventional chemotherapy. Despite the well known hepatotoxic effect of the compound (Sinusoid Occlusive Syndrome), neither of them had such adverse event, moreover the second patient safely underwent allogeneic bone marrow transplantation.

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix??) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p???=???0.002), ELN high risk (HR) (p?????0.001), lower white blood cell (WBC) count (p???=???0.040), TP53 loss, and/or mutations (p???

Background The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. Case presentation We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. Conclusions This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an ‘act’ to ‘atc’ codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3511-2) contains supplementary material, which is available to authorized users.

Outcome for elderly patients with acute myeloid leukemia (AML) is extremely poor. Intensive induction chemotherapy is often unsuitable. Sixty-six newly diagnosed AML patients (median age: 76 years), ineligible for standard therapy, were consecutively treated with low-dose lenalidomide (10 mg/day orally, days 1–21) plus 10 mg/m2low-dose cytarabine, subcutaneously, twice a day (days 1–15) every six weeks, up to 6 cycles. Complete remission (CR) rate was 36.3% according to intention-to-treat. Responding patients had a longer median overall survival than non-responders (517 vs. 70 days, P < 0.001). The achievement of CR was not predicted by bone marrow blast count, cytogenetics, molecular markers, prior MDS, white blood cell count. Conversely, by studying the global gene expression profile, we identified a molecular signature, including 309 genes associated with clinical response (CR versus no CR). Based on the expression of a minimal set of 16 genes, we developed an algorithm to predict treatment response, that was successfully validated by showing an overall accuracy of 88%. We met the primary endpoint of the study, by beating the estimated successful CR rate (P1) fixed at 30%. Moreover, CR induced by this 2-drug combo was efficiently predicted by genetic profiling, identifying a biomarker that warrants validation in independent series.

11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predictive of response and can help define therapeutic strategies. Our aim is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis by a SNP array-based genotyping approach. Methods: We performed SNP 6.0/Cytoscan HD (Affymetrix) on 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number (BioDiscovery) and R Core Team. Results: We found several genes preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p < .001) for genes mapping in these pathways: aberrant PD-1 signaling, loss of function of SMAD4 in cancer and SMAD4 MH2 Domain mutants in cancer. The pathways significantly (p < .001) deregulated in our cohort with single copy gain and homozygous amplification were: regulation of transcription and nucleic acid, negative regulation of metabolic processes, constitutive signaling by aberrant PI3K in cancer and PI3K/AKT network. In order to define driver alterations, we correlate deletions and losses with mutational data. We found losses are also targeted by mutations ( BRCA2, LRRK1), while deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations ( CASK deletion and MPP6 mutation, CDK6 deletion and PPM1D mutation, MAPT deletion and SPAG5mutation). Conclusions: We have identified new CNAs and pathways involving novel potential leukemia-related genes. The comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes in deregulated pathways deserve further investigation in order to identify new genes which could be relevant AML biomarkers. Ackn: ELN, AIL, AIRC,prog. Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY.

11611 Background: Necroptosis is a type of necrotic cell death involving several genes transcription and activation of molecular mechanisms as death receptors, interferon, toll-like receptors, intracellular RNA and DNA sensors.The process is leading by the family of receptor-interacting protein kinase ( RIPK3, RIPK2, RIPK1) and the MLKL substrate. Losses of RIPK3 or MLKL, as well as deficiency in apoptosis, could allow tumor cells to escape the immunomediated cells death (ICD). Methods: We performed SNP Arrays (Cytoscan HD and SNP 6.0, Affymetrix) on a cohort of 300 non-M3 AML patients at diagnosis and we analyzed the Overall Survival (OS) of our patients with deficiency on necroptosis pathways. Survival was analyzed with Kaplan-Mayer method and Log-Rank test. We further analyze the relevance of different prognostic factors by the use of COX-Hazard Ratio statistical analysis. Results: We find that 18 patients presented a loss of RIPK1 or MLKL (nobody presented losses in RIPK3/RIPK2) and 13/18 patients were older than 65 years old. The Overall Survival (OS) of patients with alterations in these genes is significantly lower than control group, with a median OS of 3 vs 6 month respectively (p

// Andrea Ghelli Luserna Di Rora 1 , Ilaria Iacobucci 1, * , Enrica Imbrogno 1 , Cristina Papayannidis 1 , Enrico Derenzini 1 , Anna Ferrari 1 , Viviana Guadagnuolo 1 , Valentina Robustelli 1 , Sarah Parisi 1 , Chiara Sartor 1 , Maria Chiara Abbenante 1 , Stefania Paolini 1 , Giovanni Martinelli 1, * 1 Institute of Hematology “L. e A. Seragnoli”, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy * Co-corresponding authors Correspondence to: Ilaria Iacobucci, email: ilaria.iacobucci2@unibo.it Giovanni Martinelli, email: giovanni.martinelli2@unibo.it Keywords: CHK1, cell cycle, acute lymphoblastic leukemia, DNA damage response, chemo-sensitizer agent Received: February 12, 2016 Accepted: June 30, 2016 Published: July 11, 2016 ABSTRACT During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Introduction: Therapy for acute myeloid leukemia (AML) in elderly populations (>65 years) is still a challenge for scientists and hematologists worldwide, and represents an urgent medical need. Notably, the identification and the recognition of molecular and epigenetic mechanisms involved in the pathogenesis of such a heterogeneous disease, are providing new tools for a more successful and ‘targeted’ approach. Azacitidine is a hypomethylating agent (HMA) with relevant activity in patients affected by myelodysplastic syndrome (MDS) and AML with low blast cells percentage (>30%), in terms of reduction of transfusion dependence, and improvement of quality of life. Areas covered: This review summarizes the mechanism of action, safety profile and efficacy of azacitidine in the field of elderly AML populations, providing up-to-date references on this subset of high-risk patients. Expert opinion: HMAs are the first successful treatment for elderly patients with high-risk MDS and are effective for some AML subtypes. Translational studies based on gene expression profiling and molecular sequencing, would be able to identify, in the near future, patients with a favorable profile of response to these compounds suggesting new potential treatment combinations also.

Introduction Genomic instability and complex karyotype are linked to chemoresistance, poor prognosis and early relapse rate in Acute Myeloid Leukemia (AML). Chromothripsis, a one-step catastrophic mechanism of genomic instability, could represent a driving force in the development and progression of hematological diseases and could be identified by high throughput technologies as Single Nucleotide Polymorphism (SNP) Array. We investigate the mechanisms involved in chromothripsis in newly diagnosed non M3-AML patients (patients) in order to better characterize a class of very high risk patients that could be candidate to innovative therapies. Methods We performed classical cytogenetic and microarray analysis (SNP Arrays 6.0 or Cytoscan HD Arrays, Affymetrix) in 418 AML samples. Data were analyzed by Nexus Copy Number™ and R Core Team. Chromothripsis-like patterns were confirmed by CTLP Scanner (Log Ratio ≥ 8, ≥ 10 switches, minimum segment size of 10 kb, distance between adjacent fragments ≤ 10% and 0.3 as variation from different copy number (CN) states). Overall survival was analyzed by Kaplan-Meier method and Mantel-Cox test. Results Twenty-six/418 patients (6.2%) showed chromothripsis involving different chromosomes. Chromosome 12 (23%), 17 (19%) and 5 (19%) were the most affected, followed by chromosomes 3, 6, 7, 8, 10, 11, 13, 15 and 20. Patients harboring chromothripsis had a higher median age compared with chromothripsis-negative ones (70.4 vs. 55 years, respectively, p The group of patients with chromothripsis presented a prevalence of complex karyotype (p Chromothripsis-positive patients showed an extremely poor prognosis compared with chromothripsis-negative ones (median survival of 120 vs. 526 days, respectively, p Of note, chromothripsis was associated with worst prognosis even if compared with HR karyotype features without chromothripsis (median survival of 120 and 265 days, respectively, p Finally, by comparing patients with and without chromothripsis, we identified several genes and pathways differentially altered between the 2 groups (p Conclusion Our results demonstrate that chromothripsis is a recurrent event in adult AML and independently defines a group of patients with poor prognosis. Chromothripsis is strongly associated with TP53 alterations in AML and for the first time, we define incidence and survivals of chromothripsis in a set of 418 newly diagnosed consecutive AML patients. Chromosomal abnormalities like chromothripsis could be a driving force in the development of cancer as well in hematological disease like AML, in which the genomic instability represent the main scenario. Since TP53 is known to deregulate Fanconi Anaemia genes, the deregulation of DNA double-strand break repair could sustain the maintenance of catastrophic mechanism like chromothripsis. Acknowledgment ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. MCF and GM equal contribution Disclosures Kralovics: Qiagen: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Research Funding. Martinelli:Novartis: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; MSD: Consultancy; Genentech: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Roche: Consultancy, Speakers Bureau.

7043Background: Young patients affected by non APL-Acute Myeloid Leukemia (AML) achieve complete remission (CR) using conventional induction chemotherapy in about 55-70%. The addition of Gemtuzumab...

// 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.

e23207 Background: Nowadays, Acute Promyelocytic Leukemia (APL) is a disease entity with a very high rate of cure and an estimated 2-year overall survival of 97%. Early death, rather than resistant disease so common in all other subtypes of AML, has emerged as the major cause of treatment failure, and relapse is a very rare occurrence. Methods : We collected data of all the APL referred to our institution from 2014. Within 23 patients, we encountered 20 new diagnosis and 2 relapse of APL. We analyzed blasts in samples obtained from Bone Marrow with Single Nucleotide Polymorphisms Array Cytoscan HD. Results: We compared copy number alterations in both relapsed patients with alterations detected in the pool of 20 newly diagnosed APL and we found specific signatures of CNVs for each patient. There were several copy number alterations related to each patient: the first patient presented gain of ROBO2, GRIP1, CTNNB1, SOX6, PBX1, GRIK2, CDKAL1 and loss FAF1, CREBBP, SBF1; the second patient presented gain of ROBO1, MAPK10, CADPS2, APBA1 and loss of GRIP1 and MYB. Subsequently we focused our attention on ROBO and GRIP1genes because they were alterated in both relapsed patients: ROBO proteins are associated to K channels while GRIP1 is involved in various critical functions, for example in androgen receptor binding, beta-catenin binding, glucocorticoid receptor binding, and it is also a regulator of glutamate metabolism, a well-known pathway in Leukemic Stem Cells. Conclusions: APL relapse is a very rare entity, and it is announced to become rarer with the advances in first line therapy. Molecular characteristics are hard to analyze without an effort to collect and bank samples together from multiple institutions. Since relapses, especially relapses out of follow-up period, represent a sudden life-treating condition for patients, to predict patients at higher risk of relapse we selected two candidate genes that could be involved in pathways favoring relapse. By the analysis of ROBO 1-2 and GRIP1 at the diagnosis of APL we could establish a different and strict follow-up program for patients with these alterations. Acknowledgement: ELN,AIL,AIRC,prog. Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project,HARMONY.

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.

e18521Background: AML is a disease with a high number of various mutations and genomic abnormalities; most of them have a proved prognostic value. We focused on FLT3 gene, which shows a high hetero...

e18235Background: Patients affected by oncohaematology pathology follow multiple therapies that can contribute to the drug interactions resulting in change in pharmacokinetics or pharmacodynamics o...

7044Background: Genomic alterations are proved to initiate cancer and related to prognosis in AML. We tested 101 consecutive AML patients (pts) at diagnosis to find prognostic implications of new g...

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

Abstract 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.