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Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3-4, 45%) for an incidence rate of 3.9% per patient-year. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate-2 IPSS category (HR 1.8, 95%CI:1.2-2.7; p=0.02) and spleen length ≥10 cm below left costal margin (HR 1.6, 95%CI:1.1-2.5; p=0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX-treated patients (44% vs. 20%, p

An accurate histological diagnosis may distinguish essential thrombocythaemia (ET) from early primary myelofibrosis (early-PMF), which is associated with worse outcome. Outcome of ET is also negatively affected by the presence of the JAK2V617F mutation. To investigate the impact of JAK2V617F mutation burden and histology on outcome, we collected 475 WHO-diagnosed ET (69.2%) or early-PMF JAK2V617F-positive patients followed in 4 Italian haematology centers. JAK2V617F allele burden was ≤50% in 90% and 87% of ET and early-PMF patients, respectively (P = .34). During follow-up, 32 (9.7%) ET and 18 (12.3%) early-PMF patients experienced 59 thrombotic events, and 27 patients (5.6%) and 6 (1.2%) patients evolved to myelofibrosis and acute leukemia, respectively. At last contact, 28 (5.8%) patients had died. In early-PMF compared to ET, the 10-year mortality rates (6.7% and 4.3%, P = .73), leukemic transformation rates (1.4% and 1.2%, P = .45), and thrombosis rates (16.7% and 12.2%, P = .12) were comparable. Only progression to overt myelofibrosis at 10 years was significantly worse (11.4% and 1.5%, P = .004). In multivariate analysis, a higher (>50%) JAK2V617F burden was significantly correlated with fibrotic progression and histology. Considering JAK2V617F-positive disease, a higher (>50%) JAK2V617F burden and histological classification are independent prognostic risk factors for disease progression. These findings reinforce the need for standardized detection of this mutation.

none 18 no We investigated the influence of molecular status on disease characteristics and clinical outcome in young patients (⩽ 40 years) with World Health Organization (WHO)-defined essential thrombocythemia (ET) or early/prefibrotic primary myelofibrosis (early-PMF). Overall, 217 patients with ET (number 197) and early-PMF (number 20) were included in the analysis. Median follow-up time was 10.2 years. The cumulative incidence of thrombosis, hemorrhages and disease evolution into myelofibrosis/acute leukemia were 16.6%, 8.6% and 3% at 15 years, respectively. No differences were detectable between ET and early-PMF patients, although the latter cohort showed a trend for worse combined-event free survival (EFS). Mutation frequency were 61% for JAK2V617F, 25% for CALR and 1% for MPLW515K, and were comparable across WHO diagnosis; however, JAK2V617F allele burden was higher in the early-PMF group. Compared with JAK2V617F-positive patients, CALR-mutated patients displayed higher platelet count and lower hemoglobin level. CALR mutations significantly correlated with lower thrombotic risk (9.1% versus 21.7%, P = 0.04), longer survival (100% versus 96%, P = 0.05) and better combined-EFS (86% versus 71%, P = 0.02). However, non-type 1/type 2 CALR mutations ('minor' mutations) and abnormal karyotype were found to correlate with increased risk of disease evolution. At last contact, six patients had died; in five cases, the causes of death were related to the hematological disease and occurred at a median age of 64 years (range: 53-68 years). Twenty-eight patients (13%) were unmutated for JAK2, CALR and MPL: no event was registered in these 'triple-negative' patients. Palandri, F; Latagliata, R.; Polverelli, N.; Tieghi, A.; Crugnola, M.; Martino, B.; Perricone, M.; Breccia, M.; Ottaviani, E.; Testoni, N.; Merli, F.; Aversa, F.; Alimena, G.; Cavo, M.; Martinelli, G.; Catani, L.; Baccarani, M.; Vianelli, N. Palandri, F; Latagliata, R.; Polverelli, N.; Tieghi, A.; Crugnola, M.; Martino, B.; Perricone, M.; Breccia, M.; Ottaviani, E.; Testoni, N.; Merli, F.; Aversa, F.; Alimena, G.; Cavo, M.; Martinelli, G.; Catani, L.; Baccarani, M.; Vianelli, N.

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1.

Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3-4, 45%) for an incidence rate of 3.9% per patient-year. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate-2 IPSS category (HR 1.8, 95%CI:1.2-2.7; P = 0.02) and spleen length ≥10 cm below left costal margin (HR 1.6, 95%CI:1.1-2.5; P = 0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX-treated patients (44% vs. 20%, P

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1. Overall, we have demonstrated that in MF different mutations lead to phenotypic and functional alterations in different immune subsets that may have a potential role in disease progression and susceptibility to infections.

Since low JAK2V617F allele burden (AB) has been detected also in healthy subjects, its clinical interpretation may be challenging in patients with chronic myeloproliferative neoplasms (MPNs). We tested 1087 subjects for JAK2V617F mutation on suspicion of hematological malignancy. Only 497 (45.7%) patients were positive. Here we present clinical and laboratory parameters of a cohort of 35/497 patients with an AB ≤ 3%. Overall, 22/35 (62.9%) received a WHO-defined diagnosis of MPN and in 14/35 cases (40%) diagnosis was supported by bone marrow (BM) histology (‘’Histology-based’’ diagnosis). In patients that were unable or refused to perform BM evaluation, diagnosis relied on prospective clinical observation (12 cases, 34.3%) and molecular monitoring (6 cases, 17.1%) (‘’Clinical-based’’ or ‘’Molecular-based’’ diagnosis, respectively). In 11/35 (31.4%) patients, a low JAK2V617F AB was not conclusive of MPN. The probability to have a final hematological diagnosis (ET/PV/MF) was higher in patients with thrombocytosis than in patients with polyglobulia (73.7% vs 57.1%, respectively). The detection of AB ≥ 0.8% always corresponded to an overt MPN phenotype. The repetition of JAK2V617F evaluation over time timely detected the spontaneous expansion (11 cases) or reduction (4 cases) of JAK2V617F-positive clones and significantly oriented the diagnostic process. Our study confirms that histology is relevant to discriminate small foci of clonal hematopoiesis with uncertain clinical significance from a full blown disease. Remarkably, our data suggest that a cut-off of AB ≥ 0.8% is very indicative for the presence of a MPN. Monitoring of the AB over time emerged as a convenient and non-invasive method to assess clonal hematopoiesis expansion.

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.

An accurate histological diagnosis may distinguish essential thrombocythaemia (ET) from early primary myelofibrosis (early-PMF), which is associated with worse outcome. Outcome of ET is also negatively affected by the presence of the JAK2

// Margherita Perricone 1 , Francesca Palandri 1 , Emanuela Ottaviani 1 , Mario Angelini 2 , Laura Bagli 3 , Enrica Bellesia 4 , Meris Donati 5 , Donato Gemmati 6 , Patrizia Zucchini 7 , Stefania Mancini 8 , Valentina Marchica 9 , Serena Trubini 10 , Giovanna De Matteis 11 , Silvia Di Zacomo 12 , Mose Favarato 13 , Annamaria Fioroni 14 , Caterina Bolzonella 15 , Giorgia Maccari 16 , Filippo Navaglia 17 , Daniela Gatti 18 , Luisa Toffolatti 19 , Linda Orlandi 20 , Veronique Laloux 21 , Marco Manfrini 1 , Piero Galieni 2 , Barbara Giannini 3 , Alessia Tieghi 4 , Sara Barulli 5 , Maria Luisa Serino 6 , Monica Maccaferri 7 , Anna Rita Scortechini 8 , Nicola Giuliani 9 , Daniele Vallisa 10 , Massimiliano Bonifacio 11 , Patrizia Accorsi 12 , Cristina Salbe 13 , Vinicio Fazio 14 , Milena Gusella 15 , Eleonora Toffoletti 16 , Marzia Salvucci 3 , Mirija Svaldi 18 , Filippo Gherlinzoni 19 , Francesca Cassavia 20 , Francesco Orsini 20 , Giovanni Martinelli 1 1 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology ‘L. and A. Seragnoli’, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy 2 Molecular Hematology Laboratory U.O.C of Hematology Hospital Mazzoni, Ascoli Piceno, Italy 3 Medical Genetics Unit- Hub Laboratory AUSL Romagna, Pievesestina di Cesena, Italy 4 Imaging and Laboratory Diagnostic Department, Clinical Chemistry and Endocrinology Laboratory, Hematology Unit, Oncology and Technology Department, Hospital S. Maria Nuova, IRCCS, Reggio Emilia, Italy 5 Clinical Pathology Laboratory, A.O. Ospedali Riuniti Marche Nord, Pesaro, Italy 6 Center Hemostasis and Thrombosis, Section of Medical Biochemistry, Molecular Biology and Genetics, Department of Biomedical and Specialty Surgical Sciences, University of Ferrara, Ferrara, Italy 7 Department of Medical and Surgical Sciences, Division of Hematology, University of Modena and Reggio Emilia, Modena, Italy 8 Clinical Hematology Laboratory, Department of Molecular and Clinical Sciences, Polytechnic University of Marche, Ancona, Italy 9 Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy 10 Clinical Pathology, Molecular Biology Laboratory, and Hematology/Bone Marrow Transplantation Unit, AUSL Piacenza, Piacenza, Italy 11 Section of Clinical Biochemistry and Section of Hematology, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy 12 Department of Hematology, Blood Bank and Biotechnology, Ospedale Civile Pescara, Pescara, Italy 13 UOS Molecular Diagnostics, Department of Clinical Pathology, ULSS12 Venetian, Venice, Italy 14 UOC laboratory medicine, P.O. San Salvatore, Sulmona, L’Aquila, Italy 15 Department of Oncology, Laboratory of Pharmacology and Molecular Biology, ULSS 18, Rovigo, Italy 16 Clinical Hematology, Department of Experimental and Clinical Medical Sciences, University of Udine, Udine, Italy 17 Department of Laboratory Medicine, University-Hospital of Padova, Padova, Italy 18 Department of Haematology and BMT, Healthcare Company of South Tyrol, District of Bolzano, Bolzano, Italy 19 Department of Pathology and Haematology, Treviso General Hospital, Treviso, Italy 20 Werfen, Milano, Italy 21 QIAGEN GmbH, Hilden, Germany, member of the European LeukemiaNet (ELN) Foundation Circle Correspondence to: Margherita Perricone, email: margherita.perricon2@unibo.it Keywords: JAK2 V617F mutation, myeloproliferative neoplasms, qPCR standardization, molecular diagnosis Received: November 01, 2016 Accepted: February 22, 2017 Published: March 06, 2017 ABSTRACT To date, a plenty of techniques for the detection of JAK2 V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory. A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2 V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2 V617F allele burden (AB) using both quantitative and qualitative assays. The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2 V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples. In conclusion, a qualitative approach is not sensitive enough to detect the JAK2 V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2 V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2 V617F .

Genomic and functional alterations of enzymatic activity drive cancer metabolic reprogramming along with mutations of tumor suppressors and oncogenes. IDH1/2 lesions represent a paradigmatic example in acute myeloid leukemia (AML). The study aimed to stratify AML patients based on their metabolic landscape and to map potential connections between their metabolic and genomic profile. The genomic landscape of 166 AML patients was obtained by whole exome sequencing. Variants were called by MuTect and Varscan2. Six additional cases were analyzed by targeted sequencing (SOPHiA GENETICS). Metabolites were quantified by mass spectrometry of bone marrow cells (35 CD34+ and 15 CD33+ AML from the above-mentioned cohort) and compared with CD34+ cord blood and CD33+ healthy blood cells (n=21 each, Metabolon) by Welch's t-test. In AML, 17% of somatic variants targeted metabolism-related genes: 38% were enzymes (according to Recon2), including 3% of electron transport chain genes encoded by mitochondrial DNA (COX1-3, ND1-6), 3% were AML-related genes with a known involvement in cell metabolism (e.g. IDH1/2, MYC, KRAS) and 59% were metabolic regulators, defined by gene ontology annotation. Ninety-one% of patients carried at least one mutation in a metabolism-related gene, with 43% of variants rated as damaging. The most represented pathways were lipid, carbohydrate, nucleotide (AK9, H6PD), amino acids (IDO2) and glucose metabolism (PKM, HK3). Principal component analysis of metabolic data showed a distinct profile between AML and healthy cells, with a predictive accuracy of 86% and 95% for CD34+ and CD33+ cells, respectively. Conversely, few differences were observed between CD34+ and CD33+ AML. Unsupervised hierarchical clustering clearly defined 3 AML clusters (C1-3). Moreover, 3 subgroups could be identified in C3 without ambiguous assignments. C1 was enriched for NPM1-mutated (mut) cases (83%, 33%, 27% in C1, C2 and C3, respectively, p=0.03). NPM1-mutated AML were distinguished by a 12-metabolites signature. They showed increased levels of spermidine, cytidine 2′ or 3′-monophosphate (P), thymidine 3′-monoP, uridine-2',3'-cyclic monoP and decrease of inosine 5'-monoP, suggesting altered polyamine, pyrimidine and purine metabolism. Moreover, NPM1-mut AML had reduced levels of intermediates involved in acyl carnitine, lysophospholipid, phosphatidylethanolamine and sphingolipid metabolism. Overall, mutations of metabolism-related genes are common in AML. We defined a metabolic-based classification of AML and identified a new metabolic signature based on 12 metabolites that distinguish NPM1-mut AML from wild-type cases and healthy CD34+/CD33+ cells. Major alterations in the nucleotide and polyamine pathways suggest novel potential therapeutic approaches. Supported by: EHA research fellowship award, AIRC, FP7-NGS-PTL, Fondazione del Monte. Citation Format: Giorgia Simonetti, Antonella Padella, Eugenio Fonzi, Martina Pazzaglia, Margherita Perricone, Maria Chiara Fontana, Samantha Bruno, Maria Teresa Bochicchio, Eugenia Franchini, Jacopo Nanni, Giovanni Marconi, Italo F. do Valle, Rossella De Tommaso, Anna Ferrari, Enrica Imbrogno, Claudio Cerchione, Cristina Papayannidis, Emanuela Ottaviani, Daniel Remondini, Giovanni Martinelli. Metabolic profiling defines a new characterization of acute myeloid leukemia and identifies NPM1-mutated cases as a distinct subgroup [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5279.

Background. Ruxolitinib (RUX) is the first commercially available JAK1/2 inhibitor that may control splenomegaly and systemic symptoms related to myelofibrosis (MF). Despite MF occur frequently in elderly patients (pts), no data are yet available on RUX efficacy and safety in this particularly frail population. Methods We report on 100 pts [M/F 57/43, median age at diagnosis 75.7 years, interquartile range (IQR) 72.3 - 78.0, median age at baseline of RUX treatment 77.7 years, IQR 76.2 - 80.3] with WHO-defined MF treated with RUX when aged ≥ 75 years. Data were extracted from the whole cohort of 408 pts of any age collected in a database involving 22 Italian Centers. Comorbidities were recorded at the time of diagnosis and classified according to the Charlson Comorbidity Index (CCI). Response to RUX was evaluated according to IWG-MRT criteria. Results Main clinical features after stratification according to age at RUX start are reported in Table 1. Compared to younger pts, elderly pts carried a higher number of co-morbidities and had lower hemoglobin and platelet values, thus starting RUX with lower doses. Time from diagnosis to RUX start was comparable among the two cohorts (median 15.5 months, IQR 4.6 - 66.7, vs 20.8 months, IQR 4.1 - 66.0, p=0.74). According to IWG criteria, a spleen response was achieved by 37 out of 90 (41.1%) evaluable elderly pts compared to 115 out of 272 (42.2%) pts Drug-related anemia (Hb Evolution into acute leukemia occurred in 8 (8.0%) elderly pts and in 22 (7.1%) younger pts, respectively (p=0.78), with a similar evolution-free survival from RUX initiation (p=0.35). As expected, 43 (43.0%) elderly pts and 53 (17.3%) younger pts died (p The 4-year cumulative Event-Free Survival (taking into account: RUX discontinuation, blastic evolution and death for any cause) was 30.1% (95% CI: 16.2 - 44.0) in elderly pts and 46.1% (95% CI 37.3 - 54.9) in younger subjects, respectively (p=0.002). Conclusions. Despite the elderly carried a higher number of comorbidities and were treated with lower starting and titrated doses of RUX,RUX was feasible and effective in this setting, achieving clinical responses similar to younger subjects, with comparable toxicity rates. Thus, the study do not support to restrain the use of RUX based on older age and comorbidities. Figure Figure. Disclosures Latagliata: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria; Shire: Honoraria. Bonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding. Tiribelli:Novartis: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. Cavo:Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Celgene: Honoraria, Research Funding. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria.

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease.

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

Background: Mutations in TP53 gene predict a poor prognosis in patients with AML. The reported TP53 mutation rate in AML is low (2.1%) by contrast AML with a complex karyotype (CK) is higher (69-78%) and have a poor outcome. Quite common is to found paired TP53 mutation together and a segmental 17p deletion. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the correlation with known molecular alterations and the prognostic role TP53 mutations in adult AML pts. Moreover we would identify genes/pathways that are mainly affected in the mutated TP53 group compared to the wt one. Patients and Methods: 258 adult AML pts with miscellaneous cytogenetic abnormalities and normal karyotype were examined in our Institution for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq2000 platform and were correlated with cytogenetic analysis. 124/258 samples were genotyped with SNP arrays (Affymetrix, 3 250K, 43 SNP 6.0, 78 CytoScan HD). Copy Number Alterations (CNAs) analyses were performed using Chromosome Analysis Suite (Affymetrix) and Nexus Copy Number (BioDiscovery) software. Results: Mutation analysis detected TP53 mutations on 39 patients with 48 different types of mutations (32 deleterious point mutations; 4 deletions); nine pts have 2 mutations. We found 34/48 (70%) missense mutations, 5 mutations in the splice sites, 4 deletions ,2 intronic and and 3 others mutations. The mutation rate is of 15.1%. Mostly (28/39) of the TP53 mutated pts (71.8%) had CK while only 11/39 (28.2%) mutated pts have “no CK” (P>0.0001). Alterations of TP53 were significantly associated with poor outcome (OS and EFS p Conclusions: Our data demonstrated that TP53 mutations occur in 15.1% of AML with a higher frequency in the subgroup of CK-AML (p Citation Format: Anna Ferrari, Eugenio Fonzi, Maria Chiara Fontana, Andrea Ghelli Luserna Di Rorà, Marco Manfrini, Antonella Padella, Carmen Baldazzi, Cristina Papayannidis, Giovanni Marconi, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Enrica Imbrogno, Eugenia Franchini, Claudia Venturi, Elisa Zuffa, Maria Chiara Abbenante, Giorgia Simonetti, Nicoletta Testoni, Emanuela Ottaviani, Martinelli Giovanni. Distinct pattern of alterations in tp53 mutated and wild type acute myeloid leukemia (AML) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1766. doi:10.1158/1538-7445.AM2017-1766

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.

Introduction: Myelofibrosis (MF), a clonal disorder of the hemopoietic stem/progenitor cell, is characterized by distinctive abnormalities in megakaryocyte (MK) development. A proliferation of dysplastic MK with disturbance of nuclear/cytoplasmic maturation is observed. In addition to driver mutations in Janus Kinase 2 (JAK2) and Calreticulin (CALR) genes, chronic inflammation has emerged as a key-player in MF pathogenesis. Here, we analyzed the potential contribution of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α and Tissue Inhibitor of Metalloproteinases (TIMP)-1 to MF dysmegakaryocytopoiesis. Methods: We studied 10 patients with MF at diagnosis while 6 patients had received previous treatment for MF. In all cases, therapies had been discontinued for at least two months before peripheral blood samples collection. To characterize the mutational status, JAK2V617F and CALR exon 9 mutations were assessed by quantitative PCR-based allelic discrimination assay and Next Generation Sequencing approach, respectively. To quantify MK progenitors, circulating CD34+CD41+ cells of MF patients (10 JAK2V617F and 6 CALR mutated) and controls (cord blood (CB); 8 cases) were identified by flow cytometry analysis. To evaluate the pro-inflammatory profile, IL-1β, TNF-α and TIMP-1 were measured in plasma of patients (10 JAK2V617F and 6 CALR mutated) and healthy subjects (10 cases) by ELISA. To determine whether and to what extent crucial factors of the inflammatory microenvironment may affect the in vitro growth of MK progenitors, Colony Forming Units-Megakaryocyte (CFU-MK) growth of circulating CD34+ cells from MF patients (6 JAK2V617F and 6 CALR mutated) and CB (6 cases) was assessed with a collagen-based medium in the presence or absence of TIMP-1, TNF-α and IL-1β (alone or in combination). Pure and mixed CFU-MK were quantified on the basis of CD41 immunostaining. Results: We found that, irrespective of mutational status, IL-1β, TNF-α and TIMP-1 plasma levels were significantly increased in MF patients. The frequency of circulating MF-derived CD34+CD41+ cells was significantly higher than the CB counterparts. However, a significantly higher proportion of cells was associated with CALR compared to JAK2V617F mutated patients. The growth of pure CFU-MK from untreated cells of the JAK2V617F mutated patients was significantly decreased as compared to that of CALR mutated patients and the CB counterparts. Factors alone did not stimulate the CFU-MK growth from CB CD34+ cells. Conversely, IL-1β had stimulatory activity on pure MK colony formation of JAK2V617F, but not CALR, mutated patients and the growth of MK progenitors from MF patients was significantly inhibited by TNF-α, irrespective of mutational status. TIMP-1 was ineffective. Factors in combination did not significantly modify the growth of pure CFU-MK from patients/CB as compared with factors alone. The growth of mixed MK colonies from untreated cells was absent (JAK2V617F mutated patients) or very low (CALR mutated patients and CB). Only TNF-α (alone or in combination (IL-1β+TNF-α and IL-1β+TNF-α+TIMP-1)) increases the growth of mixed MK colonies of patients harboring CALR mutation and CB. Interestingly, factors alone or in combination failed to stimulate the growth of mixed MK colonies from JAK2V617F mutated patients. Conclusions: Taken together these results demonstrate that in MF signals provided by the inflammatory microenvironment promote a mutation-associated functional dysregulation of MK progenitors and suggest a mechanism by which chronic inflammation may play a role in the development/maintenance of the abnormal megakaryocytopoiesis. Disclosures Martinelli: BMS: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau. Cavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria.

Myelofibrosis (MF) is a clonal disorder of the hemopoietic stem/progenitor cells (HSPCs). Together with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of MF pathogenesis. It is argued that the up-regulated production of pro-inflammatory cytokines selects for the malignant clone. However, the key players linking inflammation and cancer in MF are still poorly known. Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived HSPCs. Plasma TIMP-1, TNF-α and IL-1β were measured by ELISA assay. Circulating CD34+ cells from MF patients (JAK2V617F (10 cases) and CALR (10 cases) mutations) or cord blood (CB; 8 samples) were functionally characterized after incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), cell cycle and clonogenic capacity. Migration was assessed towards a C-X-C motif chemokine 12 (CXCL12) gradient in the presence or absence of the pro-inflammatory factors. MF-derived CD34+ cells were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence of the pro-inflammatory factors and then evaluated for survival. Regardless mutation status, the plasma levels of IL-1β, TNF-α and TIMP-1 are increased in MF patients and the presence of pro-inflammatory cytokines confers a survival advantage of MF-derived CD34+/CD34+CD38- cells. In addition, MF-derived CD34+ cells promoted cell-cycle progression to the S-phase. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α ± TIMP-1 impaired the erythroid clonogenic output of the CALR mutated patients. Of note, IL-1β + TNF-α ± TIMP-1 highly promoted the in vitro migration of MF-derived CD34+ cells in the presence of CXCL12. Intriguingly, MF-derived CD34+ cells revealed increased clonogenic ability after migration toward CXCL12 and IL-1β + TNF-α ± TIMP-1, suggesting a pivotal role of inflammation in the selection of the malignant clone. Finally, co-cultures of normal MSCs with MF-derived CD34+ cells sustained the survival effects of IL-1β+TNF-α±TIMP-1. Overall, our data indicate that the behavior of the MF-derived HSPCs can be influenced by regulatory signals provided by the microenvironment through the cooperation between various pro-inflammatory factors. The interplay of these pro-inflammatory factors promotes and selects the circulating MF-derived HSPCs with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach. Citation Format: Daria Sollazzo, Dorian Forte, Nicola Polverelli, Marco Romano, Margherita Perricone, Sofia Fatica, Francesco Lapi, Emanuela Ottaviani, Martinelli Giovanni, Nicola Vianelli, Michele Cavo, Maria Pantaleo, Francesca Palandri, Lucia Catani. Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4077.

Introduction: Genomic rearrangements can drive the development of cancer through different mechanisms: chromothripsis, a catastrophic mechanism of genomic instability, could be relevant for hematological disease. Aim: To discover the mechanisms underlying the pathogenesis of Acute Myeloid Leukemia (AML), we studied chromothripsis in our cohort of patients (pts). Methods: We perform SNP Array 6.0 or Cytoscan HD Array (Affymetrix) in a cohort of 104 AML pts at diagnosis. SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Seven/104 pts (6.7%) showed chromothripsis events involving different chromosomes (8, 17, 11, 5 and 16). These pts had median age of 68.5 years (range 56-76), complex karyotype and high risk disease according to ELN definition. Among the pts showing chromothripsis, we compared chromosomic abnormalities of pts with de novo (5/7) and secondary (2/7) AML. De novo AML showed a prevalence of trisomy of chromosome 8 in a non-statistical way (4/5 vs 0/2), due to the low number of cases. However, we identified significant differences in the pattern of genes altered in the two groups. De novo AML had copy number gain of PEX1, ANK1, NCOA2, ESRP1, TPD52, ESRP1, ZFPM2 and MITF (p Conclusion: Our data suggest that different pathways and genomic alterations are involved in chromothripsis events in de novo and secondary AML, which could be explained by the repeated rounds of stress underwent by leukemic cells in secondary AML cases. Cytoskeleton and microtubules formation pathways appear to be the main cellular processes implicated in chromothripsis genesis, while alterations of histone acetyltransferase, immune response and antigen presentation pathways could sustain leukemic cells after chromothripsis. Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12(L. Bolondi), FP7 NGS-PTL project. Citation Format: Maria Chiara Fontana, Viviana Guadagnuolo, Cristina Papayannidis, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Marco Manfrini, Barbara Santacroce, Margherita Perricone, Silvia Lo Monaco, Emanuela Ottaviani, Simona Soverini, Michele Cavo, Giovanni Martinelli. Chromothripsis in AML patients: A new mechanism of cancer initiation and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3582.

Introduction It is acknowledged that an accurate histological diagnosis may distinguish Essential Thrombocythemia (ET) from early Primary Myelofibrosis (early-PMF), which is projected to worse outcome in terms of survival and disease evolution into acute leukemia (AL) or overt myelofibrosis (MF). It is also accepted that the outcome of ET is related to the mutational status, with JAK2V617F mutation having a negative impact. In previous analyses, outcome data derived from the admixture of the two variables, histology and mutational status. Here, we present a large cohort of ET/early-PMF patients positive for the JAK2V617F mutation, with the aim to evaluate the impact on outcome of the sole histological definition. Methods A clinic-pathologic database of ET patients followed in four Italian Hematology Centers was created and a total of 475 WHO-diagnosed ET or early-PMF JAK2V617F-positive patients was collected. Bone marrow specimens were performed or reviewed at local institution. Baseline clinical/molecular characteristics and outcome measures (vascular complications, disease transformation/progression, overall and event-free survival) were evaluated. In all patients, JAK2V617F allele-burden was assessed in granulocyte DNA by using ipsogen JAK2 MutaQuant Kit (qPCR). The study was approved by the Ethic Committee of each participating Centers. Results Overall, 329 WHO-defined ET and 146 early-PMF patients positive for the JAK2V617F mutation were included in the study. Median follow-up was 6.6 years (range: 0.5-32.4). Compared to ET patients, early-PMF patients presented with older age (median, 57 versus 53.5yr, p=0.02), lower hemoglobin levels (median, 14.2 versus 14.5 g/dl, p=0.01), higher leukocyte count (median, 10.4 versus 9.5x109/l, p=0.01), and higher incidence of spleen enlargement (35.9% versus 13.9%, p The rate of composite outcomes (thrombosis, evolution into overt MF or AL and death) was significantly higher in early-PMF (3.1% vs 2,3% pts/yr) with a combined event-free survival of 69% versus 82% in ET patients at 10 yrs (figure 1). Conclusions. This study eliminates the confounding factor of different molecular status on outcome by focusing on a large cohort of WHO-defined ET/early-PMF patients, all carrying the JAK2V617F mutation. Overall, the study validates the clinical relevance of strict adherence to WHO criteria on prognosis, and particularly on disease progression into secondary MF. Figure 1. Combined event-free survival according to histology Figure 1. Combined event-free survival according to histology Disclosures Palumbo: Novartis: Honoraria, Other: Advisory Board. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Introduction: Novel array-based technique-single-nucleotide polymorphism (SNP) microarray can detect cytogenetic lesions mostly involving structural alterations with losses or gains of chromosomic material. These abnormalities are predictive of response and can help define therapeutic strategies. SNP microarray can also detect copy-neutral loss of heterozygosity (CN-LOH), which has a described role in Acute Myeloid Leukemia (AML) by inducing oncogene duplication, tumor suppressor inhibition and epigenetic reprogramming. Aim: To improve conventional cytogenetic analysis and identify new genes relevant to leukemogenesis by SNP array-based genotyping. Materials and Methods: We analyzed 279 AML patients (pts) at diagnosis by SNP Array 6.0 or Cytoscan HD Array (Affymetrix). Thirty-four samples were also analyzed by Whole Exome Sequencing WES (HiSeq 2000,Illumina). SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team, while WES data were analyzed by GATK and MuTect. Results: Copy Number Alterations (CNAs) were scattered across all chromosomes (chrs). All pts showed CNA events: 44.4% of CN gain, 21% of CN loss and 34.6% of CN-LOH. Single copy gains mainly affected chrs X, 1, 2, 4, 9 and 8. Duplications occurred at chrs 2, 3, 5 and 14. Heterozygous loss events were detected in chrs 3, 5 and 14, while regions of deletion were located in chrs 6, 7 and 22. The CN-LOH event was the most common event and involved chrs 1, 2, 3, 4, 5, 6 and 8. We studied the deletome profile in our cohort (Fig. 1) pf pts in order to define the minimal common deletion region. SNP array analysis showed that several genes were preferentially deleted, including ADAM5 (12,9%), PHF6 (12,2%), AGPS (10%), SOX6 (7,9%), WT1 (6,5%), CRLF2 (5,4%) and LRRK1 (4,3%); while the genes preferentially amplified were GPC3 (70,25%), FLT3 (44,8%), FGF13 (36,2%), KIT (31,54%), AFF2 (31,5%), ETS1 (26,52%), MITF (22,2%), CASK (16,5%), CDY1 (14,33%), MECP2 (14,33%), FOXP2 (14%) and SMAD4 (12,9%). Single-copy losses and deletions were enriched (p Concerning single copy gain and amplified genes, the functional pathways significantly represented in our cohort were: cancer-related (103 genes), regulation of actin cytoskeleton (60 genes), MAPK signaling (64 genes), metabolic (171 genes) and cell adhesion molecules pathways (CAMs, 5 genes). Regarding gain events, 24 genes were shared by at least 42 pts (15%), 184 genes were shared by at least 14 pts (5%) and 58 genes by at least 28 pts (10%); whereas 15 genes have duplication events (in homozygosis) shared by at least 14 pts (5%). Significant CN-LOH events included genes mapping to metabolic pathways (249 genes), pathways in cancer (103 genes), regulation of actin cytoskeleton (67 genes), calcium signaling pathways (57 genes), WNT signaling pathways (51 genes) and protein digestion and absorption (34 genes). CN-LOH events in 119 genes were shared by at least 14 patients (5%). Finally, we could distinguish 3 clusters of pts, each one characterized by a peculiar pattern of amplified or deleted genes. In order to define relevant pathogenic mechanisms in our cohort, we combined the deletome profile with WES data obtained from 34 pts. Interestingly, we found deletion of genes which are also targeted by mutations (BRCA2, LRRK1). Moreover, some deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations (CASK deletion and MPP6 mutation, CDK6 deletion and PPM1B mutation, MAPT deletion and SPAG5 mutation). Conclusion: By SNP array we have identified CNAs involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on the prognosis of AML pts. Minimal deleted regions deserve further investigation in order to identify new candidate oncogenes which could be relevant AML biomarkers. Acknowledgment: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12(L. Bolondi), FP7 NGS-PTL project. MCF and VG equally contributed to this work. Figure 1. Figure 1. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Background: Infectious complications represent one of the main causes of morbidity and mortality in patients (pts) with Primary Myelofibrosis (PMF), Post-Essential Thrombocythemia and post-Polycythemia Vera MF (PET/PPV-MF). Up-to-date, very few data are available on incidence and outcome of infectious complications. Also, risk factors of this potentially fatal complication are still to be investigated and defined. This is particularly relevant in the era of targeted therapy in MF, since an increased infectious risk has been reported in pts treated with ruxolitinib (RUX), a JAK1/2 inhibitor. Aims: To evaluate risk factors for severe infections in a large cohort of MF patients. Methods: Clinical and laboratory data of pts with MF were retrospectively collected from the database of 5 Italian Hematology Centers. Severe infections were defined according to the CTCAE. The study was approved by the Ethic Committee of each participating Centers. Results: Between 1980 and Aug 2014, 507 pts with PMF (362 pts, 71%), or PET-MF (14%) or PPV-MF were diagnosed and followed for a median follow-up of 4.2 yr (0.5-30.1). Baseline characteristics were (median): age, 66 y (range, 26-87); ≥65 y, 54%; male, 59%; hemoglobin (Hb), 11.9 g/dL (4-17.9); Hb Overall, 112 pts (22%) experienced 160 infectious events (grade 3-4, 45%), for an incidence rate of 3.9% pt-y. The cumulative incidence was 20%, 33% and 51% at 5, 10 and 20 y, respectively. Infections were: bacterial (143 events, 89%; pneumonia: 80 cases, 56%); VZV reactivations (11 events, 7%), nodal TBC (3 events, 2%), fungal infections (3 events, 2%). Infectious complications represented the causes of death in 10 (7%) out of 134 deceased pts. Among baseline features, age≥65 y (p=0.001), primary vs secondary MF (p=0.009), spleen length>10 cm below left costal margin (p=0.006), high/intm-2 IPSS (p Overall, 128 pts at intm-2/high IPSS risk were treated with RUX for a median time of 23 mos (1-41). Infection-free survival at 5 years was comparable in RUX-treated pts compared to non RUX-treated intm-2/high risk pts (76% vs 67%, p=0.82). In the RUX-treated cohort, age ≥65 y (p=0.008), JAK2 allele burden ≥75% (p=0.03) and steroids exposure before RUX (0.007) correlated with an increased infectious risk. Multivariate analysis confirmed age and corticosteroids utilization as independent negative prognostic risk factors (p=0.003 and p=0.043, respectively). Also, patients who obtained a spleen reduction higher than 50% during RUX therapy were projected to a better infection-free survival compared to non-responders (89% vs 70% at 12 mos, p=0.001) Summary/Conclusion. This large study confirms severe infections as frequent and potentially fatal events in MF. Also, this study has led to the identification of the main baseline features associated with increased infectious risk, namely baseline IPSS category and massive splenomegaly. Surprisingly, RUX therapy did not seem to significantly increase the risk of infections, despite its immunosuppressant properties. Yet, the successful use of RUX in terms of spleen response was found to correlate with a significant reduction of the probability to develop an infectious complication. Conversely, a combined or sequential use of corticosteroids and RUX may further increase the risk of infectious complications and therefore require a careful evaluation. Disclosures Martinelli: MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Introduction. Myelofibrosis (MF), an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC) with a dysregulation in JAK/STAT signalling (mutations in JAK2, MPL and Calreticulin (CALR) genes), is characterized by a state of chronic inflammation. It is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Only recently, it has been hypothesized that the sustained inflammatory microenvironment of MF can alter crucial biological processes, leading to genomic instability and cancer progression. Here we tested the in vitro functional effects of pivotal players of the inflammatory microenvironment (the extracellular ATP nucleotide and selected cytokines, such as Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)) on the HSPCs from MF patients. Methods: Circulating CD34+/CD34+ CD38- cells from MF patients (JAK2V617F (17 cases) and CALR (9 cases) mutations) or cord blood (CB; 8 samples) were phenotypically and functionally characterized after in vitro incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), phenotype (evaluation of CD63 (TIMP-1 receptor), CXCR4 and CD38 expression), cell cycle and clonogenic capacity. Migration was assessed first towards a CXCL12 gradient in the presence or absence of the pro-inflammatory factors. In parallel experiments, CD34+ cells from MF patients were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence or absence of the pro-inflammatory cytokines and then evaluated for their ability to migrate towards a CXCL12 gradient. Plasma TIMP-1, TNF-α, IL-1β and CXCL12 were measured by ELISA assay. Results: The plasma levels of TIMP-1, TNF-α, IL-1β, CXCL12 and the number of circulating CD34+, CD34+ CD38-, CD34+ CD63+, CD34+ CD184+ cells were increased in MF patients. According to mutational status, the CD34+ CD63+ cells were higher in the CALR+ patients. The survival of MF CD34+ cells was strongly stimulated by in vitro incubation with TNF-α or IL-1β as compared with the CB-derived CD34+ cells or untreated cells. By multiple cytokine combinations, IL-1β/TIMP-1, IL-1β /ATP or IL-1β /TNF-α treatments significantly promote the survival of MF CD34+ cells as compared with the normal counterparts or the untreated cells. Various combinations with IL-1β were also effective in stimulating survival of CD34+CD38- cells. IL-1β/TIMP-1 and IL-1β/TNF-α/TIMP-1, but not factors alone, significantly increased the CFU-C growth of MF patients as compared with the CB-derived counterparts and the untreated cells. Moreover, comparing CALR+ vs JAK2V617F+ patients, the colony formation of JAK2V617F+ patients was mainly promoted by the IL-1β/TNF-α treatment. Along with clonogenic capacity stimulation, exposure of CD34+ cells from MF patients to IL-1β/TNF-α/TIMP-1 significantly increases the S-phase cells, suggesting that these pro-inflammatory factors stimulated cell-cycle progression in dormant CD34+ MF cells. Migration of CD34+ cells from MF was significantly increased in CXCL12 treated cells. In addition, exposure of MF CD34+ cells to IL-1β/TNF-α, IL-1β/TIMP-1 or IL-1β/TNF-α/TIMP-1 significantly promotes cell migration in comparison with the CB-derived counterparts or SDF-1 alone. MF migrated cells in the presence of IL-1β/TNF-α significantly upregulate CD63 expression. Intriguingly, colony formation of MF migrated CD34+ cells in the presence of IL-1β/TNF-α or IL-1β/TNF-α/TIMP-1 was potently increased. Finally, co-culture systems with normal MSCs in the presence of pro-inflammatory factors revealed that MF CD34+ cells display increased migration ability toward CXCL12 gradient. Conclusions: Altogether our findings suggest that in MF the inflammatory niche plays a key role in the maintenance of the malignant clone. Thus, the interplay between the pro-inflammatory cytokines promote and select the HSPCs with higher proliferative activity, clonogenic potential and migration capability. Targeting these microenvironmental interactions may be a clinically relevant approach. D.F. and D.S. equally contributed Disclosures Martinelli: Pfizer: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy.

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 172/886 AML pts treated at the Seràgnoli 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 Number™ 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 (OS) (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p 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-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Anna Ferrari, Stefania Paolini, Carmen Baldazzi, Chiara Sartor, Maria Chiara Abbenante, Sarah Parisi, Francesca Volpato, Ilaria Iacobucci, Antonella Padella, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Giorgia Simonetti, Elisa Zuffa, Eugenia Franchini, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. Very poor outcome and chemoresistance of acute myeloid leukemia patients with TP53 mutations: Correlation with complex karyotype and clinical outcome. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B03.

AML is a heterogeneous disease with a variety of structural and numerical chromosomal and genetic alterations that provided important prognostic information capable to guide therapy and predict outcome. The reported TP53 mutation rate in AML is low (2.1%). By contrast, the incidence of FLT3 disruption is frequent (20-30%) and is an independent predictor of unfavourable outcome of acquired clinical resistance to FLT3 inhibitors. TP53 mutations in AML with a complex karyotype (CK) is higher (69-78%), but few data are available for association between the most common AML associated lesions such as FLT3, NPM1, DNMT3A, IDH2 and TP53 mutation or CK. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the molecular abnormalities, the correlation with known molecular alterations (FLT3, NPM, etc.) and the prognostic role TP53 mutations in adult AML pts. Patients and Methods: 886 AML patients were analysed for cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, DNMT3A, IDH1-2 etc). Of these, 200 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq 2000 platform (38/200) and were correlated with cytogenetic analysis. Results: 55 pts (27.5%) showed 3 or more chromosome abnormalities (CK-AML), 83 (41.5%) presented one or two cytogenetic abnormalities (other-AML) and 43 pts (21.5%) have normal karyotype (nK). In 19 cases the karyotype was not available. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations (32 deleterious point mutations; 4 deletions); seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had CK while only 6/29 (21%) mutated pts have “no CK”. Overall, between pts with CK, TP53 frequency is 41.8% (P>0,0001). 120 pts were analysed for concomitant presence of TP53 mutations and FLT3/NPM1 disruption/mutation: revealing a significant relation between pts with FLT3-ITD or NPM1 and TP53 wild-type (p = 0.043 and 0.022 respectively). As for clinical outcome alterations of TP53 were significantly associated with poor outcome (OS and EFS p WES analysis done in 38 pts (33 TP53 wt and 5 pts TP53 mutated) revealed no genes exclusively mutated in the 5 TP53 mutated pts. Conclusions: Our data demonstrated that TP53 mutations occur in 14.5% of AML with a higher frequency in the subgroup of CK-AML (p Citation Format: Anna Ferrari, Cristina Papayannidis, Elisa Zuffa, Carmen Baldazzi, Antonella Padella, Eugenia Franchini, Ilaria Iacobucci, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Francesca Volpato, Federica Cattina, Giorgia Simonetti, Maria Chiara Fontana, Maria Teresa Bochicchio, Federica Frabetti, Elisa Lani, Katia Mancuso, Beatrice Zannetti, Simona Luatti, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. TP53 mutations are mutually exclusive with FLT3 and NPM mutations in AML patients and are strongly associated with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4906. doi:10.1158/1538-7445.AM2015-4906

Abstract 2497 Introduction: The gene TP53 encoding the tumour suppressor protein p53 is among the most commonly mutated genes in human cancer. TP53 tumour-associated alterations often cause dramatic defects in p53 function and correlate with increased malignancy, dismal survival and resistance to treatment. In contrast, only a small fraction, if any, of the >200 single nucleotide polymorphisms (SNPs) of TP53 in human populations are expected to cause measurable perturbation of p53 function. Aim: Since the pattern, frequency and significance of TP53 aberrations and SNPs in adult BCR-ABL1 positive ALL have still to be investigated, in this study we used a massively parallel pyrosequencing technique to address these issues. Patients and methods: Forty-three adults with BCR-ABL1 positive ALL were analyzed (median age 63 years, range 18–84). Twenty-four cases (56%) were analyzed only at the time of diagnosis, four cases (9%) only at the time of relapse and fifteen cases (35%) at both time points. Massively parallel pyrosequencing in picoliter-sized wells was used to allow highly-sensitive deep-sequencing detecting molecular aberrations at a low burden rate. As part of the IRON (Interlaboratory RObustness of Next generation sequencing) II study network, we applied preconfigured plates including primers for TP53 (exons 4 to 11) and allowing the simultaneous screening of 11 patients, each being recognized by a unique molecular identifier. For each plate, after generating 88 amplicons, 8 per each patient, PCR products were purified using Agencourt AMPure XP beads and Biomek 3000 Laboratory Automation Workstation (Beckam Coulter) and quantified using the Quant-iT PicoGreen kit (Invitrogen). All amplicons were pooled in an equimolar ratio to generate one single library. Subsequent emulsion PCR and amplicon sequencing was performed according to the manufacturer's recommendations on the Genome Sequencer Junior Instrument (Roche Applied Science). Data were analyzed using the GS Amplicon Variant Analyzer software version 2.7 (Roche Applied Science). For the detection of variances, filters were set to display sequence variances occurring in more than 5% of bidirectional reads per amplicon in at least one sample. Results: On average, we generated 63,068 sequencing beads (key pass wells) per plate (range, 50,798-79,486) with a median read length range between 284 and 365 base pairs. The median number of generated reads per case was 5,413 (range, 687-9,604). The median number of reads per amplicon was as follows: exon 4: 275 (range, 0–888), exon 5: 222 (range, 0–1,013); exon 6: 316 (range, 84–854); exon 7: 317 (range, 5–720); exon 8: 313 (range, 0–784); exon 9: 215 (range, 0–785); exon 10: 328 (range, 0–826), exon 11: 447 (range, 0–1,511). Forward and reverse reads were homogeneously distributed allowing a sensitive detection of variances. In total, 8 single nucleotide variations were identified. All variances, except for one nucleotide substitution occurring at position 7576743 (GRCh37/hg19), were found to represent SNPs according to the NCBI dbSNP Build 137. They included: rs1042522 C/G (41/43, 95%) and rs1800370 A/G (1/43, 2%) in exon 4, rs1800372 A/G (2/43, 5%) in exon 6, rs1625895 A/G (42/43, 98%) in intron 6–7, rs12947788 A/G (3/43, 7%) and rs12951053 A/C (4/43, 9%) in intron 7–8 and rs1800899 C/T (1/43, 2%) in intron 9–10. Interestingly, in 2 cases (12%) loss of heterozygosity occurred at the relapse at the C wild-type allele of rs1042522 in leukemia cells. The same mechanism has been identified for one case at the wild-type allele of rs1625895 with the expansion of the variant form at relapse. Both rs1042522 and rs1625895 have been described to alter p53 functionality and increase susceptibility to cancers (Whibley et al.,2009). Although the role of rs12947788 and rs12951053 has not yet deeply investigated, in our study they were found in 3 cases that all relapsed. Conclusion: Comprehensive next generation deep-sequencing of TP53 by a screening assay set up within the IRON II study has demonstrated its ability to efficiently detect TP53 variant. The inactivation of the wild-type allelic forms of rs1042522 and rs1625895, altering the p53 functionality, may serve as an important background for leukemia progression in BCR-ABL1-positive ALL. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Luppi:CELGENE CORPORATION: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Introduction: Myelofibrosis (MF) is a clonal disorder associated mainly with JAK2V617F and MPL mutations. Recently, a new mutation in the gene encoding calreticulin (CALR) was discovered in the majority of JAK2/MPL negative patients. MF is burdened by a high rate of potentially life-threatening infections. The issue of recurrent and opportunistic infections is increased after the introduction in clinical practice of JAK inhibitors with immunosuppressive activity. However, the role of crucial immune cell subsets is still poorly characterized. Here, we investigated the phenotype/function of selected immune cells in MF. Specifically, we focused on circulating regulatory (Tregs) and IL-17-producing T cells (Th17 cells), monocytes and dendritic cells (DCs). Monocyte-derived DCs were also characterized. Methods: We characterized circulating Th17 cells, Tregs, monocytes and DCs of 17 untreated MF patients and 8 healthy controls (HC) by flow cytometry. Th17 cells were identified as CD4+ CD161+ CD196+ cells while Tregs were enumerated as CD4+ CD25high CD127low T cells. We also tested the in vitro suppressive activity of circulating CD4+ CD25+ Tregs with a mixed leukocyte reaction assay. Two subpopulations of circulating DCs, myeloid CD11c+ and plasmacytoid CD123+cells, were enumerated as well. In addition, after immunomagnetic selection, we tested both phenotype of circulating monocytes and their capacity to differentiate into CD14-derived immature and mature DCs, using a specific cytokines cocktail. JAK2V617F and MPL mutations were detected with RT-PCR while the presence of CALR mutations were tested with Exon 9 Next Generation Sequencing assay. Results: JAK2V617F (11 cases), MPL (3 cases), and CARL (3 cases) mutations were detected. We found that circulating CD4+CD25highCD127low Tregs were reduced in MF patients as compared with healthy controls (p=0.043), although their suppressive ability was maintained. We also found a lower number of circulating Th17 cells (p=0.0026) in MF patients. This finding was particularly evident in JAK2V617F+(p=0.008) and CARL+(p=0.03) patients. Despite their number was in the normal range, circulating monocytes from MF patients showed reduced expression of the CD86 co-stimulatory molecule. Moreover, as compared with the normal counterparts, immature monocytes-derived DCs from patients maintained low CD14 expression without upregulating the CD80 co-stimulatory molecule expression (p=0.0063). Interestingly, at variance with plasmacytoid DCs, a reduced number of circulating myeloid DCs was observed in MF patients as compared with that of HC (p=0.01). Conclusions: Here we demonstrated that specific crucial subsets of immune cells show quantitative and/or qualitative abnormalities in MF patients. These findings may be useful to better understand the increased susceptibility of these patients to infections, since Th17 cells play a role in bacterial and fungal infections while myeloid DCs regulate Th1 activity. Of note, DCs inhibition might result in increased propensity to infections and compromised immune response to cancer.In addition, since monocytes are DC precursors, alterations in their differentiation pathway may contribute to develop defective immune responses. Disclosures Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Introduction Young adults with Essential Thrombocythemia (ET) or early Primary Myelofibrosis (early-PMF) are a category of patients projected to a prolonged survival but also to an extended utilization of medical resources. Mutations, including those in the calreticulin (CALR) gene, have been reported to affect main clinical features and outcome in large cohorts of patients with Ph-negative MPNs. However, no data are available on mutational status and long-term outcome in young MPN patients. Methods A clinic-pathologic database of ET patients followed in 5 Italian Hematology Centers was created. A total of 217 WHO-diagnosed ET or early-PMF patients ≤ 40 years at diagnosis was retrieved from the general database of 2635 patients. All bone marrow biopsies were reviewed at local institution. Baseline clinical/molecular characteristics and outcome measures (thrombosis, hemorrhages, secondary MF and AL, second neoplasia, death, overall and event-free survival) were evaluated. JAK2V617F allele-burden was assessed in granulocyte DNA by using ipsogen JAK2 MutaQuant Kit (qPCR). CALR mutations were identified by next generation sequencing (NGS) approach on GS Junior (Roche-454 platform); MPL mutations were evaluated by using ipsogen MPLW515L/K MutaScreen Kit. Results Overall, 197 WHO-defined ET and 20 early-PMF (age range: 16-40, median 34) were included in the study. Mutational frequencies were 61% for JAK2, 25% for CALR, 1% for MPL and 13% for triple negative. Baseline clinical characteristics and use of antiplatelet/cytoreductive therapies were comparable in ET and early-PMF, although frequency of triple negative was higher in the early-PMF cohort. Compared to the JAK2 positive population, both CALR and triple-negative patients showed higher platelet count and lower hemoglobin and hematocrit levels (Table 1). Median follow-up was 10.2 years (range: 0.5-37.5). During follow-up, 19 (9,6%) ET and 3 (15%) early-PMF patients experienced a total of 31 thrombotic (arterial: 38%) and 12 hemorrhagic events, with an incidence rate of 0.91% and 0.39% patients/yr, respectively. The cumulative incidence of thrombosis was 0,14% and 0,24% at 15 and at 20 years, respectively. Overall, 10 patients (4,6%) and 1 (0,4%) patients evolved to MF and AL, respectively; 10 developed a second neoplasia. The cumulative incidence of disease progression into MF/AL was 0,03% and 0,13% at 15 and at 20 years, respectively. At last contact, 6 (2,7%) patients had died, at a median age of 61 years (20-71), for an overall survival of 98% at 15 years. Causes of death were related to the myeloproliferative neoplasm (disease evolution or thrombotic complications) in all patients but one. Event-free survival was similar in the ET/early-PMF cohorts considering both every event separately and all together. In univariate analysis, male sex (p=0.003), previous thrombosis (p=0.001), splenomegaly (p=0.037), JAK2V617F (p=0.019) were associated with increased thrombotic risk; in multivariate Cox analysis, only previous thrombosis and male sex remained significant (p=0.012). Baseline splenomegaly was the only predictive factor for subsequent hemorrhages (p=0.017). Abnormal karyotype was associated with secondary MF (p=0.013) and also with second neoplasia (p11x109/L, abnormal karyotype was also associated with worse survival in univariate analysis. However, in multivariate analysis only JAK2V617F mutation remained as negative predictor of survival (p=0.019). Also, multivariable analysis confirmed JAK2 mutation and splenomegaly as independent risk factors for cumulative events. Conclusions With the limitations due to the low number of early-PMF, the outcome of young adults with early-PMF and true ET seemed to be comparable. The correlation of abnormal karyotype with MF transformation and second neoplasia suggests the need for an accurate cytogenetic analysis at diagnosis. Mutational status did influence disease phenotype, in terms of baseline characteristics and prognosis. Indeed, JAK2 mutational status confirmed a negative prognostic role for thrombosis and survival, while event-free survival was significantly better in triple-negative patients. Notably, causes of death were mostly related to the hematological malignancy, pointing out the substantial impact that this generally indolent disease may acquire in young adults. Disclosures Latagliata: Novartis: Consultancy; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy; Shire: Consultancy. Cavo:Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria.

Introduction: AML is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome. The TP53 gene 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 (CK-AML) is higher (69-78%). Aims: to investigate the frequency and the prognostic role of TP53 mutations in adult AML patients (pts) focusing the screening on subgroups of pts with chromosome abnormalities. Patients and Methods: 106 adult AML pts with FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype (nK-AML, 14/106 pts) were examined. Forty-four pts (41.1%) showed 3 or more chromosome abnormalities (CK-AML), 42 (39.6%) presented one or two cytogenetic abnormalities (other-AML) and in 6 cases the karyotype was not available. Genomic DNA and/or cDNA were isolated from mononuclear AML blast cells. TP53 mutation screening was performed on all 106 AML pts, in particular in 42 from exon (ex) 2 to 11, in 48 from ex 4 to 11 and in 16 pts from ex 2 to 8. Analysis was focused on coding sequences (RefSeq GRCh37/hg19 NG_017013.2). Results: By PCR and subsequent Sanger sequencing, mutations of TP53 were detected in 23 pts (21.1%). Seven pts revealed 2 mutations. 83% of all mutated pts had CK (19/23) by contrast the frequency of mutations was very low in “no CK-AML” pts (6.5%). Overall, mutated patients included 19/44 with CK-AML (43.2%); 1/6 (16.7%) with nK and 3/42 (7.1%) with other-AML. 26 TP53 point mutations [19 missense, 1 silent, 5 intron and 1 3’ untranslated region (UTR)] and 4 TP53 deletions were found. Twenty-six out of 30 mutations/deletions (86.6%) were located in the DNA binding domain, 2 in the carboxyl-terminal tetramerization and regulatory domains and 2 in the transcriptional activation domain. All mutations in coding regions were classified by the IARC database (http://p53.iarc.fr/TP53GeneVariations.aspx) as deleterious. Of note, alterations of TP53 were significantly associated with poor outcomes in terms of overall survival and disease free-survival (P < 0.0001). Conclusions: Our data demonstrated that mutations of TP53 occur in 21.7% of AML with a higher frequency in the subgroup of CK-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 in CK-AML pts. Supported by: EuropeanLeukemiaNet, AIL, AIRC, PRIN 2010-2011, Fondazione del Monte di Bologna e Ravenna, European Union Seventh Framework Programme [FP7/2007-2013]. Citation Format: Anna Ferrari, Ilaria Iacobucci, Cristina Papayannidis, Carmen Baldazzi, Chiara Sartor, Emanuela Ottaviani, Nicoletta Testoni, Valentina Robustelli, Margherita Perricone, Claudia Venturi, Maria Chiara Abbenante, Viviana Guadagnuolo, Antonella Padella, Federica Cattina, Giorgia Simonetti, Domenico Russo, Giovanni Martinelli. Tp53 mutation screening in adult acute myeloid leukemia (AML) patients shows a strong association with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2014-570

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.

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

Introduction: Myelofibrosis (MF) is an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC). Mutations in JAK2, Calreticulin (CALR) and MPL genes are associated with MF. MF is characterized by a state of chronic inflammation and it is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Interestingly, recent findings showed that HSPCs actively sense pro-inflammatory factors. However, the key players linking inflammation and cancer in MF as well as the underlying pathogenetic mechanism are still to be defined. Extracellular nucleotides (ATP) and selected cytokines, such as Tumor Necrosis Factor (TNF)-alpha or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), are crucial factors of the inflammatory microenvironment. Here we investigated the in vitro effects of ATP, TNF-alpha or TIMP-1 on phenotype/function of HSPCs from MF patients. Methods: Circulating CD34+ HSPCs from MF patients with JAK2V617F (7 cases), CALR (3 cases) and MPL (3 cases) mutations or from cord blood (5 samples) have been phenotypically and functionally characterized at baseline or after in vitro incubation with or without increasing doses of ATP, TNF-alpha or TIMP-1 (alone or in combination) at different time points. The expression of selected hemopoietic/endothelial markers (CD45,CD133, CD41, CD105, KDR, CD184,CD44) has been analyzed on CD34+ cells by flow cytometry. The expression of the TIMP-1 receptor CD63 was also assessed. Immunomagnetically isolated CD34+ cells were stained with Annexin-V/Propidium Iodide and survival/apoptosis were analyzed by flow cytometry. Migration of purified CD34+ cells were also assayed towards a CXCL12 gradient (150ng/mL) in transwell chambers. Clonogenic assay (BFU-E and CFU-GM in vitro growth) of CD34+cells was also tested. Results: We found that the survival of MF CD34+ cells was strongly stimulated by TNF-alpha (10 ng/mL; 2,9 fold increase) as compared with that of cord blood-derived CD34+ cells (1,7 fold increase). TIMP-1 (100ng/mL) treatment also promotes the survival of MF CD34+ cells. Accordingly, MF patients show increased number and percentages of circulating CD63+CD34+ cells. However, at variance with cord blood-derived CD34+ cells, MF CD34+ cells were more resistant to the citotoxic activity of high dose ATP (1000 nM). Interestingly, when we tested ATP, TNF-alpha and TIMP-1 in combination, the survival of CD34+ cells from MF patients was mainly promoted by the TNF-alpha/TIMP-1 treatment. At variance, ATP addition reduced the TNF-alpha or TIMP-1 pro-survival activity. We also found that TNF-alpha (1,6 fold increase) and TIMP-1 (1,4 fold increase), tested alone, slightly increased the BFU-E formation as compared with that of cord blood- derived CD34+ cells (2,2 and 2,0 fold increase, respectively), without affecting the number of CFU-GM progenitors. By contrast, the number of BFU-E and CFU-GM was significantly reduced when CD34+ cells were incubated in the presence of ATP+TNF-alpha (4,5 and 2,1 fold reduction as compared with untreated cells, respectively), whereas TIMP-1 addition slightly reduced the inhibitory activity of ATP+TNF-alpha (4,0 and 1,8 fold reduction, respectively). Of note, survival and proliferation of MF CD34+ cells were independent from the occurrence of JAK2, MPL or CALR mutations. In addition, despite CXCR4 (CD184) expression of CD34+ cells was significantly increased as compared with the cord blood-derived counterparts, when we analyzed the in vitro migration of CD34+ cells toward a CXCL12 gradient, we found the migration rate of MF CD34+ cells to be highly heterogeneous, ranging from 0,9 to 2,9 fold increase as compared with the corresponding untreated samples. Interestingly, CD34+ cells from 2 out of 3 CALR+patients showed the highest migration capacity. Conclusions: Here we demonstrated that crucial factors of the inflammatory microenvironment abnormally modulate the HSPC functions of MF and may contribute to the maintenance of the malignant clone. Specifically, TNF-alpha together with TIMP-1 promote the survival of the CD34+ HSPCs of MF, while ATP/TNF-alpha combination reduces their proliferation. Interestingly, the inflammatory factors tested in combination differentially modulate the proliferation of the MF CD34+ cells as compared with the factors alone. Disclosures Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.