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Background Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. Methods Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD. Results The absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p. Conclusions Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

FMS-like tyrosine kinase 3 (FLT3) is among the most common driver genes recurrently mutated in acute myeloid leukemia (AML), accounting for approximately 30% of cases. Activating mutations of the FLT3 receptor include internal tandem duplications (ITD) that map to the auto-inhibitory juxtamembrane (JM) domain or point mutations within the tyrosine kinase domain (TKD). Several FLT3 tyrosine kinase inhibitors have been developed in the last few years, but midostaurin is currently the only one approved for the treatment of newly diagnosed patients harboring FLT3 mutations. Here we describe for the first time a novel in-frame deletion in exon 14 (JM domain) of the FLT3 gene, that we identified in a young woman with CBFb-MYH11-positive AML. We demonstrated that this novel FLT3 variant is pathogenic, since it is responsible for constitutive activation of FLT3 receptor. Finally, ex-vivo studies demonstrated that this novel mutation is sensitive to midostaurin.

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.

Adrenomedullin (ADM) is a hypotensive and vasodilator peptide belonging to the calcitonin gene-related peptide family. It is secreted in vitro by endothelial cells and vascular smooth muscle cells, and is significantly upregulated by a number of stimuli. Moreover, ADM participates in the regulation of hematopoietic compartment, solid tumors and leukemias, such as acute myeloid leukemia (AML). To better characterize ADM involvement in AML pathogenesis, we investigated its expression during human hematopoiesis and in leukemic subsets, based on a morphological, cytogenetic and molecular characterization and in T cells from AML patients. In hematopoietic stem/progenitor cells and T lymphocytes from healthy subjects, ADM transcript was barely detectable. It was expressed at low levels by megakaryocytes and erythroblasts, while higher levels were measured in neutrophils, monocytes and plasma cells. Moreover, cells populating the hematopoietic niche, including mesenchymal stem cells, showed to express ADM. ADM was overexpressed in AML cells versus normal CD34+ cells and in the subset of leukemia compared with hematopoietic stem cells. In parallel, we detected a significant variation of ADM expression among cytogenetic subgroups, measuring the highest levels in inv(16)/t(16;16) or complex karyotype AML. According to the mutational status of AML-related genes, the analysis showed a lower expression of ADM in FLT3-ITD, NPM1-mutated AML and FLT3-ITD/NPM1-mutated cases compared with wild-type ones. Moreover, ADM expression had a negative impact on overall survival within the favorable risk class, while showing a potential positive impact within the subgroup receiving a not-intensive treatment. The expression of 135 genes involved in leukemogenesis, regulation of cell proliferation, ferroptosis, protection from apoptosis, HIF-1α signaling, JAK-STAT pathway, immune and inflammatory responses was correlated with ADM levels in the bone marrow cells of at least two AML cohorts. Moreover, ADM was upregulated in CD4+ T and CD8+ T cells from AML patients compared with healthy controls and some ADM co-expressed genes participate in a signature of immune tolerance that characterizes CD4+ T cells from leukemic patients. Overall, our study shows that ADM expression in AML associates with a stem cell phenotype, inflammatory signatures and genes related to immunosuppression, all factors that contribute to therapy resistance and disease relapse.

Myelofibrosis (MF) is characterized by chronic inflammation and hyper-activation of the JAK-STAT pathway. Infections are one of the main causes of morbidity/mortality. Therapy with Ruxolitinib (RUX), a JAK1/2 inhibitor, may further increase the infectious risk. Monocytes are critical players in inflammation/immunity through cytokine production and release of bioactive extracellular vesicles. However, the functional behavior of MF monocytes, particularly during RUX therapy, is still unclear. In this study, we found that monocytes from JAK2V617F-mutated MF patients show an altered expression of chemokine (CCR2, CXCR3, CCR5) and cytokine (TNF-α-R, IL10-R, IL1β-R, IL6-R) receptors. Furthermore, their ability to produce and secrete free and extracellular vesicles-linked cytokines (IL1β, TNF-α, IL6, IL10) under lipopolysaccharides (LPS) stimulation is severely impaired. Interestingly, monocytes from RUX-treated patients show normal level of chemokine, IL10, IL1β, and IL6 receptors together with a restored ability to produce intracellular and to secrete extracellular vesicles-linked cytokines after LPS stimulation. Conversely, RUX therapy does not normalize TNF-R1/2 receptors expression and the LPS-driven secretion of free pro/anti-inflammatory cytokines. Accordingly, upon LPS stimulation, in vitro RUX treatment of monocytes from MF patients increases their secretion of extracellular vesicles-linked cytokines but inhibits the secretion of free pro/anti-inflammatory cytokines. In conclusion, we demonstrated that in MF the infection-driven response of circulating monocytes is defective. Importantly, RUX promotes their infection-driven cytokine production suggesting that infections following RUX therapy may not be due to monocyte failure. These findings contribute to better interpreting the immune vulnerability of MF and to envisaging strategies to improve the infection-driven immune response.

Introduction A large body of evidence has increasingly demonstrated that the immune tumor microenvironment (TME) critically contributes to acute myeloid leukemia (AML) development. However, current AML prognostic classifications only rely on leukemia cell-intrinsic alterations and do not incorporate immunological markers referring to TME. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is a central mediator of immune tolerance in the AML TME. This study aimed to identify IDO1-interacting genes in the AML TME and to develop a prognostic immune gene signature. Methods Biological and clinical data of 732 patients with de novo AML treated with curative intent were retrieved from public TCGA and HOVON datasets. Patients >= 65 years were excluded from survival analyses. Co-expression analysis was performed through cBioPortal on TCGA data aiming at discovering new IDO1-interacting genes. Cox regression analysis was used to identify most survival-predicting genes in order to generate a prognostic score. Differential expression (DE) analysis was performed using the nSolver software package (NanoString Technologies, Seattle, WA). Results BIN1 and IDO1 expression were negatively correlated in HOVON cases (P Conclusions Our data identify PLXNC1 as a novel IDO1-correlated gene. A three-gene immune signature that includes PLXCN1, IDO1 and BIN1 strongly predicted clinical outcome in large AML cohorts. Moreover, IDO1 and PLXNC1 expression-based DE analysis generated an immunological signature highly predictive of prognosis. In light of the emerging role of immunotherapies for AML, our findings support the incorporation of TME-associated immune biomarkers into current AML classification and prognostication algorithms. Figure Disclosures Cavo: Jannsen, BMS, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, AbbVie, Karyopharm, Adaptive: Consultancy, Honoraria. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding; Kura Oncology: Research Funding.

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) is a heterogeneous hematological disorder defined by morphological, genetic, and clinical features. Patients with AML-MRC often show cytogenetic changes, which are associated with poor prognosis. Straightforward criteria for AML-MRC diagnosis and a more rigorous characterization of the genetic abnormalities accompanying this disease are needed. Here we describe an informative AML-MRC case, showing two separate, but concurrent, chromothripsis events, occurred at the onset of the tumor, and originating an unbalanced t(5;7) translocation and a derivative chromosome 12 with a highly rearranged short arm. Conversely, despite chromothripsis has been often associated with genomic amplification in cancer, in this case a large marker chromosome harboring amplified sequences from chromosomes 19 and 22 arose from a stepwise mechanism. Notably, the patient also showed a TP53 mutated status, known to be associated with an increased susceptibility towards chromothripsis and a poor prognosis. Our results indicate that multiple chromothripsis events may occur early in neoplastic transformation and act in a synergistic way with progressive chromosomal alterations to determine a dramatic impact on disease outcome, as suggested by the gene expression profile analysis.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.

Chromosomal rearrangements involving 3q26 are recurrent findings in myeloid malignancies leading to MECOM overexpression, which has been associated with a very poor prognosis. Other 3q abnormalities have been reported and cryptic MECOM rearrangements have been identified in some cases. By fluorescence in situ hybridization (FISH) analysis, we investigated 97 acute myeloid leukemia/myelodysplastic syndrome patients with various 3q abnormalities to determine the role and the frequency of the involvement of MECOM. We identified MECOM rearrangements in 51 patients, most of them showed 3q26 involvement by chromosome banding analysis (CBA): inv(3)/t(3;3) (n = 26) and other balanced 3q26 translocations (t(3q26)) (n = 15); the remaining cases (n = 10) showed various 3q abnormalities: five with balanced translocations involving 3q21 or 3q25; two with homogenously staining region (hsr) on 3q; and three with other various 3q abnormalities. Complex rearrangements with multiple breakpoints on 3q, masking 3q26 involvement, were identified in cases with 3q21/3q25 translocations. Furthermore, multiple breaks were observed in two cases with t(3q26), suggesting that complex rearrangement may also occur in apparently simple t(3q26). Intrachromosomal gene amplification was another mechanism leading to MECOM overexpression in two cases with hsr on 3q. In the last three cases, FISH analysis revealed 3q26 involvement that was missed by CBA because of metaphases' suboptimal quality. All cases with MECOM rearrangements showed overexpression by real-time quantitative PCR. Finally, MECOM rearrangements can occur in patients with 3q abnormalities even in the absence of specific 3q26 involvement, underlining that their frequency is underestimated. As MECOM rearrangement has been associated with very poor prognosis, its screening should be performed in patients with any 3q abnormalities.

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.

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

Monitoring of minimal residual disease (MRD) by quantification of BCR-ABL1 transcript levels has become a main part of the management of patients with BCR-ABL1-positive acute lymphoblastic leukemia (ALL) in treatment with tyrosine kinase inhibitors (TKIs). The failure to achieve molecular negativity shortly after starting TKI has been demonstrated to be predictive of relapse, suggesting that an accurate measurement of low BCR-ABL1 levels may have a role in preventing hematological relapse. Despite the big efforts made by many European laboratories within the European Study Group, at the time of writing a standardized procedure to quantify and express results is still missing for BCR-ABL1-positive ALL. In this study, in order to detect with high sensitivity low levels of BCR-ABL1 transcripts, we used a new technology and a new molecular approach based on microfluidic digital polymerase chain reaction (dPCR) using Taqman chemistry and we compared obtained results with those generated by the conventional method based on reverse transcriptase PCR reaction (RQ-PCR) for BCR-ABL1 and total ABL1, with TaqMan chemistry and with Applied Biosystems instrument. We demonstrated the dPCR is high-sensitive (able to detect a single copy of BCR-ABL1) and reliable (results are comparable to those obtained by BCR-ABL1 quantification with conventional technology), allowing an accurate monitoring of BCR-ABL1-positive ALL patients in complete remission.

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.

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.

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

Next Generation Sequencing (NGS) studies identified 9 functional categories of mutations in acute myeloid leukemia (AML), with >99% of cases having at least one of those mutations (Ley et al. NEJM 2013). However, multiple genetic hits participate to AML pathogenesis, and metabolic dysregulations, as the one induced by IDH1/2 mutations, play oncogenic functions (Ward et al. Cancer Cell 2010). Aim of the study was to define novel functional categories of AML mutations affecting relevant and druggable biological processes, with focus on genetic determinants of metabolic plasticity. Out of 455 whole exome sequencing (WES) cases from onco-hematological patients collected in the NGS-PTL project, we analyzed 37 AML cases, belonging to our cohort of 239 FLT3-WT samples (886 AML total). We performed 100 bp paired-end WES (HiSeq2000, Illumina) and mapped the sequenced reads with Burrows-Wheeler Aligner. Variants where called with MuTect or GATK for single nucleotide variant (SNV) and indels detection, respectively (>90% confidence). Gene expression profiling was performed using HTA2.0 microarray (Affymetrix) on 56 bone marrow samples, including AML (≥80% blasts) and healthy controls. By WES analysis, we detected an average of 26 somatic variants per patient (range, 7 to 65). Gene ontology annotation identified 8 novel relevant functional categories of mutated genes: transcription, translation and post-translational modifications, protein degradation, cytoskeleton, cell cycle, DNA damage, cell survival and metabolism. Since metabolic pathways are promising targets for tailored therapies (e.g. IDH1/2 and glutaminase inhibitors), we focused our analysis on them. We identified 82 variants (74 SNVs, 2 frameshift and 4 nonframeshift deletions, 2 stopgains) targeting 70 genes involved in metabolism, with 78% of patients carrying at least one mutation in a metabolic gene and 35 variants rated as damaging by CONDEL algorithm. Among mutations in metabolic genes, the most represented pathways according to Recon X database were amino acids, lipids, CoA and nucleotides metabolism, transport and bioenergetics pathways. Notably, IMPDH2, a mediator of MYC-induced proliferation involved in nucleotide interconversion, was mutated and overexpressed in our AML cohort (p=0.01), suggesting a potential oncogenic function. Moreover, ALDH2, a regulator of hematopoietic stem cell functions which is involved in multiple metabolic pathways and associates with metabolic remodeling, was mutated and 2-fold downregulated in AML blasts. Seven genes were mutated in 5-8% of samples: RETSAT, HSPG2, CHPF, ABCA2, ND1, APOBR, NAAA. Among them, RETSAT, HSPG2, CHPF mutations were also predicted as “drivers” by DOTS-Finder tool. Bioenergetics pathways were affected by mutations in glycolysis and gluconeogenesis (GPI, ITPA), oxidative phosphorylation (ND1, ND4, ND5, CYTB), pentose phosphate pathway (H6PD, PGLS). Patients carrying mutations in the bioenergetics pathway showed a strong trend towards reduced overall survival, which did not associate with unfavorable molecular mutations. In conclusion, metabolism is the most represented class of mutated genes (8.6% of variants) in our FLT3-WT AML cohort after signaling, leading us to propose a novel functional category. Our data suggest that, along with mutations in established oncogenes and tumor suppressors involved in metabolic control (KRAS, TP53, MYC pathway), a number of genetic determinants participate to leukemia metabolic plasticity and oncogenic mutations of metabolic enzymes may drive leukemogenesis, impact on patient's survival and become novel targets for personalized therapies. Acknowledgements: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. GS and AP equally contributed to this work. Citation Format: Giorgia Simonetti, Antonella Padella, Ilaria Iacobucci, Italo Do Valle, Gabriele Fontanarosa, Elisa Zago, Francesca Griggio, Marianna Garonzi, Simona Bernardi, Cristina Papayannidis, Maria Chiara Abbenante, Giovanni Marconi, Giorgio Melloni, Laura Riva, Viviana Guadagnuolo, Mariachiara Fontana, Samantha Bruno, Elisa Zuffa, Eugenia Franchini, Annalisa Astolfi, Carmen Baldazzi, Elisa Dan, Barbara Sinigaglia, Michele Cavo, Nicoletta Testoni, Emanuela Ottaviani, Pier Giuseppe Pelicci, Marco Sazzini, Alberto Ferrarini, Massimo Delledonne, Daniel Remondini, Giovanni Martinelli. European Network NGS-PTL preliminary data: Whole exome sequencing identifies mutations of ALDH2, RETSAT, HSPG2, CHPF and other metabolic genes as a novel functional category in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A27.

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and of MDM2 inhibitor p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed by genotyping 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions, in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples, and 183 healthy controls. rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20–3.33; p = 7.1 × 10−3). We hypothesized that this association reflects the capability of some ANRIL polymorphisms to contribute to its transcription changes responsible for alterations of CDKN2A/B expression profiles, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility.

Background Myelofibrosis (MF) is a clonal hemopoietic stem/progenitor cells (HSPCs) disorder with mutations in 3 driver genes (JAK2, MPL, or CALR) and inflammatory microenvironment. Triple negative (TN) patients (pts) do not carry the 3 driver mutations and display a significantly worse survival-rate which can be partially due to a greater mutation complexity. Circulating microvesicles (MVs; 0.1-1 μm), most of megakaryocytes (MKMVs) and platelets (PMVs) origin, play a role in intercellular signalling and are increased in inflammation and cancer including MF. We hypothesized that in TN pts the HSPCs and/or the inflammatory microenvironment show a more aggressive and malignant phenotype. Aims To identify a biomolecular signature of TN pts by comparatively evaluating the circulating HSPCs behaviour and their functional interplay with the inflammatory microenvironment. Methods EDTA-anticoagulated peripheral blood (PB) was collected from 16 MF pts (WHO-2016 criteria-JAK2(V617F) mutated (n=10) and TN (n=6)) and 20 age/sex-matched healthy donors (HD). Driver mutation status was obtained by PCR/NGS. Plasma MVs were isolated by ultracentrifugation (45.000 rpm for 2 hours), counted by Nanosight technology and phenotypically characterized for PMVs (CD61+CD62P+) and MKMVs (CD61+CD62P-) by flow cytometry (size 0.5-0.9 µm). in vitro survival (24 hours), CXCL12-driven migration (12 hours) and clonogenic ability of immunomagnetically isolated CD34+ cells from pts PB or cord blood (CB; n=10) was performed in the presence/absence of the isolated MVs or IL-1β, TNF-α and IL-6 (alone/ in combination). Incorporation of CFSE-labelled MVs into CD34+ cells was analyzed by confocal microscopy. MicroRNA (miRs) profile analysis of the isolated MVs and Gene expression profile (GEP) analysis of CD34+ cells from 3 JAK2(V617F) mutated, 3 TN pts and 3 HD/CB was performed. Plasma inflammatory cytokines levels were evaluated by ELISA. Results We firstly compared the TN and JAK2(V617F) CD34+ cells. TN CD34+ cells showed decreased in vitro survival and clonogenic ability but comparable migration capacity (Fig. 1 A, B, C). Consistently, comparing the GEP, the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN CD34+ cells. We then investigated whether signals from the microenvironment may affect the in vitro behaviour of the TN CD34+ cells. IL-1β, IL-6 and TNF-α plasma levels were significantly increased (p Conclusions Our results demonstrate that TN CD34+ cells show in vitro defective function and are unresponsive to the inflammatory microenvironment. Interestingly, only the autologous circulating MVs promoted the malignant clone, suggesting that MVs, as signals from microenvironment, may have a pathogenetic role in TN MF and may be the target of novel therapeutic approaches. Figure 1. Figure 1. Disclosures Fabbri: Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palandri:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction Monosomy 7 (-7) and interstitial deletions of chromosome 7 (7q-) are among the most recurrent chromosomal aberrations found in myeloid neoplasms. Patients carrying these cytogenetical alterations present a poor overall survival (OS), mainly due to a low sensitivity to standard chemotherapy and a high incidence of relapse. In our study, we aimed to disentangle the biology of patients with -7/7q- and find new candidate therapeutic targets for a disease with such a dismal prognosis, by integrating wide genomic approaches. Methods We collected bone marrow samples from 487 adult patients at diagnosis, treated in 3 institutions: Institute L. & A. Seragnoli (Italy, n = 213), CEMM (Austria, n = 160), University of Michigan (US, n = 114, GSE23452). Three hundred ninety-five samples were analyzed by SNP arrays (Affymetrix™), 51 samples by mass spectrometry (Metabolon™) and 57 samples by GEP (Affymetrix™) approaches. Chi-squared, fisher's exact test and ANOVA were used to test differences in proportion and distributions. False discovery rate, Bonferroni correction, and Welch's correction were calculated whenever appropriate. Results Among the 474 patients with evaluable karyotype, 65 (13.7%) had -7/7q-; 47 (9.9%) had -7, while 18 (3.8%) had 7q-. In our sets, the median age at AML diagnosis was 64 years (21-86) and most of the subjects had a de novo AML (65.1%). WBC count at diagnosis was significantly lower in -7/7q- patients (10.4 vs 35.2 k/mm3 p Within patients tested for FLT3, NPM1 and TP53 mutation at diagnosis, 1/50 among -7/7q- patients vs 59/300 controls harbored FLT3 ITD mutation (350 patients tested, 2% vs 19.7%, p In terms of outcome, -7/7q- AML had a median of overall survival of 10.3 months (95% C.I 5.8-14.8), which accounted for 49.5 months (95% C.I. 40.5-58.4) in other AML cases. GEP data of a cohort of 57 patients (8 with -7/7q- and 49 controls) revealed that 24 genes were under-expressed in -7/7q- AML (with e-4 significance threshold, Figure 1A). Twenty-three out of 24 genes mapped on chromosome 7; one gene, COX17, mapped on chromosome 3 (Figure 1A). COX17 plays a role in the recruitment of copper to mitochondria. By metabolomic analyses, we considered quantitative data of 300 different metabolites in 32 patients (4 with -7/7q- vs 28 controls, 19 patients were excluded because samples at diagnosis were not available). In -7/7q- AML, fatty acids (sphingomyelin, 1-linoleoylglycerol), β-cytrilglutamate and the UDP were overrepresented if compared with other AML cases; on the other hand, galactiol and 1-(1-enyl-palmitoyl)-2-linoleoyl-GPC were underrepresented in -7/7q- patients. Furthermore, -7/7q- AML cells seem to accumulate 3-hydroxy-3-methylglutarate and to have lower levels of 2-Hydroxyglutarate (Figure 1C). With SNP arrays, we considered copy number alterations in 395 patients (52 -7/7q- patients vs 343 controls, Figure 1B). 5q was the most recurrent concurrent deletion, with a minimal common deleted region (MCDR) in q31.3-q33.3. Additionally, 17p (MCDR p11.2 - p13.1), 12p (MCDR p12.3-p13.1), 16q (MCDRs q11.2-q12.1, q21-q22.1 and q24.2-q24.3), 16p (MCDR p11.2) and chromosome 4 (MCDRs q34.1 and q35.2) deletions also co-occurred in -7/7q-, listed per frequencies (Figure 1B). These regions are enriched for genes controlling cell signaling, DNA transcription, post-transcriptional modifications (such as SUMOylation), mRNA splicing and cellular senescence. Conclusions SNP, GEP and metabolomic approaches gave new insights on -7/7q- AML biology, identifying 24 new genes differentially expressed in -7/7q-, and 6 MCDR associated with -7/7q- AML. Deletion of chromosome 16q and 4 were never reported in literature associated to AML. Furthermore, for the first time, we described metabolites associated with -7/7q- AML. These data may represent a useful backbone to search for candidate targets in the setting of one of the most aggressive AML subtypes. Supported by:EHA Non-Clinical Junior Research Fellowship,HARMONY,Fondazione del Monte,FP7-NGS-PTL,AIRC. *MCF and MO equally contributed &CP and GS shared the last authorship Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Soverini:Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Martinelli:Ariad/Incyte: Consultancy; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background: The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia.\ud Design and Methods: Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels.\ud Results: We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4+CD25+ Foxp3+ T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4+CD25+ T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms’ tumor protein.\ud Conclusions: These data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.

Background: The reported TP53 mutation rate in AML is relatively low (7.5-9%, TCGA) and predict a poor prognosis. In 2017 European LeukemiaNet recommended for AML to add TP53 mutations (muts) in the risk stratification. Specific chromosomal aneuploidies are closely correlated with each other and with presence of TP53 muts. Aims: Considering that TP53 mut AML pts have HRisk and no target therapy, we would identify genes/pathways that are mainly CNA-affected (Copy Number Alteration) in the mut TP53 group compared to the wt one. Patients and Methods: 358 adult AML pts were screened for TP53 muts. 219/358 samples were genotyped with SNP arrays. CNA analyses were performed using two software to confirm or integrate karyotype data. Fisher's exact test and pathway enrichment analyses were performed. Results: We detected TP53 muts in 52/358 (14.5%) pts. Mostly (34/52) of the TP53 mut pts (65.4%) had complex karyotype. TP53 alterations were significantly associated with poor outcome (OS and EFS p Citation Format: Anna Ferrari, Eugenio Fonzi, Andrea Ghelli Luserna Di Rorà, Maria Chiara Fontana, Marco Manfrini, Carmen Baldazzi, Cristina Papayannidis, Vincenza Solli, Antonella Padella, Giovanni Marconi, Stefania Paolini, Valentina Robustelli, Enrica Imbrogno, Eugenia Franchini, Perricone Margherita, Maria Chiara Abbenante, Giorgia Simonetti, Nicoletta Testoni, Emanuela Ottaviani, Michele Cavo, Giovanni Martinelli. Distinct pattern of alterations in TP53 mutated/deleted and wild-type high risk acute myeloid leukemia (AML) patients: Identification of new "targetable" genes/pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 656.

The main reason for the unfavorable clinical outcome of BCR-ABL1-positive acute lymphoblastic leukemia (ALL) is genetic instability. However, how normal B-cell precursors acquire the genetic changes that lead to transformation has not yet been completely defined. We investigated the expression of the activation-induced cytidine deaminase (AID) and its role in clinical outcome in 61 adult BCR-ABL1-positive ALL patients. AID expression was detected in 36 patients (59%); it correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors. Different AID splice variants were identified: full-length isoform; AIDDeltaE4a, with a 30-bp deletion of exon 4; AIDDeltaE4, with the exon 4 deletion; AIDins3, with the retention of intron 3; AIDDeltaE3-E4 isoform without deaminase activity. AID-FL predominantly showed cytoplasmic localization, as did the AID-DeltaE4a and AID-DeltaE3E4 variants, whereas the C-terminal-truncated AID-DeltaE4 showed a slightly increased nuclear localization pattern. AID expression correlated with a higher number of copy number alterations identified in genome-wide analysis using a single-nucleotide polymorphism array. However, the expression of AID at diagnosis was not associated with a worse prognosis. In conclusion, BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that may act as mutators outside the immunoglobulin (Ig) gene loci in promoting genetic instability.

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1–positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Δ4-7) and by removal of exons 2 through 7 (Δ2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Δ4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Δ2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Leukemic cells spontaneously secrete cytokines involved in the proliferation of the clone; in this study we evaluated the effects of all-trans retinoic acid (ATRA) on the in vitro autocrine production of cytokines by acute myeloid leukemia cells. Thirty acute nonlymphoid leukemia cases (ANLL) (10 APL and 20 ANLL of other cytotypes than APL) were studied; the in vitro secretions of IL-1 alpha, IL-3, IL-4, IL-6, IL-10, G-CSF, GM-CSF, TNF-alpha were tested with and without ATRA addition. After 5 d exposure to ATRA 10(-6) M APL-treated samples showed a significant reduction of IL-6 (p = 0.008) and GM-CSF (p = 0.03) and a significant increase of IL-1 alpha (p = 0.01) production, if compared to untreated APL samples. No difference was seen in IL-3, IL-10 and IL-4 productions; G-CSF production resulted absent in all but 3 APL cases, in which addition of ATRA determined increase in the production. Interestingly, the 3 G-CSF-producing cases did not obtain clinical remission with ATRA; GM-CSF and IL-6 were spontaneously produced by all the cases, and 7 of 10 APL patients subsequently obtained complete remission after induction. TNF-alpha was produced only in 1 case. No statistical difference was seen in all the productions obtained from other than promyelocytic acute leukemic cells, both with and without ATRA addition. However, it is noteworthy that the production of IL-6 was more than twice as high in ANLL non-APL than in APL cases. In conclusion, these data could thus suggest possible complementary mechanisms of the exhaustion of the leukemic clone upon treatment with ATRA.

We have previously reported that the antineoplastic activity of 3'-azido 3' deoxythymidine (AZT) can be increased by drugs that inhibit "de novo" thymidylate synthesis, such as 5-fluorouracil, methotrexate and hydroxyurea. In the present study we tested the combinations AZT+alpha interferon (IFN) and AZT+gamma IFN on in vitro growth of the human acute-phase chronic myeloid leukemia (CML) cell line K562. After 72 hours incubation, not only AZT+alpha-IFN but also AZT+gamma-IFN were synergistic in inhibiting K562 growth, as demonstrated by isobologram analysis of the data. This enhanced cytotoxicity was confirmed by the evaluation of [3H]AZT incorporation into cellular DNA, that was increased by 50% and 222% in the presence of alpha- and gamma-IFN, respectively. The addition of 50 mumol/l thymidine to the culture medium was able to reduce the cytotoxicity of the drug combinations to the degree observed with each compound alone; furthermore, the increased incorporation of AZT into DNA was completely reversed. These data indicate the existence of a biochemical interaction between AZT and IFNs that results in an increased cytotoxic effect. While the combination AZT+alpha-IFN is currently being tested in HIV-related malignancies, AZT+gamma-IFN is new and deserves further study in human CML acute and chronic phase models, in view of possible clinical applications.

We report a case of a 65-year old patient affected by neurofibromatosis 1, documented by the presence of germ-line mutation on the NF1 gene, who developed various hyperproliferative malignant and benign diseases. He was brought to our attention for the diagnosis of acute myeloid leukemia revealed by major fatigue and dyspnea. The disease characteristics at diagnosis were hyperleukocytosis and complex karyotype with the inversion of the chromosome 16, classifying as a high-risk leukemia. The association between leukemia and neurofibromatosis 1 is controversial and needs to be further investigated. Nevertheless, such patients present a wide number of comorbidities that make therapeutic strategies most difficult.

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

Background Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Methods The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent n-ethyl-n-nitrosourea. Results Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia. Conclusions In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0206-5) contains supplementary material, which is available to authorized users.

Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.

Abstract 76 Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases regulated by Ataxia-Telangiectasia and Rad3-related (ATR) kinases and involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Deregulation of these pathways has been previously described in BCR-ABL positive cells and involved in chemoresistance. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, in this study we aimed to investigate the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in Ph+ ALL and to determine potential biomarkers of functional inhibition. We first examined Chk1 and Chk2 mRNA expression levels in 45 newly diagnosed Ph+ ALL patients, in their paired remission samples, in 14 relapsed cases and in 3 Ph+ cell lines (BV-173, SUPB-15 and K562) by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation). Higher transcript levels of Chk1 but not Chk2 were found in newly diagnosed patients compared to remission samples (p = 0.0009 for Chk1 and p= 0.8183 for Chk2). Chk1 transcript levels were comparable between diagnosis and relapsed cases (p = 0.5728), suggesting that the ATR-Chk1 pathway is strongly activated in Bcr-Abl-positive cells. This was confirmed by phosphorylation of Chk1 Ser317 by western blotting analysis in Ph+ cell lines. We then evaluated the effect of PF-0477736 as single agent on cell viability using the Cell Proliferation Reagent WST-1 (Roche). BV-173, SUPB-15 and K562 cell lines were incubated with increasing concentration of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1–0.5 μM, with BV-173 being the most sensitive, while K562 the most resistant. These results were confirmed in primary blasts cells from a Ph+ ALL patient with wild-type Bcr-Abl and from 3 cases harboring the T315I Bcr-Abl mutation found to be insensitive to the available TKIs (IC50 ranged from 0.1–0.5 μM at 48 hours). Consistent with the WST-1 results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in both cell lines and primary cells. To test whether increased apoptosis resulted from Chk1 inhibition, we assessed the changes in phosphorylation of Cdc25c phosphatase, which is inactivated by Chk1 to prevent mitotic entry, and of γH2AX, which is increased in response to DNA damage. Western blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 and increases levels of γH2AX. Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening by amplification and subsequent sequencing of all coding exons of p53. All cell lines except for K562 and primary leukemia cells lacked mutations in the p53 gene, demonstrating that in Ph+ ALL PF-0477736 is highly effective also on p53 wild-type tumor cells. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on 3 treated Ph+ cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p < 0.05) of 211 genes including those involved in apoptosis and cell cycle (CEBPB, CUL1, Histone H1-H2A, 2B family clusters, Histone H4, DHX15, SNCB, FOS) and DNA damage, such as DNA-damage-inducible transcript 3 (DDIT3) and growth-arrest and DNA damage-inducible proteins GADD34 and GADD45a, suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in Ph+ ALL cells. In conclusion, for the first time we demonstrate in a large cohort of Ph+ ALL patients an over-expression of Chk1, providing a strong rational for its inhibition. In vitro treatment of Ph+ ALL cells with PF-0477736 alone resulted in reduction of inhibitory phosphorylation of Cdc25c, inhibition of proliferation and induction of biomarkers of DNA damage and apoptosis, suggesting that single-agent Chk1/2 inhibition may be an effective treatment strategy for Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Introduction: Novel array-based technique as SNP microarray can detect losses or gains of chromosomic material, which could be predictive of response and can help define therapeutic strategies. The aim of this study is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis, by a SNP array-based genotyping approach. Materials and Methods: We performed SNP 6.0 or Cytoscan HD (Affymetrix) in 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number v8.0 (BioDiscovery) and R Core Team. Results: Copy Number Alterations (CNAs) were scattered across all chromosomes and all pts showed CNA events. SNP array analysis showed that several genes were preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), while the genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p Conclusion: We have identified new CNAs and pathways involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes implicated in deregulated pathways deserve further investigation in order to identify new candidate genes which could be relevant AML biomarkers. Acknowledgements: ELN,AIL,AIRC,progetto Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY. Citation Format: Maria Chiara Fontana, Giovanni Marconi, Cristina Papayannidis, Eugenio Fonzi, Giorgia Simonetti, Antonella Padella, Anna Ferrari, Emanuela Ottaviani, Silvia Lo Monaco, Stefania Paolini, Simona Soverini, Giovanni Martinelli. Genomic wide microarray analysis identifies novel copy number alterations in adult acute myeloid leukemia [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 2451. doi:10.1158/1538-7445.AM2017-2451

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

Introduction PI3P is a molecule that regulate 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). AMPK plays a role as a regulator of cellular energy homeostasis. Aims The aim of the this study is to define the role of PI3P pathways and AMPK pathway in AML. Methods In this work 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. We performed Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD in all the patients; we performed 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 selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). We analyzed the gene in two different pathways. PI3K/AKT/mTOR pathway includes the following genes: pik3ca, cdkn1a, akt1, akt3, mtor and pten, pdk1,pik3r1 and irs1. The second one is AMPK pathway and it include: sesn, prkaa1, prkab1, prkag1, prkag3. Alterations in PI3K/AKT/mTOR pathway confer worst OS (p = .035) when compared with unaltered patient, but events in these pathways did not affect therapy response. Alterations in AMPK pathway confer worst OS (p Conclusions Our work investigates the role of PI3P and cAMP pathways in AML. Surprisingly, it showed that alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP pathways were associated with therapy resistance. Acknowledgement: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12, FP7 NGS-PTL project, HARMONY Citation Format: Mariachiara Abbenante, Mariachiara Fontana, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Elena Tenti, Eugenia Franchini, Anna Ferrari, Sarah Parisi, Emanuela Ottaviani, Nicoletta Testoni, Viviana Guadagnuolo, Chiara Sartor, Silvia Lo Monaco, Cristina Papayannidis, Giovanni Martinelli. Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia 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 515. doi:10.1158/1538-7445.AM2017-515

Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR) by recruiting BRCA1, BRCA2 and RAD51. Alterations in PALB2 were described in hereditary breast cancer and Fanconi Anemia (FA). Little is known in Acute Myeloid Leukemia (AML). Aim of the study is to define the frequency and interplay of PALB2 alterations with patterns of somatic mutations and copy number variants (CNVs) in AML. We genotyped 233 AML samples by Single Nucleotide Polymorphism array (SNP 6.0 and Cytoscan HD, Affymetrix) to detect CNVs. We also performed Whole Exome Sequencing (WES, Illumina) of 56 cases to detect single nucleotide variants and small indels (MuTect and Varscan 2.0). Differences in survival were assessed by Kaplan-Meier survival analysis and Breslow test. We detected PALB2 loss in 12/233 patients (5%), with a minimal common region of deletion of 6.6 Kb including exon 12, which encodes for the domain of interaction with RAD51, BRCA2 and POLH. PALB2 deletion were correlated with CN loss of MYH11, CREBBP, PLK1 and FANCA on chromosome (chr) 16 (p≤.005), deletions of TP53, NF1 and BRCA1 on chr 17 (p≤.004), 5q deletions (p≤.001) and loss of XPO1 on chr 2 (p≤.006). PALB2-loss patients were enriched for alterations in genes involved in the protein kinase pathway (p=5×10-4), JUN kinase activity (p=.0006) and, notably, in genes belonging to the chromosome breakage pathway (p=.001; TP53, BRCA1 and BRCA2). To identify mutations that co-operates with CN alterations, we integrated WES data of 56 AML patients. No mutations in PALB2 were detected. However, we identified mutations in other DDR genes, including FANCE, BRCA2, TP53 and BRCA1. Of note, alterations in the DDR genes frequently co-occurred with both mutations and CN loss of TP53, leading to homozygous loss of function of TP53. In addition, KRAS, IDH1, TET2 and BCOR mutations were mutually exclusive with PALB2 loss. Preliminary gene expression profiling data revealed a 2-fold upregulation of FANCA in samples with DDR genes alterations compared with patients with no alterations (p=.04), while the other genes of the pathway were not affected. Finally, patients harboring PALB2 loss had a poorer prognosis compared complex karyotype cases (p=.021). We here define a new subset of AML patients, characterized by the synergistic loss of key DDR genes, which may in turn result in FANCA over-expression as a mechanism to overcome the DDR deficit. Our data suggest that the leukemogenic process associated PALB2 loss integrates defects in the DDR together with enforced proliferative stimuli leading to uncontrolled proliferation of cells, which potentially accumulate genetic lesions. These results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Maria Chiara Fontana, Marco Manfrini, Giovanni Marconi, Anna Ferrari, Italo Faria do Valle, Marianna Garonzi, Cristina Papayannidis, Eugenia Franchini, Elisa Zuffa, Viviana Guadagnuolo, Samantha Bruno, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Daniel Remondini, Massimo Delledonne, Giovanni Martinelli. Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia 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 4671. doi:10.1158/1538-7445.AM2017-4671

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

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

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

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph(+) acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph(+) ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph(+) ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph(+) ALL patients. (Blood. 2008; 112:3847-3855)

none 24 si BACKGROUND: Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. DESIGN AND METHODS: In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. RESULTS: We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. CONCLUSIONS: Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia. Iacobucci I; Lonetti A; Cilloni D; Messa F; Ferrari A; Zuntini R; Ferrari S; Ottaviani E; Arruga F; Paolini S; Papayannidis C; Piccaluga PP; Soverini S; Saglio G; Pane F; Baruzzi A; Vignetti M; Berton G; Vitale A; Chiaretti S; Müschen M; Foà R; Baccarani M; Martinelli G. Iacobucci I; Lonetti A; Cilloni D; Messa F; Ferrari A; Zuntini R; Ferrari S; Ottaviani E; Arruga F; Paolini S; Papayannidis C; Piccaluga PP; Soverini S; Saglio G; Pane F; Baruzzi A; Vignetti M; Berton G; Vitale A; Chiaretti S; Müschen M; Foà R; Baccarani M; Martinelli G.

Abstract 692 Background and Aims: In chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant Bcr-Abl mutants. Mutation status of resistant patients is usually investigated by Sanger sequencing (SS) of the Bcr-Abl kinase domain (KD). Novel ultra-deep sequencing (UDS) technologies allow to conjugate higher sensitivity with the unprecedented possibility to perform instant cloning of thousands of DNA molecules. We thus decided to take advantage of an UDS-based approach in order to: 1. resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs; 2. investigate their clonal structure and evolution in relation to time and treatment. Methods: We retrospectively performed a longitudinal analysis of a total of 111 samples from 35 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs (two to four TKIs among imatinib, dasatinib, nilotinib, ponatinib) and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS; 74/111 (67%) were positive for one (n=33) or multiple (n=41) mutations. UDS of the Bcr-Abl KD was done using Roche 454 technology. UDS allowed to achieve a lower detection limit of at least 0.1% – as compared to 20% of SS. Results: Bcr-Abl KD mutation status was found to be more complex than SS had previously shown in 85/111 (77%) samples (representative examples are detailed in Table 1 ). In 33/74 (44%) samples known to harbour one or more mutations by SS, UDS revealed that up to four ‘minor' mutations with 1–20% abundance were present in addition to the ‘dominant' one(s). The type of mutations could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the current or to the previous TKI received. The higher degree of complexity was evident also when the clonal relationships of multiple mutations were reconstructed ( Table 1 ). This revealed that identical mutations may be acquired in parallel by independent populations (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). In addition, longitudinal quantitative follow-up of mutated populations revealed that: 1. complexity generally increases with increasing lines of TKI therapy; 2. with a few exceptions, double compound mutants have higher selective advantage over single mutants but also over triple; 3. however, whether a compound mutant will ultimately take over depends on TKI, treatment duration, competition with other coexisting populations - the same compound mutants behaved differently in different patients receiving the same TKI. Conclusions: 1. sequential changes in the selective pressure exerted by TKIs may result in a heterogeneous mosaic of subclones harbouring different mutations or mutation combinations; 2. the evolution of each subclone is shaped not only by its inherent sensitivity to the specific TKI administered (‘absolute' fitness) but also by the competition with all other coexisting subclones (‘relative' fitness); (nonlinear) acquisition of additional mutations dictates further dynamics of shrinkage/expansion over time; 3. information provided by SS may not always be sufficient to predict responsiveness to a TKI; 4. sensitivity of a single or a compound mutant to a TKI in vivo is dictated by more complex factors than the mere in vitro IC50 value. Table 1 . Sample TKI (line) Mutations by SS (%) Mutations by UDS (%) Mutated populations by UDS (%) CML-04 DAS (3rd, after IM and NIL) T315I (∼60) E255K (∼30) T315I (54.89) E255K (26.15) E255V (1.37) T315I (47.02) E255K (18.63) T315I+E255K (7.52) E255V (1.02) T315I+E255V (0.35) ALL-09 PON (3rd, after IM and DAS) E255K (∼70) T315I (∼50) E255K (76.25) T315I (52.19) Q252H (cag>cac) (14.20) Q252H (cag>cat) (7.49) G250E (0.95) E255K+T315I (51.60) E255K+Q252H (cag>cac) (13.94) E255K+Q252H (cag>cat) (7.34) T315I (5.75) E255K (2.52) E255K+ G250E (0.70) Q252H (cag>cac) (0.25) G250E (0.25) Q252H (cag>cat) (0.15) CML-21 DAS (2nd, after IM) Y253H (∼60) F317L (∼60) Y253H (54.90) F317L (54.40) Y253H+F317L (43.00) Y253H (11.90) F317L (11.40) Disclosures: Soverini: ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Castagnetti: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Luppi: CELGENE CORPORATION: Research Funding. Rosti: Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. 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.

Heat shock proteins (HSPs) are a group of highly conserved polypeptides involved in cellular response to heat or other physical or chemical stresses. It has been recently reported that HSPs could play a role in cellular differentiation. In this study we have evaluated, by a cytofluorimetric method, the presence of HSP-70 in HL-60 cells during treatment with all-trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA), and 13-cis retinoic acid (13-cis RA). After 1 and 3 days of incubation at 10(-7) M, HSP-70 did not show any variation compared to control; prolonging the exposure, together with the appearance of cellular differentiation along the granulocytic pathway and apoptosis, a progressive decrease of HSP-70 was observed that, after 8 days of treatment, was reduced by 40% with ATRA and by 28% with 9-cis RA compared to untreated samples, while only minimal changes were evident by incubating the cells with 13-cis RA. Reduction of HSP-70 was not associated with decreased protein synthesis, as demonstrated by [3H] leucine incorporation. Double labeling with propidium iodide showed a decrease in HSP-70 in all the phases of the cell cycle concomitant with a reduced percentage of cycling cells in ATRA-treated samples. Dot blot and Northern blot analysis demonstrated no change in HSP-70 mRNA after retinoid treatment, thus suggesting a post-transcriptional regulation of the phenomenon. This reduced production of HSP-70 caused by ATRA and by 9-cis RA, though to a lesser extent, could render the cells more sensitive to cytotoxic agents and could provide the rationale for the efficacy of ATRA + chemotherapy combinations.

Genetic heterogeneity in drug-metabolizing enzyme/transporter (DMET) genes affects specific drug-related cancer phenotypes. To investigate the relationships between genetic variation and response to treatment in acute myeloid leukemia (AML), we genotyped 1931 variants on DMET genes in 94 CD33-positive AML patients enrolled in a phase III multicenter clinical trial combining Gemtuzumab-Ozogamicin (GO) with Fludarabine-Cytarabine-Idarubicin (FLAI) regimen, with the DMET Plus platform. Two ADH1A variants showed statistically significant differences (odds ratio (OR)=5.68, P=0.0006; OR=5.35, P=0.0009) in allele frequencies between patients in complete/partial remission and patients without response, two substitutions on CYP2E1 (OR=0.13, P=0.001; OR=0.09, P=0.003) and one on SLCO1B1 (OR=4.68, P=0.002) were found to differently influence liver toxicity, and two nucleotide changes on SULTB1 and SLC22A12 genes correlated with response to GO (OR=0.24, P=0.0009; OR=2.75, P=0.0029). Genetic variants were thus found for the first time to be potentially associated with differential response and toxicity in AML patients treated with a combination of GO-FLAI regimen.

We investigated whether members of the protein kinase C (PKC) family of enzymes were involved in the nuclear events underlying granulocytic differentiation induced by 10(-6) M all-trans retinoic acid (ATRA) in HL-60 cells. PKC activity was analysed by using a serine substituted specific peptide which enabled the evaluation of the whole catalytic activity of both Ca2+ -dependent and Ca2+ -independent PKC isoforms. In parallel, the subcellular distribution of various PKC isoforms was evaluated by Western blot, immunoprecipitation and in situ immunocytochemistry analyses. The level of PKC catalytic activity in the nuclei of HL-60 cells significantly (P < 0.01) and progressively increased from 1 h of ATRA treatment onwards. Consistently, PKC-alpha and -zeta showed a striking and selective accumulation inside the nucleus upon treatment with ATRA. On the other hand, PKC-beta I and -beta II, the only two other isoforms present at nuclear level, did not show any significant modification upon ATRA treatment. The remaining PKC isoforms were not detectable inside the nucleus and showed only modest and non-significant variations, also in whole cell homogenates, upon ATRA treatment, except PKC-delta which showed a progressive down-regulation. Our data suggest that a selective nuclear translocation of PKC-alpha and -zeta might be involved in the process of granulocytic differentiation induced by ATRA in HL-60 cells.

Chromosome gain or loss, which is the hallmark of aneuploidy, occurs in about 10% of adult Acute Myeloid Leukemia (AML) cases (Farag et al. IJO 2002, Breems et al. JCO 2008), despite inducing a dramatic reduction of cellular fitness in non-malignant cells (Torres et al. Science 2007). The study aimed to identify AML-specific molecular mechanisms having a causative and/or tolerogenic role towards aneuploidy. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 38 aneuploid (A) and 34 euploid (E) AML cases, identified according to cytogenetic analysis and SNP array (CytoScan HD, Affymetrix). Variants were called with GATK, MuTect and VarScan. We also compared the transcriptomic profile of leukemic bone marrow cells from 21 A-AML and 28 E-AML cases (HTA 2.0, Affymetrix). A-AML showed a significantly higher mutation load compared with E-AML (median number of variants: 25 and 15, respectively, p Our data show a link between aneuploidy and genomic instability in AML and highlight novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid phenotype. Deregulation of the cell cycle machinery and DNA damage/repair checkpoints, either through mutations, copy number and transcriptomic alterations, cooperate with leukemogenic pathways, as KRAS signaling, to develop A-AML and overcome the unfitness barrier. This evidence suggests that a number of A-AML patients may benefit from pharmacological reactivation of TP53 and inhibition of KRAS pathway. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Citation Format: Giorgia Simonetti, Antonella Padella, Marco Manfrini, ĺtalo Faria do Valle, Cristina Papayannidis, Carmen Baldazzi, Maria Chiara Fontana, Viviana Guadagnuolo, Anna Ferrari, Elisa Zago, Marianna Garonzi, Simona Bernardi, Annalisa Astolfi, Maria Chiara Abbenante, Giovanni Marconi, Elisa Zuffa, Eugenia Franchini, Ilaria Iacobucci, Michele Cavo, Emanuela Ottaviani, Nicoletta Testoni, Alberto Ferrarini, Massimo Delledonne, Torsten Haferlach, Daniel Remondini, Giovanni Martinelli. A cell cycle-related genomic and transcriptomic signature distinguish aneuploid and euploid acute myeloid leukemia. [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 90.

Acute Myeloid Leukemia (AML) is a clonal hematopoietic disorder characterized by an abnormal proliferation and differentiation of immature blast cells in the bone marrow. SNP microarray approach has resulted in genome-wide screening for genomic alterations with information not previously achievable and also it allows to map all the genes involved in these alterations which may plays a role in oncogenesis. Our objective is to evaluate the prognostic impact of these genetic alterations on clinical outcome. We analyzed 285 AML samples at diagnosis of which 221 by SNP Array 6.0 (Affymetrix) (Affymetrix), while 64 by by CytoScan HD Array (Affymetrix). The results obtained were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 softwares (BioDiscovery). The 285 AML patients (pts) analyzed are equally distributed between both sexes, they have a median age of 60 years, include miscellaneous cytogenetic abnormalities and normal karyotype. The 44% of pts are treated with chemotherapy (CHT), the 16% of pts with low-dose cytosine arabinoside, the 20% of pts with 5-azacytidine and the 19% of pts are not treated or had only supportive therapy. The ratio between responders and non responders pts was 57/43. Each pt presented an average of 1448 events so distributed: 31% of homozygous deletions, 32% of homozygous amplifications, while irrelevant events are related to heterozygous deletions and amplifications. We also found 36% of uniparental disomy (UPD)events. Among all these macroscopic and submicroscopic alterations, we focused on JAK2 gene, which is located on human chromosome 9 at p24.1 locus. It is composed of 25 exons and encodes a 1132 amino-acid protein of 130.7 kDa. This gene was amplified in 12/285 pts (4%) and deleted in 13/285 (5%). All these pts are treated with CHT but only those characterized by JAK2 deletion are responsive in first line. We showed that the group of pts which present this deletion had a better overall survival rate than the group with the amplification of this gene (p-value < 0,01). The deletion event of JAK2 gene involved two different regions: the first region had a length of 7.429 bp involving the intron 4 and was found in the database genome variant (DGV); the second deletion presented a variable length between different pts: the smallest deleted region had a length of 3342 bp and included the exons 17, 18, 19, while the largest one had a length of 76903 bp which included the portion from exon 9 to exon 19. This second deletion has not been described in DGV and it involved the pseudokinase and kinase domain of JAK2 protein. We have identified Copy Number Variation involving important cancer genes in AML. We have identified a new JAK2 deletion involving its Pseudokinase and Kinase domain and we observed that this deletion of JAK2 correleted with overall survival. ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project Citation Format: Viviana Guadagnuolo, Maria Chiara Fontana, Cristina Papayannidis, Marco Manfrini, Antonella Padella, Giorgia Simonetti, Anna Ferrari, Giovanni Marconi, Andrea Ghelli Luserna di Rorà, Stefania Paolini, MariaChiara Abbenante, Sarah Parisi, Chiara Sartor, Emanuela Ottaviani, Giovanni Martinelli. New JAK2 heterozygous loss: A role in overall survival in acute myeloid leukemia patients. [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 4507.

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.