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The WHO recognizes tMN as a distinct disease entity. The majority of tMN arise after treatment of solid tumors with cytotoxic chemotherapy or radiation therapy. In many tMN patients, clonal hematopoiesis was demonstrated to be already present at diagnosis of the solid tumor and later gave rise to the tMN. Cases of tMN following successful treatment of AML are rare and poorly characterized. We therefore set out to study the genetic profile of these patients and evaluated if AML and subsequent tMN are two genetically distinct diseases or share a common ancestral clone. We selected cases of AML sent to our laboratory for diagnostic work-up from 2005 to 2018 who subsequently developed a tMN. To exclude events of AML relapse from the cohort, known AML driver mutations such as in NPM1 or AML defining rearrangements present at diagnosis (Dx) of AML had to be absent at Dx of tMN. Based on sample availability, whole exome sequencing (WES) was carried out for 25 pts at the time points of Dx of AML and Dx of tMN as well as on matched remission samples that were available for 19 pts. Enrichment based library preparation was performed using the xGen Exome Research Panel and sequenced (2x151bp) on a NovaSeq instrument. Data was processed with BaseSpace using the BWA Enrichment app with BWA for Alignment (against hg19) and GATK for variant calling with default parameters. Data was subsequently loaded into BaseSpace Variant Interpreter to filter and prioritize variants of interest. Only passed protein changing variants were considered with an ExAC population frequency of less than 1% for further analysis. The cohort included 13 females and 12 males, aged 28 to 77 years (median: 60 yrs). Median time to Dx of tMN following Dx of AML was 42 months (range: 8-96 months). Thirteen pts had developed tMDS and 12 pts tAML. While WES identified numerous recurrently mutated genes, we focused our further analyses on 28 genes associated with myeloid neoplasms and detected a total of 97 mutations (1-8 mutations per patient, median: 4). Following genes were mutated in >15% of pts: NPM1 (14/25 of pts), DNMT3A (9/25), TET2 (9/25), SRSF2 (7/25), CEBPA (6/25), RUNX1 (6/25), ASXL1, FLT3, IDH2 and TP53 (4/25 of pts, each). We did not identify a mutation pattern distinguishing between pts who developed a tAML or tMDS, however, pts with tAML had a significantly higher total number of mutations than those with tMDS (median of 5 vs 3 mutations, p= 0.022). When comparing matched initial AML and subsequent tMN samples for each patient, a total of 43 mutations were detected at Dx of AML only, while 25 mutations were exclusive to the tMN sample. Interestingly, 18/25 (72%) of pts harbored mutations present at both time points (Figure 1). Moreover, in 8/22 of pts with available material, mutations in DNMT3A (n=6), IDH1, SRSF2 and TET2 (n=1, each) persisted during remission. Of the 7 pts without shared mutations between time point of AML and tMN, 2 were initially positive for the PML-RARA rearrangement, and 1 patient showed a CBFB-MYH11 rearrangement at Dx of AML. Next, we compared gene mutation data with cytogenetic information available for 21/25 pts at both time points. Nine patients with a normal karyotype (NK) at Dx of AML developed chromosome aberrations at Dx of tMN. In this group, 8/9 pts harbored mutations in epigenetic modifiers (DNMT3A, TET2 and SRSF2) present both at AML and tMN. Similarly, 2/3 patients with NK AML and NK tMN shared mutations in IDH2, SRSF2 and ASXL1 at both time points. Eight pts showed either independent cytogenetic aberrations between AML and tMN or harbored chromosome aberrations at Dx of AML that were absent at Dx of tMN. Even in this group of cytogenetically independent clones, 3/8 pts harbored the same mutations at both time points. Our study provides novel insights into the pathogenesis of tMN. In the majority of analyzed cases, we detected mutations present in the AML and tMN clones of the same patient. Considering the absence of known AML driver gene mutations such as in NPM1 in the tMN sample, we speculate the presence of a common ancestral clone with mutations mostly affecting epigenetic modifiers which have previously been linked to clonal hematopoiesis. Unlike the full-blown leukemia, this clone is refractory to chemotherapy and later gives rise to the tMN. The absence of shared mutations between AML and tMN in patients with PML-RARA or CBFB-MYH11 rearrangement is in line with thought that these entities are true de novo AML that do not evolve from a clonal hematopoiesis. Disclosures Hartmann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Vetro:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Purpose : Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL. Experimental design : First, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N=40) and validation (N=78) sets. Results : s-Arg-1 was higher in patients with advanced stage (p=0.045), B-symptoms (p=0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p=0.012). Baseline levels of s-Arg-1 >200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2. Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p=0.0004 and p=0.012). Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N=63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N=46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N=9) with a 3-Y PFS of 37% (p=0.0004). Conclusions : We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a potential novel potential biomarker for HL prognosis. Disclosures Vetro: MLL Munich Leukemia Laboratory: Employment. Chiarenza:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Background: In 15-20% of CLL cases no aberrations are detected by chromosome banding analysis (CBA) and FISH due to limited resolution, lack of evaluable metaphases or presence of aberrations in loci not covered by standard-panel FISH probes. As reported in our previous study (Haferlach C. et al., ASH 2015, abs ID#79545), genomic arrays (GA) detected abnormalities in almost 20% of cases classified as normal by CBA and FISH and these showed an impact on time to first treatment (TTT) (Vetro C. et al., EHA 2016, abs ID# E1069). The CLL subgroup without abnormalities in CBA, FISH, and GA has not been characterised in detail, so far. Aims: 1) to describe CLL without abnormalities by CBA/FISH/GA by evaluating an extended gene panel, the IGHV mutation status and the B-cell receptor (BCR) stereotypy; 2) to determine prognostic impact of these factors. Patients and Methods: CLL diagnosis was based on cytomorphology and immunophenotyping according to standard guidelines. From a cohort of 1190 patients at diagnosis, 133 (11%) were selected based on normal karyotype by CBA, no abnormalities by interphase FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), centromeric region of chromosome 12 and t(11;14)(q13;q32) (IGH-CCND1) and no abnormalities by GA (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). IGHV mutation status and BCR stereotypy were determined according to Agathangelidis et al., Blood 2012, and DNA sequencing was performed for the following genes: ATM; SF3B1; TP53; KLHL6; KRAS; MYD88; NOTCH1; NRAS; POT1; FBXW7; HIST1H1E; XPO1; ITPKB; MAPK1; BIRC3; BRAF; DDX3X; EGR2; RIPK1; RPS15; CND2. Results: Median age was 66 years (range: 33-83). Median follow-up was 5.6 years, 33 patients (25%) received treatment since genetic analyses. 10-year overall survival (OS) was 76% and median TTT was 9.2 years. Mutations were observed in 53 patients (40%): SF3B1 (n=17; 13%); NOTCH1 (n=10; 8%); KLHL6 (n=6; 5%); TP53 (n=6; 5%); ATM (n=5; 4%); XPO1 (n=4; 3%); FBXW7 (n=3; 2%); MYD88 (n=3; 2%); DDX3X (n=2; 2%); POT1 (n=2; 1.5%); ITPKB (n=1; 1%); KRAS (n=1; 1%); NRAS (n=1; 1%); and no mutation in RPS15, CCND2, MAPK1, EGR2, BRAF, HIST1H1E, RIPK1, BIRC3. 6 patients had 2 simultaneous gene mutations and 1 patient had 3 (i.e. NOTCH1, ATM and TP53). A mutated IGHV status (IGHV-M) was present in 100 patients (75%) and an unmutated IGHV status (IGHV-U) in 33 patients (25%). IGHV-U was related to both the occurrence of any gene mutation (p In Kaplan-Meier analysis, IGHV-M patients did not reach a median TTT, while IGHV-U had a median of 5.1 years (p Only age showed an impact on OS (RR: 1.2 per decade, p Conclusions: 1. The CLL subset without any genomic event by CBA/FISH/genomic array is characterized by very low frequency of IGHV-U status; 2. IGHV-U subgroup showed higher gene mutation rate compared to IGHV-M subgroup, in particular higher NOTCH1, XPO1 and POT1 mutation rate; 3. BCR stereotypy is less frequent than in CLL in general. 4. IGHV-U, as well as the higher disease burden (i.e. % CLL cells), has an independent negative impact on TTT. 5. Requirement for treatment is low and prognosis very favorable in CLL without any genomic event by CBA/FISH/genomic array and a mutated IGHV status. Disclosures Vetro: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Background In the last decades, evaluation of thrombotic complications secondary to acute leukemia (AL) has been poorly investigated. Only scant data are available on management and prevention of thrombosis in this setting. We performed a multicenter retrospective study with the aim to evaluate the management of venous thromboembolism (VTE) in patients with AL and to report the most commonly adopted regimens of treatment. Materials and methods Available clinical records of out and in-patients diagnosed with AL from January 2008 to June 2013 in 7 Reference Regional Hospitals were analyzed. Cases of VTE, including thrombosis in atypical sites [Retinal occlusion (RO) and Cerebral Sinus Thrombosis (SCT)], were reported. All data were recorded in a dedicated electronic database. The patient’s basic demographic data (age, gender, race), medical history, disease-related information, and laboratory data were extracted. Instrumental diagnosis of deep vein thrombosis (DVT), pulmonary embolism (PE) and RO and SCT was performed according to ACCP guidelines. Data were collected and analysed by the IBM SPSS Software 21.0 version (SPSS, Inc., Chicago, Ill, US) and the Epi Info software, version 3.2.2, (Centers for Disease Control and Prevention). Statistical analysis of quantitative and qualitative data, included descriptive statistics, was performed for all the items. Results Over a population of 1461 patients with AL, 99 (6.8%) cases of VTE were recorded, mainly in hospitalized patients: 72 cases were associated with Acute Myeloid Leukemia (AML) and 27 with Acute Lymphoblastic Leukemia (ALL), with a mean age of 52.2 ± 15.4 years (median age: 53years). In particular the incidence/ratio over the sub-population of AML-patients was 6.0%, that is 72/1191 cases; with a mean age of 54.7 ± 14.3 years (median age: 57 years). VTE occurred during chemotherapy (CHT) in 90/99 (90%) cases, mainly during the induction phase of treatment (in 70% of cases ),the remaining 9 cases were diagnosed in concomitance with acute leukemia. In both subgroups with VT, there was no statistical significant difference between time at diagnosis of VT and time at diagnosis of AL. Treatment of VTE was mainly based on Low Molecular Weight Heparin (LMWH), in accordance with results from previous studies and current guidelines (full dosage for the first month from diagnosis and reduced dosage at 75% for the following months). Thrombocytopenia occurred in 55 patients at diagnosis of AL, in 33 cases platelets were Conclusion VTE can complicate the clinical course of AL in a not negligible percentage of cases. Anticoagulant treatment schedules and duration in patients with AL are influenced by many factors, mainly related to chemotherapy and severe thrombocytopenia. In the analyzed subset of patients, full dose treatment with LMWH for at least one month followed by a dose reduction for at least three months was appropriate. Disclosures No relevant conflicts of interest to declare.

Background: In Hodgkin Lymphoma (HL) elevated neutrophil (HL-N) count is a well-known negative prognostic factor but its biological meaning is not elucidated. Our previous work showed that HL-N are dysfunctional and can suppress T-cell activation in vitro, as consequence of increased amount of Arginase-1 (Arg-1). Aim: Investigating clinical meaning of arginase increase in HL, correlating its amount to features at diagnosis, including some semiquantitative parameters of 18-FDG- PositronEmission Tomography (PET) acquired with a novel operator-independent algorithm. Material and methods: We prospectively measured soluble Arg-1 (s-Arg-1) in 135 sera obtained from 90 patients with HL, distinguished in a training set (N=35) and a validation set (N=55) and 20 healthy participants. In the training set, blood was taken at three fixed time-points prior, during, and after first-line therapy. In the first ten patients, a correlation between s-Arg-1 and semiquantitative parameters of PET at diagnosis was explored, including the Metabolic Tumor Volume (MTV) and the Total Lesion Glycolysis (TLG). Briefly, our group developed ad hoc tool independent from the operator: PET images are represented as a graph in which the voxels are its nodes and the edges are defined by a cost function which maps a change in image intensity to edge weights. This approach is an efficient and accurate method to segment lesion in low contrast images characterized by noise and weak edges as metabolic images (Stefano, 2013). Results: s-Arg-1 was increased in HL patients compared to healthy subjects, reduced after therapy in responders and increased in relapsed patients (p In the training set, 32% patients had high s-Arg-1, 24% had positive PET-2 and were addressed to an early salvage therapy accordingly to BEACOPP scheme. A level of 205 ng/mL s-Arg-1 resulted in 83% (95% C.I. 58-96) sensitivity and 81% (95% C.I. 42-96) specificity in predicting response status in the training set (area under curve, AUC, 0.81, p=0.02). In the validation set, baseline levels of s-Arg-1>205 ng/mL resulted in 83% (C.I. 95% 62-95) sensitivity and 87% (C.I. 95% 47-99) specificity in predicting response status. Patients with s-Arg-1 ≥ 205 ng/mL had shorter PFS than patients carrying Arg-1 < 205 ng/mL (despite both groups did not reach the median, because of the short follow-up, p=0.005). In first 10 patients enrolled in the study, semiquantitative parameters of PET at diagnosis were explored: SUVmax was 12.7 (range 5.9-14.2), MTV median was45.5 (range 8.9-308.7), TLG mean was 43.7 (range 25.2-2475.3). MTV and TLG, but not SUVmax, were positively correlated to s-Arg-1 (respectively, r=0.68, p=0.003 and r=0.59, p=0.002). Conclusion S-Arg-1 is a predictor of PFS in HL and it is positively correlated with MTV in PET scans at baseline calculated with a novel operator-independent tool for imaging analysis. An update will be provided at the conference. Disclosures No relevant conflicts of interest to declare.

Introduction Our previous work showed that in Multiple Myeloma (MM) the immune function is impaired, including immunosuppressive properties of granulocytes due to their increased amount of arginase-1 and reduced phagocytic activity (Parrinello, manuscript in preparation). It is currently unknown if granulocyte dysfunction occurs in progression from MGUS to MM. Aim Providing a gene expression profile of mature granulocytes isolated from peripheral blood at the steady-statein MGUS and MM. Methods Using oligonucleotide microarrays we first evaluated the gene expression profile of granulocytes at the steady state in 5 MM, 3 MGUS and 3 healthy subjects matched for sex and age. Then, we validated the first up-regulated gene PROK-2, obtained from preliminary findings in granulocytes from peripheral blood in 85 consecutive newly diagnosed MGUS (N=45), MM (N=40) and 15 healthy subjects, in RT-PCR (validation set). Results We found 708 genes differentially expressed (467 up- and 241 down regulated) in MGUS versus healthy granulocytes at the steady state. The set of annotated, differentially expressed genes could befunctionally organized by “gene ontology” (http://www.geneontology.org/) into the following major categories: i) receptors and signal transduction (including up-regulation of CD14, Toll-like receptor 5 (TLR-5), IL-7 Receptor (CD127), IL-11 receptor, TGF-beta receptor 2, hematopoietic cells kinase (HCK), IFNAR1); ii) negative regulation of adaptive immune response (including up regulation of CD127, STAT6, IFNAR1, OSCAR, PROK-2 and down regulation of p50, p65,NFKBIA, IL8, ELK-1, HIF-1 alpha, CEBP-beta, CEBP-zeta). In MM samples we confirmed a statistically significant up-regulation of PROK-2 (a key molecule of VEGF-independent angiogenesis), CD14 (mediator hypersensitive innate immune response to lipopolysaccharide) and HCK (the hematopoietic cell kinase, involved in neutrophil migration and degranulation). In the validation set, PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics. Conclusion Granulocytic impairment is present in MGUS and worsened in MM patients due to increased expression of genes that negatively regulate adaptive immune response. PROK-2 is a key molecule involved in the granulocyte dysfunction and could be involved in the progression from MGUS to MM. Disclosures Musto: Celgene: Honoraria; Janssen: Honoraria.

Background Inflammation dominates both the histological and the clinical pictures of Hodgkin’s Lymphoma (HL) and there are several clues that accessory cells such as neutrophils and macrophages have an important role in the development and progression of the disease and are well recognized negative prognostic factors. Materials and Methods In order to identify an additional HD marker with prognostic significance, we evaluated 70 HL patients for circulating levels of Myeloid Derived Suppress Cells (MDSC), identified as CD34+, CD45+, CD11b+, CD33+, CD14- in peripheral blood by flow cytometry at diagnosis. All patients were treated with ABVD but those patients who showed an interim-PET (PET-2) positivity were switched to BEACOPP followed by autologous transplant. In addition, neutrophils (N) obtained from 15 HL patients we tested for phagocytic activity, enzymatic activity of arginase (ARG-1) (a molecule able to suppress T-lymphocytes activity), expression of ARG-1 and pro-angiogenic factor PROK-2, and suppression of healthy T-lymphocytes activation in co-culture experiments. Results In HL patients the mean absolute count of MDSC at diagnosis was higher than healthy controls (2.7±0.2 cells/uL versus 1.6±0.1 cells/uL), correlated with PET-2 positivity and identified all except one (3 out of 4) patients with relapse/progression despite a PET2-oriented therapy. MDSC count at diagnosis added further prognostic information to PET-2 since, among the 62 PET-2 negative patients, 4 relapsed, all of them with high MDSC count at diagnosis. On the whole,13 patients out of 70 (19%) presented with MDSC count higher than 4.5 cells/uL (cut off identified by ROC analysis) at diagnosis and 9 (69%) of them had a negative event: 5 were PET-2 positive and 4 relapsed within 18 months. On the contrary, only one patient with low MDSC count at diagnosis had progression of disease. In term of PFS, MDSC count and PET-2 had a similar predictive value (respectively p= In vitro studies showed an increase of ARG-1 expression in N-HL up to 100 folds and of PROK-2 up to 36 folds compared to healthy subjects matched for age and sex (p=0.001), independently from tumor load and other well-known prognostic factors. N-HL exhibited a reduced phagocytosis (73.1 ± 3.7 vs 93.2 ±1.9 %, p=0.0008) and an increased arginase activity up to 15 times compared to healthy subjects matched for age and sex. Finally, we co-cultured lymphocytes isolated from healthy subjects (h-Ly) with neutrophils isolated from fresh peripheral blood of HL patients (HL-N) or healthy subjects (h-Ne). After PHA-P stimulation, the activation markers CD69, CD25 and CD71 were increased in h-Ly while their expression was down-regulated by co-culture with HL-N (but not h-Ne) at ratio 1:4 and 1:8 at all tested time-points. Conclusion MDSC are increased in peripheral blood of HL patients at diagnosis, correlate with interim PET, and have a strong prognostic value, earlier and more easily accessible than interim PET. Neutrophils isolated from HD patients have a reduced phagocytic activity, produce an angiogenic factor (PROK-2) and high amount of arginase and are able to reduce normal lymphocytes activation. These findings represent a paradigma of how a myeloid compartment may favor the development of a lymphoid neoplasia through T-cell impairment and encompasses a prognostic significance. Disclosures: No relevant conflicts of interest to declare.

Abstract 1831 Introduction In Multiple Myeloma (MM), but not in the monoclonal gammopathy of unknown significance (MGUS), the immune function is impaired as consequence of an immunologically hostile microenvironment and cellular defects, including reduction of immuno-surveillance and T-cell immunoparesis. We conducted an study focused on the myeloid compartment in MM, and its role in the progression from MGUS to MM. Methods Between January 2009 and April 2011 peripheral blood obtained from 60 consecutive newly diagnosed MM and 70 MGUS plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis. Myeloid dysfunction was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-like mo-MDSC (CD14+/HLA-DRlow/-). Myeloid function was evaluated by phagocytic activity using a commercially available kit (Phagotest R). Further, we investigated whether MM-neutrophils were able to induce anergy in T-cells. Neutrophils isolated from healthy donor (N=6), MGUS (N=3) or MM (N=6) peripheral blood (PB) were co-cultured with T-lymphocytes obtained from healthy donors. Expression of markers of activation in response to stimulation with PHA-P for 2 hours was assessed by flow cytometry as antigen density expressed as normalized mean of fluorescence intensity (N-MFI) of CD71 at 48 hours. Results The capability of phagocytosis of in neutrophils and monocytes from MM patients at diagnosis was significantly reduced compared to healthy subjects (p After PHA-P stimulation, expression of CD71 (a marker of activation) in normal T-lymphocytes was increased (2954 ± 240.6 arbitrary units, au), and it was reduced (751.3 ± 30.48 au, p=0.0001) when co-coltured with MM-neutrophils, while no differences were evident in co-colture with MGUS- (2783 ± 206.1 au, p=0.61) or healthy donors-neutrophils (2588 ± 135.4, p=0.38). Conclusion Taken together, our findings suggest that in MM but not in MGUS there is a myeloid cell dysfunction that is correlated to impairment of T- cell arm. These alterations may have a role in the development of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 3818 Background Conventional cytogenetics is the major prognostic factor in predicting outcome of myelodysplasia (MDS), but at least 50% of patients have a normal karyotype. 5-azacytidine (5-AZA) is a DNA methyltransferase inhibitor used in treatment of high-risk MDS, which is able to delay the progression to AML and improve clinical outcomes, but targets of the methylation status are poor known. Aims To evaluate molecular changes, at genomic or proteomic level, which can be identified as additional targets of 5-azacytidine in MDS patients with normal conventional cytogenetics. Methods By reverse-phase protein microarray (RPMA), we analyzed bone marrow mononuclear cells from 19 patients affected of high risk MDS, treated with 5-azacytidine (median age 71 years, M/F=12/5). Treatment consisted of 4 cycles of 100mg flat dose for 7 days+21 days of wash-out. In 7 cases the sample after 4 cycles of therapy was matched to the sample collected before starting treatment. RPMA was used to quantitatively map 45 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. For the first 4 patients we used also a genome-wide approach based on SNP (single nucleotide polymorphism) array (6.0 Affymetrix platform) to detect copy number and allelotype data in order to identify new genetic markers in terms of SNP, CNV and LOH. Results All patients were evaluable for response one month after the 4th cycle. Three patients were refractory and progressed to AML and 1 was a late responder (after 7 cycles). All other patients experienced hematologic improvement. After 4 cycles of 5-AZA, at proteomic level we found three main signaling pathways were increased and did not correlate with the response: 1) pro-survival signaling: PLC-y-1-Tyr783 (p=0.0017), and its upstream regulators Src-Tyr416, c-Abl-Tyr735 (p=0.002) and downstream target STAT5Tyr694 (p=0.0017) were increased, without affecting proliferative pathways, such as AKT activation status on Ser473 and Thr308 or mTORSer2448. 2) Autophagy: ATG5, Beclin 1 and LC3B were significantly elevated after treatment (p values respectively At genomic level, we identified 46 losses and 25 gains. In three cases we found a deletion on chromosome 20q13.12 (from 46125 kb to 46178 kb) at level of NCOA3 gene, a transcriptional coactivator protein that contains several nuclear receptor interacting domains and an intrinsic histone acetyltransferase activity. In 2 cases we found a loss in the region 22q13 (from 40871 kb to 40910 kb), at level of MKL1 gene, encoding a nuclear protein involved in transduction signals from the cytoskeleton to the nucleus. MKL1 is involved in a specific translocation event associated with acute megakaryocytic leukemia. Moreover in one case we found a deletion on chromosome 4q24 (from 105800 kb to 107200 kb) in a region involving TET2 gene. Loss of heterozygosity 4q24 and TET2 mutations have been implicated in the pathogenesis of myelodysplastic syndromes. Another patient showed autozigosity regions with an extensive CN-LOH on chromosomes 7 and 11, involving the centromers and recently their neoplastic potential in colon cancer have been reported and never, before, observed in MDS. Conclusion More informative findings can be detected using a proteomic approach in combination with the genomic one. Autophagy activation can be considered an escape pathway promoting survival in neoplastic cells and the observation that 5-AZA does not affect proliferative pathways suggests the possibility to combine it with anti-proliferative agents. The increase of HDAC1 and HDAC3 can provide the molecular rationale for the development of a combination of 5-azacytidine with HDAC inhibitors. Disclosures: No relevant conflicts of interest to declare.