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Background Erythropoiesis‐stimulating agents effectively improve the hemoglobin levels in a fraction of anemic patients with myelodysplastic syndromes (MDS). Higher doses (HD) of recombinant human erythropoietin (rhEPO) have been proposed to overcome suboptimal response rates observed in MDS patients treated with lower “standard doses” (SD) of rhEPO. However, a direct comparison between the different doses of rhEPO is lacking. Methods A cohort of 104 MDS patients treated with HD was retrospectively compared to 208 patients treated with SD in a propensity score‐matched analysis to evaluate hematological improvement‐erythroid (HI‐E) rate induced by the different doses of rhEPO. The impact of rhEPO doses on survival and progression to leukemia was also investigated. Results Overall HI‐E rate was 52.6%. No difference was observed between different rhEPO doses (P = .28) in matched cohorts; in a subgroup analysis, transfusion‐dependent patients and patients with higher IPSS‐R score obtained a higher HI‐E rate with HD, although without significant impact on overall survival (OS). Achievement of HI‐E resulted in superior OS. At univariate analysis, a higher HI‐E rate was observed in transfusion‐independent patients (P, When compared in a propensity‐score matched analysis, standard doses of rhEPO showed similar effects to those of higher doses in anemic MDS patients. Achievement of favorable response with hematologic improvement in this clinical scenario is possible by reducing drug doses and costs.

Introduction T-cell prolymphocytic leukemia (T-PLL) is a rare T-cell-neoplasm that is characterized by uncontrolled proliferation of mature T-cell lymphocytes with a very poor prognosis. Nowadays, alemtuzumab as a single agent is the best available option for both treatment-naive and relapsed/refractory patients; however, its efficacy is hampered by various adverse events (AEs) such as infusion-associated reactions, autoimmune AEs, and opportunistic infections. Objective We conducted a monocentric, retrospective study to evaluate the safety and efficacy of bendamustine, which is a bifunctional mechlorethamine derivative with alkylating and antimetabolite activities, as first-line therapy in patients unfit for alemtuzumab. Patients and Methods We collected clinical and biological data of 7 T-PLL patients treated with bendamustine between 2014 and 2020 in our institution. Four out of seven (57%) were males, the median age at diagnosis was 73 years (65–83 years). The median time from diagnosis to treatment was 15 months. Bendamustine was administered intravenously (90 mg/m2 on days one and two, every four weeks) for up to six cycles. Results Overall response rate was 57%, 3 patients achieved complete response (43%), and one partial response (14%). One patient experienced stable disease, and two patients progressed during treatment. Five patients (71%) experienced a relapse. Median overall survival (OS) was 63.7 months (19–108); median OS for responding patients was 187 months versus 24 months for non-responding patients (p=0.093). Median progression-free survival (PFS) was 12 months (11–13). Three patients received second-line therapy: one patient was treated with gemcitabine; the others received a second bendamustine course. Two patients received a third-line therapy with combination chemotherapy: COMP (prednisone, cyclophosphamide, vincristine, myocettm) and CVP (cyclophosphamide, vincristine, prednisone). Two patients experienced infusion-associated reactions. Four patients experienced hematological toxicities: one thrombocytopenia G3, 3 neutropenia G3 requiring G-CSF administration. Four patients received co-trimoxazole prophylaxis, one received acyclovir prophylaxis. One patient experienced herpetic retinitis. Premature termination ( Conclusions Our data are consistent with scientific literature. This treatment is feasible and safe, with an acceptable toxicity profile. Although alemtuzumab remains the standard of care of this rare disease, bendamustine might be a valid option either for unfit patients or for undeferrable disease.

Clinical guidelines recommend the use of erythropoietin-stimulating agents (ESA) in anemic patients with lower risk myelodysplastic syndrome (MDS)[1][1]–[4][2] and two registration trials for ESA have just been completed.[5][3],[6][4] We conducted a retrospective study in MDS patients selected by

Myelodysplastic Syndromes (MDS) are clonal neoplasms where stem/progenitor cells endowed with self-renewal and capable of perpetuating the disease have been demonstrated. It is known that oxygen tension plays a key role in driving normal hematopoiesis and that hematopoietic stem cells are maintained in hypoxic areas of the bone marrow (BM). Hypoxia could also regulate leukemic/dysplastic hematopoiesis. We evaluated the stem cell potential of MDS cells derived from the BM of 39 MDS patients and selected under severe hypoxia. MDS cells rescued from hypoxia-incubated cultures were subjected to stem and progenitor cell assays in vitro, as well as to hematopoietic reconstitution assay in NOD-SCID mice. Incubation in severe hypoxia of cells explanted from MDS patients selected a cell subset endowed with stem cell potential, as determined in vitro. This occurred only from the BM of patients classified as IPSS low/INT-1 risk. Transplantation into NOD-SCID mice confirmed using an in vivo model that severe hypoxia selects a cell subset endowed with stem cell potential from bone marrow mononuclear cells (BMMC). derived from patients belonging to the IPSS low/int-1 risk group. Data here reported show that cells endowed with stem cell potential and capable of adapting to hypoxia and escaping hypoxia-induced apoptosis exist within MDS cell populations.

Karyotype according to the revised IPSS is a strong independent prognostic factor for overall survival (OS) in myelodysplastic syndromes (MDS), however established in untreated patients. The prognostic impact of cytogenetics and cytogenetic response (CyR) in MDS patients receiving azacitidine (AZA) remains uncertain. We examined the prognostic value of baseline cytogenetics and CyR for overall response rate (ORR) and OS in 702 AZA-treated higher risk MDS and low blast count acute myeloid leukemia (AML), including 493 (70%) with abnormal karyotype. None of the cytogenetic abnormalities had significant impact on ORR (43.9%) or complete response (15.35%), except 3q abnormalities and complex karyotypes, which were associated with a lower ORR. OS differed significantly across all R-IPSS cytogenetic subgroups (p

Venetoclax is the first BCL-2 inhibitor approved for the treatment of patients with chronic lymphocytic leukemia (CLL) alone or in combination with anti-CD20 monoclonal antibodies (MoAbs). Initial studies have shown high efficacy of venetoclax monotherapy or in combination with rituximab with an overall response rate of 79-92% in patients with relapsed/refractory (R/R) CLL. To investigate the use of venetoclax combinations in a real-world population, we designed this observational retrospective and prospective study aimed at defining the outcome of patients with R/R CLL treated with venetoclax-based regimens outside clinical trials in Italy. Efficacy (progression-free survival -PFS-, overall response rate -ORR-, complete response -CR-, measurable residual disease -MRD-) and safety (most frequent adverse events -AE-, grade 3-4 AE, laboratory and clinical tumor lysis syndrome -TLS-) data were collected within the study through a web-based platform after patients signed written informed consent. As of July 8 th, 2021 124 subjects (85 males, 39 females) were enrolled into the study (Table 1). Median age at venetoclax initiation was 70 years (44-91), median CIRS score 4 (0-14), median creatinine clearance 70 ml/min (22-123). A relevant proportion of patients presented adverse prognostic features, including TP53 aberrations [32% TP53 mutation and/or del(17p)], unmutated IGHV (77%). At the time of venetoclax initiation, patients had received a median of 2 previous therapies (1-8), with 57% previously exposed to BTK and/or PI3K inhibitors. 67/117 (57%) patients received venetoclax alone, while 50 were treated with a combination of venetoclax + rituximab (VenR), information on treatment plan was missing in 7 patients. Patients on venetoclax monotherapy compared to those receiving VenR showed a higher percentage of elevated LDH at baseline (64% vs 38%), were more heavily pretreated (median lines of therapy 3 vs 1), and had more frequently received treatment with ibrutinib (66% vs 27%) and/or idelalisib + rituximab (29% vs 4%). After a median follow-up of 13.7 months (0-41.9), the estimated 12m-PFS was 82% (CI 74-90%) (Figure 1), and the estimated 12m-overall survival (OS) was 83% (CI 76-91%). The best ORR in the whole cohort was 85% (CI 95% 76-91%) with a CR rate of 40%; best response was reached after a median of 3.9 months of treatment (range 0.6-30.5). The best ORR was not different in patients receiving VenR vs venetoclax alone (83% vs 88% respectively, p = n.s.), while the CR rate was significantly higher in those receiving VenR vs venetoclax alone (57% vs 30%, p=0.011). The ORR and CR rate in patients previously exposed to BTK and/or PI3K inhibitors were significantly lower (77% and 29% respectively), and the 12m-PFS was shorter in patients previously treated with ibrutinib compared to ibrutinib-naïve (75% vs 89%, p=0.005). Twenty subjects discontinued venetoclax (median time to discontinuation 4.1 months, range 0.4-10.8), 8 due to disease progression, 3 Richter's transformation, 7 AEs, 1 was lost to follow-up and 1 underwent planned allogeneic stem cell transplantation. Venetoclax-based regimens were well tolerated, with the most frequent AE of any grade related to venetoclax being neutropenia (79.6%), grade 3-4 in 61.9% (Table 2). In the whole cohort one grade 3 clinical TLS occurred and resolved without sequelae, and only 2/124 patients experienced laboratory TLS during ramp-up phase (both grade 1). No treatment-related death was reported. In conclusion, this analysis presents the results of one of the largest cohort of patients with R/R CLL treated with venetoclax-based regimens outside clinical trials and confirms the favourable efficacy and safety profile of the drug. In this patient population highly enriched for unfavorable disease features (1 out of 3 carrying TP53 aberrations, almost 80% with unmutated IGHV) venetoclax-based regimens were able to obtain a response in a high proportion of cases, with a very short time to best response (less than 4 months). As expected, response duration was shorter in patients previously exposed to BTKi/PI3Ki. When we compared the outcome of patients treated with venetoclax monotherapy vs VenR, the addition of anti-CD20 MoAb allowed to reach deeper responses by significantly increasing the CR rate. As the study and its data collection are still ongoing, the updated analysis of an expanded cohort with longer follow-up will be presented at the meeting. Figure 1 Figure 1. Disclosures Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Quaglia: Roche: Other: Travel grants; Janssen: Honoraria, Other: Travel grants, Speakers Bureau; Sandoz: Consultancy; AstraZeneca: Honoraria. Marasca: AbbVie: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; Janssen: Honoraria, Other: Travel grants. Sanna: Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Murru: Abbvie: Consultancy, Honoraria, Other: travel and accommodation; Janssen: Consultancy, Honoraria. Laurenti: Roche: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria; BeiGene: Honoraria. Gaidano: Incyte: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Reda: Beigene: Consultancy; Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Molica: Astrazeneca: Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Sportoletti: AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Galimberti: AbbVie, Janssen: Honoraria, Other: Travel grants; Incyte: Speakers Bureau. Mauro: AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Cuneo: Janssen: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau. Ghia: AstraZeneca: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; ArQule/MSD: Consultancy, Honoraria; Sunesis: Research Funding.

BACKGROUND: Anemia is the most frequent cytopenia in myelodysplastic syndromes (MDS), neoplastic diseases characterized by ineffective hematopoiesis and bone marrow dysplasia. MDS usually affect elderly individuals (median age in Italy 74 years), and quite rarely concern younger patients. It is of increasing interest to verify whether "young", not therapy-related MDS patients carry germline (GM) predisposition, in order to optimize therapeutic and transplant choices. The best approach is to perform Whole Exome Sequencing (WES) instead of targeted next-generation sequencing (t-NGS) panel in cases in which MDS patients have no syndromic signs or any indicative sign of predisposing alterations. AIM: We considered a cohort of 21 consecutive MDS cases with age at diagnosis < 60 years and anemia, referred to our MDS Unit for a second opinion. METHODS: After bone marrow (BM) re-evaluation and morphological confirmation of dysplasia compatible with MDS, we screened the 21 MDS patients by WES on BM-DNA or peripheral blood DNA (PB-DNA). For DNA sequencing, DNA libraries were sequenced using the NextSeq 550 Illumina System. Variant calling was carried out using GATK caller. T-NGS myeloid panel was in some cases applied to confirm somatic nature of mutations. Samples of 5/21 MDS patients and their relatives were further analyzed after WES by applying high-coverage t-NGS 86-gene custom panel for hereditary anemias on PB-DNA. WES of DNA extracted from saliva (S-DNA) samples was performed to provide a GM confirmation. In order to confirm pathogenic role in red blood cell (RBC) membrane defects of identified variants, we also performed the ektacytometry analysis that evaluates the erythrocyte deformability and the hydration status by subjecting them to an increasing osmotic gradient with constant shear stress. RESULTS: Five of 21 MDS cases (23.8%) with symptomatic anemia (3 MDS-MLD, 1 MDS-SLD, 1 MDS with isolated del5q) were identified as carriers of variants suspected responsible of RBC membrane defects in both BM/PB-DNA and S-DNA samples, where no other germline myeloid predisposing alterations were demonstrated. MDS-associated somatic variants were present in 4/5 patients (KRAS, TET2, CBL, SRSF2, U2AF1, IDH2, with VAF >25 %). High coverage t-NGS analysis confirmed the presence of a heterozygous rare missense variant in PIEZO1 in probands 1 and 5; a heterozygous rare missense variant in PIEZO1 and ANK1 in proband 2; a heterozygous rare missense variant in PIEZO1 and SPTA1 in proband 3; a complex mode of inheritance in proband 4 with causative variants in G6PD, KCCN4, SPTB, and ABCB6 genes. PIEZO1 or KCNN4 gene variants result in erythrocyte dehydration and determine dehydrated hereditary stomatocytosis (DHS). Ektacytometry analysis showed a significant difference in the osmolarity curve of these patients, confirming the pathogenic role of the variants identified. Among these MDS suspected cases, only proband 4 was diagnostically re-categorized because of lack of acquired recurrent somatic mutations and presence of multiple alterations in erythroid genes causing dysplasia of erythroid precursors, masquerading as MDS. None of the patients presented a history of unexplained anemia before the onset of MDS, consistent with what shown for DHS1 patients with well-compensated hemolysis. Inheritance pattern of the identified variants according the study of the families confirmed the segregation of the pathogenic variants. CONCLUSION: Onset of de novo MDS in unusually young age should always prompt investigation of predisposing conditions. We suggest here that co-existence of inherited RBC membrane defects with MDS may determine earlier clinical manifestation of the clonal disease. Hereditary anemias, in particular xerocytosis, may mimic MDS morphology. In our study, among the 5 "young" MDS with concomitant inherited RBC membrane defect, one case did not finally confirm MDS diagnosis by karyotype or NGS myeloid mutation analysis. This case fulfilled the diagnostic criteria for combined hereditary hemolytic anemia: DHS2, G6PD deficiency, spherocytosis, familial pseudohyperkalemia 2, with multi-locus mode of inheritance that justifies the clinical condition (splenomegaly, hemolytic crisis, ascites). Addition of genetic testing for hereditary anemias beside assessment of germline predisposing variants may allow a more precise diagnosis in controversial cases of "young" MDS. Disclosures Sanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Santini: Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Context: Standard treatment of anemia in lower-risk myelodysplastic syndromes (LR-MDS) is based on erythropoietic-stimulating agents (ESAs), but some patients are primary refractory (PR) or lose response (secondary refractory, SR). High serum erythropoietin (sEPO) is the main negative predictor, and treatment of ESA-resistant anemia is challenging, so more predictors are needed. EPO promotes differentiation of committed erythroid cells (COMe) into erythroid precursors (EP), constituted by early (EEP) and late (LEP) precursors. Flow cytometry (FC) can characterize COMe and EP. RED score is an FC parameter that assesses dyserythropoiesis. Objective: We aimed to correlate COMe, EP, and RED score with response to ESAs, WHO category, and sEPO in 44 anemic LR-MDS patients. Design: We evaluated 10 negative controls, 17 PR patients, 17 SR patients, and 10 long responders (LR, response duration >24 months). Fifty-nine percent had sEPO 500 mU/mL(H-EPO); 23% had single lineage dysplasia (MDS-SLD), 34% multilineage dysplasia (MDS-MLD), 23% ring sideroblasts (MDS-RS), and 20% deletion of 5q (MDS-5q-). FC analysis was performed on bone marrow aspirates for all subgroups at diagnosis and within SR subgroup at loss of response to ESA (SRII). We used anti-CD34, CD36, CD105, CD117, CD33, CD71, and CD45 antibodies to identify COMe, total erythroid cells (TOTe), and EP. Coefficients of variance of CD71 and CD36 were used to calculate RED score. Results: TOTe and COMe were increased in LR-MDS compared to controls (p=0.013 and p=0.04, respectively), with the exception of MDS-5q- cases. In the PR subgroup, LEP were increased and COMe were reduced compared to LR. In the SRII group, COMe were lower than at diagnosis (p=0.039). RED score was higher in PR compared to responders (p=0.011). MDS-5q- cases showed reduced TOTe (p=0.047), while MDS-RS cases had higher LEP and RED score (p=0.008 and p=0.012, respectively). Finally, LEP were increased in the H-EPO group compared to L-EPO (p=0.023). Conclusions: These data suggest that COMe seem to be positively associated with ESA response, whereas higher LEP and RED score are negatively associated and also correlate with MDS-RS and H-EPO. Finally, MDS-5q- cases displayed fewer TOTe and COMe, suggesting premature apoptosis of erythroid precursors.

Somatic mutations contribute to the heterogeneous prognosis of chronic myelomonocytic leukemia (CMML). Hypomethylating agents (HMAs) are active in CMML, but analyses of small series failed to identify mutations predicting response or survival. We analyzed a retrospective multi-center cohort of 174 CMML patients treated with a median of 7 cycles of azacitidine (n = 68) or decitabine (n = 106). Sequencing data before treatment initiation were available for all patients, from Sanger (n = 68) or next generation (n = 106) sequencing. Overall response rate (ORR) was 52%, including complete response (CR) in 28 patients (17%). In multivariate analysis, ASXL1 mutations predicted a lower ORR (Odds Ratio [OR] = 0.85, p = 0.037), whereas TET2mut/ASXL1wt genotype predicted a higher CR rate (OR = 1.18, p = 0.011) independently of clinical parameters. With a median follow-up of 36.7 months, overall survival (OS) was 23.0 months. In multivariate analysis, RUNX1mut (Hazard Ratio [HR] = 2.00, p = .011), CBLmut (HR = 1.90, p = 0.03) genotypes and higher WBC (log10(WBC) HR = 2.30, p = .005) independently predicted worse OS while the TET2mut/ASXL1wt predicted better OS (HR = 0.60, p = 0.05). CMML-specific scores CPSS and GFM had limited predictive power. Our results stress the need for robust biomarkers of HMA activity in CMML and for novel treatment strategies in patients with myeloproliferative features and RUNX1 mutations., Highlights • TET2mut/ASXL1wt genotype predicts higher complete response rate and prolonged survival in CMML with hypomethylating agents. • Conversely, RUNX1mut and CBLmut genotypes are associated with poorer outcome, independently of higher leukocyte count. • CPSS and GFM prognostic scores showed modest performance when calculated at initiation of hypomethylating agents. Somatic mutations contribute to the heterogeneous prognosis of chronic myelomonocytic leukemia (CMML). Hypomethylating agents (HMAs) are active in CMML. Response and survival in MDS and AML patients treated with HMAs is difficult to predict. We explore the predictive role of recurrent somatic mutations in a large retrospective cohort of 174 HMA-treated CMMLs. Consistent with MDS studies, we report a higher response rate in TET2mut/ASXL1wt patients. We also identify a CMML-specific molecular pattern (RUNX1mut or CBLmut) associated with shorter survival. Our results can inform treatment decision in CMML, for instance by using HMAs prior to transplant in TET2mut/ASXL1wt patients.

Chronic myelomonocytic leukemia (CMML) is a complex clonal hematological disorder classified among myelodysplastic (MDS)/myeloproliferative neoplasms. Prognosis is poor and there is a lack of effective treatments. The hypomethylating agent decitabine has shown activity against MDS and elderly acute myeloid leukemia, but there is little data focusing specifically on its efficacy in CMML. In this prospective, phase 2 Italian study, CMML patients received intravenous decitabine 20 mg/m2 per day on Days 1–5 of a 28-day treatment cycle. Response was evaluated after four and six cycles; patients responding at the end of six cycles could continue treatment with decitabine. Forty-three patients were enrolled; >50% were high-risk according to four CMML-specific scoring systems. In the intent-to-treat population (n=42), the overall response rate after six cycles was 47.6%, with seven complete responses (16.6%), eight marrow responses (19%), one partial response (2.4%) and four hematological improvements (9.5%). After a median follow-up of 51.5 months (range: 44.4–57.2), median overall survival was 17 months, with responders having a significantly longer survival than non-responders (P=0.02). Grade 3/4 anemia, neutropenia and thrombocytopenia occurred in 28.6%, 50% and 38% of patients, respectively. Decitabine appears to be an effective and well-tolerated treatment for patients with high-risk CMML.

Purpose Most anemic patients with non-deleted 5q lower-risk myelodysplastic syndromes (MDS) are treated with erythropoiesis-stimulating agents (ESAs), with a response rate of approximately 50%. Second-line treatments, including hypomethylating agents (HMAs), lenalidomide (LEN), and investigational drugs, may be used after ESA failure in some countries, but their effect on disease progression and overall survival (OS) is unknown. Here, we analyzed outcome after ESA failure and the effect of second-line treatments. Patients and Methods We examined an international retrospective cohort of 1,698 patients with non-del(5q) lower-risk MDS treated with ESAs. Results Erythroid response to ESAs was 61.5%, and median response duration was 17 months. Of 1,147 patients experiencing ESA failure, 653 experienced primary failure and 494 experienced relapse after a response. Primary failure of ESAs was associated with a higher risk of acute myeloid leukemia (AML) progression, which did not translate into an OS difference. Of 450 patients (39%) who received second-line treatment, 194 received HMAs, 148 received LEN, and 108 received other treatments (MISC), whereas 697 received RBC transfusions only. Five-year AML cumulative incidence was 20.3%, 20.3%, and 11.3% for those receiving HMAs, LEN, and MISC, respectively ( P = .05). Five-year OS for patients receiving HMA, LEN, and MISC was 36.5%, 41.7%, and 51%, respectively ( P = .21). In a multivariable analysis adjusted for age, sex, revised International Prognostic Scoring System score, and progression at ESA failure, there was no significant OS difference among the three groups. Conclusion In this large, multicenter, retrospective cohort of patients with non-del(5q) lower-risk MDS treated with ESAs, none of the most commonly used second-line treatments (HMA and LEN) significantly improved OS. Early failure of ESAs was associated with a higher risk of AML progression.

Summary Iron chelation is controversial in higher risk myelodysplastic syndromes (HR-MDS), outside the allogeneic transplant setting. We conducted a retrospective, multicentre study in 51 patients with transfusion-dependent, intermediate-to-very high risk MDS, according to the revised international prognostic scoring system, treated with the oral iron chelating agent deferasirox (DFX). Thirty-six patients (71%) received azacitidine concomitantly. DFX was given at a median dose of 1000 mg/day (range 375–2500 mg) for a median of 11 months (range 0·4–75). Eight patients (16%) showed grade 2–3 toxicities (renal or gastrointestinal), 4 of whom (8%) required drug interruption. Median ferritin levels decreased from 1709 μg/l at baseline to 1100 μg/l after 12 months of treatment (P = 0·02). Seventeen patients showed abnormal transaminase levels at baseline, which improved or normalized under DFX treatment in eight cases. One patient showed a remarkable haematological improvement. At a median follow up of 35·3 months, median overall survival was 37·5 months. The results of this first survey of DFX in HR-MDS are comparable, in terms of safety and efficacy, with those observed in lower-risk MDS. Though larger, prospective studies are required to demonstrate real clinical benefits, our data suggest that DFX is feasible and might be considered in a selected cohort of HR-MDS patients.

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a unique disorder caused by a PIG-A gene mutation in a stem cell clone. Its clinical picture can sometimes make challenging the distinction from other disorders, and especially from myelodysplastic syndromes (MDS), since both diseases correlate with cytopenias and morphological abnormalities of bone marrow (BM) cells. Recently, flow cytometry (FC) has been proposed to integrate the morphologic assessment of BM dysplasia, and thus to improve the diagnostics of MDS. Methods. In the present study, we have analyzed systematically FC data resulting from the study of BM cells from patients with PNH and MDS. Results. Our data demonstrated abnormalities in PNH beyond the deficiency of glycosylphosphatidylinositol-linked proteins and the application of a systematic approach allowed us to separate effectively MDS and PNH in a cluster analysis and to highlight disease-specific abnormalities. Indeed, the parallel evaluation of some key parameters, i.e. patterns of expression of CD45 and CD10, provided information with practical diagnostic usefulness in the distinction between PNH and MDS. Moreover, the hypo-expression of CD36 that we observed on monocytes might be related to the thrombotic tendency in PNH. Conclusions. We investigated systematically the phenotypic profile of BM cells from patients with PNH; our data provide useful antigenic patterns to solve between PNH and MDS, sometimes morphologically overlapping. Moreover, some PNH-related phenotypic changes might be involved in the physiopathology of the disease and further studies addressing this issue are warranted. © 2013 International Clinical Cytometry Society

Zinc is an essential trace element for living organisms and their biological processes. Zinc plays a key role in more than 300 enzymes and it is involved in cell communication, proliferation, differentiation and survival. Zinc plays also a role in regulating the immune system with implications in pathologies where zinc deficiency and inflammation are observed. In order to examine the experimental evidence reported in the literature regarding zinc levels in the body of patients with autoimmune disorders compared to control individuals, a systematic review and meta-analysis were performed. From 26,095 articles identified by literature search, only 179 of them were considered potentially relevant for our study and then examined. Of the 179 articles, only 62 satisfied the inclusion criteria. Particularly for Fixed Model, Zn concentration in both serum (mean effect = −1.19; confidence interval: −1.26 to −1.11) and plasma (mean effect = −3.97; confidence interval: −4.08 to −3.87) samples of autoimmune disease patients was significantly lower than in controls. The data presented in our work, although very heterogeneous in the manner of collecting and investigating samples, have proved to be extremely consistent in witnessing a deficiency of zinc in serum and plasma of patients compared to controls.

Background: Type 1 diabetes incidence presents a decreasing gradient in Europe from the Nordic countries to the Mediterranean ones. Exception to this gradient is represented by Sardinia, the second largest Mediterranean island whose population shows the highest incidence in Europe, after Finland. The genetic features of this population have created a fertile ground for the epidemic of the disease, however, as well as being strikingly high, the incidence rate has suddenly presented a continuous increase from the '50s, not explainable by accumulation of new genetic variants. Several environmental factors have been taken into account, possibly interacting with the genetic/epigenetic scenario, but there are no strong evidences to date. Methods: The present study investigated the hypothesis that geochemical elements could create permissive environmental conditions for autoimmune diabetes. An ecological analysis was performed to test possible correlations between the values of eight elements in stream sediments and type 1 diabetes incidence rate in Sardinia. Results: Analyses revealed negative associations between elements, such as Co, Cr, Cu, Mn, Ni, Zn, and type 1 diabetes incidence. Conclusions: The results suggest a possible protective role of some elements against the onset of the disease. Copyright

Introduction: Azacitidine is at present the standard of treatment of myelodysplastic syndromes (MDS). We demonstrated that MDS patients responsive to azacitidine have significantly higher expression of the azacitidine-activating enzyme uridine-cytidine kinase-1 (UCK1) in bone marrow mononuclear cells (Valencia et al. Leukemia 2014). Correlation of the expression of nucleoside transporter, activating and disactivating enzymes with clinical and in vitro response to hypomethylating drugs azacitidine and decitabine has been suggested by several authors. Yet, the crucial role of these enzymes has to be ascertained, as well as their possible different importance in determining resistance. Objectives: To confirm that the cellular expression of nucleoside metabolizing enzymes plays a major role in cellular resistance and significantly impacts on clinical response to azacitidine. Methods: Two cell lines, SKM1 sensitive (SKM1-S) and SKM1 resistant (SKM1-R) to azacitidine (Cluzeau et al. Oncogene 2014) were analyzed for expression of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 by quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes were acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Corresponding proteins were quantitated by western blotting in both cell lines. The expression of UCK1 and UCK2 was blunted by siRNAs in order to determine their role in in vitro sensitivity to azacitidine. For UCK2 silencing in SKM1-S, specific siRNAs were used (OriGene Technologies, Inc., Rockville, MD, USA). SKM1-S cells were cultured at a density of 1x106 cells/ml in 9 ml of RPMI 1940 medium. After 72 h of transfection, cells were treated for further 48h with azacitidine at the concentrations of 0,1 and 1 μM. After assessment of effective gene silencing, apoptosis and DNA methylation were evaluated by Annexin V test, MSP and pyrosequencing in cells exposed to azacitidine. In addition, the expression of nucleoside metabolizing enzymes was evaluated prospectively in IPSS high risk MDS patients treated with azacitidine 75mg/m2/7 days every 28 days. Results: SKM1-R cells did not express UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2. Corresponding proteins were also not expressed. In order to clarify the specific role of UCK2 protein in the mechanisms of resistance to azacitidine, we knocked-down UCK2. A reduction of apoptosis was observed in UCK2-silenced SKM-1 S after azacitidine 0.1 μM treatment: 31%±0,85% Annexin V-positive cells versus 21%±0,35% in non-silenced control SKM1-S cultures (P= 0.054). Hypomethylation induced by in vitro azacitidine treatment was also hampered by absence of expression of UCK2. Quite surprisingly, in MDS cases, gene expression of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 in primary cellsdid not predict different clinical response to azacitidine. Conclusions: We demonstrated that UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 and the corresponding proteins are absent in an in vitro generated azacitidine-resistant cell line SKM1-R. These abnormalities could be major determinants of the induced resistance to the nucleoside analogue. Supporting the importance of these enzymes in resistance to nucleosides, and the major role played by UCK2, its silencing induced by synthetic siRNAs significantly decreased azacitidine effects. Prospective evaluation of the predictive role of cellular expression of genes involved in azacitidine metabolism is ongoing in a larger cohort of MDS patients. Establishing the mechanisms of resistance to azacitidine could lead us to optimize treatment of MDS patients whose scarce susceptibility could be predictable. Disclosures Santini: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Other: advisory board; Novartis: Consultancy, Honoraria; Onconova: Other: advisory board; Janssen: Consultancy, Honoraria; Astex: Other: advisory board.

The nucleoside analog azacitidine (AZA) is used in the treatment of myelodysplastic syndromes (MDS), but 30–40% of patients fail to respond or relapse after treatment. Hence, to identify new molecular alterations that allow for identification of patients unlikely to respond to AZA could impact the utility of this therapy. We determined the expression levels of genes involved in AZA metabolism: UCK1, UCK2, DCK, hENT1, RRM1 and RRM2 using quantitative PCR in samples from 57 patients with MDS who received AZA. Lower expression of UCK1 was seen in patients without a response to AZA (median 0.2 vs 0.49 for patients with response to AZA, P=0.07). This difference in UCK1 expression was not influenced by aberrant methylation of the UCK1 promoter. In addition, the seven polymorphic loci found in the coding sequence were not associated with UCK1 gene expression nor AZA response. Silencing of UCK1 by siRNA leads to blunted response to AZA in vitro. The univariate analysis revealed that patients expressing lower than median levels of UCK1 had a shorter overall survival (P=0.049). Our results suggest that expression level of UCK1 is correlated with clinical outcome and may influence the clinical response to AZA treatment in patients with MDS.

Purpose: Myelodysplastic syndromes (MDS) are heterogeneous clonal diseases characterized by cytopenias as a result of ineffective hematopoiesis. Little is known about alterations in signal transduction pathways in MDS. Experimental Design: Multiparameter flow cytometry was used to evaluate the proteolytic activation of caspase-3 and the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and STAT5 specifically in defined CD34+, CD45+, or CD71+CD45− bone marrow (BM) cells from 60 MDS cases and normal controls, both at baseline and following stimulation with granulocyte colony-stimulating factor (G-CSF) and erythropoietin. Results: In CD71+CD45− cells from a subpopulation of 36 MDS cases who were predicted to be responsive by clinical parameters (endogenous erythropoietin levels, transfusion dependency, percentage of blasts in the BM), erythropoietin failed to activate ERK1/2 or STAT5 in 23 of 36 cases, but it was effective in 13 of 36 cases, although to a significantly lower degree than in CD71+CD45− cells from healthy donor BM. The erythropoietin response in vivo correlated with in vitro erythropoietin-dependent STAT5 activation in 20 of 22 cases. STAT5 was significantly activated at baseline in MDS cells compared with normal controls, whereas caspase-3 was activated in CD34+ and CD45+ MDS cells, and was activated more often in the RA and RAEB-1 MDS subtypes. G-CSF stimulation activated ERK1/2 and STAT5 equally in MDS and normal CD34+ cells. Conclusions: Abnormalities in the response to growth factors are restricted to erythropoietin stimulation in CD71+CD45− cells and correlate with the clinical response to erythropoietin. Activation of baseline signal transduction for proliferative and apoptotic signals is altered in MDS but with different patterns among the various BM subpopulations. Clin Cancer Res; 18(11); 3079–89. ©2012 AACR.

Azacitidine and decitabine are DNA methyltransferase inhibitors used to treat myelodysplastic syndromes and acute myeloid leukemias. To further characterize different mechanisms between these two agents, cellular extracts from leukemic cells untreated or treated with either drug were analyzed using 2D electrophoresis. Numerous differentially expressed proteins were identified with MALDI-TOF/TOF-MS. Cyclophilin A, Catalase, Nucleophosmin and PCNA were decreased exclusively by azacitidine, TCP1 and hnRNP A2/B1 by both drugs; alpha-Enolase and Peroxiredoxin-1 by decitabine. Interestingly, the expression of the proinflammatory protein Cyclophilin A, also suggested as marker of cell necrosis, was stimulated by decitabine. Finally, a comprehensive pathway analysis of data highlighted a relationship between the identified proteins and potential effectors.

The nucleoside analog (na) 5-aza-2’-deoxycytidine (dac) has good, but heterogeneous, efficacy in the therapy of chronic myelomonocytic leukemia (cmml). Given that no standard therapy has been identified so far for cmml, there is urgent need to identify molecular markers that could support the choice of dac therapy and identify patients more prone to respond. Recently, we demonstrated that in mds patients the expression of uck1, involved in the activation of azacitidine, may influence the clinical response to this treatment (valencia et al, leukemia 2013). In the present study, we assessed the role of dac metabolizing enzymes as well as genetic alterations common to cmml patients in response to dac in a uniformly and prospectively treated cohort of cmml patients. Methods: Patients Forty cmml patients were treated with dac 20mg/m2/day for 5 days every 28 d. DNA and rna were extracted from bm mononuclear cells of 19 pts defined as responders to dac (r), and 21 as non-responders (nr). Hematological response was evaluated according to iwg 2006 criteria. Gene mutation. the fifteen most frequently mutated genes in cmml were sequenced at a mean depth of coverage of 520x (range 169–714x). Functional studies. the role of two main genes involved in dac metabolism: dck (dac activation) and dctd (dac deactivation) was evaluated by silencing both genes with sirnas. The experiments were performed in the mds-skm1 cell line and in primary cells of 6 cmml cases exposed in vitro to dac 10um for 48h. Gene expression. the expression level of genes hent1, hent2, dctd, hcnt3, cn-ii, dck and cda, involved in dac metabolism was evaluated by qrt-pcr in 38/40 cmml patients and by rnaseq in 14/40 patients. CDNA libraries were sequenced using the hiseq 2000. The counts of endogenous genes were normalized by ercc spike-in library controls, and differential expression analysis was performed using edger (v3.4.2) glm model. The differentially expressed genes were identified at the fdr cutoff of 0.05 and absolute fold change greater than 2. Results: in skm-1 cells and in cmml primary mononuclear cells, dctd silencing increased dac- induced apoptosis (annexin v-positive cells 20.2%±0.8% vs control 13.8%±0.5%; p=0.01). dck silencing led to a decrease in dac-induced apoptosis (annexin v-positive cells 8.8%±0.1% vs control 13.8%±0.5%; p=0.05). We could not detect by rnaseq a significant difference in the expression levels of na metabolizing enzymes between responders and non responders to dac. qrt-pcr confirmed these observations. The mutational frequencies (figure) in this cohort of cmml cases were: srsf2 50%, tet2 44.7%, asxl1 39.4%, nras 18.4%, runx1 and dnmt3a 10.5%, u2af1 and tp53 7.8%, kras, jak2 and kit 5.2%, ezh2, idh1, idh2 and sf3b1 2.6%. No single genetic alteration was significantly associated with shorter overall survival or resistance to dac. Conclusions: although we could clearly demonstrate that in vitro expression of dac metabolizing enzymes influenced response to dac, clinical resistance does not appear to be directly correlated with the expression of genes involved in dac metabolism nor to specific gene mutations and is likely determined by other clinical/molecular variables that remain to be identified. Figure 1 Figure 1. Disclosures Finelli: Janssen: Speaker, Speaker Other; Novartis: Speaker, Speaker Other; Celgene: Research Funding, Speaker Other.

Background hypomethylating agents, especially AZA, have become the reference standard for the of higher risk MDS, but the prognostic value of baseline cytogenetics on response to AZA, and the impact of cytogenetic response (CyR) on outcome in responders remain uncertain. Methods We collected data from 931 MDS patients (including FAB RAEB-T/WHO AML 20-30% blasts), treated with AZA (75mg/m²/d x7d, for a median of 6 cycles [range 1-72]) in 6 centers in the US, Italy and France between January 2002 and March 2013. Median age was 70 years (range 24-91 years), and 35% of the patients were women. Cytogenetics at onset of AZA was evaluable in 878 pts (the remaining pts had cytogenetic failure), 581 (66%) of whom had abnormal karyotype, as shown in table 1. Revised (R) IPSS cytogenetic category (Shanz, JCO 2012) was very good, good, int, poor and very poor in 2%, 40%, 18%, 15% and 25% pts respectively. R-IPSS was very good, good, intermediate, poor and very poor in 1%, 4%, 17%, 35% and 43% respectively. Results 379 (41%) pts achieved hematological IWG 2006 response, including 121 (13%) CR, 86 (9%) PR, 52 (6%) marrow CR, 120 (13%) stable disease with HI. With a median follow up of 41 months, median OS was 16.5 months. Cytogenetic characteristics are summarized in table 1. In the following text, unless specified, results apply to chromosomal rearrangements occurring alone or with additional abnormalities (abn). Trisomy 8 and del(5q)/-5 were associated with significantly better CR rate (21% and 18.5% , respectively, vs 12% in other patients p=0.007 and 0.01, resp.). 3q26 was associated with lower overall response rate (ORR) (22% vs 42%, p=0.04) and only 1/27 of 3q26 pts achieved CR. None of the other cytogenetic specific abnormalities or groups (table 1) and none of the R-IPSS cytogenetic categories had any significant impact on ORR or CR to AZA. Among patients with complex Karyotype (>=3), monosomal karyotype had no influence on response to AZA. When the comparison was made versus patients with normal cytogenetics, presence of -7/del(7q), del(5q)/-5, del(17p), 3q26, monosomal karyotype and complex (>=3) karyotype had no significant impact on the response rate. Compared to other patients, patients with -7/del(7q) (p=3), those with very complex karyotype (>=5) had shorter OS (median 11.1 vs 15.4 mo, p=0.008). When the comparison was made versus patients with normal cytogenetics, presence of -7/del(7q), del(5q), del(17p), 3q26 , monosomal karyotype and complex (>=3) karyotype were associated with significantly shorter OS. By multivariate analysis (including cytogenetic R-IPSS categories, del 20q, 7/del(7q) , del(5q)/-5, del (17p) , 3q26 , complex and monosomal karyotype), only the presence of del (17p) (HR 1.54[1.14-2.10], p=0.005), -7/del(7q) (HR 1.23 [1.01-1.50], p=0.04) and del(5q) (HR 1.36[1.08-1.72], p=0.009) retained significant impact on OS. 362 pts with abnormal cytogenetics at onset of AZA had cytogenetic analysis at treatment evaluation, and results were evaluable in 327 of them (the other 35 pts had cytogenetic failure): 106 (32.4%) achieved cytogenetic response (CyR), including 82 (25%) Complete CyR (CCyR), and 24 (7.3%) Partial CyR (PCyR), while 221 (67.6%) had no CyR. Of note, among the 106 cytogenetic responders, 29 (27%) had failed to achieve any hematological response. In a landmark analysis performed 3 months after AZA onset, achievement of any CyR or of CCyR had no significant influence on survival, even when the analysis was restricted to patients who achieved IWG response. Conclusion Baseline cytogenetic pattern generally did not predict response to AZA (except the presence of +8 or del(5q), associated with higher CR rate, and 3q26 abn with fewer responses). However, cytogenetic results were strong predictors of survival, especially del (17p), -7/del(7q) and del(5q) associated with significant shorter OS in multivariate analysis. In patients with baseline cytogenetic abnormalities, achieving cytogenetic response was not associated with outcome. Disclosures: Komrokji: Celgene: Research Funding, Speakers Bureau. Santini:Celgene: Honoraria; Novartis: Honoraria; GSK: Honoraria; Janssen: Honoraria. List:Celgene: Research Funding. Fenaux:CELGENE: Research Funding.

Decitabine is a pyrimidine nucleoside analogue of cytidine and with azacytidine has been approved for treatment of MDS and non-proliferative CMML. Anyhow, few CMML cases were included in the registrative studies and CMML treatment still remains very arduous, being CMML an heterogenous disease by itself, with characteristics of both MDS and MPN and with a variable prognosis. Decitabine has been suggested to be particularly effective in CMML in a few studies. We evaluated efficacy and safety of decitabine 20mg/m2/day for 5 days every 28 days for a minimum of 6 cycles in a group of pretreated CMML patients. Evaluation of response was performed after 4 and 6 cycles of therapy, according to IWG 2006 criteria. Morphological diagnosis, evaluation of dysplastic signs and monocytic component was performed centrally after first on-site diagnosis. The mononuclear cell expression of decitabine metabolizing enzymes CDA, hENT1, DCK, and CN-II was determined. Between April 2010 and October 2011, 44 patients were enrolled from 15 Italian Hematology centers, 43 had a confirmed centralized diagnosis of CMML (M/F 30/13), and 42 were finally evaluable. According to WHO 27/42 pts were classified CMML-I with organomegaly and 15/42 CMML-II. Among these patients, 46% had received previous therapy. Proliferative CMML was diagnosed in 32/42 cases. Median age was 71 (42-84) yrs, median number of cycles 6 (1-28). Eleven patients received < 4 cycles, 16 from 4 to 6 cycles and 15 > 6 cycles. Eighty one percent of patients with CMML-I received > 4 cycles. All cycles were administered in an out-patient basis. Overall response rate (ORR) was 33%, mCR in 19%, CR in 14.3 %, PR 2.3% and SD in 34.9% and at 3 year-observation time 31% of patients are still alive (13/42). Median duration of response was 9 months. At present, 16.3% (7/42) of patients are still in treatment. Splenomegaly was significantly reduced in 28% of cases. No significative difference in response rate was present in CMML I vs CMML II, nor in proliferative CMML vs dysplastic ones. At analysis of karyotype, 57% of cases had no cytogenetic abnormality and there was no difference in RR between normal and abnormal cases, among which only 5% had complex karyotype. Median OS is 19 months (2-39). For CMML I cases OS is 26 months vs 15 months for CMML II (p Interruption of therapy with decitabine was mainly due to progressive disease (52% of drop-out cases), death (9%) and in 7% of the cases to toxicity of the drug. Grade 3/4 toxicity was in the majority of cases hematological, and present all over treatment courses. In 7/42 patients severe infections occurred, and 1/42 had grade 3/4 cardiac and gastrointestinal toxicities respectively. Conclusions CMML patients even when heavily pretreated, can be responsive to decitabine which can be administered for several cycles with good tolerability. Our data are confirming reported data on ORR and OS after decitabine therapy (Braun et al Blood 2011). OS of decitabine- responding patients is significantly prolonged. No clinical or cytogenetic characteristics seem to be predictive of response to decitabine. No significant difference in enzyme expression was demonstrated, although a trend in lower expression of hENT1 was noted for non responsive cases. Disclosures: Santini: Celgene: Honoraria; Janssen : Honoraria; Novartis: Honoraria; gsk: Honoraria.

Abstract 5052 Background: 5-azacytidine (Vidaza, AZA) and 5-aza-2'-deoxycytidine (Decitabine, DAC) are active in therapy of myelodysplastic syndromes (MDS) probably with different mechanisms of action. Azacitidine is approved by FDA and EMEA and is routinely used in therapy of higher risk MDS in Europe and in the US. Decitabine is a demethylating agent approved for treatment of IPSS INT-2 and high risk MDS by FDA but not yet from EMEA and it seems particularly active in Chronic myelomonocytic leukemia (CMML) and AML of the elderly. Difference in mechanisms of action of these 2 agents have been determined in vitro, involving nuclear acid incorporation, cell cycle and apoptosis, gene and protein expression. Patients: Nine patients (4 RAEB 2, 4 AML, 1 CMML-1) were treated with decitabine 20mg/m2 × 5 days every 28 days as single agent or in association with Gemtuzumab ozogamicin 3 mg/m2 in day 5 and 9. IPSS score at diagnosis was: 1 low, 4 intermediate 1, 4 intermediate 2. All patients received at least 4 cycles of azacitidine 75 mg/mq per seven days every 28, 44% of them reached at least Hematological improvement and received DAC as second line after relapse. Median age was 70 (62–84) and 8/9 were male. Methods: Medical records of 14 patients who received decitabine treatment were reviewed and survival was calculated by the Kaplan Meier method and differences in survival calculated by the log-rank test using SPSS software. Complete remission was defined by normo-cellular bone marrow with 1.5 mmol/L, WBC count by >1000, and platelet count by >50,000. Results: Median overall survival (OS) was 121 days, range 27–586 days. Overall response rate (CR+PR+HI) was 33.3% with mean duration of response was 3.33 months. Mean number of cycles was 4 (range 1–20). Median OS of responder patients was 12 months vs 1 month of non responder patients. OS of patients treated with DAC after AZA-resistance was 2 months vs 4 months of patients relapsed, but who reached at least HI with AZA (p>0.05). Longer history of MDS disease seemed not relevant for outcome. Therapy was well tolerated and only two patients required a long hospitalization (42 and 77 days) for sepsis due to relevant myelosuppression. Conclusions: Decitabine treatment has some activity in MDS, CMML and AML patients refractory to AZA or who relapsed after initial response to AZA, suggesting that the two supposed hypomethylating agents act via partially different molecular mechanisms. Disclosures: No relevant conflicts of interest to declare.

Abstract 1023 Background: The annual incidence rate of myelodysplastic syndromes (MDS) in the general population is estimated at around 3–5/100,000. Because of a lack of specific therapies, until recently small attention has been devoted to costs of treatment of MDS. A specific Diagnosis-Related Group is still missing. In the US the mean annual cost for MDS patients ranges from $19,811 to $51,066. Aims: To assess the annual cost of illness (COI) and quality of life (QoL) of patients with MDS in Italy. Methods: The Costo Sociale delle Sindromi Mielodisplastiche e Qualità della vita in Italia (CoSMIQ) is an observational, cross-sectional, retrospective, prevalence-based, multicenter study based on an International Prognostic Scoring System (IPSS)-stratified sample of patients with MDS ≥18 years of age seen in standard clinical practice at 7 hematologic institutions across Italy (3 in the North; 2 in the Center; and 2 in the South). Demographics, clinical history, health care and non-healthcare resource consumption, and patients' and caregivers' productivity losses were collected by physicians using clinical records and information provided by patients. COI (in Euro (€) 2010) was determined utilizing a societal perspective. QoL was assessed via EORTC QLQ-C30 and QOL-E v.2 questionnaires, the latter being a new specific tool for the assessment of health-related QoL in patients with MDS. Kendall's tau rank correlation, chi-squared test, multivariate analysis of variance (ANOVA; MANOVA) and multiple linear regressions were performed when appropriate. Results: In all, 225 of 234 patients who met the inclusion criteria were analyzed (IPSS low risk: 124 patients; intermediate-1 (Int-1) risk: 75 patients; Int-2 and high risk: 26 patients) (Table). The total COI reached €27,980.4 ± 28,322.2 (mean ± standard deviation) (Figure). Cost-drivers of COI were antianemics (lower-risk) and antineoplastics (higher-risk). The Italian National Health Service (INHS) funded the greatest share of COI (from 97.4% to 99.5%). In general, CoSMIQ patients perform worse than the general population in all EORTC QLQ-C30 domains; the total global health status (QL) domain reached 65.1 ± 22.2 (low risk: 64.2 ± 22.3; Int-1 risk: 67.4 ± 22.1; higher-risk: 62.5 ± 22.3). QL was negatively correlated with COI (p=0.049) and patients' age (p=0.089), and positively correlated with disease duration (p=0.203). Keeping the other predictors constant, RBC transfusion dependence predicts increased COI (p=0.006) and lower QL (p=0.009). Regarding the QOL-E questionnaire, the QOL-E MDS-specific domain (MDSS) was positively correlated with total COI (p=0.0002), and patients' age (p=0.348) and negatively correlated with disease duration (p=0.013). The total QOL MDSS was 27.5 ± 19.8 (low risk: 25.3 ± 19.4; Int-1 risk: 29.6 ± 19.3; higher-risk: 31.6 ± 22.2). The total treatment-outcome index domain (QOL TOI) was 49.4 ± 14.4 (low risk: 49.1 ± 14.7; Int-1 risk: 48.3 ± 14.7; higher-risk: 53.9 ± 11.7). QOL TOI was negatively correlated with total COI (p=0.379), and patients' age (p=0.0003) and positively correlated with disease duration (p=0.162). Differences by IPSS group were not statistically significant for COI (p=0.191) or for any domain of the EORTC QLQ-C30 or QOL-E questionnaires (p=0.124; p=0.467). Conclusion: MDS dramatically increases INHS budgets while negatively impacting patients' QoL. The results of the CoSMIQ study highlight the strong correlation between RBC transfusion dependence and both COI and QL. Disclosures: Lazzaro: Celgene Srl: Research Funding. Martelli:Celgene Srl: Employment, Equity Ownership.

Abstract 2799 Background. In IPSS lower-risk Myelodysplastic Syndromes (MDS), anemia is the main therapeutic challenge. Erythropoietin stimulating agents (ESAs) can frequently correct anemia, but not all patients respond, and median response duration to ESAs is around 2 years (Park, Blood 2008;111:574). Patients with deletion of chromosome 5q are usually considered refractory to ESAs. Endogenous serum erythropoietin levels have been demonstrated in only one study to be higher in patients carrying del5q than in patients with Lower-Risk MDS and different abnormalities in karyotypes. Aims. We wanted to further analyze differences in management, efficacy and tolerability of ESAs therapy between MDS with and without del5q. Patients and Methods. We studied 239 MDS patients affected by IPSS low and intermediate 1 risk disease. 82 patients carried deletion of 5q, 12/82 in association with other anomalies. MDS patients with del 5q were WHO diagnosed as 9 RA, 9 RCMD, 13 RAEB-1, 47 5q minus syndrome and 2 unclassifiable MDS and divided by IPSS risk: 49 low risk and 33 INT-1. Patients without del5q were diagnosed as follows: 59 RA, 24 RARS, 43 RCMD, 17 RAEB-1, 4 CMML and 10 unclassifiable MDS. When subdivide per IPSS risk: 90 low risk and 67 INT-1. 75% of patients with del5q were female against 37% of patients without del5q. Medical record of patient were collected and reviewed, statistical analysis were perform with SPSS software. IWG criteria for response to ESA treatment were applied. Results. Endogenous erythropoietin (EPO) level at diagnosis were significant higher in patients with del 5q (mean 599,6 mU/ml) than in others (mean 99,3 mU/ml)(p=0.002). There was no significant difference, in terms of endogenous EPO levels, either stratifying by WHO classification (p=0.996) or by IPSS score (INT-1 325.8 mU/ml, low 138.6 mU/ml, p=0.120), or blast % (p>0.05). Serum creatinine levels were correlated with endogenous EPO levels (p=0.003 Spearman's rho −0.258). Serum Epo was also correlated to age (p=0.026 Spearman's rho −0.183). No correlation was seen between number of transfusions at diagnosis and serum EPO levels and between hemoglobin levels and serum EPO levels. 18 patients (22%) with deletion of 5 q responded to ESAs treatment versus 56,8% of responding patients without del 5q (p Disclosures: No relevant conflicts of interest to declare.

Iron overload may represent an additional clinical problem in patients with Myelodysplastic Syndromes (MDS), with recent data suggesting prognostic implications. Beyond red blood cells transfusions, dysregulation of hepcidin, the key iron hormone, may play a role, but studies until now have been hampered by technical problems. Using a recently validated assay, we measured serum hepcidin in 113 patients with different MDS subtypes. Mean hepcidin levels were consistently heterogeneous across different MDS subtypes, with the lowest levels in refractory anemia with ringed sideroblasts (RARS, 1.43 nM) and the highest in refractory anemia with excess blasts (RAEB, 11.3 nM) or in chronic myelomonocytic leukemia (CMML, 10.04 nM) (P = 0.003 by ANOVA). MDS subtypes remained significant predictors of hepcidin in multivariate analyses adjusted for ferritin and transfusion history. Consistently with current knowledge on hepcidin action/regulation, RARS patients had the highest levels of toxic non-transferrin-bound-iron, while RAEB and CMML patients had substantial elevation of C-Reactive Protein as compared to other MDS subtypes, and showed lost of homeostatic regulation by iron. Growth differentiation factor 15 did not appear as a primary hepcidin regulator in this series. If confirmed, these results may help to calibrate future treatments with chelating agents and/or hepcidin modulators in MDS patients.

Abstract 606 Myelodysplastic syndromes (MDS) are affecting mainly elderly patients, and age is considered per se a negative prognostic factor. Most of the elderly patients have comorbidity that presumably negatively impact the overall survival and quality of life and may influence response to therapy. A subanalysis of a recently concluded international trial demonstrated that MDS patients aged > 75 yrs treated with azacitidine have a significantly longer overall survival respect to best supportive care treated patients (Seymour, 2010). We analyzed whether presence and number of comorbidities could have an impact on response and management of azacitidine treatment in elderly and very elderly patients treated in our Center, outside clinical trials. We evaluated outcome, survival and type of response, as well as adverse events in all patients. We analyzed 59 elderly MDS patients (IPSS INT-1 15/59, INT-2 32/59 and High 12/59) treated with Azacitidine 75 mg/kg/day sc for 5 days every 28. Mean number of cycles was 9 (range:2-42). Mean age was 69 yrs (50-82); 30% of patients (18/59) were >= 75 yrs and 39% of the latter (7/18) >= 80 yrs. We also evaluated Charlson comorbidity index (CCI) (10 patients scored == 4), the Cumulative Illness Rating Scale (CIRS) (25 patients scored == 4) and the Adult Comorbidity Evaluation-27 (ACE-27) (13 patients scored ==5). Overall response rate (HI, CR and PR) according IWG criteria 2006 was 49.1%, stable disease was obtained in 37.1 % of MDS patients. We demonstrated by Fisher test that these IWG responses did not correlate with age. We also evaluated CCI, CIRS, and the ACE-27 in relation to age and to hematological response and no correlation was showed. Hematological or non hematological adverse events were presented by 34% and 36% of patients, respectively. Adverse events were uniformly distributed independently from age. Median overall survival (OS) of our patient cohort was 21 months. Median OS in patients < 75yrs (20 monthts) and >= 75 yrs (16 months) was not significantly different (p value > 0.65). OS in patients with higher CCI, CIRS, and the ACE-27 was respectly 10 months, 6,5 months and 6 months. 5 patients presented renal failure, but treatment with standard dose aza did not worsen creatinine clearance. Very elderly patients with comorbidities may be treated with success with azacitidine, without any substantial increase in AE. Nevertheless comorbidities negatively influence overall survival. Evaluation of comorbidities with validated indexes is an useful and easily applicable tool to refine prognostic evaluation. ACE-27, although more complex, seems to give best prognostic evaluation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4250 Background and Aims. Iron overload is frequently occurring in patients with myelodysplastic syndromes (MDS), with recent data suggesting an impact on both overall and leukemia-free survival1,2. Though prolonged RBC transfusion therapy appears the main contributor, many patients develop iron overload at an early stage of the disease, before the onset of transfusion dependency. It has been postulated that an altered production of hepcidin, the key hormone regulating iron homeostasis, may play a role at this regard. Until recently, studies have been hampered by problems in the development of reliable hepcidin assays, so that only scanty and conflicting data based on semi-quantitative measurement of urinary hepcidin have been reported3,4. This study mainly focused on analyzing serum hepcidin levels in MDS patients by means of a recently validated and improved Mass-Spectrometry based method5. Patients and Methods. One hundred and thirteen consecutive patients (mean age 72.8 ± 9.2 years; 68.1% males) with different types of MDS according to the WHO classification were included in this study. To be enrolled, patients had to be previously untreated or treated only with transfusions. Besides hepcidin, in all subjects we determined serum ferritin, transferrin saturation (TS), non-transferrin-bound-iron (NTBI), along with some putative determinants of hepcidin, like GDF-156 known to be associated with ineffective erythropoiesis, and C-Reactive Protein (CRP) as a surrogate of systemic IL-6 production. Fifty-four healthy individuals (61.1% males) with rigorous definition of normal iron status were used as controls. Main Results. Biochemical markers of iron overload (ferritin and TS), but also CRP and GDF-15 were significantly higher in MDS patients than in controls, even when considering only non-transfused patients. Patients with RARS and the 5q- syndrome appeared as the most iron overloaded, having the highest levels of ferritin, TS, and NTBI. In the whole MDS population, serum hepcidin levels showed a considerable variability, with overall mean values not significantly different from controls [geometric means (gm) with 95% CIs: 5.31 (3.98-7.08) versus 4.2 (3.53-5.0) nM, P=0.28], while the hepcidin/ferritin ratio was significantly lower than in controls [10.1 (7.53-13.53) versus 52.9 (43.6-64.3), P Conclusions. Hepcidin levels are consistently heterogeneous in MDS according to different subtypes, likely as the result of the relative strength of competing stimuli. Relative inhibition by ineffective erythropoiesis (but not mediated by GDF-15) seems to prevail particularly in RARS and 5q- syndrome, and is likely to increase the risk of iron overload in these subgroups. On the other hand, patients with RAEB 1–2 and CMML appears to have hepcidin induction that could be driven by cytokines. If confirmed, these results may be relevant not only for a better understanding of iron pathophysiology in MDS, but also for possible future approach with hepcidin modulators7. References: 1) Sanz G, et al. Blood 2008;112: abs 640. 2) Alessandrino EP, et al. Haematologica 2010;95:476-84. 3) Winder A, et al. Br J Haematol 2008;142:669-71. 4) Murphy PT, et al. Br J Haematol 2009;144:451-2. 5) Campostrini N, et al. J Biomed Biotechnol 2010;2010:329646. 6) Tanno T, et al. Nat Med 2007;13:1096-101. 7) Sasu BJ, et al. Blood 2010;115:3616-24. Disclosures: No relevant conflicts of interest to declare.

Abstract 1752 Poster Board I-778 Iron overload (IO) occurs in a large proportion of patients affected by myelodysplastic syndromes; however data on the mechanisms responsible for IO and on the consequences on organ damages are scanty. IO in MDS may result both from ineffective erythropoiesis and from reiterative transfusions. The role of iron chelation therapy in MDS is deduced from thalassemia experience. We evaluated parameters of iron metabolism and erythropoiesis in 171 consecutive chelation naïve MDS patients. Patients were classified according WHO criteria, and prognostic scores evaluated according IPSS and WPSS criteria. CMML patients (n= 7) were also included in the analysis. Serum ferritin, iron, transferrin, transferrin saturation and non-transferrin-bound iron (NTBI) were measured. Growth-differentiation factor-15 (GDF-15) and hepcidin-25 were also determined, parallel to hemoglobin, endogenous erythropoietin (EPO) and LDH. All the parameters studied were highly variable within the analyzed cohort, as expected due to the great heterogeneity of MDS. As already observed, ferritin levels were higher than normal range in MDS patients (1091 ± 1135 ng/ml) and significantly increased in transfused ones (1890,55 ± 1833 ng/ml). EPO production was stimulated in all MDS patients (215, 40; range 85-1011 U/L), respect to normal range, and physiologically inversely correlated with hemoglobin levels (p Disclosures No relevant conflicts of interest to declare.

Abstract 2814 Purpose: ESA are generally the first line treatment of anemia of IPSS low and int1 (lower) risk MDS. While endogenous EPO level and RBC transfusion requirement are well defined prognostic factors of response to ESA (Hellstrom, BJH 2003), many patients (pts) are treated before they require RBC transfusions and, in a GFM large cohort of lower risk MDS with anemia, baseline EPO level was below 500U/l in 88% of the cases (Kelaidi, Haematologica 2010). In non transfusion dependent (non TD) lower risk MDS, it is therefore important to find additional simple prognostic factors of response to ESA. In a joint study of the French (GFM) and Italian (FISM) MDS groups, we retrospectively assessed prognostic factors of response to ESA in non TD lower risk MDS. Particular emphasis was put on the prognostic value of marrow dysplastic features on ESA response, suggested in a previous work (Howe, Blood, 2004). Methods: We are performing a cooperative (GFM and FISM) retrospective study in IPSS low and int 1 (lower risk) MDS diagnosed between 1994 and 2010. The present analysis focused on lower risk MDS with Hb level Results: For this work, 503 pts with adequate data have been identified. Results are presented in 228 completely reviewed cases. For morphology, there was an inter observer agreement in 85% of cases, discrepancies being resolved by taking mean values. Median values at onset of ESA in those pts were: age 75 (range 21–96), Hb 9.1g/dl (range 4,9–10), MCV 98u3 (85–127), ANC 10% marrow cells was present: for dyserythropoiesis (DE) in 188 (82%), dysgranulopoiesis (DG) in 129 (57%) for dysmegacaryopoiesis (DM) in 96 (43%) pts. WHO 2008 classification was: RA 36%/ RARS 12%/ RAEB-1 9%/ RCMD 36%/ RCUD (other than RA) 1%/ MDS 5q-5%/ unclassifiable 1%. IPSS was low in 49%, int-1 in 44%, NA in 7% pts. IWG 2006 response rate was 60,6%. In univariate analysis, Hb150 U/l (p=0.001) were significantly associated with lower response rate. Response was 61% and 58% in pts with and without DE (p=0.8), and 55% and 65% in pts with and without DM (p=0.1), and 58% in RCMD versus 69% in RA, RARS (p=0.08). DG was inversely correlated with ANC (p=0.003), was seen in 45% IPSS low and 69% int 1 (P=0.003), was correlated with WHO (RA 35%, RARS 48%, RAEB-1 60%, RCMD 85%, MDS 5q– 33%, p9g/dl and EPO Conclusion: In this population of lower risk MDS with non transfusion dependent anemia, significant dysgranulopoiesis (DG) was, in addition to Hb150 UI/l, associated with lower response rate to ESA in multivariate analysis. DG was a stronger predictive factor of non response to ESA than the WHO classification and, contrary to the findings of Howe (Blood 2004), multilineage dysplasia had only borderline prognostic value for response to ESA. We are analyzing more patients in this retrospective cooperative study, also studying other potential prognostic parameters of response to ESA including somatic gene mutations. Disclosures: No relevant conflicts of interest to declare.

Introduction The “classical” IPSS, based on cytogenetics, marrow blast percentage, and number of cytopenias, has played a major role in prognosis assessment in MDS. The recently published revised IPSS (IPSS-R), refines the original IPSS prognostic value (Greenberg et al, Blood 2012). However, its prognostic value for response to ESA has not been assessed. We analyzed it retrospectively in 456 IPSS Low-/Int-1-risk MDS patients (pts) treated with ESA in France, Germany and Italy. Results Those 456 pts had serum EPO < 500mU/ml and Hb≤10g/dl, and had received ESA (EPO alfa or beta 40000-60000 IU/week, or darbepoietin 150 - 300µg/week) for at least 12 weeks. In addition to IPSS-R parameters, age, sex, serum EPO level, serum ferritin (SF), and RBC transfusion requirement before ESA onset were assessed for response to ESA (based on IWG 2006 criteria), and overall survival (OS) from ESA onset. Characteristics of the 456 pts at ESA onset are listed in Table 1. 71% of the pts had never received RBC transfusions, with a median Hb level of 9.3g/dl (range 7.0-10) , and 29% pts had received at least 4 RBC concentrates/8wks before ESA onset (with a maximum of 12). Median SF was 357ng/ml and serum EPO 60mU/ml (range 6-483). IPSS was low in 55% and intermediate-1 in 45% pts. IPSS-R was very low in 15%, low in 61%, intermediate in 19% and high in 4% of the pts. 303 (61%) pts had an erythroid response (ER), including 72% and 52% of low and int-1 risk pts respectively (p=0.001). Using IPSS-R, 85%, 68%, 48% and 31% of pts had ER in the very low, low, intermediate and high risk group respectively (p Other prognostic factors of ER, in univariate analysis, included individual IPSS-R parameters analyzed according to IPSS-R thresholds (Hb level, platelet count, ANC, marrow blast %, cytogenetics) serum EPO level, SF (variables tested as continuous variables) and previous RBC-transfusions. In multivariate analysis, IPSS-R, serum EPO, and SF remained significantly associated with ER (p Applying 1 point to each of the following unfavorable variables of ER: serum EPO >200mU/ml (=1), SF >350 ng/ml (=1), IPSS-R (very low=0, low=1, intermediate=2 and high=3), yielded a score ranging from 0 to 4, with ER rates of 85%, 80%, 64%, 40% and 20% respectively. As expected, IPSS-R also had strong prognostic value for OS (not shown). Conclusion In this patient cohort with overall favorable prognostic factors of response to ESA according to the Nordic score (ie serum EPO =3 (using IPSS-R, serum EPO and SF) proved useful to identify pts with low response to ESA, who also have worse OS and may require alternative treatments (Kelaidi et al, Leukemia 2013). Disclosures: No relevant conflicts of interest to declare.