ALBERT

Description: Introduction DLIs represent a major therapeutic approach for relapse and mixed chimerism (MC) after allogeneic hematopoietic cell transplant (AlloHCT). DLI studies have identified several variables with impact on response and GvHD. Despite some studies having explored the role of T-cells and other cell subsets, such as mononuclear cells (MNCs), comprehensive data regarding the cellular composition of DLI and its role in GvHD remains incomplete, as the development of GvHD post DLI is often unpredictable. Herein we analyzed the cellular composition of DLI from fully human leukocyte antigen (HLA) identical sibling (HLA Id Sib) donors and its impact on the development of GvHD in patients who underwent AlloHCT for hematological malignancy, and its impact on the development of GvHD. Methods Inclusion criteria were as follows: 1) Patients ≥ 18 years-old, 2) AlloHCT, 3) HLA Id Sib donor; 4) treatment with DLI; and 5) signed informed consent of patient and donor. Exclusion criteria were: 1) unrelated or mismatched related donors, 2) HCT2 prior to DLI, or 3) GvHD at DLI. For the purpose of avoiding bias, only the cell composition of the first DLI (DLI1) was analyzed. The following cell subsets of the DLI were studied: CD3+, CD4+, CD8+, CD16+CD56+CD3+ (NKT-cell), CD3+CD45RA+CCR7+ (TN), CD3+CD45RA+CCR7+CD31+ (TRTE), CD3+CD45RA+CD95+CD27+ (TSCM), CD3+CD45RA-CCR7+ (TCM), CD3+CD45RA-CCR7- (TEM), CD3+CD45RA+CCR7- (TTE), CD3+CD4+CD25brightCD127dim (TREG), CD3+CD4+CD25brightCD45RA+CD127dim (naïve TREG). The TN, TCM, TEM and TEM compartment was analyzed for both CD4+ and CD8+. We also analyzed the MNCs, CD19+ (B-cell), CD27+CD19+ (mature B-cell), CD16+CD56+CD27- (natural killer (NK+) cell) and CD16+CD56+CD27+ (CD27+NK+cell). Results Fifty-six DLIs were infused in 36 patients; the median number of DLI was 1 per patient (range, 1-3). Diagnoses were as follows: 13 AML/MDS, 6 HL, 5 MPN, 4 NHL, 4 CLL, 3 MM and 1 B-ALL. For the study, a landmark analysis was performed from the DLI date. The median follow up from DLI was 282 days (range, 9-5,560 days). Overall response rate in relapsed patients was 29% (9 of 31 patients; 6 CR and 3 PR, most responses being observed after DLI1. Further, five patients had DLI for MC and full donor chimerism was achieved in all patients. Thirteen patients (36%) developed GvHD post DLI. Two patients had GvHD before DLI, but there was no case of GvHD at DLI. The median time interval form DLI to GvHD was 76 days (range, 7-261). As per clinical presentation, 10 patients (27%) had acute GvHD, whereas eight patients (22%) had chronic GvHD. The 6-month and 1-year cumulative incidence (CI) of GvHD was 33% and 46%, respectively. When the risk of GvHD was analyzed according to DLI cell subsets, we observed that a DLI1 containing 〉3x106 CD8+TN correlated with an increased incidence of GvHD (Figure 1a). Also, a DLI1 with 〉0.8x108 MNCs/Kg (Figure 1b), 〉2.6x106 mature B-cell/Kg, or 〉0.35x106 CD27+NK+cells/Kg were linked to the development of GvHD (Table 1). Noteworthy, CD3+, TN (both CD4+ and CD8+ combined) or CD4+TN had no impact on the development of GvHD; and a high proportion of TREG was not protective for the development of GvHD (Table 2). Finally, there was no statistically significant association between any clinical variable and GvHD. Conclusion In conclusion, in this cohort of AlloHCT patients from HLA Id Sib donors, a DLI1 containing a high proportion of CD8+TN, but not CD4+TN, increased the probability of developing GvHD. Further, a DLI1 containing a high dose of MNCs, CD27+NK+cells and mature B-cell also associated with GvHD. These data provide novel insight for the understanding of GvHD post DLI. A DLI1 containing a lower dose of CD8+TN could reduce the risk of GvHD, but this asset warrants further validation in larger cohorts, and within a controlled randomized trial setting. Disclosures Bosch: F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Introduction: Myelodysplastic syndromes (MDS) are characterized by bone marrow dysplasia and ineffective hematopoiesis. Zhou et al. showed that transforming growth factor-beta (TGF-β) signaling was constitutively activated in MDS CD34+ cells and that this over-activation and subsequent myelosuppression was based on reduced expression of SMAD7, the natural inhibitor of TGF-β, in MDS CD34+ cells (Zhou L et al. Cancer Res 2011;71:955-963). Galunisertib specifically inhibited the kinase activity of the TGF-β receptor type I (TGF-βRI) also known as ALK5 and its downstream signaling pathway theoretically replaced the SMAD7 function. Galunisertib reversed hematopoietic suppression in human MDS bone marrow assays, and in a murine model of TGF-β derived bone marrow failure. Based on these preclinical studies that demonstrate hematological improvement (HI) in MDS models following galunisertib treatment, a single-arm phase 2 part of a phase 2/3 proof-of-concept study in very low-, low-, and intermediate-risk patients with MDS was conducted. Methods: The primary objective of this study was to estimate the HI rate based on International Working Group (IWG) 2006 criteria in patients with very low-, low-, and intermediate-risk MDS by Revised International Prognostic Scoring System (IPSS-R), treated with galunisertib. Eligible patients were treated with galunisertib 300 mg/day (150 mg BID) orally for 14 days, followed by 14 days off, constituting a cycle of 28 days. Eligibility criteria permitted any prior therapy, all of which were required to be discontinued at least 28 days prior to initiation of galunisertib. Supportive therapies including ongoing transfusions were allowed. Eligibility criteria included confirmed diagnosis of MDS, anemia with hemoglobin ≤10.0 g/dL, and an Eastern Cooperative Oncology Group performance status (ECOG PS) ≤2. Safety was assessed and summarized using the Common Terminology Criteria for Adverse Events (CTCAE v4.0). Descriptive statistics were used to report baseline characteristics and response rates. Results: In this phase 2 study, 41 patients received galunisertib orally (N=39, 150 mg BID and N=2, 80 mg BID for PK comparison). Patients were 62% males. The median age was 71 years (range: 52-84), the majority of patients were classified as refractory cytopenia with multilineage dysplasia (66.7%) or refractory anemia with ringed sideroblasts (20.5%) based on WHO MDS classification. ECOG PS was 0/1 in 53.8%/46.2% of patients. Sixty-two percent of the patients received ≥6 cycles of treatment. Among the 39 patients receiving 150 mg BID, a total of 15 (38%) patients discontinued from the study within 6 cycles; one due to AE and 9 due to patient/physician decision. The most common possibly related any grade treatment-emergent adverse events (TEAEs) included fatigue (20.5%), diarrhea (15.4%), pyrexia (10.3%), vomiting (10.3%), anemia (7.7%), nausea (7.7%), urinary tract infection (7.7%), neutrophil count decreased (5.1%), and platelet count decreased (5.1%); 12 (30.8%) patients had grade 3/4 TEAEs, 4 (10.3%) were drug-related. One of the 39 patients was protocol ineligible and was removed from the efficacy analysis. Among the 38 evaluable patients in the ITT population, 14 of whom required fewer than 4 units of transfusion per 8 weeks, 10/38 (26%) patients achieved HI, defined as at least a continuous 8-week response with at least a 4-unit reduction in transfusion requirement from baseline or hemoglobin increase by at least 1.5 g/dL per 8-week period. Of these 10 patients, 4 became transfusion-independent, and 5 had transfusion reduction. In a subgroup of 24/38 patients who had a transfusion requirement of at least 4 units every 8 weeks at baseline, 9 (38%) of these patients achieved a transfusion reduction of at least 4 units. No apparent correlation between cytogenetics or MDS subtype including ringed sideroblasts and response was identified; however, only 14 patients had abnormal cytogenetics. No platelet or neutrophil responses were observed. Conclusion: Galunisertib is well tolerated in this MDS population where this ALK5 inhibitor was investigated for the first time. Patients most commonly discontinued from study treatment due to patient/physician decision and not for toxicity. The clinical endpoint of HI was observed in 26% of the ITT population, and no specific response sub-group was identified. Disclosures Valcarcel: GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Platzbecker:Boehringer: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. Díez-Campelo:Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding. Schlenk:Boehringer-Ingelheim: Honoraria; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Gaidano:MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding. Perez de Oteyza:Eli Lilly and Company: Research Funding. Cleverly:Eli Lilly and Company: Employment, Equity Ownership. Chiang:Eli Lilly and Copany: Employment. Lahn:Eli Lilly and Company: Other: Former employee. Desiaih:Eli Lilly and Company: Employment. Guba:Eli Lilly and Company: Employment, Equity Ownership. List:Celgene Corporation: Honoraria, Research Funding. Komrokji:Pharmacylics: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Incyte: Consultancy; Celgene: Consultancy, Research Funding.

Description: Peripheral blood (PB) has become the major source of hematopoietic stem cells for autologous stem cell transplantation (ASCT) in the last 15–20 years. Nevertheless, there is a subset of patients who do not mobilize adequate numbers of CD34+ cells. There are no clearly established guidelines with respect to second-line mobilization protocols. The aim of this study has been to analyze our experience as a single center with this population of poor mobilizers trying to identify clinical or biological adverse prognostic factors associated to a poor mobilization of progenitor cells into PB, results of second- and third-line mobilization procedures and outcome after the ASCT of those patients who could be autografted in terms of hematological recovery. Poor mobilizing patients were defined as those in whom the apheresis procedure could not be started because of 〈 10 CD34+ cells/ul or those in which a less than 2 × 106 CD34+ cells/kg could be collected in the first mobilization attempt. From January/2000 to January/2008, 126 patients [70 males/56 females, median age of 53 years (range, 20–70)] out of a total number of 450 patients mobilized for an ASCT in our institution (28%) were identified as poor mobilizers. Clinical diagnosis were: 29 multiple myeloma, 16 Hodgkin’s lymphoma, 48 non-Hodgkin’s lymphoma, 28 acute leukemias and 5 other diagnosis. Median time from diagnosis to mobilization therapy was 19 (range, 3–120) months and median number of chemotherapy lines received before the procedure was 2 (range, 0–5). The first mobilizing protocol was G-CSF alone (5–10 ug/kg/day sc) in 72% of the patients or the combination of chemotherapy plus G-CSF in 28% of the patients. A second mobilization procedure was attempted in 34 patients (28%) with high-doses of G-CSF alone (16–20 ug/kg/day sc) in 24 patients, the combination of G-CSF plus chemotherapy in 8 patients and the combination of G-CSG with stem cell factor (SCF) in 2 patients. A third mobilization attempt was performed in 6 patients (high-doses of G-CSF alone in 4 patients, G-CSF plus chemotherapy in 1 patient and G-CSF plus SCF in 1 patient). Sixty-nine patients (54%) were finally autografted. Median number of CD34+ cells/kg infused were 2.15 × 106/kg (range, 1.01–4.00). Median time to neutrophil recovery after transplantation was 11 days (range, 4–20). Patients with an inadequate mobilization constitute a significant clinical problem (25% of the whole population of patients with an indication of ASCT in our centre). Nevertheless, half of these patients can be rescued for an ASCT procedure with one or two more attempts. Neutrophil recovery after the autologous transplant in those patients undergoing the procedure seems to be similar to that of the group of patients with an adequate first mobilization attempt. New mobilizing agents should be investigated in order to increase the efficacy of the mobilization processes.

Description: Abstract 1181 Poster Board I-203 MNGIE is an autosomal recessive disorder of nucleotide metabolism due to TYMP gene mutations that cause loss of activity of thymidine phosphorylase (TP). As a result, thymidine (Thd) and deoxyuridine (dUrd) plasma and tissue levels increase and cause nucleotide pool imbalances. This leads to instability of mitochondrial DNA with loss of mitochondrial respiratory chain functions. Clinical consequences manifest as a multisystemic disease with severe gastrointestinal dysmotility, most severe cachexia (BMI 10-17 kg/m2 in our patients), ptosis and/or ophthalmoparesis, peripheral neuropathy and leukencephalopathy. While TP is not expressed in all tissues, cellular and plasma Thd and dUrd levels appear to be in equilibrium among all body compartments. Mononuclear white blood cells and platelets are rich in TP activity and can transiently restore TP activity upon transfusions. Therefore allogeneic HSCT was tested as a permanent replacement therapy by several teams. A coorperative group under the auspices of the WBMT collected the global experiences. So far, 10 patients underwent an attempt for HSCT between 2005-2009. One patient stopped conditioning due to toxicity and did not proceed to transplantation. Nine patients had 12 allogeneic HSCT. They were 6 males and 3 females with a median age of 28y (range 10-41y) at transplantation. All were symptomatic at time of HSCT (5 on parenteral nutrition). A variety of different conditioning regimens and GvHD prevention strategies were used. Fludarabine was included in all conditioning regimens, combined with busulfan or cyclosphoshamide, melphalan, thiotepa or TBI. T cell depletion (TCD) with ATG, alemtuzumab or in vitro TCD was performed in 10 HSCT. GvHD prevention with sirolimus or calcineurin inhibitors was used in combination with mycophenolate mofetil or methotrexate. Graft source for first transplants was peripheral blood stem cells (PBSC) in 4, bone marrow (BM) in 3 and cord blood in 2. PBSC and BM donors were HLA-identical siblings in 2, phenotypic identical parent in 1 and unrelated donors in 4 (3 with 10/10 HLA-match, 1 with 9/10-match). Engraftment was problematic. Three primary graft failures and two late graft failures were observed. A second HSCT was performed in three patients, all engrafted but two died due to TRM. The two patients without a second HSCT died from their disease. Four patients developed acute GvHD grade I-IV. To date, five patients are alive 8-48 months posttransplant, three after related PBSC-HSCT, one after unrelated BM-HSCT (10/10-HLA identical), one after two HSCTs with BM and PBSC respectively from the same unrelated 10/10-HLA identical donor. In all of these patients metabolism normalized as measured by normal Thd and dUrd levels. All surviving patients are off parenteral nutrition without further weight loss or increase in body weight. Gastrointestinal symptoms improved and in the two patients with the longest follow up a slight improvement of neurological symptoms can be observed now. Further time is needed to determine whether other disturbed organ functions will be reversible. Conclusion: Allogeneic HSCT can restore full metabolic function and halt and restore clinical signs and symptoms in this otherwise unrelenting progressive disease. Engraftment was identified as a key obstacle. Still, the optimal transplant regimen needs to be defined to improve patients' outcome. A common consensus transplant protocol and disease specific pre- and posttransplant evaluation protocol was developed with participation of all involved teams. Disclosures: Gratwohl: Amgen: Research Funding; Roche: Research Funding; Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding. Hirano:Athena Diagnostics: Speakers Bureau; Pfizer: Research Funding.

Description: Background and Aim Although new agents have been approved for the treatment of MDS, the only curative approach for these patients is allogeneic hematopoietic stem cell transplantation (HSCT). Nevertheless, in these patients this approach has only obtained 40-60% of overall survival. Somatic mutations in MDS have recently been analyzed in order to confirm clonally and also prognostic impact in MDS patients. In this regard, TP 53 mutated gene is present in MDS in less than 10% of patients and is associated with advanced disease and high-risk features. Recent studies confirms poor outcomes in patients with TP 53 mutated receiving allogeneic stem cell transplantation1,2. The present study try to analyze if the development of chronic graft versus host disease (cGVHD) could modify, due to graft versus leukemia effect, the adverse prognosis of these high-risk patients (TP53 mutated patients). Design and Methods Results of HSCT in 92 MDS patients from 5 centers in Spain were retrospectively studied. Samples were collected 1 month prior to transplant. 280ng of the genomic DNA from BM cells was screened for somatic mutations in TP53 gene. The study was done by NGS on a GS Junior Instrument (Roche) according to an amplicon sequencing design. For each sample, eight exons (4-11) were amplified with preconfigured primer plates provided within the IRON II study network. Data analysis, were carried out using the Sequence Pilot software version 3.5.2 (JSI Medical Systems) and GS Amplicon Variant Analyzer software, versions 2.7 and 2.9 (Roche Applied Science). Minimum coverage of sequenced exons was 100 reads and the sensitivity of variant detection was set to a lower limit of 〉2% for bidirectional reads. Only those variants that resulted in amino acid change in the protein sequence were considered. OS and RFS were calculated using the Kaplan-Meier method. The log-rank test was used for comparisons. All calculations were done using SPSS 18.0. Cumulative incidence of relapse was also calculated by xlstat version 2014 program. Results Median age was 54 years (17-69), 71.7% were "de novo" MDS and regarding IPSS, 53% were in the int-2/high-risk category. Other characteristics were in Table 1. In the pre-transplant evaluation, 15 patients out of 92 (16,3%) were TP 53 mutated. The mutations were located in exons 5, 6, 7, 8 and 10. These variations were present in a variable percentageof the cell population (3 to 84%). All mutations were specific nucleotide changes except for two cases. At the time of the last update, 16 patients had relapsed (17.4%) and 40 had died (43.5%). After a median follow up of 15.5 months, OS was 56.5%. Median OS for patients with mutated TP53 trend a toward to be shorter than survival for patients without mutated TP53 (median of 7 mo vs median not reached, respectively, p=0.156). Multivariate analysis for OS confirmed complex karyotype (HR 5,588, 95CI 1,794-17,407, p=0.003) and no developement of cGVHD (HR 3,531, 95IC 1,634-7,632, p=0.001) as predictors for poor outcome. Cumulative incidence of relapse was 20.3% (+/-4.3%) at 1 years. Mutational status of TP53 significantly influenced on relapse (53.3% +/-12.9% vs 13.7% +/-4% at 1 year for patients with vs without TP 53 mutation (Gray test=0.001, Figure 2). Regarding Relapse Free Survival (RFS), after a median of follow up of 17 months, RFS was 67.9% and as previously suggested, the presence of TP 53 mutation had an impact on RFS (41.7% for mutated (median RFS of 6 months) and 75% for non mutated patients (median RFS not reached), p=0.009). Multivariate analysis for RFS confirmed age (HR 1.054, 95CI 1.005-1.106, p=0.032) and TP 53 mutated (HR 3.054, 95IC 1.145-8.149, p=0.026) as predictors for lower RFS. Regarding 15 patients with mutated TP 53, 7 did relapsed and 9 had died. Developement of cGVHD showed a trend toward to improve outcome among TP 53 mutated patients, with a better OS and RFS for those developing cGVHD as compared to those who did not (OS of 55% vs 17% for patients with and without cGVHD, p=0.039, Figure 2 and RFS of 71% vs 50%, respectively, p=0.3). Conclusions Mutated TP53 pre-allo patients presents poor outcome as compared to not mutated, as previously described Bejar1 and Kim2. Nevertheless, the developement of cGVHD could overcome the adverse impact of this factor due to the developement of graft versus tumor efect, improving survival curves (OS and RFS) as compared to previous published results. Study supported by GRS-1033/A/14 P53. 1.-BŽjar, JCO 2014, 32(25). 2.-Kim, BBMT 2015, Epub ahead of print. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Sanz: JANSSEN CILAG: Honoraria, Research Funding, Speakers Bureau. Valcarcel:AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Díez-Campelo:CELGENE: Research Funding, Speakers Bureau; JANSSEN: Research Funding; NOVARTIS: Research Funding, Speakers Bureau.

Description: INTRODUCTION: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms originated in hematopoietic stem cells, characterized by citopenias, dysplasia in one or more cell lines, ineffective hematopoiesis and an increased risk of progression to acute myeloid leukemia (AML). Treatment of MDS depends on subtype and prognostic category. DNA methyltranferase inhibitors are approved for high risk MDS. Over the past decade, the application of new high-throughput technologies to the study of MDS has led to the identification of several recurrently mutated genes. These include genes producing proteins involved in RNA splicing, DNA methylation, chromatin modification, transcription, DNA repair control, cohesin function, RAS pathway, and DNA replication. There is a significant overlap between the genes mutated commonly in MDS with those found in AML. Mutation status is not widely used to select treatment in MDS. The aim of this study is to define the mutational status of MDS and secondary AML (sAML) patients at diagnosis that have been treated with azacitidine (AZA) to see if it could help to discriminate which patients will respond from those who will not. MATERIAL AND METHODS: A prospective study was performed on 36 patients with MDS and sAML treated with AZA. Genomic DNA was obtained from bone marrow at diagnosis. SeqCap EZ and KAPA Library Preparation Kit (Roche) reagents have been used to enrich DNA of 83 genes implicated in myeloid neoplasm. The customized panel has been analyzed in MiSeq Illumina platform with 150bp paired-end reads. Samples were preliminary analyzed using Illumina MiSeq Reporter and Variant Studio softwares. Data from response to treatment and survival have been collected from all patients. RESULTS:The mean depth of the targeted resequencing per base was 685-fold. After filtering all the variations obtained for quality, biological consequence and discard the known SNPs, we have obtained 162 variations, including 145 single nucleotide variants (SNV) and 17 insertions/deletions. All patients harbored at least 1 alteration with a mean of 4.5 variants per sample. The average of alterations detected in each cytological category can be observed in Table 1.Table 1.Average abnormalities detected by cytological category.Nº patientsAverage of alterations detected for patient (range)sAML104,8 (1-8)RAEB-274,9 (2-8)RAEB-1123,7 (1-6)RCDM54,4 (3-7)RCDM-RS16RARs11The most frequent altered genes have been TP53, TET2 and DNMT3A. The numbers of variations detected for each gene are represented in Table 2.Complete results, including correlation with treatment response will be presented in the meeting.Table 2.Number of variations in each gene.GeneNº of variations foundNº of diferent variationsNº of patients with variationsFrequency of variationsTP5322191952,8%TET214101027,8%DNMT3A88822,2%CREBBP75719,4%SRSF271719,4%ASXL165616,7%U2AF162616,7%EP30053513,9%STAG255513,9%CUX144411,1%ETV643411,1%MLL (KMT2A)43411,1%RUNX14438,3%BCOR3338,3%CDH133338,3%CTNNA13238,3%EZH23338,3%GCAT3338,3%MLL2 (KMT2D)3338,3%NF13338,3%PDGFRB3338,3%SH2B33338,3%TGM23238,3%UMODL13338,3%CEBPA2125,6%CSF3R2225,6%GATA22125,6%PHLPP12225,6%RAD212225,6%SF3B12125,6%SUZ122225,6%TIMM502125,6%Others*1112,8%*ABL1, BCORL1, CALR, CDH3, IDH2, KRAS, LUC7L2, NPM1, NRAS, PHF6, SF3A1, SFPQ, SMC3, TERT, WT1, ZRSR2. CONCLUSIONS: Targeted deep-sequencing technique is a good tool to study mutational profile in MDS and sAML. SNV are the most frequent type of alteration found in our cohort. The patients with sAML and RAEB-2 present more variations than patients with RAEB-1. The rest of groups are less representing to be evaluated. The most affected genes match with those described in the literature, with some exceptions that need to be studied in more detail. We expect to predict in advance which patients are going to respond when we study the correlation of mutational analysis with treatment response. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain. Diana Domínguez for her technical assistance Disclosures Valcarcel: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: INTRODUCTION: The myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). Aberrant DNA methylation is the dominant and most well-studied epigenetic alteration in MDS. Various genes, including cell cycle regulators, apoptotic genes, and DNA repair genes, are epigenetically silenced and play a role in pathogenesis and transformation to leukemia. The clinical response of MDS and AML to drugs that revert the aberrant hypermethylation, such as 5-aza-2x-deoxicitidine and 5-azacitidine (AZA), suggests that this hypermethylation could have an important role in the disease and it is not a secondary effect to other mechanisms. The aim of this study was to define the methylation pattern of DNA at diagnosis in patients with high-risk MDS and secondary AML to determine if there are some pattern predictive for response to AZA treatment or relapse. MATERIAL AND METHODS: Genomic DNA was obtained from bone marrow (n=100): 39 patients at diagnosis prior AZA treatment (24 MDS, 10 AML and 5 CMML), 10 control samples (peripheral blood of donors of bone marrow stimulated with hematologic growth factors) and 51 samples at different moments of follow-up (3 months, 6 months, 12 months and/or 18 months). Genome-wide DNA methylation profiling was performed using the Illumina Infinium 450K methylation array. We have used the IWG-2006 criteria for classifying the responders versus non responders. RESULTS: We have focused on MDS patients treated with AZA comparing methylation profiles at diagnosis and at the time of first detected response to treatment (mostly at 3 months). We performed different experiments of DNA methylation assays using RnBeads tool. The global methylation dendogram (Fig.1) clearly demonstrate that the subgroup of responders (7 patients) show strong demethylation after the treatment, while in non-responders methylation profiles has not changed that dramatically even after 6 months from the beginning of the treatment. We also have found several hundreds differentially methylated regions (DMR) at gene promoters, gene bodies and other genomic locations, which are significantly hypomethylated after the treatment in responders, while remain almost unchanged after the treatment in non responders. CONCLUSIONS: Illumina Infinium 450K methylation array serves as an informative methodology to study the methylation changes in bone marrow samples. A more detailed investigation of obtained DMR might shed light on the mechanisms of the response to AZA treatment and as result to allow detection of methylation markers of response to treatment or relapse. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain. Diana Domínguez for her excellence technical assistance. Figure 1. Bi-clustering of the methylation levels of gene promoters in responders (green: diagnosis and orange: response) Figure 1. Bi-clustering of the methylation levels of gene promoters in responders (green: diagnosis and orange: response) Disclosures Valcárcel: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Introduction: Based on the 2008 World Health Organization classification (WHO 2008), erythroleukemia is defined by the presence of ≥50% erythroid precursors in bone marrow (BM) and ≥20% myeloblasts in the non-erythroid cell population. Multilineage dysplasia is almost always present with high rates of MDS-like cytogenetic abnormalities, specially complex karyotypes. Therefore an extensive comparison with myelodysplastic syndromes (MDS) with ≥50% erythropoesis seems crucial to elucidate whether erythroleukemia and MDS with erythroid hyperplasia should be considered as different biological entities. Aim: To elucidate this issue, the outcome and cytogenetic alterations of erythroleukemia patients were studied and compared to MDS patients with ≥50% erythropoesis with

Description: Introduction: The MDS are a group of clonal hematopoietic disorders characterized by blood cytopenias and increased risk of transformation into acute myeloid leukemia (AML). The MDS predominate in old people (median age at diagnosis 〉 70 years) so that a fraction of the observed mortality would be driven by age-related factors shared with the general population rather than the MDS. Distinguishing between the MDS-related and unrelated mortality rates will help better assessment of the population health impact of the MDS and more accurate prognostication. This study was aimed at quantifying the MDS-attributable mortality and its relationship with the IPSSR risk categories. Methods: The database of the GESMD was queried for patients diagnosed with primary MDS after 1980 according to the WHO 2001 classification. Patients with CMML, younger than 16 years or who lacked the basic demographic or follow-up data were excluded. Relative survival and MDS-attributable mortality were calculated by the cohort method and statistically compared by Poisson multivariate regression as described by Dickman (Stat Med 2004; 23: 51). Three main parameters were calculated: the observed (all-cause) mortality, the MDS-attributable mortality (both as percentage of the initial cohort), and the fraction of the observed mortality attributed to the MDS. Results: In total, 7408 patients met the inclusion criteria and constitute the basis for this study. Among these patients, 5307 had enough data to be classified according to the IPSSR. Median age was 74 (IQR: 16-99) years and 58 % were males. The most frequent WHO categories were RAEB, type I or II (29% of cases), RCMD (28%), and RA with ring sideroblasts (16%). Most patients (72%) were classified within the very low and low risk categories of the IPSSR. At the study closing date (December 2014), 1022 patients had progressed to AML, 3198 had died (974 after AML) and 3210 were censored alive. The median actuarial survival for the whole series was 4.8 (95% CI: 4.6-5.1) years and 30% of patients are projected to survive longer than 10 years. The overall MDS-attributable mortality at 5 years from diagnosis was 39%, which accounted for three-quarters of the observed mortality (51%, figure). The corresponding figures at 10 years for the MDS-attributable and observed mortality were 55% and 71%, respectively. According to the IPSSR, the 5-year MDS-attributable mortality rates was 19% for the very low risk category, 39% (low risk), 70% (intermediate risk), 78% (high risk), and 92% (very high risk). On average, the incidence rate ratio for the MDS-attributable mortality increased 1.9 times (95% CI: 1.7-2.3, p

Description: Introduction Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder characterized by a heterogeneous clinical and morphological expression that shares features of both myelodysplastic syndromes (MDS) and chronic myeloproliferative disorders. In the last years therapy of CMML has undergone a change with the inclusion of the demethylating agents but data regarding their impact on the “real life” setting are still scarce. The aim of our study was to evaluate the use of the different therapies and the time to therapy in an unselected Spanish population within the ERASME study. Materials and methods The ERASME study (CEL-SMD-2012-01) is an observational, post-authorization, prospective, multicenter study that will include a total of 600 patients with MDS and CMML and follow them during a minimum of three years (or until death). The primary objective of this study is to describe the disease progression in routine clinical practice, based on the initial therapeutic strategy, in patients with newly diagnosed MDS and CMML. We present here the results of a pre-specified interim analysis with data of CMML patients enrolled in the ERASME study. Initial patient management strategy is classified in three groups: Observation (OB) & support (SP) (including blood and platelet transfusions and growth factors), active therapy (AT) (including chemotherapy, azacitidine, lenalidomide, etc) and allogenic hematopoietic cell transplant (HCT) (including those patients receiving other therapies before transplant). Results A total of 41 CMML patients (34% women) with a median age of 80 years (range 49-91) have been recruited between January 2013-June 2014. The median follow-up time was 6.7 months (range 0.4-15.1). Morphological subtypes according WHO classification were CMML-1 (blasts count 13x109/L). Karyotype was normal in 32 patients (86%). Five patients displayed cytogenetic abnormalities; 3 out of 5 patients with trisomy 8 (isolated or with one additional abnormality). The CMML-GESMD cytogenetic risk classification was low/intermediate/high risk in 83%/10%/5% of patients, respectively. The CPSS was low/int-1/int-2/high in 46%/32%/15%/5% of patients, respectively. Nine out of 41 patients were transfusion dependent at diagnosis. Median bone marrow blast count was 3% (range 0-33). Hemoglobin, platelet and neutrophil count was: 11.1 g/dL (range 7.8-16.7), 106x103/µL (4.2-415), and 3.98x109/L (range 0.48-57.2), respectively. After diagnosis, 33, 7 and 1 of CMML patients were considered candidate to SP/OB, AT and HCT strategy, respectively. The main reasons for treatment selection were risk-disease (90%), symptomatology (83%), age (73%), and comorbidities (46%).The median time to AT initiation from diagnosis for AT/OB&SP was 0.52/2.5 months (range 0.22-2.29) and (range 1.0-4.7) for each group, respectively. Patients in active therapy received azacitidine (n=2, 29%), other low-dose chemotherapy (n=4, 57%) and other therapy (erythropoietin and azacitidine) (n=1, 14%), respectively. Only one patient was considered candidate for HCT and this patient received azacitidine prior the transplant. At last follow-up, a total of 5 (12%) of patients have died (2, 29% of active therapy and 3, 9% of support group) after a median of 3.6 months (range 3.1-4.1) and 1.7 months (range 0.7-10), for each group respectively. Conclusions CMML patients were treated on an individualized therapy strategy after diagnostic evaluation and prognosis assessment. More data on disease progression in routine clinical practice may be useful in characterizing the newly diagnosed CMML patients. Our prospective study confirms that azacitidine has been considered a therapy for CMML patients, including for HCT candidates. Disclosures Off Label Use: Vidaza, erythropoietin stimulating agents, revlimid. Valcarcel:Celgene: Honoraria, Speakers Bureau. Rafel:Celgene: Employment. Garcia:Celgene, Novartis: Consultancy, Speakers Bureau.