ALBERT

Description: Background: Lenalidomide (LEN) is a reference treatment in IPSS low and in1 (lower) risk MDS patients with isolated de(5q) (MDS-del(5q)) and RBC - TD. Most low risk MDS-del(5q) patients with anemia and independent of transfusions develop TD or need of treatment for symptomatic anemia very early after diagnosis (median time to transfusion/treatment of 20 months, López Cadenas et al abstract 3180 ASH, 2016). LEN directly targets the del(5q) clone improving anemia, quality of life and survival in this subset of patients. Limited data also suggest a role of LEN in non-TD patients with del(5q) (Oliva et al Cancer Med 2015). However no prospective randomized study of LEN has been performed in this group of patients. Material: The Sintra-Rev clinical trial is a phase III European multicenter study, in low-risk MDS-del(5q) patients, with anemia (Hb

Description: Under steady-state conditions a small fraction of hematopoietic stem and progenitor cells (HSPC) circulate in the bloodstream. Circulation of HSPC follows a circadian rhythm of migration from the bone marrow (BM) to blood and possibly, lymph. The functional purpose of circulating HSPC is unclear but it has been associated with homeostatic regulation of tissue-specific reservoir of HSPC, vasculogenesis and immunosurveillance. During infectious stress, HSPC are activated by inflammation mediators and chemokines and mobilized to circulation and extramedullary tissues. Side Population (SP) cells are stem cells with pluripotent differentiation ability found in a number of adult tissues. Here we have studied whether the lymph nodes (LN) are an obligatory site of transit/homing of HSPC in inflammation. To analyze whether there is accumulation of HSPC in inflammatory lymph nodes, we first analyzed the presence of SP cells in human LN. The patient median age was 34 (range 3-89). Patient-derived LN biopsies were diagnosed by the pathologist as having reactive lymphadenitis (RL, n=35, 50.7%) or cancer (n=34, 49.3.%). Cancer diagnoses were Hodgkin’s Lymphoma (HL, n=12,17.4%), non-Hodgkin’s lymphoma (NHL, n=20, 28.9%) or adenocarcinoma (AC, n=2, 2.9%) which associate with decreasing levels of inflammation. The biopsy-derived LN cells were stained with Hoechst 33342/propidium iodide and monoclonal antibodies against CD45, CD34, CD20, CD19, CD133 and ABCG2 surface markers. Flow cytometry analysis of LN specimens identified SP cells in 82.3% of RL LN samples (1.9±1.2%; range 0-39%), 50% of HL LN biopsies (0.7±0.6%; range 0-7.7%) and 20% of NHL LN biopsies (0,03±0.01%; range 0-0.23). Interestingly, SP cells were absent from AC LN specimens. The presence of LN SP cells was significantly associated with RL versus any of the analyzed cancer groups (P

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: Abstract 2586 Introduction: Acute Myeloid Leukemia (AML) is a heterogeneous disorder arising from a clonal expansion of Leukemic Stem Cell (LSC). The characterization of LSC is crucial because it is resistant to conventional chemotherapy and is ultimately responsible for leukemic relapses. The LSC in AML is a phenotypically heterogeneous population (CD34+CD38-, CLL1 +, CD96 +…). In this sense, “Side Population” cells (SPHo342Low) are considered to be a type of stem cells that can self-renew and differentiate into tissues. SP are characterized by their ability to efflux the vital dye Hoechst 33342 through the drug ABCG2 pump. SPHo342Low cells have been described in many types of solid tumors and AML as potential LSC. The objective in this study is to analyze the frequency of SPHo342Low in AML, their phenotype and the possible prognostic impact on outcomes. Patients and Methods: Bone marrow samples (BM) obtained from 57 patients (median age 58 years, range: 4–82), diagnosed with AML between Mar-07 to Mar-12, were included. Distribution of cytogenetic risk groups was: Favorable (12.5%), Intermediate (60.7%) and Unfavorable (26.8%). NPM1mut was present in 11 cases and FLT3-ITD in 6 cases. Prior MDS was present in 10 cases. After achieving complete remission (CR) with conventional chemotherapy, allogeneic or autologous stem cell transplantation was performed in 17 and 12 patients respectively, according to individual risk and availability of donor. Eleven frail patients received as front-line, low intensity therapy with Azacytidine. We detected LSC, SPHo342Low in marrow MNCs obtained at diagnosis (N=40), at morphologic complete remission (CR) (N=21) or at relapsed / resistant (N=16) disease. For detection, 2×10(6) MNC/ml were resuspended in HBSS medium with 5 ug/ml of Ho342 dye and CD45-FITC, CD34-PE Mn-Abs, analyzing at least 1×105 viable cells in UV laser FACSVantage cytometer with the combination of filters BP 670/40 for emission in red and BP 450/30 for the blue emission. We verified SP region by inhibiting ABCG2 pump with Verapamil (50μM/mL). As controls we analyzed MNCs from BM aspirates from healthy donors (N=5). Results: In all BM samples from healthy donors, SPHo342Low population was detected accounting for 0.5% (range: 0.1 to 0.9%) and it was CD34negCD45neg phenotype in 80% of cases. SPHo342Low cells were detected in 23/40 cases (57.5%) of samples from AML diagnosis with a median of 0.08% (range 0.01–2.3%). Phenotype of SPHo342Low cells at diagnosis was CD34+CD45+/− in 36% of cases. The presence of SPHo342Low cells presented in AML at diagnosis did not statistically correlate with any prognostic clinical variables such as age, cytogenetic-molecular risk or prior MDS. Interestingly, the detection of LSC SPHo342Low at diagnosis was statistically associated to the presence of 〉0.1% of CD34+CD38- AML cells (P=0,03). In BM samples obtained from AML patients in CR, SPHo342Low cells were detected in 17/21 (81.0%) with a median of 0.17% (range: 0.1 to 0.76%), with a phenotype mostly CD34 negative. In BM samples obtained from AML patients in relapsed/refractory situation, SPHo342Low cells were detected in 14/16 (87.5%) with a median of 0.22% (range: 0.2 to 0.91%) with a phenotype of CD34+ CD45+/− in 33% of cases. Interestingly, patients who did not achieve CR, have a significantly higher percentage of SPHo342Low at diagnosis (0.42% vs. 0.06%, P = 0.044) as well as those who need more than one cycle to achieve CR (0.52% vs. 0.07%, P = 0.04). Moreover, for those patients achieving CR, persistence of Minimal Residual Disease (MRD+) was associated to a higher percentage of SPHo342Low at diagnosis (0.28% vs. 0.05%, P = 0.021). Likewise, Relapse-free survival (RFS) was significantly higher in AML patients lacking SPHo342Low at diagnosis (70 ± 18.2% vs. 43.3 ± 17.6%, P = 0.0324, Log rank test). Conclusions: Detection of LSC SPHo342Low+CD34+CD45+/− phenotype in AML at diagnosis is a common finding that is associated with increased resistance to achieve CR, clearance of MRD and lower RFS. During progression of disease this SPHo342Low+ population increases and maintains CD34+CD45neg phenotype. BM samples obtained from AML patients at CR were SPHo342Low+ CD34negCD45+/− phenotype which can be considered responsible for normal hematopoietic regeneration. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid stem cell disorders that are highly prevalent in elderly populations. MDS are characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and increased risk of transformation to acute myeloid leukemia (AML; 20–30% of patients with MDS). Around 50% of MDS patients carry at least one karyotypic aberration. The interstitial deletion of the long arm of chromosome 5 ([del(5q)] is the most common aberration, accounting for almost 30% of abnormal MDS karyotype. Various studies supports a favorable prognosis of MDS with isolated del(5q) with an excellent response to lenalidomide treatment. In order to describe the molecular events associated with MDS and del(5q) we performed whole-exome sequencing (WES)(assessing 334,378 exons) of tumor-normal paired samples from 20 MDS patients to unravel the genetic basis of MDS with del(5q). The analysis is ongoing and the complete results will be presented in the meeting. METHODS A total of 50 samples from 20 patients with MDS, with del(5q) were collected. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss del(5q)) and SNP arrays with Cytoscan HD (Affymetrix). These samples included: 20 tumor samples at diagnosis, 20 control samples and 10 samples after diagnosis, during lenalidomide treatment (5) or at the moment of relapse (5) in order to compare the genetic status before and during the treatment. Genomic DNA from tumor cells was obtained from bone marrow (BM) samples or from PB granulocytes. As a source of constitutional DNA we used CD3+T cells from each patient by isolating by magnetic-activated cell sorting. WES targeted capture was carried out on 7μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4.Libraries were sequenced on an Illumina HiSeq2000. Sequencing data will be analyzed using an in-house bioinformatics pipeline as previously reported. RESULTS Our preliminary analysis of these 20 new patients by WES confirmed our previous analyses with mutations in well described genes as ASXL1, JAK2 and TET2, but not in genes RUNX1, SF3B1 and SRSF2. In those patients we found two patients with missense mutation in TP53, one of the patients had an isolated del(5q) and is receiving lenalidomide treatment, and the other one had a complex karyotype. According to our prior analyses, in which 249 non-silent somatic variants were detected, we look forward to validate these mutations in this new series of patients. CONCLUSIONS We envision to validate these previous results with the new sequencing data of more patients with MDS and del(5q). We expect to measure somatic mutations that vary in abundance after lenalidomide treatment, potentially identifying mutations associated with resistance or relapse. ACKNOWLEDGEMENTS: This work has been supported (in part) by a grants from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras; Obra Social “la Caixa”; Sociedad Española de Hematología y Hemoterapia (SEHH)and Celgene Spain. FOOTNOTES Rafael Bejar and Francesc Sole contributed equally. Disclosures Díez-Campelo: Novartis, Celgene: Honoraria, Research Funding. Xicoy:Celgene: Honoraria. Cañizo:Celgene, Jansen-Cilag, Arry, Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. sanchez-Garcia:Celgene: Honoraria, Research Funding. Bejar:Celgene: Membership on an entity's Board of Directors or advisory committees; Genoptix Medical Laboratory: Consultancy, Honoraria, Licensed IP, no royalties Patents & Royalties, Membership on an entity's Board of Directors or advisory committees. Sole:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Cytogenetic abnormalities are found in around half of MDS patients (pts) and have both clinical impact and may be subtype-defining, e.g. in 5q-syndrome. Interstitial deletion of the long arm of chr.5 [del(5q)] is the most common aberration (almost 20% of cases with abnormal cytogenetics). Del(5q) is heterogeneous, occurring as a sole abnormality or in combination, with the deleted region often truncated within or extended and/or beyond the CDR boundaries. Isolated del(5q) is frequently shorter and confers a more favorable prognosis with regard to survival and lenalidomide (LEN) responsiveness, while del(5q) in the context of a complex karyotype (CK) imparts a poor prognosis. In addition to chromosomal lesions, somatic mutations can contribute to the pathogenesis of MDS, including del(5q). We theorized that recognition of molecular defects in MDS with del(5q) may clarify the pathogenic mechanisms behind this lesion and help explain the clinical heterogeneity. We analyzed 225 pts with myeloid neoplasia and del(5q) using WES (n= 107 samples) and targeted multiplexed PCR (top 60 most frequently mutated genes) (n =133 samples); serial analysis was performed in 15 pts studied at ≥2 time points, 11 during LEN therapy and 4 upon relapse/progression. A total of 116 samples had a CK with other lesions such as -7/del(7q) found in 31% cases, and 18% had -17/del(17p). WES (average depth 〉60x) was followed by a bioanalytic pipeline, detecting ≥1 mutated gene in 71% of cases. Candidate somatic alterations were found in 357 genes and selected for further analysis. When focused on hemizygous mutations within the retained 5q allele, CSNK1A1 mutations were the most common, found in 4 pts, while other genes were only sporadically affected. Among heterozygous mutations on the non-deleted portion of del(5q) and other chromosomes (Chr), we found several novel mutations, in addition to TP53 (n=26), DNMT3A (n=8), PRPF8 (n =8), RUNX1 (n=5), TET2 (n=5), and ASXL1 (n=4), among others. Furthermore, LOH/haploinsuffciency of genes on 7q (e.g., LUC7L2, CUX1, EZH2 and MLL3) appears to be a common defect seen in pts with non-isolated del(5q), suggesting synergistic functional defects. When functionally grouping gene mutations, DNA methylation family (8 cases) and transcription factor mutations (29 cases) were associated with advanced disease (AD) and a CK. Heterozygous mutations in TP53 (34%) or deletions involving the TP53 locus (23%) resulted in total of 42% of cases carrying either TP53 LOH or mutation. TP53 lesions were more common in pts with AD vs. low risk. (21 vs. 5 p =.0008). In contrast, TP53 mutations are found in 8-10% of cases of MDS. A total of 34 pts were treated with LEN and subgrouped into responders (n=17) vs. refractory (n=9) with an overall response rate of 65%. When mutational profiles were compared, the presence of TP53 mutations did not preclude responsiveness to LEN. CK was present in 12% of responders vs. 67% of refractory pts. The most frequent Chr abnormalities were -7/7q (0% vs. 67% in responders vs. refractory) and 17p-(6% vs. 67% in responders vs. refractory) suggestive of their role in LEN resistance. In addition to cross sectional analysis, our WES study using paired Germline/tumor samples followed by deep sequencing facilitated analyses of clonal architecture by examining clonal dynamics over time. Assessment of del(5q) clone size by allelic imbalance combined with clonal burden by VAF allowed us to reconstruct the clonal hierarchy: in 73% of cases, del(5q) appeared to be the initial defect followed by subsequent mutations (e.g., TP53, DNMT3A, IDH2). In contrast, in 24% of cases, TP53, RUNX1, JARID2, were the primary defect followed by a subclonal del(5q) events. Serial samples collected before and after therapy demonstrated that responses were associated with decreased clonal burden for del(5q) but persistence of certain mutations. In refractory cases, persistent subclonal lesions and the appearance of new lesions were associated with progression. For example, pts with TP53, LAMB4, EPHA6 progressed and acquired additional lesions such as CSMD2 or KCND2, and did not see the disappearance of TP53 alterations upon treatment. In conclusion, no unifying somatic defect was found in pts with del(5q) regardless if the deletion event was primary or subclonal. Most commonly associated lesions were not present on the retained 5q alleles but rather other chr yet modified clinical behavior, including responsiveness to LEN. Disclosures Bejar: Celgene: Consultancy, Honoraria; Alexion: Other: ad hoc advisory board; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: HCT is the only potentially curative approach that may provide long-term disease control for patients con relapsed and/or refractory acute leukemia (Re/Ref AL). However, active disease burden at HCT is associated with high relapse rates and dismal outcomes. Recently, promising results in this way have been reported using sequential treatment schedules. We performed a retrospective multicentre analysis to investigate the safety and efficacy of clofarabine (CLO) cytoreduction prior to HCT for Re/Ref AL. Spanish Agency Qualification was obtained (AAH-CLO-2013-01/EPA-OD). A total of 50 patients from 14 Spanish centres (AML 33; ALL 17) who received CLO and had an HLA-matched donor were included. Median age was 37 years (18-64); sex: 26 M, 24 F. High risk cytogenetic/molecular profile in 31.4%, Intermediate 54%, and low risk 14.6%. Patients received first-line chemotherapy (ct) regimens based on cooperative national groups (PETHEMA, CETLAM and SHOP) and 2nd lines with fludarabine-based regimens in 85%. Prior to CLO, 56.3% of patients had received 2 CT lines and 35.5% ≥ 3 lines including 6 auto-HCT and 12 allogeneic HCT. Disease status at CLO administration was primary refractoriness (29.2%) secondary refractoriness (33.3%), first relapse (10%) and ≥2nd relapse (27%). CLO was administered at 20-40 mg/m2/day for 5 consecutive days combined with ARA-C in AML cases and Citoxan-Etoposide in ALL cases. Response was assessed by IWG-2006 criteria and toxicity evaluated according to CTCAE v3. Out of 50 included patients, 16 did not underwent allogeneic HCT due to early toxic deaths (N=6) or due to lack of disease control (N=10) 1.- Response was valuable in 44 cases, achieving 11 CR and 7 PR. Effective Cytoreduction (defined as

Description: A small fraction of hematopoietic stem and progenitor cells (HSPCs) is cyclically released into the bloodstream from bone marrow (BM). The existence of HSCP-lymphatic-blood circulation with the potential to give rise to extramedullary tissue-resident myeloid cells has been described. However, the functional role and underlying mechanisms of this circulation are not known. We hypothesized that inflammation may be a mediator of HSPC mobilization to lymph. In order to determine if inflammation was related to increased circulation of HSPCs through lymph, we analyzed the HSPC content in lymph nodes (LN) of patients with lymphadenitis. Analysis of CFU and side population (SP+) cell content confirmed that LNs from patients with reactive lymphadenitis and patients with inflammatory Hodgkin's disease contained ~2-fold higher HSPCs than in patients diagnosed of non-Hodgkin's lymphoma. To identify the pattern and mechanisms that regulate HSPCs during inflammation. We analyzed the content of HSPCs in blood (PB), BM, LN, thoracic duct (TD) and other organs after systemic administration of lipopolysaccharide (LPS) and compared with the normal circadian migratory rhythms in control animals (PBS). Mobilization of BM myeloid progenitors (mostly granulo-macrophage progenitors-GMPs, but not stem cells), to LN and TD peaked as early as three hours after LPS administration, and followed a very different pattern than the mobilization kinetics in PB. By using 3D reconstruction of confocal microscopy imaging of complete LN from lymphatic endothelium reporter mice (Lyve1Cre-eGFP), we localized all myeloid progenitors in the mantle zone of secondary follicles. We note that they did not enter the follicle germ centers. To determine the molecular mechanisms at play to induce HSPC mobilization to the LN upon LPS administration, we analyzed whether the recruitment of HSPCs in LN required Traf6, a mediator downstream of the LPS/TLR4 signaling pathway. Inducible deficiency of Traf6 driven by Mx1-Cre recombinase completely abolished the migration of myeloid progenitors to LN, but not to PB, induced by LPS as compared with their Mx1-Cre;Wt littermates indicating that the mechanism controlling HSPC recruitment to the LN was distinct from the one controlling HSPC migration to PB. Using a combination of in vivo and in vitro assays, we found that Traf6 activity regulates HSPC migration in a non-cell autonomous manner that depends on its expression in a small (~1%) fraction of non-progenitor LN myeloid cells expressing high levels of Ccl19. In vivo neutralization of Ccr7, the receptor for Ccl19, results in abrogation of myeloid progenitor mobilization to LN. GMPs migrating to LN were biased to differentiate into dendritic cells within 7 days post-migration. Using Cx3cr1-GFP transgenic mice as macrophage-dendritic cell progenitor (MDP) reporters, we found that the vast majority of GMPs recruited to the LN were MDPs, and thus already poised to DC differentiation. Finally, a combination of genetic and pharmacological approaches revealed that Traf6 signaling effects are NFkB independent but dependent upon Irak1/4, Ucb13 and IKKβ resulting in SNAP23 phosphorylation and exocytosis of pre-formed cytokines from LN myeloid cells. This study identifies the cellular and molecular basis of inflammation dependent migration of DC progenitors and suggests that the mobilization of HSPCs from BM to LN results in homeostatic replenishment of highly specialized antigen presenting cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2645 Poster Board II-621 BACKGROUND AND AIMS: The PI3/Akt pathway has been implicated in the pathogenesis of a wide variety of cancers including Acute Myeloid Leukemia (AML). Activated Akt is known to function as an essential survival factor by inhibiting apoptosis through its ability to phosphorylate several targets including Bad, FoxO transcription factors, Raf-1, caspase-9 and inhibitor of apoptosis protein family (IAPs). Survivin is a member of IAP regulating both apoptosis and cell cycle progression. Survivin binds to several structural components of the mitotic apparatus and can block apoptosis by inhibiting caspases 9, 3 and 7. Recently the importance of PI3/Akt/survivin pathway in solid neoplasias such as breast or prostate cancer has been highlighted but its role in AML remains unknown. In this work we analyzed the PI3/Akt/survivin pathway in human AML. METHODS: Bone marrow samples obtained at diagnosis of 68 AML consecutive patients and K562, MV4-11 and HL-60 cell lines were included. Patients Median age was 62 years (range 8–89). Median leukocyte count was 10.3×109/L (range 0.8–285). FAB subtypes were: M0=8, M1=20, M2=13, M3=8, M4=11, M5=7 and M6=1 with the following cytogenetic findings: t(15;17)=8, t(8;21)=2, complex karyotype=8, 11q23=2, normal karyotype=36 and others=12. There were 11.3% patients with FLT3-ITD and 16.9% with NPM1 mutation. Cytoplasmic and nuclear Proteins were harvested with Q-proteome cell compartment (Qiaqen) and protein concentration assayed using Protein Assay Kit (Bio-Rad). Total Akt, Akt-pSer473 and survivin proteins were detected by Western Blot and were visualized by enhanced chemiluminescence (ECL-Plus, GE Healthcare) in Chemigenius-2 and quantified using Gene-Tools software. Cell cycle analysis was assessed by double Hoechst 33342- Pyronin Y staining and flow cytometry in FACSvantage. Inhibition experiments were done using Ly294002 at 25 μM, and Wortmaninn at 250 nM for 12 hours. RESULTS: In our series p-Ser473Akt was detected in 56% of AML marrow samples (with high levels expression in 27%) and in all cell lines. In cytoplasmic protein extracts, Survivin WT and 2B isoform were detected in 45% and 45.2% of patients respectively. All three leukemic cell lines showed only Survivin 2B expression. Interestingly, there was strong statistical correlation between the levels of p-Ser473Akt with cytoplasmic Survivin (P=.01). Inhibitors of PI3K/Akt pathway LY294002 and Wortmaninn both decreased in vitro p-Ser473Akt expression but only the irreversible action of Wortmaninn caused a marked dowregulation of cytoplasmic survivin. Meaningfully, lack of cytoplasmic Survivin was associated with an increased proportion of cells in Go cell cycle phase (11.1 % vs. 3.6%, P=.04). Moreover, cytoplasmic Survivin WT localization and high p-Ser473 Akt levels, were both significantly correlated with less unfavourable FAB leukemia subtype and cytogenetic risk (P

Description: Introduction: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. The clinical efficacy of lenalidomide in MDS patients has been extensively reviewed and although the mechanisms of action in del(5q) clone have been previously described, in vivo sequential studies of modulatory effect on T lymphocytes are lacking. Our study was conducted in patients included in the Sintra-REV Clinical Trial: Lenalidomide (Revlimid) phase III, multicenter, randomized, double-blind study versus placebo in patients with low-risk MDS (low and intermediate IPSS-1) with del(5q), with anemia (HB≤12gr/dl) and without transfusion needs. Aim: The aim of this study was to explore the effect of lenalidomide in T-lymphocytes in MDS patients with del(5q) and without transfusion dependence. Materials and Methods: Sequential study was carried out in 26 samples from 13 paired MDS patients with del (5q). Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated-patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR 〈 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite. Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; 199 of them were over-expressed after the administration of this drug (Fig 1a). Of note, none of them were observed in non-treated patients after one month. The ORA revealed significant differences in the gene expression profile of sixteen cytokines and enrichment of genes of the cell cycle pathway (35 genes). The most relevant up-regulated cytokines were: IL10, TNFSF10, IFNGand IL6. These data explain lenalidomide-induced activation of an antileukemic immune response and secretion of anti-inflammatory cytokines. Although lenalidomide has been reported to reduce the expression of IL6 secreted by myeloid cell derived from MDS clon, we have observed upregulation of this gene in T-lymphocytes. Moreover, our study showed a downregulation of MBP6 that may help to correct the anemia and also attenuate inflammation signaling in MDS patients with del(5q) (Fig 1b). In addition, the most represented up-regulated genes related to cell cycle pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C), BUB1 mitotic checkpoint genes (BUB1, BUB1B), and genes involved in cell division (CDC6, CDC7,CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells (Fig 1b). Conclusions: The immunomodulatory properties of lenalidomide can be summarized in two: a) regulation of antileukemic and anti-inflammatory cytokines production, b) activation of cell cycle and proliferation in T cells. To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. Disclosures Sanz: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Fenaux:Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Diez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Lenalidomide was administered in anemic but not transfusion-dependence patients with low-risk MDS and del(5q)