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TO THE EDITOR: Treatment of splenic marginal zone lymphoma (SMZL) is not standardized due to the lack of prospective randomized trials.[1][1][⇓][2][⇓][3][⇓][4][⇓][5][⇓][6][⇓][7][⇓][8][⇓][9][⇓][10][⇓][11][⇓][12]-[13][13] After our initial 2007 paper, we now present updated data

Introduction-Aim: Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by the prolonged and unexplained reduction in the number of peripheral blood (PB) absolute neutrophil counts (ANC). The underlying pathogenesis in CIN implicates the production of proinflammatory cytokines by activated lymphocytes and monocytes that induce excessive apoptotic death of the bone marrow (BM) granulocytic progenitor cells. Clonal hematopoiesis identified by next generation sequencing (NGS) of myeloid genes is found in 11% of CIN patients conferring an increased risk for MDS/AML transformation whereas the non-clonal patients display usually a benign course. The basis for the immune cell activation and proinflammatory cytokine production in CIN remains obscure. Based on previously reported data showing increased frequency of mutations of the MEFV gene encoding pyrin in patients with idiopathic inflammatory conditions other than typical Familial Mediterranean Fever (FMF), we sought to investigate the common MEFV mutations in a cohort of well characterized CIN patients. Patients-Methods: We have studied 50 patients fulfilling the previously reported diagnostic criteria of CIN (median ANC 1.5x10 9/L, range 0.2-1.7 x10 9/L), 44 females and 6 males with a median age of 56 years (range 25-87 years) and a long-follow-up (median 132 months, range 8-336 months) in the Department of Hematology of the University Hospital of Heraklion, Crete, Greece. Nonisotopic RNase cleavage assay (NIRCA) analysis was used as first screening method to detect MEFV exons 10 and 2 mutations in DNA extracted from PB or BM samples from CIN patients, confirmed by direct NGS analysis. These sequences contain the main disease-related mutations and polymorphisms. Results: Genetics alterations of MEFV were detected in 22 out of 50 CIN patients (44%). Pathogenic mutations (variants associated with typical or "atypical" FMF phenotype in Greek population) were identified in 10/50 CIN patients (20%). The 20% frequency of MEFV mutations in exon 10 and/or exon 2 in CIN patients is significantly higher compared to the carrier rate of common MEFV mutations in the healthy Greek population (0.7%) according to our previously reported data (PATG; Ile>Met), two patients with heterozygous A744S (GCC>TCC; Ala>Ser) and one with homozygosity, one patient with heterozygous M694V (ATG>GTG; Met>Val), one with heterozygous K695R (AAG>AGG; Lys>Arg) and one with heterozygous M680I (ATG>ATC; Met>Ile), all in exon 10, and (b) four patients with homozygous R202Q mutation in exon 2 (one patient with homozygous A744S co-mutation in exon 10) and two patients with R202Q heterozygosity combined with heterozygosity of I720M and A744S of exons 10, respectively. None of the patients displayed any symptoms/signs of FMF or other systemic inflammatory disease. No statistically significant differences were identified between MEFV mutated and non-mutated CIN patients in the severity of neutropenia or in lymphocyte, monocyte, hemoglobin and platelet counts. A significant difference was identified between the two patient groups in serum IgG (1440±264 vs 1133±245 mg/dl; P = 0.0023, Mann-Whitney test) but not IgA or IgM levels. Discussion: This study reports for the first time that 20% of unselected, consecutive patients with CIN carry mutations of the MEFV gene without clinical manifestations of FMF. Whether these patients represent atypical cases of FMF or the identified MEFV genetic alterations have a pathogenetic/modifying effect in the inflammatory responses associated with CIN is an open/novel field of research. As a first step we are currently investigating the neutrophil autophagic status, IL-1β production and the neutrophil extracellular trap (NET) formation in CIN patients with mutations in MEFV to clarify their potential effect in the immune deregulation known to characterize CIN. Disclosures No relevant conflicts of interest to declare.

Background: We have previously performed NGS analysis of genes that are recurrently mutated in myeloid malignancies in a cohort of patients with the diagnosis of chronic idiopathic neutropenia (CIN) according to previously reported criteria that largely overlap with those proposed for idiopathic cytopenia/neutropenia of undetermined significance (ICUS-N). We have thus estimated for the first time the frequency of clonal hematopoiesis in patients with CIN/ICUS-N (11.54%) and found that clonal CIN patients have a significantly higher risk of developing a myeloid neoplasm than those with no evidence of clonality (non-clonal). 1 However more longitudinal follow-up NGS studies are required for the tracking of clonal evolution and delineation of clonal CIN natural history. Aims: To conduct longitudinal follow-up NGS analyses in order to assess clonal evolution and associate the clinical significance of detected clonal aberrations with the risk of transforming to myeloid malignancy in clonal CIN clinical outcome. Methods: Genomic DNA was extracted from patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted next generation sequencing (NGS) on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies. Results: Follow-up analysis by NGS was performed in 16 clonal CIN patients (Figure 1). (Out of these 16 patients, follow-up NGS data has already been published in 9 patients, however additional timepoints were tested in 3 of them). 1 The median time between the first and subsequent analysis was 28.5 months (range 8-164 months). Ten of these patients carried the initial somatic mutations with only subtle changes in the size of clone as estimated by the variant allele frequency (VAF); the patients displayed absence of additional mutations and did not develop myeloid malignancy (Figure 1A-C, E-G, I, K, L, P). Two patients acquired a second mutation at follow-up. One of them still displayed stable disease course (Figure 1D) whereas the second eventually progressed to CMML (Figure 1H). The analysis also revealed that one patient lost the initial detected mutation at follow-up after 98 months (Figure 1J). Two patients who progressed to MDS/MPN and AML respectively, displayed a notable clonal expansion with additional mutations at the time of progression (Figure 1M and Figure 1N, respectively). Specifically, the patient who progressed to MDS/MPN acquired a mutation in JAK2 and ASXL1 while the patient who progressed to AML acquired the typical NPM1 p.L287fs mutation. The patient who developed MDS with multilineage dysplasia, carrying three mutations in DNMT3A and IDH1, showed a moderate increase in the VAF of these mutations at first follow-up (Figure 1O). The patient progressed to acute lymphoblastic leukemia (2 nd follow-up) with acquisition of additional truncating mutation in ETV6. Following treatment (3 rd and 4 th follow-up) mutation in ETV6 was lost, however the three mutations in DNMT3A and IDH1 persisted and their clone size increased. Conclusions: In the majority of patients tested for clonal evolution over time, most mutant clones appeared to be remarkably stable, with minimal VAF change, no acquisition of new molecular alterations and no progression to overt myeloid malignancy. Two CIN patients who transformed to a myeloid malignancy displayed a clonal expansion as was reflected by the increase of VAF and the development of additional mutations whereas in the third patient only a modest VAF increase was identified before malignant transformation. Finally in the patient bearing 4 mutations no progression to overt malignancy was observed after 12 months of follow-up. This ongoing study of sequential NGS analysis of CIN patients is anticipated to contribute to the better understanding and enrich further the knowledge on the natural history of this rare disease. References: Tsaknakis G, Galli A, Papadakis S et al. Incidence and Prognosis of Clonal Hematopoiesis in patients with Chronic Idiopathic Neutropenia. Blood. 2021 Jun 24:blood.2021010815. doi: 10.1182/blood.2021010815. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Less than 5% of patients with MDS present with thrombocytopenia as an isolated abnormality (MDS-IT). There have been few systematic studies on MDS-IT and data regarding its course and prognosis are conflicting. Previous studies have defined MDS-IT based on the IPSS thresholds (Hb ≥10 g/dL; ANC ≥1.8×10 9/L; PLT Methods: We identified patients who had PLT 13 g/dL (men) or >12 g/dL (women), and ANC ≥1.8 ×10 9/L, registered in the Hellenic National Registry of Myelodysplastic and Hypoplastic Syndromes which includes 2792 patients (analysis cut-off date; July 7, 2016). Patients were divided into 4 groups: group 1 had PLT 149-100 ×10 9/L; group 2, 99-50 ×10 9/L; group 3, Results: A total of 77 patients (45 men; 32 women) with MDS-IT were identified (2.9% of total MDS cohort). Of these, 28.6% were classified in group 1; 49.4% in group 2; 14.3% in group 3; and 7.8% in group 4. Median PLT count was 87 ×10 9/L (12-139 ×10 9/L), WBC count 4.6 ×10 9/L, and Hb 13.6 g/dL. Bone marrow (BM) blasts ranged from 0-9% (median, 2%). Median follow-up was 51.0 months (41.6-60.4), during which 15 (19.5%) patients died. AML developed in 9 patients (11.7%). Histologically, MDS with multilineage dysplasia (MLD) was seen in 77.6% whereas MDS with excess blasts (EB) and MDS with single lineage dysplasia (SLD) comprised 10.7% and 11.9% of cases, respectively. Most patients (73.5%) had lower-risk MDS on the IPSS-R (i.e. IPSS-R ≤3.5). Of the 59 patients with cytogenetic data, 83.1% had favorable, 13.5% intermediate, and 3.4% poor risk cytogenetics. Most (40) had a normal karyotype followed by isolated del(20q) (6). All patients with del(20q) showed a characteristic set of clinical features: age >60 years, blasts 0-3%, bilineage (erythroid/megakaryocytic) dysplasia, and increased reticulin fibrosis. There were no significant differences between any of the 4 PLT groups regarding age, sex, IPSS-R, cytogenetics, BM blasts, and histology. Median OS was 109 months (95% CI 103-115) and LFS 108 months (101-115). Our results showed no significant difference in OS (P=0.891) and LFS (P=0.871) between the 4 PLT groups. As compared with total MDS cohort, MDS-IT occurred at younger age (64.7 vs. 72.4 years, P In comparing MDS-IT with ITP, the median age at diagnosis was 66.0 years for MDS-IT and 49.0 years for ITP (P80 years. Its incidence reached a peak between the ages of 70-79 years, whereas ITP occurred at a more constant level over time (Figure 1B). Women predominated in ITP and men in MDS-IT (P=0.007). Overall, ITP was associated with more marked thrombocytopenia than MDS-IT (15.0 ×10 9/L vs. 87.0 ×10 9/L) (P Conclusions: In one of the largest reported series, we conclude that MDS-IT is associated with MDS-MLD, favorable cytogenetics, lower-risk IPSS-R, high survival rate, and a low risk of AML evolution. Our data suggest that the superior prognosis in MDS-IT than general MDS may have intrinsic genomic underpinnings as survival curves remained unchanged after correcting for age, sex, blasts and IPSS-R. Importantly, no significant differences in OS and LFS were noted between the 4 PLT subgroups, suggesting that the degree of thrombocytopenia does not correlate with mortality in MDS-IT. From the diagnostic standpoint, age 80 years and PLT Figure 1 Figure 1. Disclosures Viniou: Sandoz: Research Funding; Takeda: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Roche: Research Funding; Astellas: Research Funding; Celgene: Research Funding. Vassilakopoulos: Dr. Reddy's: Research Funding; Amgen: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Other: Travel; AbbVie: Consultancy, Honoraria; Integris: Honoraria; Pfizer: Research Funding; Roche: Consultancy, Honoraria, Other: Travel; Takeda: Consultancy, Honoraria, Other: Travel, Research Funding; Genesis Pharma: Consultancy, Honoraria, Other: Travel; Merck: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Karyopharm: Research Funding; AstraZeneca: Honoraria. Hatzimichael: Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria; Gilead: Honoraria; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Pharmathen- Innovis: Honoraria; GSK: Honoraria; Bristol Myersr Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Symeonidis: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi/Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Demo: Research Funding; MSD: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; WinMedica: Research Funding; Astellas: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GenesisPharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background-Aim: Infection from SARS-CoV-2 has emerged as new pathological entity within the global medical community. One of the earliest questions was in relation to the ability of the immunocompromised patients to clear the infection. In COST EuNet-INNOCHRON we were interested in the impact of SARS-CoV-2 infection in patients with different types of chronic neutropenia (CNP). The aim of the current study is to understand the impact of SARS-CoV-2 infection and to identify any possible characteristic patterns of the clinical course in patients with CNP. Patients and Methods: The COST EuNet-INNOCHRON Action in collaboration with the European Haematology Association - Scientific Working Group (EHA-SWG) on Granulocytes and Constitutional Marrow Failure Syndromes has conducted an online survey on SARS-CoV-2 infection in patients with CNP. The EuNet-INNOCHRON participants from different countries got access to an on-line platform fulfilling the General Data Protection Regulation (GDPR) and could register adult and paediatric CNP patients who had been infected by SARS-CoV-2 from March 2020 to June 2021. Data on demographic characteristics, type of CNP, patients' background and SARS-CoV-2 infection history (symptoms, laboratory features, radiological appearance, therapeutic approach and outcome) were collected. Results: Twenty-six patients with diagnosis of CNP, 7 males and 19 females were registered. Patient age distribution as follows: 16 patients >18 years old (y.o.)5 patients 5-18 y.o, 4 patients < 5 y.o whereas age was not available for one of the patients. Nine of the patients were diagnosed with idiopathic CNP, 7 patients with congenital neutropenia (6 of them with severe congenital neutropenia), 3 with secondary CNP, 2 with suspected autoimmune neutropenia of infancy (although antineutrophil Ab were negative), one with autoimmune neutropenia, one with drug induced neutropenia and 3 with other types of CNP. Twelve patients were on treatment with G-CSF and 6 patients had a history of previous viral or bacterial infections. Clonal Cytopenia(s) of Undetermined Significance (CCUS) was excluded in the eight patients who were investigated. Twenty-four out of 26 patients had positive PCR and one was found incidentally with positive antibodies for SARS-CoV-2. One more patient was symptomatic with history of close contact with SARS-CoV-2 infected family members. The commonest observed symptoms were fever >38 oC (19 patients), cough (10 patients), rhinorrhoea (10 patients), sore throat (6 patients), musculoskeletal pains (7 patients), taste/smell loss (5 patients), headache (5 patients), dyspnoea (4 patients), chest pain (one patient) and none of them had gastrointestinal symptoms. No other associated respiratory viral or bacterial infections were reported. Four patients who had one or more underlying conditions (immune deficiency, heart/respiratory/kidney disease) were admitted in hospital and needed anti SARS-CoV-2 treatment. Two of them had non-invasive ventilation and one of them needed admission in intensive care unit (ICU); both recovered. Another patient with Fallot's tetralogy needed mechanical ventilation in ICU and sadly passed away. No other deaths were observed. Deterioration of the pre-existing neutropenia was seen in two patients, two patients developed thrombocytopenia, one patient developed worsening lymphopenia and one anaemia. Twelve patients had chest X-ray and consolidation was found in two of them. All three patients who had chest CT scans were found with ground-glass changes. During the observation period (up to two months), no re-infection from SARS-CoV-2 was found. The Stockholm, Sweden experience is similar to the above data. One hundred fifty-four patients with CNP were followed up, for 10 months (March 1 to December 31, 2020) for SARS-CoV-2. Seventeen of these (i.e. 11 %) were infected. None needed hospitalization and there were no fatalities. Conclusion: Although the relative susceptibility of neutropenic patients to contract SARS-CoV-2 needs to be assessed with further studies, the clinical course and severity of SARS-CoV-2 infection doesn't seem to be worse in CNP patients (regardless the type of neutropenia and the need for GCSF treatment) compared to the general population. Also, like what has been observed in non-neutropenic patients, underlying comorbidities is a significant risk factor for severe disease and adverse outcome. Disclosures Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding. Palmblad: Chiesi Ltd Sweden: Honoraria; Roche Sweden: Speakers Bureau; Chiesi Ltd Candada,: Honoraria.

Myeloid-derived suppressor cells (MDSCs) are myeloid cells with immunoregulatory properties characterized mainly by suppression of T-cell responses (Bizymi et al, HemaSphere 2019). They are divided in HLA-DRlow/-/CD11b+/CD33+/CD15+ polymorphonuclear (PMN-MDSCs) and HLA-DRlow/-/CD11b+/CD33+/CD14+ monocytic (M-MDSCs) subsets and they are implicated in inflammatory and malignant diseases. Chronic idiopathic neutropenia (CIN), is a (usually benign) neutrophil disorder characterized by persistent and unexplained neutropenia following a detailed clinical/laboratory investigation including anti-neutrophil antibody testing, bone marrow (BM) biopsy and karyotype (Dale & Bolyard, Curr Opin Hematol 2017). Previous studies have shown that neutropenia in CIN is associated with increased apoptosis of BM granulocytic progenitor cells due to an inflammatory BM microenvironment consisting of oligoclonal T-lymphocytes, proinflammatory monocytes and proapoptotic cytokines. The aim of the present study is to explore the possible involvement of the MDSCs in the pathophysiology of CIN by investigating their number in peripheral blood (PB) and BM in association with their functional characteristics. We have studied 100 CIN patients and 49 age- and sex-matched healthy controls. The patients fulfilled the previously described diagnostic criteria for CIN (Papadaki et al, Blood 2003) and had mean neutrophil counts 1095.67 ± 479.52 (median 1215, range 100-1700). MDSC subsets were quantitated by flow cytometry in the PB mononuclear cell (PBMC) fraction using the combination of CD33PC7/CD15PC5/HLA-DRECD/CD14PE/CD11bFITC monoclonal antibodies and the Kaluza analysis software. MDSC subsets were also studied in the BMMC fraction of 24 CIN patients and 8 healthy controls from the study population. The T-cell suppression function of patient MDSCs was evaluated in coculture experiments of immunomagnetically sorted, CFSE stained, normal CD3+ cells with immunomagnetically sorted M-MDSCs and PMN-MDSCs from 4 patients and 4 healthy donors using recombinant human IL-2 as activating factor. CFSE staining was detected in the CD3+ cells on day 0 and day 3 of coculture and analysis was performed with the Fcs Express 7 software. Statistical analysis was performed with the Statistica software. We found that the proportion of PB M-MDSCs was statistically significant lower in CIN patients (1.45% ± 1.82%) compared to controls (3.68% ± 3.12%, Mann-Whitney test, p < 0.0001) (Figure a) whereas the proportion of PB PMN-MDSCs, although lower in patients, did not differ significantly from the controls. The proportion of BM M-MDSCs did not differ significantly between CIN patients and controls whereas the proportion of BM PMN-MDSCs was statistically significant lower in patients (13.27% ± 11.27%) compared to controls (19.49% ± 4.46%; Mann-Whitney test, p = 0.0291) (Figure b). Paired analysis showed that the proportion of PMN-MDSCs were higher in the BMMC compared to PBMC fraction in both CIN patients (13.27% ± 11.27% vs 1.14% ± 1.64%, respectively; Wilcoxon test, p = 0.005) (Figure c) and healthy controls (19.49% ± 4.46% vs 9.92% ± 9.08%, respectively; Wilcoxon test, p = 0.0118). Interestingly, the proportion of increase of PMN-MDSCs (in BMMC vs PBMC fraction) was significantly higher in patients (86.71% ± 21.26%) compared to controls (55.95% ± 38.59%; Mann-Whitney test, p = 0.0357) (Figure d). The above data indicate low production of PMN-MDSCs in CIN patients compared to controls but a trend for accumulation of these cells in patients' BM. No statistically significant difference was documented in paired analysis of M-MDSCs between BMMC and PBMC fractions in either CIN patients or healthy controls. Patient PMN-MDSCs and M-MDSCs displayed normal capacity to suppress T-cell proliferation as was indicated by the T-cell generations in coculture experiments of normal CD3+ cells in the presence or absence of patient MDSCs (Figure e). In conclusion, CIN patients display low proportion of MDSCs in the PB and lower proportion of PMN-MDSC in the BM compared to normal individuals. Patient MDSCs display normal capacity to suppress T-cell activation. The low proportions of MDSCs may sustain the inflammatory process associated with CIN whereas the accumulation of PMN-MDSCs in the BM represents probably a compensatory mechanism to suppress the inflammatory processes within patients' BM microenvironment. Figure Disclosures Papadaki: Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction. Myelodysplastic Syndrome (MDS) is a disease of the elderly. Apart from IPSS, IPSS-R and WPSS, several indexes incorporating patient comorbidities (such as the MDS CI index- Della Porta et al Haematologica 2011, the HCT-CI index - Sorror et al Blood 2005) and performance status (the GFM index- Itzykson et al Blood 2011) have been used to predict outcome in MDS patients treated with azacytidine (AZA). We sought to investigate the effect of comorbidities on the outcome after AZA in a large group of patients from the MDS registry of the Hellenic MDS Study Group. Methods. The present study has been conducted as a retrospective observational cohort one. It included high-risk MDS and low blast count AML patients treated with AZA from 26 centers in Greece from 2007 to 2018. T-test and ANOVA were used to compare scale variables between two or more groups respectively. Univariate analysis of nominal and scale survival data was performed using Kaplan-Meier survival curves and Cox regression respectively. All variables achieving p Results. We analyzed 536 consecutive patients. Patient characteristics are depicted in Table 1. The median follow-up period was 27.5±4.8 months. 371 patients received at least four cycles of AZA and 165 patients received less than 4 cycles of AZA. Patients who received ≥4 cycles of AZA did not differ from those who received To assess the prognostic significance of risk factors on leukemia free survival (LFS) and overall survival (OS), univariate and multivariate analysis for the whole population was performed, as well as a landmark analysis for patients who were treated with at least 4 cycles of AZA. ECOG performance status and the presence of peripheral blasts were independent prognostic factors for LFS and OS for the whole cohort analysis while response to AZA and the presence of peripheral blasts were independent prognosticators for LFS and OS in the landmark analysis. In addition, prior low dose cytarabine was an independent adverse prognostic factor for LFS in the landmark analysis. As regards comorbidities, neither of MDS-CI, HCT-CI and GFM systems independently predicted LFS or OS in either analysis, but eGFR with a cut-off of 45 ml/min was a strong and independent prognosticator for LFS and OS in both the standard and the landmark analysis. Kaplan-Meier survival curves regarding LFS and OS at AZA initiation and landmark analysis after 4th cycle of AZA in relation with eGFR are shown in Figure 1. Conclusion. This is the first study to demonstrate the importance of eGFR at baseline as a prognostic marker for LFS and OS in high-risk MDS and low-blast AML patients treated with AZA. The role of comorbidities and PS needs to be further evaluated in this patient group. Disclosures Symeonidis: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Vassilakopoulos:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; WinMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: 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. Panayiotidis:Bayer: Other: Support of clinical trial. Pappa:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kotsianidis:Celgene: Research Funding.

Introduction: Clinical trials in patients with high risk myelodysplastic syndromes (MDS) have shown that these patients benefit from the available hypomethylating agents 5-azacytidine and decitabine. The majority of these patients display hypercellular bone marrow, but a small proportion despite the excess of blasts, exhibit marrow hypocellularity ( Patients & Methods: This is a retrospective multicenter study from the Hellenic National MDS Registry (EAKMYS) on behalf of the Hellenic MDS Study Group. Between 1.1.2009 and 31.12.2018 a total of 1161 MDS patients who have received treatment with azacytidine have been registered. Complete patient information and follow-up were available for 989 patients, and all these have been included in the final analysis. Statistical analysis was performed and overall survival (OS) was evaluated, using Kaplan-Meier estimates (GraphPad Prism software, CA). A p value less than 0.05 was considered statistically significant. Results: Forty nine patients had a hypocellular bone marrow (hMDS), representing the 4.95% of the whole patient population. Of these patients 39 were men (5.3% of all men included in the study) and 10 were women representing the 2.98% of all women enrolled (male to female ratio 3.9). In the non-hypoplastic group, 750 were men and 358 were women (male to female ratio 2.09). The median age at diagnosis for the hMDS group was 70.8 years, compared to 72.8 years in the non-hypoplastic group. The IPSS-R prognostic risk categorization included 15 hMDS patients in the low group, 9 in the intermediate, 14 in the high and 11 in the very high risk group. Twenty-six patients (53%) of the hMDS group had bone marrow blasts between 10 and 20%, and the remaining 23 (47%) had 5-10% blasts. The patients with hMDS received an average of 10 cycles of azacytidine treatment during the follow-up period (range 2-29 cycles). The outcomes tested were overall survival and progression to AML. The median overall survival of patients with hMDS, following azacytidine treatment start, was not significantly different from the median survival of patients with non-hypoplastic MDS [20 months versus 16 months in the non-hypoplastic group (95% CI of ratio: 0.839 to 1.863). The survival curves were not significantly different between the hMDS and non-hypoplastic MDS group (p=0.32, Figure 1). Progression to AML was also evaluated. Eleven (22.4 %) hMDS patients showed disease progression to AML. Patients with hMDS had significantly prolonged estimated median time to AML transformation, compared to the non-hypoplastic MDS population (31.7 versus 22 months respectively, p Discussion and Conclusive remarks: In this retrospective study, in which a large number of MDS patients was analyzed, we showed that bone marrow cellularity does not affect the outcome in patients treated with azacyitidine. Patients with hMDS show statistically significant slower AML progression compared to non-hypoplastic MDS. Bone marrow cellularity should not be a contraindication for using hypomethylating agents as a therapeutic option, and this type of treatment can be used safely, when indicated, also in patients with hMDS. Disclosures Pappa: Amgen: Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vassilakopoulos:Merck: Honoraria; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Winmedica: Honoraria; Servier: Membership on an entity's Board of Directors or advisory committees. Symeonidis:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

INTRODUCTION: The hypomethylating agents 5-azacitidine (5-AZA) and decitabine are recently considered the most preferable treatment option for patients with intermediate-2 and high-risk myelodysplastic syndromes (MDS), by International Prognostic Scoring System (IPSS). 5-AZA responders experience improved survival both in clinical trials (AZA 001) and in the real-life setting. Thrombocytopenia is a common event in MDS, during the course of the disease; recently, severe thrombocytopenia (≤30,000 platelets/μL) has been suggested as an important factor regarding the survival of MDS patients. In the present study, we examined the potential prognostic significance of severe thrombocytopenia, in intermediate-2- and high-risk MDS patients, being treated with 5-AZA, during the first 3 years of treatment. METHODS: This retrospective study included 850 higher-risk patients (intermediate-2- and high-risk), registered in the the Hellenic MDS Registry, treated with 5-AZA from 2010 to 2018 and were followed up for a time period up to 3 years. Complete patient data were available for 225 patients. Biostatistical analysis performed in this study included Kaplan-Meier survival analysis and Cox regression. The level of statistical significance was set at a probability value of less than 0.050 (P RESULTS: The current study included 225 patients (159 male and 66 women) with intermediate-2- or high-risk MDS treated with 5-AZA, with a median age of 74 years (range: 47 - 89). WHO diagnosis included 1 (0.4%) case of RCUD, 8 (3.6%) cases of RCMD, 3 (1.3%) cases of RCMD-RS, 43 (19.1%) cases of RAEB-1, and 170 (75.6%) cases of RAEB-2. According to IPSS, 174 (77.3%) patients were classified in the intermediate-2 risk group and 51 (22.7%) patients in the high-risk group. In addition, according to IPSS-R, 24 (10.7%) patients were categorized in the intermediate risk group, 106 (47.1%) patients in the high-risk group, and 95 (42.2%) patients in the very-high risk group. All patients were evaluated regarding response to 5-AZA treatment. The initial response at 6 months was: complete remission (CR) in 40 (18.4%) patients, partial remission (PR) in 24 (11.1%) patients, hematological improvement (HI) in 35 (16.1%) patients; therefore, the initial overall response rate (CR, PR, and HI) was 45.6%. Stable disease (SD) was achieved by 56 (25.8%) MDS patients, while 62 (28.5%) patients showed progression of disease (PD) or treatment failure. Severe thrombocytopenia was not predictive of response, as shown using logistic regression analysis. However, severe thrombocytopenia predicted poor overall survival (OS) in the first 3 years of treatment with 5-AZA, as shown by the Kaplan-Meier analysis (Figure 1; P=0.016). Regarding AML-free survival, a strong trend was observed for thy unfavorable prognostic role of this severe cytopenia (P=0.096). Univariate Cox regression analysis for OS revealed a statistically significant hazard ratio (HR) of 1.6 for MDS patients with severe thrombocytopenia (HR=1.6, 95% CI=1.08, P=0.019). CONCLUSIONS: Our study showed that severe thrombocytopenia (≤ 30,000 platelets/μL) in intermediate-2- and high-risk MDS patients, treated with 5-AZA, predicts lower OS rates during the first 3 years of treatment. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

To investigate whether bone marrow (BM) stem cell compartment and/or BM microenvironment are affected by the immune insult in autoimmune cytopenias (AICs), BM stem cell reserve and function and BM stromal function were studied in 15 AIC patients. Stem cells were evaluated by means of flow cytometry, clonogenic progenitor cell assays, long-term BM cultures (LTBMCs), and limiting dilution assay for quantification of long-term–culture initiating cells (LTC-ICs). Stromal cell function was assessed with the use of preformed irradiated LTBMCs from patients and normal controls, recharged with normal CD34+ cells. AIC patients exhibited a high number of CD34+, CD34+/CD38+, and CD34+/CD38− cells; high frequency of granulocyte-macrophage colony forming units in the BM mononuclear cell fraction; high colony recovery in LTBMCs; and normal LTC-IC frequency. Patient BM stromal layers displayed normal hematopoietic-supporting capacity and increased production of granulocyte-colony stimulating factor. Data from this study support the concept that AIC patients with severe, resistant disease might be appropriate candidates for autologous stem cell transplantation.

Introduction: Chronic idiopathic neutropenia (CIN) of adults is an acquired disorder of granulopoiesis characterized by an unexplained, prolonged reduction in the number of peripheral blood (PB) neutrophils. We have previously shown that CIN pathophysiology is related to T-cell activation and increased apoptosis of granulocytic progenitor cells. Notably, in our cohort of patients we exclude cases with antineutrophil antibody activity, i.e. autoimmune neutropenia cases. Aim of the study: Preliminary data have shown the presence of minor populations of paroxysmal nocturmal hemoglobinuria (PNH) type cells in the PB of CIN patients. The present study aims to investigate further the presence of PNH type cells in the PB of a large cohort of patients fulfilling the diagnostic criteria of CIN and probe the possible association with the severity of the disease and the skewed T-cell profile. Patients - Methods: We have studied 91 adults with CIN and 55 hematologically healthy subjects, age- and sex-matched with the patients. We have used flow cytometry for the detection of PNH type cells according to the International Clinical Cytometry Society guidelines and for the identification of T-cell expansions based on the analysis of T-cell receptor beta variable (TRBV) gene repertoire. Results: CIN patients displayed increased proportion of PB PNH type FLAER-/CD24- neutrophils (0.05% ± 0.18%) and FLAER-/CD14- monocytes (1.33% ± 1.38%) compared to healthy individuals (0.005% ± 0.01% and 0.64% ± 0.66%, respectively; P=0.0044 and P=0.011, respectively). Similarly, the patients displayed increased proportion of both, type II CD235+/CD59dim and type III CD235+/CD59- (0.059% ± 0.29% and 0.065% ± 0.28%, respectively) PB PNH red blood cells, compared to healthy controls (0.01% ± 0.03% and 0.01% ± 0.03%, respectively; P Conclusion: CIN patients display minor populations of PNH type PB neutrophils, monocytes and red blood cells. The frequency of PNH type neutrophils is associated with the severity of neutropenia and it is higher among patients with skewed TRBV repertoire. These data support further the hypothesis that CIN displays common pathophysiologic features with immune-mediated bone marrow failure syndromes and should be included in this spectrum of disease entities. Disclosures Papadaki: Alexion Pharmaceuticals: Research Funding.

Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. However, there is a concurrent high prevalence of bone marrow (BM) infiltration, suggesting that BM microenvironment dynamics could have a potential involvement in disease pathology. In this regard, we aim to characterise BM derived mesenchymal stem cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if MSCs show altered properties in SMZL patients compared to healthy controls. BM MSCs were isolated from 8 SMZL patients and 10 age- and sex-matched healthy controls. MSCs were in vitro expanded and re-seeded for a total of 5 passages (P). The colony forming unit-fibroblast (CFU-F) assay was used for the estimation of MSC frequency within the BM mononuclear cell (BMMC) fraction. Ex-vivo expanded MSCs were phenotypically characterized by flow cytometry (FC) using appropriate markers. In vitro differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. The proliferative potential of ex vivo expanded MSCs was evaluated by Methyl Triazolyl Tetrazolium (MTT)-based assay and survival characteristics were studied using FC and 7-Aminoactinomycin D (7-AAD) staining. To assess the effect of patient MSCs on B cell growth, B cells were immunomagnetically isolated (Miltenyi Biotec GmbH, Germany) from peripheral blood (PB) of normal individuals, labeled with carboxy fluorescein succinimidyl ester (CFSE; Gibco Invitrogen, Paisley, Scotland) and subsequently cultured in the absence or presence of confluent layers of allogeneic BM-MSCs from SMZL patients or normal controls in the presence of CpG oligonucleotide 2006 (Invivogen, France) and IL-2 (R&D Systems, Minneapolis, MN). In a separate set of experiments, B cell survival was evaluated via FC and 7-AAD staining, after co-culturing with BM-MSCs from patients or healthy donors. Finally, to study BM-MSC capacity to chemotactically attract B-cells, transwell migration assays were set. In the bottom chambers MSCs from patients or healthy individuals were grown until confluency and then isolated B cells from PB of either patients or controls were added into the upper chamber. Twelve hours later migrated cells were enumerated. Grouped data are expressed as means± 1 standard error of the mean (SEM). MSCs were successfully expanded from all participants in the study. Adherent cells from both study groups displayed the typical spindle-shape morphology and immunophenotypic analysis at the end of P2-P3-P4 demonstrated that cultures constituted of a homogeneous cell population, typically expressing CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. SMZL-derived MSCs were similar to their normal counterparts in the capacity to differentiate towards adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.5±0.68/105 ΒΜΜCs and 7.23±0.6/105 ΒΜΜCs, respectively; P=0.0032) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs displayed defective proliferative potential as compared to their normal counterparts at P2, as evidenced by the MTT assay (P In conclusion we have shown for the first time that SMZL lymphoma MSCs are intrinsically defective in terms of proliferative potential and exert an altered modulation of B cell apoptosis and B cell chemotaxis. These preliminary results concerning the properties of SMZL MSCs merit further investigation and provide the theoretical background for exploring their potential implication in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

Expression of CD25 on blasts of AML patients has been shown to hold independent prognostic value for both survival and response to induction therapy. Patients with MDS-related AML have generally higher CD25 expression from de novo AML patients, but though there is paucity of a serviceable biomarker of outcome in MDS patients treated with azacytidine, the prognostic value of CD25 has not yet been investigated. Bone marrow samples of 61 patients with intermediate-2/high risk IPSS, high/very high WPSS and low blast count AML were obtained before and 15 days (D15) after the initiation of treatment. All patients received azacytidine in a non clinical trial setting at an initial dose of 75mg/m2 SC for 7 days on 28-day cycles. CD25 expression was assessed by 4-color flow cytometry on total CD34+ blasts, committed progenitors (Lin-CD38+CD34+) and leukemic stem cells (LSC, Lin-CD38-CD34+). Positivity was defined as a CD25 expression of ≥ 20%. Statistical comparisons were done by ÷2, one-way ANOVA and paired or unpaired t-test as appropriate, and survival with Kaplan-Meier analysis and log-rank test. Overall survival (OS) was defined as the time from azacytidine initiation to death from any cause and event-free survival (EFS) as the time from diagnosis to disease progression, relapse or death. Multivariate survival analysis was based on Cox’s proportional hazards model using a backward stepwise selection procedure with entry and removal criteria of p=0.05 and p=0.10, respectively. As shown in table 1 the cohorts of CD25- and CD25+ patients were well balanced for most known predictive factors and characteristics, except sex. Compared to CD25+ patients the CD25- ones have significantly longer OS (16.2 vs 8.8 months, respectively, p=0.04) and EFS (12.8 vs 6.66 months, p=0.04) in univariate analysis. Multivariate analysis confirmed the independent predictive power of CD25 for OS and EFS (p=0.006 and p=0.009, respectively), whereas heavy transfusion requirements (p=0.003 and p=0.002) and age>75 (p=0.02 and p=0.02) were also independent predictors. The average expression of CD25 in CD34+ blasts of all patients was 21.6%±24%. Compared to committed progenitors, LSCs displayed higher expression (19.4%±23.7% vs 24.1%±28.2%, respectively, p=0.027). Interestingly, on D15 CD25 was downregulated in LSCs (p=0.03) but remained stable in committed progenitors (p=0.8, n=18), indicating a particular sensitivity of the CD25+ subset of LSCs in azacytidine.Table 1Patient characteristics. N/A: not applicable/not available.CD25- (n=36)CD25+ (n=25)p-valueAge72,5 (53,4-83.5)72,9 (52-81.7)0.2 >6531(86%)17(68%) 15%18(50%)9(36%) ≤15%18(50%)16(54%)Transfusions ≥ 4 per month0.48 Yes23(4%)17(4%) No13(4%)8(4%)Response0.4 CR & PR12(33%)4(16%) Hematologic improvement5(14%)4(16%) Stable disease8(22%)5(20%) Failure11(31%)12(48%)Figure 1(A) OS and EFS according to CD25 positivity status. (B) OS and EFS according to transfusion requirements.Figure 1. (A) OS and EFS according to CD25 positivity status. (B) OS and EFS according to transfusion requirements. Collectively, our findings reveal an independent prognostic role for CD25 in MDS patients treated with azacytidine. In addition, the differential expression and epigenetic modulation of CD25 in the LSC compartment support the investigation of therapeutic strategies using monoclonal antibody targeting combined with epigenetic agents. Disclosures: Kotsianidis: Genesis Hellas: Honoraria, Research Funding. Spanoudakis:Genesis Hellas: Honoraria. Tsatalas:Genesis Hellas: Honoraria.

Bone narrow (BM)- derived mesenchymal stem/stromal cells (MSCs) represent the most extensively studied population of adult MSCs and are considered as the gold-standard for MSC-based clinical applications. Yet, it is now becoming increasingly clear that BM may not represent the most suitable source for MSC collection. Indeed, Umbilical cord (UC) has emerged as a more abundant and easily attainable source of MSCs and several reports have shown that MSCs can be efficiently isolated from the connective tissue that surrounds UC vessels, namely the Wharton's jelly (WJ). According to the existing literature, WJ-MSCs display typical MSC characteristics, however a head-to-head comparison with BM-MSCs is still lacking. Provided that ex vivo MSC expansion is a prerequisite for clinical MSC-applications, in the present study we seek to comparatively investigate the characteristics of WJ- and BM-MSCs, cultured under identical conditions. MSCs were isolated and expanded from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=10) after written informed consent of the family. MSCs were in vitro expanded and re-seeded for a total of 10 passages (P) and phenotypically characterized by flow cytometry (FC). MSCs were induced to differentiate in vitro to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. Relative gene expression was calculated by the ΔCt method. MSC growth characteristics were assessed by evaluating the population doubling time (DT) and by a methyl-triazolyl-tetrazolium (MTT)-assay throughout passages. Cell-cycle analysis was performed using propidium iodide (PI) staining. MSC survival was evaluated by FC with 7-Aminoactinomycin D (7-AAD) and senescence was estimated by the percentage of SA-b-gal+ cells in cultures. Moreover, MSC karyotypic stability was assessed with classic G-banding. Finally the expression of genes related to Wnt-mediated signal transduction was also investigated, using a PCR array. Total RNA was thus isolated from 6 representative BM- and 6 WJ-MSC cultures at P2. The fold change (FC) for each gene between the group of WJ- and the group of BM-MSCs was calculated with the ΔΔCt method (FC=2-ΔΔCt). WJ-MSCs displayed a spindle-shape morphology, similar to BM-MSCs. Furthermore, WJ- and BM-MSCs displayed identical immunophenotype, as evidenced by the expression of CD90,CD105,CD44,CD29,CD73 and the lack of expression of CD45,CD14,CD34,CD31. WJ-MSCs displayed superior proliferative potential compared to BM-MSCs throughout passages (p Taken together WJ-MSCs display decreased cellular senescence after extended in vitro culture, increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, as compared to BM-MSCs. The last two observations can be explained, at least partly, by the aberrant expression of Wnt-signaling molecules in WJ-MSCs. The emerging role of Wnt-signaling pathway in WJ-MSC biology is currently under investigation. Disclosures: No relevant conflicts of interest to declare.

Abstract 831 Chronic idiopathic neutropenia (CIN) is a disorder of neutrophil production usually characterized by benign and asymptomatic course and female predominance. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitor cells, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients. Recently, using flow cytometry and CDR3 spectratyping, we obtained for the first time evidence for expanded T cell populations alluding to repertore skewing in both PB and BM of patients with CIN, but more prominent in the BM CD8+ subset. Prompted by these fndings, here we significantly extended our studies of the cytotoxic T cell responses in CIN in order to obtain a comprehensive view of the role of antigen selection in CIN pathogenesis. The study included 18 patients with CIN, 17 females and 1 male, diagnosed according to the established criterial for CIN. TRBV-TRBD-TRBJ gene rearrangements were amplified on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=6) or BM (n=12) samples. PCR products were subcloned by transformation into E.Coli/TOP10F' competent bacteria and individual colonies were chosen randomly and subjected to Sanger sequencing. Sequence data were analyzed using the International imMunoGenetics information system and more particularly the IMGT/V-QUEST tool. Overall, 507 TRBV-TRBD-TRBJ gene rearrangements were analyzed (19-30/case) of which 466 were productive since they used functional TRBV genes and also carried in-frame CDR3s. A polyclonal profile was seen in only 3/18 cases (16.7%). The remaining cases were found to carry clusters of identical rearrangements corresponding to distinct immunodominant clonotypes. The frequency of each clonotype was determined by dividing the number of identical sequences by the total number of subcloned sequences analyzed. In 10/18 cases (55.5%), the dominant clonotype accounted for 12.8–22.6% of the total, whereas in 5/18 cases (27.8%) it accounted for 34.2–68.4% of the total. The TRBV28 gene was used by the dominant clonotype of three different CIN cases; the TRBV10-2, TRBV10-3, TRB19, and TRBV7-8 genes were identified in the major clonotypes of two cases each. Importantly, cluster analysis of the CDR3 sequences of all CIN cases of the present study identified 4 different rearrangements that were shared by different patients (public clonotypes). In conclusion, the present study strongly suggests that CIN may result from an autoimmune reaction directed against granulocytic progenitors triggered by a restricted range of, as yet, unidentified antigen(s). The finding of public clonotypes may indicate that public antigenic stimuli and/or shared immune processes underlie CIN development, at least for a proportion of cases. Overall, our results further support the concept that CIN may share common pathophysiologic mechanisms with other disorders characterized by T-cell and cytokine mediated suppression of hematopoiesis, perhaps representing a milder extreme within the spectrum of these acquired immune-mediated BM failure syndromes. Disclosures: No relevant conflicts of interest to declare.

Abstract 3316 Background: ITP is characterized by platelet counts Methods: This ongoing study enrolls ITP pts ≥18 years old, who have received romiplostim in clinical practice in Austria, Belgium, Czech Republic, France, Greece, Portugal or Sweden. Pts participating in another study, who initiated romiplostim prior to commercial launch, or received other TPOra or related products are excluded. Data recorded as per clinical practice, including concomitant medications, is collected for up to 2 years following romiplostim initiation. Study outcomes include pt characteristics (at romiplostim initiation), romiplostim dose, adverse drug reactions (ADRs) and bleeds, summarized for pts meeting the study eligibility criteria (Full Analysis Set; FAS). Grade 3 or 4 bleeds are classified using the WHO bleeding scale. The study, and medical writing assistance for this abstract, was funded by Amgen (Europe) GmbH. Results: As of February 2012, 237 (96%) of 248 pts enrolled were included in the FAS. Of these, 60% (143/237) remained on study, 29% (69/237) had completed the 2 year observation period and 11% (25/237) had withdrawn, with death the most common reason (17/237 [7%]). At romiplostim initiation, median (Q1, Q3) age, weight and platelet count were 62.0 (46.0, 74.0) years, 74.00 (63.00, 85.00) kg and 18.0 (8.0, 34.0) × 109/L; 33% (79/237) of pts were splenectomised, 54% (129/237) female, 32% (77/237) had been diagnosed with ITP for < 1 year (median [Q1, Q3] time from diagnosis, 3.63 [0.42, 11.59] years), and 54% (128/237) had received ≥3 prior ITP therapies. Thirty-one percent (74/237) of pts stopped romiplostim before the end of the observation period, with requirement for alternative therapy (22 [9%] pts), hemostatic platelet counts/no further treatment necessary (11 [5%]), ADR (8 [3%]) and death (6 [3%]) the most common reasons. Median (Q1, Q3) duration of romiplostim exposure was 53.6 (22.6, 94.0) weeks (maximum 106 weeks); median (Q1, Q3) observation following romiplostim initiation was 18.75 (12.20, 24.10) months. Taking the average weekly dose of all pts, the median (Q1, Q3) was 2.8 (1.5, 4.3) μg/kg/week. Median platelet counts rose rapidly during the first 4 weeks of romiplostim treatment and remained >50 × 109/L thereafter (approximately 2 years; Figure). Grade ≥ 3 bleeds were rare following romiplostim initiation (Table). The most commonly reported ADRs were headache, thrombocytosis, arthralgia, asthenia, flushing and myalgia (2.0–5.9 events per 100 pt-years). Five pts reported a total of 8 serious ADRs: 2 events each of pulmonary embolism and myelofibrosis (initial diagnosis inconsistent with ITP, myelofibrosis more likely due to the underlying disease [MDS, metastases to bone marrow]); 1 event each of deep vein thrombosis, drug ineffective (clinical symptoms, thrombocytopenia), platelet count decreased (platelets Platelet counts Summary/conclusions: With similar doses as previously reported (Kuter et al, Blood 2008), and no new safety signals, pts with ITP of varying duration and severity receiving romiplostim in clinical practice achieved sustained increases in platelet counts and a reduction in grade ≥3 bleeds. Incomplete reporting before romiplostim initiation and a shorter observation period may have led to an underestimation of the number bleeds during this period; hence the true reduction in grade ≥3 bleeds following romiplostim initiation may be greater than reported here. Disclosures: Selleslag: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Janssens:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Wadenvik:Novartis, BMS, GSK, Amgen, Alexion: Consultancy. Steurer:Amgen: Consultancy, Honoraria. Kaiafa:Amgen: Consultancy, Honoraria. Kozak:Amgen s.r.o.: Membership on an entity's Board of Directors or advisory committees. Viallard:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dillingham:Amgen: Employment, Equity Ownership. Kreuzbauer:Amgen: Employment, Equity Ownership.

Abstract 1250 The CCAAT-binding transcription factor NF-Y, consists of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YA is characterized as the regulatory subunit of the complex because of its differential expression during cell cycle. NF-YA appears to have a complex role in hematopoietic stem cell (HSC) biology. So far, however, there are no available data on the role of NF-Y in mesenchymal stem cell (MSC) biology. The aim of this study is the evaluation of NF-YA expression levels during the ex vivo expansion of human bone marrow (BM)-MSCs and the assessment of its possible role for the BM-MSC differentiation process towards the osteoblastic and adipocytic lineages. BM-MSCs were isolated from posterior iliac crest aspirates of hematologically healthy individuals undergoing orthopedic surgery after written informed consent. BM-MSCs were ex vivo expanded until passage 10 (P10). Total RNA was isolated from BM-MSCs at P2 and P10 for the evaluation of NF-YA mRNA levels. P3 BM-MSCs' ex vivo differentiation into adipocytes and osteoblasts was induced using the appropriate culture media. Mineralization was evidenced via Alizarin Red and Von Kosa staining and lipid droplets were revealed with Oil Red O staining. Total RNA and proteins were isolated from undifferentiated and differentiated BM-MSCs at various time-points. Protein levels of NF-YA were immunodetected by Western blot. The relative gene expression of NF-YA, as well as that of lineage-specific markers was evaluated by real-time PCR. All PCR results are expressed as 2−ΔCt. Adipocytic and osteoblastic differentiation of BM-MSCs was also induced following knockdown of NF-YA expression after BM-MSC transduction with lentiviral particles carrying either shRNA against NF-YA or nonsense shRNA. Total RNA was isolated from transduced BM-MSCs at several time-points during differentiation. A statistically significant (p=0.04) reduction in the mRNA levels of NF-YA was found in P10, as compared to P2 BM-MSC cultures (n=12). During adipogenic differentiation, NF-YA significantly increased (p=0.0478) at day 16, as compared to the onset of differentiation induction (day 0), (n=14). Similarly, protein levels of NF-YA also increased during adipogenesis Furthermore, NF-YA expression levels strongly correlated with the mRNA levels of adipogenesis-associated genes PPARG (r=0.74, p In conclusion we have shown for the first time, that BM-MSCs display decreased NF-YA mRNA levels after prolonged ex vivo expansion, a finding that might be associated with MSCs' senescence and/or loss of stemness., We have also shown that NF-YA does not have any major effect on BM-MSCs' osteogenic differentiation but displays a significant role in their adipogenic differentiation as was shown by (a) the increased NF-YA mRNA and protein levels during the ex vivo adipogenesis, (b) the positive correlation between the NF-YA mRNA expression levels and the adipocyte-associated genes PPARG, C/EBPA and LPL and (c) the impaired adipogenic differentiation following NF-YA knock down. Collectively our results imply a novel role for NF-YA in BM-MSCs' biology Disclosures: No relevant conflicts of interest to declare.

Abstract 3861 The microenvironment in both the bone marrow (BM) and lymph nodes has clearly been implicated in the biology of B-chronic lymphocytic leukemia (B-CLL). The non-hematopoietic components of the BM microenvironment originate from a rare population of multipotent progenitor cells, currently referred to as mesenchymal stem/stromal cells (MSCs). The latter have been shown to support hematopoiesis and to affect B cell proliferation and differentiation, thereby setting the stage for studying MSCs' putative role in CLL pathogenesis. However, this particular field of research has not been extensively investigated and the question as to whether patient MSCs differ from their normal counterparts has not been properly answered. The aim of the present study is to explore whether BM-derived MSCs from CLL patients harbor intrinsic abnormalities, which in turn might contribute to the pathophysiology of the disease. BM MSCs were thus isolated from 11 patients with B-CLL (Rai stage 0-III) and 16 age- and sex-matched healthy individuals and their quantitative, functional and cytogenetic characteristics were comparatively assessed. BM MSCs were expanded and re-seeded for a total of 6 passages. Adherent cells from both study groups displayed the same spindle-shape morphology and showed a similar expression of CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. Even though MSC cultures could be established and serially replated from all CLL patients, their growth rate over passages was significantly reduced compared to cultures generated from normal individuals (P < 0.0001). These findings were further substantiated by the MTT assay according to which the number of live cells, at a representative passage (P2), remained significantly lower in CLL patients compared to controls (P In conclusion, ex-vivo expanded B-CLL-derived MSCs harbor intrinsic qualitative and quantitative abnormalities that may be implicated in disease development and/or progression. We anticipate that our observations will contribute to delineating B-CLL biology and will hopefully provide important clues for the design of appropriate microenvironment-targeted therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3795 Both basal and cytokine-induced phosho-STAT3 and pSTAT5 levels are altered in leukemias and profiling the STAT signaling network at the single cell level provides prognostic information (Irish et al, 2004). As cell signaling interacts with the epigenome (Mohammad et al, 2010) and numerous genes involved in signaling are aberrantly methylated in MDS (Figueroa et al, 2010) we investigated the alterations of STAT3/5 signaling in MDS progenitors during azacitidine therapy. Bone marrow samples of 58 high-risk MDS and AML/MDS patients were obtained before (d0) and 15 days (d15) after azacytidine initiation and at the indicated time points. According to the IWG response criteria, patients were divided into 4 groups: response (CR and PR, n=11 and n=5, respectively), hematologic improvement (HI, n=8), stable disease (SD, n=11) and failure (F, n=23). Mononuclear cells were either left untreated or stimulated with G-CSF and GM-CSF for 15′ and then stained intracellularly with the indicated antibodies. The hematopoietic hierarchy and G-CSF receptor (CFS3R) transcripts by qPCR were assessed on immunomagnetically purified Lin-CD34+ and CD34+ cells, respectively. Statistical comparisons were done by ANOVA and unpaired or paired t-tests as appropriate. STAT signaling profiles (SPs) in CD34+ cells were grouped using complete linkage hierarchical clustering and correlated with treatment response, karyotype and transfusion rate by using χ2 or Fisher Exact tests. Unsupervised clustering of pretreatment SPs in CD34+ cells identified 2 clusters (Fig 1A) mainly differing in the degree of potentiated STAT3/5 phosphorylation. Patients in cluster I displayed weak potentiated expression of pSTAT3/5 and had marginally better response to azacytidine compared to the ones in cluster II (p=0.06), whereas there were no differences among the two groups regarding the MDS subtype (p=0.41), karyotype (p=0.45) and transfusion rate (p=0.33). We further identified a G-CSF-inducible pSTAT3/5 double positive (DP) subpopulation of MDS CD34+ cells, whose levels both at d0 and d15 of the 1st cycle were inversely associated with response (Fig 1B), whereas its kinetics were following the disease course and response to azacytidine (Fig 1C,D). The DP subset was enriched in GMPs compared to the G-CSF-unresponsive pSTAT3/5 double negative (DN) subpopulation, suggesting a higher leukemia initiating cell activity (Goardon et al, 2011), whereas the DN subset was marginally enriched in MEPs (Fig 2). Also, compared to the DN subset, the DP population exhibited decreased Ki67 expression and increased Bcl2 and p53 levels (Fig 3A), indicating quiescence and increased antiapoptotic and tumor suppressive properties, respectively. Of note, the levels of the above molecules in the two subsets remained unaltered both at d0 and d15, indicating that these subsets represent genuine cellular entities with solid properties. To determine if CSF3R expression and/or its modulation by azacitidine are responsible for the alterations of the DP population, we checked CSF3R levels in isolated CD34+ cells from patients with either absence or full expression of the DP subset because CSF3R is downregulated after G-CSF stimulation. The two groups showed identical protein and mRNA levels of CSF3R at both d0 and d15 (Fig 3B,C). In summary, we identified a G-CSF-inducible pSTAT3/5 DP subpopulation with leukemic stem cell properties, which is potentially involved in MDS biology and epigenetically modulated, as implied by its kinetics during hypomethylating therapy. More important, given the strong association of pretreatment levels of the DP subset with the response to azacitidine, the latter subset may serve both as a treatment target and response biomarker. Fig 1. (A) Heatmap of pretreatment SPs in CD34+ cells. (B) The DP subset was significantly decreased in responding patients. (C, D) Results and representative plots of the DP subset kinetics in patients with R (n=7), HI (n=3), SD (n=1) and F (n=3). Fig 1. (A) Heatmap of pretreatment SPs in CD34+ cells. (B) The DP subset was significantly decreased in responding patients. (C, D) Results and representative plots of the DP subset kinetics in patients with R (n=7), HI (n=3), SD (n=1) and F (n=3). Figure 2. (A) Cytometric and (B) cumulative analysis (n=8) of the hematopoietic hierarchy in DP and DN subsets. Figure 2. (A) Cytometric and (B) cumulative analysis (n=8) of the hematopoietic hierarchy in DP and DN subsets. Figure 3. (A) Results and histograms of ki67, Bcl-2 and p53 assessment in DP (grey fill) and DN (thick line) subsets (control, thin line). (B) Protein and mRNA expression of CSF3R in patients with absence (n=5) or full expression (n=5) of the DP population. (C) Similar CSF3R levels at d0 and d15, despite changes in the levels of the DP subset (not shown, n=5). Figure 3. (A) Results and histograms of ki67, Bcl-2 and p53 assessment in DP (grey fill) and DN (thick line) subsets (control, thin line). (B) Protein and mRNA expression of CSF3R in patients with absence (n=5) or full expression (n=5) of the DP population. (C) Similar CSF3R levels at d0 and d15, despite changes in the levels of the DP subset (not shown, n=5). Disclosures: Kotsianidis: Genesis-Pharma: Honoraria, Research Funding.

Abstract 2712 Treatment of splenic marginal zone lymphomas (SMZL) is traditionally based on splenectomy. However, preliminary data suggest that rituximab monotherapy is highly effective in SMZL patients. The aim was to assess the efficacy of rituximab administration in a large series of patients with SMZL, as upfront therapy. Fifty-eight patients with the diagnosis of SMZL were prospectively treated, between September 2003 and July 2011, with rituximab monotherapy. Patients' clinical characteristics are shown on table 1. Rituximab was given in two phases: Induction phase at a dose of 375mg/m2 per week for 6 weeks and maintenance phase at the same dose every 2 months for 1–2 years. Evaluation of response was performed 2 months after induction and 2 months after the end of maintenance phase.Table 1.Clinical and Laboratory Findings of SMZL patients at Diagnosis According to Treatment ApproachFeatures# % of patientsN58Age –median (range)64 (41–91)Sex:Male26 45Â symptoms1 2Palpable splenomegaly(cm, below left costal margin, range)10 (2–16)Lymphadenopathy*14 24Bone marrow involvement58 100Anemia (Hb4.0x109/l)27 47Thrombocytopenia (normal values20/57 35M component19/50 38Hepatitis C0International Prognostic IndexLow risk17/57 30Low-intermediate27/57 47High-intermediate12/57 21High risk4/57 7*by computerized tomography Fifty-five patients were evaluable for response. The overall response rate (ORR) after the end of induction phase was 94% (52/55) with 45% (25/55) presenting complete response (CR), 27% (15/55) unconfirmed CR (CRu) (in those who did not undergo bone marrow reevaluation), 22% (12/55) partial response (PR). The median time to hematologic and clinical response was 2 and 3 weeks respectively. 29/52 rituximab responders have already completed maintenance therapy and were evaluable for response (10 did not receive maintenance therapy due to refusal, 13 have not completed this phase yet). Evaluation of response after the end of maintenance phase disclosed that 21 patients sustained their initial response while in 7 patients a further improvement of response was documented: PR after induction phase was converted to CR after the end of maintenance phase. However one patient lost her initial response (CR) during maintenance therapy. The 5-year OS and PFS for rituximab treated patients was 94% and 68% respectively. Of the 10 patients, who did not receive maintenance, three (33%) relapsed at 24, 29 and 38 months respectively. On the other hand, among the 29 patients who have already completed maintenance therapy, 4 relapsed (14%) at a median time of 43 months (range, 35–48). None of the 13 patients who are currently receiving maintenance has relapsed so far. Maintenance phase was clearly associated with better PFS: Median PFS was 38 months for pts not receiving maintenance, but it has not been reached yet in pts receiving maintenance. At 4 years PFS was 79% vs 27% (p=0.001), for patients who received maintenance or did not, respectively. A total of 7/52 rituximab responders (13%) relapsed at a median time of 37 months (range, 24–48). 6/7 patients were retreated with rituximab and 4 of them responded for a response rate of 67%. One out of the 4 responders to rituximab reinduction experienced a second relapse 30 months later. This patient was retreated with rituximab and remains in CRu for 26 months. Three deaths were recorded in the rituximab group: 1 patient died of unrelated cause and 2 of lymphoma. Rituximab is a very effective and well tolerated therapy and may substitute splenectomy as first-line treatment for SMZL. Maintenance with rituximab might prolong the duration of response, although this warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

Abstract 272 The pathogenesis of Myelodysplastic Syndromes (MDS) is characterized by the acquisition of multiple consecutive alterations in an early hematopoietic stem cell, leading to a stepwise clonal cell expansion and disease evolution. An abnormal bone marrow (BM) microenvironment, however, might also contribute to the BM failure in MDS through defective support of hematopoiesis. The cellular elements of the BM microenvironment derive from a common progenitor cell, namely the mesenchymal stem cell (MSC). We and others (reviewed in Kastrinaki et al. Curr Stem Cell Res Ther. 2011) have recently reported that MDS-derived BM MSCs show defective proliferative potential, impaired clonogenic capacity and limited cellular expansion during passages. The pathogenetic basis of these abnormalities and their implication in the disease process remain unknown. The aim of this study is to probe the mechanisms underlying the impaired functional properties of BM MSCs in patients with lower risk MDS. BM MSCs were isolated from 12 patients with lower risk MDS aged 51 to 75 years (median 67.5 years) and 20 healthy volunteers aged 50 to 73 years (median 63.3 years). BM MSCs were expanded and re-seeded for a total of 10 passages (P). To investigate whether the decreased proliferative capacity of MSCs in MDS patients might be related to replicative senescence, genomic DNA was isolated from culture expanded MSCs at P2, P6 and P10 and telomere length was measured by means of real-time PCR using β-globin as control single-copy-gene. Telomere length is proportional to the relative telomere/single-copy-gene ratio (T/S): T/S=2−δCt(δCt=Cttelomere-Ctβ-globin). Furthermore, total RNA was extracted from culture-expanded P2, P6 and P10 MSCs and amplified by real-time PCR for the evaluation of the senescence associated genes CDKN1B (p15), CDKN2A (p16), CDKN1A (p21), pRB, TP53 (p53) and PARG1. Relative gene expression was calculated by the δCt method. Given that the Wnt signaling pathway plays a key role in MSC proliferation and differentiation, we next investigated whether the proliferative defect of MSC in MDS might be associated with abnormal expression of genes related to Wnt-mediated signal transduction, using a PCR array. Total RNA was thus isolated from 4 representative MDS and 6 normal MSC cultures at P2. The fold change (FC) for each gene between the group of patients and the group of controls was calculated with the δδCt method (FC=2−δδCt). The relative telomere values declined over the entire observation period (P2-P10) in both patient- and control-derived MSCs. T/S values remained constantly higher over passages in MDS MSCs compared to healthy individuals (F=4.362, P MDS-derived MSCs display longer telomeres and decreased expression of senescence-associated genes compared with healthy individuals. The possibility therefore that cellular aging account for the defective proliferative potential of patient MSCs seems rather unlikely On the other hand, activation of the non-canonical Wnt pathway in association with the inhibition of the canonical one can explain, at least in part, the decreased proliferative capacity of MSCs in MDS patients. Collectively our results suggest that aberrant Wnt signaling is actively implicated in the mechanisms underlying the impaired functional properties of BM MSCs in MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract 2538 Chronic lymphocytic leukemia (CLL) has a proliferation rate higher than previously recognized. Proliferation centers (PC) play an important role in the biology of CLL given the fact that they constitute its proliferative compartment. The complexity of the microenvironment of PC, as well as the molecular events taking place in the PC and their clinical significance remain to be elucidated. The aim was to identify the presence of PC in lymph node and other tissues, except the bone marrow; to analyze their proliferative status; to evaluate the expression of molecules implicated in the apoptotic process in PC, compared to their expression in the non-PC areas; and to correlate the aforementioned findings with the clinical and laboratory features in a series of CLL patients. Fifty patients, fulfilling the diagnostic criteria of CLL/SLL and in whom lymph node biopsy or other tissue material (44 lymph nodes, 5 spleens, 1 skin) were available, were enrolled in this study. Twenty five biopsies were performed at diagnosis while the rest prior to treatment initiation. All necessary clinical and biological data were recorded on each patient, including Binet stage, lymphocyte doubling time, bone marrow infiltration pattern, CD 38 expression, IgVH mutational status and FISH analysis for genetic abnormalities. PC were defined as pale areas containing large cells so called paraimmunoblasts and prolymphocytes, surrounded by a dark background of small lymphocytes. Immunohistochemical detection of the following molecules participating in the apoptotic process were studied in all tissue sections: Bcl-2, Fas, FasL, c-FLIP (in the PC and the non PC areas) as well as cleaved caspase 3 that was evaluated in the entire tumor area. ZAP-70 was also studied by immunohistochemistry while proliferation status was assessed by the Ki-67 immunostaining. The median age of our patients at diagnosis was 55 years (36–77). Twenty-nine (58%) had disease stage A, 17(34%) B and 4(8%) C, while 12% had B-symptoms, 26% elevated LDH levels and 25% presented with rapid lymphocyte doubling time. 39% needed treatment immediately after diagnosis. Proliferations centers were present and easily identified after staining with haematoxylin-eosin on lymph node and splenic sections with their numbers varying from case to case. Proliferation assessment revealed that median Ki67 proliferation index per PC was 10% (1–20%), while median Ki67 in the whole tissue section was 3% (1–8%) (p Apoptotic molecules evaluation disclosed that the expression of cleaved-caspase 3 was very low (median 0.0046%). Fas, FasL and cFLIP activity was expressed in various percentages in the PC and in the non-PC areas as shown in Table 1. Further on, in cases of overexpression of Fas and FasL, an analogous increase of cFLIP expression was not observed. Patients with elevated Ki67 proliferation index in PC area tended to express more Fas and FasL in the same areas. Strong homogenous Bcl-2 expression was observed both in the PC and in the non PC areas. Weaker Bcl-2 expression in PC compared to non PC areas was observed in 10 patients in whom a higher Fas (p=0.054) and FasL (p=0.005) was present. 48% of the cases were ZAP-70 positive. ZAP70 positivity was correlated with increased expression of cFLIP in PC (p=0.0216). The only statistically significant correlation between apoptotic molecule profile and clinical features was between an increased FasL expression in PC and B-symptoms (p: 0.0263). In univariate survival analysis, coexpression of Fas/FasL and cFLIP at increased levels in PC correlated with poor disease-free and overall survival (p=0.0031, p=0.0521 respectively).Table 1.Fas, FasL and cFLIP expression in lymph node and splenic sections of CLL patientsMoleculePC median(%)Non-PC areas (median %)P valueFas50400,1581FasL30100,0107cFLIP50400,0726 Proliferative centers in lymph node and splenic sections of CLL patients present particular proliferative and apoptotic profile. Inhibition of Fas mediated apoptosis in PC may not be attributable to cFLIP expression. Increased Fas,FasL and cFlip coexpression in PC was correlated with disease-free and overall survival. Disclosures: No relevant conflicts of interest to declare.

Abstract 4556 Members of the tumor necrosis factor / tumor necrosis factor receptor (TNF/TNFR) superfamily are involved in bone marrow (BM) homeostasis by regulating the survival, apoptosis, proliferation, and differentiation of hematopoietic progenitor and precursor cells. The CD40 Ligand (CD40L)/CD40 molecules belong to the TNF/TNFR superfamily; we have recently showed that the CD40/CD40L interaction on BM CD34+ cells accelerates the apoptotic cell death. The aim of the present study is to investigate the distribution and function of the CD40/CD40L molecules in the BM granulocytic progenitor and precursor cell populations as well as the effect of their interactions on the stromal release of cytokines related to granulopoiesis. BM samples were obtained from posterior iliac crest aspirates from 19 hematologically healthy subjects after informed consent. We evaluated: (a) the surface expression of CD40 and CD40L on immunomagnetically sorted CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells representing sequential stages of the granulocytic development, using flow cytometry in steady state conditions and following 20-hour incubation with 25ng/ml recombinant-human (rh) TNFα; (b) the proportion of apoptotic cells and the level of caspase-3 activation following CD40 engagement in the above cell populations using flow cytometry and a chromo-enzymatic method, respectively; (c) the production of granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) by long-term BM culture (LTBMC) stromal cells upon induction with rhCD40L (1μg/ml), using an enzyme-linked immunoabsorbent assay (ELISA) in culture supernatants. We found that the basal expression of CD40 and CD40L on the CD34+, CD34-/CD33+, CD34-/CD33-/CD15+ cells was 3.34%±3.90% and 1.8%±1.03%, 16.74%±14.69% and 6.96%±4.68%, 3.22%±3.36% and 2.26%±2.71%, respectively. Following incubation with rhTNFα a substantial increase was obtained in the expression of CD40 in all the above cell populations compared to baseline (37.18%±12.51%, 29.25%±14.81%, 15.84%±6.28%, respectively) (p Disclosures: No relevant conflicts of interest to declare.

Clonal/oligoclonal expansions of cytotoxic T-lymphocytes (CTLs) and quantitative/qualitative abnormalities of T-regulatory cells (Tregs) have been implicated in the pathophysiology of acquired bone marrow (BM) failure syndromes such as aplastic anemia, myelodysplastic syndromes and large granular lymphocyte proliferative disease. Chronic Idiopathic Neutropenia (CIN) is an acquired disorder of granulopoiesis characterised by increased apoptosis of BM CD34+/CD33+ granulocytic progenitor cells. We have previously provided evidence for oligoclonal T-cell expansions with possible pathogenetic significance in CIN. The aim of the current study is (a) to define and compare the magnitude of CD4+ and CD8+ T-cell responses and (b) to evaluate the number of Tregs, in the peripheral blood (PB) and BM of CIN patients. Eighty five CIN patients and 85 healthy controls, age- and sex-matched to the patients, were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1410 ± 330 neutrophils/μL, range 100–1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease.The T-cell receptor (TCR)-Vβ repertoire was analysed by flow cytometry (Vβ spectratyping) in the PB and BM CD3+, CD4+ and CD8+ cells. The size distribution of the TCR-Vβ complementarity determining region 3 (CDR3) was analysed in immunomagnetically sorted PB and BM CD4+ and CD8+ cells by PCR according to the -2 protocol using a fluorescence-based DNA sequencer (CDR3 spectratyping). Vβ family expansions were defined as above of 2 standard deviations from the mean values of the 85 healthy controls. CDR3 oligoclonal/monoclonal patterns were defined upon comparison with the normal Gaussian-type size distribution. The Treg cell frequency was defined as the proportion of FOXP3+ cells within the CD4+/CD25high PB and BM cell fraction by flow cytometry. We found that 69.41% and 82.61% of CIN patients displayed one or more predominant TCR-Vβ family expansions within the CD3+ cell fraction of PB and BM, respectively. None of the controls displayed clonal/oligoclonal expansions in either PB or BM (P

Late-onset neutropenia (LON) is a complication of Rituximab of yet unknown pathophysiology. We investigated potential underlying mechanisms in 12 patients with various non-Hodgkin lymphoma (NHL) subtypes who developed LON without identifiable causes at a median of 95 (range 67–420) days after completion of the intended treatment with Rituximab ± chemotherapy. The study also included two control groups: healthy donors (n=25) for comparative analysis of bone marrow (BM) functional parameters; NHL patients treated with similar Rituximab ± chemotherapy regimens without LON for comparative evaluation of peripheral blood and BM lymphocyte subpopulations (n=38) and BM pathology findings (n=27). Ten of 12 patients with LON and 33/38 NHL controls developed profound B-cell depletion. Inverted CD4/CD8 cell ratios were observed in 10/12 LON cases vs. 13/38 NHL controls (p1.0x109/L was identified in 8/12 LON cases vs. 8/38 NHL controls (p30%) was observed in 7/12 LON cases vs. 14/38 NHL controls (p=0.1885). BM biopsy samples from 10/12 LON cases were examined at onset of neutropenia. Mild to moderate lymphocytic infiltration with predominantly nodular and/or interstitial growth was observed in all cases. Lymphoid aggregates lacked CD20+ or CD79a+ B-cells and were composed entirely of CD3+CD45RO+CD43+ T-cells. In the control group, BM infiltration by T cells with similar features as above was observed in 18/27 cases. Seven of 10 LON patients showed moderate-to-significant hypoplasia of the granulocytic series; the remaining three exhibited hyperplasia. All ten LON patients had pronounced shift to the left, extending to maturation arrest in 6/10 cases. In contrast, 21/27 NHL control cases (78%) showed granulocytic hyperplasia; the remaining cases were either normal or showed granulocytic hypoplasia. As in the LON cases, the control NHL cases exhibited different degrees of shift to the left of the granulocytic series. Hyperplasia of the erythroid and megakaryocytic series with dyserythropoiesis and dysmegakaryopoeisis, respectively, was identified in all LON cases and most NHL controls. In all cases with repeat samples, MDS-like changes eventually resolved. Compared to healthy donors, LON patients displayed: low reserves of granulocytic progenitors associated with increased apoptosis and increased proportion of Fas-expressing cells within the CD34+/CD33+ cell compartment; Fas-Ligand and Interferon-γ mRNA expression within BM CD3+ cells; increased TNFα and IL-1β levels in long-term BM culture supernatants; accelerated apoptosis in all stages of BM granulocytic development. All LON patients were negative for granulocyte-reactive antibodies. We conclude that T-cell mediated autoimmune myelopathy/myelodysplasia associated with MDS-like changes of all myeloid series and suppression of predominantly granulocytic progenitor cell growth is critically implicated in the pathophysiology of Rituximab-related LON, at least in a subset of cases.

We have investigated the etiology of congenital neutropenia in a girl with de novo duplication of chromosome 17q25.3. She presented during the first year of life with neutropenia, episodic hypothermia, failure to thrive, and other congenital abnormalities. Peripheral blood karyotype demonstrated 46,XX,add(17)(q25.3), and molecular cytogenetic studies confirmed interstitial duplication of chr17-derived material. We excluded ELA2 mutation (the most common cause of hereditary neutropenia) and reasoned that the neutropenia and other medical problems most likely were the result of the chromosomal abnormality. In the duplicated 17q25.3 region, SOCS3 emerged as a promising candidate gene responsible for neutropenia, because SOCS3 is a well-characterized negative regulator of G-CSF-receptor signaling and, in murine conditional knockout models, acts as a physiologic negative regulator of granulopoiesis. As we had previously demonstrated that mutations of the Gfi1 transcriptional repressor are a rare cause of human neutropenia, we searched for potential Gfi1 binding sites in the SOCS3 promoter and noted a total of five, and chromatin immunoprecipitation analysis validated occupancy of the SOCS3 promoter by Gfi1 in Jurkat, HL-60, and U937 cells. Thus, several lines of evidence suggested SOCS3 as a plausible neutropenia gene. We confirmed that SOCS3 is indeed duplicated in this patient by both FISH and quantitative genomic PCR. In an effort to identify other patients with SOCS3-related neutropenia, we sequenced both exons of SOCS3 in a total of 66 patients with severe congenital neutropenia (SCN) or cyclic neutropenia (CN) and 94 patients with chronic idiopathic neutropenia of adults (CINA) in whom ELA2 mutations were absent. No coding mutations were detected, but we did identify several variants in the SOCS3 promoter and 5′- and 3′- UTRs, including a single base pair substitution (+1779AtoG) in the 3′-UTR in a SCN patient and deletion of a single G (-1318delG) occurring in a six-G tract immediately adjacent to a predicted STAT binding site in a highly conserved region of the SOCS3 promoter (found in heterozygous and homozygous form in several patients with CN and SCN). The 3′-UTR alteration was absent in 270 control chromosomes and is located within a predicted binding site for miR-449 that is well-conserved across mammalian species. In sum, we have identified a patient with congenital neutropenia and a de novo duplication of 17q25.3 defining a new candidate locus for congenital neutropenia, and the evaluation to date suggests that SOCS3 is a promising dosage sensitive candidate gene in this interval that additionally could be a key target in neutropenia associated with Gfi1 mutations.

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent cells, easily expanded in culture and able to give rise to many lineages. These characteristics make MSCs a very attractive tool for developing new strategies for clinical applications based on cell therapy. So far, the most common source of MSCs has been the bone marrow (BM). However, identification and characterization of alternative sources of MSCs is of great importance. One such alternative source is the amniotic fluid (AF), which can be collected during scheduled amniocentesis without any ethical concerns. To this end, in the present study, we introduced an improved protocol for isolating and clonally expanding fetal MSCs from second trimester amniotic fluid (AF) and we further characterized these cells based on their phenotype, pluripotency, differentiation potential and proteomic profile. The AF samples were obtained during routine amniocentesis and AF-MSCs were enriched by a modified culture protocol. The isolated MSCs expanded rapidly and exhibited differentiation potential into adipocytes and osteoblasts. More importantly, we showed that these cells can differentiate in vitro not only into cell types derived from mesoderm (adipocytes and osteoblasts) and ectoderm (neural cells) but also more interestingly into endoderm (hepatocytes) derived cells. Moreover, we documented that AF-MSCs express Oct-4 transcription factor, a marker of pluripotency, and we studied for the first time its expression over different passages by real time PCR and documented that it remained constant for at least 17 doublings. An extensive characterization of the phenotypic features of AF-MSCs by using a wide range of surface markers and flow cytometry, indicated that they are positive for all the mesenchymal stem cell markers such as CD90, CD105, CD73 and CD166 and generally exhibit a similar expression pattern to the BM-MSCs. To characterize these cells in more detail, we established the first proteomic database for human AF-MSCs. Using 2D-gel electrophoresis and matrix-assisted laser desorption ionisation-time of flight-mass (MALDI-TOF) spectrometry approach, we have generated for the first time the protein map of AF MSCs, by identifying 260 proteins and directly compared this protein profile with that of MSCs derived from BM. We further performed a similar analysis for BM-MSCs, identifying 170 different proteins and generating a reference map for these cells. The comparison of the proteomic pattern from both sources was similar. In general, 140 proteins were identified in AF-MSCs related to cell growth/maintenance, metabolism/energy pathways, protein metabolism, apoptosis, signal transduction and communication as well as transcription and transport, that are not present in BM-MSCs. The approach we initiated, is expected to facilitate systematic functional studies for these multipotent cells. One such approach could be the implementation of the proteomic analysis, during differentiation of AF-MSCs to cells derived from all three germ layers as shown in our study. Data derived from these approaches are expected to clarify the therapeutic potential of the MSCs.