ALBERT

Description: The structure of factor IX gene was analyzed in a hemophilia B patient with inhibitor. Genomic DNA, digested with a variety of restriction endonucleases, was hybridized with the cDNA and various genomic factor IX probes. A large subtotal deletion of the gene was observed. The borders of the deletion span from a approximately 125 nucleotide region within the last exon to an unknown domain at least 7.5 kb upstream from the first exon: it thus involves approximately 33 kb of the factor IX locus. The abnormal gene was inherited by the daughter of the propositus, who showed both the normal and the deleted allele.

Description: Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two- dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Description: Monoclonal antibodies (MoAbs) to myeloid differentiation antigens have a potential use in purging bone marrow of leukemia cells in autologous bone marrow transplantation (ABMT) therapy for acute myelogenous leukemia (AML). Because the efficiency of purging by MoAb and complement (C) is important to the success of ABMT, we have designed an assay to determine optimal conditions for leukemia cell lysis. In order to mimic the conditions of remission bone marrow, normal buffy coat cells were mixed with cells from the HL-60 promyelocytic leukemia cell line at a concentration that approximated the normal-leukemia cell ratio found in remission marrow. The cell mixture was treated at variable times and temperatures in the presence of C and PM-81, an IgM MoAb that reacts with both normal granulocytes and monocytes as well as with HL-60 cells. PM-81 binds to the majority of cells from 90% of patients with AML yet does not react significantly with normal stem cell populations. Because of the potential use of PM-81 in ABMT, it seemed especially important to show that the antibody was capable of mediating cytotoxicity of HL-60 cells in the presence of an excess of antigen-positive cells. A clonogenic assay that permitted the growth of HL-60 cell colonies but not normal progenitor cells in methylcellulose cultures was used to measure the efficiency of HL-60 cell lysis. We found that under certain conditions, PM-81 was capable of removing the small percentage of HL-60 clonogenic cells admixed with normal buffy coat cells. This information was useful for determining the optimal conditions for purging bone marrow of leukemia cells for ABMT.

Description: The expression of Rhesus antigens on hematopoietic progenitor cells was studied using monoclonal antibodies. Because these antibodies are not capable of lysing mature red blood cells in a complement-dependent cytotoxicity assay, fluorescence-activated cell sorting was performed. Using the monoclonal anti-Rh 29 antibody B10, 68% +/- 6% of the mature erythroid progenitor cells (CFU-E) were sorted into the positive fraction, while only 2% +/- 1% of the relatively immature erythroid progenitor cells (BFU-E), and 3% +/- 1% of the granulocyte-macrophage progenitor cells (CFU-GM) were cultured from this same fraction. Thus up to a 15-fold enrichment of CFU-E could be obtained. In two experiments more than 4% of the cells in the positive fraction consisted of CFU-E; in one experiment even more than 7% did. Using fractionated cell sorting, the Rhesus antigens appeared to have a lower density on CFU-E than HLA-DR determinants. Antibodies against the Rhesus antigens can be applied to enrich erythroid-committed stem cells and to separate mature from immature erythroid progenitor cells.

Description: Surface membrane glycoproteins (SMGs) of cells from the parental wild- type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two- dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritide) and peptide (1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pl], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55- to 60-kD neutrophil- associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross- resistance mechanism(s) for RA/purine bases but not for DMSO.

Description: The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.

Description: The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

Description: A long-term liquid culture system of hemopoietic tissue derived from adult hamster spleen has been described. These primary liquid cultures can maintain stem cell proliferation and differentiation for more than three months without secondary repopulation. A characteristic of the liquid cultures is the formation of clusters of hemopoietic cells around adherent stromal cells. Some islands were composed exclusively of megakaryocytes and adherent cells. Isolation of these clusters of differentiating megakaryocytes and their adherent cellular substrate permitted the analysis of the morphological and ultrastructural features of the interaction between cells of megakaryocytic lineage with the adherent stroma.

Description: We have used immunogold staining to locate thrombospondin (TSP) on thrombin-activated human platelets, and have compared its distribution with that of fibrinogen (or fibrin) on thrombin- and ADP-stimulated platelets. To do this, isolated platelets were incubated with monospecific antibodies to TSP or fibrinogen (fib) and the bound IgG located with a second antibody adsorbed to gold particles. Thrombin- induced secretion in Tyrode-Ca2+ was followed by both anti-TSP and anti- fib binding, with large clusters of gold particles observed on the platelet surface. Little or no labeling was observed on unstimulated platelets with either antibody. When secretion was effected in Tyrode- EDTA, anti-TSP IgG still bound to the activated platelets, but the number of particle clusters was significantly reduced. Little binding of anti-fib IgG now occurred. Platelets activated with ADP in the presence of added fib, and subsequently incubated with anti-fib IgG, showed small particle clusters over the whole platelet surface. Thrombin-stimulated platelets from two patients with thrombasthenia bound anti-TSP IgG similarly to normal platelets activated in Tyrode- EDTA. No anti-fib binding occurred. Our results suggest that fib and TSP bind to specific domains on the stimulated platelet membrane. Such sites may be responsible for the mediation of platelet surface contact interactions.

Description: Protein-heparin complexes, prepared by a water-soluble carbodiimide coupling technique, were used to produce anti-heparin antibodies in rabbits. Antiserums that recognized carbodiimide-treated heparin, but not untreated heparin, were obtained. Carbodiimide-treated heparan sulfate exhibited 10% to 20% cross-reactivity compared with a similarly treated heparin, whereas there was no cross-reactivity with five other carbodiimide-treated mucopolysaccharides. 3H-1-ethyl-3-(3- trimethylammoniumpropyl) carbodiimide iodide was used to demonstrate that carbodiimide forms a stable adduct with heparin and other mucopolysaccharides. Using an antibody fraction that eluted from 1- ethyl-3-(3-trimethylammoniumpropyl) carbodiimide iodide-treated heparin- Sepharose with 2 mol/L KI, it was demonstrated that, for the antibody population studied, the addition of one carbodiimide per heparin molecule resulted in complete epitope expression without loss of anticoagulant activity. The addition of up to eight additional carbodiimide molecules to heparin did not increase the extent of epitope formation, although anticoagulant activity was lost. Except for heparan sulfate, the addition of radiolabeled carbodiimide to other mucopolysaccharides did not result in epitope formation. These data demonstrate that antibodies to an epitope derived from heparin can be formed, that the epitope is fully expressed while anticoagulant activity is present, and that the antibody is specifically directed against an altered portion of the polysaccharide.

Description: We tested conditioned media from 12 patients with T lymphocyte neoplasms and four T cell lines for their ability to stimulate the in vitro growth of erythroid-burst-forming units (BFU-E) from bone marrow mononuclear cells in a methylcellulose culture system. Nine patients suffered from acute lymphocytic leukemia, two from chronic lymphocytic leukemia, and one from non-Hodgkin's lymphoma. The T lymphocytes were characterized by a series of monoclonal antibodies and their stage of development was correlated with their ability to produce burst- promoting activity (BPA). Conditioned media from cells classified as prothymocytes (three cases), common thymocytes (one case), mature thymocytes (three cases), and mature lymphocytes of the helper subtype (two cases) increased BFU-E proliferation four- to 19-fold over control values using normal bone marrow as target cells. Conditioned media from OKT8+ malignant T lymphocytes (three cases) did not enhance BFU-E proliferation. Conditioned media from cells classified as immature T cells stimulated CFU-GM proliferation in only one of seven cases even though they secreted BPA. Conditioned media from three of the four cell lines stimulated by phytohemagglutinin, enhanced BFU-E growth. Our results indicate that malignant cells that have characteristics of immature T cells are able to produce BPA. Studies using techniques to isolate homogeneous populations of normal T cell subsets are required to determine whether normal immature T lymphocytes have the same capability.

Description: Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.

Description: The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

Description: Leukemic blasts from 774 children with newly diagnosed acute lymphocytic leukemia (ALL) have been phenotyped by microcytotoxicity testing with a panel of monoclonal antibodies and heteroantisera as part of a Pediatric Oncology Group classification study of acute leukemia. One hundred twenty-two cases, or 16% were designated as T cell leukemia based on the reactivity of blast cells with previously well-characterized antisera (PT) against a T lymphocyte-associated antigen. Using this antisera-based definition as a standard, we looked for a monoclonal antibody combination that would be a suitable substitute. An algorithm calling for reactivity with either monoclonal antibody 3A1 or Leu-1 was a 92% sensitive and 97% specific predictor of PT reactivity. Only 27 of 755 cases of leukemia were incorrectly classified using this algorithm. Subsequently, Ficoll-Hypaque-separated bone marrow cells from 118 additional patients with ALL (21 of whom had T cell ALL) were stained by immunofluorescence using a combination of directly fluoresceinated 3A1 and Leu-1. Reactivity of 20% or more of the cells with this antibody combination was a 100% sensitive and 94% specific indicator of T cell ALL defined by PT positivity; with a higher cutoff value for positive values, or the use of supplemental tests, even this small number of false-positives could be eliminated. We conclude that this monoclonal antibody combination is a satisfactory replacement for our heteroantisera definition of T cell ALL.

Description: The effect of a zinc metalloprotease from Serratia marcescens on platelet surface glycoproteins (GP) Ib and V was analyzed. Increasing protease treatments caused progressive loss of GP Ib with appearance of the major fragment, glycocalicin, in the supernatant solution. No GP V was detected in the supernatant solution, and protease-pretreated platelets had the same capacity as control platelets to release fragment 1 of GP V in response to thrombin. The Serratia protease- pretreated platelets did show the lag before thrombin-induced dense granule secretion, characteristic of platelets modified by pretreatment with other nonstimulating proteases. Treatment with Serratia protease gives the only demonstrated selective loss of GP Ib without apparent effect on GP V. It suggests that GP V (1) does not depend on GP Ib for its association with platelets and (2) is not the substrate for protease modification of platelet function.

Description: The blood platelet is the only human cell known to have a circumferential band of microtubules. However, the mechanisms involved in assembly of the multi-looped coil, its interaction with the cell membrane to support discoid shape, and constriction into tight rings around centrally concentrated organelles in activated platelets are unknown. Separation of the microtubule rings from intact platelets would permit new approaches to solution of these questions. The present study has used simultaneous detergent extraction and fixation to isolate intact microtubule coils in significant numbers from suspended platelets for the first time. Isolated coils closely resembled the circumferential band observed in thin sections of plastic embedded platelets and in platelets prepared by the negative-stain whole-mount method. Enough microtubule coils could be recovered from suspensions of concentrated platelets to permit counting and quantitation on microscope grids. Results of this study will permit new approaches to clarification of the structural physiology of platelet microtubule coils.

Description: Twenty-two patients with hairy cell leukemia were treated with biosynthetic (recombinant) alpha-2-interferon in an open-label, single- arm efficacy study. Patients received 2 X 10(6) U/m2 recombinant alpha- 2-interferon three times weekly. Therapy was well tolerated subjectively with minimal short-term hematologic toxicity. Two patients had bacterial infections during the period of study, and one patient experienced a short-lived readily reversible rejection of a corneal transplant. Statistical comparison of the mean hematologic indices at study entry and after three to six months of therapy with recombinant alpha-2-interferon indicates a significant improvement in hemoglobin, granulocyte, and platelet counts. Bone marrow biopsies in six of 14 patients after six months of therapy showed a greater than 50% decrease in the infiltration of leukemia cells. We conclude that recombinant alpha-2-interferon is highly effective therapy for hairy cell leukemia.

Description: Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.

Description: Pregnancy in female carriers of abnormal hemoglobins with great avidity for oxygen provides a unique opportunity to assess the importance of the usual difference in oxygen affinity between fetal and maternal blood. Outcome of pregnancy was recorded for carriers of hemoglobins Bethesda, Osler, and Yakima, whose p50s (9.5, 9.1, and 12 mm Hg at pH 7.4) were far lower than that of a normal fetus (23 mm Hg at pH 7.3). Neither spontaneous abortions nor intrauterine growth retardation could be attributed to the presence of high oxygen affinity in the mothers. In vitro simulations suggested that neither maternal or fetal polycythemia alone was sufficient to adjust for perturbation of the normal situation, and increased uterine and/or fetal blood flow probably provided additional compensation.

Description: Neutrophil function was studied in a patient with polymorphonuclear leukocyte (PMN) glycoprotein-180 deficiency and in her parents. PMNs of the patient had abnormal chemotaxis, phagocytosis, adherence, surface charge, and membrane-associated events of activation. Selective defects to C3b, immunoglobulin G (IgG), phorbol myristate acetate (PMA) and N- formyl-methionyl-leucyl-phenylalanine (FMLP) are described, although C3b receptor density was normal. The parents were found to have abnormal adherence to nylon-wool fibers, abnormal transmembrane potential depolarization with PMA, and reduced amounts of glycoprotein- 180 in their PMNs. These studies provide further evidence that the oxidative burst has several different pathways for activation. They demonstrate that the absence of a single PMN surface glycoprotein is associated with a broad spectrum of PMN functional abnormalities. Finally, the observations made in the parents support an autosomal recessive mode of inheritance.

Description: Ecto-5′nucleotidase (5′NT) activity of peripheral blood (PB) lymphocytes was determined in 31 patients with serum monoclonal gammopathies (MG). Twenty-one patients had a diagnosis of multiple myeloma (MM), and ten patients had monoclonal gammopathy of undetermined significance (MGUS). The proliferative activity of the bone marrow plasma cells (LI%) was investigated in 28 of these MG patients by means of tritiated thymidine uptake evaluated by simultaneous autoradiography and cytoplasmic immunofluorescence. 5′NT activity was significantly lower in MG patients as compared with normal controls. MM patients had lower 5′NT activity than MGUS patients, but the difference was not significant. By contrast, MM had significantly higher LI% than MGUS patients. There was a linear regression of 5′NT on LI% which was statistically significant: the higher the LI%, the lower the 5′NT. Because the LI% is an accurate prognostic and monitoring factor in MG, this correlation indicates that 5′NT may be of assistance in predicting the clinical progress of MG patients. In seven MGs, the PB T and B lymphocytes were studied separately. The T cell subpopulation was 5′NT deficient compared to the normal controls, shown as a significant linear regression of T cell 5′NT on the LI%. This suggests that in MG there may be an alteration of nonneoplastic T lymphocytes correlated with tumor growth. The OKT8+ lymphocytes were mainly responsible for the 5′NT deficiency of unseparated T lymphocytes.

Description: Peripheral blood leukemia cells from patients with acute monoblastic leukemia (AMoL) were tested for killer cell activity against target cells that detected natural killer cell-mediated or monocyte-mediated spontaneous cytotoxicity. The fibrosarcoma cell line Wehi 164, pretreated with actinomycin D to induce susceptibility to lysis, specifically detects the activity of unstimulated human monocytes. In four of six cases of AMoL, high killer cell activity could be measured against this target. In three of these four cases, the killer cell activity could be assigned exclusively to the leukemic clone, based on the high leukocyte counts and the resultant dilution of normal cells, as evidenced by marker and by functional analysis. While leukemic cells with killer cell activity against Wehi 164 contained 34% to 45% cells that were positive for binding of the 63D3 monoclonal antibody, the two leukemic samples without killer cell activity contained only 1% and 12% 63D3-positive cells. Cell sorting of 63D3-positive and -negative cells from two leukemias with killer cell activity demonstrated that the killer cell activity was restricted to the 63D3-positive fraction of AMoL cells. These data demonstrate that monoblastic leukemia cells can be potent killer cells and that killing activity is linked to the 63D3- defined cell surface molecule.

Description: The Belgrade laboratory rat (b/b rat) has hereditary, hypochromic, microcytic anemia with a variety of red cell abnormalities. Although this anemic syndrome has been recently ascribed to the defective delivery of iron to the developing red cell, the basic hematopoietic defect is still unknown. In this article we present evidence that the b/b rat has an additional hematologic defect. We have found that the megakaryocyte number in the marrow of the b/b rat is decreased to one half that of the normal rat, but the maturation rate of recognizable megakaryocytes is accelerated and the size is increased. The platelet count is moderately reduced. These findings indicate that megakaryocytopoiesis in the anemic b/b rat is abnormal and suggest that the genetic defect may involve the progenitors of the megakaryocyte cell lineage. Alternatively, the megakaryocytic abnormalities may be secondary to the severe anemia.

Description: DNA from mononuclear blood and tumor cells from 33 patients undergoing bone marrow transplantation for leukemia was examined for the presence of Epstein-Barr virus (EBV) genomes by blot hybridization. Four groups of patients were studied soon after engraftment, during long-term remission, after relapse of the original leukemia, and after development of secondary B cell neoplasms. Only the cells of patients with secondary neoplasms demonstrated EBV genomes, where all five adequately studied samples were positive. Samples from all other patient categories were negative for EBV genomes. We conclude that EBV genomes do not frequently persist in normal engrafted lymphocytes or in mononuclear cells of patients suffering recurrent leukemia. These results are consistent with EBV playing a role in the genesis of secondary B cell neoplasms following bone marrow transplantation.

Description: Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.

Description: Hyperhaemolysis is a rare, poorly understood complication of red cell transfusion. We report the outcomes of SCT in 2 boys of Middle Eastern origin who developed life-threatening hyperhaemolysis each following their third transfusion for β-TM at the ages of 2.5 and 4 years. The diagnosis of hyperhaemolysis was based on laboratory evidence for haemolysis, post-transfusion haemoglobin (Hb) levels lower than pre-transfusion, positive Coomb’s tests and no allo-antibody able to be identified. Haemolysis was intravascular, extravascular and severe. Precipitous drops in Hb made transfusion unavoidable. The first boy responded to prednisolone, intravenous immunoglobulin (IVIG) and splenectomy. Haemosidderosis and fibrosis were present on liver biopsy, he was therefore regarded as a Class 3 thalassaemia patient and received hydroxyurea, azathioprine, erythropoietin, desferrioxamine, busulfan, cyclophosphamide and fludarabine conditioning prior to an HLA identical sibling SCT. He is alive and well 7 years post BMT with 30% stable donor chimerism and a normal Hb. The second child’s hyperhaemolysis failed to respond to prednisolone, IVIG, rituximab and splenectomy. Provision of the large number of suitably matched red cell units required was problematic. After receiving hydroxyurea, azathioprine, desferrioxamine, busulfan, cyclophosphamide, fludarabine, thiotepa and antithymocyte globulin (ATG) preparation for a CD3/CD19 depleted maternal haplotype peripheral blood SCT, the haemolysis finally stopped. Post-transplant severe veno-occlusive disease and multi-organ failure (MOF) required dialysis and ventilation. The maternal graft was rejected so 28 days after the first transplant he received a 4/6 mismatched unrelated cord blood transplant (UCBT) following further fludarabine and ATG, which fully engrafted. He recovered from MOF and was discharged from hospital 47 days after the UCBT, transfusion independent. On day +86 he contracted Respiratory Syncytial Virus chest infection with acute intravascular haemolysis necessitating transfusions. Fulminant liver failure developed, presumably due to iron toxicity, and death occurred on day +102, having received 112 transfusions in the 12 months since presentation. In conclusion, avoiding red cell transfusion is not always possible in hyperhaemolysis, especially in β-TM. Patients may quickly become classified as Class 3 in terms of predicting BMT outcome. Immune modulation therapy and SCT was effective in 1 case but only temporarily stopped haemolysis in the other, despite full engraftment ultimately being achieved with a mismatched UCBT. SCT should be considered early in cases of hyperhaemolysis in β-TM because it can potentially cure both and result in transfusion independence. Disclosures Off Label Use: Intravenous immunoglobulin and rituximab for treatment of haemolytic anaemia; hydroxyurea, azathioprine, fludarabine, erythropoeitin, busulphan, cyclophosphamide, thiotepa and antithymocyte globulin for use in stem cell transplantation in children with thalassaemia.

Description: Background: Post-remission treatment for AML is very aggressive and many times a SCT is needed. Comparisons between Allo-SCT and Auto-SCT ve always shown more Transplant Related Mortality (TRM) but less Cumulative Incidence of Relapse (CIR) in the first group. Our study describes, not only the long-term survival outcomes, but also the quality of life in long survivors. Methods: Retrospective study of 274 patients diagnosed with non-promyelocytic AML who underwent SCT between 1982 and 2011 in our center. Characteristics in the 162 Allo-SCT and the 112 Auto-SCT groups of patients were respectively: median age of 38 and 45 years old, secondary AML in 20% and 10%, refractory to Induction AML in 16% and 3%, pre-SCT status different from Complete Remission (CR) in 13% and 3% and year of SCT before 2005 in 53% and 86%. No significant differences between both groups were found in other risk factors as hyperleucocytosis at diagnosis or adverse cytogenetics. Results: With a median follow-up of 55 months [2-316], Overall Survival (OS) until 1997 in Allo-SCT and Auto-SCT were respectively, 40% and 61% at 1 year and 28% and 45% at 5 years (figure 1), but from 1997, 66% and 70% at 1 year and 47% and 48% at 5 years (figure 2). Disease Free Survival (DFS) until 1997 in Allo-SCT and Auto-SCT were respectively, 50% and 65% at 1 year and 38% and 46% at 5 years, but from 1997, 67% and 63% at 1 year and 52% and 47% at 5 years. In the last 15 years, no differences were found between both groups in OS nor DFS. CIR in Allo-SCT and Auto-SCT were respectively, 18% and 32% at 1 year and 24% and 50% at 5 years, without dependence on year of SCT. No relapse was observed later in any group. TRM until 1997 in Allo-SCT and Auto-SCT were respectively, 30% and 7% at 1 year and 35% and 9% at 5 years, but from 1997, 16% and 2% at 1 year and 25% and 4% at 5 years. Multivariable analysis showed that the only risk factor with a negative impact on OS was not having achieved CR at the time of SCT. Other variables as older age, hyperleucocytosis at diagnosis, adverse cytogenetics, secondary AML or sooner year of SCT lost their univariable analysis significance. Allo-SCT: From the 162 patients, 72(44%) are alive by this moment, 43(60%) with ECOG 0, 21(29%) with ECOG 1 and the other 8(11%) with ECOG 2, basically because of graft versus host disease (GVHD in 39 patients, 21 steroid-dependent and 3 refractory to any treatment). All of them have been in CR during the last 2 years of follow-up. In contrast, 90(56%) patients have died: 52(58%) because of SCT complications (20 infections, 16 GVHD, 8 toxicity and 8 mixed causes), 33(37%) because of disease and 5(5%) because of other causes. With a median follow-up of 43 months [2-316], there have been 4 secondary neoplasm, all of them solid ones, which appeared with a median of 242 months [179-311] from SCT. None of them had previously received radiotherapy. Auto-SCT: From the 112 patients, 43(38%) are alive by this moment, 32(74%) with ECOG 0 and the other 11(26%) with ECOG 1. All of them have been in CR during the last 2 years of follow-up. In contrast, 69(62%) patients have died: 45(65%) because of disease, 14(20%) because of SCT complications and 10(15%) because of other causes. With a median follow-up of 93 months [5-230], there have been 6 secondary neoplasm, 5 of them hematologic ones, which appeared with a median of 90 months [76-115] from SCT. None of them had received radiotherapy, but previously treated hematopoietic stem cells. Only one is alive at the time of last follow-up. Conclusions: In one hand, despite the high incidence of relapse in Auto-SCT in any period, OS is lower in Allo-SCT during the first years [1982-1996], although it has a tendency towards OS in Auto-SCT from 1997 because of the decrease in TRM, which is more significative in Allo-SCT. In the other hand, DFS is slightly higher in Allo-SCT during the last years [1997-2011], although the quality of life in long survivors is worse, basically because of GVHD. In summary, we have not really found differences between Allo-SCT and Auto-SCT in terms of OS and DFS in our series, so both procedures are efficient to treat AML (near 50% of the patients in both groups are alive at 5 years from SCT in recent years). The decrease in TRM until 4% at 5 years in Auto-SCT makes it a good choice, particularly for older patients without risk factors. However, the development of secondary hematologic neoplasms is a relevant fact, with an incidence of 11% and a high late mortality. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: By reducing treatment intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become feasible for elderly patients. Different reduced-intensity conditioning (RIC) regimens are available, but there is little consensus about the optimal preparative regimen to use, in particular with regard to the outcomes counterbalancing the aim of feasibility and tolerability with higher rates of relapse. Here, we retrospectively evaluate the outcome of sequential therapy employing RIC with fludarabine 30 mg/m2, cytarabine 2g/m2 and amsacrine 100 mg/m2 for 4 days (FLAMSA; Schmid C et al. JCO 2005) followed by busulfan 10 x 0.8 mg/kg (FLAMSA-Bu) compared to RIC utilizing fludarabine 5 x 30 mg/m2, carmustine (BCNU) 2 x 150 mg/m2 and melphalan 110 mg/m2 (FBM; Marks R et al. Blood 2008) in elderly patients treated consecutively at our institution between July 2005 and October 2012. We analyzed the course of 114 patients (pts) with acute myeloid leukemia (AML; n=99) or myelodysplasia (MDS; n=15) aged ≥ 59 years with 59 pts aged ≥ 66 years who were treated with either FLAMSA-Bu (n=66; n=24 ≥ 66 years) or FBM (n=48; n=35 ≥ 66 years). All patients received sero-therapy with anti-thymocyteglobuline (ATG). Median patient age was 66 years for the entire cohort (68 years FBM; 64 years FLAMSA-Bu). 36 patients (75%) of the FBM and 42 patients (63 %) of the FLAMSA-Bu group suffered from high risk disease defined as relapsed or refractory AML or refractory anemia with excess of blasts in transformation (RAEB-T). The hematopoietic cell transplantation comorbidity index (HCT-CI) was higher for the patients of the FBM group than for the FLAMSA-Bu group with 26 (54 %) versus (vs) 24 patients (36 %) scoring ≥ 2 (p 0.085). Graft source after conditioning with FBM/FLAMSA-Bu was bone marrow (1/2), G-CSF mobilized peripheral blood stem cells (40/62) and double-umbilical cord-blood (7/1). In 23 pts (20 %) HLA-matched related and in 91 pts (80 %) HLA-matched unrelated donor transplantation was performed. Engraftment failure was observed in 1 patient after FLAMSA-Bu, while engraftment was achieved in all evaluable patients of the FBM group in a median of 23 days vs 18 days after FLAMSA-Bu (p 0.003), while 7 pts with double-umbilical cord-blood transplantation where included in the FBM group vs 1 pt in the FLAMSA-Bu group. Non-hematological treatment-related acute toxicity ≥ CTC III (gastrointestinal, hepatic, cardiovascular, renal, centralnervous system) occurred in 12/48 pts (25 %) after FBM and in 18/66 pts (27 %) after FLAMSA-Bu. Incidence of severe acute (III-IV) and chronic GvHD was 22.9 %/16.6 % for FBM vs 18.2 %/19.7 % for FLAMSA-Bu, respectively. After conditioning with FBM 2/48 pts vs 9/66 pts after FLAMSA-Bu were diagnosed with a secondary malignancy (p 0.08). Non-relapse mortality (NRM) after 12 months was 26.8 % for FBM versus 25.2 % for the FLAMSA-Bu group. Incidence of relapse after FBM vs FLAMSA-Bu conditioning was 22.9 % vs 15.2 % after 1 year and 31.3 % vs 16.7 % after 2 years. Occurrence of relapse was significantly related to an incomplete or mixed chimerism (donor cells ≤ 95 % in peripheral blood and/or bone marrow) at day +30 (p 0.001). After a median follow up of 31.4 months (range 4.4-97.5) estimated overall survival (OS) and relapse-free survival (RFS) after 2 years was 55.4 % and 51.4 % for the FBM vs 58 % and 56.7 % for the FLAMSA-Bu group, respectively. Analyzing different subgroups, FBM conditioning might be favorable for pts aged ≥ 66 years when suffering from high risk AML (n=26): Within this group 1-year OS after FBM vs FLAMSA-Bu was 71.4 % vs 66.7 % (p 0.58) and 1-year RFS was 71.4 % vs 58.3 % (p 0.59), respectively. Notably, for pts at highest risk (aged ≥ 66 years and suffering from secondary or therapy-related AML; n=24) the benefit of FBM conditioning becomes more pronounced: 1-year OS after FBM vs FLAMSA-Bu was 62.5 % vs 37.5 % (p 0.26) and 1-year RFS 54.2 % vs 37.5 % (p 0.17). Both conditioning regimens are feasible, and provide similar rates of acute toxicity, NRM and GvHD. There might be evidence for a benefit of conditioning with FBM for the subgroup of “the oldest patients at highest risk”. Taking into account that there is an increasing group of ‘medically fit’ elderly patients in the field of allogeneic transplantation, prospective clinical trials are needed to investigate different conditioning regimens considering their special requirements. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Cerebral sinus venous thrombosis (CSVT) is potentially life-threatening thrombosis with mortality around 10%. Venous thromboembolism (VTE) is a common complication in children with cancer. These children have several thrombotic risk factors such as the malignancy itself, severe infections, prothrombotic medication and immobilization. The treatment of acute lymphoblastic leukemia (ALL) includes steroids and asparaginase (ASP), raising the VTE risk. In children with ALL the central nervous system (CNS) is a common localization for VTE. However, retrospective studies on small numbers of patients, larger studies and population-based data in children are scarce. The five Nordic countries, Estonia and Lithuania have a common treatment protocol for children with ALL between 1 and 18 years of age with prospective registration of toxicities, including CSVT offering a unique opportunity to study CSVT in this patient group. This is to our knowledge the largest report of children with ALL and CSVT describing the incidence, symptoms, treatment and the effect of CSVT on ALL treatment. Methods We assessed the symptoms, treatment, clinical risk factors and outcome of all children between ages 1 and 17 years at diagnosis of B-cell precursor or T-cell ALL between June 2008 and July 2013 and with CSVT. Data were collected from the patients’ medical records and the NOPHO leukemia registry. Results In total, 20 (1.9%) of the 1038 children with ALL treated according to the NOPHO ALL 2008 protocol developed CSVT. The cumulative incidence of CSVT was 2.0%. All the thromboses occurred within the first 5 months of treatment. The most common symptoms at the diagnosis of CSVT were headache, convulsions, weakness/fatigue and cerebral nerve palsy/hemiparesis/hemiplegia. The most frequent localizations for CSVT were sinus sagittalis (n=16) and sinus transversus (n=10). However, in most cases multiple cerebral veins were involved ( 70%). Median D-dimer at time of the CSVT diagnosis was 0.85 mg/L (range 0.19-4.7 mg/L) with 5 patients having normal D-dimer. We could not identify any clinical risk factors for CSVTs. CSVT was associated with steroids (treatment within 2 weeks before the diagnosis of CSVT) in 16/20 and with Pegylated asparaginase in 16/20. Fifteen patients were later screened for the inherited thrombophilic factors; one child had heterozygous prothrombin G20110A mutation and another heterozygous factor V (R506Q) Leiden mutation. Most patients (19/20) were treated with anticoagulants: mostly low molecular weight heparin (LMWH). The median treatment with LMWH was 26 weeks (range 14-119 weeks). No bleeding complications were observed in connection with LMWH. Two deaths were directly related to CSVT. Asparaginase was omitted from the treatment in 7 and delayed or reduced in 5 of the cases raising the risk for subsequent suboptimal leukaemia treatment. Of the surviving 18 patients, follow-up imaging revealed complete recanalization in 7 and partial recanalization in 7 cases. No imaging was available for the remaining 4 patients. Conclusions The incidence of CSVT in children with ALL was approximately 2%. No statistically significant clinical predictors for CSVT were identified. The mortality related to CSVT was 10%. Anticoagulation with LMWH was the treatment of choice in most cased and was well tolerated. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Busulfan (Bu) is used in the conditioning regimen for hematopoietic stem cell transplantation (HSCT). Therapeutic drug monitoring (TDM) of Bu with subsequents adjustments doses based on a “target” therapeutic concentration may reduce toxicity after HSCT. Objectives: To evaluatethe impact of TDM of Bu and clinical outcomes in patients with acute leukemia that underwent to allogeneic matched related donor (MRD) and allogeneic matched unrelated donor (MUD) HSCT. Patients and methods: From January 2009 to January 2014, we prospectively analyzed 42 patients with diagnosis of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) who underwent TDM of Bu (IV or oral) before transplantation (test dose) and TDM on 1st day of conditioning regimen. Samples were collected at 0, 30’, 60’ and subsequently every hour until 6 hours after administration of busulfan. The plasma was extracted by HPLC (High Performance Liquid Chromatography). All the patients that were submitted to TDM had a test dose 15 days to 48 hours before transplantion. The dose of Bu was adjusted during the first day of the conditioning regimen based on test dose. At the same time we analyzed 21 patients in the retrospective group who did not underwent TDM (from 2004 to 2010). Results: In the retrospective group (n=21), all of them underwent to MRD transplantation. Six (46.2%) were in first complete remission (CR1), 18(85.7%) patients received Bu and cyclophosphamide (BuCy) and the mean of age was 38 years (18-55 Yo). The median of CD34+ cells was 5.4 x 106/kg. The second group consisted by patients that received oral Bu (n= 21): 7 (33.3%) underwent to MUD transplantation, 14 (66.6%) to MRD transplantation, 8 (44.4%) patients were second complete remission (CR2), 4 (22.2%) had active disease status with a mean age of 32.7 years (14-58 Yo). Fifteen (71.4%) received BuCy and 16 (76.2%) received cells from peripheral blood. The median of CD34+ cells was 5.8 x106/kg. The median area under the curve (AUC) in 24 hours was 4950 μMol.min (3196.6- 8212 μMol.min). The third group was IV Bu (n= 21): 7 (33.3%) patients underwent MRD and 14 (66.6%) MUD transplantation, 7 (33.3%) patients were CR1, 7 (33.3%) had active disease or prior HSCT with a mean of age 52.7 years (20-74 Yo). The majority of patients received fludarabine and Bu (n=18; 85.7%) as conditioning and bone marrow was the main source. Immunosuppression was based on FK-506 and methotrexate (90.5% of patients). The median of AUC in 24 hours was 5690 μMol.min (3539.6- 8881.8 μMol.min). The cumulative incidence (CI) of sinusoidal obstructive syndrome (SOS) in the retrospective group and IV Bu were 9.5% for both, while in the group oral Bu it was at 19% (p = 0.566). The median AUC of Bu received during conditioning for those who died of SOS was lower in oral Bu than IV Bu (4872 uMol.min vs 5732 uMol.min respectively). The CI of acute graft-versus-host disease (aGVHD) at D+100 was 38.1% in the retrospective group, 40.6% in oral Bu and 42.9% in IV Bu. Chronic GVHD was 13.6% in oral Bu, 34% in IV Bu and 42.9% in the retrospective group (p = 0.142). The CI of relapse at D+100 was 19% in IV Bu, 4.8% in the retrospective group and oral Bu did not have this event. The IC of death at D+100 was 34.9% in group oral Bu, 9.5% in IV Bu and 14.3% in the retrospective (p = 0.102). The CI of relapse at 1.5 years was 35.8% in the IV Bu, 34.8% in oral Bu and 14.3% in the retrospective group. The CI of death at 1.5 years was 9.5% in group IV Bu, 53.5% in oral Bu and 34.3% in the retrospective (p = 0.015). Among patients who died until D+100, the median of AUC was 5732 μMol.min (5578.5-6818.5 μMol.min) during the conditioning for IV Bu and 4872 μMol.min (3448-8212 μMol.min) for oral Bu. The range between the AUC was large and there was no correlation with patients who died. Conclusion: In acute leukemia SOS had an impact on mortality at D+100 after HSCT (p

Description: Introduction: The application of nanoparticles in dendritic cell (DC)-based cancer immunotherapy represents a promising strategy to enhance antigen-specific T cell immune responses. This study was to investigate the effect of bPEI-SPIONs on antigen-specific T cell responses elicited by DCs loaded with apoptotic U266 myeloma tumor antigen in the presence or absence of bPEI-SPIONs. Materials and Methods: The myeloma tumor antigens were prepared as following: 1) apoptotic U266 cells by UBV-irradiation and overnight incubation (16 h irradiated cells) and 2) apoptotic U266 cells by UVB-irradiation and 2 h incubation in the absence (2 h irradiated cells) or 3) presence of bPEI-SPIONs (bPEI-SPION 2 h irradiated cells). Monocyte-derived immature DCs were activated with TNF-α, loaded with several kinds of myeloma tumor antigens 2 h after TNF-α stimulation, and cultured for 2 days. Results: Optimal concentration of bPEI-SPIONs was 16 µg/mL to uptake into tumor cells and the bPEI-SPIONs render U266 cells sensitive to UVB-irradiation through reactive oxygen species (ROS) induction pathway hence accelerated the apoptotic cell death. 2 h irradiated cells and bPEI-SPION 2 h irradiated cells released immunogenic proteins, including HSP70, HSP90, and HMGB1. bPEI-SPION 2 h irradiated cells were easily up-taken by DCs without alteration of surface phenotypes and migration capacities. DCs loaded with bPEI-SPION 2 h irradiated cells showed highest IL-12p70 production level and Th1 polarization compared to other DCs. Conclusion: These results suggest that bPEI-SPIONs are a promising tool to improve immunogenicity of myeloma tumor cells and to enhance Th1 polarization of DCs loaded with these tumor cells. Disclosures No relevant conflicts of interest to declare.

Description: ADAMTS-13 is a protease, member of the ADAMTS family (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeats-13), which cleaves the cell bound large ultrapolymeric von Willebrand factor (vWF) strings. Circulating ADAMTS-13 is primarily synthesized and released from hepatic stellate and endothelial cells. Acquired functional deficiency of ADAMTS-13, usually due to inhibitory IgG autoantibodies, results in excessive platelet aggregation and disseminated vWF/platelet-rich thrombus formation. A possible association of low levels of ADAMTS-13 Ag with arterial thrombosis and endothelial dysfunction has also been reported. Hivert and colleagues (Blood 2012;120:3214-21) and our group have shown that increased levels of vWF, the only known substrate of ADAMTS-13, are associated with poorer prognosis in patients with symptomatic Waldenstrom’s Macroglobulinemia (WM). Thus, our aim was to investigate the possible association of ADAMTS-13 antigen (Ag) levels with features of WM and possible biologic implications of the ADAMTS-13 / vWF interaction in WM patients’ prognosis. Our study included 42 patients with symptomatic WM who were treated and followed in the Department of Clinical Therapeutics of the University of Athens (Greece), from 1999 to 2012, 22 patients with asymptomatic WM and 19 healthy controls of matched gender and age. For the purpose of this study we used stored serum, which had been collected before initiation of any therapy. ADAMTS-13 antigen levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN, USA), which has a detection limit for ADAMTS-13 13 (ng/mL) with intra- and inter-Assay Precisions of

Description: Background: Extramedullary disease (EMD), strictly defined as an infiltrate of clonal plasma cells at an anatomic site distant from the bone marrow or adjacent soft tissue in a patient with underlying multiple myeloma, is an uncommon manifestation of multiple myeloma. Comparatively little is known about this disease entity, with no large case series published in the last decade. Patients and Methods: 663 consecutive patients with multiple myeloma who underwent autologous or allogeneic stem cell transplantation at a single, large, academic medical center in the United States from January 2005 to December 2011 were assessed for the presence or absence of EMD, as well as baseline demographic and biochemical characteristics, treatment regimens, and response to therapy. Results: A cohort of 55 patients with biopsy-proven EMD was identified, comprising 8.3% of the total study population. Among the patients with EMD, 13 (23.6%) were found to have EMD at the time of initial presentation, while the remainder developed EMD during the course of their illness. Patients with EMD received a median of 5 different treatment regimens during the course of their illness, most commonly with combinations of dexamethasone, thalidomide, lenalidomide, and bortezomib, as well as autologous hemotopoietic stem cell transplantation. Patients had received a median of 3 lines of therapy prior to experiencing an extramedullary relapse. Patients with EMD had markedly elevated maximum serum LDH levels (median 613.5 units/L) and low minimum hemoglobin levels (median 7.8 g/dL). Common cytogenetic abnormalities included deletion 13q, deletion 11q, t(11;14), and deletion 17p. Available immunohistochemical data suggest that EMD specimens had frequent expression of CXCR4, CD44, and CD56. The median overall survival data of these patients was 3.2 years (range, 0.9-9.5) and the median time from diagnosis of EMD to death was 0.5 years (range, 0.002-3.2). Conclusions: This report describes a large series of multiple myeloma patients with EMD who were treated in the era of stem cell transplant at a single academic medical center. Further studies to examine the molecular characteristics of extramedullary multiple myeloma are necessary to better define this entity and characterize therapeutic options that can prolong survival in this otherwise very vulnerable population. Disclosures Ghobrial: Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx Pharmaceuticals: Research Funding. Schlossman:Millennium: Consultancy. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Research Funding; Johnson & Johnson: Research Funding; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

Description: Autoimmune hemolytic anemia (AIHA) occurs in CLL at some time during the course of the disease in up to 7-10% of patients. The acute onset of AIHA may occur unrelated to therapy but has also been linked to treatment with chemo­therapeutic agents including chlor­ambucil, benda­mustine and particu­larly purine nucleosides such as fludarabine. Although the mechanism is still not well understood, chemo­therapy-induced changes in regulatory T-cells have been proposed as a trigger for autoimmunity and clinical hemolysis. In contrast to these cytotoxic therapies, ibrutinib, an inhibitor of Bruton’s tyrosine kinase recently approved for the treatment of CLL, appears to have a different mechanism of action and thus far has not been associated with AIHA in published reports. However, we report here a patient with CLL and a history of prior AIHA, who developed a recurrence of acute hemolysis after the initiation of ibrutinib. The patient is a 67-year-old man diagnosed with CLL in 2002 and treated for progressive disease with a single cycle of bendamustine in 2009. Although the lymphocytosis resolved rapidly, the hemoglobin also decreased from 14 g/dL to 5.2 g/dL by 3 weeks after the start of therapy. Due to the onset of Coombs-positive AIHA, chemotherapy was dis­con­tinued. Hemolysis resolved with prednisone therapy and did not recur after a slow taper. The CLL then remained asymptomatic until 2012 when night sweats developed at a white blood cell (wbc) count of 95,000/µL. Benda­mustine was re-started and despite a negative Coombs test prior to treatment, Coombs-positive AIHA developed again with the hemoglobin falling from normal to 7.0 g/dL within 4 weeks. After stabilization with transfusions and steroids, an additional cycle of bendamustine plus rituximab was administered without further complications and the patient’s symptoms and lympho­cytosis resolved. After the discontinuation of prednisone, hemolysis did not recur clinically although the Coombs test remained 1+ positive through early 2014. By May 2014 the wbc count had increased to 144,000/µL with the onset of a mild anemia (Hgb 12.3 g/dL) and symptomatic night sweats. Due to the history of repeated chem­otherapy-associated AIHA, alter­native therapy with ibrutinib, which had not been associated with AIHA, was instituted at 420 mg daily. However, within 2 weeks the hemoglobin decreased to 7.0 g/dL while the wbc count increased to 300,000/µL. A reticulocyte count was 16%, total bilirubin 3.2 mg/dL, haptoglobin

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

Description: Background Ixazomib is the first oral proteasome inhibitor to be investigated clinically for the treatment of MM. Phase 1 studies have shown single-agent activity and manageable toxicities in RRMM (Kumar et al. Blood 2014) and phase 1/2 studies have suggested the feasibility and activity of weekly oral ixazomib plus Rd in previously untreated MM (Kumar et al. ASH 2012; Richardson et al. ASH 2013). These findings have led to ongoing phase 3 trials of weekly ixazomib 4 mg + Rd in RRMM and previously untreated MM. However, the early-phase studies were conducted in Western pts. This phase 1, open-label multicenter study aimed to determine the safety, tolerability, and pharmacokinetics (PK) of weekly ixazomib alone or with Rd in Japanese pts with RRMM (Japic Clinical Trials Information no. 121822). Methods Primary objectives were to evaluate the safety and tolerability, including dose-limiting toxicities (DLTs) and adverse events (AEs), and the PK of ixazomib alone or with Rd. A secondary objective was evaluation of antitumor activity. Japanese pts aged ≥20 years with RRMM who had received at least 2 prior regimens, which must have included bortezomib, thalidomide or lenalidomide, and corticosteroids, were eligible. All had measurable disease and ECOG performance status of 0–2. Pts with grade ≥2 peripheral neuropathy or grade ≥2 diarrhea at study entry were excluded. Pts received ixazomib 4 mg on days 1, 8, and 15 of 28-day cycles, alone or with Rd (lenalidomide 25 mg on days 1–21, dexamethasone 40 mg on days 1, 8, 15, and 22), per the regimen used in the ongoing phase 3 trials. AEs were graded per NCI-CTCAE v4.03. Blood samples for PK analysis were taken at multiple time points prior to and after dosing on days 1 and 15 of cycle 1. Responses were assessed per IMWG uniform response criteria. Results Fourteen pts were enrolled; 8 (57%) were male, median age was 62.5 yrs (range 53–71), 4 pts were aged ≥65 yrs, median number of prior therapies was 7. Seven pts received single-agent ixazomib and 7 received ixazomib + Rd. One pt in each cohort was excluded from the DLT-evaluable population. Two patients experienced DLTs in cycle 1: 1 pt receiving single-agent ixazomib had grade 4 thrombocytopenia and grade 3 diarrhea, hypertension, hypokalemia, hyponatremia, and nausea; 1 pt in the ixazomib + Rd cohort had grade 4 thrombocytopenia and neutropenia. All events were considered treatment-related. At data cut-off (Jan 6 2014), 6 pts remained on treatment and 8 had discontinued due to: progressive disease (PD; n=3), AEs (n=3), symptomatic deterioration, and protocol violation (each n=1). At data cut-off, pts (n=14) had received a median of 6 cycles of ixazomib (range 1–21); the 7 pts in the ixazomib + Rd cohort had received a median of 4 cycles (range 1–12) of ixazomib + Rd. Thirteen (93%) pts experienced treatment-related AEs; the most common were neutropenia (71%), thrombocytopenia (71%), leukopenia (64%), lymphopenia (57%), and diarrhea (50%). There were no cases of peripheral neuropathy. Nine (64%) pts had grade ≥3 AEs; the most common were lymphopenia (50%), neutropenia (43%), and thrombocytopenia (36%). Two (14%) pts (single-agent cohort) had serious AEs (grade 2 bronchitis in 1 pt, and grade 4 thrombocytopenia and grade 3 hypokalemia in 1 pt). Three pts discontinued due to AEs; 1 due to diarrhea in the single-agent cohort, and 1 due to neutropenia and 1 due to thrombocytopenia in the ixazomib + Rd cohort. There were no deaths. PK data showed ixazomib was rapidly absorbed with a Tmax at 1.08–1.83 hrs. Terminal half-life (geometric mean) was 5.7 days for single-agent ixazomib and 5.2 days for ixazomib + Rd. There were no substantial differences in the ixazomib PK profile between the two cohorts. Thirteen pts were response-evaluable. One pt (ixazomib + Rd cohort) had a partial response; at data cut-off, this pt remained in response with a 100% M-protein reduction (unconfirmed VGPR) and duration of response of ~10.8 months. Seven pts had stable disease (including 3 with M-protein reductions of 25–50%), 2 had PD, and 3 were not assessable. Conclusions These data suggest that ixazomib 4 mg alone or with Rd is feasible and tolerable in Japanese pts with RRMM. The AEs were manageable, reflecting the AE profile seen in Western populations, supporting the use of this dose and schedule in Japanese pts. Disclosures Handa: Celgene: Research Funding; Yakult: Research Funding; Kirin: Research Funding; Chugai: Research Funding. Off Label Use: Investigational agent ixazomib for the treatment of Japanese patients with relapsed and/or refractory multiple myeloma.. Matsushima:Takeda Pharmaceutical Company Limited : Employment.

Description: Myeloma (MM) cells grow and expand almost exclusively in the bone marrow while creating a cellular microenvironment suitable for MM cell growth and survival (MM niche). In pursuing the molecular mechanisms whereby MM cells gain drug resistance in the “MM niche”, we have found that the serine/threonine kinase Pim-2 is constitutively over-expressed in MM cells, and further up-regulated by co-cultures with bone marrow stromal cells (BMSCs) as well as osteoclasts (Leukemia, 2011), and that Pim-2 is an important therapeutic target in MM for the progression of MM tumor and bone disease (Leukemia, 2014). The ABC transporter BCRP is preferentially expressed in drug resistant MM cells as well as in MM progenitors or stem cells. BCRP has been demonstrated to be phosphorylated by Pim kinases to trigger its dimerization and function; Pim inhibition may suppress the BCRP function to sensitize BCRP-expressing MM cells to chemotherapeutic agents. In the present study we therefore explored whether Pim inhibition is able to target and impair BCRP-expressing drug-resistant MM cells and MM progenitors. We analyzed an ABC transporter activity in BCRP-expressing RPMI8226 and KMS11 cells by intracellular accumulation and retention of BCRP substrates with auto-fluorescence emission, mitoxantrone and doxorubicin, in flow cytometry. Treatment with Pim inhibitors, SMI-16a or SMI-4a, increased the incorporation of these drugs into the MM cells and enhanced their subsequent intracellular retention after 6-hour incubation without these drugs, although BCRP expression on their surface was only marginally affected by the Pim inhibition. Interestingly, acidic conditions up-regulated Pim-2 expression while reducing the accumulation and retention of these drugs in BCRP-expressing RPMI8226 and KMS11 cells. However, the Pim inhibitors efficaciously restored the drug accumulation and retention reduced by extracellular acidification, and enhanced the cytotoxic activity of the BCRP substrate doxorubicin against RPMI8226 cells rather preferentially in acidic conditions. Furthermore, the Pim inhibition minimized the sizes of “side populations”, highly drug-resistant fractions with enhanced BCRP activity, and the ability of colony formation in RPMI8226 and KMS11 cells, which was more marked in acidic conditions. We previously demonstrated the in vivo effects of the Pim inhibitors in human INA-6 cell-bearing SCID-rab MM models and syngeneic mouse MM models with an intra-tibial inoculation of 5TGM1 MM cells (Leukemia, 2014). To further examine the acid-tropism of anti-tumorigenic activity of Pim inhibition, we pretreated murine 5TGM1 MM cells in vitro with or without SMI16a at pH6.8 for 24 hours, and transplanted to the tibiae in mice the same numbers of viable MM cells remaining in each treatment group. Treatment with SMI16a at pH6.8 almost completely abrogated in vivo tumorigenic capacity of 5TGM1 cells, while MM cells without the treatment rapidly grew and expanded in and outside of the tibiae, suggesting targeting clonogenic MM cells by Pim inhibition preferentially in acidic conditions. Taken together, Pim-2 may become an important therapeutic target of drug-resistant BCRP-expressing MM cells and their progenitors which appear to gain more drug resistance in acidic bone lesions. Combinatory treatment with Pim inhibitors warrants further study to overcome drug resistance in MM cells, including their tumorigenic cancer stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a devastating bone marrow (BM) cancer characterized by clonal proliferation of plasma cells. Despite the emergence of novel therapeutics MM remains a fatal disease. The tumor microenvironment plays a critical role in promoting MM growth. We have recently demonstrated that a population of BM myeloid-derived suppressor cells is involved in regulation of MM progression. These cells abundantly produce the pro-inflammatory protein S100A9. Tasquinimod (ABR-215050, Active Biotech/IPSEN) is a quinoline-3-carboxamide derivative that binds to S100A9 and blocks its interaction with receptors TLR4, RAGE, and CD147. Here we investigated whether pharmacological inhibition of S100A9 with tasquinimod inhibits MM progression. A panel of MM murine (DP42) and human (RPMI-8226, H929, and U266) cell lines was cultured in the presence of tasquinimod or vehicle control and cell viability was determined using MTT assay. Treatment with tasquinimod did not affect MM cell viability. We then evaluated the anti-tumor effect of tasquinimod in vivo in a MM syngeneic model. In this model, murine MM DP42 cells are injected i.v., home to the BM, and grow as MM that closely resembles the human disease. On day 2 after tumor cell injection mice were randomly assigned to the treatment or control groups. Treatment group received tasquinimod at a dose of 30 mg/kg/day in drinking water for 28 days. We found that tasquinimod significantly improved survival of MM-bearing mice (p

Description: Introduction: Capillary zone electrophoresis (CZE) and measurement of total immunoglobulins (total Ig) are standard techniques for the identification and quantification of monoclonal immunoglobulins (M-Ig). Heavy/light chain (HLC) pair analysis allows discrimination between Igκ and Igλ and as a result allows measurement of both the monoclonal involved and polyclonal uninvolved HLC pair. We compared measurement of M-Ig by CZE and total Ig to HLC levels. Methods: 93 samples from 8 patients with IgA intact immunoglobulin multiple myeloma (MM) were analysed. M-Ig was measured using CZE Sebia Capillarys 2 system, total IgA (tIgA) and IgAκ and IgAλ HLC concentrations on a SPAPLUS analyser. IgA HLC levels were measured with Hevylite® and compared to published normal ranges (IgAκ (g/L): 0.57-2.08, IgAλ (g/L): 0.44-2.04, IgAκ/IgAλ: 0.78-1.94). Passing and Bablok fit analysis was used to determine correlation between the assays. Results: Measurement of the involved HLC pair (iHLC) (median: 12.45g/L; range: 0.64-44.71g/L) compared well with CZE (n=34; median: 11.04g/L; range: 1.24-37.71g/L.; y= 1.2x + -2.65, R2= 0.94). Measurement of iHLC (median: 0.88. range: 0.05-21.55g/L) also compared well with tIgA measurement (n=65 median=1.44g/L; range: 0.227-21.11g/L, y=0.85x + -0.26, R2=0.98). Percent changes in iHLC concentrations from baseline through treatment compared well with CZE (n=28; y=1.59x + 0.15 R2=0.93) and tIgA (n=57; y=1.06x + 0.01, R2=0.96). Of the 34 samples with quantifiable M-protein by CZE, 32(94%) had an abnormal HLC ratio. The two discrepant samples were follow up samples from the same patient, where HLC normalised alongside total IgA entering the normal reference range. In addition, all 15 samples (15/65; 23%) where tIgA concentration was above the normal reference range all had abnormal HLC ratios. M-Ig was not detected by CZE in 48/82 (57%) samples, 46/48 of the samples (96%) had a normalised HLC ratio. In 38/65 (58%) samples, tIgA concentrations were within the normal reference range, and 34 (90%) had a normalised HLC ratio. HLC ratio for all patient samples normalised in subsequent samples following treatment. Conclusion: The measurement of M-Ig is comparable between Hevylite® and both CZE and tIgA. The presented data indicate that Hevylite® is a more sensitive test for detecting residual disease and warrants prospective studies on larger cohorts of patients. Acknowledgments:This work was partially supported by the National Science Fund (D02-35/2009). Disclosures Guenova: Novartis Pharma Sevices Bulgaria: Consultancy, Research Funding, Speakers Bureau; Roche Bulgaria: Consultancy, Research Funding, Speakers Bureau; Amgen Bulgaria: Consultancy, Research Funding, Speakers Bureau; Sanofi-Aventis Bulgaria: Consultancy, Research Funding, Speakers Bureau.

Description: Background We have a poor understanding of the vaccination immune response and outcomes in multiple myeloma (MM). As MM patients (Pts) are living longer and therapies are immunomodulatory there is an unmet medical need to further characterize the role of the immune system. A common reason for hospitalization or death in MM Pts is infection. As an initial step in MM Cancer Care Delivery Research (CCDR), we evaluated the current vaccination practice patterns in MM Pts at Aurora Health Care using the EMR and data analytics. Methods An IRB approved study reviewed MM Pts from 5/15/2012 to 5/15/2014. Data collected included demographics, influenza (FV) and pneumonia vaccination (PV) history, hospitalization episodes, cost associated with hospitalization, and admission and discharge diagnoses. Pts were considered PV positive if vaccinated within 5 years prior to study with any PV type. FV was none (no FV in 2012-2014), optimal [FV in 2012 and (2013 or 2014)], or suboptimal [FV in 2012 or (2013 or 2014)]. Data was analyzed using SAS and STATA 12. Results A total of 1131 MM Pts were identified. Race included 70% white, 13% black, and 17% mixed, other or information not available. MM median age at diagnosis was 71 and only 4% (47) had prior autologous stem cell transplantation. PV rate was 30%. FV was 55% none, 24% suboptimal and 20% optimal. There was no statistically significant difference in the rate of PV and FV when stratified vs age, gender, and race. Over two years there were a total of 662 hospitalization events involving 317 MM Pts. The total cost of hospitalization was approx $35M. The average charge per hospitalized patient was $110K (range: $2K -1.3M) with an average $52K per hospitalization encounter (range: 2K – 648K). The rate of PV and FV vaccination among Pts with index hospitalization is significantly higher than non-hospitalized patients. There was no difference in hospitalization cost based on vaccination status. (See Table 1) Discussion Vaccination rates were low and did not correlate with hospital outcomes. This may be explained as a limitation for a retrospective EMR analysis without accounting for temporal relationship of vaccines – i.e. possible vaccination after admission. Alternatively, this may indicate that our current methods of vaccination in MM are not effective. Other limitations include need for a more granular review of treatment regimens and infectious complications. Additional surrogate markers are needed to understand the effect of vaccines and the immune system on health care outcomes such as hospitalizations, cost, and survival. This will be addressed in prospective registries and immunologic studies at our center and may be queried at other health systems. Table 1 – Vaccination Status and Hospitalizations Vaccination Status % Hospitalization Events, % Hospitalization Charge, $ PV – No 70% 20% $16M PV – Yes 30% 52% $18M FV – None 55% 16% $9M FV – Suboptimal 24% 42% $13M FV - Optimal 20% 43% $12M Disclosures No relevant conflicts of interest to declare.

Description: The combination of fludarabine, cyclophosphamide and rituximab (FCR) is still currently regarded as the standard regimen for treatment of physically fit patients with chronic lymphocytic leukemia (CLL). This therapy can be associated with significant toxicity, and patient adherence to the protocol may often be difficult outside of clinical trials. This retrospective study aimed to evaluate the efficacy and safety of FCR therapy in the real life setting, with particular focus on the influence of dose reduction on treatment outcome. A total of 132 CLL patients (≤70 years of age) treated with FCR as frontline therapy from 10 medical centers, were reviewed. The majority of patients were males (73.5%, n=97) and younger than 60 years (78%, n=103). Eleven patients had Binet stage A (8.3%), 72 (54.5%) were stage B and 49 (37.1%) had Binet stage C. Results of FISH analysis were available for 99 patients, with high risk cytogenetics of del(11q) in 21 patients (21.2%) and del(17p) in 9 cases (9.1%). The majority (56.5%, n=74) received rituximab at a dose of 500mg/m2 and the rest 375mg/m2. Almost half of the patients (49.2%, n=65) were given a reduced dose of chemotherapy (

Description: Introduction: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with variable clinical course. Several studies have been conducted to predict outcome in patients with CLL and also have been going on. A proliferation inducing ligand (APRIL) has been shown to involve in survival and resistance to apoptosis in CLL, and APRIL molecule has been investigated as a prognostic marker in CLL patients. However, there are limited and controversial data regarding APRIL and its impact on prognosis in CLL. We aimed to compare serum APRIL levels in CLL patients with those of age and gender matched healthy subjects, and to investigate the relationship between APRIL and the other common prognostic factors, and to determine whether serum APRIL levels predict time to first treatment in CLL. Methods: After ethical approval and informed consent were obtained, between May and December 2012, venous blood samples were driven from 96 CLL patients’ and 25 healthy controls’, and serum APRIL levels were measured by ELISA. Demographic data and the prognostic markers were obtained from the patients’ files, and patients have been followed for a minimum of 12 months. We tested the correlation between APRIL with the, clinical and biological parameters, and used the log rank test to compare their Kaplan Meier curves. Results: Patients were divided into three groups: Treatment naive (group A, n=49), chemotherapy receiving (group B, n=25) and who had previously received chemotherapy (group C, n=22). Median APRIL level was higher in group A (2.78 vs 1.29; p=0.034) and group C (3.54 vs 1.29; p=0.001) when compared to healthy controls, but was not different in group B (1.56 vs 1.29; p=0.3) (Figure 1). Serum APRIL level in group A was negatively correlated with hemoglobin levels (r=-0.298; p=0.037) and platelet counts (r=-0.321; p=0.025) whereas no correlation with age, Rai and Binet stages, lymphocyte counts, β2-microglobulin and CD38 levels were detected. Group A patients were also divided into 2 subgroups (APRIL levels low, n=20 and APRIL levels high, n=29) using median natural logarithm of serum APRIL level as cut off. April low and high subgroups were similar with respect to demographic data and prognostic factors. Median time to first treatment was not reached in the APRIL low group, but was 104 months in the APRIL high group (p=0.13, log-rank test). Conclusions: Among the treatment naive patients, serum APRIL levels only negatively correlate with hemoglobin levels and platelet counts. These correlations seem to be associated with tumor burden rather than the prognosis, because APRIL levels were not different in chemotherapy receiving patients compared to healthy controls. Since a median survival time could not be reached in the APRIL low group, short follow up time might be an explanation why the APRIL levels did not predict the time to first treatment. In conclusion, our findings let us to think APRIL levels are not a useful marker to predict prognosis in patients with CLL. Figure 1. Median APRIL levels of CLL patients and healthy controls (ng/mL) Figure 1. Median APRIL levels of CLL patients and healthy controls (ng/mL) Disclosures No relevant conflicts of interest to declare.

Description: Bendamustine has been demonstrated to be effective for the treatment of CLL, either alone compared with chlorambucil (Knauf et al, JCO 2009 and BJH 2012) or in combination with monoclonal antibodies such as rituximab both in second or more lines (Fischer et al, JCO 2011) and in first line treatment (Fischer et al, JCO 2012). However, the relationship between its activity with clinical and biological prognosticators has been addressed only in few studies. For this purpose, we evaluated the efficacy and safety of bendamustine, in a real-life contest, on 56 patients, median age 66 years (41-80), median number of previous regimens 1 (0-3, 32% previously untreated). Bendamustine was given for a median number of 6 cycles (70-90 mg/m2), in 82% of cases with rituximab at conventional doses. Overall (ORR) and complete response (CRR) rates were 73% and 44.6%, respectively. Obviously, CRR was higher (83.3%) for 18 patients treated in first line. A significant correlation was found between lower ORR and lymphocyte doubling time 30%) of alpha-4 integrin CD49d (OR 13.0; P=0.018), an important marker of bad prognosis in CLL (Bulian et al, JCO 2014). On the other hand, no significant correlations were found between ORR and CD38, ZAP-70 or IGHV mutational status. Similarly, no significant correlations were noted between ORR and FISH cytogenetics, excluding del(17)p, or NOTCH1 mutations, thus confirming the independence of response to bendamustine from some well-known important biologic prognostic factors. In fact, multivariate analysis confirmed a significant relationship only between ORR and TP53/del(17)p (OR 0.020; P=0.0015) and concomitant rituximab (OR 0.019; P=0.0074). The estimated 1-year OS and PFS were 57% and 86%, respectively. Side effects included grade 3-4 neutropenia, infections, thrombocytopenia and anemia which occurred in 21%, 12%, 12% and 5% of patients, respectively. Grade 3-4 non-hematologic toxicity, including infusion-related reactions, heart or kidney or liver failure were found almost exclusively in elderly patients treated with bendamustine after two or more lines of therapy (12.5%). In multivariate analisys of OS, calculated from the end of treatment with bendamustine, only response to bendamustine (P=0.008) was confirmed to be an independent prognostic factor, while both the number of previous therapies and the concomitant use of rituximab demonstrated no statistical significance. These our results confirm both the activity and safety of bendamustine, particularly in combination with rituximab, also in the setting of elderly patients, often affected by two or three comorbidities. Noteworthy, this effectiveness appears to be present also in patients with unfavorable clinical and biological features, excluding del(17)p or TP53 mutations, in which the employment either of modern oral BCR inhibitors or of BH3 mimetics anti-Bcl-2 will be definitely active, also in combination with the same bendamustine. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Chemoimmunotherapy for chronic lymphocytic leukemia (CLL) has been the standard of care for initial treatment. A randomized demonstrated both a progression free survival (PFS) and overall survival (OS) advantage when rituximab was added to fludarabine (F) and cyclophosphamide (C). Alemtuzumab (Campath) (CAM), an anti-CD52 monoclonal antibody, is an effective therapy for patients with both previously untreated and relapsed CLL. Its role in combination with chemotherapy is less certain. METHODS: We conducted a multicenter phase II clinical trial of FC followed by subcutaneous CAM in previously untreated CLL. Patients were eligible if they met standard criteria for initiating therapy, or if they were asymptomatic with the prognostically adverse immunoglobulin heavy chain variable (IGHV) gene unmutated status. Patients received fludarabine (25 mg/m2/day, days 1-3) and cyclophosphamide (250 mg/m2/day, days 1-3) every 28 days for six treatment cycles, followed by a 3-8 week rest period. Disease response was assessed, including minimal residual disease (MRD) status by sensitive flow cytometry in those in complete remission (CR). Patients who achieved less than a CR were eligible to receive standard dose CAM (30 mg thrice weekly for 12 weeks); those who were in CR but MRD positive could receive reduced dose CAM (30 mg weekly for 12 weeks). The primary outcome was duration of response (DOR). Secondary outcomes included the response rates after FC and after the addition of CAM, as well as the safety profile of the regimen. RESULTS: We enrolled 25 patients from November 2004 to June 2007 at 3 centers. The median age of the participants was 62 years (range 42-75). Detailed information was available for 17 patients pre-treatment: high risk Rai stage in 9, IGHV unmutated in 9 including 4 patients who were IGHV unmutated as their indication for treatment. Five patients had trisomy 12, 4 had 13q deletion, 1 each had 17p deletion and 11q deletion, and 6 had no abnormality. One patient was excluded from the analysis due to a diagnosis of mantle cell lymphoma after eligibility review. Four patients had no response evaluation and were considered treatment failures. Seventeen (71%) patients had a CR after 6 cycles of FC, including 11 who were MRD negative, one had a partial response (PR), and two had progressive disease (PD). Four of the 6 patients who were MRD positive received CAM after FC. Two required only a single dose to become MRD negative, and 2 received 12 weekly doses. One of these patients became MRD negative. The median DOR for those achieving CR was 38 months (range 12-105 months). There were no treatment related deaths. Five patients experienced a SAE including one with febrile neutropenia, two with pneumonia, and two with autoimmune hemolytic anemia. There were ten additional treatment emergent adverse events including two that were grade 3 (mucositis and fever) and one CMV reactivation while receiving CAM. Two patients developed treatment related myelodysplasia, one died and the other underwent allogeneic stem cell transplant. There were two deaths due to Richter’s transformation. During long term follow up, there have been five additional deaths. CONCLUSIONS: The CR rate after FC was higher than that reported in prior trials of previously untreated patients and the incidence of MRD negative CR was surprisingly high. The DOR was consistent with prior experience with FC. Too few patients received CAM to draw any conclusions about its role as a consolidative therapy given subcutaneously on a weekly schedule. Both the FC and CAM therapies were well tolerated, with few adverse events associated with their use. Disclosures No relevant conflicts of interest to declare.

Description: Background Many studies have examined the disparities in cancer diagnosis, treatment and survival among different subgroups classified based on race, socioeconomic status and age. Not as many studies have examined disease characteristics in rural populations, perhaps because of the lack of a consensus in the United States regarding the definition of "rural". In these few studies, many disparities were reported in association with rural residence such as lower levels of utilization of cancer screening tests, lower likelihood of receiving guideline-appropriate therapy and shorter survival. Objectives To examine multiple myeloma disease characteristics and survival in rural patients of southern New Mexico in comparison to their urban counterparts. Methods Patients presented to Memorial Medical Center (MMC) and its associated cancer center from January 2003 to December 2013 with multiple myeloma were enrolled in the study. Demographic and clinical data were collected. The charts were also examined for evidence of offering or discussing Autologous Stem Cell Transplant (ASCT) as a treatment option with patients, and whether it was actually performed. Patient staging at diagnosis according to International Staging System (ISS) for myeloma was determined, if possible. Urban vs. rural classification was based on the Rural-Urban Commuting Area codes (RUCA) version 2.0; a census tract-based classification scheme that utilizes the standard bureau of census urban definition in combination with work commuting information. Categorization D of RUCA was chosen. It defines urban as all places that have 30% or more of their workers going to a Census Bureau-defined Urbanized Area. Results A total of 87 patients were initially enrolled in the study. Four patients were excluded because sufficient evidence to establish the diagnosis could not be verified. Two additional patients who had solitary plasmacytoma with no evidence of systemic involvement were excluded as well. Patients were classified based on their residence at the time of diagnosis as rural (29 patients) and non-rural (52 patients). There was no difference between the mean age at diagnosis between the two groups with mean being 66.20 for non-rural group and 67.77 for the rural group. The type of heavy chain protein was generally similar for both groups with 55.74 % of patients diagnosed with Immunoglobulin G heavy chain disease. The average duration of initial presenting symptom prior to diagnosis was 7.36 weeks for the whole sample, 13.6 week for the rural group, and 4.56 weeks for the non rural group, which suggests that non-rural patients were more likely to seek medical attention sooner than their rural counterparts (p=0.0037). Tobacco consumption was higher among patients in the rural group compared to non-rural group. Nearly half (47.37%) of rural patients were diagnosed at stage 3 according to ISS staging system, while only 31.03% of non-rural patients were diagnosed at the same stage. Rural patients were more likely to be diagnosed at a more advanced disease stage (p=0.063). The nature of the chief presenting problem was generally similar in both groups with the exception of the higher likelihood of patients in non-rural group to be diagnosed at an asymptomatic stage. In the whole sample, 33.33% of patients had evidence of being offered or educated about ASCT as a treatment option, and 18.52% of patients actually receiving it. There was no different between the two groups in this regard. Median survival time for the whole sample was 57 months. Patients in the rural group had a median survival of 39.03 months (95% CI 18.96- 57.99), while non-rural patients had a median survival of 68.99 months (95% CI 25.95-90.02) ( p

Description: Markers indicative of prognosis play a consequential role in the clinical management of patients suffering from chronic B-cell lymphocytic leukemia (B-CLL). Soluble CD163 (sCD163) has been shown to be a useful biomarker in a wide variety of disease entities (Moller, 2012), however its presence in B-CLL has not been addressed. Using an enzyme-linked immunosorbent assay the concentration of sCD163 was measured in peripheral blood of 30 B-CLL patients at diagnosis. The results were related to the course of disease for up to two years post diagnosis. The median level of sCD163 in the plasma was not significantly higher in the B-CLL patients (2.085 mg/L, range 0.77- 9.01 mg/L) than in an age-matched control group (1.800 mg/L, range 0.97-2.45 mg/L) (n=10) (p=0.157). As CD163 is a monocyte/macrophage specific membrane protein, the relationship between the percentage of monocytes and the level of sCD163 was relevant. The level of sCD163 did not correlate with the percentage of monocytes in peripheral blood of the patients, but as previously described, there was an inverse correlation between measured sCD163 and the CD163 surface expression as measured by mean fluorescence intensity on the monocytes (r=0.088, p= 0.658 and r= -0.476, p=0.01, respectively) (Davis et al. 2005). Elevated levels of sCD163 have been linked to bacterial infection however in neither the B-CLL cohort nor the healthy control group, a correlation was found between the levels of sCD163 and CRP concentrations (r=0.24 and p=0.23 in B-CLL, and r=0.10 and p=0.81 in healthy controls) (Knudsen et al., 2007). To test the prognostic impact of sCD163 the B-CLL patients were divided into two subsets using the highest level of sCD163 measured in the age-matched healthy control group as a cut-off. Hence, 11 of 30 B-CLL patients were assigned to an sCD163high group. In total, 7/30 (23%) patients experienced disease progression defined as need for cytoreductive treatment within the two years follow-up period. In more detail, 5/11 sCD163high patients (45%) and 2/19 patients (11%) in the sCD163low group received therapy (p=0.068). When analyzing the relevance of sCD163 in terms of predicting disease progression, there was a significant difference between patients in the two groups, indicating that patients with high concentrations of sCD163 in the plasma progressed more rapidly (p=0.029) (Figure 1A). In this cohort, CD38 expression was also of prognostic value for progressive disease (p=0.006) (Figure 1B), while b2M was of borderline significance (p=0.051) (Figure 1C). The role of mutational status as predictor of short time to treatment was not significant (p=0.21) (Figure 1D). When performing the log-rank univariate analyses on the dataset, there was an obvious increased hazard of receiving treatment for patients in the sCD163high group compared to the sCD163low patients (Table I). As expected, CD38 expression, b2M, and IgVH mutational status were albeit to varying degrees covariates in the time to treatment analyses (Table I). The data strongly indicate that sCD163 is a prognostic marker in B-CLL. The study is limited by the cohort size, however, indubitably, the results lay the ground for a larger study. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoesis, associated with cytopenias and high risk of leukemic transformations with common morbidity. MDS are hematological malignancies of unclear etiology where oxidative/nitrative stress may contribute to the pathogenesis1. The posttranslational oxidative modifications of proteins and low molecular weight compounds are induced, revealing dysbalance of redox systems in vivo. Nitration of tyrosine either in free form or bound in proteins is important marker of nitric oxide synthase (NOS) activity shift in the presence of oxidative stress in favour of superoxide formation. The aim of this work was to assess whether 3-nitrotyrosine (3-NT) serum concentrations are enhanced also in MDS patients. Methods: Serum samples were obtained using blood of either MDS patients or healthy donors. All tested individuals agreed to the study at the time of blood collection. We proposed HPLC-MS/MS method to estimate 3-NT concentration in serum samples using QTRAP 4000 mass spectrometer (ABSciex, Prague, Czech Republic). Serum proteins were precipitated using ethanol, supernatants were evaporated, reconstituted in 0.1% HCOOH/2% methanol and injected onto HALO C18 microcolumn 100x0.5 mm (ABSciex, Prague, Czech Republic). Oxidative stress in MDS patients and controls was assessed by serum malondialdehyde concentrations measured by HPLC of 2-thiobarbituric acid MDA derivative using UV detection. Results: The sensitivity of method proposed for analysis of 3-NT in sera was sufficient for estimation of differences of 3-NT in patients and control samples. We have found enhanced concentrations of both MDA and 3-nitrotyrosine in serum of MDS patients as compared with healthy donors. Discussion: Enhanced MDA concentrations in MDS patients confirmed the presence of oxidative stress in MDS patients. The reactive oxygen species may oxidize tetrahydrobiopterin, important cofactor of NOS, resulting into nitric oxide synthase uncoupling with enhanced superoxide and consequently peroxynitrite production2. It is known that methylarginines, naturally occurring inhibitors of NOS, can profoundly increase superoxide generation from uncoupled NOS. Recently, we have found significantly enhanced concentration of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome3. The observed increased concentrations of 3-NT in MDS patients correspond with assumed enhanced peroxynitrite formation as compared with controls. 3-nitrotyrosine concentrations thus could serve as a new criterion of NOS changed activity in MDS patients. Literature: 1. Farquhar MJ, Bowen DT. Oxidative stress and the myelodysplastic syndromes. Int J Hematol. 2003;77:342-350. 2. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315-424. 3. Štikarová J, Suttnar J, Pimková K, Chrastinová-Mášová L, Čermák J, Dyr JE. Enhanced levels of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome. Journal of Hematology & Oncology. 2013;6:58. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: PI3Kδ signaling is critical for the proliferation, survival and homing/tissue retention of malignant B cells. Idelalisib is a first-in-class, highly selective, oral inhibitor of PI3Kδ recently approved for the treatment of relapsed CLL in combination with R. This report summarizes the long-term follow-up of the Phase 1 combination experience of idelalisib with anti-CD20 antibodies. Methods: This Phase 1 study evaluated idelalisib for relapsed/refractory CLL continuously given at 100 mg BID (4 of the pts receiving R) or 150 mg BID (all other pts) in combination with a total of 8 infusions of rituximab (R, 375 mg/m2 weekly x 8), or a total of 12 infusions of ofatumumab (O, 300mg initial dose either on Day 1 or Day 2 relative to the first dose of idelalisib, then 1,000 mg weekly x 7, then 1,000 mg every 4 wks x 4). Pts on treatment after 48 weeks were eligible to continue idelalisib on an extension study. Clinical response was evaluated according to published criteria (Hallek 2008; Cheson 2012). Results: 40 pts (12F/28M) with a median (range) age of 66 (43-87) years and a WHO performance status of 0 (24, 60%) or 1 (16, 40%) were enrolled. 19 pts received idelalisib in combination with R and 21 with O. Adverse disease characteristics (n, %) included Rai Stage III/IV (20, 50%), bulky lymphadenopathy (23, 58%), refractory disease (15, 38%), multiple prior therapies (median 2, range: 1-9). Almost all pts (39, 98%) had at least 1 prior therapy containing R, and 3 of the 21 pts (14%) receiving idelalisib + O had received prior O. 63% of the pts receiving idelalisib + R, and 48% of the pts receiving idelalisib + O were refractory to R. Prior therapies also included alkylating agents (31, 78%, [bendamustine: 20, 50%]) and purine analogs (31, 78%, [fludarabine: 28, 70%]). Data available from 39 pts showed that 11 (28%) pts had evidence of del(17p) and/or TP53 mutations and 30 (75%) had unmutated IGHV. As of 7/15/2014, the median (range) treatment duration was 18 (0-44) months. 23 (58%) pts have completed the primary study and enrolled into the extension study. Primary reasons for study discontinuation (as reported by investigators) included disease progression (14, 35%), adverse events (AEs) (12, 30%), investigator request (3, 8%), withdrawal of consent (n=1), BMT (n=1). There were a total of 8 deaths on study: 2 deaths occurred after disease progression, and 6 pts died because of AEs (all assessed as unrelated/unlikely related to idelalisib by investigators). A total of 4 pts (10%) were continuing idelalisib treatment on the extension study at time of analysis. Selected treatment-emergent AEs (any Grade/≥Gr 3, regardless of causality) included diarrhea/colitis (55%/23%), cough (40%/3%), pyrexia (40%/3%), dyspnea (30%/3%), fatigue (25%/0%) nausea (25%/0%), rash (20%/0%), pneumonia (20%/18%), and pneumonitis (8%/5%). Elevation of liver transaminases (TA, any Grade/≥Gr 3) was seen in 30%/10%. Re-exposure to idelalisib after resolution of TA elevation generally was successful; only 1 patient discontinued the study because of (recurrent) TA elevation. Other AEs leading to study discontinuation and reported as possibly/probably related to idelalisib included diarrhea/colitis (4, 10%), pyrexia (n=1), interstitial lung disease (n=1), pneumonia (n=1), rash (n=1), psoriasis (n=1). Secondary malignancies leading to discontinuation (all reported as unrelated) were breast cancer (n=1), recurrent colon cancer (n=1), AML (n=1). There was no obvious overall difference in the toxicity reported for pts receiving idelalisib with rituximab compared to those with ofatumumab. The ORR (N=40) was 83% (33/40), with 2 CRs (5%) reported. Median PFS (N=40) and duration of response (DOR) (n=33) were 24 months. Median (range) time to response was 1.9 (range 1.7-16.9) months. Median overall survival (OS) has not been reached with a KM estimate for OS of 80% at 24 months. For the 11 pts with del(17p) and/or TP53 mutations, the response rate was 73%, and the median PFS and DOR were 20 and 24 months, respectively. Conclusions: Combinations of idelalisib with anti-CD20 antibodies such as R or O represent non-cytotoxic regimens with acceptable safety profiles and considerable activity resulting in durable tumor control in pts with relapsed/refractory CLL, including those with high risk factors such as del(17p) or TP53 mutations. A Phase 3 trial evaluating the efficacy of idelalisib in combination with ofatumumab is ongoing (NCT01659021). Disclosures Furman: Gilead Sciences: Research Funding. Off Label Use: Zydelig is a kinase inhibitor indicated for the treatment of patients with: 1) Relapsed chronic lymphocytic leukemia (CLL), in combination with rituximab, in patients for whom rituximab alone would be considered appropriate therapy due to other co-morbidities; 2) Relapsed follicular B-cell non-Hodgkin lymphoma (FL) in patients who have received at least two prior systemic therapies; and 3) Relapsed small lymphocytic lymphoma (SLL) in patients who have received at least two prior systemic therapies.. de Vos:Gilead Sciences: Research Funding. Barrientos:Gilead Sciences: Research Funding. Schreeder:Gilead Sciences: Research Funding. Flinn:Gilead Sciences: Research Funding. Sharman:Gilead Sciences: Research Funding. Boyd:Gilead Sciences: Research Funding. Fowler:Gilead Sciences: Research Funding. Leonard:Gilead Sciences: Research Funding. Rai:Gilead Sciences: Research Funding. Kim:Gilead Sciences: Employment, Equity Ownership. Viggiano:Gilead Sciences: Employment, Equity Ownership. Jahn:Gilead Sciences: Employment, Equity Ownership. Coutre:Gilead Sciences: Research Funding.

Description: The revised international prognostic scoring system (IPSS-R) improved cytogenetic prognostic classification in comparison with IPSS classification in patients with myelodysplastic syndromes (Greenberg P et al. Blood 2012, Schanz J et al. JCO 2011). Monosomal karyotype (MK) defined as the presence of at least two autosomal monosomies or one monosomy associated with structural abnormalities has been investigated in MDS patients with contradictory results regarding its independent prognostic significance (Patnaik M et al. Leukemia 2011, Gangat N et al. AJH 2013, Valcarcel D et al. JCO 2013). The aim of this study was to investigate the prognostic significance of MK in relation to IPSS-R and the presence of complex karyotype (CK). The study was conducted in 391 patients with primary MDS treated at three hematology departments in Serbia. The median age was 65 years, from 15 to 89 years. There was a predominance of male patients who made 60% (235) of patients. 229 (58 %) patients died. 87 patients (22 %) transformed to AML. The sixty eight % of patients were transfusion dependent. Disease modifying treatment was applied in 48 (12.3%) of patients (AML like chemotherapy in 37 patients, low dose ara-c in 11 patients, stem cell transplantation in five patients, and in one patient azacitidine). The rest of the patients received supportive treatment. Karyotypes were classified according to the International System for Cytogenetic Nomenclature Criteria. The inclusion of patients was based on criteria used in IPSS-R classification (a white blood cell count ≤12x109/l, an absolute neutrophil count ≤8x109/l, peripheral blood blasts ≤19%, and bone marrow blasts ≤30%). The distribution of patients according to FAB classification was as follows: 129 (32.99%) RA, 47 (12.02%) RARS, 143 (36.57%) RAEB, 48 (12.27%) RAEB-T, and 22 (5.6%) non proliferative CMML. The classification according to WHO 2008 classification was as follows: RCUD 29 (7.43%), RARS 39 (9.97%), RCMD 90 (23.01%), RAEB1 78 (19.95%), RAEB2 65 (16.62%), AML/RAEB-T 48 (5.27%), 5q- 11 (2.81%), MDS-u 7 (1.8%), CMML 1 15 (3.83%), CMML 2 7 (1.8%). IPSS-R distribution was: very low risk 38 (9.71%), low risk 108 (27.6%), intermediate risk 86 (21.99%), high risk 83 (21.23%), very high risk 74 (18.9%). Two patients could not be classified because of lack of all data. Median number of metaphases was 15, from 2 to 30. The abnormal karyotype was found in 166 (42.45%) patients. CK defined as the presence of at least three cytogenetic aberrancies was present in 32 patients (8.18%) with median survival of 5 months. CK shown prognostic significance regarding the overall survival (OS) (p = 0.00001) as well as time to AML transformation (p = 0.00014). MK was detected in 34 patients (8.69%). The patients with MK had significantly shorter OS in comparison with patients without MK (median survival 5 months versus 34 months, p 〈 0.00001, Figure 1) as well as a shorter time to AML transformation (p = 0.00006, Figure 2). If we included only the patients who have MDS according to 2008 WHO classification MK shown prognostic significance for OS (p = 0.00121) as well as for time to AML progression (p = 0.00010). The presence of MK defines the group of patients with shorter OS in high risk and very high risk IPSS-R prognostic groups (p=0.008, Figure 3). In multivariate analysis, MK shown to be independent predictor of poor survival together with age, haemoglobin concentration, platelet count and bone marrow blast cells (p

Description: Background: Severe thrombocytopenia is an uncommon event in lower risk MDS patients, but it may significantly influence the prognosis. In fact, when it occurs, major bleeding may be a life-threatening complication. No licensed pharmacologic approach is nowadays available yet for these patients. Eltrombopag seems to be a very interesting product, but its efficacy and safeness are still to be better demonstrated. Romiplostim could be suitable too, but, at present, its safety is uncertain in MDS patients. Also danazol, an attenuated androgen, seems to have some ability to increase the platelet count in this context. Patients and methods: We retrospectively reviewed 17 thrombocytopenic patients affected by MDS, treated with danazol and observed for at least 6 months. Three patients of these had a therapy-related MDS. At the starting time of danazol therapy, the IPSS was “low” or “intermediate-1” in 16 cases; “intermediate-2” in 1 case. The IPSS-R was “very low”, “low” or “intermediate” in 16 cases; “very high” in 1 case. In 14 patients the platelet count was lower than 25x109/L, in the other 3 lower than 40x109/L, but with spontaneous bleeding. The initial dose was 600 mg/day for all the patients. The IWG criteria of response (Cheson 2006) were adopted. The outcomes were observed after 3 and 6 months from the beginning of therapy. Only descriptive statistical analysis was used. Results: At the beginning of therapy, the average platelet count of the 17 patients was 22.6 x109/L (S.D. 8.8, range 6-38). After 3 months, the therapy with danazol was ongoing in 16 patients (in 1 case the drug was discontinued due to renal failure). Platelet improvement, according to IWG criteria, was observed in 8 cases (47%). The average platelet count was 45.3x109/L (S.D. 32.9, range 4-133). The only one “high risk” patient did not show response. After 6 months danazol was still ongoing in 11 patients (in 5 cases the drug was stopped for inefficacy). The response according to IWG criteria was evident in 9 patients (52% of the initial 17 patients). The average platelet count was 66x109/L (S.D. 63.9, range 11-218). Adverse events recorded were as follows: increase in transaminases in 3 cases (in 2 of these the dose was reduced to 400 mg/day); severe but reversible renal failure in 1 case (the drug was stopped); moderate increasing of serum creatinine in 1 case (the drug was reduced to 400 mg/day); reversible cutaneous rush (the drug was reduced to 400mg/day); amenorrhea in 1 case (the only fertile woman in the series); weight loss and loss of appetite in 1 case, weight gain in 1 case. Conclusions This series confirms the efficacy of danazol to improve platelet count in approximately half of patients with severe thrombocytopenia due to “low-risk” MDS. In all patients with increased platelet count, the response was clinically significant. The response may not be immediate. Actually, there was an improvement of platelet count even after three months of therapy. The toxicity profile of this drug is low. The mechanism of action of danazol in MDS patients remains unclear. Waiting for more information on the efficacy and safety of eltrombopag from the clinical trials in progress, danazol may be a good therapeutic option for these patients. Disclosures Off Label Use: Danazol in MDS patients with severe trhombocytopenia.

Description: Background Hydroxyurea (HU) is an oral chemotherapeutic agent that has been used for the treatment of sickle cell anemia (SCA) and many other conditions. Although the efficacy of HU has been established in SCA, there is ambiguity regarding the long-term adverse events of this treatment. As significant risk such as secondary malignancies or myelodysplastic syndrome (MDS) have been linked to this drug but occur in small numbers across the various indications, we planned to pool the results from HU studies used in many conditions (excluding malignant and premalignant diseases) to obtain much wanted risk estimates. Objectives To evaluate the long-term safety (carcinogenicity) of HU therapy in people with SCA or any benign diseases (excluding malignant and premalignant diseases) of any age. Search strategy We searched MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL), ongoing trials registers, and major preceding conferences. Hand searches were also conducted using reference lists from primary studies. All searches were updated to May 25 2014. Selection criteria Randomized controlled trials (RCTs) and observational studies (sample size ≥ 20 and mean/median follow up ≥ 2 years) assessing the toxicity of HU for the treatment of patients with any benign diseases were included. Data collection and analysis Two authors acted as reviewers and independently completed the search process, selected the studies, assessed study quality, and extracted data from the included studies. Authors of included studies were contacted if further information was required. Since the majority of the included studies were single-arm design with no control groups, the effect size was estimated as a proportion (events over patient-years on HU) and reported as overall cancer risk (OCR) for the included studies. All data was analyzed using comprehensive meta-analysis (CMA), version 2.0. Main results A total of 37 studies (3 RCTs and 34 observational studies) involving 3278 patients were included. We identified four benign diseases where HU has been used ≥ 2 years and met the inclusion criteria. These include SCA (26 studies), β-thalassemia (9 studies), cyanotic congenital heart diseases (one study), and renal stones (one study). HU was not associated with increased risk of cancers in the treated group with OCR of 0.2% (95% CI, 0.0-0.3%). Further to that, 12 studies or extension studies with ≥ 5 year follow up on HU showed stable result of 0.2%. This weighted effect size represent cancer incidence rate of 2 per 1000 which is not higher than the most recent published cancer incidence rate for all African-American or Whites, US 2005-2009 period. Acknowledging the differences between the two rates and they are not meant to be direct comparison, a useful representation can be extracted from them. Overall, there were 7 cases of malignancy/MDS were identified post relatively long-term use (≥ 2years) of HU in 3278 patients. In the narrative review, we identified another 13 cases of malignancies post HU use in hemoglobinopathy as described by different case reports and studies that did not meet our inclusion criteria with majority have been leukemia. The majority of the included studies had several limitations, such as small sample size, lack of comparison group, under-reporting of data and methods, and the majority having been observational studies. Authors’ conclusion The use of HU in treating patients with hemoglobinopathies does not appear to be associated with increase risk of secondary malignancies nor MDS despite being used for relatively long-term courses. However, ongoing long-term studies as well as updated national and international registries and other types of large database are highly needed to further consolidate this finding. Disclosures Off Label Use: Hydroxyurea for management of β-Thalassemia, Cyanotic congenital heart diseases, Renal stones, or Children with sickle cell anemia..

Description: Background Polycythemia vera (PV) is a subgroup of myeloproliferative neoplasm (MPN) BCL-ABL1 negative. The current therapy of PV should be aimed at preventing vascular complications and avoid increasing the risk of leukemic transformation. The therapy response monitoring is based in the European LeukemiaNet (ELN) unified definition of clinical resistance and intolerance to hydroxycarbamide in polycythaemia vera consensus process, published by Barbui T et al in Br J Haematol 2010;148(6):961-963. Objectives We conducted a study to assess in our clinical practice the aplicability of the standard criteria for resistance and intolerance proposed by ELN in patients (pts) with PV that have been treated with hydroxycarbamida (HU). Methods This is a retrospective study in a cohort of pts with PV enrolled in a single Hematology University center in South Brazil. All pts were treated according to PV guidelines, and the response monitoring was based on clinical practice. All database was compared to standard criteria proposed by ELN. Intolerance /resistance was defined by: a) need for phlebotomy to keep hematocrit 〈 45% after 3 months of at least 2 g/d of HU or b) uncontrolled myeloproliferation (ie, platelet count 〉 400 x109/L and white blood count (WBC) 〉 10 x109/L) after 3 months of at least 2g/d of HU or c) failure to reduce massive splenomegaly by 50% as measured by palpation or failure to completely relieve symptoms related to splenomegaly after 3 months of at least 2 g/d of HU or d) absolute neutrophil count 〈 1.0 x109/L or platelet count 〈 100 x109/L or hemoglobin 〈 10 g/dL at the lowest dose of HU required to achieve a complete or partial clinicohematologic response or e) presence of leg ulcers or other unacceptable HU related nonhematologic toxicities, such as mucocutaneous manifestations, GI symptoms, pneumonitis or fever at any dose of HU. Results We analyzed data from 33 patients with PV assisted in the last five years in our outpatient clinical data. The ELN criteria for resistance and intolerance were accessed in these patients. At diagnosis, 42,4% of pts were younger than 61yo, and 54,5 were male. Arterial hypertension, diabetes mellitus and dyslipidemia were identified on 21, 2 and 7 pts, respectively. Only one patient was tobacco smoker at diagnosis. Total of 5 pts showed WBC 〉 15 x109/L, and 7 pts showed platelets 〉 450 x109/L. Massive splenomegaly is a rare PV manifestation in our series, occurring in 2 pts. Five patients complained of symptoms related to PV as pruritus and vasomotor phenomena at diagnosis. Less than 5% of patients had been treated with 2 g of HU for more than 3 months. In daily practice, when the patient presented hematologic toxicity, the HU was decreased and, if the hematocrit was over 45%, an occasional phlebotomy was performed. In relation to platelets (less than 400 x 109/L) and leucocyte (less than 10 x109/L) counts, these targets were not used exclusively in the clinical practice to change treatment. The intolerance was easily discriminated in patients with leg ulcers and other non-hematological events. Conclusion These criteria were done based on an expertise consensus for international criteria standardization for clinical studies. Its application in a retrospective study, using clinical daily practice data is not adequate. The reason is that in the last years the main target to treat patients was the hematocrit above 45%, the exact number of platelets and leucocytes is still not a consensus to define resistance, so different counts had been used to guide treatment, we rarely used HU doses above 1500 mg/daily to treat our patients and massive splenomegaly was observed in very few patients. These criteria should be used most for prospective studies. Disclosures No relevant conflicts of interest to declare.

Description: MF is a neoplastic stem cell disorder in which a multipotent hematopoietic stem cell acquires a clonal proliferative advantage, and its progeny inappropriately releases fibrogenic factors into the bone marrow microenvironment, leading to secondary bone marrow fibrosis. The cytokines implicated in the pathogenesis of MF include transforming growth factor β (TGF-β), basic fibroblastic growth factor (b-FGF), and platelet-derived growth factor (PDGF). Some of these cytokines, such as b-FGF and PDGF can act as angiogenic factors. Patients with MF have higher concentrations of circulating vascular endothelial growth factor (VEGF) and b-FGF than do control subjects. In addition, a higher degree of bone marrow angiogenesis has been reported in patients with MF. Eph receptors form the largest subgroup of receptor tyrosine kinases (RTK). The Ephrins are the ligands of the Ephs and stimulate bi-directional signaling allowing cell movement and shape change. EphA3 is a member of the Eph family and is expressed mainly during fetal development. Aberrant expression of EphA3 is detected in some solid and hematologic tumors. Recently, the expression of EphA3 has been detected in bone samples from subjects with MF. Antibody targeting of EphA3 may therefore constitute a novel approach to treating MF and other hematologic malignancies. Using well-characterized normal and diseased FFPE bone marrow biopsies, an IHC assay for EphA3 expression, which was developed and validated previously for use in AML and MDS patient screening, was evaluated further for use on MPNs, such as MF, polycythemia vera (PV) and essential thrombocythemia (ET). This IHC assay for EphA3 expression was validated (intra-assay variability/precision and inter-assay variability/reproducibility established by variance of SHS for serial tissue sections) using MPNs with negative, low, medium and high intensity and/or percent positive expression of EphA3. A numerical scoring scheme (semi-quantitative simplified H-Score [SHS]) was used by a-board certified anatomic & clinical pathologist to capture tumor & non-tumor (specifically, fibroblast) reactivity of EphA3-specific monoclonal antibody. A survey of ten (10) normal bone marrow (NLBM), thirty five (35) cases of primary MF, five (5) post PV-MF cases, six (6) PV cases and four (4) cases with ET was made with the validated IHC assay. For NLBM, there was low level reactivity in a subset of immature-blast cells that represent a small fraction (1-5% or less) of the total population of nucleated cells. There was no reactivity in fibroblasts, if present. Low EphA3 immunoreactivity of NLBM samples per se is consistent with published RT-PCR data. For MPNs, significant EphA3 expression in both tumor and tumor-associated stromal fibroblasts was noted (not solely in areas of fibrosis, although not all MPN samples studied were in the fibrotic phase). Interpretation of EphA3 status in these NLBM and MPNs was used to refine the semi-quantitative SHS scheme in anticipation of setting patient sample cut-off. Between 60-70% of MF samples were deemed EphA3+ using this IHC assay for EphA3 expression. Targeting EphA3 tumor cells and/or tumor stromal fibroblasts may therefore constitute a novel approach to treating MF and other MPNs. The Phase 2 component of a clinical study is ongoing in which the activity of KB004, a non-fucosylated anti-EphA3 Humaneered® antibody, will be characterized in disease specific cohorts including AML, MDS, MF and other MPNs. Disclosures Locke: QualTek Molecular Labs: Employment. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership.

Description: Introduction: Myeloid malignant disorders are clonal diseases arising in hematopoietic stem or progenitor cells. Several somatic mutations involved in these diseases are currently known and routine molecular testing involves screening genes of therapeutic and prognostic significance. Mutational analysis of FLT3 in combination with NPM1 can be used to predict outcome and direct therapy in normal karyotype acute myeloid leukemia (AML). JAK2, MPL and CALR mutation detection complements the molecular diagnostic testing menu for myeloproliferative neoplasm’s (MPN): polycytemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In addition, these molecular markers are utilized for minimal residual disease (MRD) detection, e.g. following stem cell transplants. Little is currently known about the lineage-specific distribution of some of these markers. In this study we aimed to assess the distribution of common genetic mutations in multiple lineages (lymphoid, myeloid, monocyte, multipotent progenitors, myeloblast and erythroid) of MPN and AML utilizing fluorescent activated cell sorting (FACS). Method: Different cell lineage fractions (lymphoid (CD3+), mature (CD16+) and immature (CD16dim) granulocytes, monocyte (CD14+), erythroid(CD36+), multipotent progenitors (34+) and/ or myeloblasts (CD117+) of unseparated bone marrow and peripheral blood specimens of myeloid disorders were sorted on a BD Aria 2. The patient specimens selected were positive for either JAK2V617F, MPLW515L or CALR Exon9 insertion/ deletion (MPN’s) or for NPM1 and/or FLT3 mutations (AML; diagnostic, relapse and minimal residual disease (MRD)). Fractions were subsequently analyzed for the presence of the respective mutation by PCR and/ or bi- directional sequencing. Results: All FACS purified CD34+ progenitors, myeloid and erythroid cell fractions of MPL W515L (3) or CALR exon9 (12) positive MPN specimens demonstrated the presence of mutations, respectively. Interestingly, JAK2V617F was present in the sorted erythroid cell fraction in 5/6 MPN cases tested. However, the granulocyte cell and blast cell fraction of one polycythemia vera specimen tested negative for the presence of Jak2V617F. All lymphoid CD3+ T-cell fractions were negative. The NPM1 exon 12 mutation was uniformly detected in progenitors and all myeloid cell fractions of 3/3 diagnostic, 2/2 relapse and 1/8 MRD AML specimens. For 5/8 MRD cases all lineages tested negative. Surprisingly, for two MRD cases the mutation was observed only in the unseparated and myeloid lineages but not in CD34+ blast fraction. Similar to the above findings, FLT3 mutations were detected in multipotent progenitors, and/ or myeloblasts collections of 4/4 diagnostic specimens. However, the mutation was absent in the granulocyte and monocyte fraction of one case. No detectable signals were observed in the cell fractions of 5 MRD specimens and in the CD3+ lymphoid cell fractions of all AML cases. Conclusion: We conclude that CALR and MPL mutations are uniformly detectable in the unseparated bone marrow specimens of MPN’s as well as separated progenitor, erythroid, granulocyte and monocyte fractions. Interestingly, JAK2 mutations can be exclusively found in the erythroid lineage in PV, whereas it can be absent in the granulocyte and blast compartment. This finding may have implications on specimen processing to ensure that erythroids are retained for clinical Jak2 testing. In addition, our results support the hypothesis that CALR Exon9 mutations are early event driver mutations in comparison to JAK2V167F. Both NPM1 and FLT3 mutations, in AML, were detected in unseparated specimens as well as in the multipotent progenitors or myeloblasts at diagnosis and relapse. However, the NPM1 mutation was observed in unseparated specimens and granulocyte cell fractions of 2 residual disease cases, whereas it was surprisingly absent in the CD34+ cell lineage fractions. Conversely, FLT3-ITD was exclusively found in the progenitor cells and absent in the granulocyte lineage of one case at diagnosis. Our findings reported here may be able to assist assay development efforts for diagnostic and residual disease mutation detection in myeloid disorders. In addition, flow cytometric assessment of monitoring specimens prior to molecular analysis may be beneficial to decide if cell enrichment steps can give additional evidence for the presence of residual disease. Disclosures Burnworth: HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Angela:Hematologics: Employment. Wentzel:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.

Description: A sixty-one year-old Hispanic female with Waldenstrom’s Macroglobulinemia diagnosed in 2011 and successfully treated with 6 monthly cycles of Cyclophosphamide, Rituximab and Dexamethasone (CDR) from 12/11 through 5/12 was then put on a two-year maintenance scheme with Rituximab every three months. In February, 2014 (six months before the end of the planned treatment), she came to the ER complaining with severe headache, aphasia and blurred vision. A stroke was initially ruled out and she received Paracetamol with partial improvement. Nonetheless, symptoms re-appeared accompanied with disorientation and agitation. Antipsychotic medication was given with no improvement. On PE she was disoriented with aphasia, paraparetic and neck stiffness suggestive of meningitis. Blood tests, a MRI and lumbar puncture were performed showing leptomeningeal hyperintensity with no signs of encephalitis (Figure 1). Figure 1 Leptomeningeal reinforcement as seen in MRI. Figure 1. Leptomeningeal reinforcement as seen in MRI. CSF analysis showed WBC 64 cells/µL, (95% MNC), glucose= 9.8 mg/dL and proteins= 110 g/dL. Gram dye was negative. A geneXpert for Tuberculosis was negative. CSF cytology showed an infiltration of lymphoid neoplastic cells confirmed by cytochemistry (Figures 2a and 2b). Figure 2a: CD 20+ and 2b: kappa + neoplastic cells in CSF Figure 2a:. CD 20+ and 2b: kappa + neoplastic cells in CSF Figure 3 Figure 3. With these results a Bing Neel syndrome was diagnosed and IT Methotrexate was given for a total of 6 doses resulting in a nice reduction of the neoplastic cells. However, she relapsed in April/2014 and IV Fludarabine was started. We are planning to add IT liposomal Cytarabine. Additionally, MYD 88 gene mutation was detected. DISCUSSION: There are only 33 reported cases of Bing-Neel syndrome in the medical literature for the last 80 years and this one has been confirmed with the newest tools such as: MRI, cytochemistry and gene mutation. CONCLUSION: Bing-Neel syndrome should be suspected in every patient with Waldenstrom’s Macroglobulinemia and CNS impairment. Disclosures No relevant conflicts of interest to declare.

Description: Background: Hodgkin Lymphoma (HL) has improved markedly overtime. This study aims to assess our experience in treating advanced HL. Method: In a retrospective cohort study, patients age 〉14 with advanced HL from 2007-2010 were included. Using IPI, patients were categorized into low and high risk. ABVD and Hybrid ABVD+BEACOPP regimens were used. Result: 74 patients were included with the median age at diagnosis of 25.5 years. 50% presented with stage IV. Two different protocols were used (ABVD and Hybrid ABVD + BEACOPP). Median follow up was 30.3 months. Male gender and stage V were most often to receive hybrid regimen (odd ratio 3.74 and 6.58 respectively). When the two groups were compared, the higher risk group (mostly treated with hybrid regimen) showed similar survival rates to the lower risk group (treated mostly with ABVD) with p-values of 0.41 for OS and 0.42 for PFS. Conclusion: This study shows quite similar results to the published studies in the OS and PFS with a tendency to overcome the high-risk features by the hybrid regimen in advanced HL. However, this needs to be confirmed in a prospective randomized study. Disclosures No relevant conflicts of interest to declare.

Description: Objectives and background: The level of early MR is important for the optimization of therapy and making a decision to a switch to 2nd line therapy in patients (pts) who have not achieved an optimal response (OR). According to the recent recommendations at definition of OR on CML therapy, pts must have the level of BCR-ABL transcript gene at 10% or less and Ph-positive cells 35% or less at 3 months. But, in half of all cases of pts with BCR-ABL 〉10% at 3 months progression events happen between 3 and 6 months. The goal of our research was to investigate the prognostic impact of a large BCR-ABL transcript amount at 3 months on the subsequent response and the long-term outcome of CML pts treated frontline with IM. Methods: We have examined 185 pts, who have got IM from January 2010 up to the present. Molecular monitoring has been done regularly in all patients according to ELN recommendations. Median age was 49 years. All pts were in CP. BCR-ABL transcript levels were assessed by real-time quantitative PCR. Results: In our study 54% (100/185 cases) of pts achieved the optimal response with BCR-ABL transcript levels ≤10% at 3 months, 50,3% (93/185 cases) did it - with BCR-ABL transcript levels ≤1% at 6 months, and only 18% achieved the optimal response at 12 months. The comparative analysis has shown statistical differences in all characteristics in 2 groups of pts, who either achieved or not the optimal response at 3 months. Pts with BCR-ABL transcript levels ≤10% more often achieved CCgR at 6 months (g=0,0000), CCgR during all period (g=0,0004), MMR at 12 months (g=0,0000), MMR during all period (g=0,0012) and MR4 during all period (g=0,0000), pts had londer event-free (g=0,0432) and overall (g=0,0279) 4-year survival. Figure 1 Figure 1. In our center we have switched 6 patients to the 2nd TKI - those who didn't achieve the optimal response at 3 months. The switching showed the positive influence on loss level expression of BCR-ABL gene in 5 out of 6 patients. After that all patients achieved the optimal response in the future. For example, we had one patient with failure of IM at 3 months. We switched him the therapy to NI in 5 months after the diagnosis. As a result the patient achieved CCgR at 1,5 months, and the deep molecular response 4,5 log at 3 months. Conclusions: Early and deep responses to TKIs are predictive of long-term response and favorable survival outcomes. 3-month reduction in BCR-ABL transcript levels to 〉10% is a factor of bad effectiveness of TKI therapy and requires switching to the 2nd TKI. Timely switching to the 2nd TKIs allows us to achieve an optimal response in CML patients with level BCR-ABL 〉10% at 3 months. References: Timothy P. Hughes, Giuseppe Saglio, Hagop M. Kantarjian et al. Early molecular response predicts outcomes in patients with chronic myeloid leukemia in chronic phase treated with frontline nilotinib or imatinib. Blood, 27 February 2014 x Volume 123, Number 9. Disclosures No relevant conflicts of interest to declare.

Description: Background Patients (pts) with DLBCL who have received rituximab (R) antibody in the first-line setting, have poorer treatment outcomes when they are re-treated with R-chemotherapy in the salvage setting. Resistance to R may have developed. Ofatumumab (O) is a human monoclonal antibody against CD 20 that targets a different epitope than R. We hypothesized that replacing R with O in second line setting may overcome R resistance. The primary end-point of this ongoing phase II study is the objective response rate (ORR) following two cycles of O in combination with ICE (Ifosfamide, Carboplatin and Etoposide) or O-ICE. Secondary end points include the assessments of the safety and tolerability of the combination, progression-free (PFS) and overall survival (OS). Methods This was a multi-center, non-randomized phase II open label study. Pts with histologically confirmed DLBCL who failed or were refractory to first line R-based anthracycline therapy were eligible. Pts were accrued according to a 2-stage Simon’s Optimal Design at the National Cancer Centre Singapore and 3 medical centres in South Korea; Samsung Medical Centre, Ajou University Hospital and Soonchunhyang University Hospital. Stage 1 will enrol 24 pts and if 14 or more pts respond, we will proceed to stage 2 where another 37 pts would be recruited. Pts receive 3-weekly ICE (Ifosfamide 5g/m2 on day 2, Carboplatin AUC = 5 on day 2, Etoposide 100 mg/m2 day 1-3) and O (1000 mg), infused on day 1 and 8 of cycle 1, and thereafter only on day 1 of each cycle. A restaging scan is performed after cycle 2. Pts who are candidates for high dose chemotherapy (HDC) and autologous stem cell rescue (ASCR) would receive additional 1 or 2 more cycles of O-ICE before stem cell mobilization. Pts who are not candidates for HDC and ASCR would receive up to 6 cycles of O-ICE. Results A total of 24 pts with a median age of 54.5 years (27-75) were enrolled. Among them, 14 (58%) were male and 14 (58%) had stage III/IV disease. A median of 4 (2-6) courses of O-ICE was administered and 11 pts underwent HDC+ASCR. The ORR after 2 cycles was 58.3%; complete response (CR) 29.2% and partial response (PR) 29.2%. On treatment completion, the best ORR became 62.5% (CR = 37.5%, PR = 25%). Grade 1/2 non-hematologic toxicities, independent of relation to study drug, occurring in at least 20% of pts included nausea (50%), pain (50%), fatigue (42%), rash (42%), anorexia (33%), vomiting (33%), diarrhea (25%), edema (29%), fever (21%), pruritus (21%), cough (21%) and paresthesia (21%). Grade 3/4 toxicities included neutropenia (50%), thrombocytopenia (71%) and 4 anemia (54%). Febrile neutropenia occurred in 12.5% and no treatment related deaths were reported. The median follow up time was 8.2months (95% CI 5.3-14.0). The median PFS was 8.2months (95% CI 4.2-17.8) and the median OS was not reached (95% CI 8.6-not reached). Conclusion The study regimen met the predefined threshold of 14 responding pts in stage 1. The ORR of O-ICE was comparable to that of RICE in the CORAL study and this result is significant considering that all pts in this study had received R prior to study entry. Together with the tolerability data and promising activity, continuation of study enrolment in stage 2 has started. Disclosures Tao: Medscape: Honoraria; Takeda: Honoraria. Quek:Novartis: Consultancy, Honoraria; Bayer: Honoraria. Kim:Novartis: Research Funding; Cellgene: Research Funding; Takeda: Research Funding.

Description: We have treated a total of 30 patients with autologous T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19; 22 of 27 evaluable patients obtained either complete remissions (CR) or partial remissions (PR). Ten patients remain in ongoing CRs of 1 to 37 months duration. The CAR was encoded by a gammaretroviral vector and included the variable regions of an anti-CD19 antibody along with CD28 and CD3-zeta moieties. The first 21 patients treated on this protocol have been reported (Kochenderfer et al. Blood 2010, Blood 2012, and Journal of Clinical Oncology 2014). To enhance the activity of the transferred CAR T cells, T-cell infusions in the previously reported patients were preceded by a chemotherapy regimen of high-dose cyclophosphamide (60-120 mg/kg) plus fludarabine. In an attempt to reduce the overall toxicity of our anti-CD19 CAR treatment protocol, we substantially reduced the doses of chemotherapy administered before CAR T-cell infusions. This abstract communicates results from 9 patients with B-cell lymphoma who received a single infusion of 1x106 anti-CD19-CAR-expressing T cells/kg bodyweight preceded by a low-dose chemotherapy regimen consisting of cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2 (Table). Each chemotherapy agent was administered daily for 3 days. Eight of the 9 treated patients had DLBCL (diffuse large B-cell lymphoma) that was refractory to chemotherapy (chemo-refractory) or that had relapsed less than 1 year after autologous stem cell transplantation (ASCT). Both of these clinical situations carry a grim prognosis, with median overall survivals of only a few months. Despite the very poor prognoses of our patients, one patient with DLBCL obtained a CR and 4 DLBCL patients obtained PRs. In some patients, PRs included resolution of large lymphoma masses. Compared to our previous experience with anti-CD19 CAR T cells preceded by high-dose chemotherapy, toxicity was reduced when CAR T cells were infused after low-dose chemotherapy. None of the 9 patients treated with low-dose chemotherapy and CAR T cells required vasopressor drugs or mechanical ventilation, although some patients did have short-term neurological toxicity. Cytopenias were mild with a mean of only 1.4 days of blood neutrophils

Description: Preclinical studies previously reported by our group at ASH suggest that treatment with granulocyte-stimulating factor (G-CSF) may provide a potent and well-tolerated method to disrupt the 'leukemia cell niche'. Specifically, our data show that G-CSF treatment in mice reprograms bone marrow stromal cells, markedly inhibiting their expression of key chemokines/cytokines that support B lymphopoiesis, including stromal derived factor-1 (SDF-1/ CXCL12), interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), and interleukin-7 (IL-7). These changes in the bone marrow microenvironment result in a more than 10-fold decrease in B cells in the bone marrow, including early B cell precursors. Preliminary studies in humans suggest that G-CSF has a similar effect on the bone marrow microenvironment in healthy subjects. Since these B trophic factors also have been implicated in the maintenance of B cell acute lymphoblastic leukemia (ALL) cells, we hypothesize that G-CSF treatment in patients with acute lymphoblastic leukemia (ALL) will generate a 'hostile' bone marrow microenvironment for ALL cells, depriving them of key support signals and rendering them more sensitive to cytotoxic therapy. Herein, we specifically test whether niche disruption with G-CSF augments inotuzumab-mediated ALL cell clearance. Inotuzumab ozogamicin (Pfizer) is a CD22-calicheamicin antibody-drug conjugate. Initial clinical trials have documented activity of inotuzumab in B-ALL, with an overall response rate of 57% in relapsed or refractory B-ALL. To test this hypothesis, we developed a xenotransplantation model of human ALL using the human B-ALL G2 cell line, which expresses moderate levels of CD22. G2 cells transduced with a GFP-click beetle red luciferase retroviral construct to allow for in vivo bioluminescent imaging were injected intravenously into NOD/SCID/γc-/- (NSG) mice. The G2 cells preferentially home to the bone marrow but eventually metastasize to other organs, including the brain. Four cohorts of mice were studied (n = 9-21, each), including: 1) G2 ALL cells alone; 2) inotuzumab alone (single injection of 80 µg/kg, IP); 3) G-CSF alone (100 µg/kg/day for 14 days by continuous subcutaneous infusion); or 4) G-CSF plus inotuzumab (G-CSF was started four days prior to inotuzumab). Treatment with inotuzumab increased median survival from 25.5 days to 41 days (P 〈 0.0001). G-CSF alone had no effect on survival. However, combination of G-CSF plus inotuzumab further increased survival to 49 days (P = 0.01 compared with inotuzumab alone). G-CSF also improved survival in mice given two doses of inotuzumab (each 80 µg/kg, IP) 10 days apart. Bioluminescent imaging revealed that, in several of the mice in the G-CSF plus inotuzumab group, the bioluminescent signal was predominantly localized to the brain, with little or no signal in bone. These data are consistent with the possibility that inotuzumab may have limited penetration into the brain. Thus, the positive effect of combination G-CSF plus inotuzumab on survival may be limited by CNS disease in this model. Indeed, the addition of G-CSF to inotuzumab decreased the bioluminescent signal in bone regions on day 21 after inotuzumab by approximately 10-fold (Figure 1, P 〈 0.001). Collectively, these data suggest that G-CSF significantly augments inotuzumab-mediated ALL clearance from the bone marrow. G-CSF may provide a non-toxic and cost-effective approach to increase durable response rates to inotuzumab (and other types of immunotherapy) in patients with B ALL. Figure 1 Figure 1. Disclosures Link: Pfizer: provided Inotuzumab for preclinical studies Other.

Description: To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pores formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: I-131-Tositumomab is a radiolabeled murine monoclonal antibody that binds to the CD20 antigen on the surface of malignant and normal B lymphocytes, targeting radiation to B cells. I-131-Tositumomab has been studied with chemotherapy and in high-doses as part of conditioning regimens for autologous stem cell transplant (ASCT) in relapsed/refractory B cell lymphoma patients (pts). Non-myeloablative I-131-tositumomab was approved in the US and Canada for treatment of CD20 antigen-expressing relapsed or refractory low-grade follicular or transformed non-Hodgkin lymphomas (NHL). Since February 2014, tositumomab and I-131-tositumomab, the Bexxar¨ therapeutic regimen, is no longer commercially available. Methods: We conducted a single-center, phase I trial of I-131-tositumomab in pts with CD20+ B cell NHL with relapsed or progressive lymphoma after ASCT. Eligibility criteria included creatinine grade 3 non-hematologic adverse event. After treatment, pts were monitored weekly for 13 weeks for hematological and other toxicities with response assessments at weeks 7 and 13, and then evaluated at weeks 26, 39, and 52, then every 3 months during year 2, every 4 months during year 3, every 6 months during year 4, and yearly thereafter. Results: 12 pts were enrolled and received both dosimetric and therapeutic doses of I-131-tositumomab; 6 pts (50%) were men. Median age was 58.5 years (range: 49-66). 5 of 12 (41.6%) pts had diffuse large B cell lymphoma (DLCBL), 4/12 (33.3%) follicular lymphoma (FL), and 3/12 (25.0%) FL transformed to DLBCL. Median number of prior therapies was 4 (range: 3 - 8); median time from ASCT was 1.6 years (range: 0.3 - 6.8). 2 pts had progressive disease after ASCT (1 transformed FL; 1 DLBCL); 10 pts had relapsed from complete remission (CR) after ASCT. 7 pts received 25cGy I-131-tositumomab. The cohort receiving 25cGy was expanded to 6 due to grade 4 neutropenia (1 pt), and then expanded to 7 because 1 pt progressed prior to week 7 response assessment. 3 pts received 50cGy and 1 pt each received 65cGy and 75cGy. At all doses, I-131-tositumomab was well-tolerated. Treatment-related toxicities 〉 grade 3 were exclusively hematological and not dose dependent, including thrombocytopenia (2/12: 1 grade 3, 1 grade 4), neutropenia (2/12: 1 grade 3, 1 grade 4) and anemia (2/12: 2 grade 3). Hematologic toxicities were reversible in all cases, with median time to recovery to 〈 grade 3 toxicity 7 days. Non-hematological adverse events were mild and 〈 grade 2. The most commonly reported non-hematological adverse event was fatigue. Overall response rate at week 7 was 41.6% (5/12) with 33.3% (4/12) pts achieving CR (2 pts with FL; 2 pts with DLBCL). Median duration of response was 13.8 months. As of May 2014, 2 pts are still in CR, including 1 pt with FL at 10.3 months and 1 pt with DLBCL at 5.5 years. Median progression free survival is 6.5 months; median time to treatment failure is 4.4 months. At the median follow-up of 21.9 months (range: 10.3 – 66.3), overall survival is 68.7%; no secondary hematologic malignancies have been observed. Conclusion: This is the first study of I-131-tositumomab RIT in pts with relapsed or progressive NHL after ASCT. In pts with CD20+ DLBCL, FL, or transformed FL following ASCT, non-myeloablative I-131-tositumomab is well-tolerated and can achieve sustained complete remissions. Table 1:Dose Escalation SchemaPlatelet count〉100,000, 150,000Dose level 120 cGy25 cGyDose level 240 cGy50 cGyDose level 355 cGy65 cGy Disclosures Schuster: GlaxoSmithKline: Research Funding.

Description: AN and MM are co-senior authors. Background. Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two widely used reduced-intensity conditioning (RIC) regimens for allogeneic hematopoietic stem cell transplantation (allo-SCT). Patients and Methods. In the current survey, we compared transplantation outcomes in a cohort of 394 acute myeloid leukemia (AML) patients given grafts from HLA-identical siblings after FB (n=218; with a total busulfan dose ranging between 7.1 and 8.9 mg/kg p.o., or between 6.0 and 6.9 mg/kg i.v.) or FM (n=176; with a total melphalan dose ranging between 130 and 150 mg/m2). Patients given manipulated grafts and those given T cell depleting agents (ATG or alemtuzumab) were not included. At time of transplantation, 266 patients (68%) were in first complete remission (CR1), 69 (18%) in later CR, while 59 patients (15%) had advanced diseases. Three-hundreds and fifty-two patients (89%) received peripheral blood stem cells while the remaining 42 patients received bone marrows as stem cell source. Results. Three FB patients but no FM patients failed to engraft. Median time for reaching 500 neutrophils was 17 (1-50) days in FB patients versus 14 (9-43) days in FM patients (P

Description: BACKGROUND: Primary mediastinal B-cell lymphoma (PMBCL) are a subtype of diffuse large B cell lymphoma (DLBCL), that represent 2-4% of all non-Hodgkin lymphoma (NHL) and 6-12% of DLBCL. They affect young adults with a median age at diagnosis of 35 years old and are more frequent in females (2:1). Approximately 80% of patients have stage I or II disease at the time of diagnosis, with B symptoms, bulky mass and superior vena cava syndrome (SVCS) in 50 %. There is no standard initial therapy regimen, anthracyclines with Rituximab are the most used. Local consolidation radiotherapy (RT) is reserved for bulky disease and to complete partial remissions (PR) after chemotherapy. AIMS: To analyze prognostic and epidemiological factors, treatment administered and response and survival rates of patients diagnosed with PMBCL in our center. METHODS: Retrospective cohort study between September 2003 and June 2014. The treatment received was 6 to 8 cycles of CHOP-like chemotherapy (Cyclophosphamide, Adriamycin, Vincristine, Prednisone ± methotrexate / Intrathecal triple therapy MTX- TIT ), with or without Rituximab, and subsequent radiotherapy if necessary. The following results were evaluated by univariate analysis: overall survival (OS), disease-free survival (DFS) and incidence of relapse after diagnosis. DFS was defined as the interval of time from complete remission (CR) to relapse or last visit. Response to treatment was assessed by PET-CT. RESULTS: We observed 14 patients diagnosed with PMBCL in our hospital with a median age of 33 years (r, 21-58 years) and female predominance (Š:‰ 10:4). At diagnosis 42.8 % had B symptoms and 42.8% elevated lactic dehydrogenase. Fifty percent of patients presented with SVCS. Central nervous system and bone marrow infiltration was not observed. Early-stage disease at diagnosis (I 30% and II 53%) was observed in 83%, with IPI 1 in 78.5% of patients. The number of cycles received was 6 in 78.5 %, 8 cycles in 14.2 %, and only 1 cycle of CHOP in one patient. Altogether 71.4 % received Rituximab and 71.4 % received MTX-TIT. Ten patients (71.4%) received RT (median 36 Gy) after chemotherapy, 9 of which had initial bulky disease. We observed an overall response rate of 92.8 % after chemotherapy (57.1 % CR, 35.7% PR). After a median follow-up of 60 months (r, 4.4 to 130.8 months) 12 patients had responded to treatment, were alive, without relapse and in complete response, and one patient is currently receiving the 6º cycle of CHOP pending reevaluation (awaiting last MTX-TIT and RT consolidation) and obtained CR after the third cycle of R-CHOP-MTX-TIT. Median overall survival was not reached and median DFS was of 54.8 months. Only one patient died (mortality 7.1 %) due to influenza A in the context of postchemotherapy aplasia after the first cycle of CHOP. CONCLUSIONS: We observed prognostic and epidemiological factors similar to those described in literature, although in our series, CHOP -like chemotherapy ( ± MTX- TIT ) with or without Rituximab and RT has shown improved survival rates and 100% of CR. Consolidation radiotherapy was successfully used to complete treatment in patients that only achieved PR after chemotherapy. Rescue chemotherapy followed by autologous hematopoietic cell transplantation was not required. It is difficult to draw conclusions on the impact of the therapeutic regimen received, we believe multicenter analysis with larger numbers of patients are necessary. Disclosures No relevant conflicts of interest to declare.

Description: Objectives Patients with aggressive non-Hodgkin’s lymphoma (NHL) and acute lymphoblastic leukemia (ALL) with high risk of CNS infiltration or recurrence may receive prophylaxis or be treated with intrathecal injections (IT) of liposomal cytarabine (LC) once every 2 weeks (due to its sustained release of cytarabine). Sequential flow cytometric studies in patients with CNS involvement and treated with LC, allow to detect tumor cells as well as LC liposomes (LCL) in the CSF. The true meaning of the presence of these liposomes during the treatment with LC is still unknown. Patients and Methods In this study, we aimed to investigate the presence of LCL detected by Flow Cytrometry (FC) among 166 CSF samples of 69 patients treated with LC. Samples were taken before each administration of intrathecal LC and centrally processed. Results Results are shown in the below table. Abstract 5444. Table 1Patients with NHL/ALL, treated with Intrathecal Liposomal CytarabineNPatients69Samples166Liposomal Cytarabine Liposomes (LCL) in CSFN%Patients1319%DLBCL646%LLT215%ALL215%MCL18%MM18%CLL18%Samples2616%Males862%2616%Age66 years[33-84]LCL in CSF of patients receiving Liposomal CytarabinePROPHYLAXISNToxycityTotal nº of IT LC dosesPatients7nº of IT LC doses*3 (2-5).LCL7After 1st dose5After 2nd dose21 LLT patient: arachnoiditis, high intrathecal pressure, papillitis, without sequelae5After 3rd dose2After 5th dose1LCL in CSF of patients receiving Liposomal Cytarabine leptomeningeal disease TREATMENTNTumor: ToxycityTotal nº of IT LC dosesPatients6nº of IT LC doses*6 (4-7)LCL6After 1st dose31 DLBCL patient: Headache, dizziness and instability51 MM patient: Headache, dizziness and instability4After 3rd dose2After 5th dose11 ALL patient: Headache, dizziness and instability7 * Median [range] The thirteen patients were simultaneously treated with LC and IT + IV dexamethasone. It was found no relationship between the amount of LCL and toxicity. Conclusions: Presence of LCL in CSF is detected in 15-20% of patients receiving IT LC as prophylaxis / treatment and it does not show a direct relationship with presence of toxicity. Although the true clinical significance of the presence of LCL in CSF remains unknown, their monitoring could be useful to individualize the treatment with CL IT (dose and time between doses). Disclosures Off Label Use: Liposomal cytarabine is not approved for prophylaxys of lymphomatous meningitis..

Description: INTRODUCTION: Though widely used and accepted as a first line regimen, published data regarding R-CODOX-M/IVAC is very limited. Up to date only three prospective studies and one case series have been reported. Herein, we present our institution’s experience with the management of Burkitt’s lymphoma with a special emphasis on the R-CODOX-M/IVAC regimen. METHODS: The files and electronic records of 35 patients diagnosed with Burkitt’s lymphoma between January 2005 and June 2014 were retrospectively revised. RESULTS: The median age at diagnosis was 40 (21-86 years). Male patients constituted 71.4 (n=25). Stage IV disease was present in 60.6% (n=20), and stage I disease in 27.3% (n=9) of the patients. ECOG performance scoring was 〈 3 in 70.6% (n=24) and Burkitt’s lymphoma risk scoring (availability of complete surgical resection, stage, serum LDH and CNS involvement) 〉 2 in 58.6% (n=17). The distribution of patients to administered regimens were as follows: R-CODOX-M/IVAC 10 patients (28.6%), HyperCVAD 5 patients (14.3%), R-CHOP 9 patients (25.7%), R-CVP 2 patients (5.7%), CALGB 10002 1 patient (2.9%) and other regimens (high dose methotrexate, high dose cytarabine, vincristine-prednisolone, etc.) 8 patients (23.0%). Median overall survival (OS) was 23.5 (1-114) months and median progression free survival was 17.0 (0-114) months. Relapses occurred in 7 patients (20.0%), mortality in 12 patients (34.3). R-CODOX-M/IVAC group (n=10) was compared to other rituximab containing regimens (R-CHOP, R-CVP) (n=11) and HyperCVAD (n=5) and other rituximab-free regimens (n=9) with respect to patient and disease characteristics, DFS and OS. The disease stage, ECOG performance score and Burkitt’s lymphoma risk score were similar between the four groups (p=0.6, p=0.2 and p=0.2, respectively). However, median OS was 13 (1-42) months in R-CODOX-M/IVAC group and it was significantly lower than other regimens (p=0.04 log rank test). Median PFS was 13 (0-42) months and also lower compared to other regimens, however, the difference was not statistically significant (p=0,1 log rank test). CONCLUSIONS: Burkitt’s lymphoma is currently one of the most aggressive mature B- cell origin lymphomas and there is no consensus on the standard of care. CODOX-M/ IVAC is one of the most frequently used regimen. Adding rituximab is also known to prolong overall survival in this patient population. Though the sample sizes are too small and the drawbacks of the retrospective study design exist, the inferior OS and PFS observed in this study with R-CODOX-M within patients having similar disease characteristics should be taken into account. Further studies comparing the efficiency of currently used regimens are needed to reach a clear conclusion. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Background: Primary mediastinal B-cell lymphoma (PMBCL) is a subtype of diffuse large B-cell lymphoma originating from the thymus with its own epidemiological, clinical, immunophenotypic and prognostic features and that was included as a distinct clinical entity in the last World Health Organization classification (2008). It is more prevalent in young female, and is characterized by a large mediastinal mass, with frequent infiltration of adjacent structures. Dissemination by distant sites may be identified at diagnosis or during the disease progression. It shows many similar aspects to nodular sclerosis Hodgkin’s Lymphoma in terms of clinical, pathological and immunohistochemical features. The standard treatment is based on multidrug regimens containing anthracyclines associated with rituximab and consolidation with radiotherapy. A recent study published in the NEJM in 2013, with a single-arm treatment with infusional dose-adjusted DA-EPOCH-R with no radiotherapy in untreated PMBCL, demonstrated 97% of overall survival (OS) and 93% of event-free survival (EFS) with a median of 5 years of follow-up. Methods: We analyzed retrospectively 40 patients with PMBCL treated at São Paulo’s Cancer Institute from June 2007 to January 2014. The objectives of the study were to compare the complete response (CR), progression-free survival (PFS) and overall survival (OS) rates between two different treatment strategies. All patients were initially evaluated with blood tests, whole-body computed tomography (CT) or fluorodeoxyglucose-positron-emission tomography (PET-CT) and bone marrow biopsy. Two chemotherapy regimens were used in the patient’s treatment: 6 to 8 cycles of conventional R-CHOP 21 with or without radiation therapy (n = 23) and R-CHOP regimen with addition etoposide (DA-EPOCH-R or R-CHOEP) with or without radiotherapy (n = 17). After 4 cycles of treatment, patients were evaluated for response to determine the total number of cycles (6 or 8). Results Among the 40 enrolled patients, 65% were female with median age of 31 years (range 14 to 62 years). The median size of the mediastinal mass was 13cm in the longest axis. Half of the patients (50%) were in advanced stage (III or IV of Ann Arbor staging) and 75% were in good prognosis category of R-IPI ( 1 or 2 risk factors of the International Prognostic Index Score for non Hodgkin lymphoma). 57,5% of patients were treated with R-CHOP and 42,5% had etoposide as part of the their treatment regimen (12,5% DA-EPOCH-R and 30% R-CHOP plus etoposide (100mg/m2 D1-D3). There was no statistically significant difference in CR rate between RCHOP vs RCHOP + etoposide (86.9% vs 86.6%). There were no differences in PFS or OS for the 2 groups (p=0.8202 and 0.9410). Conclusion The addition of etoposide to RCHOP regimen appears to increase OS and PFS of patients with untreated PMBCL as previously demonstrated. In our service, where there is difficult in hospitalization for the administration of infusional regimens such as DA-EPOCH-R, it was necessary to adjust for outpatient to R-CHOEP. The comparison between the two groups (RCHOP vs RCHOEP/DA-EPOCH-R) showed no statistically significant difference in CR, OS and PFS. However, the median of follow-up of patients who received etoposide was not sufficient to analyze the data adequately. Overall Survival Figure 1. Overal survival betwen R-CHOP and R-CHOEP in PMBCL (p = 0.8202) Figure 1. Overal survival betwen R-CHOP and R-CHOEP in PMBCL (p = 0.8202) Progression Free Survival Figure 2. Progression free survival betwen R-CHOP and R-CHOEP in PMBCL (p = 0.9410). Figure 2. Progression free survival betwen R-CHOP and R-CHOEP in PMBCL (p = 0.9410). Disclosures No relevant conflicts of interest to declare.

Description: Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, and is frequently diagnosed in elderly patients. Elderly patients with DLBCL are known to have worse outcomes than younger patients with the same disease, in part due to comorbidities and poor performance status that may decrease their ability to tolerate treatment. A recent phase II study (Lancet Oncology 2011;12:460-8) demonstrated that patients with DLBCL who were older than 80 years of age had excellent tolerability while still maintaining efficacy when treated with an attenuated dose of R-CHOP called “R-mini-CHOP”. This paper prompted a change in treatment policy of very elderly patients with DLBCL at the London Regional Cancer Program. In November 2011, we began treating all DLBCL patients 〉 80 (and select patients aged 70-79 with significant comorbidities) with the R-mini-CHOP regimen rather than with standard R-CHOP or a ‘randomly dose-attenuated version’ of R-CHOP, which had been our practice prior to the policy change. Objective: This quality assurance study was undertaken in order to monitor and ensure the safety and efficacy of this practice change. Methods: Data was collected prospectively on all elderly patients with DLBCL who were initiated on R-mini-CHOP between November 2011 and January 2013. A retrospective chart review was also performed on patients 〉 80 treated at our centre in the preceding 10 years, after R-CHOP became standard treatment in elderly patients with DLBCL. We used 16 patients 〉 80 who received full dose R-CHOP between January 2010 and November 2011 as a historical control group for our prospective cohort. Results: Since November 2011, 17 patients (median age 81, range 71-90) have been treated with R-mini-CHOP and were enrolled in this quality assurance study. All 17 patients had stage III or IV disease. In contrast, only 6/16 patients in the retrospective group were advanced stage (p=0.00004). 12/17 R-mini-CHOP patients (70.6%) had an ECOG 〉 1, vs. 7/16 R-CHOP patients (43.8%, p=0.070). No dose modifications have been required in patients on R-mini-CHOP. 11/16 patients on R-CHOP underwent dose reductions due to side effects of chemotherapy. The majority of patients in both cohorts received primary prophylaxis with neupogen/neulasta. Five patients remain on R-mini-CHOP, all showing evidence of clinical response. Follow-up is too short to report on PFS or OS, but overall response rate thus far is 10/12 (83%) with 7 CRs (58.3%) and 3 PRs (25%). This compares favorably to the retrospective group who had an ORR of 13/16 (81%, p=0.30), 7 CRs (43.8%) and 6 PR’s (37.5%). One patient in each group had stable disease at the end of treatment. One mini-R-CHOP patient progressed on treatment and ultimately died of lymphoma. In the retrospective cohort, 4 deaths occurred secondary to infectious complications of chemotherapy during treatment period. C onclusions: Results with R-mini-CHOP have been encouraging so far. Despite the more aggressive disease (higher stage, inferior ECOG) in our R-mini-CHOP patients compared to the historical controls treated with R-CHOP, similar response rates are being achieved, with fewer adverse effects and better tolerability. Ongoing enrollment and longer follow-up will further help define the role of R-mini-CHOP in the very elderly patient population. Updated results with longer followup will be presented at the meeting. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Historically, the predominant treatments for CLL and MCL have included intravenously (IV) administered chemotherapeutic regimens. However, there has been a rapid growth recently in new CLL/MCL treatment options, including orally administered products. Moreover, the therapeutic profiles of these newer agents are generally improving, particularly for patients with relapsed or refractory disease. There is currently a paucity of research evaluating patients’ experiences with current and emerging treatments for both CLL and MCL. The objective of this study was to explore patients’ experiences with, and preferences for, CLL/MCL therapies. Methods Data were collected from 27 May to 30 June 2014 via a self-administered, online, quantitative survey that was developed based on qualitative interviews with CLL and MCL patients. Patients with CLL or MCL were recruited from a market research panel or were referred by a physician or other patients. Patients were eligible for the survey if they had been diagnosed with CLL or MCL by a physician, had been treated for ≥ 2 weeks in the past 12 months, and were not a clinical trial participant. Quantitative data were collected regarding treatment history, factors influencing treatment selection, opinions of treatment, experiences with IV treatment, and patient-reported health and wellness. Results A total of 75 patients (52 with CLL, 23 with MCL) completed the survey. CLL and MCL patients provided similar responses to most questions. The majority of patients were white (72%), 〈 65 years of age (92%), and privately insured (81%); 51% were female. The mean EQ-5D self-reported score for health state (0 = worst imaginable to 100 = best imaginable) was 61. Almost half of both CLL and MCL patients had received one (44%) or two (40%) lines of treatment. A much smaller proportion of patients had received three or more lines of treatment (3rd line=4%, 4th line=8%, and 5th line or more=4%). For the following questions, patients were asked to check all reasons that applied to them. The most common reasons for changing/stopping past CLL/MCL treatments were: too many side effects (38%), medication did not work to treat the disease (36%), symptoms were not relieved (33%), and work productivity declined because of side effects (26%). When asked about reasons for dissatisfaction with CLL/MCL treatments, the most commonly reported reasons were safety concerns (33%) and side effects (32%); 37% said none applied. Common reasons for satisfaction with CLL/MCL treatment were: medication is covered by my insurance (49%), convenient dosing schedule (39%), easy to use (33%), works most of the time (32%), works all of the time (31%), works better than other drugs I have tried (28%), and is affordable (28%). 65% of patients visited their doctor together with a spouse, family member, friend, or paid helper. Frequency of IV treatment was every 1-2 weeks for 67% of patients and required an average of 3 hours for infusion and 4 hours overall, including travel and waiting time. Mean satisfaction with treatment (1 = extremely dissatisfied to 7 = extremely satisfied) was 5.3. Longer duration of effect was the most important treatment feature for CLL/MCL patients. Conclusions The results of this survey show that the greatest source of patients’ dissatisfaction with their current or most recent treatment for CLL/MCL was the treatment’s safety and tolerability. Many patients receive IV treatment for CLL/MCL every 1-2 weeks, and each IV treatment requires several hours of time not only for the patient, but in nearly two-thirds of cases, also for a family member or friend who accompanies the patient. These survey results suggest that substantial unmet needs remain for the care of patients with CLL and MCL. Disclosures Schenkel: Janssen Scientific Affairs, LLC: Employment, Stock ownership Other. Naim:Janssen Scientific Affairs, LLC: Employee of Janssen Scientific Affairs, LLC at the time of the study Other. Roland:Janssen Scientific Affairs, LLC: Consultancy. Wyant:Janssen Scientific Affairs, LLC: Consultancy.

Description: Background: PTCL is a heterogeneous group of hematologic malignancies associated with a poor prognosis for most subtypes. In the relapsed setting, hematopoietic stem cell transplantation (HSCT) is the only potentially curative option for patients with PTCL. However, many patients are not able to achieve an adequate response to allow for HSCT. Belinostat (Beleodaq) is a potent pan-histone deacetylase inhibitor that was recently approved in the US for the treatment of patients with R/R PTCL. Approval was based on data from the pivotal Phase 2 BELIEF study that enrolled 129 patients with R/R PTCL (N = 120 evaluable), and demonstrated durable clinical benefit and tolerability. This analysis presents data for 12 of the enrolled patients (9 evaluable) who proceeded to HSCT following belinostat treatment. Methods: Patients with R/R PTCL received belinostat as a 1000 mg/m2IV infusion on Days 1-5 of a 21-day cycle. The primary endpoint of the study was Objective Response Rate (ORR; Complete Response [CR] + Partial Response [PR]) determined by an Independent Review Committee (IRC). We present efficacy and safety data for the subset of 12 patients who subsequently went on to HSCT. Results: Among 12 patients who subsequently proceeded to HSCT, 4 went on to receive an autologous HSCT and 8 received an allogeneic HSCT; 8 patients (67%) were female and 4 (33%) were male, and the median age was 54.5 (range 31-71) years. The median number of prior anticancer therapies was 2 (range 1-8), including 3 patients with prior autologous HSCT. The median number of belinostat treatment cycles was 2.5 (range 1-14) compared to the median of 2.0 (range 1-8) in the overall study population. Most patients in this subgroup had PTCL-Not Otherwise Specified (58.3%), angioimmunoblastic T-cell lymphoma (16.7%), or anaplastic large cell lymphoma (16.7%); 41.7% of patients had Stage IV disease. Three of the 12 patients were not evaluable for response due to insufficient histological material for confirmation by central pathologic analysis. The IRC-confirmed ORR for the 9 evaluable patients was 33.3% vs 25.8% in the study overall, and included 2 CRs, 1 PR, 2 patients with stable disease (SD) and 3 patients with progressive disease (PD). Duration of Response after transplant ranged from 41-261 days for the 3 belinostat responders. At last study contact, 2 patients had died from cardiac events (unrelated to belinostat) and 10 remained alive, with Overall Survival (OS) ranging from 8-23+ months. Most adverse events (AEs) were Grade 1-2, with two treatment-related Grade ≥3 AEs (neutropenia and prolonged QT interval); 3 serious AEs (arthralgia, lower limb fracture, and pyrexia) were reported in this subgroup. Conclusions: Belinostat was well tolerated in previously treated patients with R/R PTCL and enabled some patients to proceed to HSCT. Three patients responded and went on to HSCT following belinostat; the remaining patients had HSCT following SD (2), PD (4) or were not evaluable (3). OS was prolonged when compared to historical controls. Summary of Patients Treated with Belinostat Who Subsequently Went on to Hematopoietic Stem Cell Transplantation Sorted by Subtype and Response Table 1PatientSubtype(Stage)Prior RegimensECOGPSBelinostat CyclesIRC ResponseOS(months)DoR(days) Evaluable Patients931-003^PTCL-NOS (IIIB)3114CR11.56261907-006PTCL-NOS (IIA)5 + auto SCT02SD13.93-907-007^PTCL-NOS (IVA)402SD12.09-907-005^PTCL-NOS (IIIA)202PD13.63-140-002PTCL-NOS (IVA)817PD17.64-914-006PTCL-NOS (IIIA)4 + auto SCT02NE13.73-245-001AITL (UNK)106PR19.9141221-003ALCL ALK– (IA)2 + auto SCT011CR20.4173907-001ALCL ALK– (IVA)204PD22.87- Non-Evaluable Patients*914-002PTCL-NOS (IVB)102PD7.75-147-002^AITL (IIIB)221NE9.43-147-001Hepatosplenic TCL (IVA)103NE10.22- AITL = angioimmunoblastic T-cell lymphoma; ALCL = anaplastic large cell lymphoma; ALK = alkaline phosphatase; auto = autologous; CR = complete response; DoR = duration of response; ECOG = Eastern Cooperative Oncology Group; IRC = independent review committee; NE = not evaluable; NOS = not otherwise specified; OS = overall survival; PD = progressive disease; PR = partial response; PS = performance status; PTCL = peripheral T-cell lymphoma; SCT = stem cell transplantation; SD = stable disease; TCL = T-cell lymphoma *Lack of central pathologic confirmation resulted in exclusion from the evaluable population ^Autologous hematopoietic SCT Disclosures Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Hsu:Spectrum Pharmaceuticals: Employment. Choi:Spectrum Pharmaceuticals: Employment. Allen:Spectrum Pharmaceuticals: Employment. Visser:Sanofi: Membership on an entity's Board of Directors or advisory committees. Horwitz:Celgene: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Infinity: Research Funding; Kiowa-Kirin: Research Funding; Seattle Genetics: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Jannsen: Consultancy.

Description: Relapse remains the major cause of death in older patients transplanted for AML in first complete remission (CR1) or for patients with advanced MDS at any age. Conventional myeloablative conditioning followed by allogeneic blood or marrow transplantation is associated with significantly less relapse compared with RIC when performed in younger patients with AML or MDS, but the toxicity of this approach in older patients is prohibitive. We hypothesized that pharmacokinetic targeting to optimize busulfan (Bu) exposure, combined with the administration of AZA post transplantation would mitigate the risk of relapse while avoiding non-relapse mortality (NRM) and ultimately improve progression free survival (PFS). Here we report the results of a Bu test dose strategy targeting daily Bu exposure as determined by the area under the plasma concentration versus time curve (AUC). The primary endpoint of the study was two year progression free survival (PFS). An important secondary objective was to determine whether administration of a test dose of Bu with post test sampling would enable achievement of a daily target Bu AUC level of 4000 uM*min in at least 80% of the recipients. We used this strategy as part of a RIC regimen on a prospective multi-center phase II trial conducted by the Alliance (formerly Cancer and Leukemia Group B (CALGB)). Eligibility included patients with AML in CR1 aged 60-74 years inclusive, MDS with IPSS risk 〉 Int-2 with less than 10% marrow blasts and age

Description: 44 years old lady presented with fever and pancytopenia in March 2014.CXR showed bilateral pneumonia. Hb 7.5xg/dl, plt 42x x109/l, wbc 1.2 x19neutrophils22%, lymphocytes75%.Circulating lymphocytes with hairy cytoplasmicprojections and indented nuclei were noted. Flow cytometry showed these abnormal lymphoid cells were CD19+ve, CD5-ve, CD 23-ve, FMC7+ve, CD25+ve, CD11c+ve and CD103+ve.Bone marrow biopsy revealed a hypercellular marrow with dense infiltration of lymphoid cells of the same immunophenotype. Braf V600 mutation was detected. Cladribine or pentostatin was out of stock and import of these drugs would take at least 2 months. In view of the severe pancytopenia and on -going infection, various treatment options were discussed with, the patient .Patient decided to start on vemurafenib 960mg twice daily while awaiting cladribine. After 8weeks of treatment, the peripheral blood counts were normalized. Hb 12.2g/dl, plt 153x x109/l, wbc 3.1x109/l, neutrophils 53%,lymphocytes 40%. Braf V600 mutation was no longer detected. Vemurafenib was well tolerated and the patient received treatment mainly as outpatient. Vemurafenib was discontinued after 8 weeks and the patient then received a 5-day course of cladribine. She remained in complete remission Discussion Vemurafenib had shown to be safe and effected in hairy cell leukemia patients who were refractory to or who relapsed after purine analogs.1,2 Still to be determined are the correct vemurafenib dosing strategy, the best timing , duration and scheduling of vemurafenib. Due to unusual circumstances, our patient received 8 weeks of vemurafenib as first line therapy. The patient achieved a complete remission with minimal residual disease. Follow data is needed to see how long the patient remains in remission 1. N Engl J Med 2014; 370:286-288 2. 19th Congress of the European Hematology Association (EHA): Abstract S696. Disclosures Off Label Use: Vemurafenib as first treatment of Hairy Cell Leukemia. Wong:johnson &johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biogen-Idec: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer, MSD, Roche, BMS, Baxter, Amgen, Alexion: Research Funding.

Description: Introduction. Serum albumin (SA) has been shown to be a prognostic marker in many hematological malignancies, and in diffuse large B-cell lymphoma (DLBCL) before the introduction of rituximab. SA may be a surrogate for age, comorbid status, and disease severity. Here, we aimed to assess the role of SA as prognostic marker for overall survival (OS) in patients with newly diagnosed DLBCL treated with and without rituximab (R) containing regimens (CHOP and CHOP-like). Patients and method We collected 738 patients complete for IPI factors [age, LDH, extra nodal sites, stage and performance status (PS)] and SA recorded in the GISL database from 1998 to 2012. OS was estimated using the Kaplan-Meier method, with Cox proportional hazard model used to identify potential risk factors for time-to-event data. Optimal cut-off for SA was calculated by means of ROC curve at 5 years of follow-up. Stability of the cut-point was analyzed using bootstrap techniques. The role of SA was explored interacting SA with R and adjusting by IPI. The strength of SA as prognostic factor was evaluated counting the bootstrap frequency of inclusion (BIF, 1000 re-samples) of SA, adjusted by the IPI factors, in Cox PH regression. Results Of the 738 patients included in the study 322 (44%) were treated with R-CHOP and 416 (56%) were treated without regimens containing R. The median age at diagnosis was 60 years (range 21-89) and 45%, 25% and 30% were categorized in IPI 0-1, 2 and 3/5 respectively; and the median SA was 3.8 g/dL (range 1.0-6.4). Patients treated with R showed a greater % of high IPI 3/5 (43%) compared to those not treated with R (20%, P

Description: Introduction: Currently, there is no consensus regarding diagnosis and treatment of occult lymphomatous menigeal involvement of cerebrospinal fluid (CSF) in patients with systemic non-Hodgkin lymphomas (NHL), especially in patients with minimal number of clonal cells detected by flow cytometry (FCM) or by cytology. Aim of the study: To describe a cohort of patients with occult lymphomatous involvement in cerebrospinal fluid including the diagnostic methods as well as management. Patients and methods: CSF at diagnosis of B-NHL was examined by FCM, cytology, biochemistry and microRNA analysis in a cohort of 62 patients (systemic DLBCL N=45, MCL N=15, Burkitt lymphoma N=2) between 2010 and 2013. Occult meningeal involvement was defined by: a/ FCM: absolute positive number of clonal cells

Description: Background and purpose CCLG-2008 protocol has been carried out for more than 5 years in most parts of China. This retrospective cohort study analyzed the clinical features and the role of prognosis index on the outcomes of patients with childhood acute lymphoblastic leukemia (ALL) treated by CCLG-2008 protocol in the Children's Hospital of Soochow University, Suzhou, China. Procedure From 2009 to 2013, 379 evaluable patients were enrolled in this protocol. ALL diagnosis was made by MICM and early prognosis index including age, gender, white blood cell (WBC), immunotype, molecular findings, karyotype and prednisone response were evaluated as predictors of adverse events by using SPSS 16.0. P-values 100*109/L, P120 months, P=0.018), more adverse karyotype distribution, a poor prednisone response (P100*109/L) or increased age (〉120 months) showed higher disease-relapse risk than other groups (P=0.003, 100*109/L have the shortest survival. Figure 2 a EFS and OS of B-ALL patients with fusion gene positive or negative. Figure 2. a EFS and OS of B-ALL patients with fusion gene positive or negative. Figure 2b EFS and OS of B-ALL patients with different risk of karyotype. Figure 3 EFS and OS of patients with different response to prednisone. Figure 3. EFS and OS of patients with different response to prednisone. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Acute Myeloblastic Leukemia (AML) is the most frequent acute leukemia in the adults and its incidence increases with age. There are few studies about the demography and outcomes of AML patients in Chile and the only report belongs to a public hospital from 2000. We discuss the results of patients treated in our institution with AML non promyelocytic. Patients and Methods: Retrospective analysis of the epidemiologic, clinical and laboratory characteristics of diagnosis (cytology and flow cytometry) and treatment of AML non promyelocytic patients between 2010-2014. Statistical analysis of the data was performed using SPSS Statistics v21 software. Results: 63 patients were diagnosed with AML non M3, 52 males (66%), with a median age of 55.4 years (range: 16 - 89). Diagnosis laboratory tests (mean values and ranges) were: WBC 45.989/mm3 (range: 700 - 405.000); hemoglobin 9,1 g/dl (range: 5,2 - 14,1); platelets 75.548/mm3 (range: 10.000 - 454.000); peripheral blood blasts 38% (range 0 - 100); bone marrow blasts 74% (range 25 - 100%). The cytogenetic risk groups were: favorable (n=5, 8%), intermediate (n=33, 52%), adverse (n=8, 13%) and unknown (n=17, 27%). Of all the patients, 75% (n=47) received induction chemotherapy (CT) and 25% (n=16) palliative care. The mean age of the group with cytogenetic analysis was 51.2 years and only 8.6% did not receive consolidation CT. On the other hand, the group of patients with unknown cytogenetics had a mean age of 68 years and 57% did not receive consolidation CT. The mean survival of the CT group was 27.3 month (range: 0 - 53). By contrast, the mean survival in the palliative care group was 1 month (range: 0 - 6). The mean follow up in all patients was 13 months, (range: 1 - 55) and 17 months (range: 1 - 54) in the group that received CT. 87% (n=41) of patients with CT had febrile neutropenia with respiratory and intestinal focus most commonly identified. The induction mortality was 4,2% (n=2). Complete cytologic remission was achieved in 70% (n=33). The 3-year relapse free survival (RFS) and overall survival (OS) in the CT group were 25% and 31%, respectively. The multivariate survival analysis using Cox’s regression demonstrated that the variables that had significant impact in RFS and OS were: age at diagnosis (

Description: HSPA9, a gene located on chromosome 5q31.2, is commonly deleted in patients with myelodysplastic syndromes (MDS). MDS patients with a deletion of the long arm of chromosome 5 [del(5q)] typically present with cytopenias, including anemia, and have increased levels of apoptosis in their bone marrow contributing to ineffective hematopoiesis. Recent evidence suggests that upregulation of TP53 in MDS bone marrow cells may contribute to the cytopenias and accererated apoptosis observed in patients. While the mechanisms of TP53 activation in MDS are likely to be multifactorial, gene haploinsufficiency has been shown to contribute. Previous reports have shown that knockdown of RPS14, a chromosome 5q33.1 gene, in human CD34+ cells (or heterozygous knockout in mouse bone marrow cells) results in upregulation of TP53 and an increase in apoptosis. It is not known whether additional del(5q) candidate genes contribute to TP53 activation in del(5q)-associated MDS. In order to determine whether HSPA9 gene deletion also results in TP53 activation, we used lentiviral shRNA vectors to knockdown the expression of HSPA9 in primary human CD34+ hematopoietic progenitor cells. The HSPA9 protein level was reduced to ~20% (sh960) and ~50% (sh433) compared to the control lentiviral shRNA (shGFP). Knockdown of HSPA9 significantly inhibited the growth (fold change sh960 compared to shGFP = 0.16, p

Description: Introduction/Background Recurrent deletions of chromosome 5 and 7 are characteristic of the myeloid malignancies MDS and AML, determine prognosis and influence therapeutic decision. The pathogenetic contribution of individual genes to MDS/AML development has been shown for several of these genes. However, how and if these genetic losses may relate to therapeutic interventions or represent haploinsufficient targets has remained elusive for the most part. We hypothesized that genes found within the commonly deleted regions (CDRs) of chromosome 5/7 (chr 5/7) represent targetable vulnerabilities in malignant myeloid cells. Therefore we generated a custom RNAi library to functionally interrogate these genomic regions alone or under 5-Azacitidine (5-Aza) treatment, to identify individual genes whose silencing modulate 5-Aza responsiveness. Methods/Materials Custom siRNA plates against ~270 genes (3x sequences/gene, Qiagen) on CDRs of chr 5/7 were assembled. Genes were silenced by siRNA for 48 hours followed by 48-72 hour 5-Aza treatment after which cell viability was determined. Four cell lines (TF1, THP1, MDS-L and HEL) were investigated in parallel with combined siRNA/5-Aza incubations and appropriate 5-Aza and siRNA only control. Hits were selected as 〉 2 standard deviation changes in viability from the median log2 value of the ratio [(siRNA + 5-Aza)/(siRNA only)], normalized per plate and across the entire screen. Duplictae RNAis screens were performed. Synergy between 5-Aza and the SMO inhibitors LDE225 (Sonidegib) and GDC0449 (Vismodegib) was assessed with CalcuSyn in a panel of AML cell lines. Ex vivo viability and clonogenic re-population experiments in primary patient derived AML and MDS cells were performed with Sonidegib. Results Several genes within the Hedgehog (HH) pathway emerged as sensitizers to 5-Aza when silenced by siRNA in three of four cell lines examined. Specifically Shh siRNA sensitized to 5-Aza in both TF-1 and THP-1 cells, SMO in HEL and GLI3 in THP-1 cells. Shh silencing alone (siRNA only) resulted in reduction of viability only in TF-1 cells but not in THP-1 cells, while SMO and GLI3 siRNA alone did not result in significant reductions in viability but sensitized to 5-Aza, indicating true sensitization and an interactions between the HH pathway and 5-Aza treatment. The SMO inhibitor Vismodegib synergized with 5-Aza in drug dose response assays in a panel of four AML cell lines (TF-1, THP-1, ML-2, HL-60) with Combination Index (CI) values of around 0.6 or lower at low doses of both drugs. Similar results were also observed with Sonidegib in vitro in 5 molecularly heterogeneous AML cell lines. Given stem cell modulation capacity of SMO inhibition, we examined ex vivo proliferation and more importantly clonogenic growth capacity. Highly interesting, ex vivo viability was reduced and synergy observed by the Sonidegib/5-Aza combination to a much greater extend in CD34+ selected primary MDS and AML cells as compared to bulk or CD34 depleted (normal) MNCs. Clonogenic growth was reduced in the combination over single agent 5-Aza or Sonidegib in ~ 50% of samples assessed to date. No direct correlations to cytogenetics were observed. A clinical Phase 1b trial was designed based on these results and is currently accruing in the first cohorts. Conclusions RNAi screening of CDRs of chr 5/7 yielded potential targetable therapeutic vulnerabilities with several genes within the HH pathway sensitizing to 5-Aza. SMO inhibitors pharmacologically validate this concept in vitro and ex vivo with the potential to more selectively affect leukemia stem/progenitor cells. Clinical data will show if this is a specific effect involving chr 5/7 or a general concept in malignant myeloid cells. Thus, SMO inhibition in combination with 5-Aza may be a novel rational combination in AML and MDS. Disclosures Off Label Use: LDE225/Sonidegib on a clinical trial. Mesa:Incyte, CTI, NS Pharma, Inc., Gilead, Celgene: Research Funding.

Description: B-Cell Receptor (BCR) triggering and responsiveness play a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. In the recent past, several groups including ours have investigated the activation status of the signaling pathways originating from the leukemic BCR. Specifically we found that around 50% of CLL patients display a biochemical signature characterized by constitutive phosphorylation of ERK1/2 (pERK(+)) and constitutive nuclear translocation of NF-ATc1. These cases are unable to respond in vitro to BcR stimulation and are resistant to spontaneous apoptosis, thus resembling B lymphocytes previously anergized in vivo. Similar biochemical and functional features have been recently demonstrated in B leukemic cells persisting in the blood in patients treated with the BTK inhibitor, Ibrutinib, thereby making anergy an attractive target on the way to obtain eradication of the disease. CLL-associated B cell anergy can be specifically targeted by using different MAPK-inhibitors that have been shown to induce apoptosis selectively in the group of pERK(+) CLL. These data suggested that MAPK signalling can be efficiently inhibited in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 may represent a reliable biomarker to predict and monitor treatment response. However, even if the tested compounds were shown to be extremely efficient in inhibiting ERK1/2 phosphorylation in vitro, a lack of clinical activity was reported for many of them when tested in patients, mostly with solid tumors. In the present work, we used Trametinib, a specific MEK1/2 inhibitor, recently approved as a single-agent for the treatment of V600E mutated metastatic melanoma, and we investigated, at preclinical level, its activity in both primary CLL samples and a xenograft leukemic mouse model. Trametinib treatment completely inhibited constitutive ERK1/2 phosphorylation in 10 pERK1/2(+) samples at 3uM after 30 minutes treatment. Additionally, in 23 patients Trametinib treatment for 48 hours reduced cell viability in the cells from all 12 pERK1/2(+) patients (28,2% ± 3,5 mean survival) tested as compared to those from the pERK(-) group (11 cases, 58,1% ± 3,8 mean survival, p〈 0,0001). To strengthen our in vitro data, we evaluated the effect of Trametinib administration in the xenograft Rag2-/-gc-/- mouse model subcutaneously transplanted with the CLL cell line MEC1, characterized by specific features of anergy. Mice were subcutaneously injected with 10x106 cells and then challenged with Trametinib (oral gavage with 1mg/kg or with vehicle alone) starting from day 21 after tumour injection for 14 days. The effect of the inhibitor was monitored by tumour volume growth. Trametinb administration delayed tumour growth (p

Description: mDia1, the diaphanous homolog 1 of Drosophila in mouse, is a formin protein involving in the linear actin polymerization. Recently, our group reported that patients with del(5q) myelodysplastic syndromes (MDS) showed a significantly decreased mDia1 expression. Mice with mDia1 deficiency developed age related hematologic features mimicking human MDS. In these mice, CD14 is aberrantly overexpressed on granulocytes, which sensitized the innate immune response upon the lipopolysaccharide(LPS) injection. Importantly, chronic LPS injection accelerated the development of MDS (Keerthivasan et al Blood 2014). Here we report that 1) the mDia1 knockout (KO) mice also have a hypersensitive innate immune response to damage-associated molecular pattern molecules (DAMPs), which can be partially blocked by CD14/Toll-like receptor 4(TLR4) signaling pathway inhibitors. This is significant since release of DAMPs from necrotic or senescent cells is a common age-related phenomenon. DAMPs are also detected in cancers including MDS. Thus our study may shed lights on how the deregulated innate immune responses get involved in the pathogenesis of MDS in a more pathophysiologically relevant etiology. 2) mDia1 KO mice have an increased Gr1/Mac1 expression on the granulocytes in peripheral blood. We demonstrate that the expression levels of Gr1/Mac1 were not changed in bone marrow granulocytes, suggesting a specific up-regulation of Gr1/Mac1 levels on circulating granulocytes in mDia1 knockout mice. Mac-1, as the predominant β2 integrin on granulocytes, plays key roles in the adhesion of leukocytes to the endothelium, and regulates the cell adhesion, migration, and chemotaxis. In this respect, the mDia1 knockout mice develop serve neutropenia, which could be due to the upregulation of Gr1/Mac1 and increased binding of the cells to intercellular adhesion molecule-1 (ICAM-1). Finally, we revealed the mechanism of Gr1/Mac1 upregulation by showing that loss of mDia1 significantly affected the endocytosis of Gr1 and Mac1 on granulocytes, however, the mRNA levels of Gr1 and Mac1 were not affected. Taken together, these studies reveal a significance of loss of mDia1 in the pathogenesis of del(5q) MDS through upregulation of innate immune response and accelerated granulocyte clearance. Disclosures No relevant conflicts of interest to declare.

Description: Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA) is a DNA demethylating agent that is effective in treating MDS and CMML. However, response rates are less than 50% and the basis for poor response is currently unknown. A patient's potential to respond cannot be currently determined until after multiple cycles of AZA treatment and alternative treatment options for poor responders are limited. To address these fundamental questions, we enrolled patients on a compassionate access program prior to the listing of AZA on the pharmaceuticals benefit scheme in Australia. We have collected bone marrow from 18 patients (10 MDS, 8 CMML) at seven different stages of treatment, starting from before treatment until after six cycles of AZA treatment, and isolated high-purity CD34+ HSPCs at each stage. 10 of these patients (5 MDS and 5 CMML) responded completely to AZA while 8 did not achieve complete response. We performed next-generation sequencing (RNA-seq) of these HSPCs to identify the basis of poor response to AZA therapy. Analysis of the RNA-seq data from pre-treatment HSPCs has revealed a striking differential expression of 1148 genes between patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA therapy (Figure 1A). Using a Fluidigm nanofluidic system, we have validated the differential expression of a subset of these genes between CR and non-CR patients in two independent cohorts, totalling 67 patients, from the U.K. and Sweden. We have additionally confirmed that our gene signature does not simply segregate patients based on disease severity or poor overall survival, but rather uniquely prognosticates best AZA response. Pathway analyses of the differentially expressed genes indicates that the HSPCs of non-CR patients have decreased cell cycle progression and DNA damage pathways, while concomitantly possessing increased signalling through integrin and mTOR/AKT pathways. Using computational methods, we have determined that the expression of 15 genes (within the 1148 gene set) is sufficient to separate CRs from non-CRs across independent cohorts (Figure 1B). We have also developed a predictive AZA response algorithm that utilises the expression of these genes to identify potential complete and non-complete responders to AZA with high specificity and sensitivity (Figure 1C). Furthermore, we have identified statistically significant correlations between recurrent DNA mutations in MDS and our prognostic gene signature (SF3B1 & TET2 with CR, STAG2 and NUP98 with non-CR, p

Description: Next generation sequencing (NGS) and single nucleotide polymorphism arrays (SNP-A) contribute to more precise identification of the spectrum of somatic mutations and karyotypic abnormalities in myeloid neoplasms. The diversity of individual defects and their combinations corresponds to the tremendous clinical heterogeneity. Identification of key driver genes remains a fundamental component to understanding the immense data generated from this technology and the contributions to leukemogenesis. In this project, we evaluated 1200 cases of MDS and AML. Somatic mutations of AT rich interactive domain 2 (ARID2) were found in myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), primary acute myeloid leukemia (pAML) or secondary AML (sAML). All ARID2 mutations occurred in either frameshift (at p.S1489 and p.T1645) or nonsense (at p.E65, p.S154 and p.Q1637) configurations, consistent with loss of function. We have identified a total of 5 mutant cases, all of somatic origin. Study of clonal architecture elucidated that ARID2 mutations were ancestral events in 50% of mutant cases as defined by variant allelic frequencies. By SNP-A, a commonly deleted region on chr.12q was defined by mapping minimally affected lesions in 9 patients with MDS, MPN, sAML or pAML. Haploinsufficient expression of ARID2 was confirmed by expression array analysis in the cases with del(12q), which is compatible to the frequent incidence of heterozygous ARID2 loss-of-function mutations. Del(12q) was more frequent in high-risk phenotype with higher blast counts. In addition, significantly lower expression of ARID2 was also observed in 22 out of 183 patients with diploid 12q. Interestingly, amplification of locus was not found in any of the cases studied by SNP-A. Altogether, such lesions of defective ARID2 were pathogenic in more than 10% of cases with myeloid neoplasms. ARID2 is encoding one of the components of SWI/SNF complex and involved in chromatin remodeling. Therefore, we stipulate that other genes which function together with ARID2 might be affected with somatic mutations or deletions. Further analyses demonstrated the presence of other somatic mutations and deletions affecting SWI/SNF complex, including ACTL6B (N=53) and SMARCD3 (N=66). While SWI/SNF complex lesions were mutually exclusive, concomitant subclonal mutations in such affected cases were commonly observed in RAS pathway genes, including K/NRAS, NF1 and PTPN11. To the contrary, ARID1B, which negatively regulates chromatin remodeling, is predominantly activated in the cohort with similar phenotype. While germline mutations of multiple genes in SWI/SNF complex are reported to be associated with Coffin-Siris syndrome, no family or past history characteristic of this congenital disorder was observed in our cohort. Further clues into the function of ARID2 in myeloid neoplasms came from studying splicing dysfunction in U2AF1 mutant cases. Deep RNA sequencing in the cases with U2AF1 mutations (p.S43F and p.Q157P), showed a consistent loss of ARID2 exon 8 (predominantly noted in sAML). Two types (whole and partial) of exon skipping led to frameshift in the transcript creating a stop codon. Targeted RT-PCR confirmed the transcriptome sequencing results in primary bone marrow samples of the cases with U2AF1 but not in the corresponding wild-type cases. These results are compatible with the genetic finding that spliceosomal mutations were not concomitantly observed in the cases with SWI/SNF complex defects, suggesting misspliced transcript with nonsense decay consequences is enough pathogenic to preclude additional spliceosomal mutations. To validate functional consequences of ARID2 loss, knockdown experiment using ARID2-shRNA transduction in K562 and HL60 cell lines were performed. Knockdown of ARID2 generally demonstrated cell cycle arrest in G2 phase prior to entry into the S-phase, which was partly caused by decreased expression of CDKL3 and CCND3. Hb staining with Benzidine showed impairment of erythroid differentiation in ARID2 knockdown K562, which was confirmed by cytological examination. In sum, multiple mechanisms of defective ARID2 including somatic mutations, haploinsufficiency and phenocopy due to spliceosomal mutations can be involved in ARID2-mediated leukemogenesis. Together with the other components, novel lesions of SWI/SNF complex constitute a group of tumor suppressor genes in myeloid neoplasms. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The development of next-generation sequencing has made it feasible to interrogate the entire genome or exome (coding genome) in a single experiment. Accordingly, our knowledge of the somatic mutations that cause cancer has increased exponentially in the last years. MPNs and MDS/MPD are chronic myeloid neoplasms characterized by an increased proliferation of one or more hematopoietic cell lineages, and an increased risk of transformation to acute myeloid leukemia (AML). MPNs and MDS/MPDs are heterogenous disorders, both in clinical presentation and in prognosis. We sought to determine the genetic landscape of Ph-negative MPNs and MDS/MPD through next-generation sequencing. Methods: Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 102 patients with MPNs or MDS/MPD was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. Diagnosis included primary myelofibrosis (MF; N=42), essential thrombocythemia (ET; N=28), polycythemia vera (PV; N=12), chronic myelomonocytic leukemia (CMML; N=10), systemic mastocytosis (MS; N=6), MDS/MPD-Unclassified (N=2) and post-MPN AML (N=2). Tumor coverage was 150x and germline coverage was 60x. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). The combined output of these 3 tools was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥10% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline 〉2; excluding mutations seen in SNP databases). All JAK2 and CALR mutations were validated through Sanger sequencing. Validation of other somatic mutations is currently underway. Analysis of driver mutations was made with the Intogen web-based software, using the Oncodrive-FM and Oncodrive-cluster algorithms (www.intogen.org). Significantly mutated genes were considered as those with a q-value of

Description: Bone marrow (BM) fibrosis is a key pathomorphologic feature of patients (pts) with primary myelofibrosis (PMF) and the fibrotic phases of essential thrombocythemia (post-ET MF) and polycythemia vera (post-PV MF). The degree of BM fibrosis appears to correlate with survival. Indeed worse survival has been associated with increased BM fibrosis. The BM stromal microenvironment is important in the pathogenesis of BM fibrosis. Cellular components (fibroblasts, macrophages, endothelial cells, adipocytes), structural fibrils (collagen, reticulin) and extracellular matrix components are all forming elements of the BM stroma. Increased stromal fibrosis has been linked to abnormalities in the number/ function of megakaryocytes and platelets in hematologic diseases. Several cytokines like Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor-Beta (TGF-b) have been also linked to the pathophysiology of BM fibrosis. PDGF has been shown to increase fibroblast growth in megakaryocytes and platelets although increased PDGF did not correlate with increased production of either reticulin or collagenous fibrosis. Moreover, PMF pts have increased TGF-b levels in platelets, megakaryocytes, and monocytes. Nitric Oxide (NO) is a ubiquitous gas important in physiologic processes particularly vasodilatation. Dysregulation of NO levels has been implicated in pulmonary hypertension (PH), hemoglobinopathies, and cardiovascular diseases. In Peyronie’s disease, a localized fibrosis of the penile tunica albuginea, increased NO production by expression of iNOS decreases collagen deposition by neutralization of profibrotic reactive oxygen species and decreased myofibroblast formation. Aside from its role in maintaining normal vascular tone, NO also plays a role in fibroblast formation and collagen biosynthesis. We previously reported that ruxolitinib, a JAK1/2 inhibitor restores NO levels leading to improvement of PH in MF pts (Tabarroki et al., Leukemia 2014). We now hypothesize that plasma/serum NO level is a key regulator of BM fibrosis in MF and that ruxolitinib treatment (Tx) leads to improvement of BM fibrosis by NO modulation. Using a Sievers 280i NO analyzer we measured the plasma/serum NO level of a large cohort (n=75) of pts with myeloid and myeloproliferative neoplasms (MPN) [MDS, RARS/RCMD=8; MPN, ET=8, PV=8, MF=24, Mastocytosis=7; MDS/MPN, CMML=11, MDS/MPN-U, RARS-T=9]. Healthy subjects (n=10) were used as a control. MPN pts had low NO (nM) levels among the pts studied with the lowest level found in MF pts: MF=30.31±11.8, PV=39.0±16.1, ET=36±20.3, RARS=74.6±41.7 (P=.01), CMML=84.4±89.2 (P=.04), RCMD=163.4±103.8 (P

Description: BCD-020 (Acellbia, rituximab biosimilar candidate) was shown to be highly similar to innovator rituximab (MabThera®/Rituxan®) in terms of its quality characteristics, in vitro biological activity, as well as toxicology and PK/PD characteristics in non-human primates. International multicenter comparative randomized open-label clinical study was carried out in a period from 2011 to 2013 and involved over 30 centers in Russia, Ukraine and India. Its methodology and design complies with current EMA guidelines on similar biological products containing monoclonal antibodies (EMA/CHMP/BMWP/403543/2010). 92 patients with follicular non-Hodgkin’s lymphoma, stage I-IV by Ann Arbor, or marginal zone lymphoma, stage I-IV by Ann Arbor, ECOG 0-2, who had at least 1 measurable lesion were enrolled. According to study protocol patients with secondary transformed B-cell lymphomas or with highly aggressive types of tumor, bulky disease, severe concomitant somatic disorders and some other conditions were excluded. If a patient had previous story of chemotherapy or radiation he could be included after at least 3 weeks post-treatment. Participation of patients who were previously treated with any kind of monoclonal antibodies was prohibited. After signing standard informed consent form and completion of 28-days screening period eligible patients underwent stratification in accordance to their prognostic risk (FLIPI or IPI) and previous treatment (naïve or pretreated). Subsequently patients were randomized (1:1) into 2 groups: 46 patients were included in the main group where Acellbia (rituximab biosimilar) was administered at a dose of 375 mg/m2 as a slow IV infusion on day 1, 8, 15 and 22; 46 patients were included in the reference group where MabThera was used at the same regimen. Use of any other medicines for the treatment of lymphoma was strictly prohibited. Efficacy was assessed on the basis of computed tomography and bone marrow evaluation which were performed 1 month after the completion of treatment. Median age of patients in each group was 57.5 years (main group [50.0-64.0], reference group [47.0-65.0]). Manageable comorbidities were reported in 50% of patients in the main group and 34.78% of patients in the reference group, p=0.2053. Comparative analysis of the prognostic risk factors confirmed the equivalence of study groups. The number of pretreated patients in both groups was equal – 8 individuals per group. Statistical analysis didn’t find any difference in overall response rate in general population of patients (39.52% patients in the main group vs. 36.57% patients in the reference group, p=0.8250), as well as in population of pretreated patients (28.6% vs 37.5% respectively, p=1.00) and in population of naïve patients (42.8% vs 39.4% respectively, p=1.00). The lower limit of the two-tailed 95% CI for difference in proportions of ORR was equal to -0.17 and exceeded the predefined non-inferiority margin -0.2, which confirmed non-inferiority of Acellbia to MabThera in terms of efficacy. Treatment-associated AE of any grade were reported in 21.74% patients in both arms, in the absence of statistically or clinically significant difference (p = 0.8005). There were 2 cases of CTCAE 4.03 grade 3-4 AEs in each group. PK and PD parameters were shown to be equivalent in both study groups. Thus, study results suggest that Acellbia has same efficacy and safety in patients with B-cell non-Hodgkin’s lymphoma. Disclosures Chernyaeva: JCS BIOCAD: Employment. Ivanov:JCS BIOCAD: Employment. Isaev:JCS BIOCAD: Employment.

Description: Nontuberculous mycobacteria (NTM) are ubiquitous free living soil and water– borne organisms that cause numerous clinical syndromes including lymphadenitis, skin, soft tissue and pulmonary infections, however disseminated infection is almost exclusively in patient with severe immunocompromise (i.e:HIV, Hematological malignancy, and bone marrow transplant). Mycobacterium avium complex (MAC) is hard to diagnose as it is considered slow grower NTM. We describe a case of disseminated Mycobacterium avium-intracellulare complex infection in teenager with sickle hemoglobinopathy with unique presentation mimicking pRBCs transfusion reaction. Patient presented on three different occasions with tachycardia, hypotension and fever within 2-24 hours following pRBCs pheresis, all three episodes were investigated and were negative for transfusion reactions. Patient had central venous catheter (CVC), frequent admissions for vaso-occlusive painful episode, on hydrocortisone for adrenal insufficiency and off Hydroxyurea for two months. Diagnosis of mycobacterium avium complex bacteremia was confirmed by two positive blood cultures, whole body CT scan showed liver nodules, spleen nodules and lung nodules. Pulmonary dissemination was confirmed by Biopsy and culture, Lymphocyte markers showed severe lymphopenia with absolute CD4 count of 64. Patient underwent treatment with three month of four antibiotics followed by 9 months of three antibiotics with removal of the central line, follow up scan showed decrease size and numbers of nodules, patient started tolerating pheresis within one month of the antibiotics initiation. NTM infection should be added to the list of pathogens in sickle cell patients with CVCs and fever and should be considered in frequent pRBC transfusion like reaction with negative workup. Routine blood culture can identify rapid growing NTM but specific mycobacterial blood culture is required in case of other NTM species (slow grower). As dissemination almost always occurs in those with impaired cellular immunity, HIV testing and lymphocyte markers should be performed Removal of involved CVCs is essential for the treatment as well as appropriate antimicrobial medications. Disclosures No relevant conflicts of interest to declare.

Description: Background In sickle cell disease (SCD), profound anaemia and severe hemosiderosis cause functional and physiological abnormalities in various organ systems, including immune system. Infectious complications are common, constitute the second most common cause of mortality and a main cause of morbidity. During the haemolytic crisis, large amount of arginase (s-Arg-1) are released, potentially acting as immunosuppressor molecule. Despite its clinical impact, only a few is known about myeloid dysregulation in SCD. Aim Detecting immunological impairment at the steady state evaluating myeloid and lymphoid cells in peripheral blood of SCD patients. Material and Methods Between May and June 2014,peripheral blood obtained from 30 consecutive SCD patients at the steady state plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis. Myeloiddys function was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-likemo-MDSC (CD14+/HLA-DRlow/-), phagocytic activityof granulocytes using a commercially available kit (Phagotest R), amount of Arg-1 expressed in mature granulocytes by RT-PCR and circulating s-Arg-1 using a commercially available ELISA kit (Biovendor). Results The capability of phagocytosis of granulocytes wassignificantly reduced compared to healthy subjects (p=0.001). G-MDSC subset was not increased, while mo-MDSC subpopulation was increased in SCD (p=0.001) but not in thalasso-SCD. s-ARG-1 was increased in both SCD and thalasso-SCD (respectively 203±3 ng/mL and 248±6ng/mL, p=0.003) and positively correlated with the amount of HbS (r=0.7, p=0.002). Arg-1 expression in granulocytes was increased (20 times higher than healthy controls, p=0.002) Conclusion SCD and thalasso-SCD caucasian patients exhibiti mmunosuppressive myeloid markers including reduced phagocytosis, increased amount of mo-MDSC, Arg-1 expression in granulocytes and circulating s-Arg-1. Further analysis are ongoing to detect if the same myeloid impairment occurs during vase occlusive crisis and in a different genetic background, like in Afro-Americans. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Patients with B-cell lymphomas harboring the oncogenic MYD88 L265P mutation have poor response to standard of care regimens such as R-CHOP (Fernandez-Rodriguez et al, Leukemia, 2014, Jun 6). These patients also have not responded well to new therapies under development, including BTK inhibitors (Wilson et al, Blood 2012; 120). The MYD88 L265P oncogenic mutation has been shown to over-activate TLR7- and TLR9-mediated signaling pathways, including NF-κB, IRAK1/4 and STAT3, which in turn promote cell survival and proliferation (Lim et al., AACR 2013, Abstract #2332). Our approach to a potential treatment of B-cell lymphomas harboring the MYD88 L265P mutation is to block TLR7- and TLR9-mediated signaling through use of a TLR antagonist. IMO-8400 is an antagonist of TLR7, TLR8, and TLR9 that inhibits cell signaling and induces apoptosis specifically in B-cell lymphoma cell lines harboring the MYD88 L265P mutation (Bhagat et al, AACR 2014, Abstract #2570). IMO-8400 is currently in clinical development for the treatment of relapsed/refractory patients with Waldenström’s macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL). Methods. The current preclinical studies were designed to evaluate the combination of IMO-8400 with rituximab, which is a key component of R-CHOP. The studies were conducted in xenograft models of the activated B-cell like (ABC) DLBCL cell line OCI-Ly10 and the WM cell line MWCL-1, both positive for the MYD88 L265P mutation. For the ABC-DLBCL model, OCI-Ly10 cells were implanted s.c in NOD-SCID mice on day 0 and treatment was initiated on day 9 when mean tumor volume reached ~200 mm3. For the WM model, MWCL-1 cells were implanted s.c. in NOD-SCID mice on day 0 and treatment was initiated on day 6 when mean tumor volume reached ~250 mm3. In both models, the tumor-bearing mice were treated with PBS (control group), IMO-8400 (25 mg/kg, i.p.), rituximab (10 mg/kg, i.p.) or a combination of the two agents. IMO-8400 was administered twice per week for 3 weeks and continued further as maintenance treatment once per week. Rituximab was administered twice per week for three weeks in the ABC-DLBCL model whereas it was given only on days 3, 6, and 9 in the WM model. Results. Treatment of tumor-bearing animals with IMO-8400 or rituximab alone and in combination was well tolerated in both the models. In the ABC-DLBCL model, tumor growth inhibition compared to PBS control was 91.3% in the combination therapy group vs. 40.8% with IMO-8400 alone (p