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  • American Society of Hematology
  • 2010-2014  (6,927)
  • 1980-1984  (714)
  • 1930-1934
  • 1925-1929
  • 2012  (6,927)
  • 1980  (714)
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  • 2010-2014  (6,927)
  • 1980-1984  (714)
  • 1930-1934
  • 1925-1929
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  • 1
    Publication Date: 1980-03-01
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  • 2
    Publication Date: 1980-03-01
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  • 3
    Publication Date: 1980-03-01
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  • 4
    Publication Date: 1980-01-01
    Description: Sephacryl beads containing an immobilized aminopropylcobalamin- transcobalamin-II complex serve as foci for the adherence of L1210 murine leukemia cells. Bead-cell interaction does not occur when (A) nonderivatized beads are used; (B) transcobalamin-II is omitted or presaturated with cyanocobalamin in the preparation of the bead complex; (C) intrinsic factor replaces transcobalamin-II; and (D) the complex is removed from beads by photolysis. These observations suggest that adherence results from the ability of transcobalamin-II to form a bridge between immobilized cobalamin on the bead and receptors in the plasma membrane of the cell.
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  • 5
    Publication Date: 1980-02-01
    Description: An immunohistochemical procedure for the detection of immunoglobulin G adherent to platelets is described. The peroxidase anti-peroxidase method is used to detect antibody activity directed against platelets from normal donors in the sera from 305 individuals. These subjects were divided into three groups: group 1, patients referred for tissue typing; group 2, healthy normal females; group 3, healthy normal males. In group 1, 28% of the sera were found to be positive; in most of these a history of prior transfusions was obtained. In group 2, 7.4% were found to be positive, most having previous pregnancies. Only 1% were found to be positive in group 3, and no reason for presensitization was found. Results from the indirect immunofluorescence technique served as a control and as a means to compare the sensitivity. Under the conditions chosen, the peroxidase anti-peroxidase test was two to eight times more sensitive than the immunofluorescence technique. Specificity of the peroxidase anti-peroxidase technique was demonstrated using a monospecific anti-PLA1 antiserum. It is concluded that the peroxidase anti-peroxidase slide technique may be a useful tool in the study of platelet-related immunophenomena.
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  • 6
    Publication Date: 1980-02-01
    Description: Membrane markers expressed by a transplantable granulocytic leukemia induced in inbred strain 13 guinea pigs by N-nitroso-N-butylurea have been investigated by serologic and immunochemical methods. Although the leukemic myeloblasts have no detectable surface immunoglobulin or Fc receptors, they have been found to synthesize and express membrane antigens encoded for by the I-region of the major histocompatibility complex. Despite the presence of Ia antigens, these cells do not stimulate allogeneic T lymphocytes in the mixed lymphocyte reaction nor are they able to present soluble antigen to immune T cells. The presence of Ia antigens on immature myeloid cells suggests that these proteins may be important in cellular functions beyond those identified in the cellular immune system.
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  • 7
    Publication Date: 1980-02-01
    Description: Urinary ferritin levels were measured by a “2-site” immunoradiometric assay in normal volunteers and in patients with various hematologic disorders. The mean urinary ferritin concentration in normal subjects averaged 2.2 microgram/liter, only 3% of the serum ferritin level. Elevated urinary ferritin levels averaging 45 microgram/liter were observed in patients with hematologic malignancies, but there was a proportional increase in serum ferritin so that the urinary level still averaged only 7% of the serum value. The highest urinary ferritin values (mean 170 microgram/liter) were associated with chronic hemolytic anemia, and in these patients, urinary ferritin rose disproportionately in relation to the serum, averaging 82% of it. This higher urinary level apparently reflects increased ferritin in renal tubular cells due to glomerular filtration of unbound hemoglobin, a mechanism that is supported by a highly significant correlation between urinary ferritin and serum haptoglobin levels. In normal subjects and in patients with malignancy, the source of urinary ferritin appears different, since a highly significant correlation was observed between urinary ferritin and reticuloendothelial iron stores as measured by serum ferritin or total iron-binding capacity. In this setting, the most likely source of urinary ferritin is the iron contained in renal tubular cells, which is apparently in equilibrium with body iron stores.
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  • 8
    Publication Date: 1980-01-01
    Description: A new assay for the long-term functional capacity of hemopoietic stem cells is reported. Stem cell function in each donor of a particular genotype is assayed by mixing its marrow cells with a constant number of marrow cells from a donor with distinguishable hemoglobin and measuring the relative ability of each donor to populate stem-cell- depleted recipients. For example, cells from many different B6 donors may be assayed by mixing them with a constant number of WBB6F1 cells from a single pool and injecting them into irradiated WBB6F1 recipients. As the ratio of B6 to WBB6F1 marrow cells increases from 0:1 to 3:1 the percentage of B6 hemoglobin increases in a linear fashion. This is also found with WB and WBB6F1 or CBA and B6CBAF1 mixtures. Correlation coefficients between the percentage of hemoglobin of one donor type and the ratio of cells in the mixture of that type ranged from 0.78 to 0.98 in single experiments, and were 0.68 and 0.75 using data pooled from several experiments.
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  • 9
    Publication Date: 1980-01-01
    Description: Erythrocytes from heterozygous carriers of the high oxygen affinity mutant hemoglobin, Hb Wood, demonstrate lower rates of methemoglobin reduction than normal human red cells when incubated in the in vitro system of Beutler and Baluda. The rate of methemoglobin reduction in red cells from an individual who is heterozygous for both NADH- methoglobin reductase deficiency and Hb Wood shows the combined effects of the two mutations.
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  • 10
    Publication Date: 1980-02-01
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  • 11
    Publication Date: 1980-01-01
    Description: Leukemic cells from 20 patients with chronic lymphocytic leukemia (CLL) and 60 patients with acute lymphocytic leukemia (ALL) were studied for T- and B-lymphocyte cell surface membrane markers. B-cell markers included surface membrane immunoglobulin, erythrocyte-antibody complement rosette formation, and B-cell (a-like or HLA-DR) antigens detected by a B-cell antiserum. T-cell markers included spontaneous sheep red blood cell rosette formation and a cytotoxic reaction to a specific T-cell antiserum. Seven patients with CLL and two with ALL had dual B and T markers. We propose that dual B- and T-cell markers are more common in CLL and ALL patients than previously reported. With newer and more sensitive tests for identification of B and T cells, this observation may be recognized more frequently.
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  • 12
    Publication Date: 1980-01-01
    Description: A patient with multiple myeloma developed periodic blood neutropenia (periodicity of 15–25 days) after 3 yr of intermittent treatment with cytotoxic agents. Peaks of serum colony-stimulating activity (CSA) level coincided with valleys of blood neutrophils. Fraction of marrow neutrophils in the multiplicative pool was high during blood neutrophil valleys and low during neutrophil peaks. In contrast, the maturation storage pool exhibited the reverse pattern. An increased fraction of marrow neutrophilic cells in the multiplicative pool was in active proliferation during a blood neutrophil valley and a decreased fraction during a blood neutrophil peak. These findings suggest that the marrow granulopoiesis was regulated through CSA. The defect causing the periodicity was probably related to the reduced number of neutrophils in the marrow maturation storage pool, which in turn may be related to a reduced and/or defective granulocytic stem cell pool size consequent to the long-term administration of cytotoxic drugs and/or infiltration of the marrow by myeloma cells.
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  • 13
    Publication Date: 2012-03-15
    Description: Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α4β1-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.
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  • 14
    Publication Date: 2012-11-16
    Description: Abstract 2099 Background: While oral iron is the preferred first-line treatment for patients with iron deficiency anemia (IDA), there are patients who cannot take oral iron, do not tolerate it or do not adequately respond to oral iron. In the US and Canada, the only approved treatment options for these patients are the iron dextrans, which have boxed safety warnings and inconvenient dosing regimens. Therefore, many of these anemic patients do not receive IV iron, and remain inadequately treated and symptomatic. In the EU, several IV irons, including iron sucrose (IS), are approved for second line use. Few studies have evaluated the IV irons in head-to-head studies. Ferumoxytol (FER) is a new IV iron approved for the treatment of IDA in patients with chronic kidney disease (CKD) that is formulated to allow for bolus IV injection. This randomized, controlled trial was designed to investigate the efficacy and safety of FER compared to IS for the treatment of IDA in patients with a history of unsatisfactory oral iron therapy or in whom oral iron could not be used. Methods: The study was designed to demonstrate non-inferiority and consisted of a 14 day screening period, treatment and a 5 week follow-up period. Key inclusion criteria included a Baseline hemoglobin (Hgb) less than 10 g/dL and 〉7 g/dL, and transferrin saturation (TSAT) 〈 20%. Patients were randomized 2:1 to receive either FER, administered as 2 injections of 510 mg 5±3 days apart, or IS, administered as 5 infusions or injections of 200 mg on 5 non-consecutive days over a 14 day period. Results: A total of 605 subjects were randomized to the 2 treatment arms (FER, n= 406; IS, n=199). FER demonstrated non-inferiority to IS in the proportion of subjects with a 〉2.0 g/dL increase in Hgb at any time from Baseline to Week 5 (the primary efficacy endpoint), compared to those treated with IS, (FER, 84%; IS 81%) with the lower bound of the 95% CI [-3.89%] above the predefined non inferiority margin [-15%]. In addition at each post-treatment time point, a higher percentage of FER-treated subjects achieved a 〉2.0 g/dL increase in Hgb compared to those treated with IS. FER also achieved non-inferiority to IS in the mean change in Hgb from Baseline to Week 5 with a robust 2.7g/dL increase in Hgb compared to 2.4g/dL with IS (the lower bound of the 95% CI [0.06g/dL] was above the predefined non-inferiority margin [-0.5g/dL]); this treatment difference (0.3 g/dL) was statistically significant (p=0.0124), and FER actually achieved superiority over IS. The overall rates of adverse events (AEs) and related AEs were lower in the FER group compared to IS-treated subjects. The serious adverse event (SAE) rate was higher in FER-treated subjects (FER, 4.2%; IS, 2.5%), but no pattern or safety trend was observed to suggest a specific safety signal; treatment-related SAEs were reported in 2 (0.5%) FER-treated subjects (1 anaphylactoid reaction and 1 hypertension). Protocol-defined AEs of Special Interest (signs/symptoms of hypotension or hypersensitivity associated with IV iron use) were reported at a higher rate in IS-treated subjects compared to the FER treatment group (IS, 5.0%; FER, 2.7%). Cardiovascular AE rates were comparable in the 2 treatment groups (1.0%). Overall, the safety profile of FER was comparable to that of IS and no new safety signals were identified. Conclusion: In this randomized, controlled trial, the efficacy and safety of 2 doses of FER were shown to be comparable to IS in treating IDA patients with a history of unsatisfactory oral iron therapy or in whom oral iron could not be used. For this IDA patient population, which has limited treatment options in the US and Canada, FER may offer an important, new treatment option with a convenient 2 dose regimen. Disclosures: Off Label Use: Feraheme (ferumoxytol) injection. For treatment of iron deficiency anemia in non-CKD patients. Bernard:AMAG Pharmaceuticals, Inc.: Employment. Strauss:AMAG Pharmaceuticals, Inc.: Employment. Cressman:AMAG Pharmaceuticals, Inc.: Employment. Li:AMAG Pharmaceuticals, Inc.: Employment. Allen:AMAG Pharmaceuticals, Inc.: Employment.
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  • 15
    Publication Date: 2012-11-29
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  • 16
    Publication Date: 2012-11-16
    Description: Abstract 4439 Background: Several reports have recently been published documenting the experience of patients with Philadelphia chromosome positive chronic myeloid leukemia in the chronic phase (CML-CP) who were switched from Glivec (IM), the β-crystalline form of imatinib mesylate, to non-identical alternate copies of imatinib mesylate (IMac) including those composed of the α-crystalline form. As a result of pre-clinical tests performed during the early development of IM, the β-crystalline form of imatinib mesylate was selected for further development due to its superior stability. In one case report evaluation, 126 Iraqi patients with CML-CP were switched from IM to an IMac that was the α-crystalline form of imatinib mesylate. At the time of switch to IMac, patients had previously received 400mg IM once-daily for an average of 50 months and were at least in complete hematologic response (CHR). Post-switch, patients were initially placed on the same IMac dose and followed-up on a monthly basis; more frequently in cases of poor response or adverse events. By 3 months post-switch, 22 (17%) patients had lost hematologic response and of these patients 18 (14%) and 4 (3%) had progressed to accelerated phase (AP) and blast crisis (BC), respectively. By 6 months post-switch, an additional 20 patients (16%; 33% total) had lost hematologic response. Safety assessment for IMac in this cohort has not been previously published. Objective: To assess the clinical safety and to estimate the survival of Iraqi CML patients switched from IM to IMac. Methods: Patient case records were retrospectively reviewed for safety findings as assessed by the treating physician. A previously published Markov transition-state model was used to compare projected life-years (LYs), progression-free life-years (PFLY), and quality-adjusted life-years (QALYs) of IM patients switched to IMac vs. patients not switched. Patients entered the model after a mean 50 months of IM therapy. At that time, based on the IRIS trial results, patients were considered to have CHR (4.7%), partial (6.5%) or complete (88.9%) cytogenetic response. Patients remaining on IM transitioned within these 3 responses levels and no hematologic response, AP, BC, and death according to the original model probabilities. For patients switched to IMac, transition rates were based exclusively on response rates observed in the Iraqi study (Scenario 1) or on the Iraqi study for the first 6 months, and thereafter based on the transition rates originally in the Markov model (Scenario 2). Utilities were from the original model. Results: Non-hematologic adverse event (AE) rates observed in patients who were switched to IMac are reported (Table). Patients remaining on IM were predicted to experience 15.7 LYs, 14.5 PFLYs, and 13.4 QALYs. Corresponding numbers for patients switched to IMac were 2.4 LYs, 1.1 PFLYs, 1.4 QALYs (in Scenario 1) and 11.4 LYs, 10.2 PFLYs, and 9.6 QALYs (in Scenario 2). Results were also sensitive to response distribution at model entry. Conclusions: The clinical safety, efficacy, and tolerability of IM have been established from more than 10 years of clinical experience. Recent introduction of generic versions of imatinib (including α-crystalline form) allows assessment of whether patients switching from IM to a generic version are able to achieve the same clinical outcomes as with the branded drug. The Iraqi case report is the largest cohort currently available for such review. This case report suggests that switching from IM to an IMac that does not have the safety and/or efficacy may result in substantial loss of LYs, PFLYs, and QALYs. Robust pharmacovigilance programs may need to be established to differentiate safety findings for originator drugs vs. substandard generic versions. Disclosures: Botteman: Novartis: Consultancy. Magestro:Novartis: Employment, Equity Ownership. Manley:Novartis Pharma AG: Employment. Zernovak:Novartis Pharmaceuticals Corp: Employment, Equity Ownership. Woodman:Novaris Pharmaceuticals Corp: Employment, Equity Ownership.
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  • 17
    Publication Date: 2012-11-16
    Description: Abstract 2195 Introduction: Eltrombopag (epag), a thrombopoietin receptor agonist (TPO-RA), increases platelet counts in patients with chronic immune thrombocytopenia (cITP). TPO-RAs have been associated with varying degrees of increases in bone marrow reticulin (Brynes 2011; Ghanima 2011). Due to lack of pretreatment evaluations, the incidence and clinical significance of these findings have not been established. Inconsistencies in specimen preparation, staining, and analysis across institutions further confound conclusions. The purpose of this 2-year (y) study (NCT01098487) is to assess for bone marrow fibrosis (reticulin and/or collagen) in patients treated with epag for cITP. Baseline and 1y findings are presented. Methods: Bone marrow biopsies are being collected at baseline (before treatment with epag) and at 1 and 2y of treatment. Specimens are centrally processed and stained for reticulin (silver) and collagen (trichrome) and undergo central independent pathology review of cellularity; megakaryocyte, erythroid, and myeloid quantity and appearance; trabecular bone quality; reticulin grade; and presence of collagen (European Consensus scale-MF; Thiele 2005). Results: Baseline and 1y (10–14 months) data are available for 101 patients. Median age is 42y (18–78); 70 patients are female; 50% are Caucasian/European, 22% are East Asian, and 29% are Central South Asian. Median time since ITP diagnosis is 4.2y (0.2–45.7). All patients had received prior ITP therapy, and 8 patients had received prior TPO-RA treatment (epag [7], romiplostim [1]), the last dose ≥6 months before enrollment. At baseline, 91 patients had reticulin grade 0 (MF-0), 10 MF-1, and 0 MF≥2. At 1y, 59 patients had MF-0, 38 MF-1, 3 MF-2, and 1 MF-3 (Figure). Compared with baseline, there was no change at 1y in MF grade in 61 patients, a decrease by 1 grade in 3, an increase by 1 grade in 35, and an increase in 2 or 3 grades in 1 patient each (Table). Three patients had collagen at 1y (1 patient each with MF-1, MF-2, and MF-3). None of the 4 patients with MF≥2 had adverse events or hematologic abnormalities considered related to impaired bone marrow function, and none withdrew due to bone marrow findings. Among the 8 patients with prior TPO-RA treatment, all had baseline reticulin of MF-0 and none had collagen; at 1y, 6 remained MF-0, 1 was MF-1, and 1 MF-3 (collagen demonstrated). Cellularity was normal in 83% and 80% of patients at baseline and 1y, respectively. Other than normalization of erythroid lineage numbers, no changes occurred in marrow cellular composition. In 3 of 4 patients with MF≥2, cellularity was increased at 1y. Trabecular bone thinning was found at baseline in 28 patients (the majority with prior steroid use) and 51 patients at 1y. Discussion: 10% of patients had MF-1 at baseline. After 1y of treatment, no increase or a mild increase in reticulin was observed in 63% and 35% of patients. No patient with MF≥2 (n=4) had clinical signs or symptoms indicative of bone marrow dysfunction and none withdrew from the study. Results were similar to those reported for EXTEND, an eltrombopag extension study (median treatment duration 〉2 years; Brynes 2011). Conclusion: These data suggest that treatment with epag is generally not associated with clinically relevant increases in bone marrow reticulin or collagen. The potential association of TPO-RAs and increased bone marrow reticulin needs further study. Disclosures: Brynes: GlaxoSmithKline: Research Funding. Orazi:GlaxoSmithKline: Research Funding. Wong:Roche: Research Funding; MSD: Research Funding; Johnson & Johnson: Research Funding; Bayer: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biogen-Idec: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; GlaxoSmithKline: Research Funding; Bristol-Myers Squibb: Research Funding. Bakshi:GlaxoSmithKline: Employment, Equity Ownership. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Brainsky:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties.
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  • 18
    Publication Date: 2012-10-11
    Description: Sjögren syndrome (SS) is a systemic autoimmune disease characterized by dry mouth and eyes, and the cellular and molecular mechanisms for its pathogenesis are complex. Here we reveal, for the first time, that bone marrow mesenchymal stem cells in SS-like NOD/Ltj mice and human patients were defective in immunoregulatory functions. Importantly, treatment with mesenchymal stem cells (MSCs) suppressed autoimmunity and restored salivary gland secretory function in both mouse models and SS patients. MSC treatment directed T cells toward Treg and Th2, while suppressing Th17 and Tfh responses, and alleviated disease symptoms. Infused MSCs migrated toward the inflammatory regions in a stromal cell–derived factor-1–dependent manner, as neutralization of stromal cell–derived factor-1 ligand CXCR4 abolished the effectiveness of bone marrow mesenchymal stem cell treatment. Collectively, our study suggests that immunologic regulatory functions of MSCs play an important role in SS pathogenesis, and allogeneic MSC treatment may provide a novel, effective, and safe therapy for patients with SS. This study was registered at www.clinicaltrials.gov as NCT00953485.
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  • 19
    Publication Date: 2012-05-24
    Description: Erythroid (red blood) cells are the first cell type to be specified in the postimplantation mammalian embryo and serve highly specialized, essential functions throughout gestation and postnatal life. The existence of 2 developmentally and morphologically distinct erythroid lineages, primitive (embryonic) and definitive (adult), was described for the mammalian embryo more than a century ago. Cells of the primitive erythroid lineage support the transition from rapidly growing embryo to fetus, whereas definitive erythrocytes function during the transition from fetal life to birth and continue to be crucial for a variety of normal physiologic processes. Over the past few years, it has become apparent that the ontogeny and maturation of these lineages are more complex than previously appreciated. In this review, we highlight some common and distinguishing features of the red blood cell lineages and summarize advances in our understanding of how these cells develop and differentiate throughout mammalian ontogeny.
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  • 20
    Publication Date: 1980-02-01
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  • 21
    Publication Date: 2012-02-02
    Description: Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep caused selective apoptosis in alloantigen-activated T cells mediating host tissue injury. This effect was associated with the ability of DZNep to selectively reduce trimethylation of histone H3 lysine 27, deplete the histone methyltransferase Ezh2 specific to trimethylation of histone H3 lysine 27, and activate proapoptotic gene Bim repressed by Ezh2 in antigenic-activated T cells. In contrast, DZNep did not affect the survival of alloantigen-unresponsive T cells in vivo and naive T cells stimulated by IL-2 or IL-7 in vitro. Importantly, inhibition of histone methylation by DZNep treatment in vivo preserved the antileukemia activity of donor T cells and did not impair the recovery of hematopoiesis and lymphocytes, leading to significantly improved survival of recipients after allogeneic BM transplantation. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat ongoing GVHD and other T cell–mediated inflammatory disorders in a broad context.
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  • 22
    Publication Date: 2012-08-02
    Description: EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2flox/flox mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia–like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.
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  • 23
    Publication Date: 2012-11-16
    Description: Abstract 45 Background Homoharringtonine-based induction regimens have been widely used in China for patients with acute myeloid leukemia (AML), which have shown to improve the rate of complete remission (CR) and long-term survival. We aimed to further evaluate its efficacy and safety in treatment of de novo AML. Methods This phase 3 study was done in 17 institutions in China. Patients between the age of 14 and 59 with untreated AML were randomly assigned to receive HAA (homoharringtonine 2 mg/m2/day, days 1–7; cytarabine 100 mg/m2/day, days 1–7, aclarubicin 20 mg/day, days 1–7), HAD (homoharringtonine 2 mg/m2/day, days 1–7; cytarabine 100 mg/m2/day, days 1–7; daunorubicin 40 mg/m2/day, days 1–3) or DA (daunorubicin 40–45 mg/m2/day, days 1–3; cytarabine 100 mg/m2/day, days 1–7) regimen as induction therapy. Patients who achieved partial remission or had a decrease of blast ¡Ý60% could receive a same second induction course. All patients who had a complete remission were offered the same consolidation chemotherapy according to the cytogenetic-risk. The primary endpoints were CR and event-free survival (EFS). The trial is registered in Chinese Clinical Trial Register, number ChiCTR-TRC-06000054. Results 620 patients were randomly assigned to receive HAA (n=207), HAD (n=206) and DA (n=207) regimens. HAA or HAD regimen, as compared with DA regimen, resulted in a higher rate of CR in the first course of induction therapy (67.5% vs. 54.0%, P=0.005; 64.9% vs. 54.0%, P=0.026, respectively). The overall CR rate remained significantly higher in the HAA arm as compared with DA arm (75.0% vs. 61.9%, P=0.005). HAA or HAD regimen has similar rates of adverse events as compared with DA regimen, but was associated with significantly increased risk of induction death (5.8% vs. 1.0%, P=0.007; 6.6% vs. 1.0%, P=0.003, respectively). The EFS was greatly improved in the HAA arm (3-year EFS 35.4±3.5% vs.23.1±3.1%, P=0.002), while not significantly in the HAD arm (3-year EFS 32.7±3.5% vs.23.1±3.1%, P=0.078) as compared with the DA arm. Overall survival (OS) and relapse-free survival (RFS) did not differ significantly in the HAA or HAD arm as compared with DA arm, but an OS and RFS advantage of the HAA arm over the DA arm was observed in patients with favorable or intermediate cytogenetic profile (OS: P=0.014; RFS: P=0.022, respectively). Patients in the HAD arm with NPM1 but not FLT3ITD mutations, as compared with the patients in the DA arm, had an improved EFS (P=0.038). In intermediate cytogenetic profile, patients with mutant CEBPA had prolonged RFS in the HAA arm as compared with the DA arm (P=0.045). Conclusions Homoharringtonine-based induction regimens are associated with a higher rate of CR and improved survival as compared with DA regimen in AML. The toxicity is mild with the exception of a higher rate of induction death. Disclosures: No relevant conflicts of interest to declare.
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  • 24
    Publication Date: 2012-11-16
    Description: Abstract 3641 Background: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of lymphoproliferative disorders, and most subtypes have a poor prognosis even with aggressive chemotherapy. Romidepsin is a potent class 1 histone deacetylase inhibitor approved by the US Food and Drug Administration for treatment of patients with PTCL who have received at least 1 prior therapy and patients with cutaneous T-cell lymphoma who have received at least 1 prior systemic therapy. A phase 2, single-arm, open-label registration study (GPI-06–0002) demonstrated the clinical benefit and tolerability of romidepsin in patients with relapsed or refractory PTCL (data cutoff: Oct 2010). Here, we present an update of the efficacy of GPI-06–0002 and characterize patients who achieved long-term responses (≥ 12 months) as of Dec 2011 (median follow-up: 22.3 months). Methods: Patients with histologically confirmed PTCL (N = 130) who failed or were refractory to ≥ 1 prior systemic therapy received romidepsin 14 mg/m2 as a 4-hour intravenous infusion on days 1, 8, and 15 every 28 days for up to 6 cycles; treatment could be extended for responding patients. The primary endpoint was confirmed/unconfirmed complete response (CR/CRu) determined by an independent review committee (IRC) based on the International Workshop Response Criteria. Secondary endpoints included objective response rate (ORR: CR/CRu + partial response), duration of response (DOR), and time to progression. Disease response was assessed every 2 treatment cycles. Baseline patient characteristics by DOR (≥ 12 months vs 〈 12 months) were examined. Results: The majority of the 130 patients had stage III or IV disease (70%); 28% had bone marrow involvement. PTCL not otherwise specified (53%) and angioimmunoblastic T-cell lymphoma (21%) were the most common subtypes. Patients received a median of 2 prior systemic therapies (range, 1–8); 38% of patients were refractory to their last line of therapy. The ORR was 25% (33 of 130 patients), including CR/CRu in 15% (19 of 130) of patients. The median duration of objective response was 28 months, with the longest response ongoing at 48 months (Figure). Of the 19 patients who achieved CR/CRu, 13 (68%) had not experienced disease progression per the IRC at a median follow-up of 25.8 months. The median duration of CR had not yet been reached (range, 1–48+ months; Figure). Of the 19 patients who achieved CR/CRu, 10 were long-term responders (responses ≥ 12 months). Interestingly, heavy pretreatment (≥ 4 prior systemic therapies) did not preclude patients from achieving long-term CR/CRu: 5 of 10 patients (50%) who maintained CR/CRu for ≥ 12 months were heavily pretreated vs 1 of 9 (11%) patients with CR/CRu maintained for 〈 12 months. Long-term CR/CRu was achieved regardless of response to last prior therapy; only 2 of 10 (20%) long-term responders had an objective response on their last treatment. In contrast, 6 of 9 (67%) patients with CR/CRu for 〈 12 months responded to their last prior therapy. Furthermore, advanced disease did not preclude long-term response to romidepsin: all 10 patients (100%) who maintained CR/CRu for ≥ 12 months had stage III/IV disease vs 55.5% of those who maintained CR/CRu for 〈 12 months. Other characteristics, such as Eastern Cooperative Oncology Group performance status, International Prognostic Index score, age, sex, and race, were similar among patients achieving CR/CRu for ≥ 12 months or 〈 12 months. Conclusions: Single-agent romidepsin induced durable responses in patients with relapsed/refractory PTCL, with responses ongoing at 48 months. None of the examined patient and disease characteristics predicted failure to achieve long-term remissions. These results support the use of romidepsin in relapsed/refractory PTCL. Disclosures: Coiffier: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pro:Allos: Honoraria; Spectrum : Honoraria; Seattle Genetics : Research Funding; Celgene: Honoraria, Research Funding. Prince:Celgene : Consultancy, Honoraria, Research Funding. Foss:Celgene : Consultancy. Sokol:Celgene : Honoraria, Speakers Bureau. Morschhauser:Celgene : Consultancy, Honoraria. Pinter-Brown:Celgene : Consultancy; Allos : Consultancy. Shustov:Celgene : Honoraria, Research Funding, Speakers Bureau. Nielsen:Celgene: Employment, Equity Ownership. Nichols:Celgene: Consultancy, Employment, Equity Ownership. Horwitz:Celgene: Consultancy, Research Funding; Kyowa Hakko Kirin Pharma: Consultancy; Bristol-Myers Squibb: Consultancy; Allos: Consultancy, Research Funding; Genzyme: Consultancy; Johnson & Johnson: Consultancy; Infinity Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Research Funding.
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  • 25
    Publication Date: 2012-07-12
    Description: In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin αIIbβ3 to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-β and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.
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  • 26
    Publication Date: 2012-11-22
    Description: The Pediatric Hydroxyurea Phase 3 Clinical Trial (BABY HUG) was a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial of hydroxyurea in infants (beginning at 9-18 months of age) with sickle cell anemia. An important secondary objective of this study was to compare clinical events between the hydroxyurea and placebo groups. One hundred and ninety-three subjects were randomized to hydroxyurea (20 mg/kg/d) or placebo; there were 374 patient-years of on-study observation. Hydroxyurea was associated with statistically significantly lower rates of initial and recurrent episodes of pain, dactylitis, acute chest syndrome, and hospitalization; even infants who were asymptomatic at enrollment had less dactylitis as well as fewer hospitalizations and transfusions if treated with hydroxyurea. Despite expected mild myelosuppression, hydroxyurea was not associated with an increased risk of bacteremia or serious infection. These data provide important safety and efficacy information for clinicians considering hydroxyurea therapy for very young children with sickle cell anemia. This clinical trial is registered with the National Institutes of Health (NCT00006400, www.clinicaltrials.gov).
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  • 27
    Publication Date: 2012-11-16
    Description: Abstract 578 Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to explore the role of immunotherapeutic strategies in eradicating residual disease. A fundamental challenge to developing an effective anti-tumor immune response is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to tumor vaccines. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (CT-011) alone (Cohort 1) and in combination with a dendritic cell/myeloma fusion cell vaccine (Cohort 2) following ASCT. To date, 27 patients have been enrolled into Cohort 1, in which patients receive three infusions of CT-011 at doses of 3mg/kg given at 6 week intervals beginning 1–3 months following ASCT. Mean age of the patients is 57 years; 61% are male. 11 patients have received at least two infusions of CT-011. The remaining patients are undergoing pre-transplant therapy/transplant. CT-011 has been well tolerated, with possibly related adverse events consisting of transient grade 1–2 leukopenia, diarrhea, fatigue, arthralgia, rash, and peri-orbital edema. One patient developed grade 3 neutropenia, which resolved after two days without growth factor. Immunologic response was determined by quantifying circulating tumor reactive T cells prior to each dose of CT-011 and at 1, 3, 6 months following the last infusion, as defined by the percentage of T cells expressing IFNg in response to ex vivo exposure to autologous tumor lysate. 4 patients have completed 6 months of follow up after the third dose of CT-011, and are evaluable for immune response. CT-011 therapy was associated with the dramatic expansion of myeloma specific T cells. Mean percentage of circulating tumor reactive CD4+ and CD8+ T cells increased from 1.5 and 1.96 respectively prior to the first infusion of CT-011, to 4.26 and 8.28 respectively 1 month following the third infusion. As determined by tetramer staining in the subset of patients who are HLA A2.1, infusion of CT-011 resulted in a mean 9 fold expansion of T cells specific to the MUC1 antigen, which is aberrantly expressed by myeloma cells. Notably, immunologic response to CT-011 persists at 6 months following completion of therapy. Clinical response, as determined by time to disease progression, will be determined with longer follow up, as the median time from transplant is presently 8 months. We are initiating enrollment to Cohort 2, in which patients will be vaccinated with an autologous DC/myeloma fusion vaccine 1 week prior to each dose of CT-011. These data demonstrate that CT-011 results in the expansion of tumor reactive lymphocytes in the early post-transplant period, providing an ideal platform for combination with a tumor vaccine. Disclosures: Rosenblatt: CureTech Ltd.: Research Funding. Schickler:CurTech Ltd.: Employment, Research Funding. Rotem-Yehudar:CureTech Ltd: Employment, Research Funding. Avigan:CureTech Ltd: Research Funding.
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  • 28
    Publication Date: 2012-11-16
    Description: Abstract 687 Background: A persistently positive PET scan after just a few cycles of therapy is predictive of a poor clinical outcome in diffuse large B-cell lymphoma (DLBCL). Prior data suggested that about one third of patients remain PET positive after 2–4 cycles, with ≤ 20% 2 year progression-free survival (PFS) for such patients. A response-adapted strategy was studied in E3404 to test the hypothesis that an early treatment change to four cycles of R-ICE might reduce the treatment failure rate of patients whose PET scan remained positive after an initial 3 cycles of R-CHOP. Study Design: Previously untreated patients with DLBCL stage III, IV, or bulky II, with measurable disease, HIV negative, and with adequate organ reserve, were eligible. PET/CT scan was performed before treatment and again after 3 cycles of R-CHOP. A fourth cycle of R-CHOP was given while the scan was centrally reviewed and scored as either positive or negative for FDG-avid tumor by a single reviewer using criteria based on modifications of the Harmonization Criteria. Persistently PET-positive patients received 4 cycles of R-ICE, while PET-negative patients received 2 more cycles of R-CHOP, for a total of 6 cycles. PET/CT was performed again at the end of treatment. A ≥45% 2 year PFS for mid-treatment PET- positive patients was viewed as a promising result, with 88% power if 33 such patients were accrued. Total accrual of 99 patients was therefore planned. Results: Of 100 patients accrued, 78 were eligible; all but 1 ineligibility was based on central pathology review. Fifty-eight were male; median age was 62 years (20–74); 13% IPI 0–1, 31% IPI 2, 37% IPI 3, and 19% IPI 4–5. Seventy-four of 78 (95%) patients completed the first 3 cycles of R-CHOP. Of 72 patients continuing treatment, 67 (93%) completed protocol treatment: 83% of mid-treatment PET-positive and 93% of PET-negative patients. Of 74 patients undergoing mid-treatment PET scan, 12 (16%) were scored as positive, and 62 (84%) as negative. At the end of treatment, 13% were scored as positive, and 87% as negative. Two-year PFS, from the time of study entry, was 72% among all eligible patients. Two year PFS from the time of mid-treatment PET scan was 45% (90% CI, 21–67%) for patients scored as mid-treatment PET-positive, and 77% (90% CI 67–85%) for patients scored as mid-treatment PET-negative. The 80% confidence interval (corresponding to a test with a one-sided type I error of 10%) did not include the null hypothesis of 25%, implying that the results met the pre-specified threshold. Among all 78 eligible patients, 10 have died (13%); 5-year overall survival was 87% (90% CI 78–92%) Three-year overall survival was 67% (90% CI 40–84%) for mid-treatment PET-positive patients, and 93% (90% CI 85–97%) for mid-treatment PET-negative patients. Conclusions: The 2-year PFS for mid-treatment PET-positive patients met the threshold to be considered promising, but the confidence interval was wider than expected due to the small patient number. In addition, the inter-observer variability in the interpretation of mid-treatment PET scans as a binary variable in this study (studied and reported separately in Horning SJ et al. Blood 2010) implies that treatment modification based on early PET scanning should remain confined to clinical trials. Disclosures: Swinnen: Celgene: Consultancy; Genentech: Research Funding. Quon:Genentech: Research Funding. Advani:Pharmacyclics: Research Funding. Kahl:Roche/Genentech: Consultancy; Celgene: Consultancy; Cell Therapeutics: Consultancy; Janssen: Consultancy; GSK: Consultancy; Gilead: Consultancy. Horning:Genentech: Employment; Roche: stock, stock Other.
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  • 29
    Publication Date: 2012-06-07
    Description: Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, 〈 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.
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  • 30
    Publication Date: 2012-02-09
    Description: Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49+ subsets in BMC rejection by using β2-microglobulin deficient (β2m−/−) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind “self-MHC” were found to be the dominant subset responsible for β2m−/− BMC rejection. This provides in vivo evidence for host MHC class I–dependent functional education. Interestingly, all H2d strain mice regardless of background were able to resist significantly greater amounts of β2m−/−, but not wild-type BMC than H2b mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.
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  • 31
    Publication Date: 2012-11-16
    Description: Abstract 2237 Introduction: von Willebrand disease (VWD) has clinically heterogeneous phenotype. Routine measurements of von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) and FVIII activity (FVIII:C) do not always reflect clinical severity, especially in type 1 VWD. These assays evaluate VWF function under non-physiological static condition - lacking blood flow. We had reported that a new microchip flow chamber system, total-thrombus-formation analysis system (T-TAS®, Fujimori Kogyo, Tokyo) would be a clinically useful flow assay for VWD (ASH 2011). In this study, we extended this study for application to evaluation and hemostatic monitoring for type 1 VWD. Methods: Citrated or hirudin-added blood from 15 patients with type 1 VWD was utilized. Re-calcified citrated blood added corn trypsin inhibitor was injected to a microchip in T-TAS at a constant flow rate (240 s−1), which flow surface was coated by collagen and tissue factor (AR chip). Hirudin-added blood was injected to a microchip in T-TAS at higher shear rate (1,000 s−1) which surface was coated by collagen (PL chip). Flow pressure curve was visualized and time until reach to 10 kPa (T10) was evaluated. AR chip promoted thrombus formation by both platelet aggregation and fibrin generation, whilst PL chip promoted thrombus formation by platelet alone. Standard laboratory tests for VWD were also performed. Clinical severities of VWD patients were evaluated by using a quantitative bleeding score (BS, from −3 without any symptoms to +45 with all major symptoms) previously reported by Tosetto (JTH, 2006). Results: Fifteen patients with type 1 VWD showed low levels of VWF:Ag [median 14% (range 1.3–51%)], VWF:RCo [8% (1.6–32%)], and FVIII:C [31% (3.0–68%)]. T10 in AR chip or PL chip was 17.7 min (11-〉30) or 7.1 min (3.3-〉10) [normal control (n=20); 12.2 min (8.6–16.6) or 3.5 min (2.4–6.6), respectively], showing delayed thrombus formation in type 1 VWD. Correlations between VWF:Ag and VWF:RCo (r2=0.80, p
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  • 32
    Publication Date: 1980-09-01
    Description: A similar defect host defense mechanisms in hairy cell leukemia was defined in two patients. Surface-adherent monocytes were not detected in the peripheral blood nor were monocytes that mediate antibody- dependent cell-mediated cytotoxicity (ADCC) to isoantibody-coated human erythrocytes. In addition, lymphocytes of both patients failed to show blastogenic responses to concanavalin A (Con-A) and pokeweed mitogen (PWM) but showed a vigorous response to phytohemagglutinin (PHA). Other immunologic abnormalities were present but were either moderate in degree or were not present in both patients. In vitro lymphocyte blastogenic responses were fully restored by incubation of patients' leukocytes with a normal donor's adherent monocytes. One patient received daily allogeneic leukocyte transfusion for 4 days. This resulted in complete normalization of monocyte adherence and ADCC that persisted for several months after transfusion and was associated with hemotalogic improvement. Therapy in case 1 resulted in correction of the blastogenic responses to Con-A and PWM. Thus, a host defense defect in hairy cell leukemia has been defined in 2 patients and a preliminary result suggests that therapy with leukocyte transfusions may be useful in the postsplenectomy patient with an infectious complication and should be explored further.
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  • 33
    Publication Date: 2012-09-27
    Description: FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34+ was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.
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  • 34
    Publication Date: 2012-09-27
    Description: RUNX1 gene alterations are associated with acquired and inherited hematologic malignancies that include familial platelet disorder/acute myeloid leukemia, primary or secondary acute myeloid leukemia, and chronic myelomonocytic leukemia. Recently, we reported that RUNX1-mediated silencing of nonmuscle myosin heavy chain IIB (MYH10) was required for megakaryocyte ploidization and maturation. Here we demonstrate that runx1 deletion in mice induces the persistence of MYH10 in platelets, and a similar persistence was observed in platelets of patients with constitutional (familial platelet disorder/acute myeloid leukemia) or acquired (chronic myelomonocytic leukemia) RUNX1 mutations. MYH10 was also detected in platelets of patients with the Paris-Trousseau syndrome, a thrombocytopenia related to the deletion of the transcription factor FLI1 that forms a complex with RUNX1 to regulate megakaryopoiesis, whereas MYH10 persistence was not observed in other inherited forms of thrombocytopenia. We propose MYH10 detection as a new and simple tool to identify inherited platelet disorders and myeloid neoplasms with abnormalities in RUNX1 and its associated proteins.
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  • 35
    Publication Date: 2012-03-22
    Description: This systematic review was designed to provide more precise effect estimates of inhibitor development for the various types of F8 gene mutations in patients with severe hemophilia A. The primary outcome was inhibitor development and the secondary outcome was high-titer-inhibitor development. A systematic literature search was performed to include cohort studies published in peer-reviewed journals with data on inhibitor incidences in the various F8 gene mutation types and a mutation detection rate of at least 80%. Pooled odds ratios (ORs) of inhibitor development for different types of F8 gene mutations were calculated with intron 22 inversion as the reference. Data were included from 30 studies on 5383 patients, including 1029 inhibitor patients. The inhibitor risk in large deletions and nonsense mutations was higher than in intron 22 inversions (pooled OR = 3.6, 95% confidence interval [95% CI], 2.3-5.7 and OR = 1.4, 95% CI, 1.1-1.8, respectively), the risk in intron 1 inversions and splice-site mutations was equal (pooled OR = 0.9; 95% CI, 0.6-1.5 and OR = 1.0; 95% CI, 0.6-1.5), and the risk in small deletions/insertions and missense mutations was lower (pooled OR = 0.5; 95% CI, 0.4-0.6 and OR = 0.3; 95% CI, 0.2-0.4, respectively). The relative risks for developing high titer inhibitors were similar.
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  • 36
    Publication Date: 2012-05-17
    Description: Severe bacterial sepsis often leads to a systemic procoagulant and proinflammatory condition that can manifest as disseminated intravascular coagulation, septic shock, and multiple organ failure. Because activation of the contact proteases factor XII (FXII), prekallikrein, and factor XI (FXI) can trigger coagulation and inflammatory responses, the contact factors have been considered potential targets for the treatment of sepsis. However, the pathogenic role of contact activation in severe infections has not been well defined. We therefore investigated whether an anticoagulant antibody (14E11) that selectively inhibits prothrombotic FXI activation by activated FXII (FXIIa) modifies the course of bowel perforation-induced peritoneal sepsis in mice. Early anticoagulation with 14E11 suppressed systemic thrombin- antithrombin complex formation, IL-6, and TNF-α levels, and reduced platelet consumption in the circulation and deposition in the blood vessels. Treatment with 14E11 within 12 hours after bowel perforation significantly improved survival compared with vehicle treatment, and the saturating dose did not increase tail bleeding. These data suggest that severe polymicrobial abdominal infection induces prothrombotic FXI activation, to the detriment of the host. Systemic anticoagulation by inhibiting FXI activation or FXIIa procoagulant activity during sepsis may therefore limit the development of disseminated intravascular coagulation without increasing bleeding risks.
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  • 37
    Publication Date: 2012-11-16
    Description: Abstract 964 Background: High dose immunosuppressive therapy and hematopoietic stem cell transplantation (HSCT) has shown efficacy in severe or rapidly progressive systemic sclerosis (SSc) in phase 1 and 2 trials with durable responses in two thirds of patients (pts), while a recent phase 2 randomised trial in 19 SSc pts showed superior benefit of HSCT over iv pulse cyclophosphamide (Cy) on skin score and lung function. Methods: The ASTIS-trial (Autologous Stem cell Transplantation International Scleroderma trial) is a multinational prospective randomized controlled phase 3 trial, comparing safety and efficacy of HSCT versus Cy in early progressive dcSSc pts with disease duration of a) 4 years or less and evidence of organ involvement or b) of 2 years or less and evidence of systemic inflammation with or without major organ involvement. Major exclusion criteria were: concomitant severe SSc disease (mean PAP 〉 50 mmHg, DLCO 〈 40% predicted, creatinine clearance (CCl) 3months oral) treatment), liver failure. Pts randomized to the transplant arm underwent mobilization with Cy 2×2 g/m2 + G-CSF 10mcg/kg/d, conditioning with Cy 200 mg/kg, rbATG 7.5 mg/kg, followed by reinfusion of CD34+ autologous HSCT. Controls were treated with 12× monthly iv pulse Cy 750 mg/m2. Crossing over was allowed after 2 years. The primary endpoint was event-free survival (EFS), defined as survival until death or development of major organ failure at 2 yrs. Toxicity according to WHO criteria and progression free survival (defined as worsening of modified Rodnan skin score, functional ability, major organ function) were the main secondary endpoints. The effects of treatment were analyzed on an ITT basis and by comparing EFS using the KM survival curves (using log-rank test) and Cox models. Results: 156 pts (female 59%), from 27 centers were enrolled in 10 countries from March 2001 until October 2009 and randomized to HSCT (n=79) or iv pulse Cy (n=77). Seventy-five pts in each arm started treatment, 70 pts in HSCT and 58 pts in control groups completed treatment. The median time (Interquartile Range (IR)) from randomization to completion of treatment was 93 (43.0) days in the HSCT and 338 (41.75) days in the control groups respectively. Baseline characteristics (mean (SD)) of the pts were: age 44 (11.2) yrs, SSc duration 1.4 (1.3) yrs, BMI 24 (14.4), Rodnan skin score 25 (8), HAQ 1.35 (0.8), prior Cy therapy 22%, creat cl 116 (39.5) ml/min, LVEF 65% (8.5), DLCO 59% (14), with no significant differences between the 2 arms. With data cut at 1 May 2012, median follow-up (IR) are 33 (42.0) and 27 (34.0) months in the HSCT and control groups respectively. Forty two events occurred : 18 in the HSCT group (16 deaths and 2 irreversible renal failures) and 24 in the control group (24 deaths). Event-free survival was time-dependent with a hazard ratio at 84 months of 0.22 (95% CI 0.08–0.58, P= 0.002). Eight deaths (including 1 during mobilization and 1 after conditioning) in the HSCT group were deemed treatment-related by the independent data monitoring committee with heart failure (3), ARDS (2), multiple organ failure (2) and pulmonary oedema (1) as the causes of death. In the control group, none died from treatment causes and most deaths were due to progressive disease. Eight pts in the control arm received rescue HSCT treatment, one of whom later died from secondary acute myeloid leukaemia. Two HSCT pts received rescue iv Cy therapy. Conclusions: The ASTIS-trial is the first international, investigator-initiated, phase 3 HSCT trial in early diffuse cutaneous systemic sclerosis. The data show that despite 10% treatment-related mortality, long term event-free survival and overall survival were better in the HSCT group than in the group treated with iv pulse cyclophosphamide. (Funded by the European Group for Blood and Marrow Transplantation, European League Against Rheumatism, AP-HP, NIHR, DIGR, Imtix-Sangstat, Miltenyi-Biotec, Amgen Europe; Current Controlled Trials number, ISRCTN 54371254). Disclosures: No relevant conflicts of interest to declare.
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  • 38
    Publication Date: 2012-05-31
    Description: Human herpes virus 8 (HHV-8) or Kaposi sarcoma-associated herpes virus is the etiologic agent of Kaposi sarcoma, primary effusion lymphoma, and plasma cell-type multicentric Castleman disease (MCD). HHV-8 encodes a viral homolog of human IL-6, called viral IL-6 (vIL-6), which does not require the cellular IL-6 receptor for binding to the ubiquitously expressed gp130 receptor subunit and subsequent JAK-STAT signaling. Thus, in contrast to IL-6, vIL-6 can stimulate virtually all cells in the body. To elucidate the mechanism by which vIL-6 drives human diseases, we generated transgenic mice that constitutively express vIL-6 under control of the MHC class I promoter. The mice were found to exhibit vIL-6 serum levels comparable with those observed in HHV-8–infected patients, to contain elevated amounts of phosphorylated STAT3 in spleen and lymph nodes, where vIL-6 was produced, and to spontaneously develop key features of human plasma cell-type MCD, including splenomegaly, multifocal lymphadenopathy, hypergammaglobulin-emia, and plasmacytosis. Transfer of the vIL-6 transgene onto an IL-6–deficient genetic background abrogated MCD-like phenotypes, indicating that endogenous mouse IL-6 is a crucial cofactor in the natural history of the disease. Our results in mice suggest that human IL-6 plays an important role in the pathogenesis of HHV-8–associated MCD.
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  • 39
    Publication Date: 2012-07-26
    Description: Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 1011, 2.2 × 1011, or 4.4 × 1011 platelets/m2 per transfusion, given for morning counts of ≤ 10 000 platelets/μL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P 〈 .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts. This trial was registered at www.clinicaltrials.gov as #NCT00128713.
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  • 40
    Publication Date: 2012-10-11
    Description: Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for ∼ 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible.
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  • 41
    Publication Date: 2012-06-21
    Description: The SHIELD program for Hodgkin lymphoma in patients 60 years of age or older, prospectively evaluated clinical features and outcome in a large patient cohort (n = 175). The central element was a phase 2 study of VEPEMB chemotherapy (n = 103, median age 73 years) incorporating comorbidity assessment. A total of 72 other patients were treated off-study but registered prospectively and treated concurrently with: ABVD (n = 35); CLVPP (n = 19), or other (n = 18). Of VEPEMB patients, 31 had early-stage disease (stage 1A/2A) and received VEPEMB 3 times plus radiotherapy. Median follow-up was 36 months. Complete remission (CR) rate (intention-to-treat) was 74% and 3-year overall survival (OS) and progression-free survival (PFS) were 81% and 74%, respectively. A total of 72 patients had advanced-stage disease (stage 1B/2B/3 or 4) and received VEPEMB 6 times. CR rate was 61% with 3-year OS and PFS of 66% and 58%, respectively. Of patients achieving CR, 13% with early-stage and 5% with advanced-stage disease progressed. Overall treatment-related mortality was 7%. In patients treated with curative intent with VEPEMB, ABVD, and CLVPP (n = 157), CR linked to several factors in univariate analysis. In a Cox regression model only, obtaining CR remained significant for OS and CR plus comorbidity and age for PFS. RS-EBV status had no significant effect on outcome.
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  • 42
    Publication Date: 2012-08-09
    Description: The development of tools for the prediction of nonrelapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (HSCT) would offer a major guidance in the therapeutic decision. Recently, the Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) has been associated with increased NRM risk in several retrospective studies, but its clinical utility has never been demonstrated prospectively in an adequately sized cohort. To this aim, we prospectively evaluated a consecutive cohort of 1937 patients receiving HSCT in Italy over 2 years. HCT-CI was strongly correlated with both 2-year NRM (14.7%, 21.3%, and 27.3% in patients having an HCT-CI score of 0, 1-2, and ≥ 3, respectively) and overall survival (56.4%, 54.5%, and 41.3%, respectively). There was an excellent calibration between the predicted and observed 2-year NRM in patients having an HCT-CI score of 0 and 1-2, whereas in the ≥ 3 group the predicted NRM overestimated the observed NRM (41% vs 27.3%). HCT-CI alone was the strongest predictor of NRM in patients with lymphoma, myelodysplastic syndrome, and acute myeloid leukemia in first remission (c-statistics 0.66, 064, and 0.59, respectively). We confirm the clinical utility of the HCT-CI score that could also identify patients at low NRM risk possibly benefiting from an HSCT-based treatment strategy.
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  • 43
    Publication Date: 1980-05-01
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  • 44
    Publication Date: 2012-11-16
    Description: Abstract 3875 Introduction: Immunologic environment influences progression of lymphoid malignancies. Specifically, shifts in subsets of natural killer (NK) and T cells as well as tumor expression of inhibitory ligands may contribute to ability to evade host detection. Immune dysfunction may be particularly important in CLL/SLL, as prevalent circulating tumor cells engage in persistent, widespread interactions with immune cells; commonly-used mAb therapies (e.g. rituximab, alemtuzumab) rely upon ADCC mediated by NK cells and other innate effectors; and disease course is highly variable and not fully accounted for by tumor-intrinsic prognostic factors. Therefore, to better characterize the immune system in CLL/SLL, we prospectively assessed NK and T cell frequency, phenotype, and function in a series of CLL/SLL patients. Methods: Serial blood samples (up to 3 samples each, 3–6 months apart) were collected from 31 untreated CLL/SLL patients (median age 66) and 15 healthy age-matched controls (HC), and peripheral blood lymphocytes (PBL) analyzed directly ex vivo by multiparameter flow cytometry (160 distinct parameters evaluated, primarily on T and NK cells). NK cell-mediated natural and antibody-dependent cytotoxicity were also assessed by CD107a degranulation assay following PBL co-culture with rituximab, 721.221 EBV-transformed lymphoma cells, or both. Differences in parameters between patients and controls, or between progressors and non-progressors [categorized based on updated NCI-WG criteria (Blood 2008;111:5446)] were analyzed by Wilcoxon rank-sum test. All subjects signed IRB approved informed consent forms. Results: CLL/SLL VS. HC: CLL/SLL samples displayed a marked decrease in the ability of the cytolytic CD56dim NK cells to degranulate in response to tumor, both with or without rituximab (Table 1). CD56dim NK cells from CLL/SLL patients also displayed a more immature phenotype (↓CD57, ↓NKG2D, ↑CD27, ↓KIR) than those from HC, suggesting either a block in differentiation or elimination of the most-differentiated cells. NK cell expression of NKp44, CD69, CD62L, CD137, granzyme B, perforin, or PD-1, as well as tumor-induced NK cell production of IFNγ, did not differ. CLL/SLL patients had increased total T cells with a decreased CD4:CD8 ratio, associated with increased total number of CD8 T cells, greater activation of naive CD4 T cells and transition to a memory phenotype. Treg (CD4+CD25+FoxP3+) frequency was significantly higher in CLL/SLL patients (4.5% vs. 1.8% of CD4 T cells, p=0.005), as was PD-1 expression on both CD4 and CD8 T cells, while CD137 and ICOS expression was similar in both groups. PROGRESSORS VS. NON-PROGRESSORS: With median follow-up of 16.5 months (range 1–37), 7 of 31 patients have met criteria for progression. Compared to non-progressors, progressors showed changes in the CD56bright NK cell compartment suggestive of increased activation and accelerated differentiation, with increased expression of CD69, granzyme B, perforin, CD16, and KIR. However, no significant functional differences in NK cells, or consistent differences in T cell subsets, have been observed to date. Conclusions: CLL/SLL patients have a shift toward less mature NK cells, associated with deficits in NK cell degranulation against tumor targets, compared with healthy donors. Those CLL/SLL patients who progressed had greater CD56 bright NK cell phenotypic aberrancies than non-progressors, though these findings require confirmation with a larger cohort. Taken together, our findings support the hypothesis that immune dysfunction in CLL/SLL may be due in part to a block in NK cell differentiation or loss of more mature cells, and current studies are exploring these possibilities and potential mechanisms. Given these findings, along with the immunosuppressive changes observed in the T cell compartment (↑Tregs, ↑PD-1), these data support therapeutic strategies in CLL/SLL aimed at augmenting NK and/or T cell function. Disclosures: No relevant conflicts of interest to declare.
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  • 45
    Publication Date: 2012-11-16
    Description: Abstract 2532 Background: The clinical management of patients with pediatric B-lineage acute lymphoblastic leukemia (B-ALL) relies on combinations of multiagent anticancer drugs and risk-stratified treatment. The prognostic significance of minimal residual detection (MRD) in pediatric B-ALL has been demonstrated in multiple cohorts. Allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin or T-cell receptor rearrangements, a method for MRD detection, requires the development of patient-specific reagents and cannot detect clonal evolution. ASO-PCR also has limited coverage, with clonal rearrangements being detected in only 90% of patients. We developed the sequencing-based LymphoSIGHT platform to address these limitations. Here we report the results of a pilot study of MRD detection using both the sequencing assay and ASO-PCR in paired diagnostic and end-of-induction samples from 7 B-ALL patients. Analysis of 82 additional patients is ongoing. Methods: Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up bone marrow samples. Amplified products were sequenced to obtain 〉1 million reads (20× coverage per B-cell), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then assessed in the end-of-induction sample. A quantitative and standardized measure of MRD level among all leukocytes in the sample was determined using internal reference DNA. Following identification of IgH clonal rearrangements and MRD assessment using the sequencing assay, we examined the MRD results obtained at Boston Children's Hospital using ASO-PCR. Among the 7 patients analyzed to date, 6 patients were in complete remission at the time of the second sample; 1 patient had persistent evidence of disease. Sequencing was performed blinded to all clinical and ASO-PCR information on these patients. Results: With the sequencing platform, we detected a high-frequency IgH clonal rearrangement in all 7 diagnostic ALL samples. The leukemic clonotype that was identified at diagnosis was detected in the end-of-induction sample in each of the 7 patients. The quantitative range of the leukemic sequence in MRD samples ranged over 5 orders of magnitude. MRD results were concordant between sequencing and ASO-PCR in 5 of 7 patients. The detected MRD level differed by 〉 10 fold in 2 patients. In patient 1, sequencing detected high MRD, while low MRD was detected by ASO-PCR; this patient relapsed 1 year later while still on therapy. In patient 6, sequencing detected low MRD, while ASO-PCR detected high MRD. This patient remains in complete remission after 7 years. In patient 7, sequencing and ASO-PCR concordantly detected low MRD; this patient relapsed after completion of therapy. Conclusions: Results from the application of a high-throughput sequencing method for MRD detection in childhood B-ALL are shown. The sequencing assay does not require development of patient-specific reagents, which will reduce cost and laboratory turnaround time. This data, along with the laboratory workflow improvements, support the use of the sequencing assay as a next-generation MRD test for B-ALL. Analysis of samples from 82 patients is ongoing. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.
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  • 46
    Publication Date: 2012-11-16
    Description: Abstract 2512 Background: Tremendous progress has been made in the management of acute lymphoblastic leukemia (ALL) in children, in part, through the wide use of minimal residual disease (MRD) monitoring to guide therapeutic intensification before and after allotransplantation. Unfortunately, poor accessibility and the high costs of MRD testing have limited its use in the management of adult ALL. A universally applicable MRD quantification method has the potential to revolutionize the management of ALL in adults. Most B- and T-cell ALL patients exhibit clonal rearrangements of one or more immunoglobulin (heavy chain, IGH; light chain, IGK/IGL) or T-cell receptor (beta, B; delta, D; gamma, G) genes. Such rearrangements may be quantified in a mixture of polyclonal B or T cells by massively parallel high-throughput sequencing (HTS), enabling highly sensitive MRD quantification. Methods: Thirty-six allografted ALL patients were selected for this retrospective study based on availability of a diagnostic sample containing leukemic cells, which was necessary for validating the amplification and sequencing method with each disease clonotype. Using Sequenta's LymphoSIGHT platform, we amplified and sequenced rearranged immunoreceptor (IR) loci from genomic DNA extracted from peripheral blood (PB) or bone marrow aspirates (BM) using V and J segment consensus primers for each IR gene (IGH, TCRB, TCRD, and TCRG) and, in some cases, D segment primers for incomplete IGH-DJ rearrangements. Sequences were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high prevalence in a PB or BM sample at a time of high disease burden. MRD levels were then determined in serial samples of PB or BM and quantified using spiked-in reference sequences. A total of 227 samples with a median 442,672 input genomes (range 8,038 – 7,162,715) were evaluated by IR-HTS. Results: A clonal IGH sequence was identified in 17/36 (47%) ALL patients. Amongst patients who did not have a detectable IGH clonotype, 14/19 (74%) had clonal sequences at one or more other loci, including partial IGH-DJ rearrangements (5/14; 36%), TCRB (4/14; 29%), TCRD (3/14; 21%), or TCRG (6/14; 43%). In total, 31 of 36 (86%) ALL patients had a clonal IR sequence suitable for MRD quantification. In 4 of 5 cases without an identified clonal sequence, only PB, but no BM samples, were available. Twenty patients achieved MRD negativity following HCT and 11 did not. In the MRD negative group, 12/20 patients (60%) ultimately relapsed with a median time to clinical progression of 231 days (range 77–889), whereas 11/11 patients (100%) in the MRD positive group relapsed with a median time to clinical progression of 139 days (range 60–304). The 12 patients who relapsed after achieving MRD negativity showed a median time to molecular disease progression of 93 days (range 59–689 days). Of the 8 patients who maintained MRD negativity following HCT, 7 remain alive at a median 1542 days (range 1133–2557 days). All 22 patients with MRD detected following HCT relapsed and 21 (95%) died (median survival 387 days; range 85–1991 days) (Figure 1). The lead time between molecular disease detection by IR-HTS and clinical relapse was a median 69 days (range 0–207 days) with significant likelihood of MRD detection in a PB sample at least one, three, and six months (each p
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  • 47
    Publication Date: 1980-01-01
    Description: We studied the anti-IgG-induced agglutination of both normal and abnormal red blood cells (RBC) using a sensitive, automated antiglobulin test. Normal RBC agglutinated strongly with anti-IgG antibody, indicating that IgG was present on the erythrocyte membrane. Young RBC, recovered by centrifugation from a normal RBC population, agglutinated with anti-IgG less than the old cells, suggesting that immunoglobulin G accumulated gradually on the RBC membrane in vivo. The degree of anti-IgG-induced RBC agglutination correlated negatively with the reticulocyte count and positively with the concentration of plasma IgG. RBC from patients with hypogammaglobulinemia appeared to have a low subnormal quantity of membrane-bound IgG, whereas the reverse was the case in hypergammaglobulinemia. During hemolytic episodes, RBC of patients with hereditary spherocytosis agglutinated poorly with anti- IgG, apparently due to predominance of young RBC. RBC of patients with nonspherocytic. Coombs-negative, nonimmune hemolytic anemia usually also agglutinated poorly with anti-IgG. However, in some cases of active hemolytic anemia, decreased agglutination with anti-IgG was not observed, suggesting that these young RBC had increased amounts of membrane-bound IgG.
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  • 48
    Publication Date: 2012-11-16
    Description: Abstract 3415 Background: The incidence of venous thrombotic events (VTE) in children has risen substantially over the past decade, resulting in increasing use of anticoagulants and making it imperative that pharmacokinetic (PK) and pharmacodynamic studies be performed in children. Fondaparinux has several advantages over low molecular weight heparins including once-daily dosing, no risk for heparin-induced thrombocytopenia, and possibly reduced effects on bone mineral metabolism. A PK, dose-finding, and safety study of fondaparinux was published; however it only studied relatively short-term outcomes and adverse effects. The purpose of this study was to investigate the long-term safety, dosing, and efficacy of fondaparinux in children. Methods: The study included all children 1–18 years (yrs) old treated consecutively with fondaparinux in a single institution between September 1, 2007 and June 20, 2011. The following data were abstracted from the medical records: demographics, location of initial VTE, fondaparinux dosing and levels, bleeding events, other adverse events, status of VTE at each subsequent imaging study (complete resolution, partial resolution, no change, or progression), and VTE recurrence. Descriptive statistics are used to describe the patients and the outcomes. Results: Data from 22 patients were collected and all were available for the safety analysis while 19 were analyzed for dosing (1 excluded due to only receiving 2 doses secondary to an allergic reaction and 2 excluded for ineligible diagnoses) and 16 for efficacy (1 excluded due to allergic reaction, 1 because fondaparinux was given as prophylaxis, and 4 due to ineligible diagnoses/insufficient data). There were 11 females (F) and 11 males (M) (10 F and 9 M analyzed for dosing; 9 F and 7 M for efficacy). The mean age of the patients was 9 yrs (median: 10 yrs; range: 1–17 yrs). The mean duration of treatment with fondaparinux was 377 days (d) (median: 171 d; range: 6–1566 d). The mean dose of fondaparinux was 0.1 mg/kg/dose (median: 0.1 mg/kg/dose; range: 0.07–1.4 mg/kg/dose). Nine of 16 evaluable patients (56.3%) had complete resolution of their thrombus while 6/16 (37.5%) had partial resolution, and 1/16 (6.3%) had no change in their thrombus. Thus, 15/16 (93.8%) patients had either a complete or partial response and 0/16 had progression. The mean time to best outcome from initiation of fondaparinux was 110.5 d (median: 63 d; range: 4–487 d). Seven patients needed a total of 12 dose adjustments (one subject with 3 adjustments and one with 4) to achieve therapeutic levels. Three patients (18.8%) (2 had prior complete resolution of the initial VTE) had a recurrent VTE. Two patients were on fondaparinux at the time of recurrence and 1 was on warfarin. There were 2 major (intracranial hemorrhage- occurred prior to initiation of fondaparinux and subretinal hemorrhage) and 3 minor (all with blood in stool) bleeding events. One patient had an allergic reaction after starting fondaparinux. Conclusions: In this long-term follow-up study on children treated with fondaparinux for VTE, 95% of patients had either complete or partial resolution while the recurrence rate was in line with previous studies. There were 5 bleeding events (2 major and 3 minor), though only 1 event required the discontinuation of fondaparinux. Given the advantages of fondaparinux over other anticoagulants, this study suggests that fondaparinux could be considered a safe and effective alternative for the management of VTE in children. Disclosures: Off Label Use: fondaparinux: anti-coagulation for the treatment of venous thromboembolic events in children. Young:Biogen Idec: Research Funding; Baxter: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria, Research Funding.
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  • 49
    Publication Date: 2012-06-07
    Description: The Medical Research Council Myeloma IX Trial (ISRCTNG8454111) examined traditional and thalidomide-based induction and maintenance regimens and IV zoledronic acid (ZOL) and oral clodronate (CLO) in 1960 patients with newly diagnosed multiple myeloma. Overall survival (OS) and skeletal-related event (SRE) data have been reported for the overall trial population. The present analysis investigated optimal therapy regimens for different patient populations in Myeloma IX. Patients were assigned to intensive or nonintensive treatment pathways and randomized to induction cyclophosphamide, vincristine, doxorubicin, and dexamethasone (CVAD) versus cyclophosphamide, thalidomide, and dexamethasone (CTD; intensive) or melphalan and prednisolone versus attenuated oral CTD (CTDa; nonintensive). Patients were also randomized to ZOL or CLO. In the nonintensive pathway, CTDa produced better responses and lower SRE rates than melphalan and prednisolone. ZOL improved OS compared with CLO independently of sex, stage, or myeloma subtype, most profoundly in patients with baseline bone disease or other SREs. In patients treated for ≥ 2 years, ZOL improved OS compared with CLO from randomization (median not reached for either; P = .02) and also from first on-study disease progression (median, 34 months for ZOL vs 27 months for CLO; P = .03). Thalidomide-containing regimens had better efficacy than traditional regimens, and ZOL demonstrated greater benefits than CLO.
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  • 50
    Publication Date: 2012-07-26
    Description: Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ∼ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.
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  • 51
    Publication Date: 2012-01-26
    Description: In patients with multiple myeloma (MM), risk stratification by chromosomal abnormalities may enable a more rational selection of therapeutic approaches. In the present study, we analyzed the prognostic value of 12 chromosomal abnormalities in a series of 354 MM patients treated within the HOVON-65/GMMG-HD4 trial. Because of the 2-arm design of the study, we were able to analyze the effect of a bortezomib-based treatment before and after autologous stem cell transplantation (arm B) compared with standard treatment without bortezomib (arm A). For allanalyzed chromosomal aberrations, progression-free survival (PFS) and overall survival (OS) were at least equal or superior in the bortezomib arm compared with the standard arm. Strikingly, patients with del(17p13) benefited the most from the bortezomib-containing treatment: the median PFS in arm A was 12.0 months and in arm B it was 26.2 months (P = .024); the 3 year-OS for arm A was 17% and for arm B it was 69% (P = .028). After multivariate analysis, del(17p13) was an independent predictor for PFS (P 〈 .0001) and OS (P 〈 .0001) in arm A, whereas no statistically significant effect on PFS (P = .28) or OS (P = .12) was seen in arm B. In conclusion, the adverse impact of del(17p13) on PFS and OS could be significantly reduced by bortezomib-based treatment, suggesting that long-term administration of bortezomib should be recommended for patients carrying del(17p13). This trial is registered at the International Standard Randomised Controlled Trial Number Register as ISRCTN64455289.
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  • 52
    Publication Date: 1980-06-01
    Description: We investigated the hypoxemia associated with extreme leukocytosis in leukemic patients. In vitro experiments showed that the rate of decrease in the partial pressure of oxygen in the blood samples from such patients was proportional to the white cell count. In the presence of normal white cell count no drop in PO2 was observed. We conclude that a low arterial oxygen tension in the presence of extreme leukocytosis reflects oxygen consumption by leukocytes rather than true hypoxemia. Both normal as well as leukemic leukocytes appear to exhibit this phenomenon.
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  • 53
    Publication Date: 2012-11-16
    Description: Abstract 2287 High throughput genomic testing for blood groups allows large scale antigen typing and assessment of donor pool compatibility with chronic transfusion dependent populations, particularly thalassemia or sickle cell disease (SCD) patients, in order to decrease RBC alloimmunization. Thus, it is crucial to determine if the quantity and antigen diversity of the donor pool meets the demands to sustain these patients on phenotype/genotype extended matched chronic transfusion protocols. We calculated the most common extended RBC predicted phenotypes for the 12 major clinically significant blood group antigens, D, CcEe, K, Jka/b, Fya/b, and Ss, in patients undergoing chronic transfusion for sickle cell disease (n=203) or thalassemia (n=98). The most common phenotypes in each diagnostic group were used to determine the prevalence of these phenotypes in a single day donor inventory (n=5,000) and stratified by ethnic group (70% Caucasian, 10% African-American, 13% Hispanic, 5% Asian, and 2% Other). All patient samples were tested for the presence of the GATA mutation which disrupts erythroid expression of Fy(b), and if present deemed not at risk for Fy(b) alloimmunization. The majority of patients with SCD and thalassemia are RhD positive (97% and 90% respectively), but differ in extended Rh phenotype, with Ro (Dce) prevalent in SCD pa tients (61%), and R1 (DCe) in thalassemia patients (79%). For patients with SCD, the most prevalent antigen-negative phenotypes were 17% C-E-K-,Fy(a-),Jk(b-), S-; 9% C-E-K-, Fy(a-),S-; 5% E-K-,Fy(a-),S-; and 4% C-K-,Fy(a-), Jk(b-), S-. In patients with thalassemia, no minor antigen profile exceeded 5% of individuals. The most prevalent antigen-negative profiles were 5% E-c-K-, Fy(b-),Jk(b-),S-, and 4% E-K-. Comparison of the most prevalent antigen-negative phenotype in patients with SCD with the donors revealed only 0.06% Caucasian (n=2), but 20% of the African-American donors (n=90) were antigen-negative matches. For the second most prevalent phenotype, 0.08% Caucasian (n=3) and 33% of African-American (n=167), 2% of Hispanic (n=13), and 5% other (n=5) were antigen-negative matches. For the third and fourth prevalent phenotypes, 47% of African-American (n=233) and 23% (n=115) respectively, were antigen-negative matches, while only 0.14% (n=5) and 0.06% (n=2) of Caucasians, but 5% (n=31) and 2% (n=13) of Hispanic donors were appropriate matches, respectively. For the thalassemia patients, antigen matches for the most common phenotype were found most often in Asian (13%) and donors identifying as “other” (6%). Matches were present in only 2% of the Caucasians, 2% of Hispanics, and 1.8% of African-American donors for thalassemia patients. These results confirmed the importance and impact of African-American donors for extended antigen-matching for patients with SCD. Less than 1% of our Caucasian donors could serve as extended matching for SCD. Nearly 20% of patients with SCD are negative for a common group of antigens, which allows future donor recruitment efforts focused on extended antigen profiles of the donor. Patients with thalassemia do not have a common antigen-negative profile, but extended matching for these patients can be improved by increase recruitment of Asian donors. High throughput genotyping enables typing of large numbers of donors, and potentially the majority of the donor inventory. Analysis of antigen-negative phenotypes in the donor pool with analysis of patient groups is important for inventory management, focused donor recruitment, and improved transfusion practice by avoiding alloimmunization. Disclosures: Stassinopoulos: Cerus: Employment, Equity Ownership, Patents & Royalties.
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  • 54
    Publication Date: 2012-11-16
    Description: Abstract 173 One of the most common karyotypic abnormalities identified in myelodysplastic syndromes (MDS) is monosomy 7 (del7) or deletion of the long arm of chromosome 7 (del7q). The presence of del7/del7q carries a poor prognosis in MDS, MDS/myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML); the impact of these defects appears similar. Recently, a copy-neutral type of loss of heterozygozity (LOH also referred to as a somatic UPD) has been identified on 7q. Microdeletion on 7q corresponding to the EZH2 locus led to identification of inactivating mutations in this gene, though hemizygous EZH2 mutations are only rarely found and do not fully explain del7/7q pathogenesis. We performed a comprehensive analysis of myeloid neoplasms (N=189), using next generation whole exome sequencing technology, including MDS (N=34), MDS/MPN (N=26) or MPN (N=4) and 124 with AML (both primary and secondary). Among them, LOH7, involving del7/del7q were observed in 17% of cases (N=33). To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalyitic filtering approach, whereby paired DNA (tumor/CD3 lymphocyte) were sequenced and results aligned using Burrows-Wheeler Aligner and variants detected using GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on searching for del7/7q linked somatic mutational events involved comparisons of mutations in the area of del7q to cases diploid for this locus. We hypothesized that there may be heterozygous mutations of 7q, which could lead to functional haploinsufficiency that is also a result of del7q (haploinsuffcient theory, heterozygous mutations). Conversely, mutations may be either unique to del7q hemizygous inactivation, or shared between 7q diploid and haploid cases. In total, we found alterations in 12 genes located on chromosome 7 (6% of all alterations found). Using filtering strategies we narrowed the focus to “tier 1” mutations to avoid false positives; 11 mutated genes were found in cases with del7/7q and 2 in UPD7q. For example, novel hemzygous (but not heterozygous mutations) of an E3 ubiquitin ligase CUL1 gene were detected only in cases with del7/7q, suggesting that the wild type allele is protective. In cases with diploid 7q, 24 heterozygous alterations were observed (10 genes shared with del7/7q). The previously described EZH2 mutations were seen in heterozygous, homozygous and hemizygous configurations, but were most common in UPD7q (100%), while only 7% of del7/7q cases were positive. Notably, 5/12 mutant genes were located in commonly deleted regions (CDRs) either 7q22, 7q34 or 7q35–36. These CDRs also contain recurrently mutated lesions, including 7q22 (CUX1:n=4; STAG3:n=2), 7q34 (a splicing factor; LUC7L2: n=3) and 7q35–36 (EZH2: n=10). When we investigated the association between haploinsufficiency and heterozygous mutations, among those on del7/7q, cases with wild type forms of corresponding genes showed decreased expression. Similarly, such mutations were occasionally present in diploid configuration; here again the wild type cases showed a decreased expression. These findings suggest that mutated genes located in CDRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. EZH2 is a good example of such a gene. We also searched accessory genetic events observed on other chromosomes along with del7/7q and UPD7. By SNP-A, there were clear differences among 3 LOH7 groups, in which del7 was more associated with accessory chromosomal defects than cases with UPD7q or del7. Similarly, mutational patterns were specific to each LOH cohort. For example, while well known frequently mutated genes, such as U2AF1, TET2 and TP53, were commonly found in all 3 LOH7 groups, some specific genes, including the CSMD family, were uniquely observed in monosomy 7, not in del7q or UPD7. Similarly, LOH7q was associated with somatic mutations in SETBP1 and RUNX1. In conclusion, we detected several candidate genes that could be associated with del7/7q and UPD7. Some mutations were heterozygous in cases with diploid 7q and correlated with CDRs on del7/7q without mutation. Certain mutations are specifically observed with del7, while others are commonly observed in all categories of LOH7, including EZH2. Moreover, some genes outside of the chromosome 7 were coincidently mutated with LOH7. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.
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  • 55
    Publication Date: 2012-11-16
    Description: Abstract 2737 Introduction: ATL is prevalent in Japan and has the worst prognosis among T-cell malignancies. PTCL also has a poor prognosis with currently available chemotherapeutic regimens, and both would benefit from better treatment modality. Lenalidomide is an immunomodulatory agent with direct tumoricidal and antiproliferative activity, and is approved for multiple myeloma (MM) in combination with dexamethasone after at least 1 prior therapy and for transfusion-dependent anemia due to low- or intermediate-1-risk myelodysplastic syndromes associated with 5q deletion. We conducted a phase 1 study of lenalidomide in patients with relapsed ATL or PTCL to establish the recommended dose and schedule for a subsequent phase 2 study. Patients and Methods: This multicenter, phase 1, dose-escalation study assessed the safety, maximum tolerated dose (MTD), pharmacokinetics, and efficacy in patients with relapsed advanced ATL or PTCL. Dose-escalation was conducted according to the standard 3+3 design. Up to one PTCL patient was allowed to be included in each cohort of 3 patients. Patients in Cohort 1 received oral lenalidomide 25 mg daily on Days 1–21 of a 28-day cycle. Patients in Cohorts 2 and 3 received 25 and 35 mg/day, respectively, on each day of the 28-day cycle. Dose-limiting toxicity (DLT) was defined as febrile neutropenia lasting 5 or more days; thrombocytopenia (platelets
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  • 56
    Publication Date: 1980-01-01
    Description: Heme biosynthesis was examined in erythroid tissue of a 4-yr-old girl with severe sideroblastic anemia since infancy, as documented by the presence of intramitochondrial deposits of iron in erythroblasts. Free red cell protoporphyrin, urinary porphyrins, and activities of erythrocyte porphobilinogen synthase, uroporphyrinogen 1 synthase, aspartate aminotransferase, and pyridoxine kinase were normal or increased. Bone marrow ferrochelatase activity was normal. Activity of bone marrow delta-aminolaevulinate (ALA) synthase was markedly reduced to 7 pmole ALA/10(6) erythroblasts/30 min (normal 127 +/- 29) but was enhanced fivefold by pyridoxal phosphate (normal 0%--25% increase). Therapy with oral pyridoxine and parenteral pyridoxal-5′-phosphate did not increase effective red cell production. The sideroblastic anemia in this patient appears to be related to a congenital defect in the initial step of heme biosynthesis.
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  • 57
    Publication Date: 2012-08-02
    Description: Proteasome inhibition has emerged as an important therapeutic strategy in multiple myeloma (MM). Since the publication of the first phase 1 trials of bortezomib 10 years ago, this first-in-class proteasome inhibitor (PI) has contributed substantially to the observed improvement in survival in MM patients over the past decade. Although first approved as a single agent in the relapsed setting, bortezomib is now predominantly used in combination regimens. Furthermore, the standard twice-weekly schedule may be replaced by weekly infusion, especially when bortezomib is used as part of combination regimens in frontline therapy. Indeed, bortezomib is an established component of induction therapy for patients eligible or ineligible for autologous stem cell transplantation. Bortezomib has also been incorporated into conditioning regimens before autologous stem cell transplantation, as well as into post-ASCT consolidation therapy, and in the maintenance setting. In addition, a new route of bortezomib administration, subcutaneous infusion, has recently been approved. Recently, several new agents have been introduced into the clinic, including carfilzomib, marizomib, and MLN9708, and trials investigating these “second-generation” PIs in patients with relapsed/refractory MMs have demonstrated positive results. This review provides an overview of the role of PIs in the treatment of MM, focusing on developments over the past decade.
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  • 58
    Publication Date: 1980-07-01
    Description: The level of serum ferritin is a reliable indicator of body iron stores. Exceptions include liver disease, malignant diseases, and treatment of iron-deficiency anemia. The latter was noted in iron- deficient infants who showed a rise of serum ferritin to normal levels in the first week of treatment. To evaluate this in adults, 14 patients with iron-deficiency anemia were studied prior to and after beginning treatment with oral ferrous sulfate in standard dose, 300 mg t.i.d., or double dose, 600 mg t.i.d. Serum ferritin was assayed by radioimmunoassay. No rise occurred in the first 3 wk in 5 patients treated with standard dose, although hematologic response occurred. With double dose, 7 of 9 showed a ferritin rise in 2 days with return to subnormal levels within 6 days of discontinuing iron. This study indicates that standard treatment of iron deficiency anemia in adults does not cause a rise in serum ferritin until hemoglobin levels are normal. The early rise seen with double dose is most likely due to absorption of iron in excess of utilization for erythropoiesis resulting in temporary storage. When iron is discontinued, stores are rapidly depleted as reflected by the prompt decrease in serum ferritin.
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  • 59
    Publication Date: 2012-11-16
    Description: Abstract 1595 Background: Immunohistochemistry for Cyclin D1 (CCND1) expression is routine in cases suggestive of mantle cell lymphoma (MCL). Most MCL are t(11;14)/IGH-CCND1-positive by FISH. PCR based detection of the fusion transcript is hampered by widespread breakpoints. Only few data is available on quantitative real-time PCR (RQ-PCR) for CCND1 expression measurement. Aims: To assess CCND1 mRNA expression and correlate it with t(11;14) in mature B-cell neoplasms. Methods: We established a RQ-PCR assay for CCND1 mRNA measurement and investigated 451 cases: 142 MCL (in all cases IGH-CCND1 confirmed by FISH), 76 chronic lymphocytic leukemia (43 typical CLL, 33 CLL/PL), 20 hairy cell leukemia (HCL), 13 hairy cell leukemia-variant (HCL-v), 20 splenic marginal zone lymphoma (SMZL), 91 other mature B-cell neoplasms. CCND1 background expression was assessed in 29 pts with other hematological neoplasms and 60 healthy individuals. FISH and/or chromosome banding analysis for the t(11;14) was available in 364 pts. Bone marrow (BM, n=267) or peripheral blood (PB; n=184) samples were analyzed by cytomorphology, multiparameter flow cytometry (MFC), FISH, and RQ-PCR. CCND1 mRNA expression was given by RQ-PCR in comparison to ABL1 mRNA expression (%CCND1/ABL1). Limited dilution of high expressers into cDNA of healthy controls revealed a sensitivity of the assay of up to 0.1 %. Results: IGH-CCND1 translocation carriers had higher %CCND1/ABL1 than those without which hold true in the total cohort (mean±SD, 420.4±740.3 vs 17.8±128.3; p
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  • 60
    Publication Date: 2012-11-16
    Description: Abstract 2566 The CCAAT enhancer binding protein alpha encoded by CEBPA gene is a transcription factors involved in myeloid lineage differentiation. Somatic CEBPA mutations are associated with a favorable prognosis in patients with normal to intermediate karyotype and lacking the internal tandem duplication in the fms-like tyrosine kinase -3 gene (FLT3/ITD) or mutation in the nucleophosmin gene (NPM1). N-terminal CEBPA mutations typically result in a frame shift encoding a truncated form of the 42-kD CEBPα protein and potentially increase translation of the alternative 30-kD isoform. The 30-kD isoform functions as a dominant negative regulator of the full-length 42-kD isoform of the CEBPα protein. C-terminal mutations are generally in-frame insertion/deletions in the DNA binding or the leucine zipper domains that cause alteration of the dimerization domain (bZIP). Germline mutations in CEBPA gene are recognized as the major cause of familial acute myeloid leukemia (AML). Familial AML is inherited in an autosomal dominant manner with complete penetrance. In contrast to somatic AML, the age onset of familial AML is earlier ranging from 4 to 39 years. The overall survival of the familial AML patients with germline CEBPA mutation is ∼50%–65% better than ∼34%–50% observed with the somatic CEBPA mutations (FLT3/NPM1 negative). In all of the familial AML pedigrees reported, the mutations were frame-shift insertions/deletions in the N-terminal domain resulting in translation of a truncated form of the CEBPα protein. We have identified four novel germline sequence variants in patients with history of familial AML and unknown karyotypes (cases 1–4). The patients' age ranges from 3 to 32 years. Of the four novel variants, one variant was identified in the C-terminal region of the gene. This is the first reported C-terminal variant detected in the proband of a familial AML pedigree. The variant is out of frame insertion/deletion of ∼401 bp in the C-terminal region of the CEBPA gene (there is a deletion ‘∼171bp then an insertion of 401bp). Another novel C-terminal variation was identified in a patient with cytogenetically normal AML undergoing testing for somatically acquired CEBPA mutations (case 5). The detection of this C-terminal variation in a buccal swab sample confirmed the germline origin of the variation. In two additional cases (cases 6 and 7), we observed that one of the two sequence variations identified was no longer present after treatment, suggesting the remaining variation was germline in origin. Our results demonstrate that germline C-terminal CEBPA mutations can cause familial AML and that germline CEBPA mutations may be identified in cytogenetically normal AML patients. The possibility of germline variants should be considered in AML patients since other family members, who may be possible transplant donors for the patient, may have also inherited the same germline variant. Case AML Type Sequence variation CEBPA gene region 1 Familial AML c.142delG; p.Ala48fs N terminal 2 c.168C〉A; p.Cys56* N terminal 3 c.175G〉T; p.Glu59* N terminal 4 c.643_814delins401; p.Thr216fs C terminal 5 Somatic AML c.1073delC; p.Ala358fs C terminal 6 c.68_78del; p.Pro23fs N terminal 7 c. 938T〉G; p. Val328G C terminal Disclosures: No relevant conflicts of interest to declare.
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  • 61
    Publication Date: 1980-04-01
    Description: Normal human erythrocyte phosphofructokinase (ATP:c D-fructose-6, P-1- phosphotransferase, EC 2.7.1.11; PFK) has recently been shown to consist of a heterogeneous mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, and L4 (M, muscle type; L, liver type). In the light of these findings, we have investigated the molecular basis of the inherited erythrocyte PFK deficiency associated with myopathy and hemolysis (Tarui disease). The propositus, a 31-yr-old male, suffered from muscle weakness and myoglobinuria on exertion. He showed mild erythrocytosis despite laboratory evidence of hemolysis. In his erythrocytes a metabolic crossover point was found at the level of PFK; 2,3-diphosphoglycerate (2,3-DPG) was also significantly reduced. The PFK from the patient's erythrocytes consisted exclusively of the L4 isozyme, and there was a complete absence of the other four. The leukocyte and platelet PFKs from the patient showed normal activities, chromatographic profiles, and precipitation with anti-M4 antibody. These studies provide direct evidence that in Tarui disease the M-type subunits are absent; but the liver- and platelet-type subunits of PFK are unaffected. The paradox of mild erythrocytosis despite hemolysis reflects the decreased production of 2,3-DPG.
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  • 62
    Publication Date: 2012-03-01
    Description: Only 30% of patients who require an allogeneic hematopoietic cell transplant will have an HLA-matched sibling donor. A search for an unrelated donor will be undertaken for patients without a matched family donor. However, many patients, particularly patients of diverse racial and ethnic backgrounds, may not be able to rapidly identify a suitably matched unrelated donor. Three alternative graft sources, umbilical cord blood (UCB), haploidentical (haplo)–related donor, and mismatched unrelated donor (MMUD) are available. UCB is associated with decreased GVHD, but hematologic recovery and immune reconstitution are slow. Haplo-HCT is characterized by donor availability for transplantation and after transplantation adoptive cellular immunotherapy but may be complicated by a high risk of graft failure and relapse. A MMUD transplant may also be an option, but GVHD may be of greater concern. Phase 2 studies have documented advances in HLA typing, GVHD prophylaxis, and infection prevention, which have improved survival. The same patient evaluated in different transplant centers may be offered MMUD, UCB, or haplo-HCT depending on center preference. In this review, we discuss the rationale for donor choice and the need of phase 3 studies to help answer this important question.
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  • 63
    Publication Date: 2012-05-24
    Description: Inappropriately low expression of the key iron regulator hepcidin (HAMP) causes iron overload in untransfused patients affected by β-thalassemia intermedia and Hamp modulation provides improvement of the thalassemic phenotype of the Hbbth3/+ mouse. HAMP expression is activated by iron through the bone morphogenetic protein (BMP)–son of mothers against decapentaplegic signaling pathway and inhibited by ineffective erythropoiesis through an unknown “erythroid regulator.” The BMP pathway is inactivated by the serine protease TMPRSS6 that cleaves the BMP coreceptor hemojuvelin. Here, we show that homozygous loss of Tmprss6 in Hbbth3/+ mice improves anemia and reduces ineffective erythropoiesis, splenomegaly, and iron loading. All these effects are mediated by Hamp up-regulation, which inhibits iron absorption and recycling. Because Hbbth3/+ mice lacking Tmprss6 show residual ineffective erythropoiesis, our results indicate that Tmprss6 is essential for Hamp inhibition by the erythroid regulator. We also obtained partial correction of the phenotype in Tmprss6 haploinsufficient Hbbth3/+ male but not female mice and showed that the observed sex difference reflects an unequal balance between iron and erythropoiesis-mediated Hamp regulation. Our study indicates that preventing iron overload improves β-thalassemia and strengthens the essential role of Tmprss6 for Hamp suppression, providing a proof of concept that Tmprss6 manipulation can offer a novel therapeutic option in this condition.
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  • 64
    Publication Date: 2012-08-23
    Description: Neutrophil recruitment and extravasation at sites of inflammation provide a mechanism for host defense. We showed previously that heparan sulfate, a type of sulfated glycosaminoglycan, facilitates neutrophil recruitment based on the reduction of neutrophil infiltration in mice in which the overall sulfation of the chains was reduced by selective inactivation of N-acetylglucosamine N-deacetylase-N-sulfotransferase (Ndst1) in endothelial cells. Here we show that inactivation of uronyl 2-O-sulfotransferase in endothelial cells (Hs2st), an enzyme that acts downstream from Ndst1, results in enhanced neutrophil recruitment in several models of acute inflammation. Enhanced neutrophil infiltration resulted in part from reduced rolling velocity under flow both in vivo and in vitro, which correlated with stronger binding of neutrophil L-selectin to mutant endothelial cells. Hs2st-deficient endothelial cells also displayed a striking increase in binding of IL-8 and macrophage inflammatory protein-2. The enhanced binding of these mediators of neutrophil recruitment resulted from a change in heparan sulfate structure caused by increased N-sulfation and 6-O-sulfation of glucosamine units in response to the decrease in 2-O-sulfation of uronic acid residues. This gain-of-function phenotype provides formidable evidence demonstrating the importance of endothelial heparan sulfate in inflammation and suggests a novel enzyme target for enhancing the innate immune response.
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  • 65
    Publication Date: 2012-06-14
    Description: Carfilzomib is a selective proteasome inhibitor that binds irreversibly to its target. In phase 1 studies, carfilzomib elicited promising responses and an acceptable toxicity profile in patients with relapsed and/or refractory multiple myeloma (R/R MM). In the present phase 2, multicenter, open-label study, 129 bortezomib-naive patients with R/R MM (median of 2 prior therapies) were separated into Cohort 1, scheduled to receive intravenous carfilzomib 20 mg/m2 for all treatment cycles, and Cohort 2, scheduled to receive 20 mg/m2 for cycle 1 and then 27 mg/m2 for all subsequent cycles. The primary end point was an overall response rate (≥ partial response) of 42.4% in Cohort 1 and 52.2% in Cohort 2. The clinical benefit response (overall response rate + minimal response) was 59.3% and 64.2% in Cohorts 1 and 2, respectively. Median duration of response was 13.1 months and not reached, and median time to progression was 8.3 months and not reached, respectively. The most common treatment-emergent adverse events were fatigue (62.0%) and nausea (48.8%). Single-agent carfilzomib elicited a low incidence of peripheral neuropathy—17.1% overall (1 grade 3; no grade 4)—in these pretreated bortezomib-naive patients. The results of the present study support the use of carfilzomib in R/R MM patients. This trial is registered at www.clinicaltrials.gov as NCT00530816.
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  • 66
    Publication Date: 2012-11-16
    Description: Abstract 2223 Disseminated intravascular coagulation (DIC) represents a complex pathophysiologic syndrome where marked alterations in the hemostatic system are manifested. As a result several inflammatory mediators are up regulated through multiple mechanisms. The up regulation of inflammatory mediators such as anaphylatoxin C5a (C5a), procalcitonin (PCT), interleukin 6 (IL-6), interleukin 10 (IL-10), myeloperoxidase (MPO), C reactive protein (CRP), and circulating levels of hemostatic markers including protein C inhibitor (PCI), plasminogen activator inhibitor 1 (PAI-1), and protein C (Pr C) were evaluated in 758 subjects enrolled in a randomized, double-blind, placebo-controlled, Phase-2B study evaluating the safety and efficacy of recombinant thrombomodulin (ART-123) in subjects with sepsis and suspected DIC. Thirty healthy male and female volunteers served as the control group. Commercially available ELISA methods were used to measure the various mediators. Marked deviations in the circulating levels of these markers, as compared to controls, were noted as shown in the following table. Compared with controls, subjects in DIC showed an increase in the circulating levels of most inflammatory markers. The levels of PCT, IL-6 and CRP, where considerably higher in the DIC subjects whereas PCI, Pr C and AT exhibited slight decreases. Wide individual variations were present. The PAI-1 levels were also increased in the DIC subjects. These results are tabulated below. These results clearly indicate that inflammation and impairment of fibrinolysis play a key role in the pathogenesis of DIC Parameter Nomal (NHP Mean+SEM) DIC (Baseline Mean+SEM) % Change Protein C (% Ag) 82.5 ± 13.6 47.6 ± 23.7 −42.2% Functional Protein C (%) 83.4 ± 13.2 46.2 ± 29.8 −44.6% PCI (% Inhibition) 130.0 ± 24.6 79.4 ± 105.5 −38.9% PAI-1 (ng/ml) 35.4 ± 10.8 140.6 ± 165.6 297.1% CRP (ug/ml) 2.6 ± 0.4 48.0 ± 14.2 1736.9% C5a (ng/ml) 9.2 ± 3.2 17.2 ± 13.3 85.1% IL-6 (pg/ml) 9.3 ± 3.7 620.3 ± 1883.4 6583.9% IL-10 (pg/ml) 13.9 ± 13.1 130.2 ± 118.6 836.1% MPO (ng/ml) 16.0 ± 4.2 108.1 ± 68.6 574.6% PCT (ng/ml) 0.2 ± 0.13 21.9 ± 43.3 14514.5% Disclosures: Osawa: Asahi Kasei Pharma America Corporation: Employment. Kaul:Asahi Kasei Pharma America Corporation: Employment.
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  • 67
    Publication Date: 2012-11-16
    Description: Abstract 44 CALGB (now part of the Alliance for Clinical Trials in Oncology) performed a non-randomized phase II study testing one year (yr) of investigational maintenance therapy with decitabine for newly diagnosed adult AML patients (pts) 1 × 109/L, platelets 〉75 × 109/L, and be within 90 days after day 1 of the final consolidation. Decitabine 20mg/m2 was given IV over 1 hour for 4–5 days, every 6 weeks, for 8 cycles. 546 pts enrolled with a median age of 48 yrs (range, 17–60) and median presenting white blood count (WBC) of 12.6 × 109/L (range, 0.3–380 × 109/L). 75% achieved CR (412/546). Reasons for CR pts not receiving maintenance were mainly early relapse, alloHCT in 1st CR, inadequate count recovery, and patient refusal (Blum et al. ASH 2011). 33% of CR pts (134/412) received investigational maintenance. Of these, 46 (34%) were favorable risk; among the remaining 88 pts, 73 were consolidated with autoHCT, and 15 received HiDAC-based consolidation. The median time from initial study registration to initiation of maintenance therapy was 6.3 months. Pts receiving decitabine had a median age of 45 yrs (range, 18–60) and presenting WBC of 13.5 × 109/L (range, 0.4–221 × 109/L). Treatment with decitabine maintenance was feasible and reasonably well tolerated; the primary toxicity was myelosuppression. Preplanned dose reduction criteria for neutropenia were met after the first 20 pts had been treated. After this early timepoint, all subsequent pts who had been consolidated with autoHCT received only 4 days of decitabine per cycle (HiDAC consolidated pts continued to receive 5 days per cycle). The median number of cycles of decitabine received was 7 (range, 1–8); 46% of pts (62/134) received all 8 cycles, and 75% completed at least 4 cycles. Reasons for discontinuation of decitabine before completing 8 cycles included relapse (28%, 38/134), pt refusal (13%), adverse events (4%), and others. In this selected cohort of pts who received post-CR maintenance with decitabine, 1-yr and 3-yr overall survival (OS) were 96% and 66%; 1-yr and 3-yr disease-free survival (DFS) were 80% and 53%. 1-yr “failure-free survival” calculated from the time of registration to decitabine was 68% (70% for favorable risk, 68% for others). These results are similar to the outcomes for comparable pts enrolled on our prior CALGB study 19808 who received identical chemotherapy for induction and consolidation and then were randomized to observation or maintenance with interleukin-2 (3-yr OS 61% and 68%, 3-yr DFS 45% and 56%, for observation and IL-2 respectively). Post-consolidation maintenance with decitabine appears to provide only modest, if any, benefit overall. Correlative studies for CALGB 10503 are ongoing, including investigations into the impact of decitabine in unique molecular and cytogenetic subsets of disease, the prognostic/predictive value of aberrant methylation and other molecular markers, and assessment of minimal residual disease. Supported by CA33601 and CA128377. Disclosures: No relevant conflicts of interest to declare.
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  • 68
    Publication Date: 2012-07-19
    Description: Red blood cell transfusions have reduced morbidity and mortality for patients with sickle cell disease. Transfusions can lead to erythrocyte alloimmunization, however, with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and difficulty in finding compatible units, which can cause transfusion delays. In this review, we discuss the risk factors associated with alloimmunization with emphasis on possible mechanisms that can trigger delayed hemolytic transfusion reactions in sickle cell disease, and we describe the challenges in transfusion management of these patients, including opportunities and emerging approaches for minimizing this life-threatening complication.
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  • 69
    Publication Date: 2012-11-16
    Description: Abstract 3240 The role of pulmonary hypertension as a common and attributable cause of mortality in patients with sickle cell disease remains controversial. To assess this question and explore risk factors for death in patients with sickle cell disease we evaluated 632 patients in the Walk-PHASST pulmonary hypertension screening cohort, recruited from nine different study sites in the United States and one site in the United Kingdom. Methods: Patient characteristics and their associations with mortality were analyzed with Cox proportional hazards regression analysis. Based on data from three right heart catheterization screenings studies that have recently been published, we defined the presence of pulmonary hypertension for this analysis by a Doppler-echocardiographic measurement of the tricuspid regurgitant jet velocity (TRV) ≥ 3.0 m/s, which has a 67–75% positive predictive value for a mean pulmonary artery pressure ≥ 25 mm Hg by right heart catheterization. This therefore represents a very conservative threshold for a large population screening study. Among subjects with a measurable TRV (n=572), 64 (11.2%) had measurements of ≥ 3.0 m/sec. Among those with measurable NT-proBNP (n=582), 140 (24.1%) had measurements ≥160 pg/mL, a value associated with both pulmonary hypertension and mortality. A total of 39 (7.4%) had both high TRV (≥3.0 m/sec) and high NT-proBNP (≥160 pg/mL). Results: Over a median follow-up time of 29 months, we observed 22 deaths. 50% (N=11) of these patients had a TRV≥ 3.0 m/sec. At 24 months the cumulative survival was 83% for patients with TRV ≥ 3.0 m/sec and 98% for patients with TRV 〈 3.0 m/sec (p
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  • 70
    Publication Date: 2012-11-16
    Description: Abstract 2801 Background: Recommendations for use of erythropoiesis-stimulating agents (ESAs) in anemic patients with MDS are based on baseline endogenous erythropoietin levels and red blood cell transfusion requirements, factors which predict the likelihood of a response to ESA treatment. These recommendations for ESA use have been incorporated into quality-of-care treatment guidelines for MDS. We examined whether baseline endogenous thrombopoietin (TPO) levels and platelet transfusion requirements likewise predict response of thrombocytopenic MDS patients to treatment with romiplostim, a TPO receptor agonist. Patients and Methods: In a placebo(PBO)-controlled trial of romiplostim (randomized 2:1) in 250 thrombocytopenic [median (Q1, Q3) baseline platelet count 19.3 (12.5, 30.3) × 109/L] IPSS low/int-1 MDS patients, study drug was discontinued early due to data monitoring committee concerns that the potential small benefit seen in the reduction of bleeding did not outweigh the potential risk for disease progression to AML and that the transient increases in blast cell counts may put patients at risk for diagnosis of and treatment for AML. Hematologic improvement of platelets (HI-P, per IWG 2006) is defined as 8 consecutive weeks of an absolute platelet increase of 30×109/L (for patients with baseline platelet counts 〉20×109/L) or an increase from 20×109/L and by at least 100% (for patients with baseline platelet counts
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  • 71
    Publication Date: 2012-09-20
    Description: Platelets are vital for hemostasis because they release their granule contents in response to vascular damage. Platelet exocytosis is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), whose interactions are governed by regulators, eg, Sec/Munc18 proteins. These proteins chaperone syntaxin t-SNAREs and are required for exocytosis. Platelets contain 3 Munc18 isoforms: Munc18a, Munc18b, and Munc18c. We report that Munc18b is the major isoform and is required for platelet secretion. Familial hemophagocytic lymphohistiocytosis type 5 (FHL5) is caused by defects in the Munc18b/STXBP2 gene. We confirm a previous report showing that platelets from FHL5 patients have defective secretion. Serotonin, ADP/ATP, and platelet factor 4 release was profoundly affected in the 2 biallelic patients and partially in a heterozygous patient. Release of lysosomal contents was only affected in the biallelic platelets. Platelets from the FHL5 biallelic patients showed decreased Munc18b and syntaxin-11 levels were significantly reduced; other syntaxins were unaffected. Munc18b formed complexes with syntaxin-11, SNAP-23, and vesicle-associated membrane protein-8 in human platelets. Other potential secretion regulators, Munc13-4 and Rab27, were also found associated. These data demonstrate a key role for Munc18b, perhaps as a limiting factor, in platelet exocytosis and suggest that it regulates syntaxin-11.
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  • 72
    Publication Date: 2012-11-16
    Description: Abstract SCI-48 Stored packed red blood cells (PRBCs) have been implicated in increased morbidity and mortality in critically ill patients, patients requiring cardiothoracic surgery, and injured patients following blunt and/or penetrating trauma. Lipids are generated during the routine storage of cellular blood components and have been implicated in transfusion-related acute lung injury (TRALI) and postinjury multiple organ failure (MOF). These lipids are comprised of two classes, as denoted by their retention times on normal-phase HPLC: 1) nonpolar lipids, including arachidonic acid, 5-hydroxy-eicosotetranoic acid (HETE), 12-HETE, 15-HETE, and 5-oxo-eicosotetranoic acid (5-oxo-ETE); and 2) a mixture of lysophosphatidylcholines (lyso-PCs), including stearoyl-, oleoyl-, or palmitoyl-lyso-PCs as well as C16 or C18 lyso-platelet activating factor. Importantly, all of these lipids were identified by quadrupole LC/MS/MS. The former are derived from red blood cells, because they are generated during the routine storage of prestorage leuko- and platelet-reduced PRBCs, while the latter are platelet-derived, and are generated during storage of platelet concentrates (PC) and unmodified PRBCs but not leukocyte-reduced PRBCs (LR-PRBCs). Generation of the nonpolar lipids requires an active phospholipase, such as peroxiredoxin-6, which accumulates during PRBC and LR-PRBC storage and appears to be active and T-phosphorylated. The nonpolar lipids do not accumulate during PC storage. These lipids are released into the plasma fraction of the stored component and have been shown to: 1) prime the polymorphonuclear leukocytes (PMN) oxidase; 2) activate primed PMNs; and 3) serve as the second event in a two-event model of TRALI in both rodents and sheep. This lipid priming of PMNs occurs through activation of specific receptors, for example G2A, for lyso-PCs, and causes activation of G-protein-linked cellular receptor, G-protein release, and stimulation of kinase cascades. This results in translocation of the cytosolic oxidase components and a change from a resting PMN phenotype to a hyperactive, adherent phenotype. Conversely, stored PRBCs have also been implicated in MOF. However, acute lung injury does not occur until 72 hours postinjury, implicating PRBCs as a first event(s). The nonpolar lipids that accumulate during routine storage activate primary human pulmonary microvascular endothelial cells (HMVECs) and primary human liver sinusoidal endothelial cells (LSECs), as quantified by increased surface expression of intercellular adhesion molecule-1 (ICAM-1) and the synthesis and release of chemokines, for example IL-8. This proinflammatory activation results in PMN adherence, and such HMVEC activation occurs through activation of the BLT2 receptor and activation of a PKC-dependent kinase cascade. Stored PRBCs (day 42) may serve as the first event in a two-event rodent model of acute lung injury. We conclude that lipids generated during the routine storage of cellular blood components have proinflammatory effects in vitro and in vivo, which may explain some of the adverse events of transfusion, including TRALI and postinjury MOF, and that inhibition and avoidance of these compounds may make transfusions safer. Further research is already under way in several prospective trials, including: 1) Age of Blood Components transfused in the PICU (ABC-PICU); 2) red cell storage duration and outcomes in cardiac surgery at the Cleveland Clinic; 3) Red Cell Storage duration Study (RECESS); and 4) the Age of Blood Evaluation trial (ABLE). Disclosures: Silliman: Pall Corporation: Honoraria, Research Funding.
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  • 73
    Publication Date: 1980-01-01
    Description: A 34% response was obtained in 202 evaluable patients in the terminal phase of chronic granulocytic leukemia using combinations of hydroxyurea, 6-mercaptopurine, and corticosteroids. Twelve percent of responses were complete and 22% partial. Overall median survival was 12 wk. A 30 wk median survival for responding patients was statistically superior to the 7-wk survival for nonresponders (p less than 0.001). Response was inversely correlated with toxicity. No responses were obtained in patients sustaining both severe infectious and bleeding complications. No benefit could be demonstrated from the addition of vincristine in induction and daunorubicin for consolidation. Although the response frequency and duration of survival with this combination chemotherapy were generally superior to those previously reported by our group, the terminal phase of chronic granulocytic leukemia still remains a formidable and generally refractory disease.
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  • 74
    Publication Date: 2012-11-16
    Description: Abstract 3562 Relapsed/refractory pediatric acute lymphoblastic leukemia (ALL) remains a continuing challenge to treat with currently available therapies, and new treatments are urgently required for the management of these high-risk cases. Activating mutations in the pseudokinase or kinase domains of Janus kinases (JAKs) 1, 2 and 3 are present in approximately 10% of high-risk pediatric ALL and are associated with high expression of cytokine receptor-like factor 2 (CRLF2) and poor outcome. These mutations can lead to continuous activation of JAKs, resulting in constitutive activation by phosphorylation of downstream signaling, including the signal transducer and activator of transcription (STAT), AKT, and mitogen-activated protein kinase (MAPK) pathways. The availability of specific JAK inhibitors, developed primarily for the treatment of JAK-mutated myeloproliferative diseases (MPDs), represents an opportunity to improve the treatment options for JAK-mutated pediatric ALL. AZD1480, a potent ATP-competitive small-molecule JAK2 inhibitor that also exhibits inhibitory activity against JAK1, is in solid tumor clinical trials. The purpose of this study was to gain a greater understanding of a potential role for AZD1480 in the treatment of JAK-mutated pediatric ALL either as a single agent or in rational drug combinations, using a preclinical model of xenografts established in immune-deficient mice from direct patient explants. As part of the Pediatric Preclinical Testing Program (PPTP) we previously showed that AZD1480 administered at 10 mg/kg twice daily × 5 then at 15mg/kg once daily × 2 via oral gavage for an intended three weeks significantly delayed the progression of only one in five JAK-mutated xenografts, with no tumor regressions observed. We now show that the relative insensitivity of JAK-mutated ALL xenografts to AZD1480 is a cell-intrinsic phenomenon, since 6/7 JAK1- or JAK2-mutated xenografts exhibited ex vivo IC50 values 〉2 μM following 72 h drug exposures, as assessed by mitochondrial function cell viability (MTT) assay. In order to gain a greater understanding of the underlying mechanisms for the lack of AZD1480 single-agent efficacy against JAK-mutated xenografts we analyzed intracellular signaling pathways and their responses to AZD1480 treatment. In contrast with “Typical” B-cell precursor (BCP)-ALL xenograft cells, JAK-mutated xenografts exhibited constitutive JAK pathway activation, as assessed by increased levels of phospho-JAK1 (pJAK1), pJAK2, pSTAT1/3/5, pAKT, pMAP2K1/2 (MEK1/2) and phospho-extracellular signal-regulated kinase 1/2 (pERK1/2). Ex vivo exposure of JAK-mutated xenografts to 1 μM AZD1480 caused rapid (within 1 h) and sustained (up to 24 h) decreases in pSTATs, but minimal reduction in pMEK1/2 and pERK1/2. These results indicate that AZD1480 alone selectively inhibits JAK downstream signaling pathways, which may be insufficient to delay leukemia progression in vivo or induce cell death ex vivo. Moreover, they provide a rationale for dual targeting of the JAK and MAPK pathways to elicit synergistic anti-leukemic cell killing in JAK-mutated ALL. Ex vivo exposure of two JAK2-mutated xenografts to 1 μM of the MEK1/2 inhibitor AZD6244 (selumetinib) caused a profound decrease in pERK1/2, and the combination of 1 μM each of AZD1480 and AZD6244 resulted in reductions of both pSTATs and pERK1/2. Moreover, fixed-ratio MTT cytotoxicity assays using these two JAK2-mutated xenografts demonstrated very strong synergy between AZD1480 and AZD6244, with Combination Indices for each xenograft of 0.36 and 0.098 at the ED50; 0.23 and 0.015 at the ED75; and 0.15 and 0.002 at the ED90. This strong synergistic effect was observed despite AZD1480 and AZD6244 exerting minimal cell killing activity against the xenograft cells when used as single agents. In conclusion, our data indicate that AZD1480 is unlikely to exert significant single-agent activity in the treatment of JAK-mutated pediatric ALL, and that future efforts focusing on dual targeting of the JAK/STAT and MAPK offer a potential pathway to achieving clinical efficacy. Disclosures: No relevant conflicts of interest to declare.
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  • 75
    Publication Date: 2012-11-16
    Description: Abstract 2872 Introduction: The DNA damage response axis plays a crucial role in chemoresistance in CLL, as indicated by the prognostic impact of deletions of 17p (locus of TP53) and 11q (locus of ATM). These deletions coincide with mutations in the remaining allele, although frequency, especially for ATM varies. Functional read-outs of the p53 axis might add clinical relevant information on the actual DNA damage response. Currently, different p53 function analyses are being developed. These assays are either based on measurement of (i) RNA expression levels of a single gene (RT-PCRp21) or gene sets (RT-MLPA), or protein expression levels (FACSp53-p21) after DNA damage by irradiation or etoposide/nutlin exposition or (ii) gene expression levels at base-line (RT-PCR miR34a). To what extent these different assays correlate is currently unknown. Aim: Detailed side-by-side analysis of available p53 functional assays in well characterized CLL samples Methods: Freshly frozen PBMC's of 15 different CLL samples (CD19/CD5〉90%) were exchanged between 5 research groups that developed the following p53 functional assays: RT-PCRp21 (Ulm), RT-MLPA (bax, puma, p21 and CD95; Amsterdam), FACSp53-p21 (Paris) and RT-PCR miR34a (Salzburg/Brno). FISH-analysis (11q, 17p, 13q and 12) was performed on all samples. In addition mutations in TP53 were determined by FASAY, DHPLC and Sanger sequencing and ATM mutations were assessed by Sanger sequencing. Results: RNA expression levels showed significant correlation (p-value 〈 0,01) between the different P53 functional assays with high correlation coefficients (range: 0,7–0,94). Based on combination of FISH-analysis, FASAY and Sanger sequencing the investigated CLL samples could be distinguished into 6 different categories; 1. 17p- with TP53 mutation (n=5), 2. Sole TP53 mutation (n=1), 3. 11q- with ATM mutation (n=1), 4. 11q- in absence of TP53/ATM mutation (n=4), 5. 17p- with both TP53 mutation and ATM mutation (n=1) and 6. No 17p- and 11q- in absence of TP53/ATM mutation (n=3). All p53 functional assays showed absent to minimal induction of expression of respective target genes in samples with a cytogenetic abnormality combined with a TP53/ATM mutation (category 1, 3, 5). In contrast, samples without a cytogenetic abnormality in absence of TP53/ATM mutations (category 6) showed a marked increase in expression of respective target genes in all the assays. The patient with a sole TP53 mutation (category 2) showed a normal DNA damage response in all assays except for FACSp53-p21. Samples with an 11q- in absence of TP53/ATM mutation (category 4) showed high inter-assay variation. The two assays with predefined cut-off values (RT-MLPA and FACSp53-p21) assessed 13 samples equally (9 p53 dysfunctional and 4 p53 functional), except for 2 patients (category 2, 3 respectively). Reproducibility of these assays was tested by blinded sending of 3 samples that have been analyzed previously in this study. Except for p21, all assays showed 100% reproducibility. Discussion: For the first time a comparative side-by-side analysis of different available p53 functional assays was performed, and demonstrated strong correlations between the different assays. All assays could detect samples with expected disturbed and normal DNA-damage responses. To what extent these p53 functional assays could be of clinical relevance especially with respect to chemo-responsiveness, should be further studied in prospective studies. Disclosures: No relevant conflicts of interest to declare.
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  • 76
    Publication Date: 2012-01-05
    Description: Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferative disease of mature T and natural killer cells. The etiology of LGL leukemia is unknown. IL-15 is an inflammatory cytokine that stimulates T and natural killer cells and is critical for their survival and proliferation. IL-15 signals through a heterotrimeric receptor that is composed of a private receptor, IL-15Rα and IL-2/IL-15Rβ and γc shared with IL-2. Using a newly developed assay, we demonstrated increased levels of soluble IL-15Rα in the serum of patients with T-LGL leukemia. Furthermore, IL-15Rα mRNA levels were also up-regulated in the PBMCs of these patients. FACS analysis indicated that IL-15Rα was expressed both on monocytes as well as on some CD8+ leukemic cells of the patients. Interestingly, the mRNA levels of IFN-γ, a known inducer of IL-15Rα, were also up-regulated in patients' PBMCs. Moreover, PBMCs of some T-LGL patients proliferated at higher levels in response to exogenously added IL-15 compared with those of normal donors. In summary, our study demonstrated increased expression of IL-15Rα in T-LGL leukemia. It is conceivable that higher IL-15Rα expression may lower IL-15 response threshold in vivo and, therefore, may contribute to the pathogenesis of the disease.
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  • 77
    Publication Date: 2012-11-16
    Description: Abstract 509 Osteoblasts, the bone forming cells, are implicated in the fate of healthy and malignant stem cells. They affect self-renewal and expansion of hematopoietic stem cells (HSCs) and homing of tumor cells into the bone marrow. Here we show that constitutive activation of canonical Wnt signaling in osteoblast precursors disrupts hematopoiesis in mice by shifting the differentiation potential of HSC progenitors to the myeloid lineage which results in accumulation of granulocyte/monocyte progenitors and concomitant development of acute myeloid leukemia (AML). B-lymphopoiesis is also decreased. The AML phenotype is associated with clonal evolution at the cytogenetic level since clonal abnormalities could be detected in leukemic blasts from mice with constitutive activation of the canonical Wnt target β-catenin in osteoblast precursors (βcateninosb mice). Bone marrow transplantation experiments from βcateninosb mice to wild type lethally irradiated mice resulted in development of AML within 8 weeks following transplantation, demonstrating progression towards AML. At the molecular level, cell-specific gene inactivation mouse models demonstrate that β-catenin interacts with FoxO1 in osteoblasts to induce development of AML. Downstream signaling events that confer osteoblast signaling to normal HSCs and lead to their leukemogenic transformation will be presented. Importantly, malignancy-inducing osteoblasts, detected by nuclear accumulation of β-catenin in bone marrow biopsies, were identified in 〉 25% of patients with myelodysplasia (MDS), acute myeloid leukemia (AML) or AML arising from a prior MDS. Specifically, 15 out of 53 patients with MDS (n=17 patients), AML (n=20 patients), or MDS that had transformed to AML (n=16) chosen at random showed nuclear localization of β-catenin in osteoblasts. Of note, 12 of the 15 (80%) patients with nuclear localization of β-catenin in osteoblasts had abnormalities of chromosome 5 and/or 7, very common cytogenetic abnormalities in patients with MDS and AML. The same signaling pathways mediating AML development in βcateninosb mice were also found to be activated in osteoblasts and hematopoietic cells from the patients with nuclear accumulation of β-catenin in osteoblasts. These findings demonstrate that genetic alterations in osteoblast precursors (1) can induce AML in mice and (2) are associated with AML development in humans. They also provide a molecular basis for the leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.
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  • 78
    Publication Date: 2012-11-16
    Description: Abstract 5042 Bortezomib is a proteasome inhibitor with potent antimyeloma activity in relapsed/refractory multiple myeloma (MM) patients. We evaluated the type and factors affecting the onset of infectious complications and mortality owing to infection in MM patients treated with bortezomib-based regimens. We reviewed 139 patients with MM treated with regimens containing bortezomib in order to assess the type and factors affecting the development of severe infections. Infections occurred in 56 (40. 3%) of 139 patients and 83 (7. 8%) cases of the 1, 069 evaluable cycles. Severe infections developed in 43 (30. 9%) patients and ten patients (7. 1%) died during bortezomib-based treatment. Multivariate analysis determined lymphocytopenia grade 3–4 (OR 3. 17, 95% CI 1. 38–7. 31, p = 0. 007) and number of cycle ° Â 8 cycles (OR 3. 91, 95% CI 1. 39–11. 02, p =0. 010) as risk factors associated with increased severe infection. This study showed that MM patients who received bortezomib-based regimens are at a higher risk of severe infections within eight cycles of treatment during especially severe lymphocytopenic period. MM patients treated with bortezomib-based regimens should be closely monitored for the development of infectious complications during lymphocytopenia. Table I. Multivariate analysis of factors affecting severe infection development Parameter OR (95% CI) p-Value Age (〉 65) 1.78 (0.77–4.13) 0.180 Immunophenotype IgA 1.51 (0.60–3.77) 0.379 Lymphocytopenia (grade 3–4) 3.17 (1.38–7.31) 0.007 Neutropenia (grade 3–4) 1.25 (0.55–2.85) 0.600 Number of cycles (°Â 8 cycles) 3.91 (1.39–11.02) 0.010 Disclosures: No relevant conflicts of interest to declare.
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  • 79
    Publication Date: 2012-11-16
    Description: Abstract 5084 Background & Aims 18F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) scan has been increasingly used for initial staging and response evaluation in patients with lymphomas, and its clinical utility is well established in diffuse large B-cell lymphoma as well as Hodgkin lymphoma. However, its role remains undetermined in marginal zone lymphomas (MZL), most common type of indolent lymphoma in Korea, due to its relatively low FDG avidity. Thus, we aimed to assess the prognostic significance of PET-CT scan in patients with MZL. Patients & methods We retrospectively reviewed the medical records of a total of 194 patients with pathologically confirmed MZL in the Asan Medical Center between February 2003 and February 2011. Post-treatment FDG PET-CT scan was defined as which performed during the periods of 2 to 4 weeks after the completion of induction chemotherapy or 7 to 9 weeks after radiotherapy. [a4] Among them, both baseline and post-treatment FDG PET-CT scans were performed in 64 patients. We investigated the prognostic significance of maximum standardized uptake value (SUVmax) at baseline PET-CT and metabolic complete response. Metabolic compete response (mCR) was defined as no pathologic FDG uptake at any site in post-treatment PET-CT scan. The log-rank test was used to assess the correlation of progression-free survival (PFS) and overall survival (OS) with baseline SUVmax or the presence of mCR. Results In a total of analyzable 64 patients, histopathologic subtypes of them were as follow: extranodal marginal zone lymphoma (ENMZL=38, 59. 4%) including mucosa-associated lymphoid tissue (MALT) (n=35, 54. 7%) and bronchus-associated lymphoid tissue (BALT) (n=3, 4. 7%) lymphoma, nodal MZL (n=25, 39. 0%), splenic MZL (n=1, 1. 6%). The median SUVmax in baseline PET-CT was 4. 9 (range, 1. 3 – 18. 8). There were no significant associations of baseline SUVmax (cutoff: 5) to mCR at post-treatment PET-CT scan or survival outcomes. Patients group with high SUVmax (SUVmax 〉5. 0) showed mCR rate of 72. 7 %, and patients low SUVmax (SUVmax ° Â 5) showed mCR rate of 67. 7%, respectively. (p=0. 786). With a median follow-up duration of 46 monthss (range, 13 to 109 months), 5-year OS and PFS rate were 91% and 71%, respectively. 5-year PFS rate (76% vs. 62%, p=0. 27) did not differ between complete metabolic responders and incomplete responders. However, complete metabolic responders showed higher 5 year OS rate compared with incomplete responders (93% vs. 86%, p=0. 43) although statistical significance was not secured. Conclusion In the study cohort, baseline SUVmax was not a significant predictor of mCR, PFS nor OS. However, the patients achieved mCR at the end of induction treatment seemed to have superior survival rates than incomplete responders, which warrants further investigation. Disclosures: No relevant conflicts of interest to declare.
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  • 80
    Publication Date: 2012-11-16
    Description: Abstract 5072 Essential thrombocythemia (ET) is currently classified as a chronic myeloproliferative disorder. Anagrelide is a novel platelet lowering agent that has recently been approved for use n ET. We present the case of ET in an 8 –month- old girl, who was treated with Anagrelid for 13 months. The girl was admitted to hospital because of elevated platelet count. She had stomach pains periodically. On admission the child's condition was good but on palpation her stomach was sore and her spleen was enlarged. Laboratory investigations showed an elevated platelet count (1. 5m) and hypercalemia (6. 3mEq/l). Thrombopoetin was normal. Acesan was added to the treatment. A morphology performed after 1 month revealed further elevation of platelets (up to 2. 65m). On the basis of laboratory investigations, bone-marrow biopsy and trepanobiopsy, we eliminated an oncological disease and infectious diseases of connective tissues. On bone-marrow investigation, acquired mutation of JAK2 (V617F) tyrosine kinase was diagnosed, which confirmed ET. On account of the growing number of platelets, which was life - threatening, we decided to administer anagrelid, starting with a dose of 0. 25 mg per day. The number of platelets decreased to 1. 4m, so the dose was increased to 0. 25 mg twice daily after 3 weeks. At present, after 37 months of anagrelide treatment, the number of platelets in the child ranges between 400 and 650. The patient visits our, department for monthly check-up (morphology, biochemical tests, ECG and echocardiography). In international literature there is no information on the use of Anagrelid in ET treatment of children under 12 months old. However, on the basis of our observations and initial results of treatment, it seems that the above protocol for Anagrelid administration is safe for infants of this age. Disclosures: No relevant conflicts of interest to declare.
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  • 81
    Publication Date: 2012-11-16
    Description: Abstract 5044 In spite of the past efforts and progress made in treatment multiple myeloma (MM), most MM patients have eventually relapsed and died of the cancer. Cancer stem cells (CSCs) are considered responsible for continued growth and recurrence of cancer. The purpose of this study was to investigate the effects of anti-ABCG2 monoclonal antibody (mAb) in combination with paclitaxel-Fe3O4 nanoparticles (PTX-NPs) on CD138−CD34−MM cancer stem-like cells (MM CSCs) isolated from MM cell line JJN3. In our results, the MM CSCs expressed higher levels of the ABCG2 transporter, exhibited high proliferative, clonogenic and migratory potency, and demonstrated strong drug resistance and tumorigenicity when compared to non-CD138−CD34−MM cells. Incubation of mAb with PTX-NPs remarkably induced G/M cell cycle arrest, and increased synergistic induction of MM CSC apoptosis compared with incubation with PTX-NPs or PTX or mAb alone. More importantly, mAb in combination with PTX-NPs led to a significant reduction in the tumor volume, to a visible alleviation of murine lytic bone lesions and to a markedly increased survival rate in contrast to using a single agent in MM CSCs when it was transplanted to nonobese diabetic/severe combined immunodeficiency mice. Our study is the first to report on the anti-MM CSC activities by PTX-NPs as single agent or used together with mAb to treat MM. This finding from our study provides a rationale for future clinical trials. Disclosures: No relevant conflicts of interest to declare.
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  • 82
    Publication Date: 2012-11-16
    Description: Abstract 5064 The chronic myeloproliferative Neoplasm (NPM) are clonal hematopoietic stem cell malignancies with 3 main subtypes: polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC proliferation in the absence of erythropoietin and proliferation of myeloid lineages usually is noted, A gain-of-function mutation of Janus kinase 2 (JAK2) V617F, is identified in about 95% of patients with PV and about 50% of patients with essential thrombocytosis and idiopathic myelofibrosis. It has been shown that JAK2 exon 12 mutations can activate erythropoietin signaling pathways while these findings have been confirmed by many studies from Western countries, there are no reports from Asian countries in general and Arab countries in particular about the prevalence of the JAK2 exon 12 mutation in patients with PV and ET. In the present study, we determined the prevalence of JAK2V617F and JAK2 exon 12 mutations in patients with PV and ET in Qatar. Materials and Methods We enrolled patients with a diagnosis of PV and ET at National Centre for Cancer Care and Research in Qatar from January till June 2012. The diagnosis of PV and ET was established according to the 2008 World Health Organization criteria. The study included 82 patients. Clinical information and the CBC data at diagnosis were obtained from medical records. Pretreatment serum erythropoietin levels. Total DNA was isolated from buffy coat cells taken from peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Allele-specific polymerase chain reaction (PCR) was performed using 80 ng of genomic DNA as the template in a35-cycle PCR reaction at an annealing temperature of 58°C, as previously described. The mutant allele yields a 203-base-pair (bp) PCR product (sensitivity of mutant allele detection
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  • 83
    Publication Date: 2012-11-16
    Description: Abstract 5026 Recent data indicate that TORC2 is necessary for survival of multiple myeloma (MM) cells. Currently, drugs targeting mTOR either inhibit TORC1 alone or both TORC1 and TORC2. To identify drugs that specifically target TORC2, we performed a drug screen in a yeast-two hybrid system to identify compounds that prevented an interaction between rictor and mTOR. We identified several potential compounds and have begun to characterize their molecular activity. These compounds induced significant MM cell apoptosis when used at concentrations below 4 uM. This cytotoxic effect was accomplished by a down regulation of TOR activity which was specific for TORC2 (ie., decreased S473 AKT phosphorylation and NDRG T346 phosphorylation). Co-immunoprecipitation experiments confirmed that at least some of the compounds prevented binding of rictor to mTOR within MM cells while having no effect on binding of raptor to mTOR. In addition, myeloma cells expressing TORC2 phosphomimetic protein substrates (AKTS473D or SGKS422D) were significantly less sensitive to apoptosis as compared to the empty vector control when treated with these compounds. These data suggest that the drug-induced cytotoxicity was mediated specifically through the inhibition of TORC2 kinase activity. These results are the initial characterization of TORC2-specific drugs and support a rationale for targeting TORC2 in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.
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  • 84
    Publication Date: 2012-11-16
    Description: Abstract 5016 Activating mutations in Ras (N- and K-) are the most common mutations in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. As previously reported, we re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found the most common mutations to affect Ras oncogenes (N and K-). To test the role of Ras activation in MM pathogenesis directly, we generated mice harboring a mutant K-ras allele, Lox-stop-lox(LSL)- K-ras G12D (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre), activated specifically in germinal center (GC) cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12), phenotypes that have previously been reported for K-ras+/G12D mice. Tumor cells demonstrated successful recombination of the K-Ras locus. We showed recombination in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). No evidence of plasma cell proliferation was seen by serum ELISA, SPEP, flow cytometry or histological examination. We confirmed these results by crossing the K-ras+/G12D mice to a second strain, AID-Cre, which expresses the recombinase earlier and more specifically in the GC. We found 100% (n=20) of K-ras+/G12D/AID-Cre-YFP mice possessed visible, small benign papillomas on the ventral neck, as early as 3 weeks of age. To stimulate malignant transformation, vitamin D deficient chow and/or sub-lethal radiation was given to K-ras+/G12D/AID-Cre-YFP and negative control, K-ras+/G12D mice. Dual treatment increased the size and number of papillomas, giving a median survival of 26 weeks, compared to 52 weeks with control. However, no evidence of MM was detected by serum ELISA, SPEP, flow cytometry or histological examination. Finally, we generated AID-Cre-K-ras+/G12D triple transgenic mice in a tumor prone Arf-null background. After 13 wks, we found sarcomas in 66% (n=3) of K-ras+/G12D/AID-Cre-YFP/Arf−/− mice. Additionally, 100% (n=3) of these mice had rapid growing benign papillomas and only a minimal increase in the plasma cell compartment, compared to K-ras+/G12D/AID-Cre/Arf +/− control. Together, these data demonstrate that Ras activation is not sufficient to transform primary germinal center or plasma cells, even in an Arf-null context, and suggests that specific myeloma events, yet to be identified, are required for malignant transformation. Disclosures: No relevant conflicts of interest to declare.
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  • 85
    Publication Date: 2012-11-16
    Description: Abstract 5014 Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival. To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation. These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity. Disclosures: No relevant conflicts of interest to declare.
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  • 86
    Publication Date: 2012-11-16
    Description: Abstract 5002 INTRODUCTION: Hyperammonemia is commonly associated with severe liver cirrhosis. However, reports have described plasma cell myeloma (PCM)-induced hyperammonic encephalopathy. We present a retrospective case series of patients with PCM who present with altered mental status and hyperammonemia without signs of hepatic dysfunction over a twelve-year period. Additionally, we performed a systematic review of the literature looking for additional cases. METHODS: A retrospective chart review was performed of patients 〉18 years diagnosed with PCM between January 2000 and January 2012. A diagnosis of PCM-induced hyperammonemia was made in PCM patients with altered mental status, elevated serum ammonia levels (〉50 umol/L) and no other cause of altered mental status including hepatic dysfunction, electrolyte and metabolic abnormalities or intracranial processes. The primary outcome was to examine factors predisposing these patients to hyperammonemia. Additionally, we performed a systematic MEDLINE search using multiple myeloma, ammonia and hyperammonemia looking for case reports and series on patients with PCM-induced hyperammonic encephalopathy meeting the above criteria. Available clinical characteristics were gathered from these reports. Characteristics are presented descriptively. RESULTS: Our retrospective study included 27 individual patients diagnosed with PCM with elevated ammonia levels in the context of presenting with altered mental status. Six out of the 27 patients had hyperammonemic encephalopathy without other known etiology. The mean age was 76 years with a 5:1 male-to-female ratio. All had stage III based on the International Staging Scale (ISS). Bone marrow biopsies demonstrated 54–98% (mean 69%) plasma cell infiltration. IgA subtype was seen in 50% (n=3), IgG in 33% (n=2) and biclonal IgG/IgA in 17% (n=1). The mean ammonia level was 113 umol/L (range: 50–171 umol/L). Each of these patients had stable hemoglobin, normal electrolytes, liver and kidney function tests, and INR. No intracranial processes were detected on imaging. Three patients had improvement in mental status and decreased ammonia levels after chemotherapy; the other three patients declined further interventions. Inpatient mortality was over 66%. Our MEDLINE search revealed 20 articles originating from the United States and Japan detailing a total of 32 patients who were diagnosed with PCM-induced hyperammonemic encephalopathy. The mean age was 52 years (range 23–89 years) with an equal distribution between men and women. The average ammonia level amongst these patients was 121 umol/L (range: 50–299 umol/L). All of these patients had stage III disease by the ISS or the Durie-Salmon system. IgG was the most common subtype at 44% (n=12), followed by IgA with 37% (n=10), light chain multiple myeloma with 11% (n=3), and IgD with 7% (n=2). Of the 25 patients that received chemotherapy, 15 (60%) survived until discharge. The inpatient mortality was 40% (n=10). Those patients who did not receive chemotherapy had a lower rate of survival at 25%. CONCLUSION: Multiple myeloma hyperammonemic encephalopathy is a rare disease process that typically occurs in patients with stage III disease. Hospitalization mortality is high for these patients despite chemotherapy and even higher without chemotherapy administration. Disclosures: No relevant conflicts of interest to declare.
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  • 87
    Publication Date: 2012-11-16
    Description: Abstract 5006 Multiple myeloma (MM) is a fatal neoplasm characterized by the accumulation of malignant plasma cells within the bone marrow (BM) and the presence of a monoclonal immunoglobulin in the serum and/or urine, it was reported that patients with abnormal metaphases by conventional cytogenetics at diagnosis had active disease and a reduced survival rate compared with those normal metaphase. To further explore the cytogenetic characteristics of multiple myeloma and its correlation with clinical treatment and prognosis, 38 cases of MM were collected and chromosome specimens of bone marrow cells were prepared by 24-hour culture. R-banding technique combined with interphase fluorescence in situ hybridization(i-FISH) was used for karyotype analysis. The impact of different treatments and chromosome aberrations on progression-free survival(PFS) and overall survival(OS) time as well as related prognosis factors of MM were analyzed. The detected total chromosome aberration rate was 34. 2% (13/38), with chromosome complex aberration 53. 8%(7/13). The detection rate of R-banding technique was 23. 7%(9/38) and 21. 1%(8/38) by FISH. The most frequent chromosome aberration was chromosome 1 abnormality. The median PFS time of abnormal group was 16 months and was not reached of normal group after median follow up for 36 months(P=0. 045). Also, the median PFS time of complex abnormal group was 15 months, and the median PFS time of non-complex abnormal group was not reached(P=0. 012). The median OS time of complex abnormal group was 22 months and the median OS time of non-complex abnormal group was not reached(P=0. 041). Boretizomib or autologous hematopoietic stem cell transplantation prolonged the PFS and OS of the chromosome aberration group compared with traditional chemotherapy, but there were no significant statistical differences between the two groups(P〉0. 05). It was concluded that chromosome complex aberration is the mainly cytogenetic characteristics of multiple myeloma, and multiple numerous and structural aberrations are involved. The patients with chromosome complex aberration have poor prognosis. Chromosome abnormalities detected in metaphases from multiple myeloma (MM) cells have a clear impact on prognosis and response to therapy. Techniques to explore cytogenetic and molecular genetic changes of myeloma cells should be used to improve chromosomal aberration detection to guide clinical treatments and evaluate prognosis. Disclosures: No relevant conflicts of interest to declare.
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  • 88
    Publication Date: 2012-11-16
    Description: Abstract 4993 Background: the role of osteopontin (OPN), glyco-phosphoprotein produced by variety of tissues, has been widely investigated in the progression of many solid tumors, but on the other side, there are not many studies performed in multiple myeloma (MM). The role of Vascular Endothelial Growth Factor (VEGF) in tumor angiogenesis, as well as in MM, has been extensively analysed, but the significance of its serum level is still not well recognize. The aim of the present study was to determine the serum levels of both above mentioned cytokines and compare their values with clinical parameters of myeloma patients. Methods: serum level of OPN and VEGF was determinated by ELISA in 44 newly-diagnosed, reviously untreated myeloma patients (21 men, 22 women; median age of 69, range 44 – 86) and 24 age-matched healthy persons who consisted control group. The obtained values were correlated with main clinical parameters such as anaemia (hemoglobine value 20 g/L below the lower limit of normal), renal dysfunction (serum creatinine level above the uper limit of normal) and bone disease (any of lytic lesions or severe osteopenia with compressive fractures on standard radiographs of the bones). The level of cytokines were also correlated with serum beta-2 microglobulin as a prognostic factor related to biological behaviour of MM, and Durie-Salmon Staging System as a measure of tumor mass. Results: serum value of OPN was significantly higher in myeloma patients (median 6. 5, range 0. 3 – 21. 7) in compare to control group (medain 2. 4, range 0. 2 – 8. 9) (p
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  • 89
    Publication Date: 2012-11-16
    Description: Abstract 50 The Epstein-Barr virus (EBV) is one of the most major human pathogen that establish long-term latent, or chronic infections, which is associated with a heterogeneous group of lymphoma, including Burkitt's lymphoma, Hodgkin's lymphoma (HL), NK-T lymphomas and lymphoproliferative disease. These malignancies are subdivided into in terms of EBV latent infection pattern, with typical three types of latency: type I to type III. HL is characterized by a minority of neoplastic Hodgkin and Reed-Sternberg (HRS), which are embedded in non-neoplastic bystanders, mostly B and T cells, but also macrophages. Without these bystander cells, these HL cells are incapable of being engrafted in immunodeficient mice. In this context, the non-tumor immune cells are tumor-supportive “inflammatory niche”. Because of the complexity of interplay between tumor and tumor surrounding immune cells, the detailed mechanism and how tumor cells escape from the attack of host immune cells remains an open question. Small RNAs including miRNAs are well known intra-cellular regulatory elements of gene expression. Recently, it was reported that they are conjugated in exosomes and transferred to cells and are involved in tumor metastasis by educating tumor surrounding niche. Moreover, it was also reported that EBV-infected lymphocytes produce exosomes that contain viral encoded, EBV specific miRNAs (BART-miRNA) and that these could be transferred in host cells and decrease the levels of known cellular targets. Accordingly, we hypothesized that EBV+ tumor derived exosomal BART-miRNA might redirect tumor surrounding immune cells from tumor reactive into tumor- -supportive “inflammatory niche”, which ultimately leads to tumor progression. To this aim, first, we evaluated tumor derived viral encoded BART-miRNA in EBV+HL clinical specimens by using BART-miRNA specific probe in situ hybridization. As expected, these EBV specific BART-miRNA could be detected in HRS as well as in tumor surrounding inflammatory niche, especially macrophage. This result indicated that tumor derived EBV specific BART-miRNA could transfer to the non-tumor cells in the tumor inflammatory niche, supporting the in vivo relevance of secretary EBV specific miRNA. Next, we evaluated the properties of exosomes produced by EBV+ cells (EBV-Ex). To this aim, EBV-Ex was harvested either from the media of the type III or type I EBV-transformed lymphoid cell line. Then, by using transwell co-culture system, we tested the delivery and the effect of EBV-Ex on human peripheral blood mononuclear cells (PBMC) derived monocyte/macrophage (Mo/Mf). As a result, we detected uptake of fluorochrome dye-labeled EBV-Exo in Mo/Mf. We also confirmed exosomal BART miRNA transfer in Mo/Mf. Surprisingly, exosome from Type III latency (Type III-Ex) was relatively enriched in BART miRNA, and were potent on Mo/Mf in inducing surface CD69 expression (Fig.A). This is in contrast to that of exosome from Type I latency (Type I-Ex), in which BART miRNA were relatively vacant and were weak in inducing surface CD69 expression (Fig.A). Panels of cytokine analysis by Q-PCR revealed that type III-Ex treated Mo/Mf displayed an anti-inflammatory/immunosuppressive cytokine rich signature, especially IL-10, compared to type I-Ex treated Mo/Mf, suggesting the possibility that type III-Ex might polarize macrophage into immunosuppressive M2-like phenotype. Intriguingly, type III-Ex from BART miRNA deletion mutant derivative cell lines totally lack the type III -Ex signature. Moreover, ectopic expression of a part of BART in Type I cells changed the EBV-Ex signature from type III to type I (Fig.B), suggesting the importance of specific BART lesion in functional EBV-Ex production in terms of Mo/Mf polarization. Taken these together, secretary tumor derived miRNAs in EBV associated malignancy, specifically in EBV+HL, might play a certain role in tumor inflammation niche. EBV might utilize the exosomal machinery to secrete key viral-encoded miRNAs, through which a small number of neoplastic EBV+ cells could modulate the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.
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  • 90
    Publication Date: 2012-11-16
    Description: Abstract 4975 Background: Our group has developed a peptide referred to as HYD1 which binds VLA-4 integrin/CD44 containing complex and induces necrotic cell death in myeloma cells (Nair et al Mol Cancer Ther 2009, Emmons et al, Mol Cancer Ther, 2011). Furthermore, we previously demonstrated in a small subset of MM patients that HYD1 was more active in specimens obtained from relapsed/refractory patients, a finding that correlated with VLA-4 expression. The frequency of expression and prognostic significance of two key surface markers VLA4 (very late antigen 4) and CD44 in patients with MM remains poorly defined. Methods: We retrospectively reviewed records of patients with MM who had a complete flow cytometry panel (which includes assessment for CD44 and VLA4) at Moffitt Cancer Center between 1/1/2004 and 12/31/2009. CD44 and VLA4 were gated by CD138 expression and we categorized expression as negative, dim, moderate or bright. We collected demographic information, disease related characteristics (including ISS stage, cytogenetics, and baseline laboratory testing) and treatment and outcomes related data (prior therapies, response to first line therapy, follow up and vital status). Responses were per IMWG criteria and for the purpose of this study, a partial remission or better qualified as a response to therapy. High risk cytogenetics (HRC) was defined by the presence of one of the following; 17p deletion, t(4;14), t(14;16) and 13q deletion by metaphase cytogenetics. The study was approved by the IRB at the University of South Florida. Results: A four color flow cytometry panel was available for review on 101 myeloma patients including 57 males, median age at diagnosis was 60 (range 39–83) years. The percentage of ISS stage I, II and III at diagnosis was 36. 4%, 34. 8% and 28. 8% respectively and 28. 7% of the patients had HRC. At the time of the flow panel, 32 patients (31. 7%) were untreated and overall patients had a median of 3 prior therapies (range 0–11). 52 patients (51. 5%), 37 (36. 6%) and 12 (11. 9%) had no, dim or moderate CD44 expression respectively while 26 (26. 3%), 68 (68. 7%) and 5 (5. 1%) had no, dim or moderate VLA4 expression respectively. Subsequently both were sub-grouped according to presence (dim or greater) or absence of expression. A correlation between the expression of CD44 and VLA4 was noted (Fisher's exact p 〈 0. 001), 45 patients express both markers, 24 express neither markers, 28 express VLA4 but not CD44 while only 4 express CD44 and not VLA4 (p
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  • 91
    Publication Date: 2012-11-16
    Description: Abstract 4996 BACKGROUND: In patients with newly diagnosed multiple myeloma (MM), a gain of the long arms of chromosome 1 is found in approximately 20% of patients by cytogenetics and 40% by FISH. A few studies have not found these abnormalities to be predictive of inferior progression free survival (PFS) and overall survival (OS) on multivariate analysis. The many studies that have were prior to the use of novel therapies in induction. Currently, testing for a gain or amplification of 1q21 locus is not included in the high risk genetic abnormalities by the International Myeloma Working Group (IMWG). Recently, two groups have reported inferior outcomes with bortezomib based induction regimens followed by high dose melphalan consolidation and maintenance therapy. The gain of 1q21 was either detected by cytogenetics or gene expression profiling (GEP70) at the University of Arkansas (Waheed et al, Cancer 2011) or by FISH in CD138 selected cells by the HOVON group (Neben et al, JCO 2012). However, the outcomes of these patients when treated with a bortezomib, lenalidomide, and dexamethasone induction regimen, which is associated with an overall response rate (ORR) of 100% and VGPR or better rate of 74% is unknown (Richardson et al. Blood 2010). Here, we describe the poor outcomes of patients with newly diagnosed MM with gain of 1q21 by FISH in unselected bone marrow aspirates despite novel agent triplet therapy. METHODS: The inclusion criteria for this IRB approved retrospective study were patients with symptomatic MM starting in June of 2008 who had a gain of 1q21 by FISH in 200 bone marrow interphase cells at the time of diagnosis. Patients with 1q amplification had at least 3 or more copies. PFS and OS were calculated by Kaplan-Meier analyses. RESULTS: 23 patients met the inclusion criteria. The median age was 59. 7 (range 46–71) and twenty patients (87%) were DS III at diagnosis, 13/23(57%) were ISS Stage 3. 6/23 (26%) had hypercalcemia, 8/23 (35%) had renal insufficiency, and 19/23 (83%) had anemia. Lytic lesions were present in 17/23 (74%) of patients at diagnosis. Of note, while 4 patients had deletion 13 by cytogenetics only 2 patients had other high risk findings, one with t(4;14) and another with deletion 17. All patients were treated with novel agent induction therapy. 19/23 (83%) were treated with triplet regimens (bortezomib, dexamethasone, and either lenalidomide or cyclophosphamide i. e. VRD or VCD). Disappointingly, primary induction failure, defined by PD or SD after 3–4 cycles, was observed in 30% of all patients. Of the 17 patients who received upfront triplet VRD or VCD therapy (with or without HDM plus SCR) the overall response rate (ORR) was only 77% (13/17), and 47% (8/17) achieved VGPR or better. More specifically, 3/17 (18%) had CR, 5/17 (29%) had VGPR, 5/17 (29%) had PR, 1/17 (6%) had SD and 1/19 (6%) had PD. Of the 4 patients who received novel doublet therapy (VD or RD) upfront, only 1 had a VGPR, one had SD and 2 had PD. The responses noted were not very durable, with a median PFS of 14 months. Although 3 patients have died (after 11–13 months from diagnosis), the median OS has not been reached with a median follow up of 13 months. Plasmacytomas of the bone with soft tissue expansion were present in 48% of the patients and 3 of 23 (13%) had spinal cord compression. Extra-osseous MM within 3 months of diagnosis was observed in 5/23 (22%) patients and 4/5 did not have any other high risk cytogenetics. Three patients (13%) had CNS MM at median of 5. 3 months after diagnosis. One patient had concomitant parenchymal brain lesions, myleomatous meningitis, and intra spinal cord disease. CONCLUSIONS: Even in the era of novel agent induction therapies, in our series of newly diagnosed 23 patients with a gain of 1q21, the failure rate of induction was 30% with a median PFS of 14 months. Moreover, these patients had a particularly aggressive clinical course with both medullary and extramedullary plasmacytomas, suggesting that PET-CTs, rather than just skeletal surveys should be considered for initial staging and monitoring. Also, given an unusually high incidence (13%) of early onset CNS disease, prompt CSF evaluation and brain MRI should be performed on patients with neurologic symptoms or signs. We recommend that gain or amplification of q21 identified either by FISH (even without CD138 selection – as demonstrated here) or GEP be prospectively studied in patients with newly diagnosed MM with consideration of novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.
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  • 92
    Publication Date: 2012-11-16
    Description: Abstract 5010 Background: Multiple myeloma (MM) is a B-cell malignancy that has remained essentially incurable by conventional therapy, highlighting the urgent need for novel treatment strategies. Lenalidomide (LEN) is thalidomide analog belonging to the class of immunomodulatory drugs which the activity are related with immunomodulatory properties. LEN augments both the adaptive and innate immune system via the co-stimulation of T cells, NK and NKT cells. In addition, LEN can inhibit the frequency and function of immunosuppressor cells. Therefore, LEN could be used to enhance immune response against MM. Cellular therapy with dendritic cells (DCs) is emerging as a useful immunotherapeutic modality to treat MM. The purpose of this study was to investigate the immunomodulatory effects of lenalidomide in combination with DCs vaccine to treat MM in vivomouse model. Methods: We used the MOPC315 myeloma murin model to evaluate tumor-specific cytotoxic T lymphocytes responses by a DC vaccine in combination with LEN. After tumor growth, LEN (50 mg/kg/day) was injected intraperitoneally at three consecutive days to cover the DC vaccination. The tumor growth inhibition effect and the antitumor activity of splenocytes from vaccinated mice were evaluated to reveal the synergistic effect of DCs and LEN. Results: The combination of LEN and DC vaccine efficiently inhibited tumor growth in mouse MM model when compared to single therapeutic agent. These vaccinated mice exhibit the reduction of myeloid-derived suppressor cells (MDSC) and regulatory T cell (Treg) in spleen. Inhibition of MDSC and Treg resulted in the increasing proportion of CD4+ and CD8+T cell in the spleen. High ratio of Th1- to Th2-type cytokines was induced by LEN plus DC vaccine. LEN also enhance the innate immune response by modulating NK cell number and function. In addition, LEN also can enhance the population of effector memory T cells in the spleen of vaccinated mice. Furthermore, the treatment of LEN can down-regulate the levels of VEGF and TNF-a on tumor tissues of vaccinated mice. Conclusion: These results suggest that a treatment combining the immunomodulatory drug lenalidomide with DC vaccine can improve antitumor immunity in mouse MM model by inhibiting immunosuppressor cells and recovering effector cells, as well as superior polarization of the Th1/Th2 balance in favor of Th1 type immune response. Disclosures: No relevant conflicts of interest to declare.
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  • 93
    Publication Date: 2012-11-16
    Description: Abstract 4956 Introduction. Patients with Myelodysplastic Syndrome (MDS) are susceptible to developing iron overload as a response to the red blood cell (RBC) transfusions and ineffective hematopoiesis. This iron overload (IOL) is characterized by an increase in oxygen-reactive species accompanied by a decrease in antioxidants, and results in hepatic, cardiac and endocrine disorders, as well as an increased risk of infection. Ineffective hematopoiesis promotes iron absorption at intestinal level. This process is enhanced by the presence of mutations in the hereditary hemochromatosis gene (HFE). This study aims to define the features that accompany patients with iron overload, comparing them to a MDS population at diagnosis. Patients and methods. 34 low/int-1 MDS patients (International Prognostic Score System, IPSS) were assessed, 22 of them at diagnosis and 12 patients when IOL was developed. Peripheral blood samples were drawn after informed consent was obtained from the patient or the patient′s guardians in accordance with the Declaration of Helsinki. The analyzed parameters were: WHO classification, sex, age, blood count, number of RBC units received, iron metabolism, and mutations of the HFE gene. Liver damage was estimated by measuring Alanine transaminase (ALT) levels, and liver iron concentration (LIC) detected by Magnetic Resonance (MR). Likewise, levels of labile plasmatic iron (LPI) (eLPI Assay Kit, Aferrix) were quantified by spectrofluorimetric determination. Oxidative damage was assessed by quantifying the modified base 8-oxo-2-hydroxi-deoxiguanosine (8-oxo-dG) by means of High-Performance Liquid Chromatography (HPLC) and the O2− anion levels by flow cytometry. Results. According to the WHO classification, 72. 2% of cases with MDS and IOL assessed belonged to Refractory Sideroblastic Anemia (RSA) group, unlike 27. 3% of patients at diagnosis (p=0. 0265). In comparison to diagnosis patients, IOL subjects presented lower mean age (76 vs. 81 years; p=0. 0172), hemoglobin levels (7. 5 ± 0. 4 vs. 10. 3 ± 0. 3 g/dl; p
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  • 94
    Publication Date: 2012-02-09
    Description: Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.
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  • 95
    Publication Date: 2012-11-16
    Description: Abstract 2074 Background: Around 75% of myeloma patients have evidence of myeloma bone disease at diagnosis and most develop it at some point. Bisphosphonates - pamidronate, sodium clodronate and zoledronic acid - have revolutionised the treatment of myeloma bone disease and form a significant part of the myeloma treatment pathway. It is therefore important to understand patient perspectives of and preferences for this important treatment. Aims: To understand the information patients receive from clinicians and the impact this has on their understanding of bisphosphonates, to establish what patient preferences are for how and where they receive bisphosphonates and to determine whether patients are satisfied with the level of decision-making input they have about their bisphosphonate treatment. Results: Patients were asked to complete a 36 question survey, devised by Myeloma UK, which was available in paper copy and online. 606 patients responded to the survey of which 95.3% had received bisphosphonates. When asked about the information they were given by their clinicians when prescribed bisphosphonates, 81.7% received a verbal explanation, 23.7% received written information and 2.7% received details of a website/helpline. 30.6% used a product leaflet and 30.1% sourced a patient organisation information sheet. 80% of patients thought the information was ‘easy to understand’. When asked why they had been prescribed bisphosphonates, patients accurately reported that it was ‘to strengthen the bones’ (82.5%), ‘to delay the progression of myeloma’ (28.7%) and ‘to prevent/reduce high levels of calcium in the blood’ (15.7%). 16.6% of patients wrongly thought that bisphosphonates were prescribed ‘to heal the bones’ and 5.8% had no understanding of bisphosphonates. 51.7% of patients received their current or last bisphosphonates ‘by monthly infusion in hospital’ and 39.1% ‘by tablet form at home’. 9.2% stated ‘other’ referring to more flexible methods of prescribing. When asked how they would prefer to receive their bisphosphonates, 46.5% selected ‘by regular visits for infusion in hospital’ and 41.1% ‘by daily tablets taken at home’. Patients who preferred to receive bisphosphonates ‘by regular visits for infusion in hospital’ reported this was due to ‘confidence in quality of care in hospital’ (80.7%), ‘there is medical support on hand’ (54.4%), ‘it keeps a check on my myeloma’ (64.9%), ‘I prefer monthly rather than daily treatment’ (52.1%) and ‘I can get my blood tests done at the same time' (59.1%). Patients who preferred to receive bisphosphonates by ‘daily tablets taken at home’, reported this was due to ‘convenience’ (93.3%), ‘ease’ (71.4%), ‘it reduces hospital visits' (61.6%) and allows them to ‘avoid infusions’ (37.9%). On a scale of (1) no input and (5) full input, patients gave the level of decision-making input they had over how and where to receive bisphosphonates an average rating of 2.1 – 64.6% rated (1), 6.2% rated (2), 4.9% rated (3), 2.8% rated (4), 21.5% rated (5). When asked if they were satisfied with this level of input, 79.5% of patients were satisfied, 20.3% would have preferred more input and 0.2% less. Conclusions: Patients who received information about bisphosphonates were generally satisfied it was good quality and helped them understand why they were prescribed bisphosphonates. A significant proportion of patients did not receive a verbal explanation and/or did not receive written information from a clinician or source information themselves, which explains the high percentage of patients who did not understand why they had been prescribed bisphosphonates. Patients have different and individual preferences for how and where they want to receive bisphosphonates. Patients were polarised between their preferences for receiving their bisphosphonates intravenously or by tablet form and when responses were cross-referenced, their preferences were approximately correlated with their most recent bisphosphonate. The survey also shows approximately 1 in 5 patients would have preferred more decision-making input on their choice of bisphosphonate treatment. In the UK, there is variation in uptake of oral and intravenous bisphosphonates despite evidence showing improved efficacy of intravenous zoledronic acid over oral sodium clodronate. Further investigation is warranted to determine the link between patient's preferences and uptake of different bisphosphonates. Disclosures: Low: Novartis UK Limited: Research Funding. Morgan:Novartis UK Limited: Research Funding.
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  • 96
    Publication Date: 2012-11-16
    Description: Abstract 1178 Microparticles (MP) are submicron size fragments produced from most cells. Although their role in disease is still not known, concern has been raised over their role in RBCs and the adverse events of RBC transfusion, particularly their pro-inflammatory effects on the host. We hypothesize that MP's increase during routine RBC storage, contribute to the priming activity that accumulates in the supernatant of RBCs, and that pre-storage leukoreduction affects these processes. Methods: RBC's were separated from the whole blood of 8 healthy donors and stored in AS-5 via current U.S. industry standards. The first 5 units were split into two equal aliquots (weight) with 50% undergoing standard leukoreuction (LR) and the remaining was left as an unmodified control (NLR). The other three units were processed as standard LR-RBCs. RBC samples were taken at days (D) 1 and 42 and cell-free supernatants were separated by centrifugation and stored (-80°C). The supernatant was centrifuged at 100,000g – 120 minutes into MP and MP free (MPF) portions and MP's resuspended in equal volume of 1.25 % HSA (MP-HSA) or cell-free plasma (MP-FFP). MP fractions were incubated with specific antibodies to RBCs (CD235), WBCs (CD45), and PLTs (CD41a) and analyzed by flow cytometry employing commercially obtained counting beads. Isolated neutrophils (PMNs) were incubated with the MP and MPF fractions [10%] FINAL for 5 min at 37°C followed by activation of the NADPH oxidase with fMLF. The maximal rate of O2− production was measured as the SOD-inhibitable reduction of cytochrome c. Results were analyzed as an ANOVA with a post-hoc Newman Keuls test for multiple comparisons. Results: The total number of MP's increased during storage in all units irrespective of LR. The predominant MPs came from RBCs with PLTs representing the least (Table 1). Although the total number of MPs increased in all units, the number of MPs that specifically marked for precursor cell types decreased over storage (Table 1). The MPF fractions from stored (D.42) NLR-RBCs and LR-RBCs caused significant priming of the PMN oxidase vs. both the MPF from D.1 and the fMLF controls (Table 1). The MP fraction from NLR-RBCs did not evidence priming activity (data not shown); however, the LR-MP-HSA fractions from both D.1 and D.42 caused significant priming of the oxidase. Resuspension in plasma (LR-MP-FFP) inhibited this priming activity (Table 1). We conclude that during storage of NLR- and LR-RBCs, MPs increase; however the number of MPs associated with a specific cell type decrease but not significantly. This loss of cellular markers may be due to non-specific loss, antigen capping, internalization or proteolysis because proteases are released and increase during RBC storage. The presence of MPs from leukocytes and platelets are likely due to filtration, which is known to cause release of WBC- and PLT-derived proteins. While the majority of the PMN priming activity is in the acellular MPF supernatant in all NLR- and LR-RBC units, there is a modest amount of priming activity in the MP fraction following LR (LR-MP-HSA), which is likely a function of LR and may be inhibited by resuspension of the MPs in plasma. Disclosures: Silliman: Pall Corporation: Honoraria, Research Funding.
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  • 97
    Publication Date: 2012-11-16
    Description: Abstract 4563 The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous, with some patients requiring treatment relatively soon after diagnosis and others having indolent disease for many years. Some patients with indolent disease, however may develop more aggressive disease over time that requires therapy. To identify genetic and epigenetic changes that associate with the transition from indolent to aggressive disease, we used genomic methods to analyze sequential samples obtained from 19 CLL patients evaluated at the UC San Diego Moores Cancer Center who ultimately required treatment, as per iwCLL guidelines. For all patients, the first time point sample collection (SC1) was obtained within 1 year post-diagnosis and the second time point sample collection (SC2) was obtained within 1 year before treatment. We performed whole-exome sequencing (Agilent 50Mb capture, 100×) and methylation (450K) array analyses on leukemia cells and germline DNA. Somatic allele frequencies ranged from 〈 10% to 50%, suggesting heterogeneity within the tumor. When comparing SC1 versus SC2, we observed changes in somatic allele frequency for 6 (32%) of 19 patients, however 13 (68%) of 19 patients did not have evidence for clonal evolution at the somatic level, suggesting that the acquisition of additional somatic mutations did not drive CLL progression and that the clonal population structure remains stable throughout disease progression with multiple clones per patient. Using 450K CpG methylation arrays, we identified 52,409 sites (FDR=0.05) that changed consistently between SC1 and SC2 across 19 patients, suggesting that epigenetic changes were widespread, even without detectable somatic mutations. In summary, our results imply that CLL progression can occur in the absence of somatic mutations, but rather may reflect non-stochastic alterations in the epigenome altering RNA expression. Disclosures: No relevant conflicts of interest to declare.
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  • 98
    Publication Date: 2012-11-16
    Description: Abstract 4592 The optimal treatment of patients with chronic lymphocytic leukemia (CLL) and concomitant renal impairment is unclear. Fludarabine containing regimens are contraindicated in patients with glomerular filtration rate less then 30 ml/min. Alkylating agents are either contraindicated or require dose reduction due to their relatively large renal clearance. Treatment choice is especially difficult in cases with refractoriness to alkylating drugs. The aim of this study was to assess safety and efficacy of bendamustine monotherapy in CLL patients with concomitant chronic renal failure. Seven patients with proven diagnosis of CLL and chronic kidney disease were treated with bendamustine monotherapy. The median age of patients was 66 years (range 61 – 83). All patients were males. The median creatinine level was 183 mkmol/L (range 165 – 573), the median level of glomerular filtration rate was 39 ml/min (range 23 – 47). The causes of chronic renal failure: membranous proliferative glomerulonephritis and focal CLL infiltration – 1 case, nephrectomy for cancer and massive CLL infiltration – 1 case, nephrectomy for cancer and pyelonephritis of sole kidney – 1 case, massive diffuse CLL infiltration with no other causes identified – 1 case, drug associated tubulointerstitial nephritis with development of irreversible renal failure – 1 case, chronic gout and urolithiasis in 1 case, unknown – 1 case. None of the patients required hemodialysis. Treatment consisted of bendamustine monotherapy, administered for two days every 4 weeks. Treatment in all patients started with dose 70 mg/m2/day. If the first course was well tolerated the dose was escalated to 100 mg/m2 on the next courses. Three patients were newly diagnosed and four patients had relapsed disease, after a medium of 2 lines of therapy (range 1 – 2). Two patients were refractory to alkylating drugs. Before initiation of bendamustine 4 patients had Binet stage C, and 3 Binet stage B. Four patients received all planned 6 cycles of therapy with dose escalation to 100 mg/m2/day. In three patients treatment was stopped prematurely. In one patient treatment was discontinued after the 4th cycle because of the grade III skin rush and grade II polyneuropathy. One severely cardio compromised patient of 72 years developed grade III bradicardia after first cycle, requiring installation of cardiac pacemaker. One patient of 83 years with refractoriness to previous treatment died after 2 cycles from infectious complications. Neutropenia grade III was observed in 5 cycles in 3 patients. Aggravation of thrombocytopenia (grade II) was observed in 2 patients and aggravation of anemia (grade I) in 3 patients. In no case there was worsening of renal function. Decrease of creatinine and urea level was observed in 5 patients. Response can be evaluated in 5 patients. 2 patients achieved a nodular PR, and 3 patients achieved a PR. In conclusion, monotherapy with bendamustine can be safely used in patients with CLL and renal impairment. Doses up to 100 mg/m2 are tolerated and do not cause worsening of renal function or severe hematological toxicity. Disclosures: No relevant conflicts of interest to declare.
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  • 99
    Publication Date: 2012-11-16
    Description: Abstract 460 Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic stem cell transplantation (alloSCT). Monitoring of EBV genomes in blood using quantitative PCR (EBV qPCR) coupled with pre-emptive administration of Rituximab in response to high-level EBV reactivation has emerged as a strategy to reduce mortality from PTLD. However, the effect of pre-transplant Rituximab therapy on the risk of EBV reactivation and survival post-alloSCT is unknown. This retrospective study examined 193 consecutive adult patients undergoing T cell depleted or cord blood alloSCT at University Hospital Birmingham, UK (UHB) and Nottingham University Hospital, UK (NUH) between May 2009 and April 2011. Median age at transplant was 54 years (range 16–73 years). Conditioning was reduced intensity in 84% and myeloablative in 16%. Stem cell source was matched unrelated donor in 70%, sibling in 24% and cord blood in 6%. T cell depletion was with in vivo Alemtuzumab in 89% and ATG in 6%. Patients were monitored by EBV qPCR whole blood assay, performed every 1–2 weeks post-transplant. EBV reactivation was defined as a single positive EBV qPCR result, whilst high-level EBV reactivation was defined according to institutional thresholds; 30,000 and 10,000 EBV genomes/ml for UHB and NUH respectively. All patients with high-level reactivation were pre-emptively treated with Rituximab. Median follow-up was 23 months (interquartile range [IQR] 18–30 months), with EBV qPCR testing for a median of 8 months (IQR 4–13 months) post-transplant. The cumulative incidence of EBV reactivation, adjusting for the competing risk of death, was 41% at 2 years post-transplant. Amongst those reactivating, the median time to EBV qPCR positivity was 120 days (IQR 77–198 days). High-level EBV reactivation was observed in 34/193 (18%) patients, accompanied by PTLD in 10 patients (4 biopsy-proven and 6 probable cases). Of patients developing high-level EBV reactivation, in 30/34 (88%) the interval from first EBV qPCR positivity to high-level reactivation was less than 4 weeks, with 15/34 (44%) exhibiting high-level reactivation at first qPCR positivity. In univariate analysis, significant predictors for EBV reactivation were older age (hazard ratio [HR] 1.02 per year; p=0.04), male sex (HR 1.75; p=0.03) and T depletion with ATG (HR 4.8; p
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  • 100
    Publication Date: 2012-11-16
    Description: Abstract 4577 Patients with chronic lymphocytic leukemia (CLL) whose tumor cells harbor 17p deletions (17p-) by fluorescent in-situ hybridization (FISH) or chromosome banding analysis (CBA) are considered to have a poor survival. The disease is usually refractory to conventional chemotherapy and alternative therapeutic approaches are generally recommended. There is, however, a degree of clinical heterogeneity within 17p- CLL patients, as a significant proportion of them remain asymptomatic for prolonged periods of time. The aim of this study was to determine the prognostic value of concomitant molecular abnormalities in patients with 17p- CLL. Clinical and laboratory data were collected from 76 patients with 17p- CLL, detected either at diagnosis (de novo deletions, 39 patients) or over the course of the disease (acquired deletions, 37 patients). The cut-off used to define a positive FISH result was 10%, and complex karyotype was defined as the presence of 3 or more aberrations by CBA. We performed Sanger sequencing of IGHV, TP53 (exons 4–9), NOTCH1 (exon 34) and SF3B1 (exons 14–18), as well as high resolution copy number analysis using a SNP-array platform (CN-SNP). Both CBA/FISH and molecular studies were performed on samples drawn on the same date. Main biological characteristics, including CD38 and ZAP-70 expression or beta2-microglobulin (B2M), were also recorded. We evaluated the impact of these variables on time to first treatment (TTFT) and overall survival (OS) from sampling. TTFT was only evaluated in patients with de novo 17p-, and OS was evaluated in the whole cohort. Optimal cut-offs for FISH, B2M and copy number aberrations (CNAs) were calculated using maximally selected rank statistics, and were ≥25%, ≥3.5 mg/dl and ≥3, respectively (maxstat package, R, version 2.15.0). TP53 mutations were detected in 28/60 (47%) patients, and were more frequent in patients with ≥25% 17p- cells by FISH (64% vs 32%, p=0.029). CN-SNP confirmed 17p losses in only 19/68 (28%) patients and 90% of them also had concurrent TP53 mutations. 17p- by CN-SNP were mostly detected in patients with ≥25% 17p- cells by FISH [11/16 (69%)], compared to 6/49 (12%) patients with
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