ALBERT

Description: Vitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing.

Description: This is the first study to investigate the efficacy of intravenous iron in treating fatigue in nonanemic patients with low serum ferritin concentration. In a randomized, double-blinded, placebo-controlled study, 90 premenopausal women presenting with fatigue, serum ferritin ≤ 50 ng/mL, and hemoglobin ≥ 120 g/L were randomized to receive either 800 mg of intravenous iron (III)–hydroxide sucrose or intravenous placebo. Fatigue and serum iron status were assessed at baseline and after 6 and 12 weeks. Median fatigue at baseline was 4.5 (on a 0-10 scale). Fatigue decreased during the initial 6 weeks by 1.1 in the iron group compared with 0.7 in the placebo group (P = .07). Efficacy of iron was bound to depleted iron stores: In patients with baseline serum ferritin ≤ 15 ng/mL, fatigue decreased by 1.8 in the iron group compared with 0.4 in the placebo group (P = .005), and 82% of iron-treated compared with 47% of placebo-treated patients reported improved fatigue (P = .03). Drug-associated adverse events were observed in 21% of iron-treated patients and in 7% of placebo-treated patients (P = .05); none of these events was serious. Intravenous administration of iron improved fatigue in iron-deficient, nonanemic women with a good safety and tolerability profile. The efficacy of intravenous iron was bound to a serum ferritin concentration ≤ 15 ng/mL. This study was registered at the International Standard Randomized Controlled Trial Number Register (www.isrctn.org) as ISRCTN78430425.

Description: Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Description: Abstract 195 ADAMTS13 contains multiple free thiols on its surface, which may form disulfide bonds with surface-exposed free thiols on plasma-derived von Willebrand factor (VWF). This interaction may prevent lateral association of apposed VWF under arterial shear stress. However, the functional consequence of ADAMTS13-VWF interaction without proteolysis is not known. We hypothesize that the interaction between the C-terminus of ADAMTS13 and the C-terminus of VWF inhibits thrombus formation under shear stress. Using a BioFlux microfluidic system, we showed that under arterial shear stress, 10 dyn/cm2, fluorescein-labeled platelets from PPACK (thrombin inhibitor) anti-coagulated human whole blood adhered to collagen (type I)-coated surface in a time-dependent manner. Addition of human recombinant full-length ADAMTS13 (10 nM) into whole blood dramatically reduced the surface coverage of fluorescein-labeled platelets. Conversely, addition of an inhibitory polyclonal anti-ADAMTS13 IgGs (150 ug/ml) to whole blood dramatically accelerated the accumulation of fluorescein-labeled platelets. These results suggest that this microfluidic system is highly sensitive for the assessment of anti-thrombotic function of ADAMTS13. Under the same conditions, we were able to further show that addition of recombinant C-terminal fragment of ADAMTS13 comprising of the 5th to 8th thrombospondin type 1 (TSP1) repeats and two CUB domains (T5C) or the 2nd to 8th TSP1 repeats and two CUB domains (T2C) into whole blood also inhibited the surface coverage of fluorescein-labeled platelets on collagen-coated surface in a concentration-dependent manner. In the presence of 0.1 μM and 0.5 μM of recombinant T2C or T5C, the surface coverage of fluorescein-labeled platelets was reduced by ∼40% and ∼60%, respectively. The inhibitory activity of these recombinant C-terminal fragments was nearly abolished if pre-treated with 40 mM of N-ethylmaleimide which blocked surface-exposed free thiols. Moreover, recombinant CUB domains at the highest concentration tested (1.0 μM) did not appear to alter the surface coverage of fluorecein-labeled platelets under the same conditions. These results suggest that the C-terminal TSP1 repeats of ADAMTS13 inhibit platelet adhesion and aggretion or thrombus formation through thiol-thiol interactions between ADAMTS13 and VWF (or other proteins). We conclude that the C-terminal TSP1 repeats may modulate thrombus formation independent of proteolytic activity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 22 For many years it has been evident that it is challenging to express recombinant factor VIII (FVIII) in vitro and in vivo. In vitro studies suggest that high levels of FVIII expression can lead to cellular stress. We have demonstrated that adeno-associated viral (AAV) vector delivery results in long-term expression of therapeutic levels of FVIII in hemophilia A (HA) dogs up 10% of normal. In this model transgene expression is restricted to a portion of liver cells resulting in sustained FVIII levels. Notably, in dogs FVIII expression is sustained for periods longer than 7 years. Conceivably, the increased load per cell for cellular processing of FVIII may impact the cell's ability to synthesize and secrete functional FVIII, activation of cellular stress and the potential for immune responses. We sought to determine whether cellular stress and immune responses to the transgene are increased by overexpression of FVIII in mice by AAV vectors. Using AAV to deliver FVIII as a B-domain deleted single chain (single chain approach) or as two separate chains (light and heavy chains) (two chain approach), we analyzed dose-dependent FVIII synthesis and its cellular impact. We hypothesized that high levels of FVIII expression after AAV-mediated delivery of FVIII may induce cellular stress. HA mice were administered AAV serotype 8 (AAV8) vector in 3 groups: (1) two-chain approach (2) single chain approach or (3) AAV empty capsid (control). Three total AAV doses were administered: 1×1010 vector genomes/mouse (low dose), 5×1010 vg/mouse (mid-dose) and 2.5×1011 vg/mouse (high dose). FVIII antigen levels (heavy and light chain) and anti-cFVIII IgG antibodies were detected by ELISA at a series of time points post vector administration until the terminal time points (1, 2, 4, 8, 12 and 24 wks). As we previously observed, after single chain delivery we detect equivalent amounts of heavy chain and light chain in the circulation by ELISA that correlates with activity. However, in the two-chain approach we observe that the light chain is 〉2-fold higher than the heavy chain in the circulation and that the activity correlates with the amount of heavy chain. We observe a dose-dependent increase in FVIII expression and dose-dependent anti-cFVIII IgG antibody titers. The kinetics of FVIII expression and antibody formation are different for the two-chain delivery compared to the single chain delivery. For the two-chain delivery, we observe AAV dose-dependent peak FVIII expression within 1 week (30, 100, or 400 ng/ml, respective of dose) followed by the onset of anti-FVIII IgG2 antibodies by 4 weeks (16, 27, 52 μg/ml, respective of dose). For single chain delivery, we observe FVIII expression within 1 week and low level antibody titers by 4 weeks that increase dramatically by 12 weeks (14, 21, 38 μg/ml, respective of dose). Thus, we observe a more gradual increase in antibody titers in the single chain compared to the two-chain delivery but at late time points the immunogenicity of both approaches is indistinguishable. RNA was isolated from the livers of the mice at the terminal time points for quantitative PCR analysis of two key components of the unfolded protein response signaling network, BiP and CHOP. We compared the two-chain and single chain AAV delivery of FVIII alongside animals injected with AAV empty capsid that did not express FVIII. A tunicamycin treated positive control had an increase in BiP (54-fold) and CHOP (74-fold) expression compared to untreated HA mice. There were mild elevations (2–3-fold) of BiP and/or CHOP in some animals observed at week 1 in all three treatment groups. Only at 12 weeks post vector administration were significant increases in BiP (〉70 fold) and CHOP (〉4 fold) observed in the single chain treated group at the high dose expressing 〉300% but not the two-chain approach or empty capsid. These studies suggest that supraphysiological expression (〉100%) of BDD-cFVIII may increase the likelihood of an immune response to FVIII and cellular stress. Further studies may determine if there is a relationship between high FVIII expression, cellular stress and immune responses to FVIII. Thus, there is a threshold of FVIII expression levels that may prove unsafe. Moreover, these data are in agreement with the lack of evidence of cellular toxicity and immunogenicity in HA dogs expressing therapeutic levels of FVIII by AAV vectors. Disclosures: Lange: Bayer Healthcare: Research Funding. Altynova:Bayer Healthcare: Research Funding. Sabatino:Bayer Healthcare: Research Funding.

Description: Abstract 4184 Introduction: FACT-MM was developed with the aim to create a disease-specific, patient-reported outcomes (PRO) measure as part of the FACT measurement system to assess multiple myeloma (MM)-related symptoms. Literature review identified 52 MM specific symptoms and concerns. 13 MM expert clinicians rated these 52 items on relevance to health-related quality of life (HRQL) for MM patients and added 11 items for comprehensive PRO assessment in MM. These 63 candidate items were rated by 13 MM patients recruited through the International Myeloma Foundation website. Disease-related symptoms and concerns were described and provided by patients through free-text comments. Information from both the MM expert clinician and MM patient surveys including free-text items was analyzed and 14 highest ranked items were selected for the FACT-MM. The FACT-MM subscale (score 0–56), the FACT-G physical and functional well-being subscales (score 0–28), and the FACT-Neurotoxicity (FACT-Ntx) subscale (score 0–44) was administered to 48 E1A05 participants to assess disease and treatment-related symptoms of patients with newly diagnosed MM receiving 8 cycles of VRd versus Vd. Methods: In this study, the first three of seven sequential assessments were evaluated: baseline, cycle 5 and end of treatment (after cycle 8 or early diconstinuation). Instruments were scored as per the FACT scoring guidelines. Descriptive statistics were provided for each timepoint and changes in scores from baseline. The Wilcoxon rank sum test was used to assess differences in scores by ISS stage and ECOG PS. Pearson correlation coefficients were obtained between the scores. Cronbach's alpha was used to evaluate internal consistency of the FACT-MM. Results: At baseline and end of treatment, 46 and 41 patients, respectively, completed assessments. The FACT-MM demonstrated good to very good internal consistency (Cronbach alpha 0.79 – 0.89). The FACT-MM subscale was significantly correlated with the FACT physical and functional well-being subscales at baseline (r = 0.72), cycle 5 (r = 0.64; r = 0.49) and end of treatment (r = 0.72; r = 0.66). All scores declined modestly by cycle 5 and end of treatment from baseline. While sample size was limited, there appeared association with ECOG PS with higher scores for patients with PS 0 status. Conclusions: The 14-item FACT-MM scale is feasible for use to measure myeloma-related symptoms and has demonstrated acceptable psychometric properties based on findings from E1A05, an ECOG myeloma trial. Disclosures: Cella: Novartis: Research Funding. Fonseca:Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

Description: Abstract 5071 Background: Multiple myeloma (MM) is de second most common haematological malignancy in adults worldwide. MM is common in the elderly. While the general life expectancy is increasing an ever increasing number of patients has to be expected. In the last decade treatment strategies are changed continuously because of the introduction of novel agents and allogeneic and autologous stem cell transplantation. In randomized controlled trials, overall survival in patients up to 65 years is improving convincing. In contrast with results in older patients. We studied data of patients treated outside clinical trials derived from the population based registry in the Netherlands to recognize trends in incidence en survival during 1989–2009. Patients and methods: We included all patients with newly diagnosed MM in the period 1989 – 2009 who were recorded in the Netherlands Cancer Registry (n =16.822 ). Follow-up was until January, 2010. We calculated the yearly age-standardised incidence rates for males and females and age-specific incidence rates in 10 year age groups for both sexes separately and combined. Changes in incidence were evaluated by calculating the estimated annual percentage change (EAPC). We then calculated relative survival, which may be interpreted as disease-specific survival within a cancer patient population. Traditional cohort-based, relative survival analysis was applied for patients diagnosed during 1989–2009. Since follow-up was available until 2010, 5 year relative survival for patients diagnosed in 2004–2009 was estimated using period-based relative survival analysis. Results: The number of newly diagnosed MM cases rose between 1989 and 2009 from 631 to 968 cases respectively. Significantly more males were diagnosed than females over time (p=0.01). Furthermore the proportion of male patients increased slightly over these time periods. However, the overall age standardised incidence rate for males and females remained stable over time but was higher among males than females. The median age at diagnosis was 71 years (p10-P90 range 53–84) and stable over time (p=0.07). Incidence was highest in the 70–79 age group for both sexes. However, because of the aging population the age-specific incidence rates (ASIR) were highest for patients aged 80+ years for both sexes. Within specific age groups significant changes were seen. In the population 50–59 years, the ASIR increased from 5.0 per 100,000 in 1989 to 6.9 in 2009 (EAPC 1989–2009 = + 0.7%; 95% CI: 0.0 –1.3). A decrease was seen in females aged 80+ years from 25.1 per 100,000 in 1989 to 22.4 in 2009 (EAPC 1989–2009 = −1,0; 95% CI: −1.8; −0.2). In the overall patient population the 1-year relative survival increased only slightly from 72% to 77% between 1989 and 2009. 5-year relative survival increased from 28% to 37%. Small improvements in survival were observed for all age groups in the past two decades except for patients aged 80+ years. Relative survival decreased with increasing age. In contrast, in the group aged 40–64 years improvements are already detectable from 1994 on. In 2004–2009 the highest 5-year relative survival, 62%, was seen in patients 40–49 year of age. However the strongest improvement over time was observed among the group 50–59 years. Conclusion: Although the average annual age-adjusted incidence rate remained stable from 1989–2009, the number of newly diagnosed MM patients increased because of the aging population. Relative survival increased slowly but continuously in time for patients until 80 years of age with strongest increase seen in patients up to 64 years of age. Improvement for these younger patients is most likely caused by the introduction of novel agents based regiments as well as by the introduction of high dose chemotherapy followed by stem cell transplantation. Our findings in trends of incidence and survival of MM are similar to those reported in other western populations. Disclosures: Sonneveld: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 475FN2 Background: An IMiDs® immunomodulatory agent, Len has a dual mechanism of action: its tumoricidal effect directly leads to tumor cell death, and its immunomodulatory effect may keep the tumor in remission. A phase 3, randomized, placebo (Pbo)-controlled trial, MM-015 compares MPR-R with fixed-duration MPR and MP induction in transplant-ineligible NDMM pts. Interim results showed unprecedented reduction in disease progression risk with MPR-R (Palumbo et al, IMW 2011); this analysis focuses on pts aged 65–75 yrs in whom the greatest benefit was observed. Methods: A total of 459 pts aged ≥ 65 yrs with NDMM were enrolled. Induction consisted of nine 28-day cycles of melphalan 0.18 mg/kg (D1-4), prednisone 2 mg/kg (D1-4), and Len 10 mg (D1-21) (MPR-R and MPR) or melphalan and prednisone with Pbo (MP). After induction, MPR-R pts received Len 10 mg (D1-21) maintenance until progression; MPR and MP pts received Pbo. Pts with progressive disease (PD) could enroll in an open-label extension phase to receive Len 25 mg (D1-21) ± dexamethasone 40 mg (D1-4, 9–12, and 17–20). This analysis includes data up to Feb 28, 2011 (median follow-up, 30 mos). Results: There were 116/152 (76%), 116/153 (76%), and 116/154 (75%) of MPR-R, MPR, and MP pts, respectively, aged 65–75 yrs. Nearly 50% had ISS stage III disease, 〉 40% had β2-microglobulin 〉 5.5 mg/L, and 40% had CrCL 〈 60 mL/min. With a median follow-up of 30 mos, MPR-R reduced progression risk by 70% and significantly prolonged median PFS (31 mos) vs MP (12 mos; hazard ratio [HR]: 0.30 [95% CI, 0.20–0.45]; P

Description: POEMS syndrome is a rare clonal plasma cell disorder without standard treatment. Based on the efficacy and low toxicity of a combination of melphalan and dexamethasone (MDex) for light chain amyloidosis, we conducted a prospective study of MDex treatment for patients with newly diagnosed POEMS syndrome. Thirty-one patients (19 men) were enrolled and the median age at the time of diagnosis was 44 years (range, 32-68 years). All patients received 12 cycles of MDex treatment. Twenty-five patients (80.6%) achieved hematologic response including 12 (38.7%) complete remission and 13 (41.9%) partial remission. Of all 31 patients, the neurologic response rate was 100%, assessed by overall neuropathy limitation scale (ONLS). The initial neurologic response was observed in 24 patients (77.4%) at 3 months after treatment and the median time to maximal neurologic response was 12 months (range, 3-15 months). Moreover, MDex substantially improved the level of serum vascular endothelial growth factor and relieved organomegaly, extravascular volume overload, and pulmonary hypertension. Only 6 patients (19.3%) suffered from grade 3 adverse events during treatment. All patients are alive and free of neurologic relapse after the median follow-up time of 21 months. Therefore, MDex is an effective and well-tolerated treatment option for patients with newly diagnosed POEMS syndrome.

Description: Multiple mechanisms operate to ensure T-cell tolerance toward self-antigens. Three main processes have been described: clonal deletion, anergy, and deviation to CD4+ regulatory T cells (Tregs) that suppress autoreactive T cells that have escaped the first 2 mechanisms. Although it is accepted that dendritic cells (DCs) and B cells contribute in maintaining T-cell tolerance to self-antigens, their relative contribution and the processes involved under physiologic conditions remain only partially characterized. In this study, we used different transgenic mouse models to obtain chimeras where a neo self-antigen is expressed by thymic epithelium and/or by DCs or B cells. We found that expression of cognate ligand in the thymus enhances antigen-specific FoxP3+ cells independently of whether the self-antigen is expressed on thymic epithelium or only on DCs, but not on B cells. On the contrary, self-antigen expression by B cells was very efficient in inducing FoxP3+ cells in the periphery, whereas self-antigen expression by DC led mainly to deletion and anergy of antigen-specific FoxP3− cells. The results presented in this study underline the role of B cells in Treg induction and may have important implications in clinical protocols aimed at the peripheral expansion of Tregs in patients.

Description: Treatment-related mortality (TRM) is important in acute lymphoblastic leukemia and acute myeloid leukemia (AML); however, little is known about how TRM is defined across trials. Two major problems are related to what constitutes treatment versus disease-related cause of death and to TRM attribution (for example, death because of infection or hemorrhage). To address the former, we conducted a systematic review of randomized therapeutic pediatric acute leukemia and adult/pediatric acute promyelocytic leukemia trials and any study type focused on TRM in pediatric acute leukemia. We described definitions used for TRM. Sixty-six studies were included. Few therapeutic pediatric acute lymphoblastic leukemia studies (2/32, 6.3%) provided definitions for TRM, whereas more therapeutic pediatric AML studies (6/9, 66.7%) provided definitions. There was great heterogeneity in TRM classification. The authors of most studies relied on deaths during induction or in remission to delineate whether a death was TRM. However, 44.4% of therapeutic AML studies used death within a specific time frame to delineate TRM. We suggest that a consistent approach to defining and determining attribution for TRM in acute leukemia is an important future goal. Harmonization of definitions across the age spectrum would allow comparisons between pediatric and adult studies.

Description: Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginaseto resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

Description: Shwachman-Diamond syndrome (SDS) results from mutations in the SBDS gene, characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. Neutropenia and neutrophil dysfunction are hallmark features of SDS; however, causes for the bone defects are unknown. Dysfunction of bone-resorbing osteoclasts, formed by the fusion of monocytic progenitors derived from the same granulocytic precursors as neutrophils, could be responsible. We report that Sbds is required for in vitro and in vivo osteoclastogenesis (OCG). Sbds-null murine monocytes formed osteoclasts of reduced number and size because of impaired migration and fusion required for OCG. Phenotypically, Sbds-null mice exhibited low-turnover osteoporosis consistent with findings in SDS patients. Western blotting of Rho GTPases that control actin dynamics and migration showed a 5-fold decrease in Rac2, whereas Rac1, Cdc42, and RhoA were unchanged or only mildly reduced. Although migration was rescued on Rac2 supplementation, OCG was not. This was attributed to impaired signaling downstream of receptor activator of nuclear factor-κB (RANK) and reduced expression of the RANK-ligand-dependent fusion receptor DC-STAMP. We conclude that Sbds is required for OCG by regulating monocyte migration via Rac2 and osteoclast differentiation signaling downstream of RANK. Impaired osteoclast formation could disrupt bone homeostasis, resulting in skeletal abnormalities seen in SDS patients.

Description: Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fIIWE), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)–dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fIIWE mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fIIWE/WE mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fIIWT/WE mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl3 vessel injury. More interestingly, prothrombinWE expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombinWE also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

Description: Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TMPro mice expressing a mutant form of TM with reduced thrombin affinity and TMLeD mice lacking the N-terminal lectin-like domain. Studies of TMPro mice revealed that TM is a powerful determinant of hematogenous metastasis. TMPro mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TMPro mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

Description: Whether long-term use of vitamin K antagonists (VKAs) might affect the incidence of cancer is a longstanding hypothesis. We conducted a population-based study including all cancer- and thromboembolism-free patients of our health area; study groups were defined according to chronic anticoagulant use to VKA-exposed and control groups. Cancer incidence and cancer-related and overall mortality was assessed in both groups. 76 008 patients (3231 VKA-exposed and 72 777 control subjects) were followed-up for 8.2 (± 3.2) years. After adjusting for age, sex, and time-to-event, the hazard ratio of newly diagnosed cancer in the exposed group was 0.88 (95% confidence interval [95% CI] 0.80-0.98; P 〈 .015). VKA-exposed patients were less likely to develop prostate cancer, 0.69 (95% CI 0.50-0.97; P = .008). The adjusted hazard ratio for cancer-related and overall mortality was 1.07 (95% CI 0.92-1.24) and 1.12 (95% CI 1.05-1.19), respectively. These results support the hypothesis that anticoagulation might have a protective effect on cancer development, especially prostate cancer.

Description: Patients referred to tertiary care centers occasionally may have their diagnostic procedures repeated and have a final diagnosis that differs from that of the referring center. The aim of this study was to evaluate discordance rates and their clinical implications in the diagnosis of patients with myelodysplastic syndrome (MDS) referred to a tertiary center. We analyzed 915 patients with MDS who were referred to M. D. Anderson Cancer Center between September 2005 and December 2009. Discordance in the diagnosis was documented in 109 (12%) patients, with a majority reclassified as having higher-risk disease by French-American-British (67%) or by International Prognostic Scoring System (77%) with implications for therapy selection and prognosis calculation. These results demonstrate the complexity of the diagnosis of MDS and highlight the need for confirmation of diagnosis when in doubt.

Description: Abstract 2283 Introduction: von Willebrand factor (VWF) acts as bridging molecule between platelets and vessel wall or as a career for plasma factor VIII (FVIII). Earlier reports using flow chamber had revealed that VWF acts as an “initiator” through its interactionwith glycoprotein (GP) Ib, whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a “stabilizer” against high shear. VWD is categorized as partial or complete quantitative defect (type 1 or 3) or qualitative defect (type 2) of VWF based on laboratory tests, such as VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and FVIII activity (FVIII:C). However, the diagnosis of VWD remains difficult because the clinical and laboratory phenotype has wide-spectrum. An assay reflecting clinical phenotypes of VWD and effect of the treatment would be helpful for clinicians. Methods: We evaluated a thrombus formation with a new applicative microchip flow chamber instrument, total-thrombus-formation analysis system (T-TAS®). Citrated or hirudin-added blood from healthy volunteers (n=5) or 3 patients with VWD [symptomatic type 1 (S-1), asymptomatic type 1 (AS-1), and symptomatic type 3 (S-3)] were utilized. Normal blood was mixed with inhibitor for GP Ib (OS-1), GP IIb-IIIa (abciximab) and monoclonal antibody against for VWF (mAb-VWF). Patients' blood was infused with FVIII/VWF concentrates in (ex) vivo. Re-calcified citrated blood (450 μl) added corn trypsin inhibitor (30 μg/ml) was infused to AR chip of T-TAS at constant flow rate (240–600 s−1), which surface was coated by collagen and tissue factor. Hirudin-added blood (350 μl) was infused to PL chip of T-TAS at higher shear (1,000–2,000 s−1) which surface was coated by collagen. Flow pressure curve was plotted and time to 10 kPa (T10) was evaluated. Furthermore, flow images were recorded with a micro camera. AR chip promoted white thrombus formation (WTF) which reflected both platelet aggregation and fibrin generation, whilst PL chip did only platelet thrombus formation (PTF). Rotational thromboerastometry (ROTEM) were simultaneously investigated. Results: Both abciximab (0.5–2.0 μg/ml) and OS-1 (100–400 nM) inhibited WTF in AR chip dose-dependently. OS-1 (200 nM) inhibited WTF partially at 240 s−1 (T10 15.4±1.6 min, control 8.9±0.4 min), but completely at 600 s−1 (T10 〉30 min, control 7.5±0.2 min). In PL chip, both agents inhibited PTF at much lower dose than those in AR chip. ROTEM parameters showed little change in both agents. Using mAb-VWF, similar results to OS-1 were obtained. These data showed that GP Ib-VWF interaction was shear-dependent, consistent with earlier reports. In in (ex) vivo assays, S-1 and AS-1 had comparable VWF levels (VWF:Ag/VWF:RCo 20%/6.4% and 5.8%/3.2%, respectively) in spite of clear difference of clinical phenotype. ROTEM parameters of S-1 were rather better than those of AS-1 likely reflecting FVIII:C (60% and 7.2%). Interestingly, however, PTF in PL chip (at 1,000 s−1) showed significant delay of T10 in S-1 (9.0 min, control 4.1±0.5 min) compared to AS-1 (5.2 min) and after in vivo infusion of FVIII/VWF concentrates, T10 in S-1 was improved (5.0 min). In S-3, ROTEM parameters declined reflecting low FVIII:C (1.2%), and prolonged T10 (〉30 min) in AR chip likely reflected complete defect of VWF:Ag (

Description: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Description: Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, β-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis.

Description: Several studies have found that high levels of reactive oxidative species (ROS) are associated with stem cell dysfunction. In the present study, we investigated the role of nuclear factor erythroid-2–related factor 2 (Nrf2), a master regulator of the antioxidant response, and found that it is required for hematopoietic stem progenitor cell (HSPC) survival and myeloid development. Although the loss of Nrf2 leads to increased ROS in most tissues, basal ROS levels in Nrf2-deficient (Nrf2−/−) BM were not elevated compared with wild-type. Nrf2−/− HSPCs, however, had increased rates of spontaneous apoptosis and showed decreased survival when exposed to oxidative stress. Nrf2−/− BM demonstrated defective stem cell function, as evidenced by reduced chimerism after transplantation that was not rescued by treatment with the antioxidant N-acetyl cysteine. Gene-expression profiling revealed that the levels of prosurvival cytokines were reduced in Nrf2−/− HSPCs. Treatment with the cytokine G-CSF improved HSPC survival after exposure to oxidative stress and rescued the transplantation defect in Nrf2−/− cells despite increases in ROS induced by cytokine signaling. These findings demonstrate a critical role for Nrf2 in hematopoiesis and stem cell survival that is independent of ROS levels.

Description: Reactive oxygen species (ROS) are highly destructive toward cellular macromolecules. However, moderate levels of ROS can contribute to normal cellular processes including signaling. Herein we evaluate the consequence of a pro-oxidant environment on hematopoietic homeostasis. The NF-E2 related factor 2 (Nrf2) transcription factor regulates genes related to ROS scavenging and detoxification. Nrf2 responds to altered cellular redox status, such as occurs with loss of antioxidant selenoproteins after deletion of the selenocysteine-tRNA gene (Trsp). Conditional knockout of the Trsp gene using Mx1-inducible Cre-recombinase leads to selenoprotein deficiency and anemia on a wild-type background, whereas Trsp:Nrf2 double deficiency dramatically exacerbates the anemia and increases intracellular hydrogen peroxide levels in erythroblasts. Results indicate that Nrf2 compensates for defective ROS scavenging when selenoproteins are lost from erythroid cells. We also observed thymus atrophy in single Trsp-conditional knockout mice, suggesting a requirement for selenoprotein function in T-cell differentiation within the thymus. Surprisingly, no changes were observed in the myelomonocytic or megakaryocytic populations. Therefore, our results show that selenoprotein activity and the Nrf2 gene battery are particularly important for oxidative homeostasis in erythrocytes and for the prevention of hemolytic anemia.

Description: Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained “secondary” gene-corrected βs/βA heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Description: The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the Aγ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

Description: Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer–disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the “barbell shapes” of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure.

Description: Myeloid-derived suppressor cells (MDSCs) inhibit adaptive and innate immunity and accumulate in the blood of persons with cancer, chronic inflammation, trauma, infection, and stress. Some of the factors inducing their accumulation are known; however, mechanisms regulating their turnover have not been identified. Mass spectrometry showed prominent expression of apoptosis pathway proteins, suggesting that MDSC turnover may be regulated by Fas-FasL–mediated apoptosis. This hypothesis was confirmed by showing that blood MDSCs induced by 3 mouse tumors were Fas+ and apoptosed in response to Fas agonist in vitro and to activated FasL+ T cells in vivo. FasL-deficient mice contained significantly more blood MDSCs than FasL+/+ mice, and after removal of primary tumors MDSCs regressed in STAT6−/− and CD1−/− mice but not in STAT6−/−FasL−/− or CD1−/−FasL−/− mice. Fas+ macrophages and dendritic cells did not apoptose in response to activated T cells, indicating that Fas-FasL regulation of myeloid cells was restricted to MDSCs. These results identify a new mechanism regulating MDSC levels in vivo and show a retaliatory relationship between T cells and MDSCs in that MDSCs suppress T-cell activation; however, once activated, T cells mediate MDSC apoptosis.

Description: Abstract 41 Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related death with a majority of the reported cases secondary to the infusion of antibodies (Abs) contained within the plasma/blood component. An experimental filter that removes IgG was developed. We hypothesize that filtration of plasma with antibodies to leukocyte antigens will decrease both antibody-mediated priming of PMNs and antibody-mediated TRALI in a two-event in vivo model. Methods: Human plasma was drawn from healthy volunteers and IgG concentrations were measured before and after filtration. Plasma was obtained from two multiparous female donors: one with antibodies to HLA-A2 and to DR7 and the other with antibodies against HNA-3a. These plasma samples were filtered (F-Plas) or left as an unmodified control (Plas) and the anti-leukocyte antibodies were measured in a blinded fashion in referral labs using flow cytometry and Luminex™ beads or standard granulocyte antibody detection assays. These plasma samples were then used to prime the fMLP-activated respiratory burst, measured as the SOD-inhibitable reduction of cytochrome c (nmol O2−/min), of PMNs from HNA-3a+ donors or donors homozygous donors for HLA-A2, respectively. For the two-event in vivo modeling rats were incubated with 2 μg/ml endotoxin (LPS, S. enteritides) or saline (NS) for 2 hours (first event) and then were transfused with heat-treated human plasma that contained 25 μg/ml of an antibody against the MHC class I antigen OX27 that was either filtered (or left unmodified) prior to infusion (second event) followed by Evans Blue dye (EBD). ALI was measured as %EBD leak from the plasma into the bronchoalveolar lavage fluid. Statistical differences were measured via paired (PMN priming) or independent (in vivo TRALI) ANOVA, and data are reported as the mean ± the standard error of the mean. *=p

Description: Clinical trials have demonstrated that rituximab improves overall survival in non-Hodgkin lymphoma (NHL), except in mantle cell lymphoma (MCL). We used Surveillance Epidemiology and End Results (SEER)–Medicare data to compare survival in older MCL patients who began chemotherapy with or without rituximab within 180 days of diagnosis. Patients were followed from diagnosis (January 1999 to December 2005) until death or the end of observation (December 2007). Medicare administrative and claims data were used to identify the date and cause of death and the immunochemotherapy regimen. Of 638 patients, the mean age at diagnosis was 75 years, 75% had stage III/IV disease, 67% had extranodal involvement, and 64% received rituximab. The average length of first-line treatment was 21 weeks, with no difference between the 2 groups (P = .76). Median survival was 27 months for chemotherapy alone, compared with 37 months for chemotherapy plus rituximab (P 〈 .001). In multivariate analysis of 2-year survival, rituximab plus chemotherapy was associated with lower all-cause (hazard ratio [HR] 0.58; 95% confidence interval [CI] 0.41-0.82; P 〈 .01), and cancer-specific (HR 0.56; 95% CI 0.37-0.84; P 〈 .01) mortality. Results were similar when using the entire observation period, propensity score analysis, and limiting chemotherapy to CHOP/CHOP-like. We conclude that first-line chemotherapy including rituximab is associated with significantly improved survival in older patients diagnosed with MCL.

Description: Abstract 4127 In the age of novel targeted agents, autologous stem cell transplant (ASCT) remains the standard of care for younger patients with newly diagnosed multiple myeloma (MM), offering similar treatment responses and overall survivals as standard chemotherapeutic agents but with the added benefit of a prolonged treatment-free period. Nevertheless, a standard of care for stem cell mobilization for ASCT has yet to be determined. Even in the era of new mobilization agents such as Plerixafor, Cyclophosphamide (Cy) and G-CSF combination remains the preferred mobilizing approach for patients with MM. Several studies have shown that Cy improves the stem cell yield at the expense of increased toxicity, but whether the administration of this chemotherapeutic agent pre-transplant has any impact on the long-term event-free and/or overall survival of myeloma patients remains controversial. In this study, we present a retrospective analysis of 186 patients with newly diagnosed MM who underwent ASCT with high-dose melphalan 200 mg/m2 (HDM) between December of 2000 and 2008 at our Institution. Eighty-three patients were mobilized with single agent G-CSF and 103 patients received high dose Cy (4 gm/m2) and G-CSF combination. Patient characteristics were similar between the treatment groups, including: age, gender, disease stage, and disease status prior to transplant. However, toxicity post-mobilization with Cy/G-CSF was significantly higher compared with G-SCF alone, including: febrile neutropenia (23%), hemorrhagic cystitis (8%), GI toxicity (57%), re-hospitalization due to complications and transplant delay (14%). The overall post-transplant toxicity was similar in the 2 groups, though the treatment related mortality was slightly higher in the Cy/G-CSF arm (4% versus 2%). Post transplant responses were not significantly different in the 2 groups, with 60% of patients achieving a VGPR or better after ASCT in the G-CSF group and 49% in the Cy/G-CSF group (p = 0.33). The median event-free survivals (EFS) for the Cy/G-CSF and G-CSF cohorts were 21.6 and 22.6 months, respectively, (p = 0.62) yielding no significant difference (Figure 1). Similarly, with a median follow up for surviving patients of 34.3 and 32.7 months, the median overall survivals were 68.2 and 62.3 months (p = 0.23) for the Cy/G-CSF and G-CSF cohorts, respectively (Figure 2). This retrospective analysis confirms that the addition of high dose Cy as part of the mobilizing regimen offers no improvement on the transplant outcome for patients with newly diagnosed myeloma and should therefore only be used in cases of difficult stem cell mobilization. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1753 Background: Ruxolitinib (INC424), a potent and selective oral JAK1 and JAK2 inhibitor, has demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms, role functioning, and quality of life (QoL) in 2 phase 3 studies in patients with myelofibrosis (MF) (the COMFORT studies). These studies compared ruxolitinib with either placebo or best available therapy (BAT). This analysis compares the efficacy outcomes between the placebo arm from COMFORT-I and the BAT arm from COMFORT-II. Methods: COMFORT-I is a randomized (1:1), double-blind, multicenter study comparing ruxolitinib 15 or 20 mg twice daily (bid) with placebo, and COMFORT-II is a randomized (2:1), open-label, multicenter study comparing ruxolitinib 15 or 20 mg bid with BAT (investigator-selected therapy, including no treatment). Both studies met their primary end points with statistical significance (ruxolitinib vs control): the percentage of patients achieving ≥35% reduction in spleen volume at week 24 (COMFORT-I, P

Description: Abstract 1768 B-cell chronic lymphocytic leukemia (CLL) is characterized by deregulated expression of microRNAs (miRNAs). These post-transcriptional regulators of gene expression play a crucial role in controlling multiple cellular processes. By microarray analysis and quantitative RT-PCR we observed significantly lower levels of miR-126, miR-130a, miR-143, miR-181a and miR-326 in primary CLL cells compared to normal B cells. Transfection of synthetic miR-130a or miR-143 induced a significant reduction in cell viability of both primary CLL cells and the CLL cell line MEC-1. As autophagy is connected to cancer cell survival and resistance to apoptosis, we investigated the effect of these two miRNAs on autophagy by following the specific autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). Therefore, we generated MEC-1 cells stably expressing GFP-tagged LC3 and analyzed autophagosome formation by using an imaging flow cytometer quantifying GFP-positive dots. These experiments revealed that autophagy is induced in these cells upon starvation, and that introduction of miR-130a, but not miR-143, resulted in a reduction of autophagosome formation (see Figure). These findings were verified by LC3 Western blot analysis, and extended to primary CLL cells, showing for the first time that autophagy is an active process in these cells and that miR-130a inhibits autophagy in primary CLL cells as well. To further elucidate the molecular mechanism of miR-130a-mediated CLL cell survival and autophagy, we aimed at identifying putative target genes of this miRNA and identified ATG2B, an autophagy-related gene, as well as DICER1 and AGO4, two components of the miRNA processing machinery, as direct target genes of miR-130a in CLL cells. The relevance and role of these three novel target genes in miR-130a-regulated cell death/cell survival programs is under current investigation. Figure: Analysis of autophagy using MEC-1 cell line stably expressing GFP-tagged LC3 protein. Green dots representing autophagosomes were quantified in MEC-1/GFP-LC3 cells under starvation by imaging flow cytometry (Image Stream, Amnis). Transfection with synthetic miR-130a reduced the autophagic flux in these cells compared to scrambled negative control miRNA (NC). Figure:. Analysis of autophagy using MEC-1 cell line stably expressing GFP-tagged LC3 protein. Green dots representing autophagosomes were quantified in MEC-1/GFP-LC3 cells under starvation by imaging flow cytometry (Image Stream, Amnis). Transfection with synthetic miR-130a reduced the autophagic flux in these cells compared to scrambled negative control miRNA (NC). Disclosures: No relevant conflicts of interest to declare.

Description: The Blood and Marrow Transplant Clinical Trials Network conducted 2 parallel multicenter phase 2 trials for individuals with leukemia or lymphoma and no suitable related donor. Reduced intensity conditioning (RIC) was used with either unrelated double umbilical cord blood (dUCB) or HLA-haploidentical related donor bone marrow (Haplo-marrow) transplantation. For both trials, the transplantation conditioning regimen incorporated cyclophosphamide, fludarabine, and 200 cGy of total body irradiation. The 1-year probabilities of overall and progression-free survival were 54% and 46%, respectively, after dUCB transplantation (n = 50) and 62% and 48%, respectively, after Haplo-marrow transplantation (n = 50). The day +56 cumulative incidence of neutrophil recovery was 94% after dUCB and 96% after Haplo-marrow transplantation. The 100-day cumulative incidence of grade II-IV acute GVHD was 40% after dUCB and 32% after Haplo-marrow transplantation. The 1-year cumulative incidences of nonrelapse mortality and relapse after dUCB transplantation were 24% and 31%, respectively, with corresponding results of 7% and 45%, respectively, after Haplo-marrow transplantation. These multicenter studies confirm the utility of dUCB and Haplo-marrow as alternative donor sources and set the stage for a multicenter randomized clinical trial to assess the relative efficacy of these 2 strategies. The trials are registered at www.clinicaltrials.gov under NCT00864227 (BMT CTN 0604) and NCT00849147 (BMT CTN 0603).

Description: Various combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently, a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150+CD48−) and non-SLAM (not CD150+CD48−) cells from human umbilical cord blood CD34+ cells as well as from human and rhesus macaque mobilized peripheral blood CD34+ cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptornull, NSG) mice. We found that the CD34+ SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34+ non-SLAM population. Thus, SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice.

Description: T-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell–derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell–derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.

Description: Abstract 2542 Background: Patients with high-risk chronic lymphocytic leukemia (CLL) uniformly relapse after conventional chemo-immunotherapy, but roughly half achieve long-term disease-free survival (DFS) following allogeneic hematopoietic cell transplantation (allo-HCT). Quantification of minimal residual disease (MRD) following allo-HCT predicts post-transplant relapse (PTR) when CLL disease burden remains greater than 10e-4 (ie, 1 leukemic cell in 10,000 peripheral blood mononuclear cells [PBMC]) when quantified by allele-specific oligonucleotide quantitative polymerase chain reaction (ASO-PCR) or flow cytometry. We previously demonstrated the feasibility of MRD quantification using consensus primers to amplify all immunoglobulin heavy chain (IGH) genes in a mixture of PBMC, followed by high-throughput sequencing (HTS) and clonotypic quantification. In our prior work, we used 454 pyrosequencing technology which enabled 10e-5 sensitivity. Here, we report 10e-6 MRD sensitivity using novel Illumina-based HTS that provides better prediction of disease recurrence than ASO-PCR. Methods: We amplified IGH loci from genomic DNA extracted from PBMC (median input 2.4×10e6 cells; range 1.1–23.7×10e6) using V and J segment consensus primers. Amplified IGH molecules were then sequenced with one million or more dedicated reads using Illumina HiSeq and clones were quantified using Sequenta HTS bioinformatics. To verify 10e-6 sensitivity using this system, a clonal B cell population was diluted to 10e-6 in PBMC from a healthy donor with successful clonal detection. Disease-bearing samples (either pre-treatment or after PTR) were sequenced to verify applicability of consensus primers for each patient and to determine each patient's unique clonal IGH sequence. Thirty-seven PBMC samples from 14 patients which were either negative (n=30) by ASO-PCR or detectable below the linear limit of detection (n=7) were subjected to MRD quantification by HTS. The integrity of all samples was determined by preliminary IGH quantitative PCR. Results: CLL-specific IGH clonotypes from all 14 patients amplified successfully from samples with known disease burden, confirming the acceptability of consensus primers for all patients. Concordant MRD negativity by ASO-PCR and IGH-HTS was only observed in 14/37 samples (38%), while 16 samples (43%) were negative by ASO-PCR but detectable at the 10e-6 level using IGH-HTS (range 0.1–11 CLL IGH sequences per 10e6 PBMC genomes). Two of 37 samples (5%) exhibited concordant low-level positivity in the 10e-5 range. 4/37 samples (11%) were concordantly positive, but quantified more than or equal to 0.5log higher with ASO-PCR than HTS. One sample was positive below the linear limit of detection by ASO-PCR but negative by HTS. With median clinical follow-up of 1072 days (range 522–1986 days), one of 7 patients (14%) who exhibited MRD negativity by both ASO-PCR and IGH-HTS relapsed. All 5 patients found to have MRD negativity by ASO-PCR with concurrent MRD positivity using IGH-HTS relapsed. The association between IGH-HTS negativity and long-term DFS was highly significant (p=0.005), whereas ASO-PCR negativity was not significantly associated with DFS (p=0.47). In patients found to be MRD negative by ASO-PCR but positive by IGH-HTS, the HTS result predicted clinical relapse by a median 321 days (range 38–644 days). Conclusions: Massively parallel immunoglobulin gene sequencing using Illumina HiSeq provides a heretofore unachievable level of MRD sensitivity in peripheral blood samples from patients with CLL. Samples found to be negative or below the linear limits of detection for CLL MRD using ASO-PCR were more accurately quantified using IGH-HTS. Quantification of CLL MRD using IGH-HTS has tremendous prognostic value since achievement of MRD negativity with 10e-6 sensitivity is highly associated with long-term DFS. To further validate the performance of Illumina-based HTS, we are currently sequencing 289 archived post-transplant PBMC from 42 CLL patients. This scalable and cost-effective platform for ultra-sensitive MRD quantification using consensus primers will broadly expand the availability and utility of post-transplant MRD assessment. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Zheng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.

Description: The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

Description: Although sickle cell disease (SCD) has a variable clinical course, many patients develop end-organ complications that are associated with significant morbidity and early mortality. Myeloablative allogeneic HSCT (allo-HSCT) is curative but has been historically performed only in children younger than 16 years of age. Modest modifications in the conditioning regimen and supportive care have improved outcome such that the majority of children with a suitable HLA-matched sibling donor can expect a cure from this approach. However, adult patients have been excluded from myeloablative allo-HSCT because of anticipated excess toxicity resulting from accumulated disease burden. Efforts to use nonmyeloablative transplantation strategies in adults logically followed but were initially met with largely disappointing results. Recent results, however, indicate that nonmyeloablative allo-HSCT in adult patients with SCD allows for stable mixed hematopoietic chimerism with associated full-donor erythroid engraftment and normalization of blood counts, and persistence in some without continued immunosuppression suggests immunologic tolerance. The attainment of tolerance should allow extension of these potentially curative approaches to alternative donor sources. Efforts to build on these experiences should increase the use of allo-HSCT in patients with SCD while minimizing morbidity and mortality.

Description: Abstract 2708 Background: The simplified Mantle Cell Lymphoma International Prognostic Index (MIPI) has been shown to be a good predictor of patient survival (Blood 2008;111:558–65; Blood 2010;115:1530–1533). This post hoc study analyzed data from a randomized, phase III clinical trial investigating temsirolimus (TEM) in relapsed/refractory mantle cell lymphoma (MCL) in which TEM 175/75 (175 mg for first 3 weeks then 75 mg weekly) demonstrated significantly longer progression-free survival (PFS) vs investigator's choice of therapy (INV; 4.8 vs 1.9 months, respectively; hazard ratio [HR]=0.44; P=.0009; J Clin Oncol 2009;27:3822–9). Patients receiving TEM 175/25 (175 mg for first 3 weeks then 25 mg weekly) also had longer PFS vs INV, but this difference was not significant (3.4 vs 1.9 months; respectively; HR=0.65; P=.06). During the trial, baseline prognostic risk classification was not recorded; thus, patients were retrospectively assigned baseline prognostic scores, and outcomes were analyzed according to risk category. Methods: All patients (N=162) were classified as low, intermediate, or high risk using the simplified MIPI. The MIPI scores were based on 4 independent prognostic markers: age, Eastern Cooperative Oncology Group (ECOG) performance status, lactate dehydrogenase level, and white blood cell count. Median PFS and overall survival (OS) were calculated using Kaplan-Meier estimates, and treatment effect was assessed using log-rank statistics. P values of ≤.05 indicated significance of the treatment effect between the 2 treatment groups. As the phase III study was not powered to analyze patients according to MIPI risk categorization, statistical analyses shown are for explanatory purposes. Results: Distribution was relatively even across MIPI risk categories (55 patients low, 59 patients intermediate, 48 patients high). MIPI distributions in the 2 TEM arms were: 175/75 (n=54: 28% low, 43% intermediate, 30% high); 175/25 (n=54: 28% low, 33% intermediate, 39% high). Relative to the TEM arms, the INV arm (n=54) had a higher proportion of low-risk patients (46% low; 33% intermediate; 20% high). TEM 175/75 resulted in significant improvement in median PFS (independent assessment) vs INV in high-risk patients (P=.003) (Table); trends toward improvement were observed for intermediate-risk and low-risk patients (P=.06 in each group). By investigator assessment, TEM 175/75 improved median PFS vs INV by 7.9 months in the low-risk category (P=.0007) and by 2.8 months (P=.06) and 1.1 month (P=.001) in the intermediate-risk and high-risk categories, respectively. A trend toward longer OS was observed in the low-risk patients treated with TEM 175/75 vs INV (P=.0502). In the low-risk category, maintenance of stable disease or better response was achieved in more patients receiving TEM 175/75 (9/15 [60%]) vs INV (5/25 [20%]); objective responses were observed in 5 patients with TEM 175/75 and in no patients with INV. Patients in the low-risk category treated with TEM 175/75 received a longer duration of therapy vs INV (30.7 vs 9.0 wk, respectively). Across the duration of treatment, the average frequency of delay was once per 5.6 wk with TEM 175/75 vs once per 6.4 wk with INV. TEM was generally well tolerated. Grade 3/4 anemia, thrombocytopenia, and infection also were analyzed by patient risk category. In both the TEM 175/75 and INV groups, the selected grade 3/4 events occurred more commonly in high-risk than low-risk patients. In the low-risk category, a higher incidence of grade 3/4 thrombocytopenia and anemia was observed with TEM 175/75 vs INV. Conclusions: Retrospective risk analysis of patients according to the simplified MIPI demonstrated that TEM 175/75 was effective across patient risk categories. The greatest benefit trend was observed in low-risk patients. In this study of relapsed/refractory MCL patients, MIPI was a good predictor of survival outcome. Disclosures: Hess: Pfizer: Consultancy, Honoraria, Research Funding. Off Label Use: Torisel is licensed for treatment of relapsed and/or refractory mantle cell lymphoma and renal cell carcinoma in Europe. Torisel is licensed in the US for renal cell carcinoma. Kang:Pfizer: Employment. Moran:Pfizer: Employment.

Description: A long outstanding problem is the resolution of the full potential of hematopoietic precursors. The commonly used allotypic marker Ly5 permits the tracing of lymphoid and granulocyte-macrophage (GM) output. Here we present a novel eGFP allele that allows the quantitative analysis of red blood cell (RBC) origin at the single-cell level. The miR-144/451 locus is required for erythroid development and homeostasis. Taking advantage of the fact that miR-451 is specifically and highly expressed in the erythroid lineage, we inserted an eGFP expression cassette into the miR-144/451 locus. In miR-144/451+/eGFP animals, accumulation of eGFP is exclusively observed during terminal erythroid differentiation. Expression of miR-144/451eGFP ignites immediately before the CFU-E stage and results in strong and complete labeling of all mature RBCs in circulation. Using competitive reconstitution experiments in the Ly5 transplant model, we show that eGFP linearly correlates with Ly5 expression. Thus, the miR-144/451eGFP allele represents a novel tool for the resolution of erythroid potential.

Description: CD36 modulates platelet function via binding to oxidized LDL (oxLDL), cell-derived microparticles, and thrombospondin-1. We hypothesized that the level of platelet CD36 expression may be associated with inheritance of specific genetic polymorphisms and that this would determine platelet reactivity to oxLDL. Analysis of more than 500 subjects revealed that CD36 expression levels were consistent in individual donors over time but varied widely among donors (200-14 000 molecules per platelet). Platelet aggregometry and flow cytometry in a subset of subjects with various CD36 expression levels revealed a high level of correlation (r2 = 0.87) between platelet activation responses to oxLDL and level of CD36 expression. A genome-wide association study of 374 white subjects from the Cleveland Clinic ASCLOGEN study showed strong associations of single nucleotide polymorphisms in CD36 with platelet surface CD36 expression. Most of these findings were replicated in a smaller subset of 25 black subjects. An innovative gene-based genome-wide scan provided further evidence that single nucleotide polymorphisms in CD36 were strongly associated with CD36 expression. These studies show that CD36 expression on platelets varies widely, correlates with functional responses to oxLDL, and is associated with inheritance of specific CD36 genetic polymorphisms, and suggest that inheritance of specific CD36 polymorphisms could affect thrombotic risk.

Description: A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4+ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4+ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25+ Treg cells was similar to that achieved by in vivo depletion of all CD4+ T cells. Depletion of CD25+ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4+ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8+ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

Description: The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (βs mice). Enhanced microvascular thrombosis in βs mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from βs mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in βs mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in βs mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Description: Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.

Description: Abstract 794FN2 LBH589 is a novel pan-deacetylase inhibitor (DACi) that has demonstrated clinical activity in phase I/II studies in patients with a variety of hematologic malignancies. Our group has previously presented preliminary results of a phase I study of LBH589 in patients with myelofibrosis (MF) (Mascarenhas et al, ASH 2009, a308) while a phase II trial using higher doses of LBH589 has also been reported (DeAngelo et al, ASH 2010,a630). Both studies identified reversible thrombocytopenia as the DLT and reported evidence of clinical responses. The final results of our phase I study and the effects of extended treatment with LBH589 are reported here. We enrolled 18 patients at 3 dose levels. Fifty-five percent of these patients had PMF, 28% Post-PV MF and 17% Post ET MF; all were intermediate/high risk based on Lille classification. Twenty-five mg PO TIW was determined to be the recommended phase II dose. All patients experienced resolution of their systemic symptoms and 10/11 patients with baseline palpable splenomegaly, who were evaluable after 1 month of therapy, had a median reduction of 30%, range 0–100%. Five patients entered into an extension phase of the trial and received 〉 6 months of therapy with a mean dose of 20mg PO TIW at time of optimal response (Table 1). Of these patients, 2 were initially enrolled in the 20 mg PO TIW cohort, 1 in the 30 mg PO TIW cohort and 2 in the 25 mg PO TIW cohort. Both patients at the lowest dose achieved clinical improvement (CI) by IWG-MRT response criteria at 6 months as did one patient at the 25 mg dose. The remaining 2 patients had SD at 30 and 25 mg. A mean reduction in palpable splenomegaly at 3 and 6 months of 55% and 83%, respectively, was observed in this group. Two of these patients had marked and durable improvement in anemia (patients 1 and 4). Patient 4 achieved a near CR at 16 months with resolution of palpable splenomegaly, elimination of peripheral blood dacrocytes and leukoerythroblastosis, a 4g/dL increase in hemoglobin, improvement in overall marrow cellularity and megakaryocyte atypia with an increase in erythroid precursors and a significant reduction of reticulin/collagen fibrosis. Patient 1 was heavily transfusion dependent requiring RBC transfusions weekly to maintain a mean hemoglobin of 6.5g/dL and after 6 months on LBH589 achieved 〉50% reduction in transfusion dependence maintaining a mean hemoglobin of 9g/dL. Patient 2 had resolution of palpable splenomegaly and leukoerythroblastosis by cycle 6 and the bone marrow at cycle 26 was characterized by a reduction in marrow fibrosis from grade 4 to 1. A phase II study is ongoing, 14 patients are currently enrolled, with a planned goal of 22 patients. Pharmacokinetic and pharmacodynamic studies as well as cytokine profiling of the phase I patients are being analyzed and will be presented at the meeting. We conclude that low doses of LBH589 delivered for greater than 6 months in patients with MF are capable of ameliorating symptoms, improving clinical features and reversing pathologic marrow changes. Disclosures: Off Label Use: Oral histone deacetylase inhibitor that targets epigenetic changes in malignant myelofibrosis cells with an goal to modify the disease process.

Description: Abstract 1506 BACKGROUND: A wide range of neuropsychological sequelae, noted in long-term survivors of ALL, have been in part ascribed to cranial irradiation (CRT), especially in children who are more vulnerable than adults. We had attempted to abandon prophylactic CRT by intensifying systemic chemotherapy and protracted triple IT (TIT) in ALL-02 trial; whereas one third of patients received CRT for CNS directed therapy in the former study (ALL-97). We present here the results of JACLS ALL-02 trials especially about the effect of the CNS-directed therapy. PATIENTS AND METHODS: Between 2002 and 2008, 1139 children with newly diagnosed non-T ALL with 1–18 years of age were enrolled to JACLS ALL-02 trial. Patients with Ph+ALL and T-ALL were registered to independent protocol. The criteria for diagnosis of CNS disease of the JACLS ALL-02 trial were defined as a CSF pleocytosis of 〉 5 cells /microL and the presence of recognizable blast cells on a well-stained cytospin preparation, or the presence of cranial-nerve palsies at a diagnosis of leukemia. Treatment group was divided into 3 groups according to the BFM-style initial prednisolone (PSL) response and the modified National Cancer Institute (NCI) workshop criteria. PSL good responders were divided into standard risk (SR) and high risk (HR) by WBC 10,000 and age 10. BCP-ALL with PSL poor responders and acute mixed lineage leukemia/ acute unclassified leukemias were treated in extremely high risk (ER). The SR patients with unequivocal CNS involvement (CNS3) at diagnosis were treated as an HR group. The patients in M1 marrow at day 33 with M2/M3 at the day 15 bone marrow (BM) were assigned to shift higher risk after induction therapy. Treatment of ALL-02 consists of early phase and maintenance phase. Early phase includes induction, consolidation, sanctuary (2 courses of high-dose MTX at a dose of 3g /m2), and re-induction therapy for 15 to 26 weeks depending on risk group. Prophylactic CRT was replaced by protracted TIT (MTX, CA, HDC) in all except the patients with CNS3 at diagnosis in ALL-02 trial. The patients with CNS3 at diagnosis received CRT at a dose of 12 Gy. Depending on the risk group, protracted TIT was given during induction, intensification and maintenance in ALL-02 (12 doses in SR and 15 in HR/ER). Accumulative doses of DEX were 120 mg/m2 in SR, whereas 170 mg/m2 in HR and 670 mg/m2 in ER. Treatment duration is 24 months for any risk. The patients assigned ER were candidate for allogeneic stem cell transplantation (SCT) by the end of early phase, provided HLA-matched siblings were available. The probability of event-free survival (EFS) and overall survival were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. All statistical analyses were done according to intent-to treat methods. RESULTS: The numbers of patients at each risk in ALL-02 were 457 (40%) for SR, 543 (48%) for HR, and 139 (12%) for ER. The number of patients with CNS3 at diagnosis was 31 (2.7%). Of 1139 patients, 16 patients (1.4%) had CNS relapses of the leukemia (an isolated CNS relapse: 10 pts, a combined CNS and BM relapse: 6 pts). The 5-year EFS rate ALL-02 was 83.7% (SE=1.1), slightly better than for ALL-97 (79.3%, SE=1.7, p =0.054). The 5-year cumulative risk of an isolated and total CNS relapse was lower in ALL-02 than in ALL-97 (isolated CNS relapse: 0.9% vs 2.7%, p=0.017, total CNS relapse: 4.5% vs 1.4%, p =0.01). The proportion of CRT and SCT were 2%, 8% in ALL-02 respectively, whereas 31%, 11% in ALL-97 respectively. CONCLUSIONS: The ALL-02 trial found that CRT was abolished successfully, in all except those with CNS3 at diagnosis, with substitution of more intensive systemic chemotherapy and protracted TIT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 255 Introduction: Standard treatment of acute myeloid leukemia (AML) comprises one or two cycles of chemotherapy to induce complete remission (CR) followed by postremission treatment in order to prevent relapse of the disease (consolidation therapy). In 2003, we initiated a prospective multicenter randomized trial to investigate the impact of different consolidation strategies on long-term outcome in AML patients ≤ 60 years. Consolidation options comprised upfront allogeneic stem cell transplantation (allo SCT) in aplasia after induction therapy, autologous SCT, and three cycles of standard high-dose-cytarabine-based consolidation. For patients receiving high-dose cytarabine, the main study aim was to evaluate the benefit of adding additional mitoxantrone and amsacrine to cytarabine consolidation. Design: From 2003 to 2009, 1182 patients (median age, 48 years; range 16–60 years) with untreated AML were randomly assigned at diagnosis to different consolidation strategies after classical 7+3 induction. According to the risk-adapted treatment strategy of the trial, cytogenetically or molecular intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-identical-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. IR and AR patients with no available donor should receive autologous SCT. All favorable risk patients and patients with no available donor were scheduled for high-dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine alone while the other half received high dose cytarabine with the addition of amsacrine and mitoxantrone. Standard chemotherapy consisted of three cycles with high dose cytarabine (2 × 3 g/m2, day 1,3,5) whereas combined consolidation contained two cycles of MAC (cytarabine 2 × 1g/m2, day 1–6, mitoxantrone 10 mg/m2, day 4–6) plus one cycle of MAMAC (cytarabine 2 × 1 g/m2, day 1–5, amsacrine 100 mg/m2, day 1–5). In order to evaluate the effect of the two cytarabine based consolidation strategies, we determined overall survival (OS) and event free survival (EFS) using the method of Kaplan Meyer. Survival distributions were compared using the log rank test. Results: 1182 patients were randomized for further intervention (Arm A+B: n=582, 49.3%; Arm C+D: n=600, 50.7 %). Median follow-up was 41.4 months (95%-CI 39.3–43.6). A total number of 375 patients received allogeneic (n=322) or autologous SCT (n=53) and 807 patients were consolidated with cytarabine. Of these patients, 407 were randomized for cytarabine alone and 400were randomized to receive cytarabine plus mitoxantrone and amsacrine (MAC/MAC/MAMAC). Complete remission rate (CR) after second induction therapy was 59.1% (n=698). Between the four arms, there were no significant differences of the CR rates. Five-year OS of patients receiving high dose cytarabine alone was 47.1% (95%-CI 42.0–52.2%), for patients receiving MAC/MAMAC as consolidation therapy it was 46.8% (95%-CI 42.3–51.3%; p = 0.610). Three-year event free survival (EFS) was also not significant with 30.5% (95%-CI 26.6–34.4%) for patients receiving high dose cytarabine alone and 35.6% (95%-CI 31.7–39.5%; p = 0.059) for patients receiving MAC/MAMAC. Conclusions: According to our data, the addition of mitoxantrone and amsacrine to high dose cytarabine consolidation confers no benefit for treatment outcome in younger AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1122 The survival of replicating B cells, with DNA damage arising from oxidative stress and/or activation-induced cytosine deaminase (AID), appears in part to be under p53 control. Importantly, a common C〉G single nucleotide polymorphism (SNP) within codon 72 of p53 influences p53 function. Among other differences, p53-72R (CGC=Arginine) is notably more effective than p53-72P (CCC=Proline) at inducing apoptosis. The SNP has been linked to clinical outcome in multiple settings, including malignancy. Most individuals in the US population display heterozygosity. In this study, we have examined whether B cells, whose genomic DNA is heterozygous for the codon 72 SNP, exhibit allelic exclusion at the level of expressed RNA. This was suggested by reported evidence that p53 expression is strongly regulated by gene methylation status; mRNA of peripheral blood cells from p53-72P/R heterozygous individuals is skewed toward the representation of only one SNP, depending upon ethnic status; and a p53 intron 2 SNP, representing a potential methylation site, is in strong linkage disequilibrium with the codon 72 SNP (PLoS ONE 6:e15320, 2011). Evidence for allelic exclusion of this functionally relevant p53 SNP would suggest that not all identically-stimulated B cells have equal likelihoods of survival within p53-72P/R heterozygous individuals. To investigate this issue, we first confirmed that p53 was expressed in non-transformed human B cells replicating in response to surrogate C3d-bound antigen, IL4 and BAFF. These physiologically-relevant stimuli synergize to induce a burst of T cell-independent clonal expansion, followed by apoptosis of many of the divided progeny (J. Immunol. 175:6143, 2005). Expression of p53 was monitored by immunoblotting and flow cytometry. Consistent with a role in regulating clonal burst size, p53 protein and mRNA/protein of p53-regulated pro-apoptotic genes were significantly elevated in blasts, prior to apoptosis. This contrasted with undectable p53 protein in non-stimulated B cells. To assess whether expressed p53 within a single lymphoblast derives from one allele, i.e. demonstrates allelic exclusion, we first identified tonsil donors heterozygous for the codon 72 polymorphism. This involved PCR-restriction fragment length polymorphism (RFLP) analysis of genomic DNA, as described by others (Leukemia Research 30:1113, 2006). Subsequently, purified resting B2 cells from cryopreserved tonsil cell suspensions, determined to be heterozygous for p53-72P/R, were labeled with CFSE and stimulated as above. At d5, single B lymphoblasts were sorted into 96 well PCR plates containing lysis buffer and cDNA prepared using random hexamer primers. A p53 sequence comprising exons 2a, 3, and 4 was subjected to two rounds of PCR amplification with the following primers, Forward: 5-cagccagactgccttccg-3 & Reverse: 5-gcaagtcacagacttggctg-3. Nucleotide sequencing of PCR-amplified p53 was performed commercially (Applied Biosystems Big Dye Terminator v3.1 cycle sequencing) and analyzed by chromasPro software. In two experiments, only 39 cells of a total of 236 assayed were positive for p53. By contrast, β-actin cDNA from two rounds of PCR amplification yielded 88% of the single cell-containing wells positive for actin. These yields indicate that p53 mRNA levels within a single cell are significantly more limiting than those for actin and are consistent with quantitative PCR of cDNA obtained from activated cell pools. Interestingly, analysis of the cDNA p53 chromatogram sequence of the p53-positive single cells (n=39), whose genomic DNA was heterozygous for the p53 codon 72 SNP, showed only a single homogenous sequence: either P (n=21) or R (n=18), but never both. This was in marked contrast to the chromatogram of cDNA derived from a pool of activated B cells within each experiment. In the latter cases, two overlapping peaks indicating co-expression of p72-P and p72-R were noted. Taken together, our findings suggest that p53 mRNA expression from a single non-transformed human B lymphocyte arises from the transcriptional activation of a single allele, i.e. shows allelic exclusion. Although the mechanism for this phenomenon requires further investigation, these results imply that B cells within individuals heterozygous for the functionally important p53-p72 polymorphism might vary considerably in their resistance to apoptosis. Disclosures: No relevant conflicts of interest to declare.

Description: HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by 〉10 000-fold for both CD34+/CD38− and CD34+/CD38+ cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34+ cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

Description: Abstract 2873 International staging system (ISS) and cytogenetics are the main prognostic factors of Multiple Myeloma (MM) but they reflect biologic characteristics of disease without taking into account individual host features. On the contrary, clinical characteristics of single patient could be substantial as to various points of view. For instance, in elderly MM patients, novel therapies reduction or interruption due to toxicity represent the major cause of unsatisfactory outcome. Therefore, it was empirically suggested different schedule of drugs in these “frail” patients but, how the “frailty” should be assessed in every single patient, is still unsettled. Advanced age, poor performance status (PS) and comorbidities are usually applied to recognize the “frailty” but it is not well known which of them are really prominent and whether these parameters, adjusted for conventional prognostic factors, still affect final outcome. We analyzed a population of symptomatic MM diagnosed from 2007 to 2010 included in the Marche Region MM Registry, to assess the frequency of “frailty” features, such as age, PS, comorbidities, cytopenias, renal insufficiency (RI) and lytic bone lesions, and their role on the overall survival (OS) when adjusted for prognostic factors. Comorbidities were scored according to Charlson Comorbidity Index (CCI) that split patients in 4 categories according to number and type of comorbidity. Patients were treated with transplant or standard therapy according to their eligibility. Overall, 88% of patients were treated with new drug-based therapies and 12% with MP. Median age of the 266 patients analyzed was 73 years (range 38–90). Twenty-four percent of patients had IgA MM, fifty patients (23%) had ISS stage=3 and 29/166 (17.5%) had unfavourable cytogenetics. Regarding “frailty” measures, 38% of patients had 〉 75 years, 39% had PS=2–4, 34% had 1 or more comorbidities. The most frequent comorbidities were hypertension (35%), heart diseases (22%), diabetes (15%), neurological diseases (16%), COBP (8%), secondary malignancies (8%) and chronic renal failure (6%). CCI ≥1 was detected in 51%. Increasing comorbitities number and CCI were associated with increased age although 37% of patients aged less than 65 years had CCI ≥2. Moreover, 35% had at least 2 cytopenias, 76% had bone disease and 14% had RI. Fifty patients (19%) died during follow-up. OS at 3 years was 74%. Univariate analysis performed on the total population determined age 〉 65 years (p=0.065), PS=2–4 (p

Description: Xanthomas are a common manifestation of lipid metabolism disorders. They include hyperlipemic xanthoma, normolipemic xanthoma, and a related condition, necrobiotic xanthogranuloma (NXG). All 3 forms can be associated with monoclonal immunoglobulin (MIg). In an attempt to improve diagnosis, understanding, and treatment of this association, we retrospectively analyzed a personal series of 24 patients (2 hyperlipemic xanthoma, 11 normolipemic xanthoma, and 11 NXG) and 230 well-documented reports from the literature. With the exception of the nodules and plaques featured in NXG, the clinical presentation of xanthomatous lesions usually resembled that seen in common hyperlipidemic forms and could not be used to suspect MIg-associated xanthomas. Extracutaneous sites were not rare. The MIg was an IgG in 80% of cases. Myeloma was diagnosed in 35%. Hypocomplementemia with low C4 fraction was present in 80% of studied patients. Low C1 inhibitor serum levels were found in 53%. Cryoglobulinemia was detected in 27%. These abnormalities suggest immune complex formation because of interactions between the MIg and lipoproteins and argue in favor of a causal link between MIg and xanthomas. Monoclonal gammopathy therapy could thus be an option. Indeed, among the patients who received chemotherapy, hematologic remission was accompanied by improvement in xanthoma lesions in several cases.

Description: Abstract 223 A coding variant form of GFI1 (GFI136N) increases the risk to develop AML by 60% and is present in about 10–15 % of all Caucasian AML patients. To determine the underlying molecular mechanism and potentially develop new therapeutic approaches, we generated “knockin” mouse strains wherein the endogenous murine Gfi1 gene was replaced either by the human GFI1 variant (GFI136N, the form predisposing to AML) or by the more common form of GFI1 (GFI136S). In most hematopoietic compartments no difference was observable between GFI136N and GFI136S expressing mice; however, there was a 3–5 fold increase in the number of granulocytic monocytic progenitors (GMPs) and common myeloid progenitors (CMPs) in Gfi136N expressing (either homozygous or heterozygous) mice compared to wild-type or Gfi136S expressing mice(p≤0.01). Interestingly, both human and murine AML leukemic cells are thought to originate from GMPs and CMPs. To assess functional differences, we seeded GMPs from GFI136N or GFI136S knockin mice on methylcelluose or transplanted them into into syngenic animals. We found that GFI136N expressing GMPs proliferate faster and have an increased self-renewal capacity both in-vitro and in-vivo compared to GMPs carrying Gfi136S alleles. A gene expression array analysis showed that GFI136N GMPs have a stem cell-like gene signature with elevated levels of Hoxa9 expression and a deregulation of a number of oncogenes involved in the development of human AML such as Trib2, Tet2 or Idh2. It is of particular interest that Hoxa9, a known GFI1 target gene, was up-regulated 3–4 fold in GFI136N GMPs compared to in GFI136S GMPs (p≤0.01). It is known that high levels of Hoxa9 accelerate AML development in mice and are associated with a poor prognosis in AML patients. GFI1 is a transcriptional repressor and exerts its function by recruiting different histone modifying enzymes, in particular LSD1, which de-methylates histone 3 (H3) at lysine 4 (K4), or histone deacetylases (HDACs), which remove acetyl groups from H3K9 residues and G9a, which initiates dimethylation of H3K9. Both H3K4 methylation and H3K9 acetylation correlate with actived gene expression, whereas H3K9dimethyl correlates with repession. Chromatin-immuno-precipitation (ChIP) of Gfi1-bound chromatin from Lin−Sca1−c-Kit+ cells, which contains the GMP population, showed that GFI136N binds to a lesser degree to the Hoxa9 locus than GFI136S. This diminished binding of Gfi136N correlated with an increased H3K4 dimethylation and H3K9 acetylation as well as diminished H3K9 dimethylation across the Hoxa9 locus in GFI136N cells. It is likely that these epigenetic changes lead to the increased Hoxa9 expression observed in GFI136N GMPs. A more exhaustive ChIP-Seq analysis with antibodies recognizing H3K4dimethyl in Lin−Sca1−c-Kit+ cells from Gfi136N or Gfi136S mice showed significant epigenetic alterations throughout the Hoxa9 locus genome and at other GFI1 target genes. It is conceivable that these epigenetic alterations explain, at least in part, the changed gene expression signatures in GFI136N GMPs. To investigate the role of GFI136N in myeloid leukemogenesis, we induced the expression of a mutated form of KRAS (K12D) in both GFI136N and GFI136S mice. All mice developed a deadly myelo-proliferative disorder, but animals carrying the GFI136N allele succumbed to the disease within a significantly shorter latency period (17 against 31 days, p≤0.01) than GFI136S mice. We also transduced GFI136N and GFI136S GMPs with retroviral vectors directing the expression of either the AML1-Eto9a or the MLL-AF9 onco-fusion proteins typically found in human AML. We observed that GFI136N GMPs expressing MLL-AF9 or AML1-Eto9a generated 5–10 fold more colonies (p≤0.01) on methylcellulose and exhibited a higher replating efficiency than the respective GFI136S GMPs. Finally, AML blast cells from GFI136N heterozygous patients expressed higher levels of HOXA9 compared to AML blasts from GFI136S homozygous patients, suggesting that our mouse model reflects the disease predisposition in human patients. Our knockin mice are, to our knowledge, the first animal model for a human genetic variation that predisposes to leukemia. Based on the findings with this model, we propose that the human GFI136N variant predisposes to AML by inducing epigenetic changes affecting the expression of important regulators with oncogenic potential such as Hoxa9. Disclosures: No relevant conflicts of interest to declare.

Description: We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL–independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P 〈 .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Description: Abstract 3986 Background: Multiple Myeloma (MM) is an incurable plasma cell neoplasm. Although current lenalidomide (R) and bortezomib containing up-front regimens can now achieve overall response rates approaching 100%, patients eventually relapse with progressively refractory disease. Histone deacetylase inhibitors (HDACi), in Phase I clinical trials in patients with multiple myeloma, have shown promising activity when combined with other agents such as bortezomib. Vorinostat, (suberoylanilide hydroxamic acid; SAHA) is an oral HDACi, currently FDA approved in the United States for the treatment of cutaneous T-cell lymphoma. Here we report the findings of the combination of vorinostat (Zolinza®), lenalidomide and dexamethasone (ZRD) in multiple myeloma patients who were refractory to RD. Methods: Patients received oral vorinostat 300 mg or 400 mg once daily (days 1–7 and days 15–21), lenalidomide 10–25 mg (days 1–21) and dexamethasone 20–40 mg weekly (days 1, 8, 15, 22) in a 28-day cycle Subjects: Twenty-nine patients were treated and all were refractory to RD; 76% were refractory to at least one bortezomib containing regimen and 48% were refractory to the combination of VRD. Twenty-six patients (90%) had undergone prior high dose therapy with autologous stem cell transplant. The median number of prior therapies was 4 (range 2–13). Results: The overall response rate (ORR) was 24 % with 1 VGPR and 6 PR. The clinical benefit rate (ORR + MR) was 51% including 8 MR. Nine patients (31%) had stable disease. The median duration of response (DOR) was 4 months (range, 0–36). The median overall survival (OS) was 11 months (range, 4–36). Common toxicities including diarrhea and fatigue (all grades) were 41% and 34% respectively. The incidence of grade 3/4 neutropenia was 45 % and grade 3/4 thrombocytopenia was 34%. Conclusion: The combination of ZRD showed significant activity in patients with RD relapsed/refractory multiple myeloma. ZRD was well tolerated and is a viable option for patients who do not respond to lenalidomide-based therapy. Further, since all 3 agents are available in oral formulations, ZRD provides an additional option for those patients wishing to avoid intravenous therapy. Formal phase II studies of this combination are in preparation. Disclosures: Off Label Use: Vorinostat is an oral HDAC inhibitor and is being evaluated in the treatment of Multiple Myeloma. Bilotti:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. McNeill:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. Graef:Merck: Employment. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Speakers Bureau. Siegel:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millenium: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Consultancy.

Description: We performed nonmyeloablative HSCT in 6 patients with a newly described genetic immunodeficiency syndrome caused by mutations in GATA2—a disease characterized by nontuberculous mycobacterial infection, monocytopenia, B- and NK-cell deficiency, and the propensity to transform to myelodysplastic syndrome/acute myelogenous leukemia. Two patients received peripheral blood stem cells (PBSCs) from matched-related donors, 2 received PBSCs from matched-unrelated donors, and 2 received stem cells from umbilical cord blood (UCB) donors. Recipients of matched-related and -unrelated donors received fludarabine and 200 cGy of total body irradiation (TBI); UCB recipients received cyclophosphamide in addition to fludarabine and TBI as conditioning. All patients received tacrolimus and sirolimus posttransplantation. Five patients were alive at a median follow-up of 17.4 months (range, 10-25). All patients achieved high levels of donor engraftment in the hematopoietic compartments that were deficient pretransplantation. Adverse events consisted of delayed engraftment in the recipient of a single UCB, GVHD in 4 patients, and immune-mediated pancytopenia and nephrotic syndrome in the recipient of a double UCB transplantation. Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B-cell, and NK-cell populations and reversal of the clinical phenotype. Registered at www.clinicaltrials.gov as NCT00923364.

Description: In multiple myeloma (MM) pathogenesis, hyperdiploidy and nonhyperdiploidy are recognized as 2 major cytogenetic pathways. Here, we assessed the role of hyperdiploidy in 426 patients with monoclonal plasma cell disorders, among them 246 patients with AL amyloidosis (AL), by interphase fluorescence in situ hybridization. Hyperdiploidy was defined by a well-established score requiring trisomies for at least 2 of the 3 chromosomes 5, 9, and 15. The hyperdiploidy frequency in AL was a mere 11% compared with 30% in monoclonal gammopathy of undetermined significance (P 〈 .001) and 46% in AL with concomitant MM I (P 〈 .001). Overall, hyperdiploidy was associated with an intact immunoglobulin, κ light chain restriction, higher age, and bone marrow plasmacytosis, but was unrelated to the organ involvement pattern in AL. Clustering of 6 major cytogenetic aberrations in AL by an oncogenetic tree model showed that hyperdiploidy and t(11;14) were almost mutually exclusive, whereas gain of 1q21 favored hyperdiploidy. Deletion 13q14 and secondary IgH translocations were equally distributed between ploidy groups. We conclude that the interphase fluorescence in situ hybridization–based hyperdiploidy score is also a feasible tool to delineate hyperdiploid patients in early-stage monoclonal gammopathies and that the cytogenetic pathogenetic concepts developed in MM are transferable to AL.

Description: Abstract 98 Background: Advanced follicular lymphomas (FL) are incurable with conventional chemotherapy and there is no consensus on the best treatment approach. The SWOG cancer research cooperative group and Cancer and Leukemia Group B (CALGB) compared the safety and efficacy of two immunochemotherapy regimens for FL in a Phase III randomized intergroup protocol (S0016) that enrolled 554 patients with previously untreated, advanced stage FL between 3/1/2001 and 9/15/2008. Methods: Pts were eligible if they had advanced stage (bulky stage II, III or IV) evaluable FL of any grade (1, 2, or 3) and had not received any prior therapy. In one arm (CHOP-R), patients received 6 cycles of CHOP chemotherapy (750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, 1.4 mg/m2 vincristine, and 100 mg prednisone daily for 5 days) at 3 week intervals with 6 doses of rituximab anti-CD20 antibody (375 mg/m2 on days 1, 6, 48, 90, 134 and 141 according to the schedule described by Czuczman et al.[J.Clin.Oncol 17:268, 1999]). In the second arm of the protocol, patients received 6 cycles of CHOP, followed by a dosimetric infusion of tositumomab/iodine I-131 tositumomab and then 1–2 weeks later a therapeutic infusion of I-131-tositumomab labeled with sufficient I-131 (median 85 mCi) to deliver a total body dose of 75 cGy (CHOP-RIT). The study was designed to have 86% power to detect a hazard ratio (HR) of CHOP-RIT to CHOP-R of 0.67 for 2 yr PFS based on a one-sided.021 level test (accounting for 3 interim analyses). Results: Of the 554 enrolled pts, 532 were eligible and 526 were evaluable for toxicity (263 on each arm of the protocol). Pt characteristics (age, sex, race, stage, beta 2 microglobulin level, tumor bulk, B symptoms) were well-balanced in the two arms of the protocol. In general, both regimens were well-tolerated (Table I). Median follow-up time among patients still alive is 4.9 years. One hundred and six of 267 eligible pts on the CHOP-R arm have progressed or died compared to 86 of 265 eligible pts on the CHOP-RIT arm. The 2 year estimate of PFS was 76% on the CHOP-R arm and 80% on the CHOP-RIT arm (Figure 1). In multivariate Cox regression adjusting for the stratification factor (serum beta-2 microglobulin level), the hazard ratio for CHOP-RIT vs. CHOP-R was 0.79 (95% CI: 0.60–1.05, p=.11 [2-sided] or p=.06 [1-sided]). Twenty-six of 267 pts on the CHOP-R arm have died compared to 40 of 265 eligible pts on the CHOP-RIT arm. The 2-year estimate of overall survival was 97% on the CHOP-R arm and 93% on the CHOP-RIT arm. In multivariate Cox regression adjusting for the stratification factor serum beta-2 microglobulin, the hazard ratio for CHOP-RIT vs. CHOP-R was 1.55 (95% CI: 0.95–2.54, p=.08 [2-sided]). Conclusion: No statistically significant differences in PFS, OS, or serious toxicities are yet demonstrable with either regimen administered in this trial. However, PFS and OS are outstanding with either of the two regimens with median times to progression not yet reached for either treatment. Future studies will be needed to assess whether combining CHOP-R with RIT consolidation and with maintenance rituximab will confer additive benefit, as being evaluated in a follow-up trial (SWOG protocol S0801) that has recently completed accrual. Disclosures: Press: Spectrum: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Off Label Use: Front-line use of I-131-tositumomab for consolidation therapy in 1st remission of follicular lymphoma. Friedberg:Genentech: Consultancy, Honoraria. Czuczman:Glaxo Smith Kline: Consultancy, Research Funding; Genentech: Consultancy, Honoraria. Kaminski:Glaxo Smith Kline: Patents & Royalties. Maloney:Genentech/Roche: Consultancy, Honoraria, Speakers Bureau; Glaxo Smith Kline: Consultancy, Honoraria, Speakers Bureau. Cheson:Glaxo Smith Kline: Research Funding. Fisher:Roche: Consultancy, Honoraria.

Description: Abstract 1131 Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P

Description: Chronic GVHD is one of the most severe complications of allogeneic HSCT. The sclerotic skin manifestations of cGVHD (ScGVHD) result from inflammation and fibrosis of the dermis, subcutaneous tissue, or fascia, leading to significant functional disability. Risk factors and clinical markers associated with ScGVHD remain largely unexamined. By using a single-visit, cross-sectional design, we evaluated 206 patients with cGVHD at the National Institutes of Health. Most patients manifested severe (ie, 63% National Institutes of Health score “severe”), refractory disease (median treatments = 4). ScGVHD was detected in 109 (52.9%) patients. ScGVHD was associated with greater platelet count (P 〈 .001) and C3 (P 〈 .001), and decreased forced vital capacity (P = .013). Total body irradiation (TBI) was associated with development of ScGVHD (P = .002). TBI administered in reduced-intensity conditioning was most strongly associated with ScGVHD (14/15 patients, P 〈 .0001). Patients with ScGVHD had significant impairments of joint range of motion and grip strength (P 〈 .001). Greater body surface area involvement was associated with poorer survival (P = .015). We conclude that TBI, particularly in reduced-intensity regimens, may be an important risk factor for ScGVHD. Widespread skin involvement is associated with significant functional impairment, distressing symptoms, and diminished survival. This trial is registered at http://www.clinicaltrials.gov as NCT00331968.

Description: Abstract 1704 Background: Lenalidomide (LEN) is approved in the US for the treatment of RBC transfusion-dependent patients with IPSS Low- or Int-1-risk myelodysplastic syndromes (MDS) with del(5q), with or without other cytogenetic abnormalities. In a phase 3 trial, treatment with LEN 5 mg and 10 mg resulted in RBC transfusion independence (TI) for ≥ 26 weeks in 43% and 56% of such patients, cytogenetic response in 25% and 50%, and a significant improvement of health-related quality of life (p

Description: Abstract 1723 Background: Two multicenter studies (MDS-003/-004) found LEN leads to RBC transfusion independence (TI) in 〉 50% of pts with RBC transfusion dependent Low-/Int-1-risk MDS with del5q (List A et al. NEJM 2006;355: 1456–65; Fenaux P et al. Blood 2011;doi: 10.1182/blood-2011-01-330126). RBC-TI ≥ 8 wks with LEN was associated with significantly reduced risk of AML progression and death (Fenaux P et al. Blood 2011;doi: 10.1182/blood-2011-01-330126). Alternative therapy is required for pts failing LEN therapy. Aims: To assess predictive factors of LEN response and long term outcomes (especially after primary or secondary LEN failure) of pts 〈 65 yrs included in MDS-003/-004; ie, those in whom intensive therapies including allogeneic stem cell transplantation (ASCT) may be considered. Methods: LEN was administered as follows (all 28-d cycles): 5 mg/d on d 1–28 and 10 mg/d on d 1–21 or 1–28. RBC-TI ≥ 26 wks and cytogenetic response (CyR; IWG 2000) are reported. Overall survival (OS) and AML progression were assessed using Kaplan-Meier method. Response rates and outcomes in pts 〈 65 yrs were retrospectively compared with pts ≥ 65 yrs. Primary failure was defined as lack of RBC-TI with LEN treatment and secondary failure as relapse after achievement of RBC-TI ≥ 26 wks. Cox proportional hazards models were used to evaluate the effect of potential risk factors (ie, age, sex, time since diagnosis, FAB classification, LEN dose, IPSS risk, WPSS risk, cytogenetics, bone marrow blast %, transfusion burden, no. of cytopenias, hemoglobin level, platelet and neutrophil counts, RBC-TI ≥ 26 wks [time-dependent variable] and CyR [categorical variable]) on OS and AML progression. Logistic model was used to evaluate the effect of potential risk factors on achievement of RBC-TI ≥ 26 wks. Results: The trials included 97 (33.9%) pts 〈 65 yrs. Of these, 73.2% were female; 20.6% were IPSS Low-, 52.6% Int-1-, and 4.1% Int-2-risk; 30.9% had del5q with ≥ 1 additional cytogenetic abnormality (8.2% had complex cytogenetics). At baseline (BL), median time since diagnosis was 2.4 yrs (range 0.2–20.7) and median RBC transfusion requirement was 6 units/8 wks (range 1–15). In pts ≥ 65 yrs (n = 189) most BL characteristics were similar except IPSS risk, which was lower (36.5% Low-, 37.0% Int-1-, 5.8% Int-2-risk; p =.012). RBC-TI ≥ 26 wks was achieved by 54 (55.7%) pts 〈 65 yrs (vs 49.7% pts ≥ 65 yrs; p =.563). The median duration of RBC-TI ≥ 26 wks in responders was not estimable in pts 〈 65 yrs or ≥ 65 yrs (log-rank p =.879). None of the potential risk factors assessed was a significant predictor of RBC-TI ≥ 26 wks in pts 〈 65 yrs, possibly due to small pt number. In pts 〈 65 yrs with available follow-up cytogenetics (n = 71), CyR was achieved by 32 (45.1%) pts (vs 64.5% pts ≥ 65 yrs; p =.014). At time of data cutoff, 51 (52.6%) pts 〈 65 yrs were alive (vs 36.0% pts ≥ 65 yrs; p =.008); 29 (29.9%) pts progressed to AML (vs 20.1% pts ≥ 65 yrs; p =.077). The 1-, 2-, and 3-yr AML-progression rates were 9.7%, 15.4%, and 24.0% in pts 〈 65 yrs; and 6.0%, 17.9%, and 22.3% in pts ≥ 65 yrs (log-rank p =.308). The 1-, 2-, and 3-yr OS rates were 91.7%, 78.1%, and 66.4% in pts 〈 65 yrs; and 83.1%, 65.2%, and 49.9% in pts ≥ 65 yrs (log-rank p

Description: The “in situ” lymphomas are often incidental findings in an otherwise reactive-appearing lymph node. Notably, the risk of progression to clinically appreciable lymphoma is not yet fully known. The diagnosis of “in situ” lymphoma is feasible when immunohistochemical characterization is carried out and genetic abnormalities are assessed. “In situ” follicular lymphoma is characterized by the presence within the affected germinal centers of B cells that strongly express BCL2 protein, a finding that supports their neoplastic nature, in the absence of interfollicular infiltration. In “in situ” mantle cell lymphoma, the lymphoma involvement is typically limited to the inner mantle zone, where lymphoma cells are cyclin D1+ and weakly BCL2+, CD5+. A staging workup to exclude other site of involvement is highly recommended for the possible coexistence of an overt lymphoma. Biopsy of all sites of suspicious involvement should be mandatory. No evidence for starting therapy also in the presence of multifocal “in situ” lymphoma exists, and a “wait-and-see policy” is strongly suggested. A follow-up strategy reserving imaging evaluation only in the presence of disease-related symptoms or organ involvement appears to be a reasonable option. For patients with concomitant overt lymphoma, staging and treatment procedures must be done according to malignant counterpart.

Description: Abstract 382FN2 VWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF−/− mice. Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p

Description: Abstract 773 Peripheral T-cell lymphomas (PTCLs) are rare and heterogeneous tumors whose biology is largely unknown. Interestingly, the commonest subtypes (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) present on one hand few disease-specific molecular features and, on the other hand, several apparently common abnormalities. So far, no data are available regarding miRNA expression in these tumors. In order to identify miRNA deregulated in PTCLs, we performed an extensive miRNA profiling (by studying 379 targets on the TaqMan Array MicroRNA Cards) of 44 PTCLs (including 23 PTCLs/NOS, 12 ALCLs, and 9 AITLs) and 13 sample representative of normal T-cell sub-populations (CD4+ and CD8+, both resting and activated). In addition, for all these cases, gene expression profiles (GEPs) were generated by the Ilumina whole genome DASL-assay. TaqMan Quantitative-PCR (qPCR) was then used for validation. First, we found that PTCLs and normal T-cells could be easily distinguished based on their miRNA profile, by both unsupervised and supervised analysis. Specifically, the latter identified 91 miRNA differentially expressed in PTCLs vs. T-cells with a fold change ≥2 and a pvalue

Description: Abstract 770 Background: Waldenstrom's macroglobulinemia (WM) is a B-cell lymphoma characterized by high serum monoclonal IgM and infiltration of lymphoplasmacytic cells into the bone marrow. As with many hematologic malignancies, cytokines within the tumor microenvironment play an important role in supporting the growth and survival of malignant WM cells. IL-21 is a pleiotropic cytokine involved in the differentiation of B cells into plasma cells. During malignancy, IL-21 has demonstrated diverse effects promoting the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in WM. Our objective here was to assess the expression of IL-21 and its receptor in WM cells and to examine whether IL-21 contributes to the biology of WM. Results: When compared to normal bone marrows, immunohistochemistry revealed significant IL-21 staining in the bone marrow of patients with WM (n=5). To determine whether WM cells are susceptible to IL-21 in the microenvironment, expression of the IL-21 receptor (IL-21R) was assessed via PCR in CD19+CD138+ cells isolated by positive selection from patients with WM (n=8) and in the newly characterized WM cell line, MWCL-1. Nearly all (7/8) CD19+CD138+ WM cells expressed IL-21R, as did MWCL-1 cells. Using flow cytometry we detected expression of IL-21R protein on the surface of WM cells as well. The contribution of microenvironmental IL-21 to the biology of WM tumors was then examined. In MWCL-1 cells, IL-21 (100 ng/mL) increased proliferation by 37% (p=0.005) over untreated controls as determined by thymidine incorporation at 72 hr, and in primary WM cells, proliferation increased by nearly 50% (p=0.003). Interestingly, the immortalized B cell line, IM-9, responded to IL-21 with a significant decrease in proliferation, consistent with previous data indicating differential effects of IL-21 depending on the pathological status of the B-cell in question. IL-21 also significantly induced (p

Description: Abstract 877 Introduction: Patients with follicular lymphoma (FL) usually respond well to immuno-chemotherapy as initial treatment and can have long survival times. However, a small proportion of patients are refractory or have early relapses. Early identification of this subgroup of patients could lead to early therapeutic changes and, potentially, to a better prognosis. [18F]Fluorodeoxyglucose-Positron Emission Tomography (FDG-TEP) is widely used for the staging and restaging of aggressive lymphoma patients but little is known about the use of FDG-PET in patients with FL, except that FL is almost uniformly FDG-avid. FDG-PET is not recommended today as a routine procedure in FL patients (Cheson et al. J Clin Oncol 2007). In this first prospective, multicentric study, we evaluated the prognostic value of FDG-PET performed at mid-treatment and at the end of treatment in patients with high-tumor mass FL treated with immuno-chemotherapy in first line. Patients & methods: Patients with previously untreated FL (grades 1–3A) presenting with a high tumor burden as defined by the GELF criteria, were treated with 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) plus 2 cycles of rituximab, given every 21 days. Patients did not receive rituximab maintenance. A FDG-PET was performed before treatment, after 4 cycles of R-CHOP (interim FDG-PET: I-PET), and at the end of treatment (final PET: F-PET). FDG-PET scans were first interpreted in each centre, then centrally reviewed by 3 investigators blinded to clinical data. Positivity or negativity was rated according to the Deauville visual semi-quantitative criteria (Meignan M: Leuk Lymphoma 2009), positivity being defined as a fixation at level 4 (FDG uptake superior to that of the liver) or 5 (uptake clearly superior to liver and/or new sites of disease). Results: 121 patients were included. The median age was 57 years and the male-to-female ratio was 1.12. Ninety-three percent had Ann Arbor stage III–IV. Ninety-seven percent had a good performance status (ECOG score 0–1). The repartition according to the FLIPI was: 0–1 factors: 15%; 2 factors: 43%; 3–5 factors: 42%. The Kappa coefficient indicated a good degree of concordance between the 3 PET reviewers. The initial FDG-TEP was positive in all except 1 case out of 118 centrally reviewed cases. After central review, I-PET (n=111) was negative in 76% of patients and F-PET (n=106) was negative in 78%. With a median follow-up of 23 months, 2-year-progression-free survival rates for I-PET negative versus positive and for F-PET negative versus positive were I-PET 86% versus 61% (p=0.0046); F-PET 87% versus 51% (p

Description: Abstract 731 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure. Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure. To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders. In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Abstract 701 Diadenosine triphosphate (Ap3A) is a component of platelet dense granules, and is released into the extracellular space during the second wave of platelet aggregation following platelet activation by ADP and other agonists. Ap3A has long been thought to stimulate platelet aggregation after release into the extracellular space by providing a prolonged source of ADP via hydrolysis by a slow extracellular enzyme present in human plasma. Here, we identify NPP-4, a member of the nucleotide pyrophosphatase/phosphodiesterase enzyme family present on endothelial cell surfaces, as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion at nanomolar concentrations. We performed lumiaggregometry with citrated platelet-rich plasma in the presence of Ap3A with or without increasing nanomolar concentrations of NPP-4. In these experiments we determined that Ap3A alone in concentrations up to 80 uM initiated a primary wave of platelet aggregation that was followed by rapid disaggregation. In the presence of nanomolar concentrations of NPP-4, however, the primary and secondary waves of platelet aggregation and dense granule release are strongly induced in an enzyme concentration-dependent fashion. In contrast, mutant NPP-4, catalytically inactivated by an active site threonine to alanine mutation, had no effect on platelet aggregation and dense granule release under identical conditions. In order to clarify the structural basis of the enzymatic mechanism, we determined the high-resolution structure of NPP4 in the presence and absence of the enzymatic product, AMP. In aggregate, our studies define the biological, enzymatic, and molecular basis for NPP-4 and Ap3A activity in platelet aggregation and secretion in vitro and suggest that NPP-4 may play a role in hemostasis in vivo by augmenting platelet aggregation and release of granule contents at the site of vascular injury. With these studies we have thus established the molecular foundation for the rational development of a new class of therapeutics capable of modulating vascular thrombosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 610 Myelodysplatic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Patients develop peripheral blood cytopenias; however, the bone marrow shows increased proliferation and apoptosis. In addition to bone marrow apoptosis, a failure of differentiation contributes to reduced terminally differentiated blood cells. A significant proportion of patients with MDS will develop anemia that are refractory to treatment with recombinant human erythropoietin (EPO) and must rely on transfusions as supportive care. The use of blood transfusions as supportive care is associated with iron overload and significant morbidity. Therefore, alternative therapies to treat anemia in MDS patients are needed. Members of the TGFβ super family of signaling molecules have been implicated in erythropoiesis and represent alternative, EPO-independent targets for the treatment of anemia. ACE-536 is a soluble receptor fusion protein consisting of a modified Activin Receptor Type IIB extracellular domain linked to a human Fc domain. ACE-536 acts as a ligand trap to modulate the activity of TGFβ ligands and promote erythroid differentiation in an EPO independent manner. Subcutaneous administration of ACE-536 to C57BL/6 mice resulted in significant increases in hematocrit, hemoglobin and red blood cells compared to vehicle treated controls within four days. These effects were observed with concurrent treatment of an EPO neutralizing antibody, indicating that EPO is not directly responsible for the initial RBC response of ACE-536. BFU-E or CFU-E colony formation assays from bone marrow or spleen of mice 48 hours after ACE-536 were normal, indicating no effect on the erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by FACS analysis following 72 hours after RAP-536 (murine version of ACE-536) treatment revealed a decrease in basophilic erythroblasts and an increase in late stage poly-, ortho-chromatophilic and reticulocytes in bone marrow and spleen compared to vehicle treated mice. The data demonstrate that while EPO treatment increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. The efficacy of ACE-536 has been demonstrated in various animal models of acute and chronic anemia. In this study we investigated the effect of ACE-536 on anemia in mouse model of MDS. The NUP98-HOXD13 (NHD13) transgenic mouse carries a common translocation found in MDS patients. NHD13 mice develop anemia, neutropenia and lymphopenia at 4–7 months of age, with normal or hypercellular bone marrow. Starting at 4 months of age, mice were treated with RAP-536 (murine homolog of ACE-536) at 10 mg/kg or vehicle control twice per week for 8 months. Wild-type littermate controls were also dosed on the same schedule. As expected, at study baseline (mice 4 months of age), NHD13 mice had reduced RBC, Hb and HCT compared to wild-type littermates. The progression of anemia over the study period was reduced by treatment with RAP-536 compared to vehicle (HCT: −8.3% v. −22%, RBC: −13% v. −30%). Based on blood smear analyses, there was no indication of increased leukemic cells with ACE-536 treatment. Our data demonstrate that RAP-536 can increase hematology parameters through enhancing maturation of terminally differentiated red blood cells and can serve as a therapeutic molecule for the treatment of anemia. As anemia contributes significantly to the morbidity of patients with MDS, a mouse model was used to test the therapeutic efficacy of ACE-536 in this disease. We have shown that systemic administration of RAP-536 to MDS mice promotes increases in red blood cell mass without enhanced progression to AML. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. Disclosures: Suragani: Acceleron Pharma Inc: Employment. Mulivor:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment. Kumar:Acceleron Pharma Inc: Employment.

Description: Abstract 544 Background. In patients with unprovoked venous thromboembolism (VTE) occurring in the absence of major provoking factors, the optimal duration of anticoagulation is anchored on estimating the risk for disease recurrence in the individual patient. Evidence from several studies suggests that, at least in selected patient subgroups, the risk for recurrence may approximates the annual risk for anticoagulant-related major hemorrhage, which is estimated at 1–3%, and a recent ISTH consensus considers an annual risk of recurrence below 5% as acceptable to justify stopping anticoagulant therapy. Aim. To develop a clinical prediction guide that stratifies patients according to recurrence risk and, thereby, facilitate decisions about whether to continue or stop anticoagulation. Methods. Individual patient data meta-analysis of 7 prospective studies enrolling patients with a first episode of objectively diagnosed VTE. Eligible VTE cases were those which occurred in the absence of surgery, trauma, active cancer, immobility, or pregnancy and the puerperium. Follow-up started when anticoagulant therapy was stopped and ended when one of the following occurred: symptomatic, objectively documented, recurrent VTE; death from another cause; resumption of anticoagulant therapy for another reason; or the study ended. Predictors were identified using stratified Cox regression, and the weight of predictors was obtained after model shrinkage to correct for over-optimism. The discriminative ability of the prediction rule was estimated using time-dependent c-statistics, and was internally validated by bootstrap analysis. Results. 1818 consecutively referred cases with unprovoked VTE treated for at least three months with a vitamin K antagonist were eligible for analysis. Abnormal D-dimer after stopping anticoagulation, age 〈 50 years, male sex and VTE not associated with hormonal therapy (in women) were the main predictors of recurrence. Optimism-corrected regression coefficients were used to derive a prognostic recurrence score (DASH, D-dimer, Age, Sex, Hormonal therapy), that showed a good predicting capability (ROC area=0.71). The DASH score attributes the following points: +2 for positive (abnormal) post-anticoagulation D-dimer, +1 for age ≤ 50 years, +1 for male sex, −2 for hormone use at time of initial VTE (in women only). The annualized recurrence risk was 3.1% (95% confidence interval [CI] 2.3 – 3.9) in patients with a DASH score ≤ 1, 6.4% (95% CI 4.8–7.9) in patients with a DASH score 2, and 12.3% (95% CI, 9.9–14.7) in patients with a DASH score ≥ 3, as reported by the Kaplan-Meier recurrence-free survival plot. By considering at low recurrence risk those patients with a DASH score ≤ 1, life-long anticoagulation might be avoided in 51.6% of patients with unprovoked VTE. Conclusions. The DASH score appears to reliably predict recurrence risk in patients with a first unprovoked VTE and may be used to decide whether anticoagulant therapy should be continued indefinitely or stopped after an initial treatment period of at last three months. Patients with a DASH score ≤ 1 appear to have an annual risk for recurrence (3.1%) that may be sufficiently low to justify stopping anticoagulation in an average patient after 3–6 months of anticoagulation, whereas a DASH score ≥ 2 appears to confer a risk of recurrent VTE that may warrant indefinite anticoagulation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4660 Objectives: Menorrhagia is a common presentation of bleeding disorders, especially VWD in women. We decided to determine the frequency of these disorders in women with menstruation problems. Materials and Methods: 208 patients in reproductive age with menorrhagia were investigated for bleeding disorders in two steps. Step one includes CBC, PT, PTT and BT, which performed for all patients, and for patients who had an abnormality in step one, step two was considered. Step two includes VWAg, RCO, factors level, RIPA and platelet aggregometry. Results: Among 208 patients who investigated for bleeding disorders 53 patients (25%) had abnormalities in coagulation tests or platelet count. In our survey frequencies of bleeding disorder was VWD=14(6.73%), thrombocytopenia=13(6.25%), deficiency of factor II= 2(0.96%), factor V=1(0.48%), factor VII=3(1.44%), factor VIII=2(0.96%), Factor XI =4(1.92%), factor XII=4(1.92%), Bernard Soulier=2(0.96%). Furthermore, we found 18 patients (8.65%) who had abnormal PT, PTT or BT with no definite diagnosis. Conclusion: In this study, the most common bleeding disorder was VWD and thrombocytopenia ranked second disorders. Although other bleeding disorders are rare, in our study a number of them were found. So, we recomment the above coagulation test for women with menorrhagia. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4657 Introduction: Pregnancy is a hypercoagulable state, and thromboembolism is the leading cause of antepartum and postpartum maternal mortality. Women with thrombophilic mutations (factor V leiden, prothrombin, and MTHFR) and inherited bleeding disorders, such as deficiency of factor XIII and fibrinogen, have been shown to be at increased risk of pregnancy loss. However, the risk of miscarriage in women with other inherited bleeding disorders has been discussed controversially. Due to the lack of data, it cannot be determined if the risk of miscarriage is increased in women with von Willebrand disease (vWD). The aim of our study was to clarify the association between inherited bleeding disorders and pregnancy loss. Patients and Methods: Subjects Concerning this investigation we included 91 female patients with two [n=46] or more [n=45] miscarriages occurring prior to 28 weeks of gestation and/or stillbirth without apparent reason. The median age of the examined group at the time of first fetal loss was 29 years, ranging from 17 to 41 years. Methods At first we compiled a detailed clinical history of bleedings of all patients. Subsequently, we performed various tests to gather information regarding coagulation abnormalities and thrombophilic defects. Therefore a molecular and functional assessment of the following data was performed: Coagulation factors, vWF:Ag, vWF:RCo, phospholipid antibodies, hyperhomocysteinaemia (HHCY), protein S (PS), protein C (PC), antithrombin (AT) and FV-Leiden mutation (G1691A), FII mutation (G20210A) and MTHFR C677T. Results: In our investigated population consisting of 91 women we registered 299 pregnancies of which 240 resulted in fetal loss, 232 prior to week 28 of pregnancy and 8 stillbirths. Seven out of 91 patients (8%) were carriers of inherited coagulation disorders; vWD: n=2 (2%), FVII deficiency: n=3 (3%), thrombocytopathy: n=2 (2%). In our study collective there was no increased rate of patients with vWD. None of the patients showed a FXIII- or fibrinogen deficiency. However, 17 patients (19%) have a bleeding diathesis. In 55 patients (60%) we could detect the following thrombophilic defects: FV-Leiden (G1691A): n=10, MTHFR C677T: n=42, PS: n=1, PC: n=1, APS: n=1. Conclusion: The incidence of vWD patients in our miscarriage collective is the same as the overall incidence of vWD patients in the general population. Therefore vWD is not associated with an increased risk of fetal loss. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4654 Purpose: There is a common guideline for surgery in hemophilia A patients - to maintain their factor VIII (FVIII) levels over 100% during and 24 hours after operation by infusion of FVIII concentrates. However their responses to coagulation factors are very different from each other even with the lack of inhibitor. Thus we demonstrated the correlation between individual FVIII recovery and prognosis after surgery. Methods: We reviewed the operation cases in hemophilia A patients who had recovery tests before surgery in Kyungpook National University Hospital from January 2001 to July 2011. The blood sample for recovery test was acquired at 15 minutes after initial infusion of FVIII concentrate from the opposite arm. The patients with inhibitors were excluded. The average hospitalization period and consumption of FVIII concentrates were analyzed. Results: A total 24 surgery cases in 16 hemophilia A patients were reviewed. They were divided into 3 groups; A (recovery/expected FVIII 〉 0.8), B (recovery/expected FVIII 〈 0.8, but they were managed by immediate additional replacement of FVIII concentrates), and C (recovery/expected FVIII 〈 0.8 and the extra supplementations of coagulation factors were delayed over 3 days). Type of operation included neurosurgeries (craniotomy/removal of hematoma 3, ventriculo-peritoneal shunt 2, ventriculostomy 1), orthopedic procedures (arthroplasty 4, open reduction/internal fixation 3, bone graft 1, drilling of bone 1), and gastrointestinal operations (subtotal gastrectomy 2, appendectomy 2, hemorrhoidectomy 2, rectosigmoidectomy 1, cholecystectomy 1, herniorrhaphy 1). The total consumption of coagulation factors and the dose per body weight were lower in group A compared with B and C (respectively p=0.028 and p=0.003, CI 95%). However the hospitalization periods were not different between group A and B (p=0.956, CI 95%). And in group B, the hospitalization period, total consumption of coagulation factors, and the dose per body weight were all lower than C (respectively p=020, p=023, and p=048, CI 95%). No complication was noted in group A and B while 2 cases of hematoma infection were developed in group C. Conclusion: The low FVIII recovery under 80% of expected may contribute higher consumption of coagulation factors after surgery in hemophilia A patients. However if the immediate supplemental FVIII replacement was performed, the medical expense would be conserved without complication. Thus the individualized replacement of FVIII concentrates according to the recovery test is very important for hemophilia A patients who need operation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4673 Background: Thrombotic thrombocytopenic purpura (TTP) is a potentially life-threatening disorder. Secondary TTP was known to be more resistant to plasma exchange and have higher mortality. Limited clinical data suggest that rituximab may be effective in the treatment of acute refractory or chronic relapsing nonfamilial idiopathic TTP (ITTP) as well as refractory secondary TTP (STTP). We performed a systematic review to evaluate the comparative efficacy of rituximab in ITTP and STTP. Method: An electronic literature search of Medline, CINAHL and the Cochrane library databases using the terms “rituximab” and “thrombotic” yielded 179 articles from March 2002 to June 2011. Studies were included if they met the following criteria: TTP in a first episode or subsequent relapse and TTP refractory to plasma exchange. Exclusion criteria were prophylactic administration of rituximab to patients in remission and familial TTP. Secondary TTP cases were author-defined. Case series without extractable individual data were excluded. Two-Tailed Fisher Exact Test was used to investigate the difference in complete remission rates between ITTP and STTP. Independent-Samples Mann-Whitney U Test was applied to detect the difference in platelet recovery days. Result: 20 case series and 33 case reports including a small prospective study were eligible. Individual data of 139 patients from Europe, Asia, Australia, South and North America were analyzed. Overall, median age was 40 years (range: 1–72). Male constituted 30% and female 70%. Complete remission rate was 88% (90 out of 102) and 84% (31 out of 37) for ITTP and STTP respectively. There was no statistically significant difference in complete remission rates between the two groups (Two-Tailed Fisher Exact Test p value = 0.569). Median follow-up time was 12 months. Median platelet recovery (more than 150×109/L) from the first dose of rituximab was 8 days (range: 2–74) and 19 days (range: 7–90) for ITTP and STTP respectively. The distribution of platelet recovery days was the same between ITTP and STTP (Independent-Samples Mann-Whitney U Test p value = 0.132). Conclusion: Rituximab has the same efficacy in both STTP and ITTP in terms of response rate and platelet recovery days. It appears to be effective and well-tolerated. We recommend the use of rituximab as a salvage therapeutic modality in refractory acute or chronic relapsing nonfamilial idiopathic as well as secondary TTP. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4681 Thrombocytopenia is common finding in trauma patients. However, platelet count fluctuates widely. Therefore, Immature Platelet Fraction is being studied for whether it can complement the platelet count. Immature platelet fraction (IPF ) is a measure of reticulated platelets (RPs), which represents the state of thrombopoiesis. IPF is obtained from an automated haematology analyzer. It is proportional to reticulated platelets and expressed as percentage of Total optical count. Aim To establish cut off value of IPF (%) with increasing severity of thrombocytopenia in trauma patients which can be useful for diagnosis and monitoring of the cases. Material and methods Total 69 patients admitted in J.P.N Apex trauma centre (AIIMS) were studied within first 24 hrs after injury. Peripheral blood was collected in K2EDTA tube for measurement of platelet count using a fully automated analyzer Sysmex XE 2100 (Japan). RET channel was selected for the measurement of IPF%.The patients were categorized with platelet count between 150×103/μ l and 100×103/μ l, between 100×103/μ l and 50×103/μ l, and below 50×103/μ l. Immature platelet fraction was measured in all categories of thrombocytopenia and statistically compared. Each patient was ascribed New Injury Severity Score and SOFA (sequential organ failure assessment) score and their correlation with IPF value was assessed. ROC curve was used to establish cut off value of IPF between those with platelet count above150×103/μ l and below 150×103/μ l and between those with platelet count above 100×103/μ l and below 100×103/μ l. Results Using Mann Whitney test, the patients (n=41) with platelet count 〈 150×103/μ l showed mean IPF (%) (18.19 ± 9.43, range: 4.0 – 46.6) which was significantly higher (p 12.5% was found in patients having platelet count 〈 150×103/μ l. Similarly, the patients (n=22) with platelet count 〈 100×103/μ l showed mean IPF (%) (22.04 ± 9.94, range:10.8-46.6) which was significantly higher (p 14.95 % was found in patients having platelet count

Description: Abstract 4682 Background. aHUS is a rare and life-threatening genetic disease characterized by systemic thrombotic microangiopathy (TMA) due to chronic, uncontrolled complement activation. Genetic mutation can be identified in only 50–70% of aHUS patients. Systemic TMA manifests as endothelial injury, hemolytic anemia, and platelet consumption, leading to multiorgan damage/failure. Despite chronic plasma exchange/infusion (PE/PI), 〉50% of aHUS patients die, require dialysis, or have permanent renal damage within 1 yr of diagnosis. Off-label use of eculizumab, a humanized high-affinity monoclonal antibody against complement protein C5, was previously reported to produce clinical remission of TMA in an 18-mo-old boy with a fourth relapse of aHUS (Gruppo et al, NEJM 2009). Results of more recent 26-wk, controlled, prospective, open-label, single-arm trials have demonstrated that eculizumab is effective in aHUS patients resistant to PE/PI or patients undergoing chronic PE/PI in inducing hematologic and TMA remission, restoring renal function, and improving QoL. No difference in response to eculizumab is seen in patients with or without an identified mutation. Long-term efficacy and safety outcomes for sustained eculizumab treatment of aHUS have not yet been reported. Aim. To provide a 3-yr follow-up of our previously-reported pediatric patient regarding the long-term efficacy and safety of eculizumab treatment for aHUS. Methods. Retrospective data collection analysis. Results. A 4-yr-old boy born at 34-wk gestation, initially presented with TMA at 4 d of age: Hg 8.5 g/dL, plt 18×109/L, BUN 34 mg/dL, Cr 1.0 mg/dL, LDH 6077 U/L (normal

Description: Abstract 4665 Introduction: Multiple sclerosis (MS) is an autoimmune inflammatory and chronic demyelinating disease. Occurrence of hypercoagulable states and breast cancer in patients with multiple sclerosis (MS) has not been extensively reported. We report a case of a female MS patient with recurrent DVTs, elevated factor VIII levels, and advanced breast cancer with aggressive biologic phenotypes. Case report: A 45-year-old Caucasian female with a history of MS had a breast mass diagnosed on a screening mammogram. She was diagnosed with right breast carcinoma (2.5 × 2.5 × 1.6 cm), T2 N3 M0, which correlated to stage IIIC. She underwent a right modified radical mastectomy with 16/25 lymph nodes involved, ER/PR status was positive, HER-2/neu 0 with high Ki67. Her post-surgery treatment plan included 4 cycles of Cyclophosphamide and Taxotere chemotherapy. She also received chest wall radiation as well as tamoxifen and aromatase inhibitor (Femara) therapy. The patient had never been on hormone replacement therapy or oral contraceptives. She had bilateral salpingo-oophorectomy and hysterectomy and pathology was unremarkable. Patient was diagnosed with Multiple Sclerosis at age 27. She was treated with intermittent courses of steroids, Interferon b-1b, Interferon b-1a, and Mitoxantrone (MTX) for relapsing/remitting MS for several years until she developed left hemiparesis, hypoesthesias, and residual visual dysfunction that prompted her to start IVIG therapy and plasmapheresis. Past medical history presents recurrent DVT (in 2006) and elevations of coagulation factor VIII (355%). The patient also developed superior vena cava thrombosis in the presence of Lovenox and coumadin therapy. Discussion: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system, frequently complicated by devastating neurologic symptoms and progressive disability. Risks of DVT in bedridden or wheelchair-bound MS patients have been suggested. Previous studies show that the frequency of DVT in late-stage MS may be over 40%. Additionally, in MS patients without risk factors, it has been suggested with an autoimmune inflammatory disease that the inflammatory infiltration in MS plaques located close to small or medium-sized veins could have a role as well. The lumbar puncture could also be one of other thrombophilic factor in MS, since after dural puncture the decrease of cerebrospinal fluid pressure induces a rostrocaudal sagging effect with traumatic damage to the fragile venous endothelial wall and may trigger a venous vasodilatation with resultant stasis. Elevated factor VIII levels are a risk factor for venous thrombosis and may also be associated with the risk of arterial thrombosis in coronary heart disease. Studies show that factor VIII levels may be increased by chronic inflammation. However, elevated factor VIII levels in patients with MS have never been reported. There is no cure for MS, though there are several drugs such as immunomodulatory agents that can slow or stop its progress. However, current data show a small increased risk of breast cancer in women with MS. The size of the breast tumor was also larger for woman with MS. More specifically, the proportions of biologically aggressive phenotypes that can worsen the prognosis of breast cancer incrementally despite the biologic phenotype at diagnosis also were investigated in this group of patients. One hypothesis linking breast cancer and MS involves long-term use of immunomodulatory agents including IFNs and glatiramer. Immunomodulatory therapy may impart immune system alterations that promote enhancement of cancer cells’ ability to evade immune recognition and cancer metastasis by altering the body’s ability to conduce immunosurveillance. However, consistent with previous observations, this remains unexplained and warrants further attention. Conclusion: The Female patient with MS described in this article presents an example of recurrent DVT, Superior Vena Cava thrombosis, elevated factor VIII levels, and breast cancer with stage IIIC and biologically aggressive phenotypes. The authors concern is that there is an increased risk of hypercoagulation states and breast cancer development in patients with MS. The systematic application of long-term preventive DVT may be considered for this group of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4593 Introduction: As deletion of 17p13 (del 17p13) in Chronic lymphocytic leukemia (CLL) patients is associated with poor outcome and chemorefractoriness to alkylating agents and purine analogues, current diagnostic guidelines recommend its screening at diagnosis and prior to treatment initiation. TP53 is a tumor suppressor gene, maps to 17p13 and is inactivated through somatic mutations or by 17p13 deletion. Recent studies show that TP53 mutation is of clinical relevance in CLL patients and has practical implication for therapeutic stratification. The purpose of this study is to characterize the TP53 mutation pattern and its incorporation into treatment algorithms in CLL patients treated in our center. Materials and methods: A retrospective study was performed on a cohort of 42 patients with CLL for whom clinical, biological and fluorescent in situ hybridization (FISH) data were available. Ages ranged from 31 to 88 (median, 63 years), 17 patients were in Binet stage A, 12 in B and 13 in stage C. Treatment was not in controlled trials, and standard clinical criteria were used for the initiation of therapy. DNA was extracted from 52 available samples and TP53 exons 2 to 11 were screened for mutations by high resolution melting (HRM) (Roche LC480). Mutations were confirmed by sequencing both strands using different primer sets (Beckman, Ceq 8000) then validated by the IARC and UMD TP53 Mutation Databases. Results: Out of 42 patients, del 17p13 was observed in 11 (26%), del 11q22 in 8 (19%), del 13q14 in 13 (31%), trisomy 12 in 2 (5%) and normal FISH in 20 (47.6%). We found an overall incidence of TP53 mutations of 19% (8 of 42 patients), 1 new mutation was identified and 5 mutations were not previously reported in CLL but in other cancers according to the IARC and UMD TP53 mutation Databases (table 1). A total of 65 polymorphisms were detected with a prevalence for the g.11446C〉G polymorphism of 0.9 compared to 0.49 in published data. Amongst the 8 patients with TP53 mutations, 7 (87%) presented a 17p deletion and mutations were mainly located in the DNA binding domain (62%). 7 of 11 patients (63%) with 17p13 deletion harbored a TP53 mutation. In the absence of del 17p13 only 3% of patients (1 of 31) had a TP53 mutation (table 2). 5 of 8 patients with mutations (62%) presented with an advanced clinical stage and a median age of 56 years. Clonal evolution leading to acquisition of the TP53 mutation was not necessary related to therapy; 3 sequential samples were available for a patient with no abnormalities at diagnosis and who developed a TP53 mutation after 2 years and before therapy initiation. Another patient who was initially devoid of TP53 mutations and responded to treatment with standard chemotherapy, developed chemorefractoriness concomitant with a TP53 mutation acquisition. An interesting case was that of a patient who had been in complete remission after treatment with rituximab, fludarabin and cyclophosphamid (RFC) despite the presence of a TP53 mutation without del 17p13 at diagnosis. Sequencing demonstrated that this patient harbored a silent mutation in exon 4 g.12441C〉G. In Conclusion: This study shows similar findings to published data except for a higher incidence of patients with TP53 mutations probably due to bias selection and a low cohort of patients. We identified a new mutation and 5 other mutations not previously detected in CLL patients. As TP53 is associated with poor outcome and chemorefractoriness, and its acquisition might be a result of positive pressure selection following treatment, we think it is of utmost importance to look for TP53 mutations in parallel to FISH at diagnosis and in case of chemorefractoriness using the HRM a cheap and sensitive method followed by sequencing positive cases. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4597 Toll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well established that B-chronic lymphocytic leukemia (B-CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course. The aim of this study was to evaluate the gene expression of TLRs in 97 B-CLL patients (median age 74 yrs, range 41–89, 41 female and 57 male) and to relate it with the clinical course of the disease (median follow-up 107 months, range 36–336), in particular with infections and autoimmunity, and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations). The gene expression of TLR4 and TLR9 was evaluated in total RNA of B cell from B-CLL patients and controls (pool of 10 healthy donors) by real-time PCR performed with a model 7300 real-time PCR system (Applied Biosystems). The TLR4 and TLR9 relative quantification expression (RQ) was normalized according to GAPDH (internal control gene) and to control mRNAs. We found that TLR4 gene expression was decreased (RQ=16.1+/−1.56) and TLR9 increased (RQ=2725+/−165) in B-CLL patients vs controls (RQ=100). TLR4 gene expression was reduced in: a) stage Binet B-C/Rai II-III-IV (n=20) versus low risk cases (Binet A/Rai 0-I) (10.8+/−2.2 vs 17.4+/−1.9, p=0.048); b) patients treated with I line therapy (n=25) and II line therapy (n=14) vs untreated patients (n=58) (13.1+/−2 vs 19.2+/−2.3, p=0.05, and 8.3+/−2.1 vs 19.2+/−2.3, p=0.03, respectively); c) patients untreated but with ITT and treated with stable disease or progressive disease (n=28) vs untreated and treated with DFS〉24 months at the time of the investigation (n=69) (12+/−2 vs 17.8+/−2, p=0.048); d) patients with grade 2–4 infections (n=52), according to Common Terminology Criteria for Adverse Events, vs patients with grade 0–1 infections (13.2+/−1.5 vs 19.51+/−2.9, p=0.04); e) patients with autoimmune phenomena (n=12) vs cases without (8.8+/−2.5 vs 17.12+/−1.7, p=0.04). As far as TLR9 is concerned, we found that it was significantly reduced in untreated and treated with II line therapy patients vs patients treated with I line therapy (2549+/−183 vs 3528+/−356, p=0.01, and 1893+/−277 vs 3528+/−356, p=0.01, respectively). Finally, we found that patients with an unmutated IgVH region status (n=15) showed TLR4 gene expression significantly reduced compared with patients with mutated IgVH region status (n=29) (9.57+/−2.1 vs 18.8+/−3.2, p=0.028). (Data are expressed as mean+/−SE for all data). These results show that in B-CLL patients the reduced expression of TLR4 is consistent with a reduced ability to mount a proper immune response and it is even more pronounced in “active” patients that show high prevalence of infectious episodes and autoimmune phenomena. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 461 Aberrant DNA methylation is a hallmark of myelodysplastic syndromes (MDS), MDS/myeloproliferative neoplasms (MDS/MPN) and secondary acute myeloid leukemia (sAML). It provides a rationale for treating these malignancies with hypomethylating agents like 5-azacitidine (AZA) and decitabine (DAC). However, treatment outcomes remain limited and heavily weighed on morphologic/cytogenetic results. The discovery of novel mutations has provided important insight into the pathogenesis of MDS and related disorders. Genes implicated in epigenetic regulation, including DNMT3A, TET2, IDH1/IDH2, EZH2, ASXL1 and UTX have been found mutated in MDS, while others have also been implicated in MDS pathogenesis. There is limited data on the predictive value of these genetic defects for treatment response and disease outcome. We hypothesized that these defects are important biomarkers predictive of response to hypomethylating agents. We studied 88 patients with MDS (RCUD=2, RARS=6, RCMD=11, MDS-U=3, RAEB-1/2=29, CMML1/2=16, MDS/MPN-U=5, RARS-T=5, AML from MDS=11) who received hypomethylating agents (AZA=53, DAC=24, both=11). The median number of cycles was 7 [range 1–35], median age was 69 years (range 42–82) and median follow-up was 18 months (range 0–76). Responses were scored according to IWG criteria. DNMT3A, TET2, IDH1/2, EZH2, ASXL1, UTX, KRAS, NRAS, CBL, RUNX1, TP53 and SF3B1 were sequenced using standard techniques. Categorical variables were analyzed using Chi-square statistics. Overall survival (OS) was analyzed using Kaplan-Meier; p-values ≤ 0.05 were considered statistically significant. Mutated patients were older than wild type (WT) cases (72 vs. 68 years, p=.01) but were well matched for marrow blast %, cytogenetic risk group and cycles of hypomethylating agents received. We found mutations in 40/88 (45%) patients. Mutations were most frequent in SF3B1 (6/11; 55%), ASXL1 (13/50; 26%), TET2 (18/88; 20%), KRAS (3/34; 9%), and DNMT3A (7/88; 8%). Less common were mutations in EZH2 (2/43; 5%), TP53 (1/23; 4%), IDH1 (4/88; 5%), IDH2 (3/88; 3%), and UTX (1/36;3%). No mutations were found in CBL, NRAS or RUNX1. Based on single mutations, overall response rate (ORR) was higher in mutated vs WT patients for DNMT3A (6/7 [86%] vs 33/81 [41%]; p=.02), ASXL1 (11/13 [85%] vs 14/37 [38%]; p=.003), and TET2 (12/18 [67%] vs. 27/70 [39%]; p=.03). All heterozygous DNMT3A mutants responded to hypomethylating agents. Differences remained significant when stratified to AZA treatment alone for DNMT3A (6/7 [86%] vs 21/56 [38%]; p=.01) and ASXL1 (9/11 [82%] vs 12/29 [41%]; p=.02) but not TET2 (6/10 [60%] vs 21/53 [40%]; p=0.22). The predictive value of combined mutations were analyzed for DNMT3A, TET2 and/or IDH1/2, showing better response to hypomethylating therapy in patients who had a mutation; ORR (mutated: 18/28 (64%) vs WT: 21/60 (35%); p=.01). This difference remained significant in patients receiving only AZA (n=53); ORR was 11/18 (61%) in mutant and 11/35 (31%) in WT patients (p=.03). No differences in ORR were noted for KRAS, EZH2 and IDH1/2 mutant and WT patients. No SF3B1 mutants responded to treatment while both patients with UTX and TP53 mutations responded. The frequency of AML evolution was also analyzed and showed no difference between mutant and WT cases for TET2 (7/18 [39%] vs 22/70 [31%];p=.52), ASXL1 (4/10 [40%] vs 11/35 [31%]; p=.61), and DNMT3A (3/7 [43%] vs 26/81 [32%];p=.56). No differences in OS and progression free survival (PFS) were noted between responders and non-responders to hypomethylating therapy (28 vs 17 mos, p=.25; 16 vs 8 mos, p=.54). Comparison of survival outcomes for mutant and WT patients showed no significant difference for DNMT3A (OS: 30 vs 21 mos, p=0.43; PFS: 20 vs 11, p=.53), ASXL1 (OS: 28 vs 22, p=.68; PFS: 16 vs 10, p=.88), and TET2 (OS: 30 vs 20 mos, p=.30). PFS was better in TET2 mutants compared to WT (19 vs 9, p=.03). No survival differences were noted between mutant and WT cases who responded to hypomethylating agents for DNMT3A (OS: 25 vs 28,p=.84; PFS: 14 vs 16, p=.78), ASXL1 (OS: 10 vs 18, p=.48; PFS: 10 vs 6, p=.76) TET2 (OS: 27 vs 16, p=.79; PFS: 18 vs10, p=.19). In conclusion, DNMT3A, ASXL1 and TET2 mutations were independently associated with a better response to hypomethylating drugs. Moreover, combined mutations in DNMT3A/TET2/IDH1/IDH2 may influence the response to hypomethylating agents, especially AZA supporting its role as a predictive biomarker in MDS treatment. Disclosures: Maciejewski: Celgene and Eisai, NIH, AA&MDS Foundation: Research Funding. Tiu:MDS Foundation Young Investigator Award: Research Funding.

Description: Abstract 4609 Active NOTCH signaling is observed in an increasing number of human neoplasias including chronic lymphocytic leukemia (CLL) and represents a potential therapeutic target. We have recently reported that the γ-secretase inhibitor DAPT may not be effective in all cases of CLL (Hubmann et al; BJH 2010). The aim of this study was to evaluate the therapeutic value of a newly identified NOTCH transactivation inhibitor (gliotoxin) and to compare its efficiency with the highly selective γ-secretase inhibitor (GSI) DAPT in CLL cells. Electrophoretic mobility shift assays (EMSA) showed that gliotoxin completely blocked the formation of DNA-bound NOTCH2 in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signaling by gliotoxin was associated with down regulation of its target gene CD23 and induction of apoptosis. The IC50 of Gliotoxin was variable between patients (n=20) and ranged between 50–100 nM irrespective to their sensitivity to GSI. Short term (4 hours) exposure of CLL cells to gliotoxin revealed that gliotoxin modulates the mRNA expression of several NOTCH-related genes including JAG1, PRKCD (PKC-δ), and NR4A1. We (Shehata et al; BLOOD 2010) and others have reported on the supportive effect of primary bone marrow stromal cells (BMSC) for CLL cells. Therefore, we tested whether gliotoxin may overcome this supportive effect. FACS analysis and MTT assays showed that gliotoxin abolished the supportive effect of BMSC under co-culture conditions. The effect of gliotoxin was dose dependent and selectively induced apoptosis in CLL cells accompanied by down regulation of NOTCH2 and CD23 transcription. Western blotting analysis demonstrated that gliotoxin also decreased the phosphorylation of Akt-pSer473 suggesting a link to PI3-K signaling. NOTCH1 has been recently shown to be affected by “gain of function” mutations in a subset of CLL patients. We observed that co-culture of CLL cells with BMSC was associated with increased NOTCH1 mRNA expression which could be decreased upon exposure to gliotoxin. In addition, gliotoxin inhibited DNA-bound NOTCH1 complexes in the T-ALL cell line SupT1 which is known to express high NOTCH1 activity. This indicates that gliotoxin may target both NOTCH1 and NOTCH2 isoforms. In conclusion, the data show that gliotoxin effectively targets NOTCH activity in CLL cells in a mechanism which is independent of γ-secretase. Thus, gliotoxin might have a beneficial effect in a wider range of CLL patients and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 490 Telomeres are highly conserved protective terminal chromosomal structures consisting of hundreds of repeated TTAGGG hexamers and associated shelterin proteins. Telomeres shorten with every cell cycle, and telomere attrition has a fundamental role in cell senescence. Telomeres of leukocytes are shorter in transplant recipients than in their donors. Dyskeratosis congenita, a congenital aplastic anemia caused by mutations in the telomerase complex genes, is associated with treatment related mortality (TRM) after hematopoietic stem cell transplantation (HSCT). We hypothesized that age-adjusted pre-transplant telomere length might generally predict TRM after HSCT. Between 2000 and 2005, 178 consecutive patients underwent HSCT from HLA-identical sibling donor after myeloablative conditioning regimen (including TBI in 57 patients), mainly for hematological malignancies (n= 153) in our center. The stem cell source was bone marrow (BM) in 128 cases and peripheral blood (PB) in 50 cases. Median age at transplant was 32 years (range 3–65). Graft-versus-host disease (GvHD) prophylaxis mostly consisted of cyclosporine and methotrexate (n=149, 84%). Before HSCT, blood lymphocytes were obtained from each of the donor-recipient pair. Telomere length was assessed by real time quantitative PCR. We first determined the normal age distribution of telomere length using a group of 173 healthy French hematopoietic stem cell donors (f=-0.00833*age+1.522) as a control group. We then calculated the pre-transplant recipient age-adjusted telomere length in comparison to controls. After age adjustment, we categorized the population in quartiles (shortest telomeres for quartile 1) and analyzed the outcome post HSCT using competing risk in univariate and multivariate analyses (Fine and Gray). The mean telomere length in transplant recipients (1.05) was shorter than in the control group (1.23, p= 0.0001). After age-adjustment, patients' distribution was similar among all four quartiles except for disease severity (more high risk disease was present among patients with the shortest telomeres). The median follow-up was 51 months (range, 1 – 121 months). All patients engrafted. The median time to achieve absolute neutrophils count 〉500/ul was 18 days (range 4–45) and median time to platelet count 〉20.000/ul was 17 days (range 7–58). Cumulative incidence (CI) of acute GvHD grade II-IV was 45% (95% confidence interval [95CI] 37%–53%) and of chronic GvHD was 41% at 36 months (95CI 33%–49%). Thirty-four patients relapsed: CI: 22% at 5 years (95CI 16%–28%). There was no correlation between telomere length and engraftment, acute or chronic GvHD or relapse. The overall survival was 62% at 5 years (95CI 54%–70%). During the study, 37 patients died due to TRM. TRM rate inversely correlated with telomere length. In the first quartile, the 5-year CI of TRM was 33% (95CI 2%–22%), 20% (95CI, 8%–32%) in the second quartile; 20% (95CI, 8%–32%) in the third quartile; and 12% in the fourth quartile (95CI, 2%–22%) (p=0.06). When quartiles 2, 3 and 4 were pooled, the increased TRM in first quartile was statistically significant (p = 0.017) (Figure 1). In multivariate analysis using competing risk regression, (including age-adjusted telomeres length, disease stage, age, TBI and source of stem cells), age of the recipients (HR: 1.1, 95% CI [.0–1.1, p=0.0001] and age-adjusted telomeres [HR: 0.4, 95% CI [0.2–0.8, p=0.01]) were independently associated with TRM. The same two factors remained significant in subset analysis of patients with malignant diseases (n=154) (p= 0.0004, HR: 1.1 and p=0.018, HR: 0.43, respectively). No association was found between donor telomere length and outcome post HSCT. In conclusion, age-adjusted recipient pre-transplant telomere length is an independent biological marker of TRM after HSCT from related donors using a myeloablative conditioning regimen and cyclosporine-based GvHD prophylaxis. Disclosures: Peffault de Latour: Alexion: Consultancy, Research Funding.

Description: Abstract 4879 Background. A hematological malignant animal model is an essential tool for evaluating efficacy of anti-cancer drugs and elucidating underlying mechanism of leukemogenesis. Intraperitoneal (IP) and intravenous (IV) xenograft of acute lymphoblastic leukemia (ALL) cells have limited capacity as in vivo anti-cancer drug screening system. Purpose. In this study, we aimed to establish an ALL animal model using NOD/SCID mouse and evaluate efficiency and sensitivity of the model as a preclinical drug screening system. Materials and Methods. Firefly luciferase (fLuc)-gene introduced ALL (ALL/fLuc) cell line and patient-originated ALL cells were transplanted into a tibia of NOD/SCID mouse. We conducted a comparative analysis of intra-bone marrow (IBMT) transplanted leukemia model with IP and IV transplantation of leukemic cells. Results. IBMT of ALL/fLuc cells effectively established a bioluminescent leukemia NOD/SCID mouse model. Upon comparison of IBMT model with IP and IV transplantation models, infusing identical number of ALL/fLuc cells into NOD/SCID mice resulted in IBMT model with evaluable bioluminescent signal, but not in IP and IV models. In IBMT model, bioluminescent signals of ALL/fLuc cells emitted from peripheral blood, tibia and infiltrated organs indicated that leukemia model was established. The changes in these signals' strength reflected dose-dependent cytotoxic effects of vincristine, which allowed leukemia model with evaluable bioluminescent signal to be utilized as a preclinical drug screening system. IBMT leukemia model was also established using primary ALL cells that can provide additional insights for the development of leukemia therapeutics. Conclusion. IBMT of ALL/fLuc cells enables development of leukemia mouse model with the greater bioluminescent sensitivity than IP and IV in NOD/SCID to evaluate candidate for development of anti-cancer drug. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4773 Background: Venous thromboembolism (VTE) prophylaxis has been recommended in clinical guidelines as an appropriate strategy for hospitalized cancer patients based on evidence of reduced VTE events and reduced mortality. However, current guidelines do not specify the appropriate length of VTE prophylaxis in this population. Real world data is needed to understand the prevalence of symptomatic and confirmed VTE for this patient population to help clinicians determine strategies regarding the appropriate duration of treatment. Objective: To document the incidence of symptomatic late VTE events in hospitalized patients who have active cancer. Methods: Charts from 1134 consecutive medical patients age 〉 60 years who were hospitalized in the Calgary region and discharged between January and February 2008 were abstracted using standardized case record forms. All hospitals in the region use a common unique patient identifier number, thus enabling the tracking of subsequent patient visits to the emergency room, inpatient admissions or outpatient visits occurring anywhere in the region's acute care system. Any identified patient was followed for a subsequent visit related to VTE. Active cancer patients were defined as those who have a cancer diagnosis at hospital admission and have a planned cancer surgery or receiving cancer treatment or were receiving palliative treatment or whose cancer treatment was not specified. Records were excluded if the patient was admitted for VTE or to rule out VTE, receiving chronic anticoagulation, experiencing acute coronary syndromes, had a hospital stay ≤ 3 days, had a remote cancer history, was a surgical or orthopedic patient, or pregnant. Data was collected on the timing of VTE related events for up to 100 days post discharge. Results: A total of 358 patients met criteria over the review period. Seventy-three percent (261/358) of all active cancer patients received mechanical or pharmacological prophylaxis in hospital. Twenty-three percent of these patients were identified as requiring medical care for symptoms associated with VTE. Confirmation of VTE by diagnostic testing occurred in 4.8% (95% CI, 2.6% to 7%) while the other 18% (95% CI, 14.0% to 22%) had diagnostic tests that were negative or inconclusive. The mean length of time to confirmed first VTE event was 38.2 days post admission. Conclusion: This study demonstrates that in a real life setting 23% of active cancer patients would develop symptoms of VTE requiring a health professional's attention with 4.8% having VTE confirmed by diagnostic testing. These events occurred despite thromboprophylaxis in hospital and suggest that the risk of symptomatic VTE could be higher in real life compared to that reported in randomized clinical trials where patients are screened for asymptomatic VTE. These findings show that the prevalence of VTE warrants consideration of extended thromboprophylaxis in active cancer patients, as the benefits of extended prophylactic therapy may outweigh the risks in this population. Disclosures: Hull: sanofi-aventis: Consultancy; LEO Pharma: Consultancy; Pfizer: Consultancy; Portola: Consultancy; Merck: Consultancy; Bayer: Consultancy; Johnson & Johnson: Consultancy. Merali:sanofi-aventis: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Abbott: Consultancy; Boehringer Ingelheim: Consultancy; Genzyme: Consultancy; LEO Pharma: Consultancy; Nycomed: Consultancy; Otsuka: Consultancy. Mills:Pfizer: Consultancy; sanofi-aventis: Research Funding. Komari:sanofi-aventis: Employment.

Description: Abstract 4703 Background: Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic hematopoietic stem cell transplantation. Corticosteroids are the standard initial treatment for cGVHD. A second-line treatment is not well defined. We prospectively evaluated the effectiveness and safety of low doses of alemtuzumab and low doses of rituximab as treatment steroid-refractory cGVHD. Materials and Methods: Ten men and 5 women with cGVHD refractory to steroids and CSA were included. All patients received subcutaneous Alemtuzumab 10 mg/day/3 days and intravenous Rituximab 100 mg on days +1, +7, +14 and +21. Results: The median age was 41 years. The main organ involved were oral mucosa (86.7%), eyes (66.7%), liver (60%), skin (53%), lungs (13.3%) and intestinal tract (6.7%). The overall response was 100% at 30-day evaluation; 10 patients (67%) had partial remission (PR), and 5 patients (33%) had complete remission (CR). At 90-day evaluation, 7 (50%) patients had PR, 4 (28%) had CR; three (21%) relapsed and 1 patient not reached evaluation. Currently, 5 patients have reached the 365-day follow-up evaluation, 2 (40%) had PR, 2 had CR and 1 showed progression. The adverse effects were mainly infectious and one patient died from pneumonia. Conclusion: This combination therapy appears to be an efficacious and safe as treatment for steroid-refractory cGVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4725 Chemotherapy is widely used in treatment of myeloid leukemia, the efficancy of which, however, is often hampered by the development of intrinsic and acquired multidrug resistance(MDR), the exact mechanism of which is still unclear. Overexpression and deregulated activation of protein tyrosine kinase(PTK) are frequently observed in several types of hematologic malignancies, the abnormally signal cascade transducted by which has been demonstrated to play an important role in antiapoptosis, differentiation block, enhancing autonomous proliferation, and also in inducing drug resistance. EphB4 is also a protein tyrosine kinase, a member of the largest family of receptor tyrosine kinase, which has been found with abnormally upregulated expression or activity in many types of cancer especially solid tumor types, such as mammary adenocarcinoma, colon carcinoma, ovarian cancer and prostatic carcinoma. Here the aim of our study was to examine the expression and biological role of EphB4 in drug resistance of myeloid leukemia. Using RT-PCR assay and western blot, we tested the mRNA level in 35 myeloid leukemia patients including 4 cases of acute myeloid leukemia(AML) with primary multiple drug-resistance(MDR), 3 cases of AML which have relapsed, 5 cases of newly diagnosed AML, 5 cases of AML which got (complete remission) CR at the first chemotherapy treatment, 9 cases of chronic myeloid leukemia in chronic phase(CML-CP), and 9 cases of CML in blast crisis(CML-BC). The results showed the EphB4 mRNA level was significantly upregulated in AML bearing relapse(EphB4/β-actin ratio:0.962±0.114) or MDR(EphB4/β-actin ratio: 0.993±0.047) and also in CML-BC(EphB4/β-actin ratio: 1.001±0.060) compared with newly diagnosed AML(EphB4/β-actin ratio: 0.332±0.014), AML with CR(EphB4/β-actin ratio:0.401±0.015) and CML-CP(EphB4/β-actin ratio: 0.432±0.020). Subsequently, we examined both the transcriptional and translational level of EphB4 in several myeloid leukemia cell lines including K562, HL60,U937,KG1α and an adriamycin-resistant cell line—HL60/ADM, and drug-resistant capacity of the five cell lines was also tested by CCK-8 assay. Finally EphB4 protein expression is found to be upregulated at both transcriptional and translational level in K562, KG1αand HL60/ADM, which showed stronger capacity of resistance to gradient concentrations of adriamycin(IC50 of K562:0.451±0.037ug/ml,KG1α:0.217±0.017ug/ml, HL60/ADM: 2.663±0.102ug/ml) compared with that of HL60 and U937(IC50 of HL60:0.040±0.001ug/ml, U937:0.040±0.005ug/ml) in which little or no expression of EphB4 mRNA or protein was observed. And the most noteworthy is that HL60/ADM, which shows the strongest drug resistant capability. also bears the most amount of EphB4 at both transcriptional level (EphB4 /β-actin ratio: 1.002±0.017), and translational level (EphB4 /β-actin ratio: 0.975±0.051). These data supports a role for EphB4 in inducing drug resistance and raise the possibility that therapeutic intervention to EphB4 expression or signaling might inhibit or even reverse drug resistande in meyloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4707 Introduction. Graft-versus-host disease (GVHD) is the most common complication of allogeneic hematopoietic cell transplantation (alloHCT) and may affect the transplant outcome. Its incidence is higher when the preparative regimen used is a non-myeloablative and the stem cell's source is peripheral blood after mobilization. Objective. Demonstrate the 10 years incidence of GVHD in 2 Mexican transplant centers using peripheral hematopoietic stem cell transplantation (PHSCT) in related donors after non-myeloablative conditioning. Patients and methods. Three hundred and four patients with hematological and non-hematological malignancies that underwent outpatient PHSCT after non-myeloablative conditioning between October 1998 and July 2008 were included. The age ranged between 1 and 71 years (median of 30.5). One hundred and eighty-five patients were men and 119 women. The median of cells CD34+ infused was 4.9 × 106/kg (0.23-17.70).They received cyclosporine 4mg/kg per day and intramuscular methotrexate 5mg/ m2 in days +1, +3, +5 and +11 for GVHD prophylaxis. Results. Two hundred thirty-nine (80%) patients were successfully engrafted. The conditioning regimen was delivered as an outpatient procedure in all individuals. One hundred and fifty-four patients (64%) developed acute and/or chronic GVHD. Sixty four patients (26.7%) developed acute GVHD, 50 (20.9%) developed chronic GVHD, and 40 (16.7%) with acute GVHD progressed to chronic. Twenty seven (26%) patients who developed acute GVHD were grade III or IV, and 30 (33.2%) of chronic GVHD patients presented in the extensive way. GVHD was the cause of dead in 40 patients; 24 of acute GVHD and 16 of chronic GVHD, even when immunosuppressive therapy (high dose steroids and rituximab) was used. Last death was 24 months ago; subsequently, alemtuzumab was included in GVHD treatment. The cumulative incidence of acute GVHD was 37.6%. Conclusion. The higher incidence of GVHD reported in other studies after PBSC transplant seems to decrease if non-myeloablative conditioning regimens are used. We suggest that the combination of a reduced-intensity conditioning transplant and the outpatient procedure is the responsible for the low incidence of GVHD in our patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4713 The generation of induced pluripotent stem cells (IPSCs) from patients with specific diseases has improved the possibilities of dissecting the etiology of human pathological conditions and has opened potential new therapeutic options for the treatment of human diseases. One of the major safety concerns in the generation of hiPSCs is the persistence of copies of the reprogramming genes in the genome of iPSCs. Additionally, the use of cells from accessible tissues, like peripheral blood, constitutes preferential options for cell reprogramming, compared to other cells whose collection would require a more aggressive intervention. Here we have selected Pyruvate Kinase Deficiency (PKD) as a human disease affordable to be modeled and genetically corrected using hiPSC technology. PKD is an autosomal recessive disease that affects the erythrocyte R-type Pyruvate Kinase (RPK) enzyme by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Deficiency in the RPK, the last enzyme of the glycolisis, reduces the energetic balance of red blood cells and therefore affects the viability and function of the erythrocytes. Clinical symptoms of PKD can progress from mild to severe anemia and can be lethal in the most severe cases. Peripheral blood mononuclear cells (PB-MNC) from healthy donors and from a PKD patient were infected with five independent recombinant non integrative RNA Sendai viruses (SeV) encoding four human reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC) and the Azami green protein as reporter. After infection with SeVs, no differences in the reprogramming efficiency of healthy and PKD PB-MNC were observed (1 hES-like colony per 6,000 transduced healthy or PKD PB-MNC). Integrated SeVs were not found in PBMNC-hiPS lines, as deduced from the lost of Azami green expression and also from RT-PCR analyses of the reprogramming factors. Healthy and PKD PBMNC-hiPSCs could be cultured for more than 15 passages, indicating their self-renewal ability. The pluripotent characteristics of these PBMNC-hiPS were confirmed by the analysis of expression of endogenous pluripotent genes by Q-RT-PCR and immunofluorescence. This study confirms the feasibility to generate integration-free hiPS lines from healthy and PKD PB-MNC. These hiPSC lines constitute an invaluable tool for the development of a human model of PKD and for applying new gene therapy strategies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 470 Multiple myeloma (MM) is an incurable and fatal neoplasm characterized by the accumulation of clonal plasma cells (PC) that results in significant end organ and tissue damage. MM is preceded by either of two premalignant asymptomatic stages, monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), both of which are identified by the presence of clonally expanded abnormal PC populations. While MGUS and SMM patients' abnormal PC populations may remain stable for years, both have a life-long significantly increased risk of progressing to MM (1% and 10% per year, respectively). For this reason, a better understanding of the molecular events prompting malignant progression and increased accuracy in identifying those patients that have begun to transition to MM is urgently needed. In this study, we identified a novel MM marker, CD147 (also known as extracellular matrix metalloproteinase inducer (MMP), or EMMPRIN), that is not only over-expressed on MM PCs as compared to its premalignant counterparts, but whose increased expression correlates with the level of abnormal PC proliferation. To our knowledge, we are the first to demonstrate a role for CD147 in MM. CD147 has been shown by others to display a variety of activities and those with potential relevance to MM include stimulation of increased MMP production and angiogenesis, and playing a critical role in glycolysis via facilitation of excess cellular lactate transport. Our initial experiments revealed that MM PCs overexpress CD147 mRNA relative to MGUS PCs. Flow cytometric analysis corroborated these data and demonstrated variable expression of CD147 across the disease continuum ranging from no expression to bimodal or uniform expression. Indeed, there was a significant difference between CD147 expression on MGUS and SMM PCs compared to that on MM PCs (p=0.02 and 0.005, respectively). We next determined whether CD147 had a signaling role in these cells. Using the natural CD147 ligand, cyclophilin B (CypB), we showed that addition of CypB to either human MM cell lines (HMCLs) or CD138+ patient PCs resulted in increased PC proliferation as measured by [3H]-thymidine incorporation. In a complementary manner, addition of CD147 antibodies significantly inhibited proliferation without an effect on cell viability. By western blot analysis we further demonstrated that CypB-mediated CD147 activation leads to MAPK phosphorylation. Next, we isolated CD147+ and CD147- MM cells from patients whose tumor cells bimodally expressed this marker and assessed the response of each subset to IL-6 and CypB. The CD147+ subset was almost solely responsible for the proliferative response in all cases examined. In addition, we cultured bone marrow mononuclear cells from CD147 bimodally expressing MM patients overnight with bromodeoxyuridine before performing cell cycle analysis on the CD147+ and CD147- MM PC populations. Remarkably, the CD147+ PCs were greatly enriched for cells in the S and G2/M phases of the cell cycle, whereas the CD147- PCs resided almost entirely in the G0/G1 phase. In the final set of experiments, we employed siRNA knockdown strategies using HMCLs to definitively test the role of CD147 in MM cell proliferation. Indeed, IL-6 induced proliferation was significantly compromised following CD147 down regulation, which was not attributed to increased apoptosis. However, IL-6 mediated phosphorylation of MAPK remained robust suggesting that the IL-6 signaling pathway overall was not compromised in these cells. Finally, cell cycle analysis demonstrated that CD147 down regulation resulted in a significant increase in the number of cells in the G0/G1 phase of the cell cycle and a decrease in the number of cells in the G2/M phase of the cell cycle, as compared to cells transfected with control siRNA. In conclusion, our data suggest that the CD147 molecule plays a critical role in the biology of malignant MM PCs, particularly as it concerns MM cell proliferation, and may thus serve as a useful and attractive target for reducing the proliferative compartment of this disease. Ongoing studies are investigating additional roles for MM cell CD147 expression, e.g., its role in MMP induction in the tumor microenviroment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4708 Background: Systemic inflammation is well appreciated to play a role in the pathogenesis of acute graft-versus-host disease (aGVHD). Recent studies have focused on homeostatic mechanisms that counteract inflammation and heal damaged tissues. These include the generation of lipoxins, resolvins, and protectins, eicosanoid molecules collectively referred to as the anti-inflammatory and pro-resolving lipid mediators. These lipid mediators have biologic activities known to antagonize mechanisms underlying the development of aGVHD, including downregulation of pro-inflammatory cytokines such as TNFα. Resolvins and protectins are derived from the Ω3-polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. Diets enriched for Ω3-PUFAs (high fish oil) are protective against inflammatory conditions, whereas “Western” diets with higher Ω6-PUFA promote inflammation. Furthermore, the anti-inflammatory properties of acetylsalicylic acid (ASA) are increasingly being attributed to its ability to enhance the generation of aspirin-triggered lipoxins, epimers of endogenously produced lipoxins. Hypothesis: We hypothesized that a diet enriched for Ω3-PUFAs, ASA ingestion or the injection of a purified aspirin triggered lipoxin would mitigate the development of aGVHD and provide a survival advantage in a major histocompatibility mismatched mouse model of aGVHD. Methods: The C56BL/6→(C57BL/6 × DBA/2)F1-hybrid mouse model was used to induce lethal aGVHD. Five groups (n=10) were established and treated as follows: Recipients were fed the diets for 8-weeks before transplantation. The Ω6-diet (control) was enriched to 2% total FA with arachidonic acid (ARASCO, Martek). The Ω3-diet was enriched to 1% each of EPA (ROPUFA-30, DSM Nutritionals) and DHA (DHASCO, Martek). Chromatography was used to confirm the fatty acid (FA) composition of each diet, and the FA content (Ω6 and Ω3 tissues stores) of the mouse livers (n=4/diet) after 8-weeks of feeding. Diets were the same for each group pre- and post-transplant. Using identical methodology, a second experiment was performed in which the recipients were treated as follows: Ω6-diet plus 15-epi-LxA4 100 mcg/kg/day, i.p. daily from day 0 until death (n=10). Survival post-transplant was the primary outcome. Mice were euthanized when they showed signs of GVHD-associated morbidity and had reached their humane endpoint as defined in our animal protocol. Results: Compared to mice fed the Ω6 or Ω6 plus ASA diets, the Ω3 and Ω3 plus ASA diets resulted in a higher hepatic content of both EPA and DHA after 8-weeks of feeding, suggesting differential tissue stores of these Ω3-PUFAs immediately before transplant in the mice fed the Ω3-diets (mean EPA as % of total FA: 0.22% and 0.19% vs. 0.92% and 1.03%, respectively; mean DHA as % of total FA: 1.58% and 1.79% vs. 6.99% and 8.15%, respectively: p

Description: Abstract 2237 Chronic inflammation has been recognized as a major factor in the development and progression of multiple cancers. A prime example of this is the strong association between colitis and colon cancer. However, the specific factors that regulate disadvantageous immune processes in the context of inflammation-associated cancers remain poorly defined. Growing evidence suggests that hemostatic system components, traditionally associated with the maintenance of vascular integrity and prevention of blood loss, also directly regulate inflammatory processes. Furthermore, thrombin and several thrombin targets (e.g., PARs, fibrinogen, factor XIII) have been shown to regulate tumor cell proliferation and apoptosis, support metastasis, and protect tumor cells from innate immune surveillance mechanisms in other experimental contexts. A logical extension of these findings is the hypothesis that thrombin, as a master regulator of both inflammatory processes and tumor cell biology, is a major determinant of the progression of inflammation-driven cancers such as colitis-associated colon cancer (CAC). To test this hypothesis, we induced CAC in mice carrying prothrombin levels 50% of normal (fII+/−) and WT mice in parallel using an established two step protocol consisting of azoxymethane (AOM) and dextran sodium sulfate (DSS) exposure. The modest diminution in prothrombin levels imposed by the fII+/− genotype resulted in a dramatic diminution in the number of colonic adenomas formed after AOM/DSS challenge relative to WT mice. In order to determine if the diminution in adenoma formation observed in fII+/− mice was coupled to thrombin function, wildtype mice challenged with AOM/DSS were treated with daily i.p. injections of hirudin, a direct thrombin inhibitor, or saline carrier. Similar to the finding in mice with a genetically-imposed diminution in circulating prothrombin, hirudin treatment significantly blunted adenoma formation. To determine if reduction of thrombin generation improved the inflammation preceding the development of colonic adenomas, we used a novel, highly-specific factor XI antisense oligonucleotide “gapmer” (ISIS Pharmaceuticals) to inhibit hepatic factor XI synthesis prior to DSS challenge. Gapmer-mediated diminution of fXI levels to ∼15% of normal resulted in a dramatic improvement in colitis related symptoms. Gapmer-treated mice had less intestinal bleeding and weight loss associated with DSS challenge relative to mice treated with a control oligonucleotide. Consistent with these gross observations, microscopic analyses of colonic tissue showed that fXI gapmer treatment significantly limited mucosal ulceration. Factor XI gapmer treatment also significantly diminished local levels of several inflammatory cytokines known to play a role in colon cancer progression (i.e., IL-6, IL-1β, IL-12). These results demonstrate that thrombin is a crucial driver of the pathogenesis of colitis-associated colon cancer and suggest that therapies directed at thrombin or thrombin generation could treat or prevent inflammation-driven colon cancer. As pathological inflammation has been estimated to account for as many as 1 in 5 cancer-related deaths, thrombin-directed therapies could have broad applicability to multiple malignancies. Disclosures: Mullins: Baxter: Consultancy. Monia:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. Palumbo:Novo Nordisk: Research Funding.