ALBERT

Description: BACKGROUND Patients with hemophilia seldom need joint arthroplasty today, due to decrease of bone and cartilage damage under factor prophylaxis started early childhood. However, hemophilia patients with inhibitors often develop severe joint damage as a result of ongoing bleedings. It is a formidable challenge to manage post-operative bleedings with bypassing agents in such cases. There have been only a few reports of major hip surgery in hemophilia A with inhibitors. Here we report a case of successful total hip replacement in an 18 year old adolescent boy with high titer inhibitor since infancy under recombinant factor VIIa (rVIIa) coverage. PATIENT The patient has been followed up with high titer inhibitor ranging between 35- 95 BU since he was three years old and received mostly on demand treatment with either rVIIa (NovoSeven®) or activated prothrombin complex concentrate (APCC) (FEIBA®). Prophylaxis with APCC and rVIIa were also given for 3 years and 6 months, respectively. During the last two years, he experienced frequent bleedings in his right hip joint and developed crippling joint damage and assessed as grade IV according to the World Federation of Hemophilia grading system leading to severe chronic pain requiring opiate analgesics, shortened right leg and difficulty in walking and climbing. Preoperative inhibitor titer was 64 BU. Intravenous rVIIa was given in a dose of 270 mcg/kg, 30 minutes before surgery, repeated in 120 mcg/kg dose two hours after the first dose and continued in 90-120 mcg/kg doses during the first 24 hours. Intraoperative hemostasis was excellent and only two units of red cell transfusion was needed. However, he developed severe bleedings at the surgery site in the second day of surgery, which was partially controlled with 270 mcg/kg rVIIa. Four units of red cell transfusion was given and tranexamic acid 4x1 g daily was started. Since 120 mcg/kg rVIIa in every 2-3 hours did not stop bleeding, we added 50IU/kg APCC twice daily, rVIIa was reduced to 90 mcg/kg and tranexamic acid was stopped. No thromboprophlaxis was given. Although bleeding gradually decreased eventually over the next 3 days, he developed high fever with increased CRP level and re-bleeding at the surgery site at the fifth day suggesting wound infection. Broad spectrum antibiotics combined with bypassing agents was continued for 21 days during which no bleeding was noted. At 1 month, he had excellent joint function and clear surgery site. He is still receiving only APCC prophylaxis 50 U/kg three days weekly. Treatment cost was calculated as postoperative changes in rVIIa and APCC doses related to the size of vials and patient weights. Total amount of treatment cost was approximately 610.000 $ US for rVIIa and 66.000 $ US for APCC during hospitalization. CONCLUSIONS Total hip replacement is rarely performed in young hemophilia patients with high titer inhibitor due to difficulty in management of bleedings and high cost of treatment product. However, it can be unavoidable in severely crippled patients, incapable of doing daily activities and suffering from intense pain. Our experience in the present case is encouraging for performing hip arthroplasty in such cases. No excessive bleeding occurred during surgery under rVIIa treatment. Combined use of rVIIa and APCC for secondary bleedings during post-operative period is safe and effective. High cost of surgery emphasize the need for development of suitable prophylaxis regimes in children with inhibitor to prevent crippling arthropathy. Disclosures No relevant conflicts of interest to declare.

Description: Large heterogeneity in bleeding phenotype is observed among patients with severe hemophilia A. Some severe patients do not require regular replacement therapy, so to address the factors influencing phenotypic Heterogeneity in Patients with Severe Hemophilia A seems particularly important. We first initiate a cohort study 0f 274 patients with severe hemophilia A who were treated at the hemophilia treatment centre at Nanfang hospital, then 209 patients were enrolled and their bleedings were recorded, finally 12.9% (12/209) of the patients were found to be mild phenotypes, which annual bleeding rate(ABR)were less than 6 times. We then use Flow-cytometric analysis to detect the platelet-derived microparticles (PMPs) among patients with mild phenotypes, patients with severe phenotypes (ABR more than 24) and the normal control group, megamix beads (0.5μm; BioCytex, Marseille, France), CD42a-PE, CD42b-PE, CD41b-PE (GPIX, platelet MPs [PMPs], BD, NJ, USA) were involved, then the results showed that there was no difference in the level of PMPs between hemophliac patients and the normal control group (P=0.317), however, the level of PMPs in patients with mild phenotypes were significantly higher than patients with severe phenotypes (3.65±1.38 vs 1.13±0.64, P

Description: Background: Administration of rFVIIa is used as a method of choice in the treatment of hemophilia pts with inhibitors. Besides, rFVIIa may be useful in thrombocytopenic pts with inefficiency or impossibility of platelet transfusions due to alloimmunization or religious beliefs. Aim: The aim of the present study was to compare the effects of rFVIIa on hemostasis in thrombocytopenic pts and hemophilia pts with inhibitors. Patients and Methods: rFVIIa was used in 20 pts with thrombocytopenia (I group) and in 20 hemophilia pts with inhibitors (II group). Indications for use of rFVIIa in thrombocytopenic pts were: gastrointestinal and intra-abdominal bleeding, intracranial hemorrhage, insertion of the central venous catheter, lumbar puncture, bleeding from the femoral artery puncture site, epistaxix and pulmonary hemorrhage. In thrombocytopenic pts blood platelet count varied from 1*109/l to 72*109/l (median 15 *109/l). The media dose of rFVIIa in thrombocytopenic pts was 79 μg/kg (ranges from 50 to 144 μg/kg). All hemophilia pts received a single dose of rFVIIa (90 μg/kg). All pts received rFVIIa (Generium, Russia). APTT, Quick prothrombin test (QPT), FVII:C level, endogen thrombin potential (ETP) and Thromboelastography (TEG) parameters (R, MA) were evaluated before and after rFVIIa administration. The median and interquartile range (IQR) were calculated as descriptive statistics. Mann-Whitney U test and Mood's median test were used to evaluate group differences. Statistical analyses were performed with SAS 9.1 (using the Npar1way procedure). Results: rFVIIa was effective in all hemophilia pts. Administration of rFVIIa in 15 min shortened but did not correct APTT, increased QPT and ETP, FVII:C (Table 1). Prior to treatment the hemophilia pts have no clotting determined by TEG, but after rFVIIa administration TEG demonstrated close to normal pattern. In thrombocytopenic pts after rFVIIa administration bleeding stopped in 51% pts, decreased in 44% and did not change in 4% pts. In thrombocytopenic pts maximal hemostatic effect was achieved within 1 hour after rFVIIa administration (Table 1). 1 case of ischemic stroke was obtained. The maximum effect of rFVIIa in thrombocytopenic pts coincided with decrease of plasma activity of FVII perhaps due to its consumption. Conclusion: In the most hemophilia pts with inhibitors the response was achieved within 15 min of administration of rFVIIa. In thrombocytopenic pts haemostatic effect of rFVIIa was delayed and reached within 1 hr, good response was achieved in 51% pts. Figure 1. Parameters of hemostasis before and after rFVIIa administration expressed as a median (IQR) Figure 1. Parameters of hemostasis before and after rFVIIa administration expressed as a median (IQR) Disclosures No relevant conflicts of interest to declare.

Description: The differentiation and maturation of erythroid cells require highly regulated patterns of gene expression and metabolism. Mitochondria are critical for heme biosynthesis and iron metabolism in erythroid cells, yet their regulation during normal erythroid maturation remains largely unexplored. Here we measured global protein and mRNA expression in primary human fetal liver and adult bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) and differentiated erythroid precursors (proerythroblasts or ProEs) by mass-spectrometry-based quantitative proteomics and RNA-seq analysis, respectively. In-depth proteomic profiling resulted in identification and high-quality quantification of proteins encoded by 14,502 genes, accounting for 72.4% of the total annotated protein-coding genes in humans. Through unbiased and comprehensive comparison of the proteomic and transcriptomic dynamics between primary HSPCs and committed erythroid precursors, we discovered a number of previously unrecognized regulatory pathways essential for normal erythropoiesis. Importantly, we identified key pathways related to mitochondrial biogenesis, including ATP biosynthesis, electron transport chain, oxidative phosphorylation, and cellular respiration, whose protein expression was substantially induced during erythropoiesis. Strikingly, the increases in protein expression were not paralleled by changes in mRNA expression of these genes, suggesting that they are regulated through post-transcriptional mechanisms. Consistent with the enhanced mitochondrial protein expression, we observed substantial increases in mitochondrial membrane potential (MMP) and intracellular ATP level during early erythroid differentiation before clearance of mitochondria at terminal maturation stages. We next profiled metabolite levels in HSPCs and erythroid precursors at various differentiation stages, and observed progressive progressive alterations of metabolites from the tricarboxylic acid cycle and other mitochondrial pathways during erythroid maturation. Furthermore, we identified two principal mitochondria-associated transcription factors, mitochondrial transcription factor A (TFAM) and Prohibitin 2 (PHB2), whose protein but not mRNA expression was substantially increased during erythroid maturation. Depletion of TFAM or PHB2 expression markedly impaired erythroid differentiation from primary human CD34+ HSPCs. TFAM or PHB2-deficient cells displayed impaired erythroid differentiation, proliferation and hemoglobin expression, reduced mitochondrial mass, membrane potential and ATP synthesis, and increased apoptosis. Pharmacological inhibition of mitochondrial activities by targeting mitochondrial complex I (metformin, phenformin, and rotenone), complex III (antimycin A), or complex V (oligomycin) in CD34+ HSPCs impaired erythroid differentiation in a dose-dependent manner, consistent with an essential role of mitochondria for normal erythroid development. To elucidate the regulatory mechanisms underlying the developmental control of mitochondrial biogenesis, we measured protein synthesis rate in CD34+ HSPCs and differentiated erythroid cells by analyzing the incorporation of the methionine analogue HPG (L-homopropargylglycine). The rate of HPG incorporation was the lowest in undifferentiated CD34+, and increased by 1.8 and 2.5-fold in CD71+CD235a- and CD71+CD235a+ erythroid progenitor cells, respectively. Consistent with the progressive increase in protein synthesis, we observed a gradual increase of mTORC1 signaling, as measured by the phosphorylated eIF4-E binding protein (4E-BP1) and S6 kinase (p70S6K), during erythroid differentiation of CD34+ HSPCs. Inhibition of mTORC1 signaling by active-site mTOR inhibitors markedly impaired expression of mitochondria-associated proteins, mitochondrial mass and membrane potential, and erythroid differentiation of CD34+ HSPCs. Thus, our results support a model that mitochondrial biogenesis is highly regulated through mTORC1-mediated activation of protein translation during erythroid lineage specification. Our studies also suggest a novel mechanism for proper regulation of mitochondrial biogenesis by post-transcriptional machinery, and may have direct relevance to the hematological defects associated with human mitochondrial diseases and aging. Disclosures DeBerardinis: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Peloton Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Description: Study objectives We aim to evaluate disease characteristics and treatment practices of pediatric pts. with Immune thrombocytopenia (ITP) in Russia. Materials and methods The ITP Registry was a multicenter, prospective, observational cohort study. Inclusion criteria: diagnosis of primary ITP, informed consent of the patient/guardians. Exclusion criteria: secondary or congenital thrombocytopenia. Data from medical records were registered in the e-CRF in average every 3 months. Descriptive statistics were used. Patients were registered since June 2011 till June 2014. Results Ninety-three pediatric pts, 46 male (49.5%) and 47 female (50.5%) with a median age 8.4 yrs (range 0.5-17.8) from 5 centers in various regions of Russia were included. The mean observation period reached 17.1 ± 6.5 mo (range1.4 to 28.6 months). Seventy (75.3%) pts had acute and 24.7% pts had insidious disease onset. The presence of trigger factors for ITP development was found in more than half of the cases (in 61.3% of patients), they are listed in Table 1. Table 1. Triggers N % No triggers 36 38.7% Infection 46 49.5% Vaccination 8 8.6% Other 3 3.2% Total 93 100% Median disease duration at enrollment was 1.07 years (range 0 to 16.7 yrs). ITP duration shorter than 5 years at the enrollment was reported in 89.2% pts, up to 1 year - in 43 (46.2%), 1- 5 years - in 40 (43%), 5-10 years - in 8 (8.6%), 〉10 years - in 2 (2.2%) pts. Newly diagnosed ITP was reported in 35 (37.6 %) pts, persistent ITP - in 12 (12.9 %), chronic ITP - in 46 (49.5 %) pts.Median platelets count was 12,0 x 109/L (range 0.0 - 72.0 109/L). Ninety-two (98%) pts experienced hemorrhagic manifestations during the course of ITP: skin hemorrhages - in 98.9%, oral bleeding - in 15.1%, epistaxis - in 36.6%, gastrointestinal bleeding - in 1.1%, intracranial bleeding - in 1.1%, hematuria - in 1.1%, and other hemorrhages - in 9.7% of pts. Relationship between hemorrhagic syndrome and platelet count at the enrollment is provided in table 2. Table 2. Relationship between hemorrhagic syndrome and platelet count (at enrollment) Hemorrhage highest grade according to WHO Platelet count (visit 1) Total pts / % 〈 30,000 30,000 -50,000 〉50,000 0 3 5.5% 3 5.5% 49 89.1% 55 100% 1 11 40.7% 5 18.5% 11 40.7% 27 100% 2 5 62.5% 0 0% 3 37.5% 8 100% 3 2 66.7% 1 33.3% 0 0% 3 100% Total 21 22,6% 9 9.7% 63 67.7% 93 100% Severe course of ITP after enrollment was observed in 12 (13%) pts (of whose 6 (6.5%) had clinically significant hemorrhage at the disease onset and 6 (6.5%) had new clinically significant hemorrhages during follow-up period. Refractory ITP at enrollment was reported in 9 (9.7%) pts and was associated with the resistance to the first-, second- and subsequent lines of therapy. At enrollment 42 (45.2%) pts received specific treatment for ITP. Before enrollment, splenectomy was reported in only 1 (1.1%) 14-years old patient who had a complete response. During the study, splenectomy was performed in 6 (6.6%) pts with chronic ITP; the duration of the disease at the time of splenectomy varied from 2 to 10 years, with average duration of 4.69 years (median - 4.5 years). Complete response to splenectomy was observed in 3 (50%) pts, a partial response - in 2 (33.3%), no response - in 1 (16.7%) patient. Loss of response to splenectomy was not reported. During the study, severe ITP was reported in 8 (8.7%) pts, 41 (44.6%) pt had various hemorrhagic manifestations of ITP at least at 1 visit, grade IV hemorrhagic syndrome was not reported. Thirty-eight (41%) pts received 1-st line treatment: glucocorticosteroids (GCS) - 23 (60.5%) pts, IVIG - 5 (13.2%), alfa-interferons -16 pts (42.1%). Twenty-three pts (24.7%) received second-line therapy: GCS - 1 (4.3%), IVIG -1 (4.3%), immunosupression - 1 (4.3%), rituximab - 2 (8.7%), romiplostim - 11 (47.8%), eltrombopag - 14 (60.9%). Conclusion For the first time new information on the features of the disease and patterns of management of pediatric pts with primary ITP in Russia was obtained in a prospective study. Interestingly, the preferred therapy for the 2nd or subsequent lines are TPO receptor agonists used outside the approved indications in research institutions, based on published clinical trial data. Splenectomy rate before and during the study was only 7.5% (7 pts) with chronic ITP; in 1 child (14.3%) splenectomy was ineffective. Low acceptance of splenectomy suggests TPO-mimetics as potential second-line therapy. In total, good disease control is achievable in the majority of pediatric pts with ITP. Disclosures Off Label Use: use of TPO-mimetics in children.

Description: Background: Atypical hemolytic uremic syndrome (aHUS) is a rare, genetic, life-threatening disease predominantly caused by chronic, uncontrolled complement activation that leads to thrombotic microangiopathy and renal and other end-organ damage. The aHUS Registry, established in April 2012, is an observational, noninterventional, multicenter, global initiative to collect information on patient outcomes regardless of treatment approach. It facilitates availability of follow-up data for eculizumab. Methods: Patients with clinical diagnoses of aHUS (irrespective of identified complement abnormality or treatment) are eligible. Demographic, medical/disease history, and treatment outcomes data are collected at enrollment and prospectively thereafter. Results: By June 30, 2015, 826 patients enrolled (Table). Overall, 54.7% of patients, including 45.1% of pediatric and 62.7% of adult patients, were female. Patients were most commonly enrolled after their first TMA event. Thrombosis occurred more frequently in adult than pediatric patients. Nonrenal conditions, including gastrointestinal, cardiovascular, central nervous system, and pulmonary, were common in both age groups and occurred in 11.0%‒20.3% overall. Eculizumab was administered to 57.3% of patients, of whom 87.3% were treated prior to enrollment. Conclusions: Registry baseline characteristics demonstrate differences between pediatric and adult patients with aHUS, notably frequencies of thrombosis. Nonrenal conditions are frequent in both age groups. Ongoing and future analyses will further enhance understanding of aHUS history and progression. Additional clinical sites are encouraged to enroll patients to facilitate knowledge acquisition and optimization of patient care and quality of life. Disclosures Licht: Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Achillon: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ardissino:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ariceta:Alexion Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cohen:Astellas: Consultancy; Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Merck: Consultancy; Genentech: Research Funding. Gasteyger:Alexion Pharma International SàRL: Employment, Equity Ownership. Greenbaum:Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ogawa:Alexion Pharmaceuticals: Employment, Equity Ownership. Kupelian:Alexion Pharmaceuticals: Employment, Equity Ownership. Schaefer:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vande Walle:Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Frémeaux-Bacchi:Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Stenotrophomonas maltophilia is an important nosocomial pathogen, particularly in immunocompromised patients, especially in patients with hematologic diseases. Methods: We reviewed the clinical characteristics and prognosis of patients with S. maltophilia bacteremia over a five-year period from January 2010 to December 2014. Species identification was performed using the automated Vitek 2 compact system (bioMe rieux). Results: The incidence of S. maltophilia bacteremia was 25.1 per 10 000 admissions in our study. Thirty-four patients (median age: 34 years; 64.7% males) with S. maltophilia bacteremia were analyzed. The S. maltophilia bacteremia related 30-day mortality was 44.1%. Risk factors associated with mortality in patients with S. maltophilia infection in the univariate and multivariate analysis were represented in Tables I and II. In the univariate analysis, risk factors included T〉39.0¡æ, septic shock, respiratory failure and non-remission after treatment for primary hematological diseases (P 39.0¡æ 75% 16.7% 2.490 1.318-4.704 0.005 Septic shock 90.0% 25.0% 2.544 1.473-4.393 0.001 Respiratory failure 100% 20.8% 4.672 2.366-9.225 0.000 Treatment outcome for hematological diseases Remission 10.0% 85.7% 0.247 0.116-0.526 0.000 HR, hazard ratio; CI, confidence interval; HSCT, Hematopoietic stem cell transplantation Table 2. Multivariate analysis of prognostic factors associated with mortality from S. maltophilia bacteremia Factor HR 95%CI P-value Respiratory failure 2.688 1.297-5.569 0.008 Remission after treatment for hematological diseases 0.367 0.153-0.879 0.025 HR, hazard ratio; CI, confidence interval Table 3. Susceptibility pattern of the 34 patients with Stenotrophomonas maltophilia bacteremia Antimicrobial agents S (%) I (%) Ceftazidime 24(70.6%) 1(2.9%) Cefoperazone 19(44.1%) 6(17.6%) Sulbactam and Cefoperazone 20(58.8%) 5(14.7%) Piperacillin 7(20.6%) 6(17.6%) Piperacillin-Tazobactam 11(32.3%) 7(20.6%) Amikacin 6(17.6%) 0(0%) Ciprofloxacin 28(82.4%) 1(2.9%) S, susceptible; I, intermediately susceptible. Disclosures No relevant conflicts of interest to declare.

Description: Background Platelets not only play an important role in the initiation of hemostasis and thrombosis, but also participate in the immune and inflammatory response. Most studies focus on the platelets-bacteria interaction and demonstrate that bacteria are capable of binding to, aggregating and activating platelets. Human platelets are reported to express several groups of TLRs, which participate in the inflammation process and monitoring host infection. Recent data from our laboratory demonstrated that Group B streptococci (GBS) could induce platelet aggregation and up-regulate the expression of CD62P and further study showed that platelet TLR2 might involve in the activation. GBS, or streptococcus agalactiae, is one of the most common cause of life-threatening sepsis, pneumonia and meningitis in neonates, pregnant women, the elderly and immunocompromised adults. Therefore, illuminating the mechanisms of GBS-induced platelet activation is important for providing the basis for platelets in defense against infection and immunity. Since increasing reports have shown that the PI3-K/Akt signaling pathway regulates platelet activation and hemostasis, so it is possible to research the TLR2 related signaling pathway. Methods 1. Platelets were from healthy volunteers (all genders, 25-52 years old) who had not taken any anti-platelet drugs (like aspirins, clopidogrel and abciximab) during the previous 30 days. GBS 639 were isolated from patient's venous blood. 2. Platelet aggregation, the expression of platelet CD62P and PAC-1 were used as the indicator of platelet activation. GBS-induced platelet aggregation was assayed by light transmission; platelet TLR2, CD62P and PAC-1 expression were determined by flow cytometry; AKT and AKT phosphorylation expression were determined by RT-PCR or western blot assay. In some experiments, platelets were pre-incubated with PI3-K specific inhibitors LY294002 or anti-TLR2 monoclonal antibody. 3. Statistical analysis: Data are reported as the mean ± SD. Treatment groups were compared with the appropriate control (s), and statistical significance was examined using the two-tailed t-test. Differences were considered significant when P

Description: Introduction: A reactive, polyclonal increase in marrow plasma cells in pts with HIV is relatively common, reported to occur in 25% of cases in one series (1) and recently noted to be "usually seen" (2). However, quantification of the degree of marrow plasmacytosis has not been well described. Methods: Pts with a diagnosis of HIV and who had a bone marrow aspirate and biopsy performed at our institution over a 10 year period (2/2004-9/2014) were reviewed. Pts with a diagnosis of myeloma were excluded. A 500 cell count was performed on each marrow aspirate and plasma cell percentage determined. Results: 65 pts were identified; 9 were excluded due to dry taps, so there were 56 evaluable marrow aspirates. Reasons for performing the aspirate and biopsy included: lymphoma staging (NHL in 11/65, HL in 4/65); abnormal SPEP (2/65); abnormal MRI (1/65); FUO (14/65), Kaposi's sarcoma/fevers (3/65), pancytopenia (15/65), thrombocytopenia (6/65), neutropenia (2/65) and anemia (10/65). The average marrow plasma cell percentage was 4.6%, with a range from 0- 21% (median 4%). Only 7/56 (12.5%) had 〉10% plasma cells. Of these 7 cases, only one had an identifiable cause (multicentric Castleman's) for the plasmacytosis. The 6 others had no neoplastic or infectious etiology, except for HIV itself (including one with 21% plasma cells). All had polyclonal plasmacytosis. Conclusions: In this small series of HIV marrows the degree of plasmacytosis was generally mild (average of 4.6%, which is barely above the upper limit of normal range of 3.5%). However, there were a minority of cases which exhibited significant plasmacytosis (as high as 21%) without any cause other than HIV. This suggests that, while significant marrow polyclonal plasmacytosis is usually not seen in HIV, it may occur as a result of HIV itself without any other identifiable cause. 1) Karchen DS and Frost AR. The bone marrow in HIV-related disease: Morphology and clinical correlation. Am J Clin Path. 1991; 95(1): 63-71. 2) Leibman HA and Tulpule A. "Hematologic manifestations of HIV/AIDS". Chapter 159. Hematology: Basic Principles and Practice, 6th edition. 2013. Disclosures No relevant conflicts of interest to declare.

Description: Objective: Very elderly MDS patients (≥75 years) have limited therapeutic options and are usually ineligible for allogeneic stem cell transplantation. We aimed to study the impact of available MDS therapies on very elderly MDS patients and their correlation with patient demographics, performance status(PS) disease characteristics and patient outcomes. Methods: We performed a retrospective analysis of MDS patients ≥75 years diagnosed and treated at the University of Alabama at Birmingham from 2008 to 2014, with a minimum followup of 12 months. We analyzed demographics, ECOG PS, karyotypic risk categories as defined by the IPSS scoring system, blast percentage, IPSS and R-IPSS scores and overall survival (OS) in this population. We stratified patients based on therapy into two groups - the hypomethylating agent (HMA) group (defined as receiving therapy with ≥ 1 cycle of HMA; Azacitidine or Decitabine or both) and the non-HMA group, which included treatment with supportive transfusions, erythropoietin stimulating agents (ESAs), lenalidomide and cytotoxic chemotherapy. We analyzed group differences for all the parameters mentioned above using chi square test for categorical variables, and t test and Mann Whitney u-test for mean and medians respectively. In addition, OS was examined using Kaplan Meier curves using the log rank test. We used univariate and multivariate analysis to examine the effects of variables of interest on OS.All results were considered statistically significant at α=0.05 level. Results: The study population included 58 patients of which 35 patients were males (60%). Median age was 78 years. Forty patients (71%) of patients had good, 6% had intermediate and 23% had poor karyotypic profiles by the IPSS scoring system. ECOG ≥2 was observed in 44% of the patients with no significant differences in both groups. Average IPSS and R-IPSS scores were 1.2 and 4.5 respectively. Median OS for the entire study population was noted to be 15.5 months (7-34m). There were 25 patients in the HMA group and 33 patients in the non-HMA group. The blast percentage was higher in HMA group (20.5% vs 9.4%) compared to non-HMA group. More patients had a good karyotypic profile in the non-HMA group when compared to HMA group (80% vs 60%). There was a statistically significant difference between the mean IPSS and R-IPSS prognostic scores in non-HMA and HMA group (0.9 vs 1.7, p=0.010 and 3.5 vs 5.5, p=0.002) respectively. There was no significant difference in median overall survival between the non-HMA and HMA group (16.5 m (7-53) vs 15.5 m (5-19) p=0.278) respectively but the mean survival rates between non-HMA and HMA group were statistically different (32.81 vs 15.85, p=0.034). According to the log rank test, a statistical difference (p=0.027) in survival estimates was observed between the two groups on Kaplan Meier curve, where the HMA group had a significantly shorter survival compared to the non-HMA group. In the univariate analysis for the entire sample, higher IPSS score; R-IPSS score, and higher blast percentage were associated with increased rate of events. Moreover, rates of events were found to be lower in patients who did not receive HMA therapy (HR - 0.45, p=0.033), however in multivariable analysis, only higher blast percentage was associated with increased rate of events (HR - 1.06 p=0.025 95% CI - 1.004-1.11). Patients in the HMA group received average of 7.8 cycles. After stopping HMA therapy, 10 patients received other therapies including cytotoxic chemotherapy, hydroxyurea and lenalidomide, 4 were enrolled in a clinical trial, 9 received supportive transfusions and ESAs while 2 died immediately afterwards. Conclusion: Our study did not find a difference in median OS between patients who received HMA therapy versus non-HMA therapy in this population of very elderly MDS patients. Patients who received HMA therapy had a higher risk karyotypic profile, increased blast percentage and higher IPSS and R-IPSS scores. The average number of HMA cycles they received was 7.8, indicating adequate therapy. However, we could not evaluate transfusion needs, hospitalizations or other quality of life measures in these 2 groups. In conclusion, further studies need to be done to better evaluate various MDS therapies and their impact on quality of life and survival in this very elderly population with a higher comorbidity burden, possibly limiting the benefit of these treatments typically seen in younger MDS patients. Disclosures Borate: Genoptix: Consultancy; Seattle Genetics: Research Funding; Gilead: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Speakers Bureau. Erba:Millennium/Takeda: Research Funding; Jannsen (J&J): Other: Data Safety and Monitoring Committees; Ariad: Consultancy; Millennium/Takeda: Research Funding; Celgene: Consultancy, Speakers Bureau; Astellas: Research Funding; Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy; Astellas: Research Funding; Incyte: Consultancy, Speakers Bureau; Pfizer: Consultancy; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Sunesis: Consultancy; Incyte: Consultancy, Speakers Bureau; GlycoMimetics: Other: Data Safety and Monitoring Committees; Jannsen (J&J): Other: Data Safety and Monitoring Committees; Amgen: Consultancy, Research Funding; Celator: Research Funding; Novartis: Consultancy, Speakers Bureau; Daiichi Sankyo: Consultancy; Sunesis: Consultancy; Seattle Genetics: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; GlycoMimetics: Other: Data Safety and Monitoring Committees; Celator: Research Funding.

Description: Introduction The rate of venous thrombo-embolism (VTE) in patients with sickle cell disease (SCD) is estimated to be 4 - 8%; the incidence of catheter-associated VTE in this cohort is 0.16-0.99 per 1000 catheter days. Possible mechanisms for VTE in patients with SCD include hyperviscosity, ischemia reperfusion injury and chronic vascular inflammation. Additionally, patients with SCD exhibit decreased plasma levels of natural anticoagulants such as proteins C and S compared to healthy adults. A retrospective review at our institution found that the incidence of central venous line (CVL)-associated VTE in children with SCD was 0.12/1000 catheter days. On multivariate analysis, CVLs were identified as the only independent risk factor for VTE. Among patients with CVL, we found an association between bilateral CVLs and VTE (Odds Ratio [95% CI] = 10.3 [1.1-92.2]). Children with SCD frequently require CVLs for intravenous access or chronic erythrocytapharesis to reduce SCD-related complications. It is unknown, however, if erythrocytapharesis may also disrupt their natural anticoagulants levels and further increase their risk for subsequent VTE. We hypothesized that erythrocytapheresis may contribute to the pro-coagulant phenotype by altering patients' natural anticoagulants levels. Methods As part of a quality improvement initiative we measured antithrombin (AT), protein C antigen and activity, protein S antigen (total and free) and activity prior to being placed on the apheresis circuit and after completion of exchange in patients with SCD (0-21 years of age) undergoing chronic erythrocytapheresis who had a CVL (between January 1, 2009 and January 31, 2015). D-Dimer levels were obtained pre exchange only. Protein C and S levels were measured using ELISA based assays; protein C and S activities were measured using a clotting based assay and AT was measured using a chromogenic assay. The resulting levels, pre and post-erythrocytapheresis were analyzed using a paired nonparametric Wilcoxon rank test. Results Eleven patients were eligible for this study (8 females, 3 males). Median age at the time of erythrocytapheresis was 14 years (±3.65 SD) years. Indications for erythrocytapheresis included primary/ secondary stroke prevention or recurrent acute chest syndrome. All patients had bilateral CVLs in place. Four of the included patients had a history of VTE; 2 patients were on anticoagulation with low molecular weight heparin at the time of the study. The mean value for total protein S antigen (65u/dL; normal 〉72u/dL), free protein S antigen (53u/dL; normal 〉70u/dL), and protein S activity (51IU/mL; normal 65-138IU/ml) were all abnormal pre- erythrocytapheresis and decreased significantly following erythrocytapheresis (52u/dL, 44u/dL, and 44IU/mL respectively). Mean protein C antigen (71u/dL; normal 〉55u/dL), protein C activity (71%; normal 55-111%), and AT activity (104%; normal 77-132%) were within the normal range pre-erythrocytapheresis and decreased significantly post-erythrocytapheresis. We demonstrated significantly lower levels of protein C antigen (p=0.01), protein C activity (p=0.01), total protein S antigen (p=0.005), free protein S antigen (p=0.036), protein S activity (p=.04), and AT activity (p=.004) following erythrocytapheresis (Figure). D-Dimer levels were elevated in 6/11 patients (54.5%). Conclusions Pre- and post-erythrocytapheresis levels of natural endogenous anticoagulants investigated in a cohort of patients with SCD undergoing chronic erythrocytapheresis demonstrated that levels of all investigated proteins were significantly lower in patients following erythrocytapheresis. D-dimer levels were elevated in the majority of patients pre exchange but the importance of this finding is unclear. Given the relatively short half-life of the natural anticoagulants (2-3 hours for protein C and 36-60 hours for AT and protein S), it is unclear if this acute decrease of natural anticoagulants is clinically relevant. Further investigation is needed to determine if this acute decrease may contribute to an increased VTE risk in children with SCD undergoing apheresis. Figure 1. Figure 1. Disclosures Woods: Biogen: Other: Educational Grant. Dunn:Novo Nordisk: Consultancy, Other: Educational Grant; CSL Behring: Consultancy, Other: Data Safety Monitoring Board; Bayer: Consultancy, Other: educational grant; Biogen: Consultancy, Other: Educational grant, Research Funding; Baxalta: Consultancy, Other: educational grant, Research Funding; Ohio State University: Employment; Pfizer: Other: Grant Review Board.

Description: Introduction: the involvement of iron in a wide range of metabolic processes make it one of the essential elements for living organisms (Inamura et al., 2005). Anemia of chronic disease (ACD), also termed anemia of chronic inflammation, is the most prevalent anemia in subjects suffering from chronic diseases such as cancer and Chronic Kidney Disease (CKD). A central mechanism by which chronic disease causes anemia is the retention of iron in the reticuloendothelial system, causing a functional iron deficiency and consequently an insufficient iron supply for erythropoiesis. Hepcidin is a primarily liver-derived peptide that orchestrates body iron homeostasis and its expression increases in response to elevated iron stores, inflammation and ER stress (Maliken et al. 2011). In those conditions produced Hepcidin bind to the cellular iron exporter, Ferroportin (Fp1), resulting in Fp1 internalization and degradation with subsequent reduction of cellular iron release (Theurl et al., 2014). Some studies showed that pharmacological inhibition of Hepcidin could reverse cellular iron retetion and improve anemia in different models of inflammatory anemia (Steinbicker et al., 2011, Theurl et al., 2011). Moreover recent scientific publications suggest also a role of other dietary supplements in regulating Hepcidin, reducing its concentration and thus increasing circulating iron in blood (Zughaier et al. 2014). Sucrosomial Iron¨ (Sideral¨) is a new and still unique preparation of ferric pyrophosphate, useful for treatment of iron deficiency related anemia. Aim: we have previously performed a clinical study in which we showed that Sucrosomial iron is able to increase Hemoglobin level in CKD patients (Pisani et al., 2014). On the basis of these results we have investigated the role of Sucrosomial Iron¨ in inflammation process. In particular, we studied the capability of Sucrosomial Iron¨ to reduce Hepcidin release in LPS -induced inflammation made in the hepatoma cell line (HepG2). Results: Cells were incubated with LPS, treated with Sucrosomial Iron¨ and then analyzed for Hepcidin production in terms of protein expression at 6 and 24h after treatment with Sucrosomial Iron¨. Results showed that Sucrosomial Iron¨ is able to significantly reduce Hepcidin level both 6 and 24 h after sucrosome treatment compare to others iron formulations (Figure 1A-B). Materilas and Methods: Sucrosomial Iron¨ preparation of ferric pyrophosphate convered by a; LPS: Lipopolysaccharides from Escherichia coli (Sigma-Aldrich); Empty matrix preparation of phospholipids plus sucrose esters of fatty acids. Conclusions: This evidence should be considered as a preliminary investigation on the effect of Sucrosomial Iron¨ on the production of Hepcidin during chronic inflammation. Bibliography Inamura J et al. Upregulation of hepcidin by interleukin-1 in human hepatoma cell lines. Hepatology Research 33 2005 198-205. Maliken BD et al., The Hepcidin Circuits Act: Balancing Iron and Inflammation, Hepatology. 2011 May ; 53(5): 1764-1766; Theurl M et al. Hepcidin as a predictive factor and therapeutic target in erythropoiesis- stimulating agent treatment for anemia of chronic disease in rats Haematologica. 2014 Sep;99(9):1516-24. Epub 2014 Jun 3. Theurl et al. Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats. Blood. 2011;118(18): 4977-84. Steinbicker AU et al. Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation Blood. 2011 May 5;117(18):4915-23. doi: 10.1182/blood-2010-10-313064. Epub 2011 Mar 10. Zughaier SM et al. The role of vitamin D in regulating the iron-hepcidin-ferroportin axis in monocytes. J Clin Transl Endocrinol. 2014 Mar 21;1(1):19-25. Pisani et al. Effect of oral liposomal iron versus intravenous iron for treatment of iron deficiency anaemia in CKD patients: a randomized trial. Nephrol Dial Transplant. 2015 Apr;30(4):645-52. Epub 2014 Nov 13. Figure 1. This graph shows the level of Hepcidin produced by LPS treated HepG2 cells 6 hours after treatment with iron compounds. Figure 1. This graph shows the level of Hepcidin produced by LPS treated HepG2 cells 6 hours after treatment with iron compounds. Figure 2. This graph shows the level of Hepcidin produced by LPS- treated HepG2 cells 24 hours after treatment with iron compounds. Figure 2. This graph shows the level of Hepcidin produced by LPS- treated HepG2 cells 24 hours after treatment with iron compounds. Disclosures Tarantino: Pharmanutra s.p.a.: Employment. Brilli:Pharmanutra s.p.a.: Employment.

Description: Background: Multiple myeloma is an incurable disease with poor survival rate. Recently, novel treatments have focused on addressing unmet needs among heavily pre-treated patients who have failed prior therapies. The purpose of this analysis was to describe the characteristics of patients who are heavily pre-treated vs patients who were not, within 2 years of initiating MM therapy. Results of this analysis will help to understand the characteristics, including treatment patterns and burden, of MM patients who progress through lines of therapy over a relatively short time period. Methods: Using a US administrative claims database (Truven Health MarketScan® Database), adult patients (age ≥18) were included in the analysis if they had 1) ≥2 MM diagnoses codes (ICD-9 203.0x) on different dates between Jan 1, 2005-Mar 31, 2014 (study period); 2) MM treatment between Jan 1, 2007-Mar 31, 2012 and within 90 days of an MM diagnosis code (date of the first MM treatment set as the index date); and 3) continuous eligibility for medical insurance 24 months pre/postindex. Patients were excluded if they had 1) MM treatment during 24 months preindex; or during 24 month pre/post index had 2) moved from commercial insurance to Medicare; 3) non-MM chemotherapy; 4) pregnancy-related or stem cell transplant codes; or 5) HIV diagnosis during study period. An algorithm using dispensing dates and days supply was developed to define line of therapy (LOT) and double refractory. A new LOT was identified where there was a ≥60 day gap with no treatment, a ≥60 day gap followed by retreatment with the same drug, or where there was a change in therapy based on 30 day windows and the summed days supply of medication was 〉60 days. Refractory events were defined when a PI or IMiD had days supply for 4 LOT were 39.8%, 33.0%, 15.4% and 6.1% and 5.7%, respectively. A single refractory event occurred in 66 (6.0%) patients and 2 (0.2%) patients were identified as double refractory. There were 80 (7.2%) patients who were heavily pre-treated. The heavily pre-treated group had a similar percent of males (56.3%) as the non-heavily pre-treated group (54.9%; p=0.82). The heavily pre-treated group was slightly younger (66.1 vs 70.9 years; p=0.0008) and had a lower percent with Medicare coverage (55.0% vs 71.1%; p=0.0024). The heavily pre-treated group had a substantially higher percent of regimens that included both PI and IMiD in the 1st LOT (28.8% vs 7.4%; p

Description: Introduction: Multiple myeloma (MM) is a disease of aging. The prognosis of older adults with MM is influenced by the presence of geriatric syndromes, including dependence in daily activities and comorbidities. Falls, another common geriatric syndrome, are associated with greater risk for severe toxicity of chemotherapy and survival in older adults with solid tumors. Among older adults with MM, the prevalence of falls and factors predictive of falls are yet unknown. Thus, we sought to determine the prevalence of falls in a cohort of older adults with newly diagnosed MM and examine associations between falls and functional status, comorbidities and self-reported health. Methods: Using data from the linkage of the Surveillance, Epidemiology, and End Results (SEER) national cancer registry with the Medicare Health Outcomes Survey (MHOS), we identified unique patients with a diagnosis of MM in the SEER registry who participated in the MHOS survey, which includes individuals who are enrolled in Medicare Advantage organizations. An item inquiring about falls was present in the MHOS survey starting in 2006. For this analysis, participants (pts) were included if they completed the MHOS baseline survey within 1 year of their diagnosis of MM. Baseline characteristics were examined with descriptive statistics. Associations between falls and patient-reported data on function, comorbidities and self-rated health were examined using Student's t-test, Pearson Chi-square or Fisher's exact test, as appropriate. We identified 1327 unique patients, of whom 376 completed their baseline MHOS survey within 1 year of diagnosis. Of these, 190 provided responses to the item regarding falls and are included in this analysis. Results: The median age of the cohort was 77 years (range 47-97). The cohort was diverse, with 58.9% white race, 19.5% Asian/Pacific Islander, 11.6% Hispanic/Latino and 10.0% black race. Half (50.0%) were male, 48.4% female, and 1.6% unknown gender. Over one-quarter (25.2%) of pts reported a fall within the prior 12 months. Fallers were more likely to report a history of congestive heart failure than nonfallers (22.7% vs 7.9%, p=0.012); the remaining comorbidities examined (coronary artery disease, stroke, chronic obstructive pulmonary disease and diabetes) were not associated with falls. Of those who reported 2 or more weeks of depression in the past year, 41.4% reported a fall, compared with only 20.1% of those who did not report depression (p=0.004). Fallers were more likely to report limitations in moderate activities (81.2% vs 62.1%, p=0.015) and in climbing several flights of stairs (89.1% vs 64.9%, p=0.001). Pts who reported numbness in their feet some, most or all of the time were numerically but not statistically more likely to report a fall (35% vs 21.9%, p=0.070). Compared with nonfallers, fallers reported more days in the past 30 days when their physical health was not good (15.8 vs 10.0 days, p=0.024), more days in the past 30 days when their mental health was not good (10.7 days vs 4.1 days, p=0.002) and more days in the past 30 days when their health interfered with their daily activities (14.3 vs 7.0 days, p=0.001). Pts who had fallen were more likely to report that their health was fair or poor than those who had not fallen (67.4% vs 33.8%, p

Description: Introduction: Aberrant DNA methylation is frequently found in hematologic malignancies where it is associated with altered gene expression. DNA hypomethylating agents (DNMTi), e.g. 5-aza-2'-deoxycytidine (DAC), are used for both global and gene-specific in vivo demethylation and offer a therapeutic option in myelodysplastic syndromes (MDS) and AML. DNMTi have already been utilized to upregulate suppressed fetal hemoglobin (HbF) in adult patients (pts) suffering from hemoglobinopathies. Here we systematically investigated the potential of DAC for in vitro induction of erythroid differentiation as well as HbF expression in the bipotent myeloid leukemia cell line K562 and in vivo in a clinical treatment situation in MDS pts. Methods and Results: We treated K562 cells with non-toxic concentrations of DAC (100 nM, three 24 hour pulses), hemin (50 nM) and phorbol myristate acetate (PMA, 5 nM). DAC treatment led to morphological changes indicating erythroid but not megakaryocytic differentiation. This was confirmed by benzidine staining where DAC (13% positive cells) and hemin (58%) but not PMA treated cells (0%) became positive for hemoglobin synthesis. Lack of CD41 detection by FACS analysis for DAC and hemin indicated absence of megakaryocytic differentiation. Transcriptome profiling by mRNA expression arrays (Affymetrix GeneChip® HG U133 Plus 2.0) revealed highest similarity between hemin and DAC treatment by unsupervised hierarchical clustering, followed by vehicle control and untreated cells. The transcriptome of PMA treated cells clustered most distantly to all other treatments. Both, DAC and hemin induced moderately balanced up- and downregulation of transcripts to an almost identical extent. 1414 transcripts were 〉2 fold upregulated and 1505 were 〉2 fold downregulated upon DAC treatment, whereas 1548 were up- and 2404 were downregulated in hemin treated cells, respectively. The extent of transcriptome dynamics was considerably stronger upon PMA treatment, where 4196 and 3780 transcripts were up- and downregulated, respectively. When intersecting transcriptome changes between the 3 drug treatments (Fig. 1), 368 out of 1548 (23.7%) upregulated transcripts in hemin treated cells were concordantly upregulated upon DAC treatment. The overlap of upregulated transcript was lower compared to PMA treated cells (14.9%). GO analyses of upregulated transcripts identified terms related to erythropoesis and iron metabolism among the top regulated groups of transcripts in DAC treated cells whereas terms related to megakaryocytic differentiation did not show significance. Particularly strong differences of transcripts were observed for a1-, a2-, Ag-, e- and z-globin expression upon DAC and hemin treatment, whereas b- and d-globin were expressed at low levels. These changes were not observed for PMA treated cells. Induction of a- and Ag-globin on mRNA level resulted in enrichment of a- and Ag-globin protein to 15.8% of total cellular protein amount, and consequently in HbF formation in K562 cells as assessed by reversed phase and anion exchange chromatography. HbF levels in peripheral blood were measured from 16 MDS pts, median age 74 years (range 66-78) also treated with a 3-day DAC schedule. Median HbF fraction at baseline was 0.4% (0.1-3.9%) of total hemoglobin with 6 pts (37.5%) exhibiting increased HbF levels (〉1%) already before treatment. In 13/16 (81%) pts, increase of HbF with a median increment of 1.2% (range 0.3-3.7%) was observed. In 3 pts, HbF decreased over the treatment course. Median number of courses until maximum increment was 3 (2-6). HbF levels in 2 pts with AML and 1 with pancreatic cancer treated with nucleoside analogues without demethylating activity (cytosine arabinoside and gemcitabine, respectively) according to standard chemotherapy protocols served as control group and did not show comparable increments. Conclusions: We describe an erythroid differentiation program, from transcriptome level to HbF protein formation, induced by the hypomethylating agent DAC in the bipotent cell line K562. This DAC-mediated differentiation process is specific for erythropoesis but not megakaryopoesis. This is substantiated by in vivo upregulation of HbF upon DAC adminstration in MDS pts. Therefore, we propose to utilize HbF expression as potential biomarker during DAC treatment. Figure 1. Intersection of 〉2 fold upregulated transcripts in K562 cells upon drug treatment. Figure 1. Intersection of 〉2 fold upregulated transcripts in K562 cells upon drug treatment. Disclosures No relevant conflicts of interest to declare.

Description: Background: Induction chemotherapy for acute myeloid leukemia (AML) is more intensive than many other cancer treatments, and may be associated with a different symptom burden. Little is known about the most prevalent symptoms during AML induction, nor how they change over time, and with remission status. Similarly, little is known about the trajectory of quality of life (QoL) and distress scores in this population. We aimed to learn more about the natural history of these issues via a prospective, longitudinal, observational patient-reported outcomes study. Methods: We enrolled 43 inpatients with AML at initiation of induction chemotherapy, and assessed their symptoms, quality of life (QoL), and distress weekly during their month-long hospitalization for induction, and monthly thereafter, using 3 validated instruments: Patient Care Monitor v2.0 (PCM); Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu); and NCCN distress thermometer (DT). We used descriptive statistics and ANOVA to analyze results. Results: Mean age of study participants was 59.4 (SD 13.4); 21 (49%) were female. Patients were mostly high-risk for recurrence, with 25 (58%) being ≥60 years old, 19 (44%) having high-risk cytogenetics, and 10 (23%) having relapsed disease. Among relapsed patients, the mean number of prior treatments was 2.7 (SD 1.3). At the time of this analysis, 5 patients (18%) had gone on to receive a stem cell transplant. As expected, symptoms were most prominent during the second and third weeks of treatment. However, across all 4 weeks of induction patients consistently reported 5 symptoms at a moderate or severe level (scores of 4 to 6, or 7 to 10 out of 10, respectively), including: poor appetite (35%), dry mouth (37%), difficulty sleeping (38%), dysgeusia (44%), and fatigue (56%). Other prominent moderate-to-severe symptoms included diarrhea (35%), daytime sleepiness (30%), and nausea (27.5%), despite standard supportive care. Mean QoL by FACT-Leu worsened substantially from week 1 (121.8, SD 27.6) to week 2 (108.2, SD 26.3), and then slowly recovered thereafter, improving to better than baseline by month 3 and continuing to improve throughout 1-year of follow-up (p

Description: Background Multiple myeloma (MM) is the second most common hematologic cancer in adults. Novel agents, including thalidomide, bortezomib, and lenalidomide, have led to improved overall survival (OS) in MM, but the disease remains generally incurable with the majority of patients inevitably relapsing after front-line therapy (FLT). In a multi-center observational analysis of 383 MM patients (treated between 2007 and 2010), median progression-free survival (PFS) and OS from treatment after first relapse were 13 and 35 mos (Durie ASCO 2012; abs 8095). According to NCCN guidelines, retreatment with primary therapy may be considered if relapse occurs more than 6 mos after discontinuation of primary therapy, otherwise a switch in regimen is recommended (NCCN MM Guidelines v.4 2015). Treatment patterns and outcomes in RRMM remain to be elucidated. We examined treatment use, retreatment and therapy switch patterns, and outcomes among patients with RRMM initiating second-line therapy (SLT). Methods We conducted a retrospective cohort study using a large, national, US claims database. Adult patients with MM who initiated cancer-specific systemic therapy between Jan 2008 and Feb 2014, without evidence of transplantation, were identified. Newly diagnosed MM patients were followed from the first claim for MM. Continuous enrollment in the health plan for 12 mos pre-diagnosis (with no claim for MM) through at least the start of SLT was required. FLT began with the first claim for MM-directed systemic cancer therapy. Unique agents administered within 90 days of FLT initiation constituted a regimen. Continuation of FLT regimen (or part thereof) or monotherapy within 3 mos of the end of the initial regimen was considered part of FLT. SLT after first relapse for RRMM was identified accordingly: 1) a treatment gap 〉6 mos between end of FLT and start of a second regimen (retreatment or switch), 2) start of a follow up regimen (retreatment) with a treatment gap of 〉3 and up to 6 mos after end of FLT, or 3) a switch to another drug combination after FLT regimen. Analyses were conducted from the first claim for SLT. SLT ended at the earliest of start of a new drug, death, or end of study period (Feb 2014). MM drug combinations were based on all unique MM systemic therapy agents received within start and end date for SLT. Vital status information was ascertained from the Social Security Death Index. Results We identified 249 patients with RRMM receiving SLT. Approximately half (49%) were male; 29% and 43% were aged 65-74 and 75 years or older, respectively. SLT regimens were: lenalidomide±dexamethasone (R±d, n=70 [28%]), bortezomib±d (V±d 61 [25%]), other (58 [23%], see Table 1), V+other (25 [10%]), R+other (14 [6%]), V-cyclophosphamide±d (VC±d, 13 [5%]), and VR±d (8 [3%]). The switch and retreatment patterns from FLT to SLT are depicted in Table 1. Among patients with treatment-free interval (TFI, time from end of FLT to start of SLT) 〉6 mos (n=64) vs ≤6 mos (n=185), 18 (28%) vs 17 (9%) were retreated with the same primary regimen in SLT, respectively. Median duration of SLT was 6.3 (95% CI: 5.6, 6.9) mos (Fig. 1) and of FLT was 6.8 (95% CI: 6.0, 7.8) mos. The 1-year OS probability from initiation of SLT for RRMM was 82% (95% CI: 76%, 86%). Conclusions Among patients with RRMM treated in the USA, V- and R-based regimens were the most common at first relapse. The vast majority of patients (91%) with TFI ≤6 mos switched therapy at time of relapse in concordance with NCCN guidelines. The relatively short duration of SLT (median 6.3 mos) in this study compared with PFS from treatment after first relapse (median 13 mos, Durie ASCO 2012; abs 8095) in a cohort of patients with RRMM, suggests that the majority of patients discontinue SLT prior to disease progression. These findings highlight the need for newer SLT regimens that are effective and more sustainable compared with current treatment choices. Table 1. Switch and Retreatment Patterns from FLT to SLT among Patients with RRMM. SLT V-based R-based VR-based Other FLT V-based 41 (41%) 49 (58%) 4 (50%) 15 (26%) R-based 33 (33%) 13 (15%) 2 (25%) 12 (21%) VR-based 9 (9%) 6 (7%) 2 (25%) 19 (33%) Other* 16 (16%) 16 (19%) 0 (0%) 12 (21%) Total 99 (100%) 84 (100%) 8 (100%) 58 (100%) *Other combinations consisted of a subset of the following agents: cyclophosphamide, melphalan, vincristine, doxorubicin, interferon-alpha, pomalidomide, thalidomide, carfilzomib, dexamethasone, prednisone Disclosures Romanus: Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Jhaveri:Sanofi: Equity Ownership; Takeda Pharmaceutical Company Limited: Equity Ownership; Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Labotka:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Henk:Optum (a consulting firm retained by Takeda to conduct the reasearch pertaining to this abstract): Employment. Seal:Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment.

Description: Introduction: Anemia is the most common hematological abnormality in patients with cancer and hematological malignancies, and is associated with poor prognosis and outcomes that have a detrimental impact on the patient's condition and quality of life (QOL). Erythropoiesis-stimulating agents (ESA) represent a good treatment option in order to increase the hemoglobin level in patients with anemia. Anemia can also be treated by red blood cell transfusion, but this has a transient effect and is associated with risks such as exposure to infectious agents, iron overload, or transfusion-related acute lung injury. ESA also have safety concerns, including the established increased risk of venous thromboembolic events. However, they are currently the only therapeutic alternative to transfusions. We performed a prospective observational study in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematological malignancies, with the primary objective of evaluating the effect of a new ESA biosimilar, epoetin zeta (Hospira) on patient QOL. Secondary objectives included hemoglobin (Hb) and platelet (Pt) recovery, safety, overall survival (OS) and relapse incidence. Results of this study were compared to two reference populations, one receiving epoetin beta (Roche) and one control group not treated with ESA. Here, we present preliminary results for the secondary objectives. Materials and methods: The study included adult patients with Hb level ≤11g/dl occurring after all types of allo-HSCT for any hematological disease (Table 1). Epoetin zeta (30,000 IU) was administered s.c. once per week for up to 6 months, and Hb levels were monitored weekly. Injections were stopped once the Hb level reached 12g/dl without transfusion. If after 4 injections, no improvement was observed, doses were doubled, and if after 8 injections, no improvement was observed, the patient was withdrawn from the study. The QOL was measured at baseline and at 1, 2, 3 and 6 months by the Functional Assessment of Cancer Therapy-Anemia (FACT-An) scale. Epoetin zeta responders were defined as having Hb level ≥12g/dl (complete response, CR) or a ≥2g/dl increase (partial response, PR) compared with baseline value, in the absence of transfusion. Patients receiving epoetin zeta (group 1) were compared to a similar population receiving epoetin beta with the same procedures (group 2) and to a matched population not treated with ESA (group 3), taking into account the following variables: sex, age, diagnosis, disease status at allo-HSCT, conditioning regimen and HSC source. Results: Between December 2011 and September 2014, 58 patients (from 168 screened) were included in group 1, and compared to 59 patients in group 2 and 65 patients in group 3. The main exclusion criteria were ESA contra-indication and patient refusal. Patients in group 1 had lower Hb baseline levels compared to group 2; patient characteristics for each group are summarized in Table 1. The median number of injections/patient was 10 (range: 6-14) in group 1 and 8 (range: 2-28) in group 2. The cumulative incidence of CR was 80% in group 1 and 71% in group 2. The median time to achieve CR was 48 days (range: 35-70) in group 1, and 39 days (range: 14-180) in group 2. Eight patients withdrew due to ESA inefficacy in group 1 and 8 in group 2. Adverse events were all thromboembolic: 2 events in group 1 and 5 events in group 2, compared to 2 events in group 3 (p=0.34). The multivariate analysis studying different confounding factors on the cumulative incidence of CR showed a significant positive impact of younger age (p=0.001), and a negative impact of being female or having major ABO incompatibility. We did not find any significant difference in terms of OS and relapse rate between the 3 groups. Conclusion: We describe here, for the first time, preliminary data for ESA biosimilar epoetin zeta (Hospira) in allo-HSCT patients showing comparable efficacy and safety to an existing ESA, epoetin beta (Roche) with no impact on OS and relapse incidence, compared to a control group. The QOL and transfusion evaluations as well as a cost-effectiveness study are ongoing and results will be presented. Disclosures Nicolini: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ariad Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: A gap in trust between patients with sickle cell disease (SCD) and medical providers is well recognized, largely originating from repetitive requests for opioid analgesics. Although expert clinical care guidelines in sickle cell disease are available, they rarely address measures by which this endemic gap in trust may be narrowed to fulfill the goals of medicine. We hypothesized that an increased familiarity with the patient as an individual through exposure of physicians to first-person narratives of the life-world of SCD may allow reformulation of perceptions and narrow the gap between physicians and patients. In a pilot study, extended first-person narratives of the illness experience elicited from patients with SCD with a history of recurrent hospitalizations (n=10) point to the individualized impact of pain, illness and stigmatizing disruptions in life-building efforts imposed by SCD together with the recurrent conflicts with medical caregivers, particularly after transition from pediatric to adult medicine. Patient-elicited narrative fragments such as "You are constantly fighting with people who are supposed to be making us feel better", "You don't know me, I am a church boy!" and "Put down your microscopes and talk to us" reveal the yearning by patients for individual integrity to be acknowledged, absorbed and interpreted by physicians. Additional narrative fragments such as "I hate myself. I hate my life", "Why am I broken?", "you don't want to be the girl with jaundice", "I had to leave work for 2 months because I was hospitalized, it killed me, it killed me", "you never know when it's going to be the last day, the last moment", exhibit the woundedness and fragility of existence experienced by patients. Narratives elicited separately from physicians caring for SCD (n=5) reveal anxiety, fear and distrust of the reported pain experience and opioid requests, and point toward deeper schisms that disparities may define: "I was very scared when I took care of my first sickle cell patient", "I came in with a bias already", "a similar frequent flier who had the same kind of behavior", "you have to be careful with empathy, people can take advantage of it", "I am not an enabler", "My grandfather told me about these people". Yet when the same physicians were invited to read and reflect on transcripts of the life-world stories of patients, reshaped perceptions of the stigmatized patient are revealed; "without knowing the story, you can't put it all together", "this would change the way I view treating them", "I will have more patience now" as examples. However, "Why can't they listen to our story? It could help them to be better patients," suggests that additional studies of the mutual intersections of patient and physician narratives are warranted and that these can offer insights and pathways toward mitigating distrust and conflict between medical care providers and patients with SCD. Disclosures No relevant conflicts of interest to declare.

Description: PURPOSE: To semi-quantitatively assess the evidence on the value of ultrasound (US) for assessment of hemophilic arthropathy (HA) in children and adults. We sought to provide the answer to the following questions: (1) Are currently available US techniques accurate for early diagnosis of pathological findings? (2) Can treatment reduce the incidence of US-detectable findings in HA? (3) Do US scores correlate with clinical/radiological constructs in the evaluation of HA? (4) Are US findings associated with functional status of joints? METHODS: Articles were screened using MEDLINE (n= 519), EMBASE (n= 493), and the Cochrane Library (n=24) (1946-2015). Two independent reviewers assessed the reporting quality and the methodological quality of articles by using the Standards for Reporting of Diagnostic Accuracy (STARD) and the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tools respectively. 4 different US scanning protocols for assessment of hemophilic joints were compared based on scanning times and anatomical landmarks. 6 US scoring systems were also compared according to number of soft-tissue and osteochondral parameters evaluated. RESULTS: Out of 16 full-text articles, 9 diagnostic accuracy studies (417 patients with hemophilia A, B and von Willebrand's disease) were evaluated for reporting and methodological quality using STARD and QUADAS-2 assessment tools respectively. Seven studies were of moderate reporting quality and 2 of low reporting quality. When using QUADAS-2, 1 study was of high, 3 of moderate, 2 of low, and 3 of very low methodological quality. Out of 9 diagnostic accuracy studies, 1 evaluated HA in ankles, knees, elbows, and shoulders while 3 evaluated ankles, knees, and elbows and only 2 evaluated ankles and knees. 2 more studies focused solely on knees and 1 on shoulders. Six US interpretation scores were reviewed and compared. All 6 articles included synovial hypertrophy in their evaluation. 5 articles incorporated cartilage modification while only 2 articles assessed hemosiderin deposition in their evaluation. Among these 6 scores, 4 were of moderate reporting quality, 1 of low and 1 of very low reporting quality. When using QUADAS-2 however, only 1 study was of high, 1 of moderate, 2 of of low, and 2 of very low methodological quality. Four US scanning protocols were also assessed, all evaluated the joints in both prone and supine positions. 3 suggested an extended scanning procedure of up to 30 minutes per joint, while 1 proposed a more simplified procedure. Two scanning protocols evaluated knees and ankles, while one focused on elbows. Only one scanning protocol included ankles, knees, and elbows in its assessment. CONCLUSIONS: There is insufficient evidence (Grade I) to recommend US as an accurate technique for early diagnosis of pathologic findings, to demonstrate that US scores correlate with clinical/radiological constructs, that treatment can reduce the incidence of US-detectable findings in HA, and to prove an association between US findings and the functional status of the joint. Further studies are required to establish standardized US scanning protocols and scoring systems and to determine US as a valuable tool for early diagnosis of hemophilic arthropathy (HA) in children and adults. ACKNOWLEDGEMENTS: This work was funded by Novo Nordisk Health Care AG. Disclosures Abadeh: Novo Nordisk Health Care AG: Other: Funding. Blanchette:Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Other: Data Safety Monitoring Board; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxter Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Data Safety Monitoring Board, Research Funding. Doria:Novo Nordisk Health Care AG: Other: Funding.

Description: Background: Venous thromboembolism (VTE) is an established complication of cancer, with a high incidence in patients with both lymphoma (5-15%) and gliomas (15-20%). There is minimal data describing the incidence of VTE in patients with CNS lymphoma. Known VTE risk factors including obesity, immobility, and hospitalization are present in those with central nervous system (CNS) lymphoma; however, concern for bleeding complications limits the use of pharmacologic prophylaxis. The aim of this study is to characterize the frequency of VTE in adult patients with CNS lymphoma treated at a large academic medical center. Methods: The University of California, San Francisco (UCSF) Cancer Registry was queried for all cases of adult CNS lymphoma diagnosed from 2008-2014. Chart review was performed and patients were excluded if the primary treatment occurred elsewhere. Demographics, histology, treatment and mortality data were examined. The presence of VTE was defined by radiographic evidence of a pulmonary embolism (PE) or deep vein thrombosis (DVT). Results: 134 cases of adult CNS lymphoma were reported at UCSF between 2008-2014; 18 patients were excluded. The average follow up time was 3.8 years from date of diagnosis. The mean age at diagnosis was 63. Of the 116 patient included in the study, 77 (66%) were identified as having Primary CNS Lymphoma and 39 (34%) had Secondary CNS Lymphoma including the following subtypes: Diffuse Large B-cell, Burkitt's, Mantle cell, Marginal zone, and NK cell lymphoma. There were 34 cases of VTE (29.3%): 12 were PE and 22 were DVT, 32 (94%) patients were hospitalized at the time of VTE diagnosis. Twenty eight (82%) were symptomatic. Ten (29%) were line-associated VTE. Fifty percent of VTE cases occurred during cycle 1 (18%) or cycle 2 (32%) of chemotherapy and 27 patients (79%) received systemic steroids within 30 days of VTE diagnosis. The median time from diagnosis of CNS lymphoma to VTE was 70 days. Confounding variables including age at diagnosis, race, sex, smoking history and histology were studied and no difference was found between the VTE and non-VTE groups. Notably, body mass index was significantly higher in the VTE group (29.85, 95% CI: 26.5-33.2) as compared to the non-VTE group (25.66, 95% CI: 24.76-26.55, p=0.019). There was a trend toward shorter time to death after the diagnosis of CNS lymphoma in the VTE group (382 days, 95% CI: 106-657) vs. the non-VTE group (699 days, 95% CI: 348-1049, p= 0.18). There was no difference in overall survival (log rank p=0.09). Conclusions: Here we report a remarkably high frequency of VTE (29%) in CNS Lymphoma patients treated at a large academic center. While there was no difference in age, sex, race, smoking history or histology, those who developed VTE did have a significantly greater BMI. Because many patients receive eight cycles of high dose methotrexate as treatment for CNS Lymphoma, our determination that half of the VTE episodes occurred during the first two cycles may be useful in defining the optimal timing of VTE prophylaxis in this high risk population. Most (82%) of the VTE reported in this study were symptomatic, which suggests VTE may have a significant impact on quality of life. There was a trend toward shorter time to death after diagnosis of CNS lymphoma in the VTE group, suggesting VTE may affect mortality. Larger, prospective studies are needed to characterize the timing and incidence of VTE in patients with CNS Lymphoma, but this study reports one of the largest single institution experiences in the literature. Disclosures Rubenstein: Genentech: Research Funding; Celgene: Research Funding.

Description: Introduction The introduction of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML). In addition to blocking their main target kinase, the BCR-ABL oncoprotein, several studies have reported that TKIs could also have secondary effects on the immune system and lymphocyte behavior. The aim of this study was to assess the bone marrow (BM) lymphocyte status at diagnosis and during different first-line TKI therapies and correlate it with treatment responses. Methods Altogether 105 first-line TKI treated patients were included in the study (imatinib n=71, dasatinib n=25 and nilotinib n=9) and samples from 14 healthy bone marrow donors served as controls. BM aspirate samples were taken from patients at the diagnosis and at 3, 6, 12 and 18 months after the TKI therapy start, and MGG-stained BM aspirate slides were examined for cellularity and individual cellular proportions. Treatment responses were evaluated with standard karyotyping and real-time quantitative PCR. Patients were divided in different groups according to ELN criteria based on their therapy response at 12 months. In addition, multi-color flow cytometry was done from both BM and peripheral blood (PB) samples using 5 different antibody panels including markers for T, B, NK and regulatory T cells. Results We found an early (3 months) expansion of BM lymphocytes during all different TKI therapies (imatinib median lymphocyte count 20%; dasatinib 21%; nilotinib 22%; healthy controls 12%, p

Description: Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR. It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations. Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs. RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. Disclosures No relevant conflicts of interest to declare.

Description: Extracorporeal photopheresis (ECP) is an effective frontline therapy for patients with leukemic cutaneous T-cell lymphoma (L-CTCL), but the mechanisms of action are not fully understood. To elucidate molecular mechanisms underlying the efficacy of ECP, we used Agilent Whole Human Genome Microarrays to examine blood transcriptional profiles in L-CTCL patients after ECP therapy. Ten L-CTCL patients including 5 clinical responders and 5 non-responders were studied. Their peripheral blood was collected before ECP (baseline), at Day 2, and one month post-ECP. Total RNA extracted from peripheral blood mononuclear cells was assayed with Whole Human Genome Oligo Microarrays (4 × 44 K) (Agilent, Santa Clara, CA). The differentially expressed gene analysis (DGA) was done using the paired t-test with Benjamini- Hochberg correction (P value 〈 0.05) between post-ECP and baseline. The fold change of gene expression between post-ECP and baseline were calculated from the normalized values. Hierarchical clustering of differentially expressed genes was performed with the Pearson correlation. The DGA between responders and non-responders were cross-compared. Canonical biological pathways were identified using Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City, CA). Differentially expressed gene profiles were different in responders from non-responders. As indicated in Figure 1, there were more genes differentially regulated in responders than in non-responders post-ECP at both Day 2 (549 genes in responders versus 66 genes in non-responders) and at one month (472 genes in responders versus 95 genes in non-responders). Among 472 differentially expressed genes in responders at one month post-ECP, almost twice as many genes (313) were down-regulated compared to up-regulated genes (159). The top down-regulated genes were IL-1β, EGR1, CCL3, CCL3L3, and CXCL2. The down-regulated genes were mainly related to functions of platelets, immune and/or stress responses, and chromatin remodeling. The upregulated genes were mainly related to functions of the nucleolus and included USP34, POLR3F, ZNF529, C22orf35, and BAT2D1. The ingenuity pathway analysis revealed that the top 5 pathways affected by ECP at one-month in responders were 1) integrin signaling; 2) granulocyte adhesion and diapedesis; 3) signaling by Rho Family GPTases; 4) agranulocytes (lymphocyte, monocyte and macrophage) adhesion and diapedesis; and 5) triggering receptor expressed on myeloid cells 1 (TREM1) signaling (Table 1). In contrast, these pathways and genes were less affected in non-responders. Of note, a comparison of all DGA results indicated that the responder group overlapped in the differentially expressed genes between Day 2 group (RD2) and one month group (RM1), but had few genes in common to the non-responder group (NM1). There were 94 genes consistently downregulated among RD2 and RM1 while only 6 genes were found in common between the RM1 and NM1 group. Similarly, 61 genes were consistently upregulated in group RD2 and RM1 while only 3 genes were found in common between the RM1 and NM1 group. In summary, the blood transcriptional profiling by this study identifies a signature of genes and pathways relevant to clinical response to ECP in L-CTCL patients. These findings expand our understanding of molecular mechanisms of ECP. Further validation of these genes and pathways is warranted in the future studies. Table 1. Top canonical pathways affected by ECP in L-CTCL patients responded to ECP at one-month Canonical Pathways Downregulated genes Upregulated genes Integrin Signaling 15/201 (7%) ITGA2B, MAP3K11, ITGA5, MYLK, ITGB3, MYL9, PARVB, AKT1, RHOB, CAPN1, ACTN4, CTTN, ARPC4, ACTN1, ITGB5 2/201 (1%) ITGB1, PPP1R12A Granulocyte Adhesion and Diapedesis 14/179 (8%) CSF3R, ICAM1, PPBP,ITGA5, CXCL5, SDC4, CCL3, ITGB3, GNAI2, CLDN5, CCL3L3, IL1B, CXCL1, CXCL2 1/179 (1%) ITGB1 Signaling by Rho Family GTPases 13/236 (6%) SEPT5, MAP3K11, ITGA5, MYLK, GNAZ, CDC42EP2, GNAI2, MYL9, GNG11, GNA15, RHOB, GNB2, ARPC4 3/236 (1%) ITGB1, DIAPH3, PPP1R12A Agranulocyte Adhesion and Diapedesis 13/190 (7%) ICAM1, PPBP, ITGA5, CXCL5, SDC4, CCL3, GNAI2, MYL9, CLDN5, CCL3L3, IL1B, CXCL1, CXCL2 1/190 (1%) ITGB1 TREM1 Signaling 7/76 (9%) ICAM1, AKT1, NLRP12, ITGA5, IL1B, CD83, CCL3 2/76 (3%) ITGB1, NLRC3 Figure 1. Differentially expressed genes post-ECP between responders and non-responders Figure 1. Differentially expressed genes post-ECP between responders and non-responders Disclosures Duvic: Therakos: Research Funding. Ni:Therakos: Research Funding.

Description: Mechanistic target of rapamycin complex 1 (mTORC1) is a central integrator of nutrient and growth factor inputs that controls cell growth in all eukaryotes. Rapamycin and its analogs (rapalogs) have been approved for the treatment of relapsed mantle cell lymphoma. A large proportion of aggressive B-cell lymphoma patients, however, respond poorly to rapalogs. The second generation of mTOR inhibitors function as ATP-competitive inhibitors (TORi), directly targeting the mTOR catalytic site. TORis have been proven to be more effective than rapalogs in cancer treatment. However, the mechanism underlying the cytotoxic effect of TORis in aggressive B-cell lymphomas remains unclear. In this study, we demonstrated that TORi-induced apoptosis is predominantly dependent on loss of mTORC1-mediated 4EBP phosphorylation. Knocking out Rictor, a key component of mTORC2, or inhibiting p70S6K has little effect on TORi-induced apoptosis. In contrast, increasing the EIF4E:4EBP ratio by either overexpressing EIF4E or knocking out 4EBP1/2 protected lymphoma cells from TORi-induced cytotoxicity. Furthermore, down-regulation of MCL1 and BCL-XL expression plays an important role in TORi-induced apoptosis whereas BCL-2, in cells with high expression, confers resistance to TORi treatment. Based on the mechanism study, we demonstrated that BH3 profiling, primarily NOXA and HRK stimulation, can effectively predict the cytotoxicity of the TORi in lymphoma cells. Also, in combination with pro-apoptotic drugs, especially BCL-2 inhibitors, the TORi exerted powerful anti-tumor effects both in vitro and in vivo. Taken together, this study provides mechanistic insight in TORi treatment in aggressive B-cell lymphoma and identified a mean to predict and improve its effectiveness clinically. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The haematological parameters, regulation of erythropoiesis, erythropoietin and body iron stores have been variably studied in dwellers and transient visitors from high altitude. Even in individuals born at high altitude, transient movements out of high altitude can significantly change these parameters. The role of this genetic adaption (reduced erythropoiesis) in Tibetans is well described (Moore LG et al, High Alt Med Biol 2001). There are scarce studies on true native highlanders and 'Ladakhi' populace from India which is geographically co-located to Tibet but of different ethnic origin (Wu TY et al, Zhongguo Ying Yong Sheng Li XueZaZhi. 2013). Objective: To study complete blood counts including red cell and platelet indices in native highlanders and influence of high altitude living on body iron stores and serum erythropoietin levels in true native highlanders. Methodology: True native highlanders in this study are defined as individuals born at altitudes above 11500ft with no descent to lower altitudes ever in their life. Baseline anthropometric data and peripheral oxygen saturations were collected. Haematological work up included total haemoglobin, haematocrit, total/ differential leucocyte count, platelets, red cell and platelet indices done by Sysmax® automated counter at Leh, Ladakh and serum erythropoietin/ ferritin levels at PGIMER, Chandigarh. A total of 1328 children were screened of which 402 children (stratified by age 4-17years) were identified as true native highlanders. Guardians of 12 children didn't consent for the haematological evaluation. Results: Study population included 197 males and 193 females. The mean age of study population was 127.58 + 39.64 months (range 35-254). The mean BMI was 18.7+2.5 kg/m2 (range 12.86-30.45). The mean peripheral oxygen saturation was 90.35 + 3.583 %. The haematological parameters of the study population are described in table 1. There was statistically significant difference between males and females in haemoglobin concentration, RBC count, haematocrit, platelet count and platelet distribution width (p

Description: BACKGROUND The Hedgehog (Hh) signaling pathway, which plays an important role during embryogenesis, can be reactivated in a wide range of cancers. Vismodegib, a selective hedgehog pathway inhibitor, demonstrated antitumor activity in medulloblastoma and basal-cell carcinoma (BCC) patients and has been approved by the FDA and EMA for the treatment of BCC. Preclinical data indicate that Hh pathway can also be activated in lymphoid malignancies and that its inhibition has antitumor activity (Dierks et al, Nature Med 2007; Singh et al, Leukemia 2012; Decker et al, Blood 2012). METHODS We conducted a phase II trial testing vismodegib in patients with relapsed/refractory lymphoma and CLL. Vismodegib was given orally at the dose of 150mg/day until disease progression or unacceptable toxicities for a maximum of one year. The primary objective was to evaluate the efficacy of vismodegib as measured by the best overall response rate (ORR) during the treatment period. RESULTS Between February 2013 and January 2014, 31 patients were recruited including diffuse large B-cell lymphoma (DLBCL, N=12), indolent lymphoma (iNHL, N=6), primary CNS lymphoma (PCNSL, N=10) and chronic lymphocytic leukemia (CLL, N=3). Patient characteristics are summarized in Table 1. We found that Hh signaling pathway (measured by IHC and/or PCR based on the expression of SHH, PTCH 1 and 2, SMO, GLI 1, 2 and 3, and ABCG2) was frequently activated at baseline in lymphoma patients. PK analysis demonstrated bioavailability of vismodegib in blood and CSF (median concentration at day 28 = 13398 [6970-20700] and 323 [99-717]ng/L, respectively). Nevertheless, none of the patients responded to vismodegib therapy, except for one. This patient had an iNHL (grade 3a follicular lymphoma). He experienced a partial remission after 2 cycles that lasted for 4.8 months. Interestingly, this patient had the strongest expression of GLI (1, 2 and 3) by PCR compared to all the other patients tested. All patients discontinued treatment prematurely (Table 1), mostly due to disease progression (90.3%). Adverse events (AE) and serious AEs (SAE) related to vismodegib were observed in 51.6 and 12.9% of the patients, respectively. SAEs related to vismodegib were diarrhea, vomiting, lung infection, hypoglycemia, pulmonary embolism, and skin rash (1 patient each). CONCLUSIONS Despite frequent Hh pathway activation, treatment with vismodegib did not show significant clinical efficacy in patients with lymphoma or CLL. This study was supported by Roche. Table 1. DLBCL iNHL PCNSL CLL Patients (N) 12 6 10 3 Median age (years) 74 74.5 65 74 Median number of prior therapies (min-max) 3 [1-6] 2.5 [1-4] 2 [1-3] 3 [1-4] Median duration of treatment (months) 1.7 1.8 1.2 1.9 Best overall response (CR/uCR/PR) 0% 16.7% (N=1*) 0% 0% Median PFS (months) 1.7 2.2 1.2 3 Median OS (months) 5.4 21.3 16.4 19.4 (*) Duration of response = 4.8 months Disclosures Off Label Use: Vismodegib in lymphoid malignancies. Haioun:Roche: Honoraria. Thieblemont:St. Louis Hospital, Paris, France: Employment. Casasnovas:Roche: Consultancy, Research Funding; Takeda: Consultancy; Gilead: Consultancy. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Salles:Roche: Honoraria, Research Funding.

Description: Increasing resistance to chemotherapy means that approximately 30% of patients afflicted with peripheral T-cell lymphoma will develop a relapsed/refractory form and eventually succumb to the disease. To date there is no clearly established second-line therapy and investigation of new therapies is necessary to address this unmet medical need. Masitinib is an oral tyrosine kinase inhibitor that potently and selectively targets c-Kit and platelet-derived growth factor receptor (PDGFR). Studies in human T-cell lymphoma have identified aberrant expression of PDGFR-alpha and that this kinase fosters peripheral T-cell lymphoma cell proliferation via an autocrine loop. Masitinib may therefore exert an antiproliferative and pro-apoptotic action on abnormal T-cells. In addition to this direct mechanism of action, masitinib can elicit antitumor effects by acting on mast cells and macrophages. By inhibiting mast cells, masitinib reduces the release of pro-tumoral M2-polarizing cytokines as well as factors favoring metastasis and angiogenesis. Masitinib can also promote macrophage infiltration into tumors, inducing an anti-tumoral Th1 immune response. Inhibition of cell proliferation was observed following single-agent masitinib treatment in the OSW canine T-cell lymphoma line, with an IC50 of 0.005 μM, suggesting that it could be used efficiently for the treatment of T-cell lymphoma. This hypothesis was further substantiated by a case study in a companion dog with T-cell lymphoma treated with masitinib monotherapy 12 mg/kg/day, reporting a complete response following 3 weeks of treatment with increased quality of life. This preliminary observation was repeated in two separate independent studies, each comprised of 11 dogs with T-cell lymphoma. A first study reported an overall response rate (ORR) of 73%, including 3/11 dogs with complete response (CR) and 5/11 dogs with partial response (PR) after 3 months of treatment. A second study reported an ORR of 45%, including 2/11 dogs with CR and 3/11 dogs with PR after 3 months of treatment. Meta-analysis of these data (n=23) showed that masitinib treatment of canine T-cell lymphoma resulted in a CR in 6/23 dogs (26%) and in a PR 8/23 dogs (35%), resulting in an ORR of 61%. Naturally occurring tumors in dogs have more clinical and biological similarities to human cancers than any other animal cancer model, hence these data provide strong medical plausibility for masitinib in the treatment of human peripheral T-cell lymphoma. The above in vitro and in vivo data led to initiation of a multicenter, randomized, open-label, three-parallel group, phase 2 study to evaluate the combination of masitinib plus dexamethasone with or without gemcitabine in patients with relapsed or refractory peripheral T-cell lymphoma. A recent decision to accelerate to a phase 3 study was based on an observed survival benefit for masitinib treated patients when compared with the control arm, and an acceptable safety profile; passage to phase 3 was validated by the independent Data Safety Monitoring Board with data blinded to sponsor and investigator. Specifically, pooled data from all masitinib-treated patients in the phase 2 stage (n=34) estimated a median overall survival (OS) of 9.0 months, which compares favorably against the literature benchmark median OS of 5.5 months for documented chemotherapy in this indication. This phase 3 study is currently open for patient recruitment in at least 14 countries and has OS as the primary endpoint. If successful, masitinib could provide a new treatment option in relapsed or refractory peripheral T-cell lymphoma. Disclosures Off Label Use: Masitinib is a new orally administered tyrosine kinase inhibitor that targets mast cells and macrophages, important cells for immunity, through inhibiting a limited number of kinases. Based on its unique mechanism of action, masitinib can be developed in a large number of conditions in oncology, in inflammatory diseases, and in certain diseases of the central nervous system. In oncology due to its immunotherapy effect, masitinib can have an effect on survival, alone or in combination with chemotherapy. Through its activity on mast cells and consequently the inhibition of the activation of the inflammatory process, masitinib can have an effect on the symptoms associated with some inflammatory and central nervous system diseases and the degeneration of these diseases.. Marçais:AB Science: Consultancy. Dubreuil:AB Science: Consultancy. Moussy:AB Science: Other: CEO.

Description: INTRODUCTION Follicular lymphoma (FL) may, over time, transform into an aggressive lymphoma, usually diffuse large B-cell lymphoma (DLBCL). Transformed follicular lymphomas (tFL) have a worse prognosis due to poorer response to treatment than primary DLBCL. The incidence of transformation is estimated in ~3% per year, although it varies largely between different studies (24%-70% overall). These differences are mainly due to different criteria to define tFL, to lack of evidence of tFL by biopsy, absence of clonality studies discarding secondary de novo NHL, studies performed in the pre-Rituximab era, or different follow-up times among studies. With all this pitfalls, the actual incidence of transformation remains an open question. The aim of the present study is to analyse the incidence and prognostic impact of transformation in patients with FL in a large retrospective series of the Spanish group of Lymphomas (GELTAMO). PATIENTS&METHODS A total of 1763 patients from 19 Spanish centres diagnosed of FL between 2000 and 2011 were recruited in the study. Data were obtained from the database of centres willing to participate in this study. True tFL (FL to DLBCL) were recorded. From the original cohort, FL IIIb, composite FL+DLBCL, discordant FL (FL in bone marrow and DLBCL in adenopathy or viceversa), and downgrading tFL (DLBCL at diagnosis and relapse of FL) were excluded. Patients with inadequate follow-up were not considered. Therefore, 1611 patients (grade I, II, and IIIa) were finally included. This study was approved by the Salamanca University Hospital Ethic Committee. RESULTS One hundred and ten patients (median follow up of 6 years) were transformed to DLBCL. Cumulative incidence of transformation at 5, 10, and 15 years was of 5%, 9%, and 14%, respectively. With a median follow up of 75.9 months (2 to 179), median time to transformation was 66 months, ranged 1-179. Considering survival from diagnosis of FL, tFL patients had a shorter OS than non-transformed (19% vs. 69%, p

Description: Introduction: Treatment of relapsed/refractory (rr) Non-Hodgkinxs lymphoma of B-cell type (B-NHL) is still challenging and new and more effective therapies are urgently warranted. Blinatumomab, a bispecific T-cell engager (BiTE®) antibody construct, engages CD3+ cytotoxic T cells, resulting in T-cell expansion and lysis of CD19+ B cells. In a prior phase I study, blinatumomab treatment resulted in an overall response rate (ORR) of 69% and 55% in diffuse large B-cell lymphoma (DLBCL), respectively for patients treated at 60 μg/m2/d. Based on this study we here present a single-center analysis on long-term outcome of patients with B-NHL after treatment with blinatumomab. We here report first preliminary data on long-term follow-up of patients with rr NHL after blinatumomab treatment. Methods: Patients with rr B-NHL who achieved an objective response (PR or CR) upon blinatumomab treatment were eligible for long-term follow-up analysis. Kaplan-Meier estimates for progression free survival (PFS) probability were calculated from first infusion to relapse or death. Patients without an event were censored at last follow-up. This study was conducted in accordance with the Declaration of Helsinki. Results: Out of 38 treated patients (17 follicular lymphoma (FL), 14 mantle cell lymphoma (MCL), 4 DLBCL, 3 others) 22 received the effective target dose of 60/90 μg/m²/d. 47,4% (CR/PR=28,9%/18,4%, n=38) and 68,2% (CR/PR=40,9%/27,3%, n=22) of the patients experienced response, respectively, whereas only 18,8% of the patients who were not treated with the effective target dose responded to blinatumomab (CR/PR=12,5%/6,3%, n=16). 6 patients are still in ongoing remission. Median overall survival (OS) of all 38 patients was 1560 days, median PFS 204 days and treatment free survival (TFS) 233 days. Median OS in the 22 patients treated with the target dose was 1793 days, median PFS 492 and TFS 752 days, compared to only 412 days median OS, 46 days PFS and 82 days TFS in the patients, who did not receive blinatumomab in an effective dosage. Conclusion: Blinatumomab has the potential to induce durable remissions in patients with rr NHL. Our data suggests that treatment at target dose of 60 μg/m²/d is an important prerequisite to achieve long-term remission. Figure 1. Kaplan Meier analysis shows significantly longer PFS in patients on target dose Figure 1. Kaplan Meier analysis shows significantly longer PFS in patients on target dose Disclosures No relevant conflicts of interest to declare.

Description: Background While kidney disease (KD) is a well described complication of multiple myeloma (MM), occurring in up to 40% of patients, the incidence, pathological manifestations, and clinical correlations associated with KD in patients with Waldenström's Macroglobulinemia (WM) or IgM MGUS remain to be clarified. Methods Out of 1,738 patients with consensus criteria defined WM (N=1,655) or IgM MGUS (N=83) diagnosis who were evaluated in the WM clinic at our institution from 2001-2015, we selected those individuals with at least one of the following abnormalities: serum creatinine ≥1.3; estimated GFR (eGFR)

Description: Background Recent studies have shown that the concurrent expression of MYC and BCL2 protein evaluated by immunohistochemistry (IHC) in patients with de novo diffuse large B-cell lymphoma (DLBCL) is associated with worse survival when treated with standard R-CHOP, but the effect of intensive chemotherapies for such patients is unknown. Thus, we evaluated the impact of the co-expression of MYC and BCL2 protein among patients with advanced DLBCL, who were treated with a dose-intensive immunochemotherapy followed by up-front autologous stem cell transplantation (ASCT). Patients and Methods This is a retrospective analysis of patients with de novo DLBCL, who were categorized into high/high-intermediate risk by the age-adjusted International Prognostic Index (aaIPI). They were consecutively treated with the R-Double-CHOP regimen, consisting of rituximab (375 mg/m2, day -2), cyclophosphamide (750 mg/m2, day 1, 2), doxorubicin (50 mg/m2, day 1, 2), vincristine (1.4 mg/m2 [maximum 2.0 mg/body], day 1) and prednisolone (50 mg/m2, day 1-5) followed by consolidative high-dose chemotherapies at our institution from 2001 to 2013. MYC and BCL2 protein were measured by IHC assay using formalin-fixed paraffin-embedded tissue specimens for all available cases. Cut-off values of positivity for MYC and BCL2 protein were set as 40% and 50% of stained tumor cell, respectively. Lymphomas showing concurrent positivity for MYC and BCL2 protein were defined as "Double expressor lymphoma (DEL)". Results A total of 40 patients with a median 53-years (range 19-68) of age were analyzed. Twenty-one patients were at high risk and the other 19 patients were at high-intermediate risk by aaIPI. Cell of origin (COO) subtypes classified by Hans algorithm consisted of 14 germinal center B-cell (GCB) type and 26 non-GCB type. Totally, 10 (25%) patients were categorized into DEL. The overall response (OR) and the complete response (CR) rates to R-Double-CHOP for all patients were 93% and 83%, respectively. The OR and the CR rates were not significantly different between the DEL group and the non-DEL group (100% vs 90%, and 80% vs 83%, respectively). The proportion of patients proceeding to ASCT was not significantly different among these groups (50% vs 60%). With a median 52 months (range 3-155) of follow-up, the 3-year progression-free survival (PFS) and the overall survival (OS) rates for all patients were 55% and 72%, respectively (Figure a, b). Both the PFS and the OS were significantly worse in the DEL group than in the non-DEL group (Figure c, d). As for aaIPI and COO subtyping, either high/high-intermediate risk or GCB/non-GCB subtype were not significantly associated with the outcome of PFS or OS. Conclusion The concurrent expression of MYC/BCL2 protein in advanced DLBCL was associated with shorter remission duration and worse survival despite similar susceptibility to the treatment when a dose-intensive immunochemotherapy was applied. Our findings suggest that patients with advanced DEL may not benefit from dose-intensified therapies, and therefore need highly discrete strategies. Disclosures Miura: Astellas Pharma Inc.: Honoraria; Celgene K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; Meiji Seika Pharma: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Hatta:Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Celgene K.K.: Honoraria. Iriyama:Brystol-Myers K.K.: Honoraria. Takei:Kyowa Hakko Kirin CO., Ltd, Japan: Research Funding; Bristol-Myers K.K.: Research Funding; Nippon Kayaku Co.: Research Funding; Shionogi & Co.: Research Funding; Meiji Seika Pharma: Research Funding; Astellas Pharma Inc.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; TEIJIN PHARMA LIMITED: Research Funding; CSL Behring K.K: Research Funding; Japan Blood Products Organization: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; TORII, PHAMACEUTICAL CO: Research Funding; Alexion Pharmaceuticals: Research Funding; YAKULT HONSHA CO., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; TAIHO PHARMACEUTICAL CO., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO. LTD: Research Funding.

Description: The development of the NCCN International Prognostic Index (NCCN-IPI) for patients with DLBCL treated in the rituximab era improves discrimination when compared to the original IPI model. The aim of the present study is to validate the results of the NCCN-IPI in a large independent series of patients in a different geographical area. Materials & Methods. This nation-wide retrospective study includes 2156 patients with de novo DLBCL diagnosed in 20 (mostly) large academic Spanish centers within the Grupo Español de Linfomas y Transplante de Médula Osea (GELTAMO) network between 1998 to July 2014. Patients had to be ≥ 18 years-old, treated with rituximab plus chemotherapy (R-CHOP or variants and also more intense treatments) and a minimum of 1 year of follow-up; all histological subtypes of DLBCL and primary extranodal cases were acceptable, with the only exclusion of primary testicular or CNS sites. In the whole series the scoring of the IPI and NCCN-IPI indexes were used and 5-year Overall Survival (5y-OS) estimated with the Kaplan-Meier method and compared with the log-rank test. Results. Debugging the database resulted in a final working series that included 1885 patients. The demographics of the series were comparable to the NCCN series: NCCN/GELTAMO male gender(%) 54 vs 50.4, Age(y) 57 vs 60, LDH〉1(%) 50 vs 54.7, Ann Arbor stage III-IV (%) 59 vs 62.5, ECOG PS≥2(%) 11 vs 30, extranodal disease(%) 36 vs 40.7. The IPI scoring (1760 patients) significantly separated the four risk groups, low (LR, 33.6% of the patients), low/intermediate (LI, 22.7%), intermediate/high (HI, 25.1%) and high (HR, 18.6%) with significantly different (p

Description: Introduction: MYC/BCL2 double hit lymphoma (DHL), defined as a large B-cell lymphoma with concurrent MYC and BCL2 translocations, is the most common type of DHL. Although multiple studies focused on DHL have been published, several issues regarding impact on prognosis remain controversial including: 1) history of low grade B cell lymphoma; 2) morphology (diffuse large B-cell lymphoma [DLBCL] versus B cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma [BCLU)]; 3) Absence or low expression of MYC or BCL2; 4) MYC translocation partner gene; and especially 5) most effective therapy. The aim of this study was to attempt clarify the prognostic importance of these factors in DHL. Methods: 157 patientsdiagnosed with MYC/BCL2 DHL between 2003 and April 2015 at two institutions were included in this study. MYC and BCL2 gene rearrangement were confirmed by FISH using a MYC breakapart probe and BCL2 and IGH dual color dual fusion probes. BCL6/3q27gene status was tested either by FISH using breakapart probe or by karyotype. MYC partner gene was identified by karyotype. MYC/BCL2 DHL cases were identified if they had rearrangements of MYC and BCL2 but not BCL6. Positive for MYC or BCL2 by immunohistochemistry was defined by 〉40% and 〉50% of lymphoma cells showed positive expression, respectively. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Fisher's exact test was used to compare the clinicopathologic features. Statistical analysis was performed using SPSS 23 software. Results: There were 103 men and 54 women with a median age of 61 years (range, 18-87). 110 patients had de novo disease and 47 patients had a history of low-grade B-cell NHL, mostly follicular lymphoma. The clinicopathologic features were similar (P〉0.05) between patients with a history of low-grade B-cell NHL and patients with de novo NHL, and therefore analysis was performed on all 157 DHL cases. Using the 2008 WHO classification, there were 91 DLBCL, 61 BCLU, and 5 composite lymphoma (4 DLBCL + follicular lymphoma and 1 DLBCL + B-lymphoblastic lymphoma). 99% of cases had a germinal center B-cell immunophenotype by the Hans algorithm. MYC expression was observed in 39/47 (83%) and BCL2 in 129/141(91%) of cases. MYC and BCL2 dual expression was present in 34/46(74%) cases. Of the 39 cases assessed, the MYC translocation partner was IGH in 13, IG light chain in 19, and a non-IG gene in 7 cases. 144 patients had complete treatment information: 61 received the R-CHOP regimen initially, 31 R-EPOCH, 29 R-HCVAD, and 23 various other chemotherapy regimens. 39 patients also received stem cell transplant (SCT) including 31 autologous and 8 allogeneic. 62 patients reached complete remission (CR) after initial chemotherapy. The median overall survival was 19 months. In a univariate analysis that evaluated 17 clinicopathologic parameters including those mentioned in introduction, extranodal sites of disease, bone marrow involvement, CNS involvement, advanced stage, and high/high-intermediate International Prognostic Index score were associated with a worse overall survival (OS, P

Description: Background: The aim of this study was to conduct an exploratory serum proteomic profiling study of diffuse large B-cell lymphoma (DLBCL) using mass spectrometry (MS) analysis to identify potential biomarkers that may provide further clues to disease mechanisms. Methods: Serum samples from 57 chemotherapy-naive male DLBCL cases and 30 healthy controls matched by age and BMI from a San Francisco Bay Area case-control study were divided into single-use aliquots and stored at -80°C until proteomic analysis. Serum samples were depleted of the 12 most abundant proteins by filtration on immunoaffinity spin columns prior to MS analysis. Proteomic analysis was performed using a GeLC approach. Specifically, equal amounts of protein from serologic specimens were separated by one-dimensional denaturing gel electrophoresis. Each lane was cut into six equal MW fractions followed by in-gel digestion with trypsin. The resultant peptides were analyzed using nano-liquid chromatography MS. Because MS is more qualitative than quantitative, the most significant and biologically relevant proteins of interest were chosen for further confirmation by ELISA in the "sandwich" configuration to specifically and quantitatively measure protein concentrations. ELISA assays were performed on unprocessed serum samples without depletion of the major serum proteins, providing a direct, one-step measurement, thereby avoiding any artifacts due to uncontrolled parameters common in preprocessing. Statistically significant differentially expressed serum peptides between the cases and controls as detected by MS were further analyzed using Systems biology/Pathway analysis. Results: MS data computed as normalized spectral counts of peptides revealed 1,207 protein IDs with 〉99% confidence, and 44 statistically significant candidates that were differentially expressed between the DLBCL cases and controls. We confirmed 4 [adiponectin (AdipoQ), extracellular matrix protein 1 (ECM1), CD14, and SerpinA3 (ACT)] of the 6 top candidates chosen for further confirmation by ELISA, which were elevated by 68.8, 62.9, 33.6 and 28.8 %, respectively, in DLBCL sera compared to controls. Adiponectin is an adipocyte-derived cytokine that regulates the metabolism of lipids and glucose. There is accumulating evidence that adiponectin also exhibits pro- and anti-tumorigenic activity, depending on the tumor type. Our study confirms previous reports of significantly elevated adiponectin levels in patients with adult non-Hodgkin lymphoma (NHL) and DLBCL, childhood NHL, and Hodgkin lymphoma compared to controls (PMID: 22547160). CD14 is a myeloid differentiation marker found primarily on monocytes and macrophages. Soluble CD14 levels have been positively associated with AIDS-related NHL risk (PMID: 23169327). ECM1 regulates cell migration, invasion and stem-like properties via induction of EMT progression and cancer stem cell formation. High ECM1 levels have been detected in various epithelial cancers, including invasive breast ductal carcinoma, esophageal squamous carcinoma, and gastric and colorectal cancer. ECM1 also has been associated with invasiveness and poor prognosis in thyroid and breast cancer. ACT is a metastasis-associated protein. Elevated ACT levels in serum have been associated with poor overall survival in acute leukemia (PMID: 24179540). The proteins identified in this study were critically analyzed using pathway analysis tools, and were found to be strongly associated with FcR signaling, and response to kinase activation including MAPK, membrane PTK, and neurotrophin TRK. These activities, and the proteins identified appear to involve IL-6 (Jak1/Stat3), ESR1/ CREB1, RXR1/ PPAR1, NF-kB, and HIF-1 signaling pathways at different stages of disease vs. controls. Based on these data, the predicted downstream effectors include regulation of immune processes that involve acute-phase and inflammatory responses, and regulation of lipid metabolism and transport. Conclusions: Through serum proteomic profiling studies, we have identified 3 novel (ECM1, CD14, and ACT) and 1 previously reported biomarker (adiponectin) of DLBCL that may have potential biological relevance in the pathogenesis or progression of the disease. Prospective epidemiology and clinical studies will be needed to determine whether these markers are involved in disease etiology and their possible prognostic value. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION: We recently described the first case of the evolution of inv(16) AML on the background of a clonal hematopoiesis due to a germline CBL mutation (defining the CBL syndrome), and we identified possibly cooperating mutations by exome sequencing (Becker et al. Blood 2014;123:1883-6). Among the mutated genes was PTPRT, encoding a protein tyrosine phosphatase that inhibits STAT3 activity and is commonly mutated in cancer (reviewed by Zhao et al. Oncogene 2015;34:3885-94). Here, we investigated the co-occurrence of mutated PTPRT with other mutated genes by single cell genotyping in order gain insights into the clonal architecture and sequence of mutation acquisition. METHODS: Exome sequencing of the bulk specimens was previously described; germline or somatic origin of mutations was verified in skin fibroblasts (Becker et al. Blood 2014;123:1883-6). For single cell genotyping, Ficoll-enriched bone marrow aspirates were DAPI stained, and single cells were placed into each well of a PCR plate using a MoFlo high speed cell sorter (Beckman Coulter). Genomic DNA was amplified by whole genome amplification (WGA) using the REPLI-g Mini Kit (Qiagen) according to a modified protocol, and subjected to PCR and Sanger sequencing of the respective mutation loci. As WGA can lead to allele dropout (ADO), we also sequenced single nucleotide polymorphisms (SNPs), that were identified by CytoScan HD array (Affymetrix) to be heterozygous in the sample and that were located nearby the respective mutation loci. RESULTS: Exome sequencing allows prediction of a possible clonal architecture based on the variant allele frequencies (VAFs). VAFs of the mutations identified in the AML were as follows: KIF14 p.V341I (VAF 51%), TMEM125 p.D113N (51%), MIOX p.W225R (46%), CAND1 p.E584* (39%), NID2 p.D319N (38%), ARF3 p.N101S (36%), PRSS16 p.R491C (36%), PTPRT p.T844M (33%), DOCK6 p.R1872_K1873insP (33%), ADAM12 p.A222V (21%), CMIP p.T323M (15%) and MYOCD p.D283N (7%); due to its germline nature, all leukemic cells harbored the CBL p.D390V mutation. In order to verify the co-occurrence of mutations in a clone and thus the clonal architecture, we performed single cell genotyping of the mutations in PTPRT as well as CAND1 and DOCK6. CAND1 and DOCK6 were selected in addition to PTPRT since their comparable VAFs did not allow identifying the sequence of acquisition. Moreover, CAND1 and DOCK6 were affected by likely deleterious mutations and were previously found mutated in AML. To control for ADO, we included the heterozygous SNPs rs2867061 (PTPRT), rs1252402 (C AND1), and rs12980863 (DOCK6) in our analyses. We analyzed 19 single cells for the 6 mutations and SNPs. This resulted in 102 successful sequencing reactions, and yielded informative results for at least 2 mutations in 12 cells and for all 3 mutations in 5 cells. Based on the concurrent presence of the wild-type allele at the mutation locus and ADO at the SNP site, 18 mutation analyses were judged to be inconclusive. Overall, our analyses confirmed that the mutations in CAND1, PTPRT and DOCK6 occurred together in the same clone. Moreover, based on the identification of cells with the presence of both CAND1 and DOCK6 mutations but presence or absence of PTPRT mutations, respectively, we concluded that PTPRT mutations were acquired after the mutations in DOCK6 and CAND1. CONCLUSION: Single cell genotyping verified the co-occurrence of PTPRT, CAND1 and DOCK6 mutations in the same AML clone and revealed a clonal hierarchy, as the PTPRT mutation was acquired after the mutations in CAND1 and DOCK6. These insights into the clonal architecture and evolution had not been possible solely based on exome sequencing and suggest that the sequential expression of mutated PTPRT may cooperate with mutated CBL and inv(16) at a late stage of AML development. Disclosures No relevant conflicts of interest to declare.

Description: Hodgkin lymphoma (cHL) is a malignant lymphoid neoplasia that shows aberrant expression of cytokines, including IL-10 and TNF-a. Single nucleotide polymorphisms (SNPs) in genes encoding IL-10 and TNF-a or their regulatory proteins, such as NFkB, may impact cHL pathobiology. In this study, SNPs in the promoter regions of genes encoding for IL-10 (SNP/pIL-10 -592, rs1800872; and SNP/pIL-10 -1082, rs1800896), and TNF-a (SNP/pTNF-a -238, rs361525; and SNP/pTNF-a -862, rs1800630), as well as in the intronic region of the NFkB1 gene (SNP/iNFkB1, rs1585215) were genotyped in 73 patients with cHL and evaluated against clinical and laboratory prognostic parameters for the disease. SNPs/pIL-10 AA were significantly associated with higher leukocyte and lower lymphocyte counts at diagnosis. In case of TNF-a, SNP/pTNF-a -238 AG was associated with EBV infection while SNP/pTNF-a -862 CC presented more frequently with leukocytosis. SNP/iNFkB1 AA generally had stage IV and extranodal disease at diagnosis. Nonetheless, none of the studied SNPs had effect on treatment outcome. This study shows that some SNPs genotypes for IL-10 and TNF-a genes are associated with prognostic parameters in cHL; furthermore, this is the first time that the SNP/iNFkB1 (rs1585215) was implicated in clinical features of the disease. Disclosures No relevant conflicts of interest to declare.

Description: The optimal source of donor hematopoietic stem cells (HSC) is controversial. Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood (G-PB) has replaced bone marrow (BM) as the most common allograft source in adults but is associated with donor morbidity and higher rates of chronic graft versus host disease (GVHD) compared to BM. The CXCR4 antagonist plerixafor (Px) mobilizes HSC into the PB (Px-PB) faster than G-CSF and preliminary data suggest both quantitative and qualitative differences in allograft content that may impact clinical outcomes. We sought to assess the efficacy and safety of transplanted allografts collected following mobilization with Px alone in HLA-identical sibling transplantation. This was a Phase II, two-strata, multi-center prospective trial (NCT01696461) to evaluate Px-PB allografts prior to reduced intensity conditioning (RIC) and myeloablative conditioning (MAC) based hematopoietic cell transplantation (HCT). Patients aged 18-65 years with an HLA-ID sibling donor and a hematological malignancy suitable for HCT were eligible. The primary objective was to determine the proportion of donors whose cells could be successfully mobilized and collected with a sufficient CD34+ cell dose using Px as the sole mobilizing agent. Px mobilization was considered successful if ≥ 2.0x10^6 CD34+ cells/kg recipient weight were collected in no more than two leukapheresis (LP) collections. All donors receiving Px were included in the analysis of the primary objective based on the intention-to-treat principle. Secondary objectives included the incidence of acute and chronic adverse events in donors, rates of hematopoietic engraftment, donor chimerism, rates of acute and chronic GVHD, non-relapse mortality (NRM), progression free survival (PFS) and overall survival (OS) for the recipients. From July 2013 to December 2014, 64 donor/recipient pairs were enrolled at 12 centers. Donors received Px at 240μg/kg subcutaneously 4 hours prior to LP. LP was performed processing at least 4X blood volume for up to two consecutive days (a third day was allowed for low CD34+ cell yields after 2 LP procedures) to achieve a target CD34+ cell dose of ≥ 4.0 x 10^6/kg recipient weight with a minimum goal of ≥ 2.0 x 10^6/kg. All allografts were cryopreserved. GVHD prophylaxis included cyclosporine or tacrolimus in combination with methotrexate, mycophenolate mofetil, or sirolimus. G-CSF was given routinely post HCT only to MAC recipients. Patient demographics are provided in Table 1. The median donor age was 56 years (18-65). 64% of the donors were male. Donors underwent one (23%), two (72%), or three (5%) LP procedures. 63 of 64 (98%) donors achieved the primary objective. The median total CD34+ cell dose/kg recipient weight collected within 2 days was 4.6 (0.9-9.6). Maximal donor toxicity following Px injection and LP was grades 0 (30%), 1 (52%), 2 (17%), and 3 (2%). Bloating, flatulence, abdominal pain, headache, paresthesisas, injection site reaction, and dizziness were the most commonly observed toxicities. Bone pain was not observed. The one grade 3 toxicity was a vasovagal episode felt related to LP and unlikely to Px. Toxicities typically resolved within a week of LP. The median follow up is 6.3 months. Median days to ANC (〉0.5 x10^9/L) and Platelet count (〉20 x 10^9/L) recovery were 13.5 (10-148) and 19 (1-76) after MAC and 14.5 (0-25) and 18 (0-141) after RIC, respectively. The cumulative incidence of acute GVHD grades 2-4 and 3-4 at day 100 were 47% (95% CI: 30-64) and 9% (95% CI: 2-22) after MAC and 19% (95% CI: 6-38) and 5% (95% CI: 0-18) after RIC. Probability of NRM at day 100 was 4% (95% CI: 0-13) and 0% after MAC and RIC, respectively. The probability of OS at day 100 was 97% (95% CI: 88-100) and 90% (95% CI: 78-98) after MAC and RIC, respectively. In conclusion, this is the first multi-center trial to demonstrate that as an alternative to G-CSF, Plerixafor rapidly, safely, and effectively mobilizes sufficient numbers of CD34+ cells from HLA-ID sibling donors for HCT following both RIC and MAC regimens. Engraftment was generally prompt and early results of secondary endpoints in recipients are encouraging. Longer follow-up and more extensive analysis of donor allografts and recipient outcomes will be presented at the time of the meeting. Research support was provided in part by Genzyme, a Sanofi Company. Table 1. Characteristics of recipients Table 1. Characteristics of recipients Disclosures Chen: Bayer: Consultancy, Research Funding. Devine:Genzyme: Research Funding.

Description: Background: Loss of major histocompatibility Class II antigens (MHCII) in diffuse large B-cell lymphoma (DLBCL) is associated with a decreased CD8+ tumor-infiltrating T-lymphocyte (TIL) response and poor patient survival. Transcription of the MHCII gene complex is under the control of the master transactivator, CIITA, which in part is regulated by histone acetylation. We tested the hypothesis that combination of histone deacetylase inhibitor vorinostat with standard chemotherapy will enhance MHCII expression and improve patient outcome in DLBCL. Methods: SWOG S0806 was a phase I/II open label trial of vorinostat given at 400 mg po daily on days 1-9 (subsequently reduced to days 1-5) combined with Rituximab-CHOP (R-CHOP) at standard doses, given on day 3 of a 21-day cycle for 8 cycles. Eligibility criteria included having newly diagnosed advanced stage DLBCL, international prognostic index (IPI) of at least 1, and lack of known CNS involvement or HIV. Primary endpoint of phase I was to establish maximum tolerated dose (MTD) of vorinostat with standard R-CHOP. Primary endpoint of phase II was to estimate 2-year progression free survival (PFS). Translational endpoints included correlation of pre-treatment acetylation status of histones, expression of MHCII genes, and percentage of TIL to PFS; and correlation of cytokine profile to response and outcomes. Results: Phase I was open in 5 SWOG institutions and enrolled 11 patients. There were only 2 patients who had dose limiting toxicities in the first cycle - grade 3 febrile neutropenia and grade 4 hypokalemia - allowing phase II to proceed with the original vorinostat dosing of 400 mg daily days 1-9, at all SWOG institutions. However, excess rates of febrile neutropenia and sepsis were seen upon further follow up, and the study was amended to reduce the duration of vorinostat to days 1-5. A total of 72 patients were enrolled in phase II, of which 8 were ineligible and 2 withdrew consent prior to treatment. For the remaining 62 patients, median age was 64 years, 92% had stage III/IV disease, 39% B symptoms, 61% elevated LDH, 39% had more than 1 extranodal site of involvement, with IPI breakdown of 13/26/47/13/2%. Notable grade 3-4 non-hematologic toxicities included febrile neutropenia (39%), sepsis (18%), fatigue (15%), hypokalemia (11%), hyponatremia (10%), and small bowel perforation (3%). Grade 3-4 hematologic toxicities included neutropenia (60%), anemia (35%), and thrombocytopenia (35%). There was one death in phase I from sepsis and multi-organ failure at the end of 8 cycles of treatment, but no deaths from toxicity in phase II. Overall response rate was 81% (95% CI: 69-90%). With median follow-up of 24.3 months, estimate of 2-year PFS is 72% (95% CI: 58%, 81%) and of 2-year OS is 85% (95% CI: 74%, 92%). Analysis of the panel of 30 cytokines performed on matched serum specimens of 40 patients showed correlation of baseline elevated IL-2R levels with worsened PFS and OS, and correlation of decrease in Epidermal Growth Factor level with improved PFS and OS. Results of immunohistochemical stains for expression of MHCII genes and percentage of TIL will be reported at the meeting. Conclusions: The regimen of vorinostat-R-CHOP achieved 2-year PFS estimate of 72%, which is slightly more than 68% expected from R-CHOP alone per IPI adjusted historical rate, but less than an IPI adjusted target of 78% that would be sufficient to warrant further investigation. It also resulted in unexpected excess rates of febrile neutropenia and sepsis. This regimen cannot be recommended for the broad DLBCL population. Current studies are focused on finding biomarkers of response to histone deacetylase inhibitors. Disclosures Persky: Gilead Sciences, Inc: Speakers Bureau. Off Label Use: vorinostat in diffuse large B-cell lymphoma. Barr:Abbvie: Consultancy; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding.

Description: Introduction: With improvements in transplant strategy and supportive care, hematopoietic cell transplantation (HCT) is increasingly being used to treat older patients (60-80 years). Frailty is a well-accepted phenomenon in the geriatric population characterized by diminished physiological reserve and increased vulnerability to stress, with strong associations with falls, disability, hospitalization and mortality. Since HCT is a substantial stressor, we hypothesized that the prevalence of frailty and pre-frailty will increase in the immediate post-transplant period, and that frailty will be associated with early (1y) post-transplant non-relapse mortality (NRM). Methods: We conducted a prospective, longitudinal study of 96 patients undergoing HCT at age 40y or older between February 2014 and April 2015, and serially performed a multi-domain geriatric assessment (incorporating function, comorbidity, cognition, psychological state, social activity/support, and nutritional status) in additional to demographic, transplant and disease related variables. Frailty was defined by presence of 3 or more of the following criteria: unintentional weight loss, exhaustion, weakness, slow walking speed and low physical activity; pre-frailty was defined by presence of 1 or 2 of the criteria. Geriatric assessments were performed pre-HCT and at 100 days, 6 months and 1 year post HCT. The prevalence of frailty and pre-frailty was evaluated at each time point in older (60-74y, n=48) vs. younger (40-59y, n=48) recipients. Longitudinal assessments (baseline, 100 days, 6 months, 1 year) of ordinal frailty measures were assessed using random effects and non-linear mixed logistic regression models. Impact of frailty on 1 year NRM was estimated using cumulative incidence estimates. Results: Older patients were more likely to undergo reduced intensity conditioning, and less likely to be employed at HCT. The prevalence of frailty increased with time since HCT: The prevalence of frailty was 8% at baseline and increased to 39% by 6 months (

Description: Mammalian microRNA expression is dysregulated in many human cancers, including hematologic malignancies. MicroRNAs are small non-coding RNAs that repress their target genes by binding to the 3' UTR sites in their respective mRNA targets, leading to transcript degradation and/or preventing translation. MicroRNAs play a variety of physiologic roles within the hematopoietic compartment, and a few microRNAs have even been shown to drive leukemogenesis when deleted or expressed at abnormally high levels. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a poor prognosis due to increased cellular proliferation and survival of the hematopoietic stem and progenitor cells (HSPCs). MicroRNA expression has been shown to be highly dysregulated in FLT3-ITD+ AML, however the functional relevance of many of these microRNAs on leukemic phenotypes remains unclear. In this study, we took an unbiased approach to determine which microRNAs, and which of their putative targets, are involved in FLT3-ITD+ AML cellular growth. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of FLT3-ITD+ leukemia cells (MV4-11 cells) over a 23-day time course. By comparing library representation from genomic DNA isolated from the final time point versus the initial time point, we were able to identify a number of protein-coding gene and microRNA candidates that either promoted or suppressed FLT3-ITD+ AML cellular growth. Our screen identified both evolutionarily conserved and non-conserved human microRNAs that function to suppress or promote FLT3-ITD+ AML cellular growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. We also performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in FLT3-ITD+ cells. We validated one of our targets, miR-155, as a critical regulator of FLT3-ITD+ AML cell growth in vitro. miR-155 knockout cells displayed a competitive growth disadvantage compared to miR-155 wild type cells. Further analysis revealed that deletion of miR-155 in MV4-11 cells led to decreased STAT5 activation, a key signaling intermediate known to promote cell survival and proliferation in FLT3-ITD+ AML. Finally, we found that miR-155 promotes FLT3-ITD-mediated myeloproliferation in vivo. FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis compared to their FLT3-ITD miR-155+/+ counterparts. This phenotype was attributed to miR-155's role in promoting proliferation of the HSPC and myeloid progenitor cell compartments in the bone marrow. Our CRISPR-Cas9 screen identified a subset of microRNAs that regulate FLT3-ITD+ cell growth, and extensively validated miR-155 as a promoter of FLT3-ITD+ cell proliferation. These findings were validated both in vitro and in vivo, and suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use could rapidly advance our understanding of how microRNAs contribute to human hematological diseases. Disclosures No relevant conflicts of interest to declare.

Description: Assessing potency of mesenchymal stem/stromal cells (MSC) used for immunologic applications such as the treatment of GVHD or other inflammatory disorders has been a challenge. Expression of PD-L1 or production of indoleamine-pyrrole 2,3-dioxygenase (IDO-1) has been proposed as potential potency markers. To screen for other pathways involved with suppression we undertook proteomic analysis of IFN-γ stimulated MSC. MSC isolated and expanded from normal healthy donors to 70-80% confluence were treated overnight with human rIFN-γ (30ng/ml). MSC were harvested using TrypLE Select and then immunologic and proteomic studies were performed. IFN-γ exposure increased a) MSC expression of IDO-1 and PD-L1, b) MSC suppression of 3rd party T lymphocyte proliferation, c) MSC inhibition of development of IFN-γ producing T lymphocytes, and d) MSC promotion of Treg expansion. Cellular proteomic changes that occur with IFN-γ exposures were studied in paired samples of control and IFN-γ treated MSC. Samples were prepared using a modified Filter Aided Sample Prep (FASP) and digests were separated using 2D liquid chromatography and analysed by tandem mass spectrometry. Data was processed with the Global Proteome Machine and only proteins with at least two confident peptides were reported. A total of 7621 proteins were identified of which 5575 were seen in all samples and 232 proteins were significantly upregulated in the IFN-γ treated cells relative to their controls. The proteomic analysis identified constitutive proteins seen in MSC. The upregulated proteins were significantly enriched (p

Description: Acute myeloid leukemia (AML) was diagnosed in approximately 18,860 people in the U.S. in 2014, and caused an estimated 10,460 deaths. AML is a heterogeneous clonal disorder with highly varied molecular abnormalities. To permit better prognosis and the development of novel targeted therapies, there is an urgent need to elucidate new molecular targets in AML. Previously, via The Cancer Genome Atlas (TCGA), we had analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b and demonstrated that low miR-199b levels correlate with worse survival outcomes, especially M5 subtype; however, their correlation with molecular mutations remained unknown. Herein, we attempt to determine the potential cooperative role of NPMc and IDH1 mutations in miR-199b-low AML and investigate the hematological phenotype resulting from miR-199b global knockout mice. Molecular mutations including FLT3-AML, NPMc and IDH1 were analyzed among miR-199b high and low AML cases. Significant correlations in terms of association and survival outcomes were observed for NPMc and IDH1 mutations. Among n=128 AML patients analyzed for miR-199b/NPMc, n=63 were miR-199b-high samples and, of these, n=2 were positive for NPMc mutation. Importantly, of the n=65 miR-199b-low samples, n=41 were positive for NPMc mutation (Fisher p value = 1.941e-08, odds ratio 19.6112). For IDH1 mutation, among the n=139 AML patients analyzed for miR-199b/IDH1, n=59 were miR-199b-high samples (with n=5 positive for IDH1 mutation) whereas n=80 were miR-199b-low samples (with n=25 positive for IDH1 mutation, Fisher p value = 0.01175, odds ratio 3.663005). Combined survival data analysis for miR-199b and NPMc+/IDH1+ (n=169 AML cases) showed worse survival in low miR-199b patients compared to AML patients negative for both mutations with high miR-199b (Chisq = 10.6 on 2 degrees of freedom, p = 0.00491). To define the functional role of miR-199b in myelopoiesis we generated a miR-199b knockout mouse strain via the CRISPR technique. Molecular confirmation of miR-199b silencing, both at the gene and transcript levels,were carried out by PCR-sequencing and RT-qPCR. Peripheral blood analyses at various time points was performed for n=4 mice each; miR-199b knockout mice exhibited a significant increase (p =0.018) in neutrophil levels (1.03 X103/uL) compared to wild type mice (0.72 X103/uL). Eosinophil and lymphocyte levels were also significantly elevated compared to wild type mice (p

Description: Aneuploidy causes a proliferative disadvantage, mitotic and proteotoxic stress in non-malignant cells ( Torres et al. Science 2007). Chromosome gain or loss, which is the hallmark of aneuploidy, is a relatively common event in Acute Myeloid Leukemia (AML). About 10% of adult AML display isolated trisomy 8, 11, 13, 21 (Farag et al. IJO 2002), or either an isolated autosomal monosomy or monosomal karyotype (Breems et al. JCO 2008). This evidence suggests that tumor-specific mechanisms cooperate to overcome the unfitness barrier and maintain aneuploidy. However, the molecular bases of aneuploid AML are incompletely understood. We analyzed a cohort of 166 cytogenetically-characterized AML patients (80 aneuploid (A-) and 86 euploid (E-)) treated at Seràgnoli Institute (Bologna). Aneuploidy was significantly associated with poor overall survival (median survival: 13 and 26 months in A-AML and E-AML respectively; p=.006, Fig.1). To identify AML-specific alterations having a causative and/or tolerogenic role towards aneuploidy, we integrated high-throughput genomic and transcriptomic analyses. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 70 samples from our A-AML and E-AML cohort of 166 patients. Variants where called with MuTect or GATK for single nucleotide variant and indels detection, respectively. AML samples were genotyped by CytoScan HD Array (Affymetrix). Gene expression profiling (GEP) was also conducted on bone marrow cells from 24 A-AML, 33 E-AML (≥80% blasts) and 7 healthy controls (HTA 2.0, Affymetrix). We detected a significantly higher mutation load in A-AML compared with E-AML (median number of variants: 31 and 15, p=.04) which was interestingly unrelated to patients' age (median age: 63.5 years in A-AML and 62 years in E-AML, Xie et al, Nat. Med. 2014). C〉A and C〉T substitutions, which are likely mediated by endogenous 5mCdeamination, were the most frequent alterations (Alexandrov et al. Nat. 2013). However, aneuploidy associated with an increased variability in terms of mutational signatures, with the majority of A-AML displaying 3 or more signatures compared to few E-AML cases (p=.04). WES analysis also revealed a specific pattern of somatic mutations in A-AML. A-AML had a lower number of mutations in signaling genes (p=.04), while being enriched for alterations in cell cycle genes (p=.01) compared with E-AML. The mutated genes were involved in different cell cycle phases, including DNA replication (MCM6, PURB, SSRP1), centrosome dynamics (CEP250, SAC3D1, HEPACAM2, CCP110), chromosome segregation (NUSAP1, ESPL1, TRIOBP), mitotic checkpoint (ANAPC7, FAM64A) and regulation (CDK9, MELK, ZBTB17, FOXN3, PPM1D, USP2). Moreover, genomic deletion of cell cycle-related genes was frequently detected in A-AML. Notably, ESPL1 which associated with aneuploidy, chromosome instability and DNA damage in mammary tumors (Mukherjee et al. Oncogene 2014) was mutated and also upregulated in A-AML compared with E-AML (p=.01), the latter showing expression levels comparable to controls. Among the top-ranked genes differentially expressed between A-AML and E-AML, we identified a specific signature characterized by increased CDC20 and UBE2C and reduced RAD50 and ATR in A-AML (p

Description: Blastic plasmacytoid dendritic cell neoplasm is a clonal disease derived from precursors of plasmacytoid dendritic cells (pDC). It is a rare neoplasm involving the skin which may or may not be associated from the outset with a leukemic component. The disease invariably progresses to aggressive leukemic dissemination, leading to a differential diagnosis with acute leukemia. In 2004, we set up a French network to recruit biological data at diagnosis. Diagnosis was according to recommendations (Swerdlow et al, 2008), with, in addition, a mandatory panel of pDC markers (Garnache-Ottou et al, 2009) detected by flow cytometry or by immunohistochemistry on infiltrated blood, bone marrow or cutaneous lesions. In total, 109 cases of BPDCN were included in 35 hospitals (2000-2013). BPDCN is more prevalent in men (sex ratio 4.4/1) and in elderly subjects (median age: 63 years; 7 patients were

Description: The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Disclosures No relevant conflicts of interest to declare.

Description: Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in vivo. A large inter-individual variation in ASNase pharmacokinetics is observed in patients. While elevated ASNase levels are associated with an increase in adverse events, underexposure, frequently caused by antibody mediated clearance, seriously reduces therapeutic efficacy. To date, it is not possible to predict pharmacokinetics of ASNase in individual patients and therefore current therapeutic protocols are supported by frequent monitoring of ASNase levels and adjustments of administration schemes. We used an in vivo imaging approach to study ASNase biodistribution and pharmacodynamics in a mouse model and provide in vitro and in vivo evidence that identifies the endo-lysosomal protease Cathepsin B in macrophages as a critical component of ASNase degradation. Results/Discussion Mice were injected with 111Indium-labeled ASNase and biodistribution was monitored by quantitative microSPECT/CT scans and ex vivo analysis of organs using a gamma counter. Over time, ASNase accumulated in the liver and particularly the spleen and the bone marrow. We hypothesized that macrophages in these organs, efficiently take up the ASNase, thereby rapidly clearing the active enzyme from the blood. Immunohistochemical analysis confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To confirm the importance of macrophage populations in ASNase clearance, we depleted mice from phagocytic cells by injection of clodronate liposomes, and studied ASNase biodistribution and kinetics. Indeed, clodronate pretreatment significantly diminished the accumulation of ASNase in the liver, spleen and the bone marrow while doubling the circulatory half-life of serum ASNase activity. We conclude from these experiments that macrophages determine the pharmacokinetics of asparaginase, which raises the question whether rapid clearance of the drug by bone marrow resident macrophages will negatively affect the depletion of asparagine in the bone marrow niche. We previously linked a germline mutation in the gene encoding endosomal protease Cathepsin B to strongly diminished asparaginase degradation in a pediatric ALL patient. To connect the macrophage mediated clearance to the proposed role of Cathepsin B in ASNase degradation, we studied the contribution of this protease in macrophage-mediated degradation of asparaginase. We used cell lines to show that Cathepsin B expression is induced during differentiation from monocytes towards macrophages. This is consistent with our finding that macrophages, but not monocytes, are capable of degrading ASNase. Furthermore, we used both chemical inhibition and RNAi mediated knockdown of Cathepsin B to show that this protease is required for ASNase degradation in these macrophages. Finally, by comparing Cathepsin B knockout mice with wildtype littermates, we demonstrated that loss of Cathepsin B activity significantly delayed clearance of serum asparaginase, consistent with a prominent role for this lysosomal protease in ASNase turnover. In conclusion, by using in vivo imaging we showed that asparaginase is efficiently cleared from the circulation by macrophages. In particular, bone marrow resident macrophages may provide a protective environment for leukemic cells by effectively removing the therapeutic protein from the bone marrow niche. However, both the prominent role of macrophages and the importance of the lysosomal protease Cathepsin B in asparaginase clearance, may allow the rational design of a next generation asparaginase. Disclosures Metselaar: Enceladus Pharmaceuticals: Employment, Equity Ownership.

Description: In patients with acute promyelocytic leukemia (APL), in spite of recent advances, early treatment related mortality (TRM) still remains a challenge and is largely related to the associated coagulopathy. It is well recognized that conventional coagulation parameters used in the clinic do not accurately predict the risk of adverse outcomes like major bleeding or thrombosis. There is limited data on the clinical utility of whole blood viscoelastic assays and extended coagulation parameters for evaluation of coagulopathy in patients with APL. Consecutive patients with PML-RARA positive APL were enrolled. Blood samples were collected prior to infusion of blood products on day 1 and again on day 4 for thromboelastometry done on whole blood and activated with recombinant tissue factor at a final dilution of 1:50,000 (ROTEM analyzer, Tem Innovations GmbH, Basel) and an extended panel of coagulation parameters done by immunoassays (TAT complex, protein C, ATIII, PIC complex, tPAIC, d-dimer and thrombomodulin). Platelet count and conventional coagulation parameters, which were prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and thrombin time (TT), were measured daily for the first 14 days (more frequently as clinically indicated). Patients received platelet transfusions to target a platelet count of 30x109/L (in the absence of bleeding). Fresh frozen plasma and cryoprecipitate transfusions were done to target a normal PT and aPTT and maintain a fibrinogen level above 140 mg%. Major bleeding, thrombosis and death during induction therapy were the outcome variables. Of the 61 patients enrolled, 11 were excluded (3 did not continue treatment at our hospital, 2 died within 48 hours of admission, and 6 took discharge against medical advice before completing induction). Of the remaining 50 cases that were evaluated, 40 were newly diagnosed and 10 were relapsed. The median age was 34.5 years (range 9-68). 25 were males (50 %). The WBC count was above 10 x 109/L in 20 patients (40%). The median platelet count was 28 x 109/L (range 4-339). There were 13 (26%) deaths in induction. Six patients had a major bleeding event. Five patients had a thrombotic event. Of the evaluated conventional coagulation parameters, extended coagulation parameters, platelet counts and ROTEM parameters at diagnosis (ROTEM data not available at diagnosis in 2 cases) only the ROTEM parameter of maximum clot firmness (MCF) as a continuous variable was significantly associated with death (p=0.012). On univariate cox regression analysis with death as the outcome, MCF≤ 30mm was a poor prognostic marker (hazard ratio of 13.24, 95%CI 1.71-102.19; p =0.013). On multivariate cox regression analysis including high WBC count (〉10 x 109/L), low platelet count (〈 40 x 109/L) and history of relapse, MCF ≤30mm was an independent poor prognostic variable (HR: 11.89; 95% CI 1.43 - 98.75; p 0.022). Four of the 6 major bleeding events, four of five thrombotic events and all deaths related to coagulopathy occurred in patients with MCF ≤30mm. The descriptive statistics of patients with MCF 〉30mm (n=21) vs those with MCF ≤ 30mm (n=27) are shown in table 1. Figure 1 illustrates the type of discordance one can see between conventional coagulation parameters and ROTEM values. This study suggests that the MCF value from a ROTEM assay at diagnosis is probably a better measure of the hemostatic defect at diagnosis in APL. Patients with APL with low MCF on ROTEM at diagnosis are at an increased risk of death during induction therapy despite an adequate blood product replacement strategy which was based on conventional coagulation parameters. In these patients, there is a need to develop novel and possibly a more intensive replacement strategy. Table 1. Comparison of baseline characteristics and clinical outcomes. Variables MCF ≤30 (n = 27)N (%) /Median (range) / Mean±SD MCF 〉 30 (n=21)N (%) /Median (range) / Mean±SD p value Age (years) 40(12-68) 27(9-48) 0.006 WBC 〉10 x x109/L 17(62.96) 2(9.52) 0.003 Platelet count (x109/L) 17(4-89) 45(10-339) 0.001 PT in seconds 14.5(11-87.5) 11.8(10.1-14.8) 0.004 aPTT in seconds 28(23.2-77.9) 30.2(22-53.6) 0.104 Fibrinogen in mg% 134.8(24.7-393) 172.2(14.7-549.5) 0.066 Major bleeding 4(14.8) 1(4.8) 0.368 Thrombosis 4(14.8) 1(4.8) 0.369 Death 12(44.4) 1(4.8) 0.003 Disclosures No relevant conflicts of interest to declare.

Description: Introduction: MRD positivity after induction/consolidation therapy in pts with de novo ALL has been shown to carry a very negative impact on outcome. However, the significance of MRD status in the salvage setting has not been extensively studied. Methods: We evaluated 130 pts with R/R B-cell ALL pts who received first (n=68), or second (n=62), salvage therapy between 2010 and 2015. Salvage therapies included single agent inotuzumab ozogamicin (INO; n=75), blinatumomab (n=20), or INO in combination with mini-hyper-CVD (n=35) [Jabbour E et al; EHA 2015]. Of the 130 pts treated, 78 (60%) responded and were assessed for MRD by six-color flow cytometry on marrow samples with a sensitivity of 0.01%. Morphologic responses were defined as follows, complete response (CR); disappearance of all disease with neutrophils ≥ 1.0 X 109/L, platelet 〉 100 X 109/L and blasts ≤ 5%, CRp; CR without platelet recovery, CRi; CR without platelet and/or neutrophil recovery. Results: The clinical characteristics of the 78 responding pts with R/R ALL are summarized in Table 1. Overall, MRD negativity was achieved at response in 41 pts (53%). Among the 41 pts who responded to single agent INO (12 CR, 26 CRp, 3CRi), 17 (41%) achieved a negative MRD status. Among the 11 pts who responded to blinatumomab (9 CR, 2Cri), 8 (73%) achieved a negative MRD status. Among the 26 pts who responded to INO in combination with mini-HCVD (21 CR, 4 CRp, 1CRi), 16 (62%) achieved a negative MRD status. Forty-four pts received allogeneic stem cell transplantation (ASCT): of those, 21 pts were MRD negative and 23 pts were MRD positive at the time of response. Median follow-up was 19 months (2-55). Overall, there was a trend for more durable morphologic responses in MRD negative pts compared with MRD positive pts: the median complete remission durations (CRD) were 17 months and 8 months, with a 2-year CRD rate of 47% and 28% respectively (Table 2). The median event-free survival (EFS) was 12 and 6 months, respectively; the 2-year EFS rates were 32% and 8%, respectively. Similarly, there was a trend for better overall survival (OS) with a median of 17 months and 9 months for pts with negative and positive MRD, respectively; the 2-year OS rates were 36% and 27%, respectively. No difference in outcome was reported whether pts were censored or not at the time of ASCT. Conclusion: In pts with R/R ALL, the achievement of negative MRD in addition to the morphologic response confers an improvement, although not statistically significant, in response duration and survival. Larger number of pts with longer follow-up is needed to validate these findings.Table 1.Clinical Characteristics of Pts (n=78)N (%)/Median [range]Age (years)38 [18-87]Sex (Male)50 (64)Performance Status1 [1-3]WBC (x 109/L)3 [0.3-38]% PB Blasts0 [0-83]% BM Blasts60 [8-97]CytogeneticsDiploid20 (26)t(9;22)4 (5)t(4;11)7 (9)Miscellaneous39 (50)Not done/ Insufficient metaphases8 (10)SalvageInotuzumab Ozogamicin41 (53)Blinatumomab11 (14)Inotuzumab Ozogamicin + mini-HCVD26 (33)Number of prior therapies1 prior therapy46 (59)2 prior therapies32 (41) Table 2. Response Rates and Survival by MRD Status MRD Negative (n=41) MRD Positive (n=37) p CR 24 18 n/a CRp 16 14 n/a CRi 1 5 n/a CRD, median (m) 17 8 0.63 2-year CRD rate (%) 47 28 EFS, median (m) 12 6 0.06 2-year EFS rate (%) 32 8 OS, median (m) 17 9 0.18 2-year OS rate (%) 36 27 Disclosures Cortes: Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BerGenBio AS: Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. DiNardo:Novartis: Research Funding.

Description: Reactivation of herpes zoster occurs in patients treated with immunosuppressive drugs but is generally not considered an infection associated with either acute myeloid leukemia (AML) or with its intense myelosuppressive treatment. Our institution has been using arsenic trioxide (ATO) since 1997 in all relapsed APL patients and more recently has included it in our front line APL regimens. Here we present a retrospective analysis investigating if ATO treatment is associated with herpes zoster reactivation and whether acyclovir can prevent this in APL patients as well as those with other malignancies on clinical trials. Methods: We evaluated records of patients treated at Memorial Sloan Kettering Cancer Center dating back to 1970, and selected patients with electronic medical records who received ATO treatment either as conventional therapy or as part of a clinical trial (n=213). We also selected records of any patient with a diagnosis of AML over the same period who had not received ATO for use as a control group (n=3366). Results: Among 213 patients who received ATO therapy, 27 (12.7%) were diagnosed with herpes zoster a median of 23 days after the initiation of therapy. This cohort included 76 APL patients, 11 (14.5%) of who were diagnosed with herpes zoster a median of 15 days after the start of therapy (1 days - 5 years). Among the 3366 patients in our AML cohort, 122 (3.6%) were diagnosed with herpes zoster a median of 230 days after the initiation of chemotherapy (2 days - 11 years). In univariate analysis we found a significant association between the likelihood of herpes zoster and ATO treatment (p 〈 0.01). Patients receiving ATO treatment had a greater than 3 fold odds of herpes zoster compared to non-arsenic treated patients, with a trend toward accelerated development of zoster at cumulative dosages 〉 1000mg (likelihood ratio test p = 0.28). Acyclovir was strongly protective in both younger and older patients, with an overall relative hazard of 0.07 (likelihood ratio p 〈 0.01). In a multivariate logistic regression analysis, we found that only arsenic use and older age at diagnosis contributed significantly to the likelihood of herpes zoster diagnosis (p = 0.01), but a diagnosis of APL did not (p=0.89). Conclusion: ATO is now part of the standard of care for treatment of patients with APL. We observed that the incidence of herpes zoster was more common among patients receiving ATO, with a trend toward greater risk at higher cumulative doses. There is evidence to suggest that ATO results in T-cell dysfunction (Morzadec, Bouezzedine, Macoch, Fardel, & Vernhet, 2012; Thomas-Schoemann et al., 2012), which is consistent with the observation that T-cell depleting diseases such as HIV and T-cell directed immunosuppressives are also associated with higher rates of herpes zoster. We therefore suggest including acyclovir prophylaxis in all patients receiving ATO, and continue prophylactic treatment up to 6 months after completion of therapy. Disclosures Douer: Gilead: Consultancy.

Description: T cell acute lymphoblastic leukaemia (T-ALL) is a rare form of leukaemia that accounts for approximately 15% of paediatric ALL cases. Unfortunately, approximately 20% of patients do not achieve long term remission as a result of failure of therapy to eradicate the disease. T-ALL is a highly heterogeneous disease that displays a spectrum of immunophenotypes, chromosomal aberrations and gene expression profiles. This heterogeneity has prompted research into more targeted therapies, with the aim of overcoming drug resistance often found with standard chemotherapeutic regimens. Here, we build upon use of the drug Parthenolide (PTL), which has shown promise in treatment of T-ALL and other leukaemias such as BCP-ALL and AML, in combination with ABT-263, a BCL-2 family antagonising agent. Bone marrow samples from 10 T-ALL cases, taken at diagnosis, were treated with PTL in vitro for 24 hours then viability was assessed using the annexin V / PI flow cytometric assay. Variable cytotoxic effects were observed in samples treated with PTL (1-10µM), with half maximal inhibitory concentrations ranging from 2.6-10 µM. At the highest dose tested, the proportion of surviving cells ranged from 5.79-56% (median 35.33%). BM from 5 of these samples was used for whole genome microarray (WGA) analysis. We compared gene expression in bulk ALL and in specific subpopulations, known to have leukaemia initiating capacity in vivo; CD34+/CD7+, CD34+/CD7-, CD34-/CD7+ and CD34-/CD7- cells. WGA data demonstrated that CD34+/CD7- was the only subpopulation to express significantly lower levels (5.38 fold) of the pro-apoptotic gene Bcl-2L11 (BIM) compared to the unsorted bulk T-ALL cells, p=0.006. Interestingly, we have previously shown that CD34+/CD7- cells from a few patients were resistant to PTL treatment in vivo compared to unsorted cells. To validate these results, mRNA and relative protein quantification was performed by qPCR and western blotting in bulk material from 8 of the 10 samples, 3 of which had been analysed by microarray for BIM expression. We found that the gene and protein expression levels of BIM were negatively correlated with PTL resistance in vitro, p≤0.0001 and p=0.049 respectively. This suggests that reduced BIM expression is related to PTL resistance. We next evaluated the effects of combining PTL and ABT-263 on T-ALL cells in vitro. ABT-263 is a BH3 protein mimetic, like BIM it promotes apoptosis by blocking the inhibitory effects that BCL-2 anti-apoptotic proteins have on pro-apoptotic proteins. The effects of combining the drugs were assessed in 7 of the original 10 samples. Unsorted ALL cells were incubated with PTL and ABT-263 for 24 hours, before viability was analysed by flow cytometry and drug synergy was calculated via the Chou Talalay method. This drug combination showed enhanced cytotoxicity to T-ALL cells compared to PTL (p=0.0282) or ABT-263 (p=0.0358) alone. Moreover, the highest combined dose tested (2.5µM PTL with 0.25µM ABT-263) killed 86.1±9% cells cf 71.8±18% with ABT-263 alone and only 21.7±11% with PTL alone. The combination also showed synergism with a combination index value below 1 in all doses tested. Previous findings in our laboratory have shown that in vivo PTL treatment eliminated childhood leukaemia in NOD/LtSz-scid IL-2Rγc null (NSG) mice, in most cases tested. It may be possible to further enhance this toxicity using ABT-263 alone or in combination with PTL. NSG mice were inoculated with unsorted T-ALL cells and leukaemia was allowed to establish until levels in peripheral blood (PB) exceeded 0.1%. NSG mice were subsequently treated orally for 21 days with 100mg/kg of ABT-263 or placebo and leukaemia burden was monitored weekly in PB aspirates. Twenty-eight days following commencement of treatment, leukaemia burden in the placebo treated group was 80.73±2.94% and the animals were electively culled. In contrast, disease burden was significantly lower in the treated animals at this stage (35.2±2.1%, p=0.004). ABT-263 treatment has significantly improved survival of all xenografts to date, (P

Description: Objective: The overall survival of pediatric acute myeloid leukemia (AML) has been around 70% on recent protocols, but the treatment causes severe toxicity. Age and body mass index (BMI) at diagnosis have been associated with outcome in AML. We investigated if toxicity and outcome were associated with age and BMI in the NOPHO-AML 2004 protocol (2004-2013). Methods: We reviewed toxicities and follow-up information of NOPHO AML-2004 registered in the database, including all protocol patients from the Nordic countries and Hong Kong who completed first induction (n=318). For children 2 years or older BMI standard deviation (SD) for age and sex was calculated and categorized (underweight: 1 SD (n=56)) according to the World Health Organization. Toxicities were registered after each block until end of treatment, stem cell transplantation, relapse or death. The age and weight groups were compared using cox-regression for the cumulative incidence of toxicity and the overall (OS) and event-free (EFS) survival. All estimates of age as risk factor were sex and ethnicity adjusted and estimates of weight as risk factor sex, ethnicity and age adjusted (age stratified for the survival analysis). Results: The cumulative incidence of treatment-related mortality was 4.6% (95%-CI 2.3-7.0%). Older children (10-17 years) were at higher risk of developing sepsis with hypotension (adjusted hazard ratio (HR) 2.3 (95%-CI 1.1-4.6) p=0.02) compared to children from 2-9 years. Overweight children (compared to healthy weight) also had a higher risk of experiencing sepsis with hypotension (adjusted HR 2.1 (95%-CI 1.0-4.3) p=0.051) and severe abdominal pain (adjusted HR 1.7 (95%-CI 1.0-3.0), p=0.048). The 5-year EFS and OS for the 318 patients were 50% (95%-CI 44-56%) and 71% (95%-CI 65-76%) respectively. Outcome depended on age. Infants (1 SD for age) at diagnosis did not appear to be prognostic marker for EFS (adjusted HR 1.10, 95%-CI 0.55-2.16, p=0.80) or OS (adjusted HR 1.01, 95%-CI 0.35-2.93, p=0.99), but in children from 10-17 years there was a trend for overweight being a positive prognostic marker for EFS (figure 1) and OS (figure 2). Conclusion: Older age and overweight was associated with increased toxicity during treatment for pediatric AML. Infants had superior EFS and older children had inferior OS compared to children from 2-9 years. In contrast to previous pediatric reports we found overweight at diagnosis was not a poor prognostic marker for survival, in contrast in older children there was a trend for overweight being a positive prognostic marker. These findings suggest different pharmacokinetics of chemotherapeutic drugs in adolescences compared to younger children and warrant further studies. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. [18 F]-Fludarabine is a promising novel positron emission tomography (PET) radiotracer for lymphoid malignancies. The rationale supporting its development is the high selectivity of fludarabine uptake within lymphoid cells irrespective of their cycle activity, and the fluorine atom within the drug, which replaced by a [18 F] confers the positron-emitter property. Pre-clinical studies with [18 F]-fludarabine (Dhilly M et al, Mol Imaging Biol 2014,16:118-26; Hovhannisyan N et al, EJNMMI Res 2015,5:23) showed a marked tumor uptake in lymphoma-bearing mice. The aim of this study was to describe anatomical sites with abnormal [18 F]-fludarabine uptake in patients with B-cell chronic lymphocytic leukemia (B-CLL), in whom [18 F]-FDG PET imaging appears to be less informative than in other lymphoid malignancies. This study was designed as a clinical proof of concept. Methods. [18 F]-Fludarabine was produced according to a method already described (Guillouet S et al, Mol Imaging Biol 2014,16:28-35). [18 F]-Fludarabine PET/CT (Discovery RX VCT 64, GE Healthcare) was performed in 5 patients (51-70 years old) with a diagnosis of B-CLL (according to current recommendations), without suspicion of Richter's syndrome. Successive partial body scans (skull vertex to mid-thigh) were acquired for 250 min after intravenous injection of [18 F]-fludarabine with an activity of 4MBq/kg. PET images were analyzed by drawing volumes of interest (VOI) over the uptake sites on a late scan and projected onto all co-registered scans of the same subject. The intensity of tracer uptake was evaluated with the maximum standardized uptake value (SUVmax). The images of [18 F]-fludarabine PET/CT were visually compared with conventional modalities (high-resolution CT) and [18 F]-FDG PET in one patient. Results. No adverse event was recorded during and after the procedure. In the 5 patients studied, the uptake of [18 F]-fludarabine coincides with sites expected to be involved following conventional clinical and CT scan staging (i.e. lymph nodes, spleen, tonsils). Additionally, [18 F]-fludarabine PET/CT displayed a significant uptake in hematopoietic bone marrow, and unexpected uptake in the medulla of some bones (femur, humerus, sphenoid, calvarium), and in Peyer's patches. SUVmax were significantly greater in involved sites in comparison with normal tissues or organs (Figure 1). Within the involved sites, [18 F]-fludarabine demonstrated a wide range of uptake which would indicate heterogeneity and differing micro-environments. For instance, the most active sites of the case illustrated in Figure 1 (scan period 30-50 min) are lymph nodes (SUVmax 7.4), spleen (SUVmax 6.3) and bone marrow (SUVmax 3.8) with aortic uptake (SUVmax 1.5) as background level. Conclusion. These preliminary results showed a clear specificity of this novel radiotracer for lymphoid tissues. [18 F]-Fludarabine PET/CT appears to be a promising tool for the diagnosis and follow-up of B-CLL. These scans indicate variable activity within proliferation sites, information otherwise not accessible by current non-invasive procedures. Further studies for a more accurate comparison with [18 F]-FDG PET, evaluation of response during and after treatment and correlation with B-CLL prognostic criteria are underway. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Objective: To analyse systematically the clinical and biological characteristics of 2080 myelodysplastic syndrome patients in our laboratory from 1984 to 2013 and to reveal the unique features of MDS patient in our area. Methods: 1. Conventional cytogenetics were performed to investigated the cytogenetics changes in 2080 MDS patients. All patients were classified according to the FAB criterion, in which, 1493 cases were reclassified according to the WHO (2008) criterion; and 550 patients' outcomes were evaluated according to the International Prognostic Scoring System, WHO classification-based Prognostic Scoring System (WPSS) and the revised International Prognostic Scoring System (IPSS-R). 2. We analysed the clinical, cytogenetic characteristics and survival of 2080 MDS patients by statistical methods. Results: 1. According to the FAB criterion: 1040 (50.0%) patients with RA, 135 (6.5%) patients with RARS, 691 (33.2%) patients with RAEB, 145 (7.0%) patients with RAEB-t, and 69 (3.3%) patients with CMML. The median age was 51 years old (range, 5-93 years old). The ratio of male and female was 1.54. 40.3%(839/2080) patients had clonal chromosome abnormalities, in which 277 (13.3%) patients with complexed karyotype. The rate of karyotype abnormalities was higher in RAEB than that in other subtypes. Survival analysis show that the subgroup with RA had a longer median survival duration than the subgroup with RAS, RAEB, RAEB-t, their median survival duration was 50 months, 32 months, 13months and 16 months, respectively. 2. According to the WHO (2008) criterion: 220 patients (14.7%) with RA/RN/RT/RCUD, 75 patients (5.0%) with RARS, 385 patient (25.8%) with RCMD, 14 patient (0.9%) with 5q- syndrome, 282 patients (18.9%) with RAEB-1, 306 patients (20.5%) with RAEB-2, 211 patients (14.1%) with MDS-U. The ratio of male and female was 1.51 (898/595) and the median age was 54 years old (range, 6-93 years old). In all patients, the median hemoglobin level was 70g/L (11~167 g/L), the median platelet count was 51.5×109/L (2~1045 ×109/L) and the median WBC count was 2.65×109/L (0.11~52×109/L). The rate of clonal chromosome abnormalities was 42.1% (628/1493), in which 216 (14.5%) patients with complexed karyotype. There was statistically significant difference in the rate of chromosomal abnormalities among different subtypes (P

Description: The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with 〉90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p

Description: Introduction: Current advances in allogeneic hematopoietic cell transplant (alloHCT) may warrant a paradigm shift in managing children with sickle cell disease (SCD). This study characterizes the clinical outcomes and health care utilization (HCU) of alloHCT for pediatric SCD. We hypothesize that early alloHCT will have improved clinical outcomes, and decreased HCU. Methods: The Center for International Blood and Marrow Transplant Research database was used to identify children 21 years or less with alloHCT for SCD in the United States. Patient data included comprehensive research forms (CRF) from 2000-13 and transplant essential data (TED) forms from 2000-11. CRFs provided clinical risk factors associated with overall survival, graft failure, grade III-IV acute GVHD, and GVHD related event free survival (GREFS) - the survival free of graft failure, chronic GVHD, or death. Risk factors included age, gender, performance status, year of alloHCT, prior SCD complications and therapy, CMV status, donor type, conditioning regimen, and GVHD prophylaxis. Due to low event rates and sample size, only univariate analysis of risk factors was performed. TED data was merged with Pediatric Health Information System (PHIS) inpatient data using a probabilistic merging algorithm to determine risk factors and clinical outcomes associated with HCU. Available PHIS adjusted cost data was used to determine the total adjusted cost for all inpatient admissions per patient per hospital day. To standardize these costs, the total adjusted cost per 30 hospital days was calculated for each patient and used as the primary HCU outcome. HCU outcomes were analyzed for the alloHCT year, day 0 to day +365. Results: CRF data for 161 patients showed an overall survival at 2 years of 90% (95% confidence intervals [CI] 85-95%): 96% (95% CI 89-100%) for related and unrelated cord blood transplant (CBT), 94% (95% CI 86-98%) matched siblings (MSD), and 74% (95% CI 54-90%) matched unrelated donors (MUD, p=0.002). All deaths occurred among children with pre-alloHCT complications of SCD, and deaths were due to organ failure (37.5%), infections (25%), GVHD (6.25%). Risk of death was significantly higher for children ≥10 years old (HR 21, p=0.003) and MUD compared to MSD (HR 5.88, p=0.005) but lower with cyclosporine A (CSA) GVHD prophylaxis versus FK506 (HR 0.33, p=0.031). 75% of deaths occurred before day +42. Cumulative acute GVHD incidence at day 100 was 14% (95% CI 9-20%)and was associated with age ≥10yrs (HR 2.63, p=0.035). Chronic GVHD incidence was 31% (95% CI 23-38%) at 2yrs, and factors associated were age ≥10yrs (HR 1.92, p=0.034), MUD vs MSD (HR 2.53, p=0.017), and CSA vs FK506 prophylaxis (HR 0.48, p=0.018). Chronic GVHD risk increased significantly after 2006 (HR 2.81, p=0.018). The 2yr GREFS was 64% (95% CI 56-71%). Age ≥10yrs (HR 2.2, p=0.005), MUD (vs MSD, HR 3.00, p=0.002) and CSA prophylaxis (vs FK506, HR 0.49, p=0.011) were significantly associated with this outcome. Among the 175 patients with combined TED and PHIS data, the median total adjusted cost was $117,393 per 30 hospital days per patient (range: $36,244-$515,640) during the alloHCT year with a median of 53 hospital days per patient (range: 16-304). Age ≥10yrs and HCU were not significantly associated (p=0.775). MSD had the lowest HCU compared to CBT and unrelated transplants (p

Description: Introduction/ Background : Chronic lymphocytic leukemia (CLL) is a heterogeneous disease which can present as an aggressive and life threatening leukemia or as an indolent form that will not require treatment for decades. This heterogeneity has important consequences which will impact on clinical approaches, treatment strategies, and survival times from diagnosis. Prognostic markers such as expression of specific proteins in or on CLL cells (ie, CD38, 70-kD ζ-associated protein or CD49d), cytogenetic abnormalities (del 13q, del 11q, del 17p and trisomy 12) quantified by FISH and immunoglobulin heavy chain (IgVH) gene mutation have all been very useful. Futhermore, patients with early-stage disease, with biologically aggressive disease and shorter survival times can be distiguished. However, these prognostic tests are expensive and require considerable technical expertise and equipment and thus are not available to many patients with CLL living in developing countries. Therefore less expensive prognostic markers are needed. In this study, we evaluated the prognostic significance of smudge cells percentage on a blood smear in CLL patients. Patients and Methods : In this prospective study, 42 untreated patients with CLL have participated after signing a consent form. Patients were seen at our center between July 2011 and May 2015. Patients were diagnosed on the basis of an absolute lymphocyte count greater than 5.109/L and a demonstration of monoclonality using flow cytometry (panel comprising CD19, CD5, CD22, CD23, FMC7, and surface Ig). Staging was done according to the Binet staging system. CD38 surface expression was determined by flow cytometry in all patients. The cytogenetic abnormalities : del 13q, del 11q, del 17p and trisomy 12, were performed by FISH and available for 25 patients.Smudge cells were defined as broken cells with no intact cytoplasm and a disrupted nuclear membrane (Figure 1). The smudge cell percentage is estimated by counting 200 lymphocytes and/or smudge cells; the smudge cell number is then divided by total number of cells counted (smudge cells + intact lymphocytes) and multiplied by 100. Each slide was evaluated by 2 hematologists and the blood smears were prepared using a manual wedge method. Categorical variables were analyzed using the χ2 test or Fisher exact test (p

Description: Myelodysplastic syndrome (MDS) is mainly a disease of elderly population. Chronic cytopenias, especially anemia, frailty comorbidities, and age may alter the physical status significantly. Only a few proportions of patients can achieve long-term cure. In particular, but not limited to patients with low risk MDS; palliative/supportive care is the mainstay of the treatment. Erythropoietin-stimulating agents (ESAs) and red blood cell transfusions are the treatment options for patients suffering from anemia. Except one study, ESA-responders had a better quality of life (QoL) in three studies. Hematologic improvements should be assesses by patient-reported outcomes (PROs) as well as objective measures. Validated PROs those were used to assess QOL in patients with MDS included Functional Assessment of Cancer Therapy- Anemia (FACT-An) and European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC-QLQ C30) questionnaires and a MDS-specific questionnaire, QoL-E. All patients with MDS were screened. Patients with low-risk MDS were included in the study. One physician completed FACT-An, Hematopoietic Stem Cell Transplantation-Comorbidity Index (HCT-CI), and G8 frailty questionnaires in all patients. Demographic data were collected from patients' chart records. A total of 66 patients were screened. Fourteen patients were excluded due to high-risk MDS or indefinite diagnosis. In one patient, informed consent could not be obtained. Finally, 51 patients were included in the study. Median age was 66 years old (interquartile range [IQR]: 55-77). Twenty-one out of 51 patients (41.2%) were male. Most prevalent MDS subtype was MDS-refractory anemia (47%). All patients had very-low/low (86.3%) or intermediate-risk (13.7%) MDS according to age-adjusted IPSS-R (IPSS-RA). Median time from the diagnosis of MDS was 113 (IQR: 53-170) weeks. Twenty-eight patients (54.9%) were transfusion-dependent. Ten patients had a high transfusion burden, which was defined as transfusion requirement ≥4 units (U) over 8 weeks. Median transfusion duration was 112 (IQR: 31-173) weeks for transfusion-dependent patients. Median red blood cell transfusion during eight weeks was 1.5 (IQR: 0-4.5) U. Median hemoglobin concentration was 10.0 (7.9-11.3) g/dL for all patients. A total of nineteen patients (37.3%) were ESA-user/responder. Most of the patients (80.4%) had a low (

Description: Background: Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism. Methods: We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1). Results: Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells. Conclusion: These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. Disclosures No relevant conflicts of interest to declare.

Description: Objective To analyze the concentration of growth differentiation factor 11(GDF11) in peripheral blood of patients with myelodysplastic syndrome (MDS), so as to evaluate the relationships between these changes and erythropoiesis functions and to explore the role of GDF11 in the pathogenesis of MDS. Methods The concentration of GDF 11 in peripheral blood was detected by enzyme-linked immuno sorbent assay in 44 MDS patients and 10 normal controls from September 2014 to June 2015 at our hospital. The percentage of nucleated erythrocyte (CD235a) in bone marrow was detected by flow cytometry. The correlation between these changes and erythropoiesis functions, including red blood cell count, hemoglobin, reticulocyte (RET%), hematokrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC) and late erythroblast in bone marrow were evaluated. Results (1)The concentration of GDF11(128.67±47.62)in high-risk MDS patients was significantly higher than that of low-risk MDS patients (65.96±36.55,p

Description: Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule and expresses on activated T cells, B cells and myeloid cells. PD-L1, a primary ligand PD-1, is mainly expressed on T cells and primary B cell surfaces and plays a role in the differentiation and apoptosis of these cells, induces a coinhibitory signal in activated T cells, promotes T cells apoptosis, incompetence and functional exhaustion. The expression of PD-1 is high in patients with hematologic malignancies and a high expression of PD-L1 is found on hematologic malignancies cells. To further know the characteristics of PD-1 and PD-L1 in patients with MDS, we detected theexpression of PD-1 and PD-L1 in peripheral blood (PB) and bone marrow (BM) samples from 25 RAEB and 10 RARS patients using Real-time PCR. The PD1 and PD-L1 expression levels of 13 PB and 8 BM samples from normal individuals were as controls. The PD-1 levels of PB samples from 25 RAEB patients [42.40(4.5-173.96)%] were significantly higher than that from normal controls [32.32(19.45-41.38)%, P=0.026]. While the level of PD-1 in 10 RARS patients was comparable to that of normal controls and RAEB patients (P=0.401 and P=0.352). Compared to normal controls [23.72(3.23-39.2)%], the median PD-1 level of BM from 10 RAEB patients[36.81(12.14-151.52)%] showed an increasing tendency, but the difference was not statistically significant (P=0.062). PD-L1 expression levels of PB samples from RARS patients were significantly lower than that of normal controls (P=0.009). There were no significant difference between RAEB patients and normal controls about the PD-L1 expression level in PB and BM samples (P=0.248 and P=0.181) and between RAEB and RARS patients about PD-L1 expression level in PB samples (P=0.243). PD-1 level in BM samples from 11 RAEB patients with remission was (31.32±15.75)% and it was lower significantly than that before treatment [(59.94±47.44)%, P=0.034]. After progression or transformation, the expression level of PD1 (54.72±37.27)% increased again and was higher than that in remission (P=0.028).There were also no significant difference on PD-L1 expression among before treatment, bone marrow remission and progression or transformation (P〉0.05). In 8 RAEB patients transformed to AML after treatment, PD-1 level has a decreasing tendency(P =0.05)and the change of PD-L1 has no significant difference (P〉0.05). In conclusion, PD1 mRNA level increased significantly in patients with RAEB and the changes of PD-1 was associated with the evolution of the disease after treatment with demethylating agents. The level of PD1 may be used as an indicator to determine the efficacy, but the changes of PD-L1 was not found regularity. Disclosures No relevant conflicts of interest to declare.

Description: Background. Chromosomal abnormalities (CA) are the most prognostic factor in acute myeloid leukemia (AML). However, it is still not clear whether the cytogenetic risk groups established for patients (pts), treated by standard chemotherapy, are so optimal for pts undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Recent studies have confirmed negative effects of both monosomal and complex karyotypes (MK, CK) on outcomes after alloHSCT [1,2]. Meantime, some investigators consider that immune effects of alloHSCT can reduce negative impact of these CA [3]. Aim. To evaluate impact of the CA in AML pts with adverse cytogenetic risk group on outcome after alloHSCT. Material and Methods. In this study, outcomes of alloHSCT, which were performed in a single institution between 2009 and 2014 years for 97 AML pts have been analyzed. Patients and transplantation characteristics are presented in Table I. The probabilities of overall survival (OS), leukemia free survival (LFS), cumulative incidence of relapse (CIR) were evaluated for different cytogenetic groups. Results. According to univariateanalysis, the probabilities of 4-year OS in pts with 5q-, KMT2A translocations and monosomy 7 were 66%, 59% and 56%, respectively. At the same time, OS in pts with CK, 7q- and 3q26 rearrangements were lower i.e. 33%, 25% and 25%, respectively (p=0.01). Multivariate analysis showed, that clinical stage at HSCT, age and HSC source are independent predictors of OS in AML pts (Table 2). Four-year LFS were various in pts with different CA. The higher LFS was noted in pts with 5q- and KMT2A translocations (66% and 52%, respectively), whereas lower LFS were in pts with 3q26 rearrangements, CK, monosomy 7 and 7q- (0, 18%, 23% and 37%, respectively) (Fig. 1). Besides, LFS distinguished between groups with CK+ and CK- (18% vs 41%, p=0.008), as well as with MK+ and MK- (17% vs 30%, p=0.04). Multivariate analysis evidenced clinical stage at HSCT, cytogenetic groups, MK and number of transplanted CD34+ cells to be independent predictor of LFS in AML pts (Table 2).Cumulative incidence of relapses in pts transplanted in remission (n=42) was higher in those with CK+ (55% vs 14%, p=0.03) and MK+ (75% vs 31%, p=0.013). Discussion. The study showed that 4-year OS in AML pts with 5q-, KMT2A translocations and monosomy 7 significantly distinguished from those with CK, 7q- and 3q26 rearrangements. Furthermore, OS depended on clinical stage at HSCT, patient's age and HSC source. On the other hand, EFS differed from all above-mentioned cytogenetic groups, including MK. Finally EFS depended on clinical stage at HSCT and number of transplanted CD34+ cells. Conclusion. On the basis of this data, a conclusion may be drawn that alloHSCT in AML pts with adverse CA should be performed at complete remission, with bone marrow as HSC source and enough number of transplanted CD34+ cells. Reference. 1. Hemmatti et al. Eur J Haematol. 2013; 92:102-10. 2. Fang et al. Blood 2011; 118:1490-4. 3. Guo et al. Biol Blood Marrow Transplant 2014; 20: 690-5. Table 1. Patients and Transplant characteristics Patients, n (%) 97 (100) Gender, n (%)MaleFemale 53 (54.6)44 (45.4) Age at HSCT, median, (range) years 25 (1.5-60) Cytogenetics, n (%) 3q26 rearrangementsDeletion 5q (sole)Monosomy 7 (sole)Deletion 7q (sole)KMT2A translocationComplex karyotype ³3 CA Monosomal karyotype 4 (4)10 (10.3)12 (12.4)4 (4)11 (11.3)56 (58)18 (18.5) Time from diagnosis to HSCT, median (range) days 477 (47 - 3482) Clinical stage at HSCT, n (%)CR1³CR2Active disease 29 (30)13 (13)55 (57) HSC source, n (%)Bone marrowPeripheral bloodBM+PB 54 (56)34 (35)9 (9) Conditioning regimen, n (%)MANon-MA 35 (36)62 (64) Donor type, n (%)HLA-id siblingMatched unrelatedHaploidentical 17 (17.5)53 (54.6)27 (27.8) Number of transplanted CD34+ cellsmedian (range), x106/kg 6.1 (1.5-17.9) Table 2. Multivariate analyses HR 95% CI P Overall survival Clinical stage at HSCT 2.65 1.72-4.09 0.00001 Median age6x106/kg 1.77 0.91-3.42 0.08 Leukemia-free survival Clinical stage at HSCT 2.59 1.8-3.7 0.0001 Cytogeneticgroups 1.31 1.03-1.69 0.031 Monosomal karyotype 1.88 0.97-3.66 0.044 Median number of transplanted CD34+ cells〉6x106/kg 2.78 1.51-5.11 0.0003 Figure 1. Leukemia-free survival for AML patients with different cytogenetic groups after alloHSCT Figure 1. Leukemia-free survival for AML patients with different cytogenetic groups after alloHSCT Disclosures No relevant conflicts of interest to declare.

Description: LK, SS Equal contributions VG, JTP Equal credit as senior authors Prolyl hydroxylase 2 (encoded by EGLN1), the principle negative regulator of HIF-1 and HIF-2 (encoded by EPAS1), is an iron dependent enzyme (Kaelin WG et al. Mol Cell, 2008); thus iron deficiency can augment hypoxic responses (Zhang X et al Blood Cells Mol Dis, 2014). Because EPAS1 mRNA has a 5' iron regulatory element, iron deficiency may inhibit the translation of HIF-2α and downregulate its target genes (Sanchez M et al Nat Struct Mol Biol, 2007). The Partnership for Anemia: Clinical and Translational Trials in the Elderly conducted a trial to determine the efficacy of IV iron in patients with borderline iron deficiency (Price E et al. Blood Cells Mol Dis, 2014). We measured expression levels of HIF1A, EPAS1, EGLN1 and HIF target genes in platelets and granulocytes. Nineteen patients were treated with 200 mg IV iron sucrose weekly for 5 weeks. Blood was obtained at screening, one week after the second dose of IV iron and 8 weeks after completion of iron therapy. Granulocytes and platelets were isolated and their RNA reverse transcribed. Transcripts were quantified by real-time qT-PCR, and normalized to HPRT gene. The threshold cycle for the expression of each gene was calculated and compared to 5 healthy controls. We found a positive correlation between granulocyte and platelet mRNA of HIF1A, EPAS1 and EGLN1, and these relationships achieved a one-sided significance level of 0.1 or stronger for most data points for HIF1A and EPAS1 (Table 1). There was also a positive correlation for granulocyte and platelet mRNA of 5 of 7 hypoxia response genes (P of 0.1 or stronger in many comparisons; the strongest being for VEGF and PDK1). Table 1. Spearman correlation rho between granulocyte and platelet mRNAs (N = 17). Gene Screen TV2 FU2 Regulators of the hypoxic response HIF1A .52** .21 .40* EPAS1 .40* .54** .67** EGLN1 .28 .30 .09 Responders to the hypoxic response GLUT1 .44** .00 .07 HK1 .25 .20 .36* PDK1 .24 .55** .41* VEGF .68** .33* .62** FOXO3 -.10 .22 .45** BNIP3 .03 -.19 -.08 BNIP3L .15 -.05 .20 *one-sided P

Description: Introduction: The treatment of choice for newly diagnosed patients with advanced diffuse large B-cell lymphoma (DLBCL) is R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) [Feugier P, Van Hoof A, Sebban C et al. Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d'Etude des Lymphomes de l'Adulte. J Clin Oncol 2005; 23: 4117-4126]. Anthracycline-based treatment is also superior to other drugs in controlling disease and prolonging survival in elderly patients [Hershman DL, McBride RB, Eisenberger A et al. Doxorubicin, cardiac risk factors, and cardiac toxicity in elderly patients with diffuse B-cell non-Hodgkin's lymphoma. J Clin Oncol 2008; 26: 3159-3165], however these individuals frequently have concomitant cardiac comorbidities such as congestive heart failure (CHF) that preclude the use of these agents. Methods: A search of the Moffitt Total Cancer Care™ database between January 1st 2008 and December 31st 2014 identified a cohort of 854 adult patients with a diagnosis of DLBCL. We performed a retrospective chart review of these patients and identified 40 individuals with documented CHF prior to the initiation of chemotherapy, due to either systolic (ejection fraction less than 50%) or diastolic dysfunction. The primary aim was to determine the chemotherapy regimens given to patients with DLBCL and CHF at Moffitt Cancer Center, and their related cardiovascular and oncologic outcomes. The study was approved by the University of South Florida Institutional Review Board. Results: 3 out of the 40 patients did not receive any chemotherapy and were excluded from the analysis. The median age was 71 years (range 21-93) with a median follow-up time of 19 months. Baseline characteristics are represented on table 1. Table 1.NPercentage (%)Sex1129.7FM2670.3Race38.1African AmericanAsian12.7White3389.2ECOG PS924.3011643.22821.6NA410.8Baseline Ejection fraction2156.8=50%1643.2Systolic Heart Failure1540.5NoYes1848.7UNK410.8Diastolic Heart Failure616.2NoYes1643.2UNK1540.5Ann Harbor Stage616.2III513.5III924.3IV1745.9 25 patients (67%) received R-CHOP or R-CHOP like (R-EPOCH) chemotherapy. The remaining patients received non R-CHOP regimens, being R-ICE (n=3), R-Hyper-CVAD (n=1), R-MTX (n=2), R-Bendamustine (n=2), R-CVP (n=2), R-CEOP (n=3). We observed an association between the type of treatment (R-CHOP vs non R-CHOP) and the type of heart failure, with diastolic CHF patients being more likely to receive a R-CHOP chemotherapy. (Table 2) Table 2.R-CHOPnon R-CHOPp-valueOR95% CINPercentage (%)NPercentage (%)Diastolic HFYes1588.2120.00.00530(2.14, 421.12)No211.8480.0Systolic HFYes731.811100

Description: Background L-asparaginase (L-ASP) is a key drug in the treatment of acute lymphoblastic leukemia (ALL). However the toxicity profile, especially hypersensitivities up to acute allergic reactions is a major drawback. GRASPA (eryaspase (proposed INN) or E-Coli L-Asparaginase encapsulated into red blood cells) is a new product under development with the aim of improving the tolerance of this enzyme. Asparagine is actively transported through the membrane of red blood cells (RBC) where it is hydrolyzed by the encapsulated L-ASP, the erythrocytes acting as "bioreactors". The RBC membrane shields against the anti-L-ASP antibody then avoiding binding to encapsulated L-ASP. Recently, a Phase III pivotal study of GRASPA in combination with COOPRALL chemotherapy protocols in patients with relapsed ALL demonstrated highly significant safety profile and clinical activity compared to control. However, there is an unmet medical need for patients who cannot receive current formulations of L-ASP. An expanded access program has recently been initiated in France to provide access for treatment with GRASPA in patients who are unable to receive other forms of L-ASP. Methods: This is a non randomized multicenter open label study, currently initiated in France. The primary objective of the EAP is to evaluate the tolerability of GRASPA. Patients under 55y of age presenting with de novo, relapsed or refractory ALL who are at risk to receive any other available L-ASP formulation are enrolled into this program. Patients with known allergic reactions to E.Coli L-ASP are also eligible. GRASPA is administrated every 2 to 3 weeks at a dose equivalent to 150 IU/kg of L-ASP during all chemotherapy courses intended to contain an asparaginase. Chemotherapy protocols are given according to the Investigator's choice. Patients are assessed regularly for safety and tolerability. The primary endpoint is tolerability; Key secondary endpoints include asparaginase activity, asparagine depletion, and clinical remission rates. An independent Safety Monitoring Board (DSMB) is set up, which will assess toxicities on yearly basis. Results As of time of June 2015, 13 patients were enrolled into the program. The first DSMB meeting reviewed the outcome of the first 7 patients enrolled into the program. Of the 7 pts (range 3 - 49 years), 5 males and 2 females were enrolled. Four pts presented with refractory disease and 3 with relapse, with all patients had evidence of allergies to 2 prior asparaginases (double allergies). There were 2 pts presenting with limiting toxicities, in the form of myelosupression, and streptococcal infection. There was no modification to the protocol recommended by the first DSMB An updated safety and clinical activity information on all patients will be provided. Conclusion: The EAP provides a potential treatment alternative for ALL patients, who are unable, or at risk of developing hypersensitivity reactions to prior asparaginases. The initial results from this program suggests that GRASPA is well tolerated, and may have a potential benefit in patients with double allergies. The program will be expanded to other European countries Disclosures Bertrand: ERYTECH Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dombret:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Recher:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Research Funding; Amgen: Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salako:ERYTECH Pharma: Employment. Godfrin:ERYTECH Pharma: Employment. El Hariry:ERYTECH Pharma: Employment.

Description: Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure, congenital abnormalities and predilection towards development of hematopoietic malignancies, including acute myeloid leukemia (AML). Congenital biallelic disruption of the FA/BRCA signaling network causes Fanconi anemia and somatic mutations within the same genes are increasingly identified in a variety of malignancies in non-FA individuals, consistent with the critical role of this signaling pathway in FA and in the general population. The FA/BRCA tumor suppressor network orchestrates interphase DNA-damage repair (DDR) and DNA replication to maintain genomic stability. Additionally, we and others have demonstrated that the genome housekeeping function of FA/BRCA signaling extends beyond interphase: loss of FA/BRCA signaling perturbs execution of mitosis, including the spindle assembly checkpoint (SAC), centrosome maintenance, cytokinesis and resolution of anaphase DNA bridges. Interphase errors exacerbate mitotic abnormalities and mitotic failure promotes interphase mutagenesis. Consequently, we had demonstrated that primary FA patients' cells accumulate genomic abnormalities consistent with a dual mechanism of impaired interphase DDR/replication and defective mitosis. Previous detailed studies had elucidated multiple mechanisms of interphase DDR-dependent assembly and activation of the FA complex at DNA damage sites to arrest the cell cycle and repair DNA lesions. However, the signaling cross-talk nodes between the FA and mitotic checkpoint pathways remain to be discovered. In this study, we identified functionally relevant mitotic signaling defects resulting from FANCA deficiency via a synthetic lethal kinome-wide pooled shRNA screen in primary patient-derived FANCA -deficient cells compared to isogenic FANCA -corrected cell line. Bioinformatics analysis of our screen results followed by secondary validation of selected hits with alternative shRNAs and small-molecule inhibitors revealed conserved mitotic signal transduction pathways regulating the SAC and centrosome maintenance. Our super-resolution structured illumination (SR-SIM) microscopy coupled with deconvolution imaging revealed that a fraction of FANCA co-localizes with key SAC kinases at mitotic centrosomes and kinetochores, consistent with the role of FANCA in centrosome maintenance and the SAC. Co-immunoprecipitation assays identified the biochemical interaction between FANCA and an essential SAC kinase whose loss is synthetic lethal with FANCA deficiency, providing first insights into the interactions between FA signaling and the canonical SAC network. Together, our study has unraveled functional and biochemical connections between FANCA and the centrosome/SAC kinases, consistent with the essential role of FANCA in cell division. Our ongoing work is aimed at mechanistically dissecting molecular links between these two key tumor suppressor signaling pathways in more detail. We hypothesize that impaired FANCA/SAC cross-talk may contribute to genomic instability in FA-deficient cells and provide opportunities to selectively kill FANCA-/- cells. Disclosures No relevant conflicts of interest to declare.

Description: Objective: Increasing evidence indicates a critical role of the hematopoietic microenvironment in the pathophysiology of myeloid malignancies. Mesenchymal stem/progenitor cells (MSPCs) represent an indispensible cellular component of the bone marrow microenvironment and play an important role in supporting and regulating the proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs). Abnormal biological characteristics of MSPCs involved in the initiating and development of hematopoietic disorders, such as chronic myelogenous leukemia and myelodysplastic syndromes. However, the characteristics and the role of primary MSPC defects in the pathogenesis of chronic myelomonocytic leukemia (CMML) remain poorly understood. Methods: MSPCs were generated from bone marrow aspirates of patients with CMML and that of healthy controls. To investigate the frequency of the MSPCs in the bone marrow of CMML patients and healthy controls in vivo, a colony-forming unit fibroblast (CFU-F) assay was performed. The phenotypic evaluation of the MSPCs was performed with flow cytometric analysis after staining with a cocktail of cell surface markers, including CD34, CD45, CD29, CD44, CD73 and CD105. To determine the functional difference between the CMML- and control-MSPCs, senescence, cell proliferation, and cell cycle were assessed by EDU incorporation assays, β-galactosidase activity, and flow cytometric analysis (following BrdU/7AAD staining, respectively. To determine the multi-lineage potential of MSPCs, MSPCs were subjected osteoblast, chondrocyte and adipocyte differentiation cultures. The hematopoietic supportive activity was assessed using cobblestone-area forming cell (CAFC) assays by coculturing of CD34+ cord blood cells with CMML- or control- MSPCs. Furthermore, to evaluate the soluble factors of these MSPCs in HSPC supportive activity, the transwell assay was performed with a subsequent flow cytometric analysis further determine the multi-lineage differentiation alteration. Cytokine array was used to determine the cytokines/growth factors in the MSPC conditioned media. Results: The expression levels of the surface markers were identical between CMML-MSPCs and control MSPCs. The frequency of CFU-F is identical between healthy control and CMML patients. However, CMML-MSPCs exhibited a poor proliferative capacity compared to controls MSPCs. CMML MSPCs exhibited an increased senescence and a S phase retardation in the cell cycle as assessed microscopically and flow cytometrically, respectively. Differentiation assay revealed that CMML MSPCs had increased chondrocyte differentiation and reduced adipocyte differentiation, while no alteration of osteoblast differentiation was noticed. In vitro co-cultures of MSPCs and CD34+ umbilical cord blood cells demonstrated that CMML-MSPCs had an impaired hematopoietic supportive capability as compared to control MSPCs as evidenced by reduced numbers of cobble-stone and reduced numbers of CFU-C. Furthermore, a reduced CFU-C and a reduced CFU-GEMM were observed in CAFC and transwell cultures containing CMML-MSPCs compared to that of control MSPCs. In contrast, an increased myeloid cells (CD33+/CD14-/CD15+) and an increased CFU-GM were observed in CD34+ cells and CMML-MSPC co-cultures as compared to that in CD34+ cells and control-MSPC co-cultures. Cytokine array showed that the conditional media of CMML-MSPCs has lower levels of multiple cytokines (IL-6, IL-8, and GRO-β) compared to that of control MSPC conditional media. Conclusion: Collectively, our study indicated that MSPCs of CMML patients had reduced proliferation, increased senescence and reduced cytokine production, while differentiation was not markedly altered. CMML-MSPCs display less hematopoietic supportive activity. Furthermore, CMML-MSPCs induced CD34+ cell differentiation favoring granulomonocytic lineage over erythroid cells. Our study suggests that the cell autonomous defects of CMML-MSPCs contribute to the pathogenesis of CMML and the interaction between hematopoietic compartment and microenvironment could serve as targets for therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Description: Key Points NKR-P1B is involved in NK cell tolerance and MHC-I-independent missing-self recognition of Clr-b-deficient target cells. The NKR-P1B:Clr-b system plays a role in tumor surveillance and immune escape in the Eμ-myc transgenic mouse model of B-cell lymphoma.

Description: Key Points Prospective analysis of antigen-specific B/T-cell immunity in natural history of human premalignancy. Stemness antigens and ICPs may be targets for cancer prevention.

Description: Key Points RGS18 acts as a brake on persistent or inappropriate platelet activation after it is released from binding sites in resting platelets. Control of free RGS18 levels provides a mechanism for coordinating signaling networks in platelets.

Description: Key Points Polyphosphate-activated coagulation factor XII drives prostate cancer-associated venous thrombosis. Targeting the polyphosphate/factor XII pathway reduces procoagulant activity in prostate cancer patient plasma and may permit safe anticoagulation.

Description: Key Points Factor V and protein S are required for sepsis mortality reduction and suppression of inflammatory gene expression by activated protein C. The R506Q mutation (Leiden mutation) abrogates the anti-inflammatory cofactor function of factor V for activated protein C.

Description: Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow–derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

Description: Cancer cells frequently overexpress tissue factor (TF) and become procoagulant. This conversion may be driven by genetic transformation, including through the expression of the oncogenic epidermal growth factor receptor (EGFR) and its mutant, EGFRvIII, present in glioblastoma multiforme (GBM). Here we show that the EGFRvIII-dependent GBM cell transformation is associated with the onset of the simultaneous overexpression of TF, protease-activated receptors 1 and 2 (PAR1 and PAR2), and ectopic synthesis of factor VII (FVII). Efficient generation of factor Xa by these cells still requires exogenous FVIIa. However, as a result of EGFRvIII-dependent transformation, GBM cells become hypersensitive to TF/PAR-mediated signaling and produce ample angiogenic factors (vascular endothelial growth factor and interleukin-8) on exposure to FVIIa and PAR1- or PAR2-activating peptides. Thus, oncogenes may cause complex changes in the ability of GBM cancer cells to interact with the coagulation system, thereby exacerbating its influence on angiogenesis and disease progression.

Description: Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I2 (PGI2), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in Gi2α that blocks RGS/Gi2 interactions to examine the consequences of lifting constraints on Gi2-dependent signaling without altering receptor:effector coupling. The results show that the Gi2α(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca++]i, Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Description: Constitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized β-arrestin recruitment site. Similar to PAR2−/− mice, TF cytoplasmic domain–deleted (TFΔCT) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TFΔCT mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TFΔCT and TFΔCT/PAR2−/− mice, and increased tumor vessel diameters of TFΔCT mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer.

Description: Key Points We conducted a phase-2 study in newly diagnosed PCNSL utilizing R-MPV and HDC with ASCT. Excellent disease control and OS (2-year PFS: 79%) were observed, with an acceptable toxicity profile and minimal neurotoxicity.

Description: Abstract 3783 Introduction: Dengue fever is the most prevalent mosquito-borne viral disease, and its clinical signs and symptoms can vary widely. Since hemorrhagic manifestations are common, hematologists are often included in the care of these patients, and face a difficult challenge trying to discriminate dengue fever from other more common viral infections. Laboratory tests used in this context are less than optimal. The expected findings of leukopenia and thrombocytopenia are absent in many cases, thus delaying the suspicion for dengue and the ordering of confirmatory serological tests. Leukocyte morphology changes in numerous medical conditions, including infections. The VCS technology available in the LH and DxH hematology analyzers is capable of automatically quantifying leukocyte morphologic characteristics: cell volume by voltage impedance (V); cytoplasmic and nuclear density by radio frequency conductivity (C); and cytoplasmic granularity and nuclear complexity by laser light scatter (S). The mean and standard deviation of each parameter is calculated. This data is collected for neutrophils, lymphocytes, monocytes and eosinophils separately. Previous studies have demonstrated the significant value of these VCS parameters in improving the laboratory diagnosis of malaria and septicemia, both in adults and neonates. The objective of this study was to compare the performance of the VCS parameters with that of traditional hematological tests used in the diagnosis of dengue fever. Methods: We included in this study 174 patients who were clinically suspected to have dengue fever, as indicated by the clinicians ordering serological testing. Of these, 92 patients were positive and 82 negative by serology. Using the Mann-Whitney U test, we compared the mean VCS values, as well as total leukocyte counts, leukocyte differential counts and platelet counts, between both groups. For those tests that showed a statistically significant difference between dengue positive and dengue negative patients, their diagnostic performance was compared by ROC curve. Finally, we separated patients with normal platelet and WBC counts, likely to be misdiagnosed based on these traditional laboratory tests, and evaluated the diagnostic performance of the VCS parameters in this subgroup. Results: Tests that showed a statistically significant difference (p

Description: Key Points Normal plasma VWF multimers act as a cofactor in the factor I–mediated cleavage of C3b to iC3b and inhibit complement activation. Large VWF multimers, including ultra-large VWF multimers, do not have factor I cofactor activity and permit complement activation.

Description: The DNA-binding zinc finger transcription factors Gfi1 and Gfi1b were discovered more than 20 years ago and are recognized today as major regulators of both early hematopoiesis and hematopoietic stem cells. Both proteins function as transcriptional repressors by recruiting histone-modifying enzymes to promoters and enhancers of target genes. The establishment of Gfi1 and Gfi1b reporter mice made it possible to visualize their cell type–specific expression and to understand their function in hematopoietic lineages. We now know that Gfi1 is primarily important in myeloid and lymphoid differentiation, whereas Gfi1b is crucial for the generation of red blood cells and platelets. Several rare hematologic diseases are associated with acquired or inheritable mutations in the GFI1 and GFI1B genes. Certain patients with severe congenital neutropenia carry mutations in the GFI1 gene that lead to the disruption of the C-terminal zinc finger domains. Other mutations have been found in the GFI1B gene in families with inherited bleeding disorders. In addition, the Gfi1 locus is frequently found to be a proviral integration site in retrovirus-induced lymphomagenesis, and new, emerging data suggest a role of Gfi1 in human leukemia and lymphoma, underlining the role of both factors not only in normal hematopoiesis, but also in a wide spectrum of human blood diseases.

Description: MicroRNAs are small noncoding RNAs that regulate cellular development by interfering with mRNA stability and translation. We examined global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were up-regulated and 81 were down-regulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were up-regulated most strongly in murine megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly up-regulated during mouse and human megakaryopoiesis but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing inflammatory cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously.

Description: Key Points Immune responses to FVIII sequence variants encoded by ns-SNPs do not contribute appreciably to inhibitor development in African Americans. African American HA subjects with an intron-22 inversion had a 2- to 3-times-higher inhibitor incidence than whites with the same mutation.

Description: Therapeutic targeting of virus-encoded proteins using cellular immunotherapy has proved successful for Epstein-Barr virus (EBV)–associated posttransplant lymphoproliferative disease. However, the more limited repertoire and immunogenicity of EBV-encoded proteins in other malignancies such as Hodgkin lymphoma and extranodal natural killer (NK)/T lymphoma has been more challenging to target. The immunosubdominant latent membrane protein 2 (LMP2) is considered the optimal target in such Latency II tumors, although data relating to its expression in T/NK malignancies are limited. In addressing the validity of LMP2 as an immunotherapeutic target we found that LMP2-specific effector CD8+ T cells recognized and killed EBV-positive NK- and T-cell tumor lines, despite an apparent absence of LMP2A protein and barely detectable levels of LMP2 transcripts from the conventional LMP2A and LMP2B promoters. We resolved this paradox by identifying in these lines a novel LMP2 mRNA, initiated from within the EBV terminal repeats and containing downstream, epitope-encoding exons. This same mRNA was also highly expressed in primary (extra-nodal) NK/T lymphoma tissue, with virtually undetectable levels of conventional LMP2A/B transcripts. Expression of this novel transcript in T/NK-cell lymphoproliferative diseases validates LMP2 as an attractive target for cellular immunotherapy and implicates this truncated LMP2 protein in NK- and T-cell lymphomagenesis. This study is registered at clinicaltrials.gov as NCT00062868.

Description: Key Points Platelets stimulate proliferation of HepG2 cells, which requires uptake of platelets by the HepG2 cell. Platelets stimulate HepG2 cell proliferation in part by transfer of RNA from the anucleate platelet to the nucleated HepG2 cell.

Description: There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

Description: Key Points Pom-Dex is active and well tolerated in adverse cytogenetic patients with early RRMM, particularly in those with del(17p). Pom-Dex prolonged OS in adverse cytogenetic patients with early RRMM.

Description: Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apcmin allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apcmin mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apcmin bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apcmin mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).

Description: Background: TG-0054 (burixafor) is a potent and selective antagonist of human chemokine receptor CXCR4 that inhibits the binding of stromal-derived factor 1 (SDF-1). Interruption of the CXCR4/SDF-1 interaction prevents sequestration of CD34+ stem cells to the bone marrow and subsequently mobilizes these cells into the peripheral blood within 1 to 3 hours of drug administration. Materials and Methods: An open-label, phase II pilot trial was conducted in patients with multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), or Hodgkin's lymphoma (HL) to evaluate the safety and stem cell mobilization of TG-0054 in combination with G-CSF. We planned to treat twelve patients with subcutaneous injections of 10 µg/kg/day G-CSF in the afternoon for 4 days. On the morning of Day 5, patients received 3.14 mg/kg TG-0054 and underwent large volume (18-24L) leukapheresis approximately 2 hours post-drug infusion. Patients were allowed by protocol to undergo leukapheresis for 1-5 days to obtain the predetermined target of ≥5.0 x 106 CD34+ cells/kg. 9 of 12 patients have been treated thus far with a plan to treat 3 additional patients. Results: A planned interim analysis revealed that6 of the 9 patients treated thus far collected more than 10 x 106 CD34+ cells/kg in 1 leukapheresis session. 2 patients required 2 days to achieve the study endpoint, and 1 outlier patient who had received Revlimid only 1 week prior to peripheral blood CD34 analysis failed to mobilize stem cells until he was allowed 2 more weeks to recover from his Revlimid treatment. Burixafor was well tolerated, and there were no adverse events that were attributed to the drug. All of the patients engrafted promptly after melphalan (7 patients) or BEAM (2 patients) conditioning regimens. Conclusion: Burixafor in combination with G-CSF is a potent and well-tolerated mobilizer of stem cells into the peripheral blood, and with the exception of 1 outlier, was able to mobilize 〉5.0 x 106 CD34+ cells/kg in 1-2 leukapheresis sessions in all patients treated thus far (median 1 day). This contrasts with our historical controls that required a median of 2-3 days to achieve a collection of ≥5.0 x 106 CD34+ cells/kg. A total of 12 patients will be treated on this pilot study. These encouraging results warrant the further testing of this drug in a larger randomized clinical trial. Disclosures Hsu: Taigen Biotechnology: Employment. Chang:Taigen Biotechnology: Employment. Schuster:Taigen Biotechnology: Research Funding.

Description: Abstract 2825 Mantle cell lymphoma (MCL) constitutes one of the lymphomas with poorest prognosis at relapse and there are no effective salvage. The activity shown by the combination Gemcitabine and Oxaliplatin in several types of limfoma, along with its “in vitro” synergistic effect, made this regimen an attractive regimen for salvaging patients with MCL. Against this background, we performed an off-label pilot study in order to assess efficacy and toxicity of the combination R-GemOx in relapsed or refractory patients with MCL. For this, 27 patients (70% male, median age 70 years) diagnosed with MCL between November 2004 and January 2010 were included in this study. Inclusion criteria were adequate performance status, confirmed diagnosis of MCL and relapse or refractoriness to the previous treatment. The regimen consisted of Rituximab 375 mg/m2 on day 1, Gemcitabine 1000 mg/m2 and Oxaliplatin 100 mg/m2 on day 2, every 14 days, up to 8 cycles. Dose and interval were adjusted according to hematological and extrahematological toxicities. Median number of previous regimens was 1 (range 1 to 3), being EPOCH-R (n=14), R-CHOP (n=6), and RFC (n=3) the most frequently used induction therapies. Twelve patients relapsed after prior CR, 10 progressed after achieving a PR, whereas 5 were refractory to therapy. At inclusion, 85% of patients were in advanced (III/IV) clinical stage, 40% had bone marrow infiltration, 28% gastrointestinal involvement, and 17% cavum infiltration. Median number of cycles administered was 8 (range, 3 to 8). Doses were reduced in 9 cycles and delayed in 15 cycles. Neutropenia grade 3–4 was observed in 9 cycles and thrombocytopenia grade 3–4 in 6. Hepatotoxicity grade 1–2 in 5 pts and grade 3 in 1, sensitive neurotoxicity grade 1–2 in 12 pts, and renal impairment grade 2 in 1 patient. After completion of the treatment, 21 pts (77%) were considered in CR/uCR, 1 (4%) achieved a PR, 1 a SD, whereas 4 PD. Six patients subsequently received a stem-cell transplantation (4 allogeneic, 2 autologous). Thirteen pts are still alive and out of progression. Ten pts have died (8 due to progression and 2 due to acute GVHD). With a median follow-up of 23 months (range: 3–57), PFS and OS at 2 years are 41% and 58%, respectively. The R-GemOx combination showed a significant activity in relapsed or refractory pts with MCL with a very acceptable toxicity profile. These results prompted us to conduct a multicenter phase II clinical trial that is now ongoing. Disclosures: López: Roche Farma: Research Funding, Travel Support. Bosch:Roche Farma: Research Funding.