bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of NFAT Transcription Factors in NK Cell Reactivity
Authors: Rothfelder Kathrin , Märklin Melanie , Wild Julia , Dörfel Daniela , Kanz Lothar , Müller Martin R , Salih Helmut R.
Published: November 2019

NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), as highlighted by its important role in T and B cell development and function. With regard to NK cells, available data indicate that NFAT is dispensable for development. However, several lines of evidence including the observation that the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, influence NK reactivity implicate a role of this family of transcription factors in NK cell reactivity and function. Here we employed different genetic mouse models on the C57BL/6 background to directly study the functional role of NFAT in NK cells.

We found that except for NFAT3 mRNA and protein of all family members (NFAT 1, 2, 4 and 5) was expressed in resting NK cells of wild type (WT) mice with NFAT1, 2 and 4 being most abundantly detectable. When we employed NK cells with knockout (KO) of NFAT 1, 2, and 4 in comparative in vitro analyses, we surprisingly found that lack of NFAT resulted in enhanced NK cell activation, degranulation and release of immunomodulatory cytokines like IFN-γ after co-culture with YAC-1 target cells as well as increased production of granzyme B and perforin after cytokine activation. The inhibitory effect of NFAT on NK cell effector function was further confirmed in vivo by employing WT and germ line NFAT KO animals in the syngeneic B16 melanoma model, which revealed a significantly reduced metastatic burden in NFAT KO mice. Depletion of NK cells in this model system in turn resulted in increased metastasis, however, with WT animals displaying significantly higher metastatic burden compared to NFAT KO mice. As this pointed to the fact that NFAT influences metastasis via both NK-dependent and independent mechanisms, we further generated mice with a NK cell-specific (conditional Ncr1-Cre dependent) NFAT2 KO. When these animals were employed again in analyses of B16 lung metastasis, comparative analyses with WT animals confirmed the inhibitory effect of NFAT on NK tumor immunosurveillance.

Taken together, these results provide the first direct evidence for the functional involvement of NFAT in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function.

Disclosures

No relevant conflicts of interest to declare.
Quinine-Induced Thrombotic Microangiopathy (Q-TMA): Frequency, Presenting Features, Outcomes
Authors: Page Evaren E , Vesely Sara K. , George James N.
Published: November 2019

Q-TMA is an acute, severe, immune-mediated, drug-induced disorder. Q-TMA is suspected when symptoms suddenly begin within hours following quinine (Q) exposure. Diagnosis of Q-TMA is established by the history of recurrent acute symptoms following recurrent Q exposures and/or by documentation of Q-dependent antibodies reactive with platelets and/or neutrophils. The Oklahoma TTP-HUS Registry enrolls all patients for whom plasma exchange (PEX) is requested for suspected TTP or HUS. Since 1995, when routine measurement of ADAMTS13 activity began, the Registry has diagnosed 78 patients with acquired TTP (ADAMTS13 <10%). During this time we have also diagnosed 17 patients with Q-TMA; 2 additional patients were diagnosed before 1995. Seventeen of these 19 patients were tested for Q-dependent antibodies; all were positive. Nine patients had a history of recurrent acute symptoms with recurrent Q exposure, including the 2 patients not tested for Q-dependent antibodies. Q exposure was a pill in 18 patients, tonic water in one. Remarkably, 18 patients were women; all 19 patients were white. Common presenting symptoms were fever, chills, nausea and vomiting. No patients had focal neurologic abnormalities. All patients had microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and acute kidney injury. Eight patients had elevated serum alanine aminotransferase (231-1345 U/L). Three patients had neutropenia (absolute neutrophil counts, 184-486). Two patients had coagulation abnormalities suggesting disseminated coagulation (DIC).

One patient died from complications of the central venous catheter insertion, performed for PEX and dialysis; all other patients recovered normal platelet counts. Three of the 18 surviving patients had end-stage renal disease (2 had kidney transplants). The median estimated glomerular filtration rate (GFR) for the other 15 patients, at 2.7-19.2 years (median, 10.2) after recovery, was 36 ml/min (range, 19-98). Only two patients had normal GFR (≥90 ml/min). Eleven patients had chronic kidney disease, defined by GFR <60 ml/min. Seven of 18 patients have died 4.1-12.7 years (median, 7.8) following recovery at ages 59-87 years. Conclusion. Quinine can cause severe immune-mediated toxicities involving multiple organ systems (Am J Hematol 2016; 91: 461). Q-TMA is an acute disorder causing severe kidney injury and, in some patients, also liver toxicity, neutropenia, and/or DIC. Q-TMA is not rare. During 20 years, we enrolled 17 Q-TMA patients compared to 78 patients with acquired TTP. Chronic kidney disease is a common long-term outcome. Explicit questions are required to discover the association of systemic symptoms with quinine ingestion.

Table Table.

Disclosures

No relevant conflicts of interest to declare.
Pharmacokinetics of Subcutaneously Administered CB2679d/ISU304 in Wild-Type and Hemophilia B Mice
Authors: Hong Seung-Beom , Levy Howard , Jung Jae Yong , Park Minkyung , Seo A Rim , Seo So Hyeon , Madison Ed
Published: November 2019

The rapid clearance of factor IX (FIX) necessitates frequent intravenous IV administrations to achieve effective prophylaxis for patients with hemophilia B (HB). Subcutaneous (SC) administration would be a preferred route of administration but has been limited by low bioavailability and potency of the marketed FIX products. CB2679d/ISU304 with enhanced biological properties was developed using a rational protein design approach and has resistance to inhibition by ATIII, increased affinity for FVIIIa, increased catalytic activity and resultant 20-fold enhanced potency in vitro (clotting activity) and in vivo (the tail clip model) and 8-fold increased duration of aPTT activity in vivo compared to recombinant wild-type FIX dosed at the same mass.

ISU304 (4622 IU/mg) was injected into HB mice SC with a single dose ISU304 at 0.02, 0.05 or 0.15 mg/kg and sampled at 4, 6, 8, 24 hours. Groups of wild-type mice received ISU304 0.02, 0.05 or 0.15 mg/kg SC and BeneFIX (273 IU/mg) 0.15 mg/kg SC, and sampled at 0.25, 1, 4, 8, 24, 48, 72, and 96 hours. Daily SC injection in HB mice of ISU304 at 0.05 mg/kg was sampled at 24, 48, 72 and 96 hours. FIX antigen was measured using a sandwich ELISA and FIX activity was measured using a one-stage clotting assay on Stago Compact. Pharmacokinetics of FIX was performed using PKSolver.

There was a dose-dependent increase of plasma FIX antigen with SC ISU304. Mass-based pharmacokinetic profiles of ISU304 (t1/2, 18 hours; Tmax,8 hours, Bioavailability, 19-22%) were similar to those of BeneFIX (t1/2, 20 hours; Tmax, 8 hours, Bioavailability, 16%). Due to ISU304 high specific activity, SC dose of ISU304 yields much higher FIX activities in mouse plasma compared with the same mass dose of BeneFIX.

Daily SC dosing of ISU304 230 IU/kg reached steady-state plateau FIX 8% activity after three injections.

The bioavailability and increased potency of CB2679d/ISU304 facilitates the initiation of the Phase 1 subcutaneous dosing study in individuals with hemophilia B.

Figure 1 Figure 1. Table 1 Table 1. Figure 2 Figure 2.

Disclosures

Hong: ISU Abxis: Employment. Levy:Catalyst Biosciences: Employment. Jung:ISU Abxis: Employment. Park:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Madison:Catalyst Biosciences: Employment, Equity Ownership, Patents & Royalties.
Identification and Characterization of a New Mutation in Coagulation Factor VII (Carmel mutation), with Different Reactivity Towards Tissue Factors of Different Origin
Authors: Cassel Aliza , Rosenberg Nurit , Muhammad Emad , Dazdik Rima , Berl Miriam , Preis Meir
Published: November 2019

Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder manifested by bleeding tendency in homozygotes or compound heterozygotes. The bleeding tendency is variable and does not always correlate with the measured amount of FVII. More than 30 mutations causing FVII deficiency have been identified. The aim of this study was to identify the molecular defect in a family with FVII abnormality and to characterize the functional defect and its clinical consequences.

An 80 year old woman showed various prolonged prothrombin time (PT) dependent on the origin of tissue factors (TF) in the reagents, in the range of 5-18%. FVII activity measured using placenta human TF was ~4% and less than 1% using recombinant human TF or rabbit brain TF. Direct sequencing of PCR-amplified FVII genomic DNA exons demonstrated a single homozygous nucleotide exchange G>A in exon 6 predicting missense mutation Tyr164Cys, in close vicinity to the active site of the protease and to the binding site of FX.

Sequencing of exon 6 in six members of the family comprising three generations was performed. All of them exhibited low activity of FVII. Direct sequencing of PCR-amplified exon 6 demonstrated that her son, daughter and grand-daughter are heterozygote to the novel mutation, Tyr164Cys, henceforth, Carmel mutation. The grand -daughter (third generation) was identified as a compound heterozygote carrying in addition an Ala244Val mutation along with a Arg353Gln polymorphism, a well-known linkage previously characterized in the Israeli population, inherited from the paternal side. Her FVII activity was 3-7% and 8% FVII antigen. In the individuals heterozygote for Tyr164Cys we detected 41-54% activity and 46-66% antigenic FVII.

The measurement of VIIa in the proband and her grand-daughter could not demonstrate any activity in plasma, possibly because of perturbation of the interaction between the mutated FVII and TF or FX which are part of the assay. The heterozygote for Tyr164Cys showed approximately 30% FVIIa of normal controls .

Despite the dramatic laboratory presentation, the clinical manifestation of the Tyr164Cys genotype is very mild, suggesting that a low level of FVII is sufficient to prevent bleeding. The homozygous patient reached an old age, gave birth to two children and did not suffer any major bleedings during her life. The compound heterozygote presents a similar phenotype with slightly higher FVII activity and antigen, no measurable FVIIa activity and mild clinical presentation. Further investigation into this novel mutation is needed in order to characterize the interaction of the FVII with TF, phospholipids and FX and to understand the reason for such low amount of FVII protein in plasma.

Disclosures

No relevant conflicts of interest to declare.
Late Effects in Survivors of Infantile Acute Leukemia Are Less Severe Than Expected: A Study of the L.E.a Program
Authors: Gandemer Virginie , Bonneau Jacinthe , Oudin Caroline , Berbis Julie , Bertrand Yves , Tabone Marie-Dominique , Ducassou Stephane , Chastagner Pascal , Brethon Benoit , Dalle Jean-Hugues , Thouvenin Sandrine , Poiree Maryline , Plantaz Dominique , Kanold Justyna , Sirvent Nicolas , Lutz Patrick , Hamidou Zeinab , Baruchel Andre , Leverger Guy , Auquier Pascal , Michel Gerard
Published: November 2019

Purpose

The long-term impact of treatment in infants with acute leukemia (AL) is poorly understood and of concern.

Patients and methods

We analyzed the incidence, in the L.E.A. cohort, of and risk factors for late sequelae in the survivors of infant AL diagnosed before the age of one year, according to group: type of AL, transplantation, sex, and age ≤ or > 6 months. Of 113 survivors, 55 had had lymphoblastic AL and 58 myeloblastic AL. The median follow-up was 11.7 years [1.9-34.7]. Sixty-three were treated with chemotherapy only (and four with central nervous system irradiation) and 50 received transplantation (with a chemotherapy-based regimen except for three).

Results

The most frequently observed sequelae included problems of height, body mass index, and gonadal or thyroid function (Figure 1). We found a mean of 1.3 +/-0.1 sequelae per patient. In multivariate analysis, treatment after the age of six months significantly decreased the incidence of major growth failure (OR 0.38, p = 0.04) and low weight (OR 0.3, p = 0.02) (Figure 2). Transplants performed in first remission did not affect the occurrence of late effects (p = 0.2) (Figure 3). Parent-reported child quality of life (QoL) scores were similar among groups and to the normative population. Adults had diminished QoL as measured by psychosocial subscales relative to French norms but with no difference between groups.

Conclusion

Altogether, these results highlight the need for comprehensive follow-up with hormone replacement therapy and psychological intervention. Our results in a large cohort with very limited use of irradiation, did not show an increased risk of late sequelae relative to older children. We conclude, that transplantation should be considered when warranted, as there is no increased risk of late effects.

Disclosures

No relevant conflicts of interest to declare.
A Novel Prognostic Index for Ocular Adnexal Lymphoma
Authors: Meeks Marshall W. , Park Henry S. , Yu James B. , Roberts Kenneth B. , Foss Francine , Wilson Lynn D.
Published: November 2019

Background: Ocular adnexal lymphoma (OAL) is a rare malignancy with a heterogeneous patient population. We sought to identify prognostic factors for OAL and to incorporate them into a novel prognostic index by using a large national database.

Methods: We identified patients diagnosed with histologically confirmed OAL in 1973-2013 using all registries of the Surveillance, Epidemiology, and End Results database. Patients with regional or distant disease at diagnosis were excluded. Indolent histologies were defined as follicular, marginal zone, small lymphocytic not otherwise specified, and lymphoplasmacytic. We determined the association between OAL histology, orbital site, and overall survival (OS) using adjusted proportional hazards analysis and Kaplan-Meier estimates. Relative survival (RS) was calculated relative to a matched cohort of patients categorized by race, gender, and age using the SEER*Stat Program.

Results: Among the 3,070 patients included, 941 (30.6%) had conjunctival tumors, 418 (13.7%) had lacrimal gland tumors, and 1711 (55.7%) had orbital-not otherwise specified tumors. Indolent histology was present in 2,073 (67.5%) patients. The 10-year OS and RS were 61% and 86%, respectively. In univariable and multivariable models, conjunctival site location (P<0.001) and indolent histology (P<0.001) were both associated with superior OS. Three primary OAL prognostic groups were identified: Group I (indolent histology, conjunctival location), Group II (indolent histology, non-conjunctival location or aggressive histology, conjunctival location), and Group III (aggressive histology, non-conjunctival location). The adjusted 10-year hazard ratios for OS were 1.4 (95% CI, 1.1-1.7, P<0.001) and 3.1 (95% CI, 2.5-4.0, P<0.001) for groups II and III, respectively, compared to group I. Groups I, II, and III had 10-year OS of 75%, 61%, and 37%, respectively, and 10-year RS of 96%, 89%, and 55%, respectively.

Conclusions: Both conjunctival location and aggressive histology were independent predictors of survival. Our proposed prognostic index could have implications on OAL staging and management, though it must first be externally validated.

Disclosures

No relevant conflicts of interest to declare.
Chemotherapy-Induced Nausea and Vomiting (CINV) - First Results of a German Non-Interventional Study with NEPA - a Netupitant/Palonosetron Fixed Dose Combination
Authors: Karthaus Meinolf , Klare P , Heilmann V , Klausmann G , Schilling Joerg
Published: November 2019

Introduction: Platinum and anthracyclin chemotherapy regimens are frequently used in hematology pts and compromised by severe acute and delayed CINV. NEPA, a fixed dose combination of the long lasting NK1-receptor antagonist (RA) netupitant and the pharmacological distinct 5HT3-RA palonosetron, has been approved by FDA and EMA for the prevention of acute and delayed CINV receiving highly emetogenic cisplatin- and AC-based as well as moderately emetogenic chemotherapy. A German non-interventional study is currently investigating NEPA's efficacy and impact on quality of life in adult cancer patients by PRO and physicians' personal assessment under real life conditions.

Objectives: Primary objective is to evaluate quality of life (QoL) in adult patients receiving NEPA for CINV prevention. Secondary endpoints are efficacy and safety of NEPA.

Methods: This open label, non-interventional, prospective, national, multicenter study evaluates CINV prevention and patients' QoL with NEPA in 2,500 pts receiving either highly or moderately emetogenic cytostatics on up to 2 consecutive days. NEPA is prescribed in accordance with the EU marketing authorization. QoL is evaluated by the validated FLIE questionnaire for 3 chemotherapy cycles 24 hours before, within 24 hours after and additional 4 days after chemotherapy. Efficacy is documented via patient diaries for the same time period. Safety and physicians' overall satisfaction is reported via eCRF. The interim analysis reports on the first 20% of the pre-planned number of patients.

Results: Between June 2015 and July 2016 a total of 583 patients were enrolled. Median age was 56 (range of 28-88) with 521 out of 583 patients that completed documentation. A total of 291 patients (56%) received AC-based chemotherapy. Efficacy, evaluated by physicians' personal assessment on a 4-point scale, was rated very good or good for 486 (90.7%), 409 (93.4%) and 350 (92.9%) patients in cycle 1, 2 and 3, respectively. Complete response was analyzed based on 87 completed patient diaries. Here, 88.5% reported no emesis and no rescue medication in the acute phase (0-24h post chemotherapy), while 85.1% and 79.3% reported complete response in the delayed (>24-120h post chemotherapy) and overall phase (0-120h post chemotherapy). A high percentage of patients did not suffer from any emesis (94%, 99% and 93% in acute, delayed and overall phase). The study is ongoing.

Conclusions: NEPA proved to be highly effective for prevention of chemotherapy-induced emesis and nausea. Patient reported outcome was rated very good or good in more than 90% of Ctx cycles in the acute as the delayed phase of highly and moderately emetogenic Ctx including cisplatin or anthracyclin based therapy.

Disclosures

Karthaus: HELSINN: Honoraria; RIEMSER: Honoraria. Schilling:RIEMSER: Honoraria.
Regulation of the Erythroid Iron Deprivation Response By a TfR2-Scribble-Epor Integrated Module Coupling Iron Sensing with Receptor Presentation
Authors: Khalil Shadi , Delehanty Lorrie L , Holy Maja , Grado Stephen , Bullock Grant C. , Goldfarb Adam
Published: November 2019

Iron deficiency in humans causes desensitization of erythroid progenitors to erythropoietin, the principal cytokine for erythroid survival, proliferation, and differentiation. This nutrient deprivation response acts in a lineage-selective, non-apoptotic manner to restrain expansion of the tissue responsible for the majority of iron consumption, thereby triaging iron utilization under conditions of deficiency. The molecular basis for this response is incompletely understood but involves transferrin receptor 2 (TfR2) and aconitase enzymatic activity as extra- and intracellular iron sensors. Here, we identify a multi-component, integrated module connecting these two elements of erythroid iron sensing with endo-lysosomal trafficking, receptor surface delivery, and configuration of EpoR signaling. Specifically, iron and aconitase activity modulate the rate of TfR2 catabolism by cathepsin protease activity. Scribble, a conserved regulator of receptor transport and signaling, binds TfR2 and serves as a downstream effector through to its co-catabolism. Scribble levels in turn influence surface delivery of the erythropoietin receptor (EpoR), which physically interacts with both Scribble and TfR2. The TfR2-dependent downregulation of Scribble associated with iron deprivation diminishes surface EpoR density and skews its signaling from JAK-STAT to Akt pathways. Mice with surface-trapped, endocytosis-defective EpoR fail to develop anemia in response to iron deficiency. These findings establish a tissue-specific nutrient sensing pathway relevant to the pathogenesis of human anemias and their resistance to erythropoietin therapy.

Disclosures

No relevant conflicts of interest to declare.
The Prognostic Role of Hypertrabeculation By Cardiac Magnetic Resonance in Thalassemia Intermedia Patients
Authors: Pepe Alessia , Macaione Francesca , Giuliano Pietro , Pistoia Laura , Barone Angelica , Sarli Roberto , Roccamo Gaetano , Maggio Aurelio , Positano Vincenzo , Renne Stefania , Assennato Pasquale , Meloni Antonella
Published: November 2019

Background. Differentiation of left ventricle non-compaction (LVNC) from hypertrabeculated LV due to a negative heart remodeling in thalassemia intermedia (TI) can depends on the selected CMR criterion. The recently proposed Piga's criterion (NC/C ratio threshold of >2.5, Am J Haem 2012) seems to have a low specificity to identify the true LVNC in TI. Anyway, the Piga's criterion could well detect easy a negative heart remodeling in TI patients.

Purpose: To assess prospectively whether the Piga's criterion has a prognostic role for adverse cardiovascular outcomes in TI patients.

Methods. We studied prospectively by CMR 168 TI patients (81 males, mean age 38.32 ±11.61 years) consecutively enrolled in the Myocardial Iron Overload in Thalassemia (MIOT) Network. Eight patients were excluded because a cardiac complication was present at the first CMR.

Using Piga's criterion the study population was divided into two groups: patients with Piga's positive criterion (n=18, 11.2%) and with Piga's negative criterion (n=143, 88.8%).

Results. Mean follow-up time was 57.50 ± 21.87 months. Sixteen cardiac new events were recorded: 1 heart failure (HF), 10 supraventricular arrhythmias and 5 pulmonary hypertension (PH). The patients with Piga's positive criterion had a significant higher risk of developing arrhythmias (hazard ratio-HR= 5.35 ; 95%CI=1.51-18.98; P=0.009). The figure shows the Kaplan-Meier survival curve.The positivity for the Piga's criterion was not predictive for PH or for cardiac complications globally considered.

Conclusions. Based on our data a NC/C ratio >2.5 provides

Figure Figure.

Disclosures

Pepe: Chiesi Farmaceutici and ApoPharma Inc.: Other: Alessia Pepe is the PI of the MIOT project, that receives no profit support from Chiesi Farmaceutici S.p.A. and ApoPharma Inc..
The Methylcytosine Dioxygenase TET2 Regulates CD8+ T Cell Memory Differentiation
Authors: Carty Shannon A. , Gohil Mercy , Banks Lauren B. , Johnson Matthew E , Stelekati Erietta , Wells Andrew D , Wherry E. John , Koretzky Gary A. , Jordan Martha S
Published: November 2019

DNA methylation is one of the major epigenetic mechanisms that control T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, intermediates in active DNA demethylation. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. However, following acute viral infection with LCMV-Armstrong, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting antigen-driven cell expansion or effector function. Integration of genome-wide methylation analysis and expression data suggest that TET2 loss leads to hypermethyation of the PRDM1 genomic locus (encoding Blimp-1) and alters the relative expression of Blimp-1 and Bcl-6, two antagonistic transcriptional repressors known to direct CD8+ T cell memory differentiation. Together, our data indicate that TET2 is an important regulator of CD8+ T cell fate decisions.

Disclosures

No relevant conflicts of interest to declare.
Are Direct Oral Anticoagulants Plasma Concentrations Associated with the Risk of Postoperative Bleeding? Results from the Real Life Cohort
Authors: Olivera Pável , Cortina Vicente , Pons Verónica , Canals Tania , Johansson Erik , Bosch Francesc , Santamaría Amparo
Published: November 2019

Background

The perioperative management (PM) of direct oral anticoagulants (DOACs) is controversial. The role of assessing DOAC plasma levels in order to ensure a safe use of these anticoagulants is still unknown.

Aims

To examine the association between DOACs plasma concentrations obtained before surgery and the risk of postoperative bleeding in the perioperative setting.

Methods

From June 2014 to December 2015 we have consecutively included 99 patients treated with DOACs and referred to our Unit for PM. Management was performed following the PM recommendations from the Catalan Thrombosis Working Group (Tromboc@t) . Bleeding events were classified following the ISTH criteria. Plasma concentrations were measured in the day of invasive procedure using the Technoclone anti-Xa assay from Technoclone (Vienna-Austria) for Rivaroxaban and Apixaban, and the Direct Thrombin Inhibitor Assay from IL (Bedford-MA-USA) for Dabigatran; in each case, specific calibrators were used. Patients were systematically followed 30 days after the surgical procedure.

Results

A total of 99 patients were recruited. Median age was 76 years (range: 61-94) and 51 (51.5%) were female. Among them, 23 patients received dabigatran, 40 rivaroxaban and 36 apixaban. As per the risk scores, 66.7% of the patients had a CHA2DS2-VASc score >3, 57.6% had a HAS-BLED score >3, and 51 (51.5%) were considered high-risk procedures. Total bleeding events occurred in 23 patients (47.8% minor, 30.4% non-major clinically relevant, and 21.7% major bleeding). The median plasma NOACs concentration was 38.3 ng/ml (0.8-226 ng/ml), with 32 patients having levels >30 ng/mL. HASBLED score > 3 was associated with an increased risk of bleeding events within 30 days (hazard ratio (HR)= 3.9, 95% CI= 1.14-13.4, P=0.03). Plasma DOAC levels > 30 ng/ml were not significantly associated with an increased risk of bleeding events (HR=2.17, 95% CI=0.862-6.67, P=0.10). Major bleeding (n=5) was probably associated with the risk of the procedure than to the DOAC plasma concentrations.

Conclusion

In our cohort we found significant association between the individual bleeding risk before surgery with the risk of postoperative bleeding. In spite of that, this study will continue to reevaluate PM in high-risk procedures according to plasma DOAC levels.

Disclosures

No relevant conflicts of interest to declare.
More Efficient Exchange of Sickle Red Blood Cells Can be Achieved By Exchanging the Densest Red Blood Cells
Authors: Thibodeaux Suzanne , Irwin Leah , Jamensky Lita , Schell Kevin , O'Doherty Una
Published: November 2019

Background: In sickle cell disease, it is well established that cells containing two mutated hemoglobins (SS) are denser than cells containing wild-type hemoglobin. We asked whether we could more efficiently exchange red blood cells (RBCs) in sickle cell anemia patients by exploiting the denser property of RBCs containing hemoglobin SS compared to wild-type RBCs.

Methods: To probe this question, we performed a series of experiments using the waste bags from sickle cell patients as simulated patients. We exchanged the simulated patient with one RBC volume using recently expired ABO compatible RBC units on a COBE Spectra apheresis instrument. We measured hematocrit and hemoglobin S levels in the simulated and control patient bag before and after the exchange. In the experimental scenario, we programmed the COBE Spectra to exchange the bottom half of the RBCs by indicating that the hematocrit was half of the true hematocrit (e.g. 21% when the hematocrit of the bag was 42%). For control exchanges we programmed the COBE Spectra to exchange the entire RBC volume by programing the hematocrit to be the true one (for this example 42%).

Results: The percentage of hemoglobin S was more effectively diminished in our modified exchanges that targeted dense RBCs than in control exchanges. Our experimental exchanges were also more effective than expected for a 1 red blood cell volume exchange by Poisson calculation (n=5). In an optimized experiment, hemoglobin S was reduced from 23.7% to 1.3 % after exchanging 1 RBC volume using our modified approach while it was reduced from 23.2 to 5.4% using the control or routine exchange parameters. The same volume of donor RBCs (1 RBC volume) was used to exchange our experimental and control simulations. We obtained a 95% reduction of hemoglobin S in our experimental conditions and a 77% reduction in our control conditions. The instrument was programmed to compensate for RBC depletion with the modified RBC exchange. The compensation is necessary to maintain a constant hematocrit since more RBCs are present in the bottom half than top half of the centrifuged RBCs.

Conclusions: It is possible to exchange sickle cell anemia patients more effectively by taking advantage of the fact that RBCs containing hemoglobin SS are denser than normal RBCs. Using waste products from sickle cell anemia exchanges provides an opportunity to safely optimize exchange parameters. This approach should allow us to: 1) achieve a higher reduction in hemoglobin S in patients, 2) achieve the previous levels of reduction using fewer donor units and/or 3) combine red cell exchange with other therapies for sickle cell disease, such as hydroxyurea, by taking advantage of the differential densities and selective depletion of red blood cells containing different levels of hemoglobin F.

Disclosures

No relevant conflicts of interest to declare.
Chromatin Accessibility Identifies CTCF As a Gatekeeper of Stemness Functions in Human Hematopoietic Development
Authors: Takayama Naoya , Murison Alex , Takayanagi Shin-ichiro , Zandi Sasan , Penrod Nadia , Mitchel Amanda , Kennedy James A. , Ng Stanley W.K. , Xie Stephanie Zhi-Juan , Minden Mark D. , Dick John E. , Lupien Mathieu
Published: November 2019

Hematopoietic stem cells (HSC), at the apex of the blood hierarchy, generate a series of increasingly committed progenitors and ultimately produce terminally differentiated cells at the bottom of the hierarchy. Although HSC and their immediate downstream, multipotent progenitors (MPP) have full multilineage differentiation capacity, only HSC have the capacity for long term self-renewal. Thus comparison of HSC with downstream progenitors that exhibit gradual fate restriction by assuming the identity of a mature blood cell is central to defining how the stemness state is established and how lineage commitment is executed. Interestingly, few genes are differentially expressed between human HSC and MPP, suggesting that some important stem cell mechanisms are in a permissive state and therefore difficult to identify simply by differential gene expression analysis. We hypothesized that these permissive genetic regions could be identified by chromatin analysis. To identify permissive genomic regions, we delineated regions of accessible chromatin through ATAC-seq assays in HSC, MPP, committed progenitors and seven mature lineages from human Cord Blood. DNA recognition motif analysis across accessible chromatin in each developmental stage identified both known and unknown candidate master regulatory transcription factors for each step of lineage development.

The DNA recognition motif for CTCF, a crucial effector of chromatin interactions, is enriched in accessible chromatin associated with progenitors but not in HSC, even though CTCF expression is not significantly different among stem and progenitor populations. Knockdown of CTCF in HSC by lentiviral vector system increased the proportion of HSC in G0 cell cycle and consequently reduced the number of mature cells generated in both conventional and high sensitivity single cell assays. The development of differentiated lymphoid and megakaryocytic lineages was affected. Few effects were seen when CTCF was silenced in MPP or more downstream progenitors. Collectively, our results suggest that CTCF is a "gate keeper factor" that regulates transition from dormant to active stages in HSC, and might control stem cell pool throughout life.

In parallel to normal blood hierarchies, AML is also composed of a hierarchy with leukemia stem cells and non-stem-like leukemia cells; LSC and non-LSC fractions sorted from 24 AML patients have also been subjected to ATAC-seq. Currently we are comparing HSC and non-HSC with LSC and non-LSC fractions to identify the similarity and differences between normal HSC and LSC. Overall, our study is revealing insights into the stemness state of normal and leukemic stem cells in primary human tissues.

Disclosures

Takayanagi: Kyowa Hakko Kirin: Employment.
Hospital Volume Does Not Influence in-Hospital Mortality of Autologous Hematopoietic Cell Transplant Among Multiple Myeloma Patients in the United States
Authors: Giri Smith , Dhakal Prajwal , Mehta Kathan Dilipbhai , Bhatt Vijaya R.
Published: November 2019

Introduction:

Hospital volume and provider experience affects survival among patients with hematologic malignancies (Giri et al Blood 3359-60). Whether hospital volume affects outcomes of autologous hematopoietic cell transplant (autoSCT) among patients with multiple myeloma (MM) remains unclear.

Methods:

We utilized the Nationwide Inpatient Sample (NIS) database to identify all adults >18 years diagnosed with MM who underwent autoSCT in 2009-2011. NIS is the largest all-payer inpatient database in the US that captures about 20% of all US hospitalizations. Hospitals were divided into quartiles, based on the annual number of autoSCT performed, and classified into high volume (above 75th percentile) and low volume centers (below 25th percentile). In-hospital outcomes including inpatient mortality, infection, mechanical ventilation, and costs of hospitalization were compared between the two groups. All p-values were 2 sided, and the level of significance was chosen at 0.05. Statistical analysis was done using STATA 13.0 (StataCorp, College Station, TX).

Results:

A total of 2,750 autoSCTs were reported among patients with MM during the study period. The characteristics of study population included mean age of 58.7 ± 8.7 years, 56% males (n=1547) and 72% whites (n=1822). No significant difference existed in in-hospital mortality rate (0.86% vs. 1.59%; p=0.183) between high volume (≥178 autoSCTs per year) versus low volume centers (≤56 autoSCTs per year). The rate of fungal infection (5.32% vs. 4.94%; p=0.75), herpes simplex virus infection (1.58% vs. 1.59%; p=0.98), and the need for mechanical ventilation (1.87% vs. 1.27%) was similar between the high volume and low volume centers. Higher rates of stomatitis (57% vs. 46%; p<0.01), use of total parenteral nutrition (12.37% vs. 6.21%) and neutropenic fever (33.6% vs. 23.5%; p <0.01) were noted in high volume versus low volume centers. The cost of initial hospitalization was similar in the two groups (mean $ 161,085 vs. $ 154,161; p value 0.17).

Conclusion:

Our study demonstrates a low risk of inpatient mortality without center effect for autoSCT in MM in the recent years. The risks of fungal and herpes simplex virus infection were also similar between high volume and low volume centers. Higher rates of stomatitis, use of total parenteral nutrition and neutropenic fever were noted in high volume versus low volume centers. The reasons for these differences are not clear from our study but may relate to possible differences in patient characteristics or conditioning chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
18F-FDG-PET/CT and Osteolytic Bone Disease in Multiple Myeloma
Authors: Østergaard Brian , Mortensen Julie R. , Nielsen Anne L , Asmussen Jon T. , Gerke Oke , Plesner Torben , Høilund-Carlsen Poul Flemming , Abildgaard Niels
Published: November 2019

Introduction: FDG-PET/CT is a promising methodology for staging, prognostication, and response evaluation in multiple myeloma (MM). The number of focal FDG lesions and the intensity of FDG uptake (standard uptake value, SUV) at diagnosis are informative of disease aggressiveness. Osteolytic bone disease is a hallmark of MM. Hypothetically; increased focal metabolic activity will precede, induce and correlate to osteolytic lesions. We aimed to elucidate the association between focal FDG positive lesions and osteolytic bone disease in MM patients.

Methods: Twenty-two newly diagnosed MM patients, 16 males and 6 females aged 53-81years, were prospectively enrolled and studied with a standardized baseline FDG-PET/CT at diagnosis. A nuclear medicine and a radiology specialist evaluated the PET and CT parts separately and independently. Focal FDG positive lesions were assessed by dedicated software (ROVER™, ABX, Germany) to yield volume and SUV measures. All osteolytic lesions identified on CT were noted by size and localization.

Results: FDG-PET and CT together identified a total of 390 lesions. We found more osteolytic lesions on CT (335) than FDG-positive lesions (169); the concordance between the modalities was 30%. 34% (114/335) of lytic lesions were FDG-positive, whereas ⅔ (114/169) of focal FDG-PET positive lesions were lytic according to CT. We found a significant correlation between higher FDG uptake measured as SUVpeak and osteolytic lesions >10 mm, and focal lesions with high FDG uptake had more pronounced osteolysis than focal lesions with less FDG uptake. Still sparse follow-up data did not allow analysis of the predictive value of focal PET positivity and the risk of development of future osteolysis. More compulsive data will be presented at the meeting.

Conclusion: FDG-PET/CT offers dual information including metabolic activity (FDG-avidity) and degree of lytic bone disease (low dose whole body CT) in MM. About ⅔ (221/335) of osteolytic lesions were FDG-negative, whereas ⅔ (114/169) of FDG-positive lesions had a lytic component.

Disclosures

Plesner: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.
Interleukin-6 Drives Multiple Myeloma Progression By up-Regulating of CD147/Emmprin Expression
Authors: Hu Jinsong , Lei Li , Wang Yanmeng , Wang Ke , Hu Xiaoyan , Wang Aiying , Vanderkerken Karin
Published: November 2019

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD147 is expressed in a variety of tissues, and is involved in a number of physiological and pathological processes. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. However, it is still unclear what mechanism is involved in the dysregulation of CD147 expression in MM. In this study, we found that CD147 can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCIH929 were stimulated with 20ng/mL of IL-6 for 24 hours, we found that the expression of CD147 on MM cell membrane was significantly up-regulated, as determined by flow cytometric analysis. Moreover, when using the IL-6 autocrine MM cell line U266, CD147 level was found to be continuously increased in serum-starved MM cells in a time-dependent manner. Interestingly, the increase of CD147 in U266 cells was found to be abrogated when using neutralizing antibody to block the secreted IL-6. Next, we investigated the molecular mechanisms involved in the IL-6-mediated upregulation of CD147 in MM cells. We confirmed that IL-6 can activate the JAK/STAT3 signaling pathway, inducing the phosphorylation of STAT3 at Tyrosine 705 in RPMI8226 and MM1.S cells. Further studies using ChIP analysis demonstrated that there is a STAT3 binding site at the position −659 to −650 in the promoter of the CD147 gene. In addition, when inhibiting of STAT3 phosphorylation with a specific inhibitor S3I-201, the CD147 expression in MM cells was accordingly decreased. Finally, we found that siRNA mediated knock-down of CD147 in RPMI8226 and MM1.S cells can decrease the proliferation and migration of MM cells induced by IL-6 and Cyclophilin A (CypA). In conclusion, our data provide evidence that IL-6 can up-regulate CD147 expression in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM.

Disclosures

No relevant conflicts of interest to declare.
Molecular and Biological Characteristics of Tumour Substrate in Patients with Extramedullary Multiple Myeloma
Authors: Firsova Maya V. , Mendeleeva Larisa P. , Kovrigina Alla M. , Deineko Natalia L. , Solovev Maxim V. , Nareyko Maria V. , Kuzmina Larisa A. , Savchenko Valery G.
Published: November 2019

Introduction. At the time of the diagnosis of MM the incidence of extramedullary disease (EMD) varies from 7 to 18%, and in case of à relapse, it is observed in more than 20% of patients. Clinical progression of MM with EMD is characterised by highly aggressive tumour, worse prognosis and often by resistance to therapy, low overall survival rate.

Aim of the study. To evaluate and to compare molecular and biological characteristics of tumour cells in bone marrow and extramedullary plasmacytoma in primary MM patients.

Materials and methods. The study enrolled 28 patients with primary MM (18 males, 10 females) 23-59 years old (median value: 52 years old). The disease was diagnosed in accordance with the IMWG criteria (2014). All patients underwent bone marrow trephine biopsy. 13 patients had EMD including 10 patients with soft-tissue plasmacytomas accompanied by cortical layer involvement and the tumour mass spreading from bone. 3 patients had extramedullary foci in stomach, brain, soft submandibular tissues. In all cases a tumour biopsy was taken which confirmed the presence of plasma cell infiltration. Paraffin block slices from trephine biopsy material and tumour biopsy material were used to perform an immunohistochemistry analysis with an antibody panel to CD56, Ki67, CXCR4, CD 166.

Results. The immunohistochemistry analysis revealed more frequent expression of CD56 (80% vs. 38.5%, p=0.05) and high Ki-67 expression (53.3% vs. 0%, p = 0.002) in bone marrow of MM patients without EMD in comparison with those with plasmacytomas. At the same time patients without EMD demonstrated CXCR4 expression by myeloma cells in bone marrow, that was significantly higher compared with MM patients with plasmacytomas (100% vs. 46.2%, p = 0.001) (Table 1).

Immunohistochemical parameters of plasma cells in bone marrow and plasmacytoma of MM patients with EMD were compared. A higher level of CD56, Ki-67, CXCR4 expression in plasma cells from tumour biopsy was observed, although the differences were not statistically significant. The CD166 expression level in bone marrow and plasmacytoma was similar.

Conclusion. Immunohistochemistry analysis of the tumour substrate revealed that MM patients with and without EMD demonstrate different molecular and biological characteristics of bone marrow. Lack of CXCR4 expression in bone marrow may be the evidence of poor prognosis for the MM patients with the higher risk of EMD occurrence.

Disclosures

No relevant conflicts of interest to declare.
Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines
Authors: Bleker de Oliveira Mariana , Eugenio Angela Isabel , Fook Alves Veruska Lia , Zanatta Daniela , Yamamoto Mihoko , Strauss Bryan Eric , Colleoni Gisele W. B.
Published: November 2019

Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12).

Disclosures

No relevant conflicts of interest to declare.