bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute Treatment Of Pulmonary Embolism With Rivaroxaban - Real Life Data From The Prospective Dresden Noac Registry (NCT01588119)
Authors: Köhler Christina , Werth Sebastian , Tittl Luise , Beyer-Westendorf Jan
Published: October 2019

Background and Objectives

In the EINSTEIN PE study rivaroxaban (RX) was found to be as effective as warfarin in the treatment of acute pulmonary embolism (PE) with superior safety. However, study results need to be confirmed in unselected PE patients in daily care.

Patients and Methods

Using prospectively collected data from a large regional registry of patients treated with novel direct oral anticoagulants (NOAC) in the district of Saxony, Germany, we evaluated the rate of recurrent VTE, other cardiovascular complications and bleeding events in patients receiving rivaroxaban for acute PE. In this ongoing registry, a network of 239 physicians enrols up to 2500 daily care NOAC patients who receive central prospective follow up (FU) by the registry office at day 30 day and quarterly thereafter to collect efficacy and safety data. All outcome events are centrally adjudicated using standard scientific definitions.

Results

Until July 31th 2013, 2249 patients were enrolled. Of these, 72 patients received RX for acute PE treatment (demographic data in table 1). Registry patients were older than the EINSTEIN PE population (67.3 vs. 55.8 years), 55.6% were female and 23.6% were treated for a recurrent VTE. During follow-up, unplanned rivaroxaban discontinuation rates were low (around 5%; table 1). So far, only one recurrent VTE event occurred (1.7 events per 100 patient years). One patient experienced non-fatal ischaemic stroke within 4 weeks after PE diagnosis (1.7 events per 100 patient years). Bleeding complications were frequent but only 2 major bleeding (non-fatal vaginal bleeds) occurred (3.3 events per 100 patient years). During follow-up three patients died of underlying diseases but none of these deaths were related to VTE or bleeding complications.

Conclusion

Acute PE treatment with rivaroxaban in daily care is effective, safe and well tolerated with low rates of unplanned treatment discontinuation. Thromboembolic and major bleeding complications are rare and seem to occur predominantly in the early phase of PE treatment.

At ASH, updated results from our registry will be presented

Disclosures:

Werth: Bayer Healthcare: Honoraria. Beyer-Westendorf:Pfizer: Research Funding, Speakers Bureau; Boehringer Ingelheim: Research Funding, Speakers Bureau; Bayer Healthcare: Research Funding, Speakers Bureau.
Ex Vivo Prothrombin Complex Concentrates and a Specific Antidote Are Effective In Reversing Dabigatran-Induced Coagulopathy In Pigs
Authors: van Ryn Joanne , Spronk Henri M.H. , Rossaint Rolf , Grottke Oliver
Published: October 2019

Introduction

New oral anticoagulants are effective alternatives to warfarin; however, no specific reversal agents are currently available for life-threatening bleeding or emergency surgery. This study measured the effects of prothrombin complex concentrate (PCC), activated PCC (aPCC) and recombinant factor VIIa (rFVIIa) and compared this to reversal with the specific antibody fragment for dabigatran after high-dose dabigatran etexilate (DE) treatment in a porcine model of trauma, to assess reversal of anticoagulation.

Methods

Studies were performed in 5 male pigs after ethical approval. DE was given orally for 3 days (30 mg/kg bid) and, on the 4th day, dabigatran was infused at 0.77mg/kg/h for 30 min followed by 0.52 mg/kg/min for 60 min. The infusion was administered immediately before trauma (blunt liver insult). Blood samples were taken before dabigatran infusion and 60 min post-injury. Two doses of PCC (30 and 60 IU/kg), aPCC (30 and 60 IU/kg) and rVIIa (90 and 180 μg/kg) and two doses anti-Dabi Fab (30 and 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by thromboelastometry (ExTEM assay) and PT and dabigatran levels with diluted TT. Data were analyzed by ANOVA (± SEM).

Results

Oral DE prolonged clot formation (CT: 389 ± 128 sec; CFT: 159 ± 39 sec) and PT (27 ± 9 sec), but coagulopathy was more severe after achieving high dabigatran plasma levels (943 ± 147 ng/ml) and inducing trauma (CT: 2378 ± 480 sec; CFT: 3374 ± 625 sec; PT: 167 ± 40 sec). At this time point, 30 IU/kg PCC and 30 IU/kg aPCC significantly accelerated clot formation (PCC: CT 416 ± 70 sec, CFT 250 ± 91 sec; aPCC: CT 525 ± 126 sec, CFT 309 ± 60 sec; p<0.05 vs. untreated samples). These interventions also shortened PT (PCC: 53 ± 14 sec; aPCC: 66 ± 16 sec; p<0.05 vs. untreated samples). Higher doses of PCC and aPCC had no further effect on hemostatic parameters. rFVIIa had no impact on coagulopathy at any time point. Addition of the specific antidote to dabigatran completely normalized all coagulation parameters to baseline levels.

Conclusion

The ex vivo addition of either PCC or aPCC is effective in reducing coagulopathy in a porcine model of high-dose dabigatran therapy and trauma. However the specific aDabi-Fab is the most promising agent to reverse the anticoagulant effects of dabigatran. The potential for reducing blood loss remains to be investigated.

Disclosures:

van Ryn: Boehringer Ingelheim Pharma GmbH & Co. KG: Employment. Spronk:Boehringer Ingelheim Pharma GmbH & Co. KG: Research Funding. Rossaint:Boehringer Ingelheim: Research Funding. Grottke:Boehringer Ingelheim: Research Funding; CSL Behring: Consultancy.
Chromatin Remodeling Enzyme CHD7 Negatively Regulate Hematopoietic Stem Cell Function
Authors: Hsu Jingmei , Huang Hsuan-Ting , Lee Chung-Tsai , Yu Shuqian , Zon Leonard I. , Speck Nancy A.
Published: October 2019

ATP-dependent chromatin remodeling enzymes alter histone/DNA interactions, and are involved in the regulation of transcription, chromosome segregation, DNA replication and repair. We identified Chromodomain Helicase DNA binding protein 7 (CHD7) as a negative regulator of hematopoietic stem cell function. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Although several cases of T and B cell immunodeficiency in a subset of CHARGE syndrome patients have been reported, no previous role for CHD7 in hematopoiesis had been proposed.

We show that morpholino knockdown of Chd7 in zebrafish embryos results in an increased number of runx1 and c-myb expressing hematopoietic stem and progenitor cells in the aorta/gonad/mesonephros (AGM) region, and this effect is cell autonomous as determined by blastula transplantation. Heterozygous germline Chd7 deletion in mice also results in an increased number of phenotypic hematopoietic stem and progenitor cells (Runx1+c-kit+CD31+) in the AGM region. Downstream lineages such as myeloid and erythroid cells are expanded in zebrafish, and similarly conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre results in myeloid lineage expansion and increased granulocyte/monocyte progenitors. Consistent with these results, microarray analysis of murine CD48- CD150+ Lin- Sca-1+ c-kit+ (SLAM LSK) phenotypic long term repopulating HSCs shows up-regulation of genes in several subclasses of the myeloid lineages. Interestingly, although CHD7 deficient mouse bone marrow had a normal frequency of SLAM LSK cells, it had a two-fold higher frequency of functional LT-HSCs as determined by whole bone marrow, purified LSK and SLAM LSK limiting dilution transplants. ChIP-seq performed in human CD34+ hematopoietic stem and progenitor cells show CHD7 localizes to genes encoding many key hematopoietic transcription factors including MYB and RUNX1. The most abundant transcription factor motif under CHD7 genomic binding sites is a RUNX motif, indicating that CHD7 and Runx1 function together. We show CHD7, Runx1 and c-Myb interact both physically and genetically. CHD7 function in hematopoietic stem cells is dependent on both Runx1 and c-Myb since the increase in hematopoiesis in fish upon morpholino knockdown of Chd7 is abolished when either Runx1 or c-Myb is mutated. In summary, our study identifies CHD7 as a novel evolutionarily conserved negative epigenetic regulator of HSCs and progenitors through its interaction with Runx1 and c-Myb.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Depletion Causes Rapid But Transient Activation Of Mouse Bone Marrow Long-Term Hematopoietic Stem Cells To Participate In Stress Thrombopoiesis
Authors: Shao Lijian , Hou Di , Feng Wei , Chang Jianhui , Ware Jerry , Hauer-Jensen Martin , Cheng Tao , Zhou Daohong
Published: October 2019

Hematopoietic stem cells (HSCs) are responsible for the production of various lineages of blood cells throughout life. To ensure the longevity of HSCs and prevent their premature exhaustion, a small population of HSCs termed long-term HSCs (LT-HSCs) is maintained in quiescence under homeostatic conditions. Whether LT-HSCs can exit quiescence rapidly in response to hematopoietic stress to replenish blood cells is not known, and thus was investigated in the present study in an acute but transient platelet depletion mouse model induced by intravenous injection of anti-GPIbα antibody. The results from our study show that in response to platelet depletion LT-HSCs can exit quiescence promptly and then proliferate rapidly to participate in stress thrombopoiesis probably via an alternative differentiation pathway. The mechanism by which platelet depletion causes LT-HSC activation is not due to a direct effect of the antibody on LT-HSCs or activation of platelets, but is attributed to a transient reduction of thrombopoietin (TPO) resulting from the acute depletion of platelets. However, the activated LT-HSCs return to quiescence when blood platelet counts are almost back to normal levels without compromising their function. These findings suggest that there is a very efficient and sensitive feedback regulatory circuitry between quiescent LT-HSCs and platelets, which allows LT-HSCs to directly and promptly respond to hematopoietic stress resulting from an acute loss of platelets. In this response, TPO may function as a switch that can rapidly turn on LT-HSCs to participate in stress thrombopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Oridonin Upregulates All-Trans Retinoic Acid Receptor Alpha and Induces Differentiation Of NB4 Cells Though NF-Kb Pathway
Authors: Yan Hua , Cao Yang , Wei Wei , Zhang Nan , Wu Ying-Li , Chen Guo-Qiang
Published: October 2019

Nuclear retinoic acid receptors (RARs) function as ligand-dependent transcriptional regulators, often heterodimerized with retinoid X receptors. They play critical roles in a variety of biological processes, including development, differentiation and homeostasis. However, it is still not very clear about the mechanism of RARα catabolism. Almost all cases of acute promyelocytic leukemia (APL) express oncogenic fusion protein involving the RARα due to chromosome translocations. Despite the great success of ATRA therapy, it is still important to search for a variety of promising compounds to enhance the pharmacologic activity of ATRA and alleviate its adverse effects. And some of these compounds may be useful as a probe to investigate the mechanism of RARα catabolism.

Our previous studies showed that oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, could have an ability to accumulate RARα, enhance ATRA-induced differentiation in ATRA-sensitive as well as -resistant APL cells, and induce apoptosis which is initiated by reactive oxygen species (ROS) production. Based on those discoveries, we supposed that inhibition of RARα degradation may be responsible for these serial actions. Herein, we used Ori as a probe to explore how Ori stabilized RARα and its potent mechanisms. Firstly, our results showed that Ori upregulated RARa in a dose- and time- dependent manner in NB4 cells while anti-oxidant agent N-acetyl-L-cysteine (NAC) addition completely blocked this effect, indicating that RARa was stabilized via Ori-induced intracellular ROS formation. Secondly, we found H2O2, which could increase ROS level also, upregulated RARa in a dose- and time- dependent manner. More of interest, we found that both Ori and H2O2 upregulated RARa through NF-kB-signaling, proved by the accumulate evidence that either NF-κB pathway inhibitor or knockdown of P65 or inhibition IkBa phosphorylation could block the RARa stabilization. Whereas, P65 overexpression or knockdown of IkBa or some NF-κB pathway activating agents like tumor necrosis factor alpha (TNFa) and lipopolysaccharide ( LPS) could increase the RARa level.

Collectively, these findings suggested that Ori is an extraordinary tool to enhance the pathophysiological and potential therapeutic roles of RARα expression though NF-kB pathway. Moreover, an approach combining ATRA with Ori warrants further investigation as a therapeutic strategy for APL patients.

Disclosures:

No relevant conflicts of interest to declare.
Histones To The Cytosol: Role Of Nuclear Protein Export In Erythroid Nuclear Condensation
Authors: Figueroa Amalia A , Venkatesan Srividhya , Hattangadi Shilpa M
Published: October 2019

Erythropoietin-refractory anemias and red cell aplasias are rare but devastating disorders in children, with no known treatment other than chronic transfusions and bone marrow transplant, with high morbidity and mortality. New, improved treatments for such anemias are severely limited by incomplete understanding of critical terminal erythroid processes such as enucleation. Our long-term goal is to understand the regulation of erythroid nuclear condensation culminating in enucleation in order to increase our basic knowledge of important cell biology concepts like nuclear export and nuclear condensation during erythropoiesis and ultimately help optimize new treatments for anemia.

Proteomic examination of normal pycnotic nuclei from mouse fetal liver (definitive adult erythropoiesis) revealed a striking decrease in almost all nuclear proteins, suggesting that nuclear protein export occurs prior to extrusion. Expression of the nuclear exportin, Exportin 7 (Xpo7) is highly erythroid-specific—utilizing a tightly-regulated erythroid-specific promoter, induced during erythropoiesis, and abundant in late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe disruption of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation (manuscript submitted), suggesting that protein export is crucial to the process of nuclear condensation. Nuclei in Xpo7 knockdown cells were larger and less dense than controls. Strikingly, DNA binding proteins such as histones H2A and H3 accumulated in the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in cells lacking Xpo7.

We have confirmed by chromatin immunoprecipitation and RACE-PCR that there is an erythroid-specific promoter (and start-site) for Xpo7, whose expression is not conserved in lower vertebrates or in primitive mouse erythroblasts. We hypothesize that the erythroid-specific function of Xpo7 therefore may account for the fact that red cells are not enucleated in these other biological systems. We have also performedimmunoprecipitation on the erythroid and non-erythroid forms of Xpo7 and determined several erythroid-specific cargo proteins of Xpo7 that are involved with other types of chromatin condensation (including Rcc1 which is involved in chromosome condensation). We are starting to build a model for how Xpo7 facilitates the removal and replacement of histones in the erythroblast nucleus in order to allow adequate nuclear condensation for extrusion.

Disclosures:

No relevant conflicts of interest to declare.
Direct Cell-To-Cell Interactions Between Osteocytes and Multiple Myeloma (MM) Cells Upregulate Sost and Downregulate OPG Expression In Osteocytes: Evidence For Osteocytic Contributions To MM-Induced Bone Disease
Authors: Delgado Calle Jesus , Bellido Teresita , David Roodman G. David
Published: October 2019

Osteocytes are the most abundant bone cells, comprising more than 95% of the cells in bone. They are embedded into the bone matrix, but extensively communicate among themselves and with cells on the bone surface and the bone marrow through the osteocytic lacunar-canalicular network. Osteocytes secrete sclerostin, the product of the Sost gene, an antagonist of Wnt signaling that potently inhibits bone formation. Osteocytes are also a major source of pro- and anti-osteoclastogenic cytokines that regulate osteoclastogenesis and bone resorption, including RANKL and osteoprotegerin (OPG). Recent evidence suggests that the bone remodeling compartment is disrupted in multiple myeloma (MM) allowing close contact of MM cells with bone cells including osteocytes. However, the consequences of these interactions and the contribution of osteocytes to MM bone disease are unclear. Therefore, we determined if interactions between MM cells and osteocytes regulate osteocytic gene expression. We found that co-culture of murine MLO-A5 osteocytic cells with human JJN3 MM cells up-regulated murine Sost mRNA expression 2-3 fold as early as 4h, which remained elevated up to 24h. Consistent with Sost upregulation induced by MM cells, the expression of OPG, a Wnt target gene, was decreased by 30-50% in MLO-A5 cells, resulting in an increased RANKL/OPG at 4h. Culture of JJN3 cells in the top and MLO-A5 cells in the bottom of Boyden chambers abolished both upregulation of Sost and downregulation of OPG mRNA expression in osteocytic cells, demonstrating the requirement of direct contact between MM cells and osteocytic cells. Human Sost and OPG mRNA transcripts were not detected in any of these experiments, demonstrating lack of contribution of MM JJN3 cells. These findings demonstrate that direct interactions between osteocytes and MM cells upregulate the expression of the bone formation inhibitor Sost in osteocytes, which in turn decreases Wnt signaling, reduces osteocytic OPG expression increasing the RANKL/OPG ratio. We propose that increased Sost/Sclerostin expression contributes to the exacerbated bone resorption and the decreased bone formation that characterizes MM induced bone disease.

Disclosures:

No relevant conflicts of interest to declare.
Effect Of Low Dose Bortezomib On Bone Formation In Smoldering Myeloma Patients
Authors: Zangari Maurizio , Berno Tamara , Salama Mohamed E , Sana Sherjeel , Talamo Giampaolo , Pena Karen , Miller Scott , Zhan Fenghuang
Published: October 2019

Background

Bortezomib has been shown to produce an anabolic bone effect (increase bone ALP and osteocalcin) in relapsed/refractory patients. This study examined the bone anabolic effect in patients with smoldering multiple myeloma (SMM) who, with a historical median age of 67 years, have frequent evidence of osteopenia not associated with lytic bone disease. SMM is usually followed expectantly without therapy. The overall risk of progression to active MM has been estimated up to 20% in the first year from diagnosis (Kyle et al, 2007). The primary aim of this trial is to determine the effect of a course of low-dose Bortezomib on bone remodeling and on disease progression.

Methods

The dose of bortezomib used in this trial of 0.7mg/m² is the lowest dose which has shown efficacy in the 3 largest monotherapy trials with bortezomib. Patients enrolled in this study had serum M protein ≥ 3 g/dL and/or bone marrow plasma cells ≥ 10% with absence of anemia, renal failure, hypercalcemia, and lytic bone lesions. Patients received 9 cycles of bortezomib given on days 1, 8, 15, 22 every 42 days. No bisphosphonates were allowed during the trial, Vitamin D supplements were allowed.

Results

Seventeen patients (9 males) with a median age of 61 years were enrolled in the study. Fourteen had IgG paraprotein and 3 had an IgA. Two participants did not complete the treatment; the first because of development of a skin rash and the second for personal reasons. A total of 11 patients completed the protocol. Four are still receiving treatment. At the time of this analysis with a median follow-up of 20 months, all patients are alive and none has progressed (Figure 1). The mean M component at baseline, end-of-study, and at most recent follow-up are shown in Figure 2. The treatment was well tolerated by the patients. One patient developed reversible grade 3 neuropathy and a drug related rash and only 3 grade 3 hematological adverse events were recorded (15%). Bone densities by DEXA scan were obtained at baseline, end of study, and yearly thereafter. Out of 17 patients, 13 had bone density T-scores before and after treatment. Six patients (46%) showed an improvement in hip T-score (mean T-score improvement 0.41, range 0.1-1.35). In the overall group, mean T scores improved by 38% (range, 3-200%). T score in lumbar spine improved in 3 patients (23%), who had a mean T-score improvement of 0.2 (range, 0.05-0.43).

Conclusions

The use of low dose Velcade in smoldering myeloma patients was well tolerated and produces significant increases in bone density in 46% of participants.

Disclosures:

Zangari: Millenium Pharm.: Research Funding. Off Label Use: Use of Bortezomib in smoldering myeloma. Salama:Eli Lilly and Co: Research Funding.
The Impact Of Thymoglobulin Prior To Pediatric Unrelated Umbilical Cord Blood Transplantation On Immune-Reconstitution and Clinical Outcome
Authors: Lindemans Caroline A , Chiesa Robert , Amrolia Persis J , Rao Kanchan , Nikolajeva Olga , de Wildt Arianne , Gerhardt Corinne E , Gilmour Kimberley , Bierings Marc , Veys Paul , Boelens Jaap Jan
Published: October 2019

Introduction

In vivo T-cell-depletion generated by the use of Thymoglobulin (ATG) within the conditioning regimen might contribute to the delayed immune-reconstitution observed after unrelated umbilical cord blood transplantation (UCBT). We studied the impact of early, late and no ATG on immune reconstitution and clinical outcome.

Methods

127 Children receiving an unrelated UCBT in London or Utrecht between 2006 and 2011 were included and divided in 3 groups: late ATG (day -5 to 0, n=48), early ATG (day -9 to -5, n= 33) and no ATG(n=46). The no ATG group received MMF and CsA as GVHD prophylaxis, while the ATG groups both received CSA and prednison as prohylaxis, Endpoints studied were survival, early and late immune-recovery, infections and GvHD. Subset analysis CD3+, CD4+ and CD4+naïve, B and NK cell numbers were prospectively measured during post-transplant follow-up at 1, 2, 3, 6 and 12 months post-UCBT.

Results

The probability of survival was not significantly different between the groups: 71% +/- 8% (no ATG), 68% +/- 9% (early ATG) and 61% +/- 7% (late ATG). There were no significant differences in engraftment and primary or secondary graft failure. Immune reconstitution as defined by CD3+, CD4+ and CD4+ naïve T-cell counts were significantly higher (p<0.001) in the no ATG group at 1, 2, 3, 6, and 12 months post-UCBT with a log more T cells than in the ATG groups. The median CD3+ T-cell count in the no ATG group at 1, 2, 3, 6 and 12 months post UCBT was 340 x10e6/L, 720 x 10e6/L, 535 x 10e6/L, 940 ± 194 x 10e6/L, and 1860 x 10e6/L respectively. In the no ATG group significantly less viral reactivations (p=0.021) were noted. A higher probability (p<0,001) of severe acute GvHD (31%+/-9%) was found in the no ATG group compared to 18% +/- 9% for the early ATG and 5% +/-4% for the late ATGgroup. This was not associated with a higher probability of chronic GvHD or non-infectious lung injury in the no ATG group.

Conclusion

Cord blood transplantation without Thymoglobulin (ATG), in the context of MMF and CsA post-transplant prophylaxis, is associated with higher GVHD risk but with a surprisingly good immune reconstitution and with an engraftment and survival similar to the groups with ATG. The findings of improved immune-reconstitution, associated with lower viral reactivations, albeit at the cost of increased rates of acute GvHD (but not chronic GvHD), suggest that omitting ATG in cord blood transplantation may be important to prevent viral reactivations, especially in those at high risk for these.

Disclosures:

No relevant conflicts of interest to declare.
Low Psoas Muscle/BMI Ratio Is a Bad Prognostic Index Of Non-Relapse Survival After Allogeneic Stem Cell Transplantation
Authors: Kim Soo Jeong , Kim Yundeok , Hwang Doh Yu , Hyun Shin Young , Jang Ji Eun , Lee Jung Yeon , Cheong June-Won , Kim Jin Seok , Min Yoo Hong
Published: October 2019

Background

Physical and functional status affect the survival after allogeneic stem cell transplantation. There are several indices to measure risk of complication and non-relapse mortality after stem cell transplantation. Karnofsky score and ECOG score are the mostly used indices for measuring functional status. However they are measured subjectively by observer and can be changed in brief time. Recently a few reports documented relationship between the mortality and sarcopenia in various malignant or non-malignant diseases. Therefore we tried to measure the patient’s functional status by measuring psoas area in abdominal CT scan which was taken before allogeneic stem cell transplantation.

Patients and Methods

The medical records of total 83 patients from August 2010 to April 2012 were retrospectively reviewed. Every patients took abdominal CT scan 2 weeks to 1 month before transplantation according to the protocol for pre-transplant workup of Severance Hospital (Seoul, Korea). The area of bilateral psoas muscle at the level of upper margin of L4 vertebral body was manually measured by one trained observer using polygon tool of CENTRICITY Radiology RA1000 program (GE Healthcare Integrated IT Solutions, IL, USA).

Results

Male patients were 49 and female patients were 34. Median age was 37.0 years (range 15-60). Underlying diagnosis was AML (n=31), ALL (n=24), SAA (n=7), MDS (n=5), lymphoma (n=7), CML (n=6) and others (n=3). Myeloablative conditioning was applied in 54 patients. Stem cell source was mostly peripheral blood (n=68). 8 patients received HLA-mismatched transplantation. The total non-relapse mortality was 20.4%. 32.5% of patients had higher BMI than 25 kg/m2. In whole patients, psoas muscle area didn’t affect non-relapse survival. However, when low psoas area/BMI ratio show significant correlation with non-relapse survival (median 733 days vs. 966 days, P=0.009). In univariate analysis for non-relapse survival, age, pre-transplant disease status, and psoas/BMI ratio showed statistical correlation. And in multivariate analysis psoas/BMI ratio was the sole prognostic index.

Low psoas/BMI ratio is related to increased treatment related mortality in obese patients. This index can be used in part to estimate risk of transplant in obese patients.

Disclosures:

No relevant conflicts of interest to declare.
Loss Of Canonic Notch Signaling In The Microenvironment Causes a Myeloproliferative Syndrome Via Derepression Of The miR155 Promoter
Authors: Wang Lin , Zhang Huajia , Rodriguez-Rodriguez Sonia , Cao Liyun , Parish Jonathan , Zollman Amy , Kamocka Malgorzata , Boswell H. Scott , Srour Edward F. , Chitteti Brahmananda Reddy , Manshouri Taghi , Verstovsek Srdan , Kapur Reuben , Cardoso Angelo A. , Carlesso Nadia
Published: October 2019

The Notch signaling pathway is an important regulator of normal hematopoiesis, and is involved in leukemogenesis. Although the cell-autonomous roles of Notch signaling on hematopoietic cells have been extensively studied, its contribution to the regulation of the bone marrow (BM) niche has not been investigated yet. In our study, we used a Notch loss-of-function model in which deletion of the DNA-binding domain of RBPJ by Mx1-Cre results in the loss of signaling from all Notch receptors, in both hematopoietic cells and stromal cells of the BM microenvironment. In this model, we observed that loss of RBPJ-dependent Notch signaling in the hematopoietic microenvironment leads to a lethal myeloproliferative-like disease. Transplantation studies revealed that the microenvironment (ME) of RBPJ KO mice is necessary for the development and maintenance of the disease. Kinetics of engraftment of BM cells by intravital microscopy of the BM niche showed that WT donor cells engrafted more rapidly and had a greater expansion when transplanted into the RBPJ KO ME than in WT ME. Analysis of the serum and of supernatants from BM stroma showed increased cytokine levels in the RBPJ KO mice compared to controls, in particular G-CSF and TNF-alpha. Histological analysis of BM and spleen of RBPJ recipients showed increased microvascular density and increased CD31+ endothelial cells (EC) by IHC, preceding the onset of disease, suggesting an activated state of the endothelium. Specific deletion of RBPJ in the endothelium by using Tie2CreER model confirmed a critical contribution of EC to the myeloproliferative disease. We observed also that RBPJ KO BM-derived stroma exhibit increased expression of inflammatory microRNAs, in particular of miR155, a microRNA involved in inflammation and cancer. miR155 promoter analysis indicated the existence of two RBPJ binding sites, and direct interaction of RBPJ and the miR155 promoter was confirmed by ChIP assay. Loss of transcriptional repression by RBPJ in BM stromal cells induced miR155 upregulation and increased cytokine levels supporting greater expansion of myeloid progenitors. Double knock-out of RBPJ-/- and miR155-/- mice validated the requirement of miR155 for the hematopoietic changes due to RBPJ deletion. Finally, Analysis of patient’s samples affected by myeloproliferative neoplasia showed elevated levels of miR155 in the BM, suggesting miR-155 as a potential therapeutic target in myeloproliferative disorders.

Disclosures:

No relevant conflicts of interest to declare.
The clinical significance of STAT3 mutation, LDH and β2-MG in T-cell large granular lymphocytic Leukemia
Authors: Qiu Zhi-Yuan , Fan Lei , Wang Li , Qiao Chun , Wu Yu-Jie , Zhou Jian-Feng , Xu Wei , Li Jian-Yong
Published: October 2019

Background

T-cell large granular lymphocytic leukemia (T-LGLL) is a rare lymphoproliferative disorder of cytotoxic T cells (CTLs) and often associated with autoimmune disorders. The signal transducer and activator of transcription 3 (STAT3) is an oncogene, and its activation plays a key role in cell signaling transduction pathways in many types of cancer. The aim of the current study was to analyze the characteristics of T-LGLL.

Methods

We did this by determining the mutation status of STAT3 in 28 patients presenting with T-LGLL and evaluating STAT3 status in association with serum level of lacticdehydrogenase (LDH) and β2-microglobulin (β2-MG). Flow cytometric analysis for immunophenotype and TCR variable β-chain (Vβ) was performed in the patients.

Results

FC-Vβ analysis was performed in 26 patients, and 22 (84.6%) patients had a restricted Vβ reactivity pattern, with predominance of a single Vβ mAb reactivity. There was no significant difference between serum LDH levels, gender, age or symptoms at diagnosis (P=0.062), lymphocytosis, anemia (P=0.057), thrombocytopenia, splenomegaly, LGL count or STAT3 mutation status. However, high β2-MG levels (P=0.005), neutropenia (P=0.018) and pure red blood cell aplasia (PRCA) (P=0.001) all displayed a significant association with STAT3 mutations. In univariate analysis, treatment-free survival (TFS) was affected by STAT3 mutation status (P=0.008) and β2-MG (P=0.006). In multivariate analysis, only anemia (P<0.001) was found to be an independent risk factor of shorter TFS.

Conclusions

We found an incidence of STAT3 mutations of 21.4% in patients presenting with T-LGLL and two mutations, E616V and V671F, had not been previously reported. Our results further demonstrate the remarkable correlation of STAT3 mutation with anemia, neutropenia and β2-MG. β2-MG and LDH is commonly used laboratory indicators, and detecting of them is helpful in estimating clinical characteristics and guiding treatment. We conclude from our data that FC-Vβ analysis is a fast and relatively economical alternative approach that can be used as a powerful tool in screening patients with suspected T-LGLL. Our study not only confirmed some of the previously reported observations, but it also provided new information on clinical associations, laboratory findings, prognostic factors, therapy and outcome in these patients.

Disclosures:

No relevant conflicts of interest to declare.
Costimulation Through GITR Increases Follicular Helper T Cell Formation and Leads To Control Of A Chronic Viral Infection
Authors: Pascutti Fernanda M , Poplonski Tomasz , Van Lier René A.W. , Brandao Claudia , Nolte Martijn A.
Published: October 2019

The glucocorticoid-induced TNF receptor family-related protein (GITR) is an important costimulatory receptor on T cells. We have previously shown that enhanced costimulation through GITR increases the formation of both effector and regulatory CD4+ T cells. Here we explored whether it could also affect humoral immunity and T cell help to B cells. Although development of mature B cells was not affected in GITRL transgenic (tg) mice, we found that the number of follicular helper T cells (CXCR5+ PD1+ CD4+ T cells, Tfh) was significantly increased, including the absolute number of Tfh-B cell conjugates, as revealed by ImageStream analysis. Tfh from GITRL tg mice had normal expression levels of ICOS, SLAM and CD44 and slightly lower levels of CD62L and CCR7 compared to wild-type (WT) littermates. Interestingly, Tfh from GITRL tg mice produced more IFN-g and IL-10, which was accompanied by a biased antibody repertoire (decreased IgG3 and increased IgA, IgG2a and IgG2b). Since Tfh have been implicated in the late control of viral replication, we infected WT and GITRL tg mice with LCMV Clone 13. Surprisingly, at day 30 after infection, we could not detect viral genome in spleen and liver from GITRL tg mice, while WT mice were still infected. Also, PD-1 expression was strongly decreased on virus-specific CD8+ T cells, which correlated with faster viral clearance. All in all, these results indicate that GITR-mediated costimulation enhances the control of chronic viral infections, by boosting and modulating Tfh cell responses.

Disclosures:

No relevant conflicts of interest to declare.
A Novel and Highly Selective Dual PI3K/mTOR Kinase Inhibitor VS-5584, Shows Promising Therapeutic Potential For The Treatment Of Multiple Myeloma
Authors: Mustafa Nurulhuda , Hart Stefan , Chng Wee-Joo
Published: October 2019

Currently, most multiple myeloma (MM) patients experience relapse and develop resistance to standard treatments. A recent study showed that patients who relapsed had poor outcomes, with an overall survival of only 6 months and an event-free survival of 1 month. The PI3K/mTOR/AKT pathway represents a critical target in MM because it stimulates proliferation, survival, and drug resistance of MM cells. VS-5584 is a novel agent, with specific and equipotent activity against mTOR and all 4 Class I PI3K isoforms, without relevant activity on 400 other lipid and protein kinases. Here we report that VS-5584 is highly efficacious against a wide panel of MM cells including Velcade- and Doxorubucin- resistant cell lines. This efficacy is maintained even in the presence of additional MM growth factors, IL-6 and IGF-1, and seems independent of PTEN status in the cell lines. Importantly, VS-5584 shows similar efficacy in patient myeloma cells and preferential tumor cell targeting compared to healthy peripheral blood mononuclear cells. Further testing in a myeloma xenograft mouse model further confirmed the potency of this compound in vivo. We have also observed synergistic activity in combination with both MM clinical therapeutic Dexamethasone, and novel anti-MM candidate Panabinostat. Comparing the basal expression profile of hypersensitive (H929) vs less sensitive (OPM2, U266) cell lines have identified the interferon alpha/beta pathway as a marker for association with sensitivity. Just recently, VS-5584 has been reported to evidence very favourable pharmaceutical and pharmacological properties in a wide range of solid tumors resistant to standard care therapies. Taken together with our data, this offers a compelling rationale for its clinical development as a single or combination therapy in multiple myeloma.

Disclosures:

No relevant conflicts of interest to declare.
Modulated Cyclophosphamide-Based In Vivo T-Cell Depletion Promotes Engraftment With Minimal Gvhd and Low Toxicity In Fanconi Anemia Patients
Authors: Thakar Monica S , Walters Mark C. , Sandmaier Brenda M. , Storb Rainer , D. Flowers Mary E. , Deeg H. Joachim , O'Donnell Paul , Woolfrey Ann E. , Kiem Hans-Peter
Published: October 2019

Building on a successful non-myeloablative conditioning regimen developed in Seattle (Blood 2003), Luznik and O´Donnell et al created a protocol that incorporates post-transplant cyclophosphamide (CY) after human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT) (BBMT 2008). This method both promotes engraftment while selectively-depleting alloreactive donor T cells to prevent graft-versus-host disease (GVHD). We have previously shown that Fanconi Anemia (FA) patients can be treated with CY 60 mg/kg in a conditioning regimen with minimal toxicity (BBMT 2007), thus we adapted this post-HCT CY strategy for in vivo T-cell depletion in patients with FA. Between 2008 and 2012, four patients from three North American centers with FA and severe marrow failure in the absence of HLA-matched donors underwent HLA-haploidentical HCT. All four patients were referred for transplantation with minimal to no transfusion burden and all were in excellent clinical condition with HCT-CI scores of 0-2 and Lansky scores of 90-100%. Median age at transplant was 9.7 (6.9-11.9) years old. Patients were transplanted at a median of 1.6 (range, 0.6 -7.1) years after FA diagnosis. Conditioning consisted of fludarabine (150 mg/m2) and 2 Gy total body irradiation; one patient also received CY (10 mg/kg), which was deleted in subsequent patients to decrease the risk of mucositis. Marrow was infused on day 0, followed by post-grafting immunosuppression with CY (25 mg/kg/day, days +3, +4), mycophenolate mofetil, and cyclosporine, the latter two beginning at day +5 with plans to continue until days +35 and +180, respectively. Full donor engraftment was seen in all patients. Two patients developed acute grade I GVHD and none of the four patients has developed chronic extensive GVHD to date. With a follow-up of 5 years, 1 year, 11 months, and 9 months, all four patients are alive with stable, full donor chimerism, and are transfusion independent. While two patients required cyclosporine beyond day +180, only one patient currently remains on low-dose immunosuppression for treatment of limited chronic skin GVHD, which has now resolved. Our results confirm that modulated post-HCT CY can be used in patients with FA to promote engraftment across histocompatibility barriers. Despite concerns for both excessive toxicity related to CY and severe GVHD related to minimizing the dose of post-transplant CY, none of the FA patients in our small series experienced these problems. Our findings also suggest that transplant should not be delayed when there is lack of an HLA-matched donor. FA patients with few comorbidities and minimal transfusion burden can successfully undergo this HLA-haploidentical HCT approach.

Disclosures:

Off Label Use: MMF.
Association Of Chemokine Receptor CCR5, CCR6 and CCR7 Expressions On T Lymphocyte Subsets In Recipients After Allo-HSCT With Acute GvHD
Authors: Wang Meng , Ren Han-Yun , Dong Yu-Jun , Liang Ze-Yin , Qiu Zhi-Xiang , Liu Wei , Wang Li-Hong
Published: October 2019

Introduction

To study the correlation of chemokine receptors expression on T lymphocyte subsets in recipients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with acute graft-versus-host disease (acute GVHD).

Methods

Forty-four recipients of family donor allogeneic hematopoietic stem cell transplantation were included in this study. According to the severity of acute GVHD, the recipients were divided into Grade II-IV acute GVHD group (n=16) and Grade 0-I acute GVHD group (n=28). Additionally,fifty healthy donors were also included in this study as the control group (n=50). The expression of chemokine receptors (CCR2,CCR5,CCR6,CCR7,CCR9 and CXCR3) on CD4+ and CD8+T cells in the peripheral blood after transplantation were detected using flow cytometry (FCM) respectively. The differences of the chemokine receptors expression between each group were compared.

Results

In both Grade 0-I acute GVHD group (on the 30th day after transplantation) and Grade II-IV acute GVHD group (at the peak of GVHD), CCR5 expression was significantly higher than that in the control group (p<0.01), and the expression of CCR7 was significantly lower than that in the control group (p<0.01). However, compared with the Grade 0-I acute GVHD group, the Grade II-IV acute GVHD group showed lower CD4+/CD8+ cell ratio (0.36 ± 0.30 vs 0.76 ± 0.61, p=0.003), higher CD4+CCR5+ ratio (65.20 ± 20.17% vs 50.82 ± 28.59%, p=0.048), decreased expression of CCR6 and CCR7 on CD4+ T cells (17.02 ± 12.43% vs 34.69 ± 18.58%, p=0.006; 20.28 ± 4.37% vs 25.45 ± 10.47%, p=0.025).

Conclusions

Chemokine receptors on T cell subsets are involved in the pathogenesis of acute GVHD. Expression of chemokine receptors can be used as a powerful marker for early diagnosis and differential diagnosis of acute GVHD. And targeting chemokine receptors might be the new direction of prevention and treatment of acute GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Higher Counts Of Plasmacytoid Dendritic Cells In The Allograft Are Associated Increased Risk Of Acute Gvhd After Stem Cell Transplantation
Authors: Vescovi Gonçalves Matheus , Yamamoto Mihoko , Kimura Eliza Y. S. , Renzi Colturato Vergilio Antonio , Ikoma Maura Valerio , Mauad Marcos , Pedro de Souza Mair , Alvarenga Gomes de Oliveira Paulo Guilherme , Vaz Carvalho Ana Paula , Kerbauy Fabio R. , Hamerschlak Nelson , Novis Yana , Ginani Valeria Cortez , Seber Adriana , Rocha Vanderson , Orfao Alberto , Rodrigues Celso Arrais
Published: October 2019

Background

Recent studies suggest that higher circulating dendritic cell counts after hematopoietic stem cell transplantation (HSCT) may be associated with lower risk of graft versus host disease (GVHD) and mortality. Plasmacytoid dendritic cells (pDC) have tolerogenic properties and may explain such results. However, only a few studies with conflicting results analyzed pDC counts in the allograft, mainly in the related HSCT setting.

Objectives

To compare pDC counts among different cell sources [umbilical cord blood (UCB), bone marrow (BM), and peripheral blood (PBSC)] and to correlate the pDC content on the graft with main HSCT outcomes.

Methods

Plasmacytoid dendritic cells (pDC: lineage negative, HLA-DR+ and CD123+) were quantified by multiparametric flow cytometry in the graft just before its infusion. Overall, 77 patients (49 male; median age 21y, range 1-74y) receiving UCB (n=26), BM (n=34) or PBSC (n=17) HSCT from unrelated (n=67) or related donors (n=10) were studied. The most common diagnosis was acute leukemia (ALL, 30 cases; AML, 22). Most patients received myeloablative conditioning regimens (n=47, 61%). Antithymocyte globulin was used in 30 patients (39%) and total body irradiation in 41 (53%). Median follow up time was 15 months (4-33).

Results

Median time to neutrophil engraftment was 19 days (range: 11-49) and 35 days to platelet engraftment (range 2-176). The median percentage of pDC on the graft was 0.20% of non erythroid nucleated cells (range: 0.02-0.67) and no differences were noticed among sources. Cumulative incidence (CI) of grade II-IV acute GVHD at 100 days was higher on patients receiving grafts with higher percentages of pDC (22% for patients with less than 0.15%pDC graft content vs. 52% for patients receiving grafts with higher counts, p=0.026). There was no impact of graft pDC content on mortality, relapse, chronic GVHD or overall survival. In a multivariate analysis, graft pDC content remains an independent risk factor for acute GVHD [HR: 3.0, CI (95%): 1.15-7.8].

Conclusion

Higher pDC graft percentages were associated with increased risk of acute GVHD, but had no impact on non-relapse mortality or survival. The precise role of pDC on immunity after HSCT deserves further investigation and might be related not only to pDC biology itself but to the chronology of the pDC interaction with host antigens.

Disclosures:

No relevant conflicts of interest to declare.
Erythrocytosis and Normal Erythropoietin Levels In a 3 Year Old Female With High-Risk Neuroblastoma
Authors: Barr Erin Kelly , Ross Mary Elizabeth , Al-Rahawan Mohamad M.
Published: October 2019

Introduction

Erythrocytosis has been reported in a number of cancers as a result of tumor production of Erythropoietin (EPO). There has been one reported case that we are aware of in a pediatric patient with neuroblastoma (Wang LY, et al. J Pediatr Hematol Oncol. 2003 Aug;25(8):649-50). Erythrocytosis in a pediatric neuroblasotoma patient with normal EPO levels, which is the subject of this case report, has not been reported previously.

Case

A three year-old female presented with a right sided abdominal pain of one week duration as well as constipation and decreased appetite of one month duration. Radiology studies showed a large abdominal mass, which turned out to be a poorly differentiated high-risk neuroblastoma on an incisional biopsy. For the first three months of this patient’s therapy and despite intense chemotherapy she was noted to have abnormally high hemoglobin levels. The peak hemoglobin level was 18 g/dL but she maintained hemoglobin levels greater than 14g/dL, which was greater than the 97th percentile for her age group (Dallman PR, et al. J Pediatr. 1979 Jan;94(1):26-31). Testing for EPO when the hemoglobin level was elevated showed a level of 8.1 mIU/ml, which was within the normal range for the patient’s age group (Krafte-Jacobs B, et al. J Pediatr. 1995 Apr;126(4):601-3). After tumor resection but before the conclusion of the myleosuppressive chemotherapy, hemoglobin levels fell below 10 for the majority of time.

Discussion

Although there has been a case reported of a pediatric patient with neuroblastoma having erythrocytosis, this is the first case where the patient was actually found to have normal EPO levels. Case reports looking at EPO and erythrocytosis in children with Wilms tumors have reported a couple cases of children who had normal EPO levels (Lal A, et al. J Pediatr Hematol Oncol. 1997 May-Jun;19(3):263-5). This report showed no recurrence of polycythemia when the tumors were removed in these patients. The authors concluded that tumor or surrounding renal tissue was at some point producing EPO despite their inability to detect it.

Conclusion

We speculate that neuroblastoma tumors and possibly other pediatric tumors may be releasing a novel erythropoietic cytokine. Hormone-like cytokines have been reported with other cancers, such as hypercalcemia produced by PTH related peptide in breast and lung cancers. More research is needed to determine what, if any, novel EPO-like hormones are being released by neuroblastoma cells resulting in erythrocytosis despite normal EPO levels.

Disclosures:

No relevant conflicts of interest to declare.