bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Red Cell Phosphatidylserine Exposure Correlates with Enhanced Thrombin Generation In Sickle Trait Patients with End Stage Renal Disease
Authors: Brittain Julia , Derebail Vimal K. , Mooberry Micah J. , Ataga Kenneth I. , Kshirsagar V. Abhijit , Key Nigel S.
Published: October 2019

Abstract 2665

Introduction:

Phosphatidylserine (PS) exposure by red blood cells (RBCs) is increased in sickle cell disease, where it is thought to contribute to anemia, inflammation and activation of coagulation. RBCs from subjects with sickle cell trait (SCT) may also express more PS compared to controls. We recently reported a high prevalence of SCT in African Americans with end-stage renal disease (ESRD) - a state marked by anemia, profound inflammation and coagulation activation. No study has yet examined RBC PS exposure, inflammation or coagulation activation in African Americans patients with SCT and ESRD.

Objective:

To determine whether RBC PS exposure is elevated in ESRD patients with SCT receiving hemodialysis compared to patients with normal hemoglobin genotype, and whether markers of coagulation activation and inflammation are similarly elevated. The relationship between RBC PS exposure, thrombin generation and inflammation was also examined.

Methods:

We performed a cross sectional study of 10 patients with documented SCT and 9 race-matched controls. Both groups were receiving in center hemodialysis. The percentage of RBCs demonstrating PS exposure was determined by annexin-V binding and flow cytometry detection. Plasma thrombin/anti-thrombin (TAT) complexes and C-reactive protein (CRP) levels were determined by ELISA. Mann-Whitney U tests were used to examine differences between these variables among the groups. Medians and interquartile ranges (IQR) are shown. Spearman's correlation coefficient was used to assess the relationship among the variables tested.

Results:

Consistent with previous reports in ESRD patients, we found markedly elevated RBC PS exposure among both groups; however, patients with SCT had significantly elevated PS exposure compared to those without SCT (6.65%, [IQR: 5.1, 8.8] vs. 3.0%, [IQR: 2.1,4.2], p = 0.002). Furthermore, patients with SCT had higher TAT levels compared to those without SCT (16.1 ng/L [IQR: 11.9, 28.4] vs. 5.5 ng/L [IQR: 3.8, 8.6], p<0.0001). We also noted increased plasma CRP levels in those patients with SCT compared to control (33.7 mg/L [IQR: 22.8, 62.8] vs. 11.2 mg/L [IQR: 2.2, 19.5], p = 0.016). In patients with SCT, there was a striking correlation between PS exposure and TAT levels (r = 0.897, p<0.0001), but no correlation was noted between PS exposure and levels of CRP (r=-0.17, p = 0.63).

Conclusions:

These data suggest that patients with SCT trait in ESRD may be subject to an increased risk of thrombotic complications related to increased PS exposure on RBCs.

Disclosures:

No relevant conflicts of interest to declare.
HLA Class I and EBV Positive Classical Hodgkin Lymphoma In the Chinese Population
Authors: Xin Huang , Diepstra Arjan , Gao Zifen , Visser Lydia , Vos Hans , Kooistra Wierd , Poppema Sibrand , Hepkema Bouke , Van Den Berg Anke
Published: October 2019

Abstract 2688

In Caucasian populations, the tumor cells of Epstein Barr virus (EBV)-positive classical Hodgkin Lymphomas (cHL) patients more frequently express HLA class I and HLA class II molecules compared to EBV-negative cHL patients. Since HRS cells also express other components of the antigen presenting pathway it can be anticipated that HRS cells in EBV-positive cHL should be able to present EBV derived antigenic peptides. In on our previous genotyping study we demonstrated that the HLA-A*02 allele was associated with a reduced and the HLA-A*01 allele with an increased risk of developing EBV-positive cHL. Intuitively, this can be explained by the generally acknowledged lack of HLA-A*01 restricted immune responses to latent EBV peptides. In this study, we analyzed HLA class I expression and the HLA association in relation to EBV in Asian cHL patients.

Formalin-fixed and paraffin embedded (FFPE) tissue blocks were available for 145 cHL patients and from 79 controls from 5 hospitals from the Northern part of China. Hematoxylin & Eosin-stained sections were used to reclassify the histological subtypes according to the WHO classification. EBV status was determined by EBER in situ hybridization. Membranous expression of HLA class I was detected by immunohistochemistry using antibodies against HLA class I (HC-10) and ß2-microglobulin. DNA was isolated from FFPE samples to detect the HLA-A*02 allele by quantitative PCR. 23 cHL cases and 5 controls were excluded due to poor DNA quality.

Positive EBV status was observed in 40% (58/145) of the Chinese cHL patients. As expected, the percentage of EBV-positive cases was much higher in the mixed cellularity subtype (71%) than in the nodular sclerosis subtype (16%; p<0.001). Expression of HLA class I was observed in 79% of the EBV-positive cHL patients and in 30% of the EBV-negative patients (p<0.001) consistent with the previous findings in the Dutch population. The HLA-A*02 allele was detected in 71% of the EBV-positive cHL and in 64% of the EBV-negative cHL patients (NS). In the controls HLA-A*02 was observed in 67% of the cases.

In this Chinese population, the tumor cells of EBV-positive cHL more frequently retained membranous HLA class I expression, similar to the Caucasian populations. The inverse correlation between presence of the HLA-A*02 allele and positive EBV status, as observed in Caucasians, is not present in these northern Chinese cHL patients. Differences in ethnic background might explain discrepancies in HLA-A association with EBV-positive cHL in different populations.

Disclosures:

No relevant conflicts of interest to declare.
Interleukin-2 Enhances Angiogenesis and Preserves Cardiac Function Following Myocardial infarction
Authors: Bouchentouf Manaf , Williams Patrick , Forner Kathy-Ann , Cuerquis Jessica , Boulassel Mohamed Rachid , Paradis Pierre , Routy Jean Pierre , Schiffrin Ernesto , Galipeau Jacques
Published: October 2019

Abstract 2786

Previous studies demonstrated that the addition of some cytokines improved cardiac recovery in experimental models of myocardial infarction (MI). The aim of the study is to test the hypothesis that pharmacological stimulation of lymphoid cells using recombinant human interleukin (rhIL)-2 improves angiogenesis and preserves heart function after MI. A single IV injection of rhIL-2 two days following MI improved by 27.7% the left ventricular (LV) fractional shortening of immune competent (C57Bl6) mice, but had no effect on cardiac function of immune-deficient (NOD SCID IL2Rγnull) mice. Immunohistochemical analysis of C57Bl6 cross sections of heart revealed that collagen deposition was reduced by 23.1% and that capillary density was enhanced in the scar area and the border zone of the infarct respectively by 22.4% and 33.6% following rhIL-2 injection. In addition, rhIL-2 enhanced 1.6-fold the in vivo endothelial cell proliferation index and 1.8-fold the number of NK cell infiltrating the infarcted heart, but had no effect on the number of cardiac CD4 and CD8 cells. In vitro, rhIL-2 activated NK cells enhanced cardiac endothelial cell proliferation by 17.2%. Adoptive transfer of IL-2-activated NK cells enhanced cardiac angiogenesis. Here we show that a single IV injection of rhIL-2 positively impacted cardiac function by improving angiogenesis through a process involving NK cells.

Disclosure:

No relevant conflicts of interest to declare.
The Novel JAK2 Inhibitor NVP-BSK805 Has Cytotoxic Activity on Malignant Plasma Cells
Authors: Burger Renate , Rademacher Franziska , Staudinger Matthias , Peipp Matthias , Güunther Andreas , Gramatzki Martin
Published: October 2019

Abstract 2993

In multiple myeloma (MM) and plasma cell leukemia, activation of the JAK/STAT pathway is induced by interleukin (IL)-6, which is produced and secreted into the tumor microenvironment primarily by stromal cells. Upon binding of IL-6 to its specific alpha-chain receptor, dimerization of the gp130 signaling subunits leads to activation of associated JAK kinases and STAT transcription factors. In particular, STAT3 has been shown to be essential for myeloma cell growth and survival. NVP-BSK805 (Novartis) is a novel substituted quinoxaline JAK2 inhibitor tool compound which displays more than 20-fold selectivity for JAK2 over the other JAK family members and more than 100-fold selectivity over a panel of additional kinases (Baffert et al., Mol Cancer Ther 9:1945, 2010). The study presented here aims at growth inhibitory effects of NVP-BSK805 in malignant plasma cells. NVP-BSK805 inhibited the growth of six human myeloma cell lines displaying dose-dependent activity with IC50 concentrations between 2.6 μ mol/L and 6.8 μ mol/L. Among the cell lines, IL-6 dependent INA-6 cells were most sensitive to the inhibitory effects of the compound: both IL-6 and bone marrow stromal cell induced proliferation as measured by [3H]-thymidine uptake was completely inhibited at 4 μ mol/L and IC50 concentrations were less than 1 μ mol/L. Viability of the stromal cells was not significantly affected. NVP-BSK805 concentrations as low as 0.5 μ mol/L were sufficient to yield a marked reduction of IL-6 induced STAT3 phosphorylation and complete abrogation at 2 μ mol/L, thereby blocking essential survival signals. Accordingly, treatment of INA-6 cells with NVP-BSK805 for 48 hours led to significant apoptosis starting at 2 μ mol/L with a 30% increase in annexin V-positive cell numbers compared to DMSO controls. Importantly, NVP-BSK805 showed potent cytotoxic activity on plasma cell-enriched primary tumor samples from patients with extramedullary plasma cell disease that are highly responsive to IL-6: in 3 out of 4 tumor samples the IC50 concentrations were between 0.5 μ mol/L and 0.6 μ mol/L. These studies are extended to combinations of NVP-BSK805 with PI3K, mToR, MAPK, HDAC, and IGF-1R inhibitors in order to optimize targeted therapy strategies facing different pathway alterations in individual myeloma patients. In INA-6 cells, synergistic activity was found combining NVP-BSK805 with rapamycin and the MEK1 inhibitor U0126. Preclinical in vivo studies are ongoing. Our results with NVP-BSK805 substantiate the use of JAK inhibitors as a therapeutic strategy for patients with MM. Since results from studies with pan-JAK inhibitors such as pyridone 6 indicate involvement of additional JAK kinases, the choice of the optimal compound will depend on its JAK family selectivity and the biology of JAK signaling in MM.

Disclosures:

No relevant conflicts of interest to declare.
Identification of a Novel T-ALL Entity with NKX2-1/NKX2-2 Rearrangements
Authors: Homminga Irene , Pieters Rob , Langerak Anton , de Rooi Johan , Stubbs Andrew , Verstegen Monique , Vuerhard Maartje , Buijs-Gladdines Jessica , Kooi Clarissa , Klous Petra , Van lierberghe Pieter , Ferrando Adolfo , Cayuela Jean-Michel , Verhaaf Brenda , Beverloo Berna , Horstmann Martin A. , de Haas Valerie , Wiekmeijer Anna-Sophia , Pike-Overzet Karin , Staal Frank , de Laat Wouter , Soulier Jean , Sigaux Francois , Meijerink Jules P.P.
Published: October 2019

Abstract 3139

To identify novel oncogenic pathways in T-cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular cytogenetic analyses. Using unsupervised and supervised analyses, we identified a novel T-ALL entity that was associated with high expression of cell cycle genes and was accordingly denoted as the proliferative cluster. Patient samples in this cluster were devoid of known oncogenic rearrangements, and clustered together with TLX1-rearranged cases in unsupervised cluster analysis suggesting pathogenic mechanisms are conserved between these cases. Alike TLX1-rearranged cases, patients within this cluster were associated with cortical thymic arrest and expressed CD1 along with CD4 and CD8. Bioinformatic analyses identified NKX2-1 as strong outlier gene that was ectopically expressed in most patients samples belonging to this cluster. One patient sample had high expression of the homologous NKX2-2 gene. Molecular-cytogenetic analyses including the Chromatine Conformation Capture on Chip (4C) method revealed various rearrangement variants of the NKX2-1 locus at 14q13 in 6 patients, including inversions to the T-cell receptor alpha/delta locus (TRAD@) at 14q11, an inversion to the IgH@ locus at 14q32 and a translocation to the TRB@ locus at 7q34. A translocation of NKX2-2 to the TRAD@ locus was identified in the case that ectopically expressed NKX2-2. All these rearrangements were novel, and have not been observed before in human cancer. NKX2-1 rearrangements could be validated in an independent T-ALL cohort. Our data strongly imply that NKX2-1/NKX2-2 represent novel oncogenes for human T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Chromosomal Abnormalities in Bone Marrow Lymphocytes of MM Patients
Authors: Marun Carina S. Debes , Belch Andrew , Pilarski Linda M.
Published: October 2019

Abstract 4045

Multiple Myeloma (MM) is characterized by accumulations of chromosomal abnormalities throughout the course of disease. Recurrent IgH translocations such as t(4;14) or deletion (del)13 occur during early stages of disease or perhaps as originating events. Other abnormalities appear to be acquired late in the disease process, after development of frank MM, including p53 deletion (Ch17) and amplification of CKS-1B (Ch1). We have previously identified drug resistant clonotypic B lymphocytes in MM that have malignant characteristics and can xenograft MM to immunodeficient mice.

Using an automatic scanning system we scanned bone marrow (BM) cytospin slides stained with May-Grünwald Giemsa to identify lymphocytes and to determine whether or not they share the same chromosomal abnormalities that are found in autologous plasma cells (PC). For 200 MM patients, we performed interphase FISH using commercial probes, to detect deletion of chromosome 13, any translocations in 14q32, t(4;14)(p16;q32),t(11;14)(q13;q32), deletion of p53 and a custom probe to detect amplification of CKS-1B. For patients harboring a given chromosomal abnormality in their PC, we found the identical abnormalities in BM lymphocytes for 35% of patients having del13, 30% having t(4;14) and 23% having t(11;14). Likewise we detected amplification of locus 4p16, 11q13, 14q32, trisomy 1 and trisomy 17 in 45%, 15%, 30%, 37.5% and 44% respectively in the lymphocytes of patients demonstrating those same abnormalities in the PC. In a more limited series of experiments using immuno-FISH, we observed that BM lymphocytes with chromosomal abnormalities expressed CD20. In contrast, amplification of CKS-1B or p53deletion were undetectable in lymphocytes from patients having these abnormalities in their PC. We are currently analyzing the impact of abnormalities present in BM lymphocytes with regards to the clinical parameters and survival. An update of these results will be given at the meeting. Given the compelling evidence that B cells comprise a clinically important compartment of the MM clone, this work suggests that fundamental genetic events shared by all compartments of the MM clone include recurrent IgH translocations and Ch13 deletions. However, further clonal expansion occurring during disease progression and after relapse may be restricted to the PC population, characterized by acquisition of p53 deletions and CSK1B amplification. Furthermore in overt MM, clonal expansion may simultaneously occur on at least two levels, firstly that of the B cells which persistently give rise to PC, and secondly that of PC themselves as they continue clonal expansion in the absence of contributions from the B cells. Abnormalities such as del13 or IgH translocations characterize early events in the disease while p53del or amplification of CKS-1B may occur as progression events restricted to PC.

Disclosures:

No relevant conflicts of interest to declare.
Feasibility and Efficacy of ABVD In Elderly Hodgkin Lymphoma Patients: Analysis of Two Randomized Prospective Multicenter Trials of the German Hodgkin Study Group (HD10 and HD11)
Authors: Böll Boris , Görgen Helen , Fuchs Michael , von Tresckow Bastian , Engert Andreas , Borchmann Peter
Published: October 2019

Abstract 418

About 20% of all patients with first diagnosis of Hodgkin Lymphoma (HL) are older than 60 years. They have a poor prognosis mainly due to an increased toxicity of chemo- and radiotherapy. Although resulting in better disease control, aggressive regimens as BEACOPP are not feasible in this cohort of patients due to a treatment related mortality of up to 20%. Therefore, ABVD is considered treatment of choice for elderly HL patients, although prospective studies are lacking and current concepts mostly rely on cohort analyses. We therefore analyzed feasibility, safety and outcome of patients older than 60 years with early favorable- or early unfavorable-stage HL treated with 4 cycles of ABVD within the HD10 and HD11 trials of the GHSG. Sixty-eight and 49 elderly patients with a median age of 65 and 64 years were treated in HD10 and HD11, respectively. Early termination of protocol therapy was documented in 18% of HD10 patients, but only in 8% of HD11 patients, resulting in a lower relative total chemotherapy dose (RCD) in HD10. The relative dose intensity (RCD divided by total relative chemotherapy duration) was much lower in both studies compared to younger patients, due to more toxicity-related therapy delays and dose reductions, as 4 cycles of ABVD caused WHO grade III/IV toxicities in 67% (HD10) and 69% (HD11). Overall efficacy was significantly lower than in younger patients with an overall response rate of 90% in HD10 and 92% in HD11. The rate of relapsing patients was the same as in younger patients in HD11 (14%), whereas in HD10 it was much higher in the elderly (12%) which was mainly due to late relapses. Overall 22% and 37% of the patients died in HD10 and HD11, respectively (median observation time: 92 months). Besides other causes as cardiovascular disease (7%) or secondary neoplasia (5%), there was a high rate of deaths due to insufficient HL-control (5%) and treatment-related toxicities (5%). The 5-year PFS estimates for elderly patients were 79% (95% CI, 67% to 87%) in HD10 and 69% (95% CI, 54% to 80%) in HD11 compared to 96% (95% CI, 93% to 97%) and 86% (95% CI, 83% to 88%) for younger patients in HD10 and HD11, respectively. In conclusion, 4 cycles of ABVD are effective in elderly HL patients; however, treatment-related toxicity is high.

Disclosures:

No relevant conflicts of interest to declare.
Effect of CMX001 on Levels of DNA Virus In Patients After Allogeneic Stem Cell Transplantation.
Authors: Gabriel Don A. , Whitley Julia , Rao Kamakshi , Horst Charles van der , Serody Jonathan , Coghill James , Sarantopoulos Stefanie , Armistead Paul , Wood William , Shea Thomas C.
Published: October 2019

Abstract 4550

Latent viral infections occurring in the post bone marrow transplant period remain a significant clinical problem. New and effective antiviral drugs (AVD) are needed to provide adequate therapy for infected patients. We report the use of a new oral AVD, CMX001 (Chimerix Inc. Durham, NC) to treat DNA virus infections in six severely ill patients.Pt #123456Adeno Blood (PCR Cycles)Pre: 30.6Pre: 33.1Post: 37.3Post: NegBK polyoma virus Urine (log copies/mL)Pre: 4.89Pre: 4.86Pre: 6.54Pre: 9.06Post: 7.88Post: 5.99Post: 7.55Post: 7.16BKV BloodCleared after 1 dosePre: No valueCleared at 6 doses;Post: detectableHHV6 Blood (PCR Cycles)Pre: 42Post: 39.9Dose (mg/kg) biw22444 reduced to 32StatusDied GVHDDied GVHDDied GVHD + SepsisDied GVHDAliveAlive

All patients had grade 2 or greater gut graft versus host disease (GVHD) during or after CMX001 treatment. The Table shows viral levels before and after treatment with CMX001 twice/wk. Pt 1's AV cycle number fluctuated and never cleared. A higher cycle number indicates less virus. Pt 2 cleared AV from blood after first dose, but urine, stool, gut biopsies remained AV positive. Pt 3's HHV6 plasma PCR levels fluctuated, but the pericardial fluid levels had cleared at autopsy. Pt 4 did not have pre dosing blood BKV PCR levels taken and his subsequent blood BKV low level was measurable (below the positive limit level). Interestingly, however, the patient's BKV related hemorrhagic cystitis (HC) symptoms resolved after 4 doses of CMX001. Pt 5's BKV HC symptoms also resolved after 4 doses of drug, and his positive blood viral level cleared after 6 doses. Pt 6 discontinued drug because of abdominal pain and the need for NPO, but his hemorrhagic cystitis symptoms had abated. No specific toxicities were documented in these severely ill patients with gut GVHD, but gut pain and transient thrombocytopenia were observed. In summary, one of 2 pts with AV cleared their blood. BKV was not cleared from urine, but 2/4 cleared BKV from blood. One patient with HHV6 did not clear blood DNA viral levels, but did clear pericardial fluid. In both cases of HC, symptoms resolved. Thus, CMX001 shows promise as new antiviral drug given its ability to decrease DNA viral levels in this small group of critically ill patients.

Disclosures:

No relevant conflicts of interest to declare.
Cytogenetic Abnormalities In the Spleen In CLL Patients.
Authors: Norin Stefan , Wallblom Ann , Aggarwal Mohit , Sander Birgitta , Kimby Eva
Published: October 2019

Abstract 4618

Background:

In CLL the prognostic importance of cytogenetic abnormalities in bone marrow or peripheral blood is well-known. However data is lacking on the frequency and importance of the aberrations in other tissues such as lymph nodes and spleens.

Aims:

To develop a method to assess the chromosomal aberrations commonly seen in CLL (13q-, 11q-, 17p- and +12) on paraffin-embedded tissue sections from spleens and to relate the findings to data on abnormalities found in bone marrow or peripheral blood.

Material and methods:

24 out of 61 CLL patients splenectomized between 1990–2009 have so far been analyzed. Paraffin-embedded sections were obtained from stored tissue material from spleen. Fluorescence in situ hybridization (FISH) was used to detect cytogenetic abnormalities and the results were compared with analyses made on blood or bone marrow. Monosomy 12, an abnormality rarely occurring in CLL was used as a surrogate marker to avoid false-positive results for deletions due to incomplete nuclei. In all but one case this was below 20%. Therefore the cut-off for the deletions (13q-, 11q- and 17p-) were set to 30%. For +12 we used 5%. Data on flow cytometry for quantification of the CLL clone in the spleen was available in 13 patients with a median of 71% clonal cells (range 11–86%). Indications for splenectomy were splenomegaly (n=13), AIHA (n=8) or ITP (n=3). All but five patients had received chemotherapy before splenectomy including purine analogs (n=7), alemtuzumab (n=1) or both (n=2) The remaining patients were treated with alkylators and/or anthracycline-containg regimens.

Result:

In all but one patient, FISH results from the splenic sections could be obtained with cytogenetic aberrations detected in 18 of 23 patients (78%). In 11 patients a single abnormality was detected, whereas multiple aberrations were present in 7 cases. The most common aberration was 13q- (70%), followed by 17p- (22%), 11q- and +12 (17% each) (Table 1). In 8 cases FISH samples from blood/bone marrow were available from time points both pre- and post- splenectomy and 6 additional cases had FISH data before splenectomy. All FISH abnormalities affecting >20% CLL cells in blood or bone marrow were also found in the spleen. In contrast an additional 17p clone was found in one patient and in two patients the 13q- and 17p- clones were significantly larger in the spleen. Remarkably one patient had both homo- and heterozygous 13q- clones in blood and in the spleen. In repeated blood samples nine years after splenectomy only a heterozygous clone remained.

Conclusion:

FISH on paraffin-embedded sections is a useful tool for the evaluation of genetic abnormalities. Clonal evolution does appear to occur in the spleen in CLL and with splenectomy selected clones might be obliterated.

Disclosures:

Kimby: Roche, Bayer-Schering, Mundipharma: Membership on an entity's Board of Directors or advisory committees, lecturer.
Can Human Umbilical Cord Mesenchymal Stem Cells Inhibit Acute Graft-Versus-Host-Disease In Murine Allogeneic Bone Marrow Transplantation?
Authors: Guo Juan , Li Xiao , Chang Chunkang
Published: October 2019

Abstract 4703

Objective:

The purpose of this study was to investigate the therapeutic potential of Human umbilical cord mesenchymal stem cells (HUCMSCs) from normal donors for preventing acute GVHD following allogeneic bone marrow transplantation (BMT).

Methods:

In this study, a new method of separating and culturing HUCMSCs was used. The human umbilical cord tissue was digested by collagenase II for 2h. And after being passaged to P3, more HUCMSCs could be obtained. In the control group, after being lethally irradiated, DBA/2(H-2Kd) mice were transferred bone marrow (BM) and splenocytes (SC) from C57BL/6 (H-2Kb) mice. Recipients in the HUCMSCs-treated group were adoptive transferred HUCMSCs, BM and SC at day 0 and were transferred only HUCMSCs at day 3 again. The two groups were compared in weight, survival, histopathological specimens, lymphocyte subgroups and serum cytokine analysis.

Results:

In vitro, IFN-γ treated HUCMSCs have a strong inhibitory effect on the proliferation of CD4 + T cells (P<0.05), but untreated HUCMSCs cannot obviously inhibit the proliferation of CD4 + T cells (P>0.05). IDO is involved in the immunosuppression of HUCMSCs. In the HUCMSCs-treated group, 50% of the mice survived over 30 days after BMT, but in the control group all mice died within 18 days. The mice treated with HUMSCs exhibited light symptoms of aGVHD after day 14. There were higher IFN-γ and IL-12 levels by day 7 in serum of mice that received HUCMSCs compared to those without HUCMSCs-treatment, while the IL-4 levels showed no significant difference between the two groups. The proportion of CD3+NK1.1+NKT cells in splenocytes of HUCMSCs-treated group is higher in day 21 compared with in day 7.

Conclusion:

Using the new method of digesting human umbilical cord tissue by collagenase II for 2h, we obtained stable and pure HUCMSCs. IDO is involved in the immunosuppression of HUCMSCs. After the xenogeneic transplantation of HUCMSCs to the aGVHD model of mice, HUCMSCs can regulate cytokines such as IFN-γ, IL-12 to increase CD3+NK1.1+NKT cells.

Disclosures:

No relevant conflicts of interest to declare.
An Open-Label, Single Ascending Dose Phase I Study of F-627, a G-CSF Dimer, In Healthy Male Subjects
Authors: Yan Xiao Qiang , Hodsman Peter G , Huang Zhi Hua , Sun Rou Yun , Sun Nai Chau , Huang Yu Liang
Published: October 2019

Abstract 4722

The management of severe neutropenia in cancer patients after high-dose chemotherapy remains a challenge, although recombinant human G-CSFs including filgrastim, lengogratim and pegfilgrastim have been widely used. We developed F-627, a mammalian expressed recombinant human G-CSF dimer to treat severe neutropenia. In a cyclophosphamide-induced neutropenia monkey model, F-627 significantly reduced the duration and lessened the severity of neutropenia compared to monkeys treated with G-CSF or pegylated G-CSF. We report the phase I results of F-627 in healthy male subjects receiving ascending single dose at 30, 60, 120 and 240 ug/kg by subcutaneous injection. The primary objective of the phase I study was to assess the safety and tolerability of F-627. The secondary objective was to evaluate the pharmacokinetics and pharmacodynamics (PK/PD) responses. F-627 was well tolerated in healthy male volunteers. Dose-dependent PD responses including increased WBC, ANC and CD34 levels in peripheral blood were demonstrated. These results indicate that the mechanism of action of F-627 is similar to the monomer recombinant hG-CSFs, while the mode of action of F-627 is different with unique PK/PD properties in human.

Disclosures:

No relevant conflicts of interest to declare.
Heart Function Disorders In Sickle Cell Disease
Authors: Heinen Charlotte , Fijnvandraat Karin , Peters Marjolein , Heijboer Henriette , Blom Nico , Kuipers Irene
Published: October 2019

Abstract 4815

Abstract

Background:

Sickle cell disease (SCD) is a hereditary anemia characterized by chronic hemolytic anemia and vaso-occlusive episodes, associated with a high susceptibility for infections and damage to all vital organs. Cardiologic effects of SCD are dilatation and hypertrophy of the left ventricle and pulmonary hypertension. The prevalence of these cardiac complications in children with SCD living in the Netherlands is unknown. The role of hemolysis in this process is still unclear. Therefore, the objectives of this study are: 1) to evaluate the prevalence of cardiac abnormalities in children with SCD living in the Netherlands, 2) to analyse the association between cardiac abnormalities and the severity of anemia and hemolysis.

Methods:

Cross-sectional study, including 92 children with SCD (genotype HBSS, HBSC or HBS-beta 0) treated at the Emma Children's Hospital AMC. None of the children had cardiac abnormalities unrelated to SCD. Echocardiograms and electrocardiograms were performed in a stable state. All cardiac values were corrected for BSA. Echocardiographic values 20% above or under the average of an age matched control group were considered abnormal. Laboratory hematological parameters were obtained within one year of the cardiac imaging. All statistical analyses were performed with SPSS 18 software, using Pearson correlations and T-tests. Forced-entry multivariate regression analysis was conducted with hematological parameters, age and SCD genotype as independent variables and cardiologic parameters as dependent variables.

Results:

The study group had a mean age of 13 years (SD 5,5) and contained 49 boys (53,3%). All children had a normal shortening fraction. Almost half of the children (39%) had a dilated left ventricular internal diameter in diastole and systole. Dilatation was inversely correlated with the Hb level. When analysed with multivariate analysis, both Hb and bilirubin were independently associated with left ventricular dilatation, suggesting that hemolysis deteriorates cardiac structure by a mechanism separate from the effect of anemia. There was no association between the left ventricular dilatation and age.

Conclusion:

Pediatric sickle cell patients have significant dilatation of the left ventricle, an abnormality that is significantly and independently associated with the severity of anemia and bilirubin levels, but in spite retain a normal shortening fraction.

Disclosures:

No relevant conflicts of interest to declare.
Study of the Quantity and Function of Regulatory T Cells In the Hemocytopenic Patients with Positive BMMNC-Coombs Test
Authors: Fu Rong , Chen Jin , Shao Zonghong , Wang Jun , Li Lijuan , Liu Hui
Published: October 2019

Abstract 4875

Objective

To investigate the quantity and function of regulatory T cells in the IRP, then explore the significance of Treg cells in the pathogenesis of IRP. Methods Sixty-two patients with IRP and twenty-four healthy donors were enrolled in this study. The ratios of CD4+CD25+/CD4+ and CD4+CD25+CD127low/CD4+ in bone marrow were examined with flow cytometry. The levels of IL-2, ATGF-β were tested with ELISA. The expressions of FoxP3 and Galectin-10 mRNA in BMMNC were measured by semiquantitive RT-PCR. Results The levels of IL-2, ATGF-β in bone marrow of untreated or recovered IRP patients (5.64±1.70, 6.19±2.53; 1.79±0.67, 1.86±0.76) were significantly lower than them of normal controls (7.91±3.71,2.48±0.94) (p<0.05); The ratio of CD4+CD25+/CD4+ cells in bone marrow of untreated IRP patients (22.46±9.47) was significantly lower than that of recovered IRP patients or normal controls (27.10±7.08, 30.59±8.58) (p<0.05); The ratio of CD4+CD25+CD127low/CD4+ cells in bone marrow of untreated IRP patients (7.18±2.72) was significantly lower than that of recovered IRP patients or normal controls (9.07±4.67, 10.44±3.24) (p<0.05). The relative mRNA expressions of FoxP3 and galentin-10 were 0.34±0.25, 0.69±0.51, 0.82±0.65 and0.66±0.11, 0.74±0.11, 0.76±0.09 in three groups, respectively. The expressions of the two factors in untreated IRP patients were significantly lower than them in recovered IRP patients or normal controls (p<0.05). Conclusions There exist abnormalities in quantity and function of Treg cells in IRP patients which might play important role in the pathogenesis of IRP.

Disclosures:

No relevant conflicts of interest to declare.
Cytoprotective Autophagy Induction by Imatinib Mesylate In Non-BCR-ABL Expressing Cells
Authors: Miyazawa Keisuke , Ohtomo Tadashi , Moriya Shota , Naito Munekazu , Kuroda Masahiko , Itoh Masahiro , Tomoda Akio
Published: October 2019

Abstract 4937

Autophagy is an intracellular degradation system that delivers cytoplasmic constitutions to the lysosome. Intracellular proteins and organelle are sequestered by the autophagosomes, then delivered to the lysosome and degraded. It has been reported that combining imatinib mesylate (IM) with an autophagy inhibitor such as chloroquine resulted in enhanced elimination of CML clones in vitro and in vivo (Bellodi C et al. J. Clin. Invest. 2009). We here report that IM induces the cytoprotective effect in non-BCR-ABL expressing cells along with autophagosome formation.

Treatment with IM resulted in an increased viable cell number of non-BCR-ABL expressing leukemia cell lines by inhibiting spontaneous apoptosis. IM did not show any effects on cell-cycle progression. When HL-60 cells were cultured in the presence of 3–30 μM of IM for 24–96 hr, up to a 1.6-fold increase in the viable cell number was observed in a dose-dependent manner. Furthermore, when HL-60 cells were cultured either under serum depletion or at lower concentrations of FBS, the cytoprotection by IM became more pronounced as compared with the cells cultured with 10% FBS. As well as in BCR-ABL expressing CML cell lines, electron microscopy revealed an increased autophagosomes after IM treatment in non-BCR-ABL expressing cells. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). IM attenuated the cytotoxic effect of cytosine arabinoside and bortezomib in leukemia cells.

Inhibition of autophagy by knockdown of LC3 gene using shRNA in HL-60 as well as complete knockout of atg5 in the Tet-off atg5-/- MEF system attenuated the cytoprotective effect of IM. This indicates that the effect is at least in part dependent on autophagy. However, the cytoprotection by IM was not medicated through supression of ROS production via “mitophagy” or reduction of ER stress via “ribophagy”. The cytoprotection with autophagy was still detectable in the HL-60 cells after knockdown of c-abl gene and in an ABL deficient-HL-60 cell line. This indicates that the proapoptotic function of ABL kinase is not involved. In addition, this effect was still observed in the presence of cycloheximide, suggesting that the effect is not mediated through translation process.

Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of targeting tyrosine kinase(s) for induction of cytoprotective autophagy.

Disclosures:

No relevant conflicts of interest to declare.
Bone Marrow Cells From Low-Risk Myelodysplastic Syndrome Patients Show Features of Enhanced Autophagy
Authors: Wang Yuan-yuan , Chen Zi-Xing , Cen Jian-nong , Shen Hong-jie , Qi Xiao-fei , Yao Li
Published: October 2019

Abstract 4961

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). Beclin 1, a gene plays an important role in autophagy, has been recognized as a tumour suppressor. The aberrant expression of Beclin 1 has been correlated to the development and progression of cancer. However, The function and expression of Beclin 1 in MDS is largely unexplored.

The present study aimed to investigate Beclin 1 expression and its correlation with IPSS in bone marrow cells from Patients with Low-Risk MDS. We have analyzed the expression of Beclin 1 in BM mononuclear cells (BMMNCs) from patients of Low-Risk MDS (31 cases), 22 AML(22 cases) and healthy control (9 cases) by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence and western blot analysis.

The expression level of Beclin 1 mRNA is significantly higher in BMMNCs in patients with Low-Risk MDS(mean± SD=6.26±4.87) when compared to healthy controls (mean± SD= 1.52±1.37), and the IPSS score was negatively correlated with the percentage of Beclin 1 positive cells. A markedly decreased expression of Beclin 1 was observed in AML (mean± SD=1.12±1.04) compared with healthy controls. The amount of Beclin 1 protein determined by either western blotting or immunofluorescence was markedly increased in MDS compared with that in controls (P<0.05). These results indicated that the cells from Patients with Low-Risk Myelodysplasia show features of enhanced autophagy, suggesting that the biological phenotype of autophagy, in addition to apoptosis and senescence, may also participate in the development of MDS and its progression to AML.

Disclosures:

No relevant conflicts of interest to declare.
Expression of Differentiation Antigens on Bone Marrow Myeloid Cells of the Patients with Myelodysplastic Syndromes and it's Clinical Significances
Authors: Shao Zonghong , Li Lijuan , Fu Rong , Wang Huaquan , Yue Lanzhu , Liu Hui , Wang Jun , Wang Honglei , Ruan Erbao , Qu Wen , Liang Yong , Wang Guojin , Wang Xiaoming , Liu Hong , Song Jia , Wu Yuhong , Xing Limin , Guan Jing
Published: October 2019

Abstract 4967

Objective

To study the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients.

Methods

Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality of these antigen expression and the prognosis of MDS cases were analyzed.

Results

The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The “right hook”, “sickle” and “retroflex 7” shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+(0.39±0.34)and CD16-/CD16+(1.33 ±0.77)of high-risk MDS group were significantly higher than those of control group (0.07±0.05 and 0.39 ±0.31 respectively) (P<0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while their MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ (3.88%±3.07%), CD11b- HLA-DR- (16.23%±15.59%), CD16-HLA-DR- (41.12%±24.53%), CD11b+CD16- (33.53%±17.26%) and CD13+CD16- (44.51%±21.99%) granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P<0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double antigen positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double antigen positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases correlated directly with their IPSS (international prognostic scoring system) (r=0.690, P=0.000) and WPSS (WHO adapted prognostic scoring system) (r=0.651, P=0.000) scores.

Conclusion

There were abnormal expressions of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity of these abnormal expressions was correlated with their prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.

Disclosures:

No relevant conflicts of interest to declare.
Apoptosis on U266 Cells Induced by Brucine by Death-Receptor Pathway
Authors: Ma Yanping , Qiao Zhenhua
Published: October 2019

Abstract 5022

Objective

Multiple myeloma (MM) is an incurable hematological malignancy with high incidence in the elderly. The currently chemotherapeutic drugs show severe side effects, dose-limiting toxicity and development of resistance. The study in order to observe the Anti-proliferative of brucine which is a novel plant anti-cancer agents on multiple myeloma cell line-U266 and find the mechanism of apoptosis.

Method

U266 cells was cultured in 90% RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, and maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells (5×104) were plated in the presence or absence of the brucine (0, 0.05, 0.1, 0.2, 0.4 mg/ml) in 96 well culture plates for 24–72 h. The anti-proliferative response of brucine was assessed by MTT assay. Nuclear morphology of the treated cells was observed using Fluorescence microscope. The mitochondrial membrane potential of treated cell were measured by flow cytometry. The expression change of caspase-3 was detected using RT-PCR.

Results

The apoptotic effect of brucine show a dose and time dependent manner (p<0.05). The 50% inhibiting concentration of brucine was 0.16 mg/ml. Brucine treated cells show significant feature associated with apoptosis by Fluorescence microscope. The mitochondrial membrane potential using flow cytometry revealed no significant decline at different concentration. In addition, Brucine induced U266 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9 (p<0.05).

Conclusions

At 0.4mg/ml concentration range brucine can induce apoptosis in U266 cells. Brucine induces the apoptosis of U266 cell via death-receptor pathway.

Disclosures:

No relevant conflicts of interest to declare.
Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin
Authors: Kowalska M. Anna , Rauova Lubica , Hayes Vincent , Cines Douglas B. , Bougie Daniel W. , Aster Richard H. , Krishnaswamy Sriram , Poncz Mortimer
Published: October 2019

Abstract 721

Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient‘s antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT.

Disclosures:

No relevant conflicts of interest to declare.
Telomerase Gene Screening and Telomere Overhang Detection in Chinese Patients with Acute Myeloid Leukemia
Authors: Yan Siyi , Han Bing , Wu Yongji , Zhou Daobin , Zhao Yongqiang
Published: October 2019

Abstract 1467

Telomeres are complex structures capping the ends of all eukaryotic cell chromosomes. Loss-of-function mutations in telomerase complex genes reduce telomerase activity and shorten overall telomere length in leucocytes, and they can clinically manifest as aplastic anaemia and dyskeratosis congenita, which predispose to acute myeloid leukemia. Telomeres are constituted of double-stranded tandem TTAGGG repeats followed by a 3' G-rich single-stranded overhang, a crucial telomeric structural component responsible for the t-loop formation. Loss of telomerase function also leads to short telometric overhangs, potentially resulting in chromosome instability.

In this study, we screened bone marrow samples from 69 Chinese patients with acute myeloid leukemia (AML, excluding AML-M3) for variants in telomerase reverse transcriptase(TERT) and telomerase RNA component(TERC) gene. We also investigated the length of telomeric overhangs in those patients and 46 healthy individuals, using Southern blot analysis. Cytogenetic status, disease severity and short time survival rate in patients with AML were evaluated. Our cohort included 2 M1,48 M2, 5 M4, 5 M5 and 9 secondary AML. Age was 47(13–77),medium follow-up time was 4 months (2–10 months). 3 TERT mutations (m896G>A, m1079C>G and m1451G>A) but no TERC mutation was identified in patients with AML. Patients carrying TERT mutations had very short telomeres, critical short overhangs and poor response to the induction chemotherapy and died in their follow-up period. In contrast to overall telomere length, which shortened with ageing, telomeric overhang lengths were constant overtime and much shorter in patients with AML compared to those of normal controls(P<0.001). In AML cohort, those who have shorter overhangs did worse than those with normal ones. Multivariant analysis showed telomeric overhang length, as well as unfavorable chromosome abnormalities, served as an independent prognostic marker for AML patients(P<0.0001). In conclusion, we report 3 mutations in TERT gene patients with AML, which may cause short telomere and overhang and account for poor prognosis in those patients. Short overhang length may be an independent factor for poor response and shorter survival in patients with AML. These findings would have to be confirmed in large, prospective studies.

Disclosures:

No relevant conflicts of interest to declare.