bluuebunny/crossref_metadata_2025 huggingface dataset:
New Interleukin-15 Superagonist (IL-15 SA) Significantly Enhances Graft-Versus-Tumor Activity After Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Bailey Cavan P , Sauter Christopher , Panis Michelle M , Budak-Alpdogan Tulin , Wong Hing , Flomenberg Neal , Alpdogan Onder
Published: October 2019

Interleukin-15 (IL-15) is a pleiotropic cytokine, which plays various roles in the innate and adaptive immune system, including the development, activation, homing and survival of immune effector cells. IL-15 has been previously shown to increase CD8+ T and NK cells number and function in normal mice and recipients of stem cell transplantation. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To overcome this, a new IL-15 mutant (IL-15N72D, J. Immunol, 2009; 183:3598) has been developed, with increased biological activity. Co-expressing IL-15N72D, in conjunction with IL-15RαSu/Fc produced a biologically active and highly potent IL-15 superagonist complex (IL-15SA, also known as ALT-803, Cytokine, 2011; 56:804). We evaluated the effects of IL-15-SA on immune reconstitution and graft-versus-tumor (GVT) activity in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Lethally irradiated BALB/c recipients were transplanted with T-cell depleted (TCD) bone marrow (BM) cells from B6 mice. IL-15 SA was administered via IP injection in two doses on days +17 and +24 after transplant. Animals were sacrificed at day 28. Administration of IL-15 significantly increased the numbers of CD8+ T cells and NK cells. IL-15 SA also augmented interferon-γ secretion from CD8+ T cells. We observed similar activity in B6CBA→CB6F1 transplant model. Interestingly IL-15 SA upregulates NKG2D and CD107a expression on CD8+ T cells. IL-15 SA administration also specifically increased slow-proliferative CD8+ T-cell proliferation in conjunction with robust IFN-γ and TNF-α secretion in CD8+ T cells in recipients of CFSE (carboxyfluorescein succinimidyl ester) labeled-T-cell infusion, whereas there was no effect on CD4+ T-cell proliferation. We then tested the anti-tumor activity of IL-15 SA in three different tumor models; murine mastocytoma (P815), murine B cell lymphoma (A20) and murine renal cell carcinoma (Renca). We found that IL-15 SA administration enhanced GVT activity against P815 and A20 in recipients of allogeneic HSCT though this activity required a low-dose T cell infusion with HSCT. Interestingly, augmented GVT activity against to Renca after IL-15 SA administration in recipients of allogeneic HSCT did not require T cell infusion.

We conclude that IL-15 SA is a very potent cytokine complex for enhancing CD8+ and NK cell reconstitution and function after HSCT, which would be a candidate for post-transplant immunotherapy.

Disclosures:

Wong: Altor Bioscience: Employment.
Impact Of Renal Insufficiency and Obesity On Safety and Outcomes Of BEAM Conditioning
Authors: Afifi Salma , Adel Nelly G. , Zhang Zhigang , Shi Weiji , Moskowitz Craig H. , Matasar Matthew J.
Published: October 2019

Introduction

BEAM (carmustine, etoposide, cytarabine and melphalan) is a standard conditioning regimen for patients with NHL undergoing autotransplant. Guidelines do not address whether patients with marginal renal function or obesity should undergo dose-adjustment to limit toxicity, and practices vary among centers.

Methods

We reviewed all patients who had BEAM autotransplant at MSKCC, 9/2003 - 12/2011; none received dose adjustment for chronic renal insufficiency [CRI, creatinine clearance (CrCl) 29-59 ml/min] or obesity (BMI>30). Outcomes included OS, relapse risk, transplant length of stay (LOS), and incidence of toxicities including mucositis, febrile neutropenia, and organ damage (acute renal failure by AKIN criteria; pulmonary toxicity requiring oxygen or corticosteroids; heart failure requiring inotropic support). Uni- and multivariate analysis evaluated association with baseline renal function and BMI, as well as age, histology, prior therapy, disease response, and comorbidity (hematopoietic stem cell transplant comorbidity index ≥4). Logistic regression was used for toxicity endpoints; linear regression and ANOVA were used for LOS; and Cox regression model and Gray’s competing risks were used for OS and relapse risk.

Results

343 patients received BEAM during the study period; median (range) age=56 (17-73), estimated CrCl=92 (29-208), and BMI=27.6 (16-47). 12% had CRI and 33% were obese (12% BMI≥35, 3% BMI≥40). Stepwise multivariate analysis showed CRI (OR 2.9, P<0.01) and obesity (OR 2.5, P<0.01) were associated with risk of organ damage independent of comorbidity and histology. Mucositis and febrile neutropenia were not associated with CRI or obesity. CRI (P=0.03), but not obesity, was associated with longer LOS. Relapse risk was associated with histology (PTCL vs. DLBCL, HR 2.3, P=0.01) and response at transplant (PR vs. CR, HR 2.3, P<0.01), but not CRI or obesity. Obesity was not clearly associated with overall survival (HR 0.62, P=0.10, NS), but CRI was associated with worse overall survival (HR 2.2, P<0.01) independent of disease status (for PR, HR=2.0, P=0.01), histology, prior therapy, or comorbidity (P=NS). However, 100d mortality was uncommon in patients with preserved (6/303, 2%) or impaired (2/40, 5%) renal function (P=NS).

Conclusions

Patients with CRI or obesity experience increased risks of acute organ damage, and those with CRI have inferior OS, but 100d mortality remains low in both groups. Our findings do not support routine dose-modification of BEAM for obesity or moderate renal insufficiency.

Disclosures:

No relevant conflicts of interest to declare.
CD34+ Selected Cells For The Treatment Of Poor Graft Function Following Allogeneic Stem Cell Transplantation
Authors: Stasia Alessandra , Ghiso Anna , Raiola Anna Maria , Galaverna Federica , Varaldo Riccardo , Gualandi Francesca , Dominietto Alida , Pozzi Sarah , Luchetti Silvia , Pogliani Enrico Maria , Bacigalupo Andrea
Published: October 2019

Background

We have previously defined poor graft function (PGF) as 2 or 3 cytopenic lines (Hb<10 g/dl, neutrophil count <1,0 x 10^9/L, platelet count <30 x 10^9/L), lasting for at least 2 consecutive weeks post-transplant, beyond day +14, with transfusion requirement, associated with hypoplastic-aplastic bone marrow, in the presence of complete donor chimerism and in the absence of severe GVHD and relapse (Larocca 2006). We have also shown that PGF can be treated with the infusion of donor CD34+ cells, selected from mobilized peripheral blood. (Larocca 2006) .

Aim of the Study

To update the 2006 study in 41 patients.

Methods

All 43 patients received a boost of CD34+ selected peripheral blood stem cells (PBSC) without prior conditioning and without GvHD prophylaxis. The median age of patients was 37 years (18-60y). The median number of CD34+ PBSC infused was 3.45 x106/Kg at median days of 140 days from 1st HSCT.

Complete response, or tri-lineage recovery was defined as achieving Hb >10 g/dl, ANC > 1000 x109/L, platelets > 100.000 x 109/L. A partial recovery was defined as transfusion independence, without a complete hematologic recovery. The median follow up was 1245 days.

Results

Tri-lineage recovery was seen in 31/41 (76%) and 3/43 patients became transfusion independent, for an overall response of 83%). The median time for complete hematological recovery from CD34+ boost infusion was 183 days.

There was no influence on tri-lineage recovery of the following factors : dose of CD34 cells (</> 3.3) (78% vs 72%), nor patient age (</>35 years) 75% vs 76%, nor donor type (HLA id sib 83%, UD 79%, family mm 68%). All the patients who achieved tri-lineage recovery are alive and disease free (28/41 patients). In this cohort 13 patients died due to relapse 9/14 (64%) or GVHD 2/14 (14%) or other causes 2/14 (14%). The overall actuarial survival is 63% with a median follow up of 1245 days (94-4151 days).

Interpretations and Conclusions

We confirm that infusion of CD34+ selected PBSC is associated with a high rates of tri-lineage recovery, with low risk of acute or chronic GVHD in patients who develop poor graft function following an HSCT. Tri-lineage recovery or achieving transfusion independency have an interesting impact either on disease free and overall survival.

Disclosures:

No relevant conflicts of interest to declare.
Gene Polymorphism Profile May Predict Late Complications In Patients Undergoing Allogeneic Stem Cell Transplantation
Authors: Gil Lidia , Wojtaszewska Marzena , Mol Anna , Lewandowski Krzysztof , Styczynski Jan , Komarnicki Mieczyslaw
Published: October 2019

Gene polymorphism analysis may predict infection development, especially invasive fungal disease, in recipients of allogeneic stem cell transplantation (alloSCT), as previously reported (Gil et al., BMT 2013). It is also suggested that gene polymorphism may contribute to non-infectious complications occurrence.

Objective

Analysis of late complication after alloSCT with respect to gene polymorphism profile.

Material and Methods

Analysis for infectious or non-infectious complications was performed in a cohort of 126 adult patients (pts) with acute leukemias (94), chronic leukemias (10) or lymphoid malignancies (22) who underwent alloSCT and survived at least 100 days. All pts and their sibling (58) or unrelated (68) donors were previously screened for TLR4, IFNG, TNFR2 and DECT1 single nucleotide polymorphisms (SNP). The primer for TLR4 SNP was designed using Lasergene Primer Select. IFNG, TNFR2 and DECT1 polymorphisms were screened with published primers with PCR conditions common to all reactions. Patients were conditioned with myeloablative (58) or reduced intensity (68) regimen and grafted with 3.8 (0.8-5.2)x10^6/kg of CD34+ cells. Standard definitions for relapse, graft versus host disease (GVHD) and infections (neutropenic, bacterial, fungal and viral) were used.

Results

Infectious complications that occurred after 100 days posttransplant included bacterial infection in 28 (22%), CMV infection in 19 (15%) and invasive fungal infection in 12 (9%) pts. Late onset acute GVHD 2-4 grade was seen in 15 (12%), while chronic extensive GVHD in 18 (14%) pts. Relapse was diagnosed in 30 (24%) pts. Secondary malignancies were observed in 2 pts. Over a median follow-up of 34.5 (range; 6-85.5) months, 90 (71%) pts were alive with median survival 35.5 (95% CI=8-87) months. Among genetic factors by multivariate logistic regression analysis donor DECT1 SNP was significant for fungal infection development (p=0.016, HR 5.7); chronic GVHD development (p=0.038, HR=4.4) and death from chronic GVHD (p=0.028; HR=4.8). Additionally, donor INFG SNP was significant for CMV occurrence (p=0.019; HR=5.5). None of studied polymorphisms had impact on the outcome. Among transplant-related factors (age, sex, diagnosis, disease stage, dose and source of stem cells, type of transplant, intensity of conditioning, neutropenia duration) only alloSCT from unrelated donor was significant for late CMV infection (p=0.47; HR=3.9) and bacterial infection (p=0.036; HR=4.4) development.

Conclusion

Our results suggest that gene polymorphism profile may predict late infectious and non-infectious complications in patients undergoing alloSCT. It may influence stratification of pts to offer an individualized therapy.

Disclosures:

No relevant conflicts of interest to declare.
Intracranial Hemorrhage and Mortality In 1461 Patients After Allogeneic Hematopoietic Stem Cell Transplantation For 6-Year Follow-Up: Study Of 44 Cases
Authors: Zhang Xiaohui , Han Wei , Chen Yuhong , Chen Huan , Liu Daihong , Xu Lanping , Liu Kaiyan , Huang Xiaojun
Published: October 2019

Although cerebral complications and causes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) were well documented, assessment of incidence rates and risk factors of intracranial hemorrhage (ICH) following allo-HSCT are less discussed. The clinical data of 1461 consecutive patients undergoing allo-HSCT in Peking University Institute of Hematology from January 2005 to June 2011 were retrospectively analyzed. Among these patients, 44 developed intracranial hemorrhage (ICH) and matched 176 patients control subjects were accrued. ICH was verified by computed tomography (CT) scan in all patients. Among the 1461 patients, 44 patients (3.0 %) developed ICH, including 29 patients (65.9%) with multiple location hemorrhage, 4 patients (9.1 %) with subdural hematoma (SDH), 8 patients (18.2%) with subarachnoid hemorrhage (SAH), and 3 patients (6.8%) with other hemorrhage in the brain parenchyma. The median time of appearance for ICH was 129 days (1-450). Survival after 6 year was significantly reduced in patients who developed ICH complications compared with control (47.1% vs. 75.7%,p<0.001 ).Multivariate analysis showed that donor type, systemic infections, III-IV aGVHD, platelet count, fibrinogen level were the independent risk factors for ICH among allo-HSCT patients. The ROC curve analysis showed a cutoff value of 13.2×109/L for platelet count and 129.5 g/L for fibrinogen level in patients with ICH. The transplantation-related mortality rate in the ICH and control group were 50% and 22.2%, respectively(p<0.001).ICH is common cerebral complication after allo-HSCT which associated with high mortality and decreased overall survival rate. After allo-HSCT patients with severe thrombocytopenia plus low level of fibrinogen and/or III-IV aGVHD appeared to be at particularly high risk of ICH.

Disclosures:

No relevant conflicts of interest to declare.
Single Cord Blood Transplantation Combined With An HLA Mismatched Third Party Donor For High-Risk Hematological Patients With HIV Infection
Authors: Kwon Mi , Kuball Jurgen H , Ellerbroek Pauline , Balsalobre Pascual , Serrano David , Buno Ismael , Gayoso Jorge , van Besien Koen , Petz Lawrence D. , Diez-Martin Jose Luis , Wensing Annemarie
Published: October 2019

Background

The safety and usefulness of unrelated umbilical cord blood (CB) stem cell transplantation (SCT) in patients candidates for allogeneic SCT and HIV infection is unknown. Single CB with the co-infusion of CD34+ cells from a third party HLA-mismatched donor (TPD), or Haplo-cord SCT, has shown to reduce the period of post-transplant neutropenia and related early complications associated with single CB transplantation. This platform could potentially reduce the risk of early infections in this particular group of patients. On the other hand, the use of a cell source homozygous for the CCR5 delta32 allele mutation, which confers high resistance against HIV-1 acquisition, is of particular interest in HIV+ patients.

Methods and Results

We report here on the first two patients with HIV infection and high-risk haematological malignancies who underwent haplo-cord SCT in two different centers: (Case 1) a 53 year-old male patient with high-risk MDS; (Case 2) a 34 year-old male with Burkitt lymphoma in CR2. Both CB transplants were performed using the haplo-cord platform with the co-infusion of mobilized and selected CD34+ cells from a TPD. Conditioning regimen were myeloablative in both cases. Case 1 had the additional potential benefit from the use of a CB unit with the homozygous CCR5delta32 mutation. Both cases achieved neutrophil and platelet engraftment similarly to previous haplo-cord SCT performed in HIV negative patients, as well as full CB chimerism. However, case 1 developed beside a severe lung infection, relapse of the underlying malignancy 2 months after SCT and died consequently. Case 2 presented one episode of bacterial sepsis with good response to antibiotics, and one episode of CMV reactivation controlled with valgancyclovir. Thirteen months after SCT, this patient is alive and in CR. Extensive analyses of viral and immunological compartments were performed showing the early viral dynamics in both patients.

Conclusions

CB SCT is feasible in patients eligible for allogeneic SCT and HIV infection. The haplo-cord strategy with the co-infusion of auxiliary cells from a TPD seems to offer a fast neutrophil engraftment similarly to HIV negative patients. Therefore, this strategy should be considered in patients with HIV infection and indication for allogeneic SCT, whenever CB is the source of choice and especially if a CCR5 mutated unit is available.

Disclosures:

Kuball: Miltenyi: GMP product development Other.
Effect Of Genotype On Pulmonary Hypertension In Patients With Thalassemia
Authors: Teawtrakul Nattiya , Ungprasert Phuangpaka , Pussadhamma Burabha , Prayalaw Patcharawadee , Fucharoen Supan , Jetsrisuparb Arunee , Pongudom Saranya , Sirijerachai Chittima , Chansung Kanchana , Wanitpongpun Chinadol , Chuncharunee Suporn
Published: October 2019

Introduction

Pulmonary hypertension is one of the major complications in patients with thalassemia. The prevalence is higher among patients with non - transfusion dependent thalassemia (NTDT) than those patients with thalassemia major. Patients with NTDT have distinct genetic subgroups. Therefore, the effects of different genotype groups on pulmonary hypertension in patients with NTDT were assessed.

Methods

A cross-sectional study was conducted in patients with NTDT aged ≥ 10 years old at Srinagarind University Hospital and Udonthani Hospital. Pulmonary hypertension was defined as a tricuspid regurgitation velocity > 2.9 m/s by trans-thoracic echocardiography. All patients gave consent and the protocol was approved by the Ethics committee of Faculty of Medicine, KhonKaen University and Udonthani Hospital. Clinical characteristics and laboratory data that literature indicated as risk factors for pulmonary hypertension were collected. Genotypes were determined by hemoglobin typing and DNA analysis. The effect of genotype group on pulmonary hypertension was evaluated by using multivariate logistic regression analysis.

Results

Two hundred twenty two patients were recruited, 3 patients were excluded (1 patient with congenital heart disease and 2 patients with valvular heart disease). In a total of 219 patients, pulmonary hypertension was found in 24 patients (10.96%). All patients were categorized into 2 groups according to genetic data that included: 1) β-thalassemia (140, 63.93%) 2) α-thalassemia and combined α and β-thalassemia (79, 36.07%).Genotype groups were statistically and significantly associated with pulmonary hypertension based on the adjusted odds ratios after adjustment for other factors. Patients with β-thalassemia had a statistically significant higher risk for pulmonary hypertension compared to patients with α-thalassemia and patients with combined α and β-thalassemia had an odds ratio of 9.47 (p=0.036).

Conclusion

Genotype group is an independent risk factor for pulmonary hypertension in patients with NTDT. Echocardiography should be routinely recommended in patients with β-thalassemia. In patients with α-thalassemia and combined α and β-thalassemia, pulmonary hypertension is uncommon. Therefore, echocardiography should be only performed when these patients have a clinical indication.

Abbreviation

Hb CS = Hemoglobin Constant spring, Hb Pakse´= Hemoglobin Pakse´

Abbreviation

PHT= pulmonary hypertension, AOR= adjusted odds ratio, 95% CI= 95% confidence interval

Disclosures:

No relevant conflicts of interest to declare.
Platelets Impair NK Cell Immunosurveillance Of Metastasizing Tumor Cells By Altering Surface Expression and Shedding Of Ligands For The Activating Immunoreceptor NKG2D
Authors: Raab Stefanie , Kropp Korbinian Nepomuk , Steinle Alexander , Kanz Lothar , Kopp Hans-Georg , Salih Helmut R.
Published: October 2019

NK cells play an important role in the immunosurveillance of tumor cells. The mechanisms leading to NK cell activation are described by the ‘missing-self’ and “induced-self’ hypotheses, implying that cells with low or absent expression of MHC class I and stress-induced expression of ligands for activating receptors like e.g. NKG2D (NKG2DL) are preferentially recognized and eliminated by NK cells. Besides the direct interaction with their target cells, NK activity is further influenced by various other hematopoietic cells. In mouse models, thrombocytopenia impairs metastasis, and this is reversed by additional depletion of NK cells. However, the knowledge regarding the molecular mechanisms by which platelets influence NK cells is still fragmentary. We recently reported that release of TGF-β by platelets upon their interaction with (metastasizing) tumor cells downmodulates NKG2D on NK cells (Kopp et al., Cancer Res. 2009; Placke et al., J Innate Immun. 2011). Moreover, platelets transfer “healthy” MHC class I to the tumor cell surface. Thus, platelets may facilitate metastasis by interfering with both, “induced and missing self’ NK cell recognition. Here we provide evidence for a yet unknown mechanism by which platelets further impair NKG2D-mediated immunosurveillance. Tumor cells were incubated with platelets from healthy donors resulting in coating of tumor cells and activation of the platelets, or treated with platelet-derived soluble factors (releasate) obtained either by tumor cell-induced platelet activation (TCIPA) or the platelet agonist thrombin. Presence of platelet derived factors derived either from coating of tumor cells or contained in platelet releasate substantially reduced NKG2DL surface expression on tumor cells. This was paralleled by enhanced levels of soluble NKG2DL in culture supernatants, indicating that platelet-derived factors mediate NKG2DL shedding from the tumor cell surface. Diminished NKG2DL surface expression resulted in decreased NKG2D-dependent cytotoxicity of NK cells as revealed by blocking experiments using NKG2D antibody and NKG2DL-specific F(ab)2 fragments targeting the specific modulated NKG2DL. Our data thus identify induction of NKG2DL shedding as novel mechanism by which interaction of platelets with metastasizing tumor cells impairs NK cell immunosurveillance.

Disclosures:

No relevant conflicts of interest to declare.
Histone Deacteylase Inhibitors (HDACi) Suppress Toll-Like Receptor-Induced Dendritic Cell Maturation and Alter Secretion But Not Gene Expression Of Polarising Cytokines By Dendritic Cells
Authors: Dickinson Michael J. , Davenport Alexander , Petrone Pasquale , Prince H Miles , Ritchie David , Neeson Paul
Published: October 2019

Aim/Background

HDACi induce cancer cell apoptosis and are in use for T-cell lymphoma. Prior studies have indicated that HDACi are immune-suppressive. In this study, we address this issue by examining the effects of two different HDACi on human monocyte derived dendritic cell (hmDC) biology.

Methods

hmDC were activated by TLR stimuli (LPS or poly:IC). HDACi effect on TLR-induced hmDC activation was assessed by up regulation of co-stimulatory molecules CD80, CD86 and CD83 along with secretion of IL-12p70, IL-10 and IL1β. To address the mechanism for drug-induced altered cytokine secretion mRNA for IL-12p70, IL-10 and IL1β were assessed followed by mir-155 mRNA and SOCS-1 protein. The functional outcome was assessed in an allogeneic MLR.

Results

At doses sub-optimal to induce myeloma cell death, both drugs significantly inhibited TLR-induced hmDC increase in CD80 and CD83, but not CD86. TLR-induced secretion of IL-10 and IL12p70 was reduced while IL-1β secretion was increased. This occurred regardless of drug sequencing. HDACi did not significantly alter expression of IL-12p70, IL-10 or IL-1β mRNA despite changes in protein levels. To reveal the mechanism for the HDACi-induced hmDC IL-12p70 defect, the upstream molecules miR-155 and SOCS-1 were examined. Interestingly, panobinostat but not romidepsin induced mir-155 down-regulation and SOCS-1 protein increase. Subsequently, allogeneic T-cells co-cultured with HDACi/TLR-ligand-treated hmDC secreted lower levels of cytokines (decreased secretion of IFN-γ, IL-2, TNF, IL-4 and IL-17) compared to those allogeneic T cells co-cultured with control hmDC.

Conclusion

In this study, we show that whilst HDACi impair DC maturation and modulate subsequent T cell responses. Our findings suggest that panobinostat induces the IL-12p70 effect via the mir-155/SOCS-1 pathway, whereas romidepsin induces the effect via a mir-155 independent pathway. This data suggests that HDACi affect the hmDC maturation response to pathogen, and that these drugs may alter the ability of treated patients to raise an effective immune response to pathogens or, indeed, tumour antigens.

Disclosures:

No relevant conflicts of interest to declare.
Antiphospholipid Antibodies Stimulate Release Of Endothelial Cell Microparticles Enriched In IL-1β
Authors: WU Meifang , Kundu Suman , McCrae Keith R.
Published: October 2019

Introduction

Microparticles are submicron vesicles released by activated or apoptotic cells. Previous studies from our laboratory and others have demonstrated elevated levels of endothelial cell (EC)-derived microparticles in the plasma of patients with antiphospholipid antibodies (APLA), which are associated with thrombosis and recurrent fetal loss. The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. APLA/anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner and stimulate the release of EC microparticles. Here, we demonstrate that these microparticles are enriched in active interleukin-1β (IL-1β) and may directly stimulate EC activation.

Methods

EC were treated with β2GPI and anti-β2GPI antibodies and microparticles were isolated by ultra-centrifugation. The treated EC, released microparticles and microparticle-free supernatant were analyzed for interleukin-1β (IL-1β) expression by western blot. In addition, we assessed the ability of microparticles derived from EC exposed to control IgG or anti-β2GPI antibodies, or microparticle-free supernatant that had been adsorbed with immobilized β2GPI to remove residual anti-β2GPI antibodies, to activate EC. Activation was defined by increased expression of cell surface E-selectin and phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4).

Results

Incubation of EC with β2GPI and anti-β2GPI antibodies stimulated the expression of pro-IL-1β. Interestingly, while EC contained exclusively pro-IL-1β, microparticles derived from these treated cells contained only the active, cleaved form of IL-1β. Moreover, extracellular IL-1β was found exclusively in microparticles, and was absent from microparticle-free conditioned medium Finally, IL-1β-containing microparticles derived from anti-β2GPI antibody-treated EC, but not microparticles released from EC exposed to only control IgG activated additional EC. Conditioned medium from anti-β2GPI antibody-treated EC that had been pre-adsorbed with immobilized β2GPI to remove residual anti-β2GPI antibodies also failed to stimulate EC activation.

Conclusions

EC exposed to β2GPI and anti-β2GPI antibodies stimulate the expression pro-IL-1β, which undergoes cleavage to an active form and is packaged in microparticles. These microparticles may directly induce activation of EC or synergize with anti-β2GPI antibodies to activate EC. We hypothesize that delivery of IL-1β and perhaps other inflammatory cytokines by microparticles derived from anti-β2GPI-exposed EC or monocytes may induce a systemic vascular inflammatory state in which anti-β2GPI antibodies may more readily stimulate thrombotic events.

Disclosures:

No relevant conflicts of interest to declare.
Platelet-Derived ERp57 Mediates Platelet Incorporation Into a Growing Thrombus By Regulation Of The αIIbβ3 Integrin
Authors: Wang Lu , Wu Yi , Zhou Junsong , Ahmad Syed S. , Mutus Bulent , Garbi Natalio , Hammerling Gunter , Essex David W
Published: October 2019

ERp57 is a member of protein disulfide isomerase (PDI) family. Although previous studies using antibodies have indicated ERp57 mediates platelet aggregation, the specific role of platelet-derived ERp57 in hemostasis and thrombosis is unknown. In this study we addressed this issue using a megakaryocyte/platelet specific ERp57-knockout mouse model (PF4-Cre/ERp57fl/fl). Although PF4-Cre/ERp57fl/fl mice had normal platelet counts and platelet glycoproteins expression, they had significantly prolonged tail-bleeding times, and thrombus occlusion times with FeCl3-induced carotid artery-injury. Using a mesenteric artery thrombosis model we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Upon stimulation with convulxin and thrombin, platelets lacking ERp57 had defective activation of the αIIbβ3 integrin and platelet aggregation. Addition of exogenous wild type ERp57 corrected the defect in aggregation implicating surface ERp57 in platelet aggregation. Moreover, using recombinant ERp57 mutants we found that the second active site of ERp57 targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn2+-treated platelets lacking β3 was substantially decreased, suggesting a direct interaction of ERp57 with αIIbβ3. Surface ERp57 activity in human platelets increased in response to stimulation with thrombin, concomitant with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

Disclosures:

No relevant conflicts of interest to declare.
An Analysis Of Ph-Like ALL In Japanese Patients
Authors: Kiyokawa Nobutaka , Iijima Kazutoshi , Yoshihara Hiroki , Ohki Kentaro , Kato Motohiro , Fukushima Takashi , Kikuchi Akira , Fujimoto Junichiro , Hayashi Yasuhide , Koh Katsuyoshi , Manabe Atsushi , Ohara Akira
Published: October 2019

Introduction

“Ph-like acute lymphoblastic leukemia (ALL)” or “BCR-ABL1-like ALL” is a recently identified subtype of high-risk B-cell precursor (BCP)-ALL with poor outcome and high frequency of IKZF1 deletion. They exhibit a gene expression profile similar to BCR-ABL1-positive (Ph1-)ALL but do not have BCR-ABL1 and other common chimeric genes. Recent study from others has identified rearrangements involving tyrosine kinase genes, such as ABL1, PDGFRB, JAK2, and CRLF2, by employing transcriptome sequencing using next generation sequencer. In the present study, we intended to explore the biologic characteristics and clinical outcomes of Ph-like ALL cases in Japanese patients.

Method

We investigated gene expression of 235 BCP-ALL cases enrolled on Tokyo Children Cancer Study Group (TCCSG) L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using both clustering analysis and Gene Set Enrichment Analysis (GSEA), a computational method that ascertains whether a given gene set is significantly enriched in a list of genes ranked by their correlation with a phenotype of interest. Four-year event-free survival (EFS) rate of the entire cases was 83.2%.

Results

Real-time PCR analysis revealed that 155 cases did not have common chimeric genes (“chimera-”), including BCR-ABL1, ETV6-RUNX1, E2A-PBX1, MLL-AF4, MLL-AF9, and MLL-ENL. Among “chimera-” cases, 11 (4.7% of the entire BCP-ALL cases) patients were identified as ALL with gene expression profile similar to that of Ph1-ALL by clustering analysis. Their 4-year EFS rate and the frequency of IKZF1 deletion were 66.7% and 80.0%, respectively.

On the other hand, GSEA detected 26 ALL cases with Ph-like signature (11.1%), including 9 cases those identified to have Ph-like phenotype by clustering analysis, and they exhibited 4-year EFS rate of 58.3% and IKZF1 deletion frequency of 38.1%. Interestingly, GSEA also revealed that 7 cases simultaneously possess ETV6-RUNX1-like signature. When we excluded 7 cases having ETV6-RUNX1-like signature and 5 cases with higher NOM p-value than 0.05, 14 cases were remained and they represented a highly unfavorable outcome (event-free survival 38.1%), while the frequency of IKZF1 deletion was 41.7%.

We performed transcriptome sequencing of ALL cases with Ph-like phenotype using next generation sequencer and identified rare and novel chimeric fusions involving tyrosine kinases, such as ABL1, as well as CRLF2-related chimeric fusions.

Conclusions

We identified the presence of ALL subset having Ph-like phenotype in Japanese patients. They represent unfavorable outcome. However, the diagnosis of “Ph-like ALL” varies according to the method to identify Ph-like phenotype. Therefore, the definition of “Ph-like ALL” needs more consideration.

Disclosures:

No relevant conflicts of interest to declare.
Serine 559 Of Glycoprotein Ibα Is Phosphorylated By Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression, and Signaling Of Glycoprotein Ib-IX
Authors: Zhang Jie , Zhang Weilin , Liu Guanglei , Zhao Lili , Yan Rong , Dai Kesheng
Published: October 2019

Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate glycoprotein (GP) Ib-IX-dependent platelet adhesion, and simultaneously trigger signaling cascades leading to integrin αIIbβIII activation, platelet spreading, and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and play key roles in regulating the VWF binding function of GPIb-IX. Several phosphoserine containing motifs in GPIb-IX complex, such as Ser609 of GPIbα and Ser166 of GPIbβ, have been confirmed to bind to 14-3-3ζ and involved in the regulation of VWF binding function. We have recently identified a novel 14-3-3ζ binding site located in the filamin A binding domain of GPIbα at Ser559. Phosphorylation of GPIbα at Ser559 not only is important for 14-3-3ζ binding, but also plays a key role in the regulation of VWF binding function of GPIb-IX. However, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIbα and purified GPIbα could be phosphorylated at Ser559 by the catalytic subunit of protein kinase A (PKA). Ser559 phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced VWF binding to the CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIbα (S559A) with alanine, while reduced in the CHO cells of Ser166 of GPIbβ (S166A) with alanine. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser559 of GPIbα inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIbα is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX.

Disclosures:

No relevant conflicts of interest to declare.
The Presence Of 1 / 8 HLA Mismatch Do Not Hamper Survival After Allogeneic Stem Cell Transplantation Using Immunoprophylaxis With Sirolimus-Tacrolimus
Authors: Parody Rocío , López-Corral Lucía , Godino Oriana Lopez , Cadenas Irene García , Lourdes Vazquez , Martino Rodrigo , Martinez Carmen , Solano Carlos , Martinez Ariadna Perez , Barba Pere , Valcarcel David , Falantes Jose F. , Márquez-Malaver Francisco J , Caballero Dolores , Perez-Simón Jose Antonio
Published: October 2019

Several studies from the IBMTR have shown that overall survival (OS) is reduced by 10% for each mismatched at obligatory locus (A,B,C or DRB1) after allogeneic hematopoietic stem cell transplant (Allo-HSCT) (Lee et al. Blood 07). In the setting of 7/8 mismatched allo HSCT, different T-cell depletion strategies are frequently used in an attempt to decrease GVHD assuming a higher risk of infections and relapse. Data from the IBMTR are mostly based on series of patients who received Cyclosporine /Tacrolimus (TKR) plus methotrexate as immunoprophylaxis. As far as we know, the effect of sirolimus (SRL)/TKR combination on overall outcomes of transplant, regardless HLA mismatched, has not yet been evaluated.

With this background, we retrospectively analysed a series of 151 adult patients who received SRL/TKR based reduced intensity (RIC)-alloHSCT in 6 Spanish centres, from January 2007 to September 2012 (median follow up in alive patients 19 months, range 4-47), comparing overall outcomes according HLA compatibility in obligatory locus A,B,C, DRB1: 8/8 (n:129) vs 7/8 (n:22). No in vivo or ex vivo T depletion was used.

Patients characteristics are not significantly different between both groups. Median age was 53 years (17-70) and the two most common diseases (56% of cases) were AML and NHL. The most frequent stem cell source was peripheral blood (94%) and 76% transplants were from volunteer unrelated donors. Forty nine per cet of patients had active disease at the time of transplant.

Comparing 8/8 vs 7/8 alloHSCT, non statistically significant differences were found in 3 years-OS (56,4% vs 69%, p=0.7), 1 year-non-relapse mortality [12 % (7-20) vs 14% (5-42), p=0.8], and 3 year-relapse rate: 28% (19-43) vs 20% (9-50), p=0.8, respectively. Cumulative incidence of acute GVHD was significantly higher in 7/8 alloHSCT [68% (49-94) vs 41% (32-51), p=0.004] but no differences were observed with respect to neither III-IV acute GVHD (5 vs 9%, p:0.6) nor global (35 vs 51%, p=0.6) and extensive (20 vs 30%, p=0.9) chronic GHVD in 8/8 vs 7/8, respectively.

The present study shows favorable outcomes of SRL/TKR combination in the setting of RIC alloHSCT, with an overall survival in the range of 55-70%%, and non significant differences in overall outcomes regardless the presence of any mismatched at obligatory locus. Comparative studies with a larger sesries is desirable to confirm these results.

Disclosures:

No relevant conflicts of interest to declare.
A Myeloablative Conditioning Regimen With Fludarabine Demonstrates Good Results In UCBT With Hematologic Malignancies, Especially Acute Lymphoblastic Leukemia
Authors: Tong Juan , Zimin Sun , Huilan Liu , Liangquan Geng , Changcheng Zheng , Tang Baolin , Kaidi Song , Wen Yao , Xin Liu , Ding Kaiyang
Published: October 2019

Objectives

We retrospectively analyzed the safety and efficacy of a myeloablative conditioning regimen without anti-thymocyte globulin (ATG) or total body irradiation (TBI) but with fludarabine (FLU) in unrelated cord blood transplantation (UCBT) for 30 patients with hematologic malignancies.

Methods

The myeloablative conditioning regimen consisted of FLU, busulfan (BU) and cyclophosphamide (CY). All of the patients received Cyclosporine (CSA) and mycophenolate mofetil (MMF) as graft versus host disease (GVHD) prophylaxis.

Results

With this conditioning regimen, we achieved high engraftment rates (96.7%) and rapid hematopoietic reconstitution. Acute GVHD occurred in 12 cases of the 29 engraftment patients (41.4%), and 6 cases (20.7%) were of grade III-IV. Chronic GVHD only occurred in 1 of 28 evaluable patients (3.6%). Twenty-three patients (76.7%) became infected, and 3 cases (10.0%) died of severe infections. Cytomegalovirus (CMV) reactivation occurred in 70.0% of the patients, but no CMV diseases were observed, nor did any patients die of CMV infection. The cumulative incidence of relapse (6.7%) was significantly reduced, and none of the acute lymphoblastic leukemia (ALL) patients relapsed. The 3-year overall survival (OS) and event-free survival (EFS) rates were 73.3% and 70.0%, respectively, representing satisfactory survival. The 3-year OS and EFS of the ALL patients was 75.0%.

Discussion

This conditioning regimen resulted in a high engraftment rate, rapid myeloid reconstruction and a low incidence of infection. Although there were many patients with high-risk disease and disease progression, the regimen resulted in low relapse rates and good survival. None of the ALL patients relapsed after UCBT, indicating that this conditioning regimen could be applied to more patients with ALL.

Disclosures:

No relevant conflicts of interest to declare.
High Plasma Level Of Regenerating Islet-Derived 3-Alpha (Reg3a) Protein Predicts Grade 3-4 Lower Gastrointestinal Acute Graft-Versus-Host Disease and Poor Prognosis
Authors: Sun Aining , Cai Chengsen , Chen Guanghua , Wang Jun , Depei Wu
Published: October 2019

Objective

To explore the feasibility of applying plasma Reg3α protein level for the diagnosis and prognosis of the lower gastrointestinal acute graft-versus-host disease(LGI-aGVHD).

Method

One hundred and three patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) in our hospital from December 2011 to December 2012 were included in this prospective study. Peripheral blood samples were collected from allo-HSCT recipients at -9d, 0d, +14d, +28d, at the time when they developed aGVHD, and at the one and four weeks after aGVHD therapy. The plasma concentrations of Reg3α protein were measured by ELISA assay.

Results

Of the 103 patients, 17 subjects never developed aGVHD symptoms (no-aGVHD), 27 subjects presented with non-aGVHD associated diarrhea, 10 subjects presented with isolated skin aGVHD, 17 subjects developed grades ±-II LGI-aGVHD, 32 subjects developed grades III-‡W LGI-aGVHD. The plasma concentrations of Reg3α in patients with LGI-aGVHD vs non-aGVHD diarrhea were 111.5(54.7-180.2) vs 23.9 (14.5-89.5) ng/ml respectively, with the difference being statistically significant (P<0.001). The plasma concentrations of Reg3α in 17 patients who had no response to therapy at 4 weeks for grades III-‡W LGI-aGVHD and 7 patients who experienced a complete or partial response were 137.2(51.7-205.4) and 679.4(122.3-896.8) ng/ml respectively, with the difference being statistically significant (P=0.028). All of the patients who had no response to therapy died of aGVHD-associated multiple organ failure. The area under the ROC curve was 0.902 when plasma concentration of Reg3α was set at 87.73 ng/ml. The sensitivity was 81.48% and the specificity was 82.86% when the critical value was used in diagnosis of grades III-‡W LGI-aGVHD. The probability of grades III-‡W LGI-aGVHD had statistical difference above and below 87.73 ng/ml after allo-HSCT( P<0.001).

Conclusion

The high level of plasma Reg3α protein after transplantation predicts grades III-‡W LGI-aGVHD. In grades III-‡W LGI-aGVHD patients receivingimmunosuppressive treatment at four weeks, the high level of plasma Reg3α protein correlates withs a poor prognosis. The plasma concentrations of Reg3α can be used as a specific biomarker of LGI-aGVHD.

Disclosures:

No relevant conflicts of interest to declare.
TPO exerts a protective effect on iron-overload induces apoptosis in cardiomyocytes via mitochondrial pathways
Authors: Yang Mo , Chan Shing , Ye Jie yu , ChiFung Chan Godfrey
Published: October 2019

Thalassaemia companied with iron-overload is common in Hong Kong. Iron overload induced cardiomyopathy is the commonest cause of morbidity and mortality in b-thalassaemia patients. One of the causes of cardiac failure is chronic iron overload of blood transfusion. Some studies showed that iron overload can cause toxic effect in heart cells. Iron-overload induced cardiomyopathy damages are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes. Our previous study showed TPO has cardiac protective effect (Li et al, Circulation, 2007). In this study, we demonstrated firstly that iron induced oxidative stress can cause apoptosis in cardiomyocytes. By using H9C2 cells, we showed that iron increased reactive oxygen species (ROS) production (n=3) and reduced cell viability in a dose-dependent manner (0-0.6 mM) (n=6). Apoptotic cells were found to be significantly increased under iron treatment (0.3 mM, 72 hrs) in the AnnexinV/PI assay (n=6). The expression of active caspase-3 significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/mL). The ROS production was significantly decreased with the addition of TPO at 50 ng/mL (n=3). Dot-plot analysis of AnnexinV/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6), indicating a trend in attenuation of the drop of mitochondrial membrane potential. Our findings suggest that iron-overload lead to generation of ROS which further induces apoptosis in cardiomyocytes via mitochondrial pathways and TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and mitochondrial pathway in cardiomyocytes.

Disclosures:

No relevant conflicts of interest to declare.
A New Mutation Of The ALAS2 Gene In a Chinese Family With X-linked
Authors: Cui Rui , Zhu Guoqing , Xu Zefeng , Zhang Yue , Huang Gang , Xiao Zhijian
Published: October 2019

X-linked sideroblastic anemia (XLSA; OMIM 301300) is one of the most common inherited sideroblastic anemia, which results from mutations in the erythroid-specific isozyme of 5-aminolevulinate synthase. An amino acid (AA) substitution for arginine at the 452 AA position of the ALAS2 protein is the most frequent mutation, which has been found in approximately one-quarter of patients with XLSA. Here we first report a 3-generation pedigree including 10 individuals with a K156E substitution in ALAS2 gene in China. A 34 year-old man, complained with a microcytic anemia (hemoglobin (Hb) 84 g/dl, MCV 63.8 fl and MCH 16.5pg), increased ferritin serum (3123ng/ml) and transferrin sat (93%), and dyserythropoiesis with 50% of ring sideroblasts (RS) in BM. A diagnosis of XLSA was made. On treatment with 300mg/d pyridoxine and iron chelation therapy with deferoxamine allowed a correction of anemia and reduction of the S-ferritin (1096 ng/ml) in the proband. The peripheral blood samples had been extracted from proband and his family members. All exons containing exon-intron boundaries of ALAS2, SLC25A38 and GLRX5, the promoter region and intron 8 of ALAS2 were amplified and directly sequenced. He is a heterozygous for ALAS2 K156E substituion. The pedigree of the proband's family is shown in Figure 1, while hematologic and iron status data for the family members are reported in Table 1. All 4 heterozygous females from this family showed marginally increased red-cell distribution width (RDW) without phenotypic expression.Figure 1The pedigree of the probandFigure 1. The pedigree of the probandTable1Clinical and hematological features of the pedigreeSubjectSex/age,yHb g/dLMCV fLRDW%Ferritin μg/LALAS2I-1F/6811585.221.4136.7+II-1F/381288912.532.89-II-2M/348463.832.83123+II-3F/3213691.914.816.8-III-1M/2015288.212.1123.6-III-2F/1812384.813.321.84-III-3M/71449212.951.9-III-4F/101338513.716.8+III-5F/812681.418.831.2-

In summary, this is a novel K156E substitution in ALAS2 gene discovered in a 3-generation pedigree in China. Furthermore, early diagnosis of XLSA allows preventing the complications of the iron overload by chelators or by iterative phlebotomies.

Disclosures:

No relevant conflicts of interest to declare.