ALBERT

Description: Key Points Novel crosstalk between SMO and NF-κB representing additional level of NF-κB regulation independent of genetic constitutive activation. SMO activates NF-κB by recruiting Gαi and Gα12 to activate PKCβ/CARMA1 and assembling CARMA1/BCL10/MALT1/TRAF6 to SMO.

Description: Key Points Elevated Jun signaling promotes lymphoma growth and dissemination to extranodal sites. Jun-regulated genes mediate the interaction of malignant cells with stromal cells and adhesion to extracellular matrix proteins.

Description: Abstract 1120 Background: Leukoreduction (LR) is a process by which leukocytes (WBC) are filtered from blood or blood component, as the leukocytes may increase the incidence of alloimmunization to human leukocyte antigens and may cause febrile nonhemolytic adverse transfusion reactions. Current transfusion practice also requires that, at minimum, blood selected for transfusion to a patient be checked (phenotyped) to be antigen negative to the existing alloantibodies in the patient's serum. Human Erythrocyte Antigen (HEA) Beadchip™ (Hashmi, G. et al. Transfusion, 47, April 2007, 736–747) has been adopted by blood centers and transfusion services for routine antigen-negative screening. A recent prospective study (Klapper, E. et al. Transfusion, 50, March 2010, 536–546) conducted in four large hospital transfusion services concluded that it is theoretically feasible to establish an inventory of DNA tested donor components from existing hospital inventories to result in the provision of more extensively matched RBC components than is the current standard of practice. Here described is a HEA LR eMAP-S BeadChip™ platform developed based on a combination of a novel DNA extraction protocol and a novel proprietary assay platform for Human Erythrocyte Antigens determination from leukoreduced blood samples. Methods: The BioArray Solutions HEA LR eMAP-S BeadChip Kit uses the novel Elongation Mediated Multiplexed Analysis of Polymorphisms in Solution (eMAP-S) technology (Lin, X. et al., 2010). The multiplex PCR amplifies 21 DNA fragments covering 24 allele variants associated with 32 human erythrocyte antigens plus one mutation for hemoglobin S. The PCR, is then followed by clean-up, multiplex allele specific primer extension (ASPE), and on-Beadchip™ detection and read-out by using the AIS-400 Array Imaging System. Verification studies was performed using 203 leukoreduced blood segments collected from various blood centers and transfusion services in US. A set of 30 blood samples was also obtained from before leukoreduction (whole blood, WBC count: 2308–6052 cells/ μL) and after leukoreduction (LR blood, WBC count:0.36-2.63 cells/μL), DNAs from LR blood samples were extracted by a novel DNA extraction method (BAS) using commercial reagents ((Qiagen, Inc., Valencia, CA). To evaluate the performance of HEA LR eMAP-S Beadchip™ Kit, the extracted DNA samples from before and after LR were analyzed by both HEA LR eMAP-S Beadchip™ and commercially available HEA 1.2 Beadchip™ Kit along with 88 whole blood DNA samples and positive controls with known HEA calls and analyzed for 10 red blood group systems (Rh, K, JK, FY, MNS, DO, Lu, Yt, Di, Co); and one mutation associated with hemoglobinopathies. Phenotype results obtained from HEA LR eMAP-S BeadChip™ Kit during the studies were compared to the data from the HEA 1.2 BeadChip™ Kit. Results: DNA was successfully extracted from 30 donor DNA samples before (DNA ng/ul: 20–56) and after LR (DNA ng/ul: 8–20) and analyzed using HEA LR eMAP-S Beadchip™ Kit with 100% concordance in HEA phenotype results. The WB DNAs were further analyzed using HEA 1.2 Beadchip™ kit and the results showed 100% concordance with LR DNA results. In addition, results from positive controls (HEA Ref-pA, HEA Ref-pB and cell line DNAs) all produced correct calls, and HEA LR eMAP-S assay and HEA 1.2 assay testing results on 88 donor whole-blood DNAs showed 100% concordance. Conclusion: HEA LR eMAP-S Beadchip™ Kit could be used for reliable determination of human erythrocyte antigens for leukoreduced blood samples. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3255 Introduction: Microvascular abnormalities underlie much of the morbidity observed in sickle cell disease (SCD). Adherence of sickle erythrocytes and leukocytes to the microvascular endothelium leads to vaso-occlusion and end organ ischemia/reperfusion injury. Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the recruitment of monocytes to sites of endothelial activation or injury and thus contributes to the pathophysiology of vascular disease. In SCD patients, circulating MCP-1 concentrations are elevated during both steady state and vaso-occlusive crisis. However, the relationship between MCP-1 and microvascular abnormalities in SCD patients has not been assessed. In the present study, we measured a panel of circulating cytokines including serum MCP-1 and hematological biomarkers including serum ferritin in SCD patients who were being monitored by computer-assisted intravital microscopy (CAIM) to assess severity of microvasculopathy. Methods: The study included 31 steady state SCD patients (22 females, 9 males) ranging in age from 4–61 years. MCP-1 concentrations were measured by Luminex multiple analyte profiling. Ferritin concentrations were measured by chemiluminometric immunossay. The conjunctival microvasculature was assessed using CAIM. A severity index on a scale of 0–15 was calculated as described previously (Cheung et al, Am J Hematol 85:899, 2010) to quantify the degree of microvasculopathy observed among the patients. Results: The mean ± SD MCP-1 concentration was 445 ± 253 pg/ml (range: 122–1180 pg/ml), mean ± SD ferritin concentration was 1675 ± 1883 ng/ml (range 18–5825 ng/ml), and mean ± SD severity index was 5.0 ± 2.5 (range: 0–9). By multiple regression analysis, with controlling for age and gender, MCP-1 was directly correlated with severity index (p = 0.046), and serum ferritin was directly correlated with MCP-1 (p = 0.019). Conclusions: Monocyte recruitment to the site of endothelial injury involves chemotactic signaling mediated in part by MCP-1, levels of which rise in SCD. Our results indicate that MCP-1 is associated with and may directly contribute to the microvasculopathy observed in SCD patients. Furthermore, we find that MCP-1 levels are correlated with ferritin concentrations. Ferritin reflects body iron stores, and an increase of iron stores in SCD resulting from chronic blood transfusion as well as increased iron absorption associated with hemolysis may thus lead to monocyte/macrophage recruitment to the vascular endothelium and contribute to vasculopathy. Measurements of MCP-1 and ferritin may also serve as surrogate biomarkers of microvasculopathy severity and inflammation. Disclosures: Green: Emisphere - Consultancy : Consultancy; Teva Pharmaceuticals - expert testimony: Consultancy.

Description: Abstract 246 Introduction: Inflammation and abnormal adhesion of sickle red blood cells (RBCs), leukocytes and platelets to the vascular endothelium are postulated to play a central role in the pathogenesis of vaculopathy associated with sickle cell disease (SCD). Dysfunctional endothelial cells in the SCD vaso-occlusive process display vasoconstriction, proinflammatory and prothrombotic changes. Sickle RBCs may damage or activate the endothelium via enhanced expression of cell surface adhesion molecules such as vascular cell adhesion molecule (VCAM), intercellular adhesion molecules (ICAM), platelet endothelial cellular adhesion marker (PECAM), E- selectin, and P-selectin. In addition, SCD modulates high levels of circulating soluble adhesion molecules especially during the sickle cell crisis. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is an endothelial cell receptor for oxidized low-density lipoprotein. The enhanced expression of LOX-1 in endothelial cells has been identified in a variety of pathologic conditions including atherosclerosis, diabetic vasculopathy, hyperlipidemia and inflammation. The purpose of this study is to investigate changes in the expression of LOX-1 and its potential role in the pathogenesis of SCD vasculopathy. Methods: Using real time quantitative PCR, we analyzed LOX-1 gene expression in cultured human coronary endothelial cells (HCEC) following static incubation with sickle RBCs. We also measured circulating soluble LOX-1 (sLOX-1) concentrations by sandwich ELISA assay in SCD patient plasma. The statistical analysis was performed using Student's t-test. Results: LOX-1 gene expression in HCEC was significantly increased by incubation with sickle RBCs compared with normal RBCs. Upregulation was detected after 1 hour of incubation, and reached a peak after 6 hours. We studied 48 SCD (hemoglobin SS) patients (26 female, 22 male); vs 17 healthy (hemoglobin AA) control subjects (12 female, 5 male). The SCD cohort comprised pediatric and adult patients in steady-state (33 patients) and vaso-occlusive crisis (VOC; 15 patients). The concentration of circulating sLOX-1 protein in plasma of SCD patients (mean: 3.05±2.53 ng/mL; range 0.30 – 11.30 ng/mL) was significantly higher (p=0.0046) than in control healthy subjects (mean: 1.27±0.81 ng/mL). In the 15 SCD patients with VOC, sLOX-1 concentrations were higher, (mean: 3.65±2.40 ng/mL). Conclusions: Our study reveals that LOX-1 gene expression in endothelial cells is upregulated by incubation with SCD erythrocytes. Baseline circulating sLOX-1 levels are elevated in SCD patients compared with healthy controls. sLOX-1 levels are further elevated in VOC. Enhanced LOX-1 expression in endothelial cells may play a role in the pathophysiology of SCD vasculopathy. Studies of sLOX-1 in SCD may provide new insights into risk stratification, and may lead to novel therapeutic strategies for sickle cell patients with acute vascular complications. Disclosures: Green: Emisphere - Consultancy: Consultancy; Teva Pharmaceuticals - expert testimony: Consultancy.

Description: Abstract 4403 Background: DNA analysis of human platelet antigens is known to improve the diagnosis and treatment of neonatal alloimmune thrombocytopenia and minimize the risks of post transfusion purpura and refractoriness to random donor platelet therapy. The availability of reliable molecular platforms such as HPA eMAP assays analyzing HPA-1, -2, -3, -4, -5, -6, -7, -8, -9, -11, and -15, are routinely used and eliminate the need for reference sera or large platelet volumes. New generations of HPA assays (HPA eMAP-S Beadchip™ Kit) described here are developed by identification of specific allele in solution and detection of targets by a generic probe array called the Universal BeadChip™ by imaging, HPA-13 was also incorporated in the panel. These assays are easily automatable due to fewer protocol steps. Methods: The BioArray Solutions HPA eMAP-S BeadChip Kit uses the novel Elongation Mediated Multiplexed Analysis of Polymorphisms in Solution (eMAP-S) technology (Lin, X., et al., 2010) to identify the presence or absence of the selected alleles associated with a given phenotype. We amplified 10 DNA fragments covering 12 allele variants which are associated with 12 platelet antigens by using multiplex PCR and HPA gene specific primer pairs. PCR was followed by multiplex allele specific primer extension (ASPE) and Beadchip detection by using the AIS-400 Array Imaging System. To evaluate the overall performance of HPA eMAP-S BeadChip™ Kits, a total 552 clinical samples with known HPA eMAP phenotype and/or serology were analyzed to determine polymorphisms associated with common human platelet antigen (HPA-1, -2, -3, -5, -15) and less common human platelet antigen (HPA-4, -6, -7, -8, -9, -11, -13). For internal study, 176 clinical samples were analyzed at BioArray Solutions (BAS). For the external study, 186 and 190 (total 376) donor samples were tested at two sites and analyzed by BASIS™ software. Phenotype results obtained from the HPA eMAP-S Beadchip™ Kit during the studies were compared to the data generated from the HPA eMAP BeadChip™ Kit and/or serology. Results: For internal and external studies, the concordance between the HPA eMAP-S Beadchip™ kit and HPA eMAP Beadchip™ kit was 99.99%. The discordant call (0.01%) on HPA-1b antigen was investigated. One HPA-1 discordant sample was sent out for sequencing and re-analyzed by using increased amount of DNA from DNA precipitation. The result from sequencing is consistent with the result from HPA eMAP Beadchip™ Kit. The HPA eMAP-S result from higher concentration DNA is also consistent with the result of sequencing. The discordance in the original HPA eMAP-S assay was attributed to less DNA input in the multiplex PCR, which will be corrected in the Package Insert of HPA eMAP-S Beadchip™ Kit. Conclusion: HPA eMAP-S Beadchip™ Kit could be used reliably for the determination of human platelet antigens using DNA analysis. Disclosures: Hashmi: Immucor: Employment.

Description: Introduction Chimeric antigen receptor (CAR) T -cell therapy has demonstrated high response rates among patients with B cell malignancies yet remission durability and safety could be improved. We have developed a novel double-chain chimeric receptor Synthetic T Cell Receptor and Antigen Receptor (STAR) consisting of 2 protein modules each containing an antibody light or heavy chain variable region, the T Cell Receptor (TCR) a or b chain constant region fused to the OX-40 co-stimulatory domain, with the 2 modules linked by a self-cleaving Furin-p2A sequence that allows the modules to be proteolytically separated and reconstituted (Fig. 1A). Here, we report pre-clinical and first-in-human phase I trial results of CD19 STAR-T cell therapy for CD19+ R/R B-ALL. Methods Peripheral blood (PB) mononuclear cells were obtained from healthy donors and patients for the pre-clinical and clinical studies, respectively. T-cells were transduced with the STAR lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR T-cell antitumor function. For the clinical trial, from Dec. 2019 to Jun. 2020, 18 CD19+ R/R B-ALL patients (M/F 10:8) with a median age of 22.5 years (range: 6-68) were enrolled (NCT03953599). Patients received a conditioning regimen of IV fludarabine (25mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single STAR T-cell infusion. Once patients achieved complete remission (CR), they were given the option to proceed to consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT) or not. Results In preclinical studies, we found CD19 STAR T-cells to be superior to conventional CAR (BBz CAR) measured by the following parameters: 1) faster/stronger T-cell activation within 3 hours (76.67±2.621% vs 46.4±9.318%; p=0.0253); 2) higher cytokine production (4100.92±174.4 pg/ml vs 2556.78±563.39 pg/ml; p