ALBERT

Description: Introduction Pure red cell aplasia (PRCA) is a severe consequence of major and bi-directional ABO-mismatched allogeneic stem cell transplantation (allo-SCT), likely the result of persistent isoagglutinin-producing host plasma cells that have escaped pre-transplant conditioning or graft vs. plasma cell effect (Aung et. al. 2013). PRCA, defined as anemia with reduced reticuloytosis and absence of red cell precursors in the bone marrow at 60 days after transplantation, complicates about 10-20% of all ABO-mismatched allo-SCTs; however, optimal treatment remains unknown. We report 5 cases from our institution of ABO mismatched allo-SCT complicated by PRCA treated with therapeutic plasma exchange (PEX) with or without the anti-CD20 monoclonal antibody rituximab. Report Patient characteristics are shown in Table 1. Indications for transplantation included refractory multiple myeloma, acute myeloid leukemia, and myelodysplastic syndrome (table 1); four of five patients underwent myeloablative conditioning, and the fifth received a reduced-intensity regimen due to age and co-morbidities. Prophylaxis for graft-versus-host disease consisted of cyclosporine alone, cyclosporine and prednisone, or tacrolimus and prednisone. ABO mismatching was major in four patients and bi-directional in one patient; all donors and recipients were Rh(D) positive. In all five patients, neutrophil engraftment occurred between days 11-15 after transplantation, with failure of red cell engraftment by day 60. Response Intervention included PEX in all patients, performed every-other-day (table 1). Three patients also received adjuvant rituximab in addition to PEX. Resolution of PRCA, which we defined as transfusion independence, presence of erythroid precursors on bone marrow biopsy, and Òmixed fieldÓ on blood type crossing, occurred in four out of five patients (mean 95 days). In two patients (patients 3 and 5) PEX and rituximab led to transfusion independence in less than 30 days after initiation of PEX. In one patient (patient 2), red cell engraftment occurred 87 days after initiation of PEX; another patient (patient 4) required 10 sessions of PEX and 2 cycles of rituximab (4 weekly doses separated by 6 months) to achieve transfusion independence 253 days after initiation of PEX. One patient (patient 1) had persistent PRCA despite 10 sessions of PEX and died of disease relapse 398 days after transplantation. In one patient (patient 2), tapering of immunosuppression was attempted in conjunction with PEX and led to resolution of PRCA; in the other four, withdrawal of immunosuppression was either not clinically indicated or unsuccessful in resolving PRCA. Conclusion We report five cases of PRCA after ABO-mismatched allo-SCT in the setting of major or bi-directional ABO incompatibility treated with PEX with or without rituximab, with four out of five patients responding to this intervention. This case series demonstrates the potential efficacy of PEX and rituximab for the treatment of PRCA. However, the optimal number of sessions of PEX, timing of this intervention, dosing and schedule of rituximab, and appropriate patient selection still remain unknown. A prospective study is planned. Table 1. Patient characteristics and treatment outcomes Patient Age Disease Graft Conditioning Recipient/Donor ANC〉500 Treatment Outcome/TTE 1 48M IgG MM PB MURD MyeloablativeBu/Cy O+/A+ F D+15 PEX 10 sessions PRCA unresolved Died 398 days after transplant of recurrent disease 2 56 M MDS PB MURD MyeloablativeFlu/Bu/ATG O+/B+ M D+11 PEX 10 sessions Withdrawal of immunosuppression Resolution of PRCA 87 days 3 58 M AML PB MRD MyeloablativeFlu/Bu O+/A+ M D+13 PEX 10 sessions Rituximab 2 doses Resolution of PRCA 13 days 4 49 M AML PB MRD MyeloablativeBu/Cy B+/A+ F D+11 PEX 10 sessions Rituximab 8 doses (2 cycles of 4 weekly doses separated by 6 months) Resolution of PRCA 253 days 5 70 M MDS PB MRD RIC Flu/Bu O+/A+ M D+15 PEX 7 sessions Rituximab 4 doses Resolution of PRCA 15 days M: male; MM: multiple myeloma; MDS: myelodysplastic syndrome; AML: acute myelogenous leukemia PB: peripheral blood; MRD: matched related donor; MURD: matched unrelated donor; Bu: busulfan; Cy: cyclophosphamide; Flu: fludarabine; RIC: reduced intensity conditioning regimen; F: female; ANC: absolute neutrophil count; PEX: plasma exchange; TTE: time to red cell engraftment after PEX. Disclosures No relevant conflicts of interest to declare.

Description: Introduction A major challenge in the development of effective myeloma (MM) therapy is addressing tumor heterogeneity, with the presence of sub-clones that exhibit resistance to standard therapy. An ongoing area of investigation focuses on identification of myeloma initiating cells that demonstrate greater capacity for self-renewal and serve as a potential reservoir for disease recurrence. It has been postulated that MM cells arise from a primitive B cell precursor population distinct from the more differentiated malignant plasma cell population. The critical feature of these myeloma-propagating cells is thought to be the ability to efficiently recapitulate MM in immunocompromised mice. MUC1 is an oncoprotein aberrantly expressed in malignant cells, including multiple myeloma, that interacts with multiple transcription factors, such as NF-κB and the β-catenin/TCF4 complex, that regulate cell survival and proliferation critical for malignant transformation. We have previously demonstrated that MUC1 is expressed by AML leukemic blasts, as compared to normal hematopoietic stem cells, and blockade of MUC1 signaling prevents establishment of leukemia in immunocompromised animals. In the present project, we identify a unique population of CD34+/MUC1+/CD138+/CD20+ cells in primary MM bone marrow samples that exhibit features of myeloma initiating cells, as manifested by high levels of enzymatic ALDH activity, the ability to efflux Hoechst dye represented as “side population” (SP), and the ability to establish disease in immunocompromised mice. Of note, MM engraftment of unselected primary myeloma cells in a xenograft model has a low success rate, and typically requires the introduction of an artificial stromal support network. Methods and results Bone marrow aspirates were obtained from newly diagnosed MM patients using an established protocol approved by the IRB. Expression of MUC1, myeloid and lymphoid markers was assessed using multicolor flow cytometric analysis. While MUC1 shows only a minimal expression (

Description: Introduction: Despite recent advances in the treatment of multiple myeloma (MM), curative outcomes remain elusive with standard therapies. MM is characterized by immune dysregulation and a loss of tumor-associated T cell surveillance contributing to disease evolution. Cancer vaccines have emerged as a promising immunotherapeutic strategy that seeks to reestablish anti-tumor immunity through the effective presentation of tumor associated antigens in the context of positive costimulation and activation signals. DCOne is a cell based tumor vaccine derived from an acute myelogenous leukemia cell line, differentiated toward a dendritic cell (DC) phenotype (DCPrime, Netherlands). DCOne strongly expresses WT-1, PRAME, RHAMM, survivin, and MUC-1. Since MM cells commonly express these tumor-associated antigens as well, we explored the efficacy of DCOne in inducing myeloma-specific immunity. Methods/Results: We first investigated the capacity of DCOne to polarize T cells toward an activated phenotype. Peripheral blood mononuclear cells (PBMCs) derived from MM patients were cultured in the presence or absence of DCOne and pulsed with whole cell DCOne lysate 24 hours prior to analysis. Intracellular levels of IFN-γ and perforin in CD8+ T cells and intracellular levels of IL-10 in CD4+ T cells were assessed via FACS analysis. Following 10 days of co-culture, we observed an increased percentage of CD8+ IFN-γ+ T cells and an increased percentage of CD8+ perforin+ T cells in PBMCs co-cultured with DCOne versus those not co-cultured with DCOne (10.8% versus 3.3%, p=0.02, n=11 and 6.6% versus 1.7%, p=0.045, n=10, respectively). In contrast, exposure of PBMCs to DCOne did not alter the percentage of CD4+ IL-10+ T cells (p=0.53, n=8). Given the observed increase in IFN-γ- and perforin-positive CD8+ T cells after co-culture of MM PBMCs with DCOne, we investigated whether CD8+ T cells co-cultured with DCOne exhibited enhanced killing of autologous tumor cells in a standard cytotoxic T lymphocyte (CTL) assay measuring Granzyme B activity by FACS analysis. DCOne potently induced CTL-mediated killing of autologous MM cells as determined by an increased percentage of CD8+ Granzyme B+ cells in stimulated versus control cells (27.4% versus 12.5%, p=0.05, n=3). We next investigated the mechanism of action by which DCOne elicited a MM-specific response. We postulated that the allogeneic cell line would induce immune activation in part through the transfer of antigen to PBMC-derived antigen presenting cells; these antigen presenting cells would in turn be capable of eliciting response via MHC restricted presentation of antigen to autologous T cell populations. Consistent with this hypothesis, we demonstrated that 32% of patient-derived PBMCs exhibited uptake of DCOne RNA following co-culture (n=3). Uptake of DCOne RNA by patient-derived PBMCs was completely abrogated with Transwell separation. Next, we examined how patient-derived PBMCs take up DCOne antigens. Using Western Blot analysis, we found that MUC-1 and survivin are expressed in extracellular vesicles (EVs) derived from DCOne lysate. Thus, we conclude that DCOne antigens are trafficked via EVs secreted by the DCOne parent cell, whereupon they are taken up by MM patient-derived PBMCs. Conclusion: In conclusion, when cultured in vitro with MM patient-derived PBMCs, DCOne results in increased IFN-γ- and perforin-positive CD8+ T cells, as well as induction of autologous tumor killing by these cells. DCOne RNA is taken up by MM patient-derived PBMCs and trafficked to the extracellular environment via EVs. DCOne demonstrates in vitro efficacy as an "off-the-shelf" strategy for stimulating MM-specific immunity. A clinical trial is being planned. Disclosures Kruisbeek: DCPrime: Employment, Other: Founder, CSO, and CEO of DCPrime. Van Wetering:DCPrime: Employment. Arnason:Gilead: Consultancy. Rosenblatt:DCPrime: Research Funding; BMS: Research Funding; Astex: Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding.

Description: Introduction: The ADAMTS13 assay is routinely ordered as part of the work-up for thrombotic microangiopathies (TMAs). While severe deficiency of ADAMTS13 activity (≤10%) is specific for the diagnosis of idiopathic TTP and identifies a group of patients who respond well to treatment with therapeutic plasma exchange (TPE), the implications of mild-to-moderate deficiency (11-66%) remain unclear. This study aims to elucidate the presenting characteristics, clinical outcomes and diagnoses, and responses to TPE in TMA patients with non-critically low ADAMTS13 activity. Methods: Two hundred-fifty two adult TMA patients from 3 large academic medical centers over a 10-year period were identified based on the presence of microangiopathic hemolytic anemia (schistocytes on smear, platelets

Description: Introduction: Thrombotic thrombocytopenic purpura (TTP) is a hematologic emergency that requires the immediate initiation of therapy without access to the results of definitive testing and often in the setting of limited prior clinical experience. In this context, a simple, validated, and rapidly deployable diagnostic scoring system could be a powerful tool. Methods: We studied 214 consecutive adults admitted to 3 major teaching hospitals between 2004 and 2014 with thrombocytopenia (range: 5 to 149,000/μL) and schistocytes suspicious for TTP (derivation cohort). Twenty-nine different laboratory and clinical parameters were evaluated to identify those most highly associated with an ADAMTS13 activity level of ≤10%. Cutoffs for continuous variables were determined by generating receiver operator curves and identifying the cutoff point associated with the maximum Youden Index. Those parameters most associated with a low ADAMTS13 level by univariate analysis (P≤0.1) were included in a logistic regression to create a parsimonious predictive model. The resulting model was validated through a prospectively collected internal cohort (n=40) and an external cohort (n=147) drawn from a different academic medical center. Results: Four individual laboratory parameters (platelet count

Description: Key Points Costimulatory blockade using abatacept represents a novel therapeutic approach for the treatment of cGVHD. Abatacept resulted in a clinical response in 44% of patients with both decreased prednisone use and T-cell PD-1 expression in responders.

Description: Introduction: Chronic graft versus host disease (cGVHD) remains a major source of morbidity and mortality following allogeneic transplantation. While corticosteroids remain first line therapy for cGVHD, they are associated with significant toxicity, and a substantial proportion of patients fail to completely respond. Treatments for steroid-refractory cGVHD are limited. While the pathophysiology of chronic GVHD is complex, activated T cells play a critical role, driven by allo-antigen stimulation. As such, inhibition of T cell activation via blockade of co-stimulation has potential as a therapeutic target in cGVHD. Abatacept is a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 and a fragment of the Fc domain of human IgG1 that has been modified to prevent complement fixation and antibody-dependent cellular cytotoxicity. Abatacept is the first drug in a class of agents termed "selective co-stimulation modulators." The CTLA-4 moiety of Abatacept binds specifically to CD80 and CD86 and down-modulates the CD28-mediated co-stimulation of T cells. We conducted a phase I clinical trial was conducted to evaluate the safety, clinical and immune effects of Abatacept in patients with steroid-refractory cGVHD. Methods: The study followed a 3+3 design with two escalating doses of Abatacept to determine the maximum tolerated dose (MTD): 3 mg/kg and 10 mg/kg. Dose-limiting toxicities (DLTs) were defined as Grade 3 or 4 toxicities judged to be probably or definitely related to Abatacept. Infection was not considered a DLT. Abatacept was administered for a total of 6 doses. Doses 1-3 were administered at two-week intervals. One month following Dose 3, Abatacept was given at four-week intervals for three doses (Doses 4-6). Inclusion criteria included recipients of allogeneic bone marrow or stem cell transplantation with myeloablative or reduced intensity conditioning, with cGVHD defined by NIH consensus criteria. Patients must have had treatment with ≥ 0.5 mg/kg/day of prednisone for at least 4 weeks. Patients with active malignant disease relapse or other active malignancy and patients with uncontrolled infection were excluded. Peripheral blood was drawn prior to each dose of Abatacept and following completion of therapy to assess the effect of treatment on circulating T cells. PD-1 expression on circulating T cells, and T cell expression of interferon gamma versus IL-10 was assessed by multichannel FACS analysis. Results: 17 subjects were treated. Three patients were treated at a dose of 3 mg/kg without DLT. Three evaluable patients completed treatment on cohort 2, at a dose of 10mg/kg without DLT. A forth participant withdrew consent following one dose of treatment and therefore is not evaluable. Ten patients were treated on an expansion cohort at a dose of 10mg/kg. We observed one grade 4 pulmonary infection, and three grade 3 pulmonary infections which resolved. Other Abatacept related adverse events included grade 2 gastritis (n=1), grade 2 pain (n=1), and grade 1 diarrhea (n=2), fatigue (n=2), rash (n=1), and skin pain (n=1). Of the 16 evaluable patients, 7 (44%) achieved a clinical partial response as defined by improvement of two disease systems based on the 2011 NIH consensus criteria. Abatacept resulted in a 51.3% reduction in prednisone usage in clinical responders with a mean baseline dose of 27mg compared to a mean dose of 14mg 1 month following the 6th dose of Abatacept (p = 0.01). PD-1 expression on circulating CD4+ and CD8+ T cells increased from a mean of 3.4% and 2.7% respectively at baseline to a mean of 8.9% and 7.6% respectively at one month following the 6thdose of Abatacept in clinical responders (n=3; p