Setting Of Bone Marrow Transplantation Research Articles

Sports in Pediatric Oncology

Malignant disease and anticancer therapy dramatically affect daily life activities and participation in grassroots and high-performance sports. Specifically in childhood and adolescence such activities are relevant factors of individual development and social life. This review focuses on the inherent reduction of normal physical activity in pediatric oncology because this cutback additionally contributes to the level of burden of malignancies. Maintaining normality requires detailed analyses of disease-related and therapy-related restrictions and their justification. Relevant efforts should be stepped up to maintain physical activity levels during pediatric cancer therapy. Another aspect addresses direct therapeutic implications. Feasibility studies, nonrandomized as well as randomized investigations addressed therapeutic effects in acute hospital care, in bone marrow transplant settings, and in outpatient therapy. The overall summary shows positive effects on clinical and psychosocial outcome. Even if the basis of the data for children is still limited, there will be no doubt about a general impact of physical activity on acute side effects as well as late effects. In the areas of tension between context-related restrictions, the right to maintain normality wherever possible and the positive therapeutic and psychosocial perspectives of sports, strong efforts are needed to support physical activity wherever indicated, clarify contraindications, and overcome structural limitations.

Efficacy of Subcutaneous Bortezomib in the Management of Patients with Multiple Myeloma or Relapsed Mantle Cell Lymphoma

The identification of the ubiquitin–proteasome system as a new therapeutic target has been one of the most recent successes in cancer treatment. The development and clinical approval of the first-in-class proteasome inhibitor, bortezomib has revolutionized the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). In MM, bortezomib is now integrated in induction, conditioning, consolidation, maintenance, and salvage treatment protocols. Bortezomib-based regimens provide high remission rates and confer significant survival advantage compared to conventional chemotherapy in both the bone marrow transplant and non-transplant setting. In MCL, overall response rates in patients who have received at least one prior therapy range from 30 to 45%, even in chemotherapy resistant patients. Clinical trials to further improve the sequencing of bortezomib-containing combination therapies are ongoing. Until recently, intravenous injection was the standard route of bortezomib administration. However, severe adverse side effects, peripheral neuropathy in particular, were observed in up to 16% of MM patients and up to 54% of MCL patients treated with intravenous bortezomib, with grade 3 and 4 in 11 and 12% of patients, respectively. Moreover, complete remission rates, if at all, are low and duration of response is short both in MM and MCL. These limitations may be overcome by changing the method of bortezomib administration as well as by rationally combining bortezomib with other therapeutic agents. Indeed, recent data demonstrate that subcutaneous bortezomib administration is non-inferior to intravenous administration, with an improved systemic safety profile, good local tolerance, and a more convenient route of administration. Based on these data, subcutaneous bortezomib injection was approved as a supplemental new drug application for all approved indications in MM and MCL after at least one prior therapy. More than 30 clinical trials in MM and MCL are currently ongoing to evaluate the efficacy and safety profile of subcutaneous bortezomib also in induction, maintenance, and salvage therapy.

Notch signaling in hematopoietic cell transplantation and T cell alloimmunity

Notch signaling can regulate both hematopoietic progenitors and alloimmune T cells in the setting of allogeneic bone marrow or hematopoietic cell transplantation (allo-HCT). Ex vivo culture of multipotent blood progenitors with immobilized Delta-like ligands induces supraphysiological Notch signals and can markedly enhance progenitor expansion. Infusion of Notch-expanded progenitors shortened myelosuppression in preclinical and early clinical studies, while accelerating T cell reconstitution in preclinical models. Notch also plays an essential role in vivo to regulate pathogenic alloimmune T cells that mediate graft-versus-host disease (GVHD), the most severe complication of allo-HCT. In mouse allo-HCT models, Notch inhibition in donor-derived T cells or transient blockade of Delta-like ligands after transplantation profoundly decreased GVHD incidence and severity, without causing global immunosuppression. These findings identify Notch in T cells as an attractive therapeutic target to control GVHD. In this review, we discuss these contrasting functions of Notch signaling with high translational significance in allo-HCT patients.

De Novo Inflammatory Bowel Disease and Its Mimics After Organ Transplantation

Diarrhea is a common symptom after solid organ transplantation or hematopoietic stem cell transplantation, with a reported prevalence up to 72%. One of the uncommon causes for diarrhea in the posttransplant setting is development of de novo inflammatory bowel disease (IBD). The incidence of posttransplantation de novo IBD was shown to be higher than that in the general population (206 versus 20 per 100,000 cases annually). The frequency seems to be much higher following orthotopic liver transplantation than the transplantation of other solid organs. De novo IBD has also been described in the setting of bone marrow transplantation though not as commonly as after SOT. While IBD is considered an immune-mediated disorder and responds favorably to immunosuppressive, de novo IBD or IBD-like conditions can occur in the posttransplant period despite antirejection immunosuppressive therapy. Damage or pathogen-associated molecular pattern molecules and their associated ongoing inflammation within the transplanted organ and the recipients' intestine have been implicated as possible etiologies. Various viral, bacterial, and protozoal infections can mimic IBD in postorgan transplantation. Common IBD mimickers in the postbone marrow transplant setting are graft-versus-host disease, infectious enteritis/colitis, and less commonly "cord colitis" that is described in detail below. In this article, we discuss the epidemiology, clinical features, and outcomes of de novo IBD after transplantation and highlight their differences in presentation, diagnosis, and management.

Spontaneous Subcapsular Splenic Hematoma Associated with Filgrastim in a Patient Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

To report the development of a spontaneous subcapsular splenic hematoma following filgrastim administration in a patient undergoing an allogeneic hematopoietic stem cell transplant. A 60-year-old female with myelodysplastic syndrome was admitted for a reduced-intensity allogeneic hematopoietic stem cell transplant from an unrelated donor. She received filgrastim 5 μg/kg starting on day 1 to accelerate neutrophil recovery. On day 5, she began reporting severe left chest-wall pain. Contrast-enhanced computed tomography of the abdomen/pelvis revealed a spontaneous subcapsular splenic hematoma. Upon discontinuation of filgrastim, the pain fully resolved. The patient was subsequently rechallenged with filgrastim, which led to recurrence of the left-sided chest-wall pain. Filgrastim was discontinued and the patient reported resolution of the pain. Filgrastim has been associated with splenic hematoma and splenic rupture, predominantly in healthy donors undergoing mobilization of peripheral blood stem cells. Although splenic rupture attributed to filgrastim in a patient undergoing allogeneic hematopoietic stem cell transplantation has been reported, to our knowledge, this is the first case report to establish causality. Using the Naranjo probability scale, an objective causality assessment revealed that the adverse drug event was highly probable. Although filgrastim-induced splenomegaly, splenic hematomas, and splenic rupture are rare, clinicians working in the bone marrow transplant setting should be cognizant of the potential of growth factors to cause these adverse events.

Overlapping roles for endothelial selectins in murine hematopoietic stem/progenitor cell homing to bone marrow

Selectins are carbohydrate-binding adhesion molecules that are critically involved in leukocyte recognition of endothelium. The endothelial selectins have been implicated in homing of hematopoietic stem and progenitor cells (HSPCs) to the bone marrow (BM) during bone marrow transplant (BMT), but the precise roles of individual selectins in this process have never been defined. BMT of lethally irradiated mice lacking both endothelial selectins (E/P KO) with limiting numbers of wild type BM cells rescued significantly fewer E/P KO than WT recipients, but higher numbers of transplanted WT cells rescued E/P KOs in a dose-dependent fashion. Short-term homing assays confirmed a substantial defect in HSPC homing to BM in E/P KO mice. In contrast, BMT of E-selectin null or P-selectin null mice at limiting cell number uniformly rescued greater than 95% of the transplanted animals. Consistent with these functional results, flow cytometric analysis revealed both E-selectin ligands and P-selectin ligands on distinct subsets of HSPC. These results demonstrate overlapping functions for the endothelial selectins in HSPC homing to BM in the setting of BMT, and define a novel aspect of HSPC heterogeneity linked to selectin ligand expression.

Management of Chemotherapy-Induced Nausea and Vomiting

Chemotherapy-induced nausea and vomiting (CINV) is associated with a significant deterioration in quality of life. The emetogenicity of the chemotherapeutic agents, repeated chemotherapy cycles, and patient risk factors significantly influence CINV. The use of a combination of a serotonin 5-HT3 receptor antagonist, dexamethasone and a neurokinin 1 (NK1) receptor antagonist has significantly improved the control of acute and delayed emesis in single-day chemotherapy. Palonosetron, a second-generation 5-HT3 receptor antagonist with a different half-life, a different binding capacity and a different mechanism of action than the first-generation 5-HT3 receptor antagonists appears to be the most effective agent in its class. Aprepitant, the first and only agent clinically available in the NK1 receptor antagonist drug class has been used effectively as an additive agent to the 5-HT3 receptor antagonists and dexamethasone to control CINV. Rolapitant and netupitant are other NK1 receptor antagonists that are currently in phase III clinical trials. Despite the control of emesis, nausea has not been well controlled by current agents. Olanzapine, a US-FDA approved antipsychotic, has emerged in recent trials as an effective preventative agent for CINV, as well as a very effective agent for the treatment of breakthrough emesis and nausea. Clinical trials using gabapentin, cannabinoids and ginger have not been definitive regarding their efficacy in the prevention of CINV. Additional studies are necessary for the control of nausea and for the control of CINV in the clinical settings of multiple-day chemotherapy and bone marrow transplantation.

Romiplostim resistance in secondary failure of platelet recovery

Romiplostim, a thrombopoietin mimetic, is FDA approved for the treatment of thrombocytopenia in patients with refractory immune thrombocytopenia. Given the success of thrombopoietin agonists in the treatment of immune thrombocytopenia, data in other clinical settings are emerging. In the bone marrow transplant setting, secondary failure of platelet recovery and persistent thrombocytopenia are associated with increased mortality. In this case, romiplostim was attempted in a chronic lymphocytic leukemia patient who underwent a matched, unrelated donor, non-myeloablative stem cell transplant. This patient experienced profound thrombocytopenia despite dose escalation. Anti-thrombopoietin or anti-romiplostim antibodies were not detected by specific assays. Furthermore, the patient remains in disease remission with full chimerism. This case demonstrates that heavily pre-treated patients with limited bone marrow reserve may not respond to thrombopoietin agonists.

A Review of the Prevention of Nausea and Vomiting Induced by Chemotherapy

Chemotherapy-induced nausea and vomiting (CINV) is associated with a significant deterioration in quality of life. The emetogenicity of the chemotherapeutic agents, repeated chemotherapy cycles and patient risk factors significantly influence CINV. The use of a combination of 5-hydroxytryptamine-3 (5-HT3) receptor antagonists, dexamethasone and a neurokinin-1 (NK-1) receptor antagonist has significantly improved the control of acute and delayed emesis in single-day chemotherapy. Palonosetron, a second-generation 5-HT3 receptor antagonist with a unique mechanism of action, appears to be the most effective agent in its class. Aprepitant, the only agent clinically available in the drug class of NK-1 receptor antagonists, has been used effectively as an additive agent to the 5-HT3 receptor antagonists and dexamethasone. Despite the control of emesis, nausea has not been well controlled by current agents. Olanzapine, a US Food and Drug Administration-approved antipsychotic, has emerged in recent trials as effective for the prevention of chemotherapy-induced emesis and nausea and for the treatment of breakthrough emesis and nausea. Additional studies are necessary for the control of nausea and for the control of CINV in the clinical settings of multi-day chemotherapy and bone marrow transplantation.

Casein Kinase 1 Alpha Maintains Normal and Leukemic Stem Cells by Regulating p53 Activity

Abstract 209In order to identify novel approaches to the targeting of acute myeloid leukemia (AML), we performed a pooled in vivo shRNA screen on murine leukemic stem cells (LSCs) targeting factors related to Wnt-signaling. We found that silencing of casein kinase 1 alpha (Csnk1a1), a serine-threonine kinase and a critical negative regulator of beta catenin, dramatically depleted murine LSCs in vivo. This is a surprising result since beta catenin is essential for MLL-AF9 AML. Validation experiments with shRNA vectors co-expressing GFP recapitulated the result from the pooled screen and confirmed efficient knockdown of both the Csnk1a1 transcript and protein. To rule out off-target effects of the Csnk1a1 shRNAs, we co-expressed the shRNAs with a Csnk1a1 cDNA mutated at the shRNA binding sites, and observed a complete rescue of the proliferative defect. Additionally, we demonstrated that a kinase dead form of Csnk1a1(D136N) failed to rescue this proliferation defect. These results indicate the specific effect of these hairpins on Csnk1a1 function in leukemia cells.The role of Csnk1a1 in normal hematopoietic stem and progenitor cells (HSPCs) is not known. We introduced the Csnk1a1 shRNA vectors into HSPCs and followed GFP over time in a bone marrow transplantation setting. Over a 24-week period, we observed a 3–4 fold depletion of GFP positive donor cells with two independent Csnk1a1 shRNAs compared to control. In contrast, the same shRNAs resulted in a 20–25 fold depletion of leukemia cells in vivo over a 2-week time period, suggesting that leukemia cells are selectively dependent on Csnk1a1.To more rigorously study Csnk1a1 in hematopoiesis, we generated a Csnk1a1 conditional knockout mouse (loxP sites flanking critical exon 3) and crossed it with the Mx1-cre mouse, allowing for hematopoietic specific inducible Csnk1a1 excision. In competitive bone marrow transplantations, Csnk1a1(−/−) donor cells exhibited a severe competitive disadvantage resulting in a 20-fold depletion of donor cells over a 12-week period. Interestingly, Csnk1a1(−/−) donor cells were devoid of myeloid lineage cells, suggesting that Csnk1a1 is particularly important for the generation or survival of myeloid cells. Moreover, in line with our shRNA results, we found that Csnk1a1(−/−) cells were resistant to MLL-AF9 mediated transformation, demonstrating that Csnk1a1 is essential also for leukemia initiation.To identify critical targets of Csnk1a1, we performed gene expression profiling of Csnk1a1 silenced cells. We identified enrichment of a p53 signature using Gene Set Enrichment Analysis (FDR= 0.001). Induction of p53 and its target p21 was confirmed by western blots in both Csnk1a1 silenced leukemia cells and in Csnk1a1(−/−) bone marrow cells. Furthermore, we demonstrated that p53(−/−) leukemia cells are resistant to the proliferative defect induced by Csnk1a1 silencing.We next tested whether D4476, a small molecule casein kinase inhibitor, would exhibit selective anti-leukemic effects. Whereas treatment of LSCs with D4476 inhibited their proliferation (IC50: 7μM), concentrations up to 40μM had minimal effects on normal HSPCs. Confirming the specificity of the compound, we found that cells carrying Csnk1a1 shRNAs were sensitized to D4476 in a dose dependent manner. In contrast, overexpression of Csnk1a1 desensitized leukemia cells for D4476 treatment, suggesting that D4476 kills leukemia cells in a Csnk1a1 dependent manner. Finally, we mixed 10,000 HSPCs with 10,000 LSCs and treated them ex vivo with either D4476 or DMSO control for 48 hours followed by injection into lethally irradiated mice. Whereas exposure to the drug caused prolonged latency of disease with some recipients never developing leukemia, there was no significant effect on HSPC donor cell chimerism at 8 weeks post transplantation compared control, indicating limited toxicity from the drug.In summary, these findings identify Csnk1a1 as critical for maintaining both normal HSCs and LSCs via modulation of p53 activity. Importantly, LSCs were significantly more sensitive to small molecule inhibition of Csnk1a1, suggesting that Csnk1a1 may be an attractive new drug target in AML. Disclosures:No relevant conflicts of interest to declare.

Targeting PKCθ in alloreactivity and graft-versus-host-disease: unanswered questions and therapeutic potential

Protein kinase C isoform θ (PKCθ) is a key modulator of TCR signaling and mediates activation of NF-κB, NF-AT, and AP-1 transcription factors. Although in vitro studies of PKCθ-/- T cells have shown impaired activation responses, in vivo studies indicate that PKCθ requirement is not universal. While PKCθ is important in induction of experimentally induced autoimmune diseases in mice and generation of Th2 responses, it is not essential for induction of T cell proliferative and cytotoxic responses against influenza virus, LCMV, and vaccinia virus. The context-specific involvement of PKCθ in T cell responses suggests that inhibition of PKCθ may be beneficial in some but not all situations. In the bone marrow transplantation (BMT) setting, we have shown that graft-versus-host-disease (GVHD) cannot be induced in the absence of PKCθ. However, graft-versus-leukemia effects and T cell ability to clear virus infection remains intact. Therefore, PKCθ is a potential therapeutic target in BMT, inhibition of which may prevent GVHD while retaining anti-tumor and anti-infection responses.

A New Approach to Predict the Chance of Infectious Complications Following Autologous Hematopoietic Stem Cell Transplantation: Mannose-Binding Lectin ELISA

Abstract 4492 Introduction:Haematopoietic stem cell transplantation associated immuncompromised state carries high risk of infectious complications. Gram-positive cocci are responsible for the majority of the post-transplant bloodstream infections. Viral and invasive fungal infections can be significant causes of morbidity.Mannose-binding lectin (MBL) is involved in innate immune response. MBL is an acute phase protein, synthesized in the liver. MBL binds microbial surface carbohydrates and mediates opsonophagocytosis directly and by activation of the lectin complement pathway. MBL also functions as co-receptor of Toll-like receptor. Serum MBL level is genetically determined and quite stable. MBL deficiency is a result of impaired assembly or stability of multimers.In patients who received high dose chemotherapy/transplantation, the innate immunodeficiency is an additive risk factor for infectious complications.According to literature, significant association was shown between low concentrations of MBL and serious infections. MBL is a potential modifier of susceptibility to infection in patients who have chemotherapy-induced neutropenia. Furthermore, infections might also compromise the engraftment of stem cells and the development of cell-lines might be prolonged. Patients and methods:The association between serum MBL level and frequency, severity and occurrence of infections has been studied in 127 patients following autologous stem cell transplantation (ASCT). Subgroups, i.e. multiple myeloma, non-Hodgkin and Hodgkin lymphoma were formed and the infectious complications have been compared.A double-monoclonal antibody sandwich ELISA system (BioPorto, Denmark) was used, which is a sensitive method for determining the MBL antigen levels in the sera.The range of MBL level in healthy population varies between 5 and 5000 ng/ml, <100 ng/ml is defined as MBL deficiency. MBL antigen levels were measured following transplantation, in a period without the presence of active infection. Results:18 patients (out of 127) proved to be MBL deficient. The median time of the onset of first infection was day +5 in MBL deficient, while day +15 among non-MBL deficient patients following transplantation. More infections were found among MBL deficient patients (2.44 vs 2.28 infectious episodes/patient). When patients with more and less than 500 ng/ml serum MBL level were compared, similar trends were seen, but the difference was not significant.The occurrence of absolute MBL deficiency was not different between patients with malignant hematological diseases and the 294 healthy controls (14.5% vs 14%). Interestingly, MBL serum levels were significantly higher in the examined patients with malignant hematological diseases compared to healthy controls. Conclusions:MBL deficiency may predispose to infections. To our best knowledge, this is one of the first reports regarding MBL deficiency in bone marrow transplant settings. Our MBL deficient patients had a greater number of severe infections and experienced their first severe infection earlier, compared to nondeficient patients following ASCT. The measuring of MBL may be helpful in antibiotic treatment, in case of MBL deficiency earlier and more intensive treatment may be indicated.Table 1The distribution of infections by MBL levelsTotalMBL <100 ng/mlMBL >100 ng/mlnumber of patients12718109patients with infections11916103infected/total (%)93.788.8994.5number of infectious episodes29244248infectious episodes/ one patient2.32.442.28development of first infection (day, mean)13.75.315mean follow-up325.9311.5328.3bloodstream-infection26 (8.9%)3 (6.8%)23 (9.3%)fever, high CRP, severe mucositis70 (24%)12 (27.3%)58 (23.4%)respiratory tract infection72 (24.7%)16 (36.3%)56 (22.6%)viral infection22 (7.5%)2 (4.5%)20 (8.1%)oral mycosis13 (4.5%)1 (2.3%)12 (4.8%)GI tract disease39 (13.4%)7 (16%)32 (13%)elevated CRP32 (11%)2 (4.5%)30 (12%)other17 (5.8%)1 (2.3%)16 (6.5%) Disclosures:No relevant conflicts of interest to declare.

Absence of STAT1 Signaling in Host Hematopoietic Cells Leads to Enhanced Gvhd Induction and Involves Increased Expression of MHC Class II and Reduced PD-L1 Expression on Dendritic Cells,

Abstract 4026Interferon-g/STAT1 signaling plays a critical role in regulating dendritic cell activation and function. Blockade of IFN-g signaling leads to reduced DC activation and impaired anti-tumor and acquired adaptive immunity. We recently reported that lack of IFN-g/STAT1 in donor lymphocytes leads to reduced GVHD induction in both MHC- and mHA-mismatched mouse BMT models. In this study, we addressed the role of host STAT1 in the regulation of GVHD. Wildtype or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad). GVHD was induced using either BALB/c or B6 donor spleen cells. We unexpectedly observed that absence of STAT1 in recipient mice led to increased GVHD-associated mortality in both MHC-mismatched (MST 5 vs. 8, p=0.01) and mHA-mismatched (MST 11 vs. 23, p<0.01) BMT settings. The enhanced GVHD induction was found to be associated with increased activation (expression of CD69 and CD25) and allo-antigen driven proliferation of donor CD4 and CD8 T cells as determined by CFSE-dilution. As host APCs have been reported to being crucial for induction of GVHD, we phenotypically and functionally characterized STAT1 deficient DCs. Our studies revealed that STAT1-deficient bone marrow-derived dendritic cells (BMDCs) which were maturated in the presence of LPS showed significantly increased MHC class II, CD86, CD80 and CD40 expression compared with wildtype BMDCs. Furthermore, STAT1-deficient BMDC showed significantly increased direct allo-stimulatory capacity resulting in increased responder cell proliferation as determined by standard MLR assays using 3H-Thymidine uptake assays as well as CFSE-dilution studies. STAT1−/− BMDCs significantly promoted CD44+CD62L- expression in responder CD4 and CD8 T cells compared to wild type BMDCs (all p<0.001). The increased MHC II expression in STAT1-deficient DC was further confirmed in host CD11b+ and CD11c+ cells following GVHD induction in vivo. To determine whether non-hematopoietic cells in STAT1−/− host contribute to the increased GVHD induction, we created radiation chimeras in which STAT1 was only deficient in the hematopoietic compartment by transplanting 129.STAT1−/− BMC into 129.STAT1+/+ recipients following lethal irradiation. 120 days later GVHD was induced using fully MHC-mismatched BALB/c donor splenocytes. Similar to STAT1-deficient recipients STAT1−/− ®WT chimeras showed enhanced GVHD induction compared to STAT1+/+®WT chimeras (MST 11 vs. 5, p<0.05).To determine the mechanism underlying the enhanced expansion of donor T cells in response to stimulation with STAT1-deficient APC, we hypothesized that STAT-deficiency may impair expression of the T cell inhibitory molecules Programed Cell Death-Ligand1 or-2 (PD-L1,-L2) on APC. We therefore studied the expression of PD-L1 and PD-L2 expression on wildtype and STAT1-deficient DC. Indeed, were able to demonstrate that absence of STAT1 significantly suppressed PD-L1 expression on BMDCs upon in vitro LPS stimulation (Mean Fluorescence Intensity 167.2± 15.9 vs. 532.5±7.6, p<0.001) and also in vivo tested on day+ 6 post-BMT in the mHA-mismatched setting. In line with these results using in vitro stimulation we could demonstrate significantly reduced Activation Induced Cell Death (AICD) in activated B6.SJL CD69+ CD4 and CD8 cells stimulated with 129.STAT1−/− BMDCs compared to cells stimulated with 129.STAT1+/+ BMDCs (10.6±1.5% vs. 28.2±1.9 % for CD4; 13.0±0.7% vs. 30.5±1.1% for CD8 respectively, p<0.001 for all). Importantly, blocking IFN-g with neutralizing antibodies significantly increased MHC class II, CD86 expression and reduced reduced PD-L1 expression on BMDCs upon LPS stimulation. In summary, our data suggest two mechanisms how the absence of STAT1 signaling in host hematopoietic cells may promote the development of GVHD: First, increased expression of MHC II and co-stimulatory molecule in STAT1-deficient APC may lead to enhanced activation and proliferation of donor lymphocytes. Second, absence of STAT1 in maturated host DC inhibits PD-L1 expression thus leading to reduced AICD of activated donor lymphocytes. These findings suggest that STAT1-signaling modulates host APC function and shapes the GVH-response by causing increased allo-antigen-specific donor T cell activation, survival and proliferation. Disclosures:Lentzsch:Centocor Ortho Biotech: Research Funding; Genzyme: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Research Funding.

Dll4 Blockade Modifies the Bone Marrow Vascular Niche and Improves Donor Bone Marrow Cells for Bone Marrow Transplant

Abstract 1321The Delta:Notch signaling pathway regulates multiple aspects of hematopoietic cell differentiation and function and more recently Delta-like 4 (Dll4) has been shown also to regulate adult neo-vascularization (angiogenesis), namely in tumors, which led to the development of therapeutic strategies targeting Dll4 for the treatment of different cancers. Nevertheless, the current therapeutic strategies to target Dll4, which were suggested to result in non-functional vessels in the tumors, appear also to affect the vasculature of normal organs. Given the importance of the Bone marrow (BM) vascular niche in regulating hematopoiesis, we hypothesized Dll4 targeting might also affect BM function. In the present study, we describe 2 means of targeting Dll4: one using Dll4 specific neutralizing monoclonal antibodies (systemic treatment), and another using VECadCreERT2Dll4lox/lox mice, where Dll4 is specifically ablated on VE-Cadherin expressing cells. Using these 2 models, we investigated if Dll4 targeting affected the BM vascular niche and consequently affected hematopoietic recovery following irradiation and BM recovery in a transplant setting.First, we show that the genetic ablation of Dll4 on VE-Cadherin+ vessels (VECadCreERT2Dll4lox/lox mice) and also anti-Dll4 antibody treatment of WT sub-lethally irradiated mice (300rad) does affect the BM vascular niche, resulting in increased megakaryocytes (CD41+), CD31+ and VE-Cadherin+ vessels, without significant changes on CD105+ vessels. Interestingly, the systemic anti-Dll4 treatment of non-irradiated and sub-lethally irradiated WT mice increased the BM and peripheral blood myeloid (CD11b+) content and promoted hematopoietic progenitor cell (Sca1+Flk1−) mobilization, as determined by FACS analysis. The genetic model (VECadCreERT2Dll4lox/lox mice) showed no changes in hematopoietic cells percentage or mobilization.However, BM recovery in a transplant setting was improved in both models. Lethally irradiated WT mice transplanted with anti-Dll4 treated BM following sub-lethal irradiation or BM from VECadCreERT2Dll4lox/lox mice showed faster hematopoietic recovery than control mice transplanted with WT BM alone.Taken together, we show that Dll4 blockade perturbs the BM vascular niche, increasing VE-Cadherin+ and CD31+ vessel number and also megakaryocyte BM content, both in the setting of Dll4 genetic ablation (using VECadCreERT2Dll4lox/lox mice) and also using a systemic approach to target Dll4. However, these vascular changes do not, at least following sub-lethal irradiation, significantly affect the BM recovery or affect the normal distribution of the hematopoietic populations. Nevertheless, the significant effects observed in a BM transplant setting (both where donor had been exposed to the systemic anti-Dll4 blockade and where Dll4 had been specifically ablated on VE-Cadherin+ vessels) suggest the existence of cell autonomous (and BM vascular niche-autonomous) effects of Dll4 targeting which deserve further detailed analysis. These data also suggest targeting Dll4-expressing vessels in the setting of BM malignancies may have therapeutic benefit, since it appears to have limited effects on normal BM/hematopoietic function. We are currently investigating the therapeutic efficacy of Dll4 targeting using the genetic model and the antibody-based approaches, in the setting of malignant myeloma and also acute leukemias. Disclosures:No relevant conflicts of interest to declare.

Notch Signaling in Graft-Versus-Host Disease

Abstract SCI-14 Notch is a highly conserved signaling pathway with multiple functions in health and disease. In the hematopoietic system, Notch was first described for its essential role at early stages of T cell development in the thymus and for its involvement in T cell acute lymphoblastic leukemia. In addition, Notch is being increasingly recognized as a potent regulator of antigen-driven mature T cell responses with context-dependent effects. These effects are influenced by the regulated expression of Jagged and Delta-like Notch ligands in antigen-presenting cells under the control of Toll-like receptors and other innate signals. We are investigating the importance of the Notch pathway in T cell alloimmunity in the setting of allogeneic bone marrow or peripheral blood stem cell transplantation (allo-BMT). After allo-BMT, alloimmune T cell responses mediate beneficial graft-versus-tumor (GVT) activity as well as detrimental graft-versus-host disease (GVHD). Using the pan-Notch inhibitor DNMAML and other genetic models of Notch inhibition, we have discovered an essential function for Notch signaling in donor-derived alloreactive T cells. In multiple mouse models of allo-BMT, infusion of Notch-deficient T cells as compared to wild-type T cells led to dramatically reduced GVHD severity and lethality. This effect was apparent for both CD4+ and CD8+ T cells and did not involve global immunosuppression, since Notch-deficient T cells proliferated normally and expanded in host lymphohematopoietic organs. However, Notch-deficient donor T cells failed to accumulate in the gut, a key GVHD target organ, and produced markedly decreased amounts of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. In parallel, Notch inhibition was associated with increased accumulation of FoxP3+CD4+ T cells. Decreased cytokine production could not be explained by a classical T helper differentiation defect. In contrast to their defective induction of GVHD, Notch-deficient T cells remained capable of mediating cytotoxic and anti-tumor responses both in vitro and in vivo, leading to preserved GVT activity even against large numbers of tumor cells. We are currently investigating the molecular and cellular mechanisms of Notch action in alloreactive T cells. We are also exploring the role of individual Notch receptors and ligands at the alloimmune synapse in vivo. Altogether, our findings identify Notch inhibition in donor T cells as a novel strategy to induce beneficial immunomodulation rather than global immunosuppression after allo-BMT. Disclosures: No relevant conflicts of interest to declare.

Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning

In order to develop minimally toxic bone marrow transplantation (BMT) protocols suitable for use in a wider range of indications, it is important to identify ways to enhance BM engraftment at a given level of recipient conditioning. CXCL12/stromal cell-derived factor-1α plays a crucial physiological role in homing of hematopoietic stem cells to BM. It is regulated by the ectopeptidase dipeptidyl peptidase IV (DPPIV; DPP4) known as CD26, which cleaves dipeptides from the N-terminus of polypeptide chains. Blocking DPPIV enzymatic activity had a beneficial effect on hematopoietic stem cell engraftment in various but very specific experimental settings. Here we investigated whether inhibition of DPPIV enzymatic activity through Diprotin A or sitagliptin (Januvia) improves BM engraftment in nonmyeloablative murine models of syngeneic (i.e., CD45-congenic) and allogeneic (i.e., Balb/c to B6) BMT (1 Gy total body irradiation, 10–15 × 106 unseparated BM cells/mouse). Neither Diprotin A administered in vivo at the time of BMT and/or used for in vitro pretreatment of BM nor sitagliptin administered in vivo had a detectable effect on the level of multilineage chimerism (follow-up >20 weeks). Similarly, sitagliptin did not enhance chimerism after allogeneic BMT, even though DPPIV enzymatic activity measured in serum was profoundly inhibited (>98% inhibition at peak exposure). Our results provide evidence that DPPIV inhibition via Diprotin A or sitagliptin does not improve engraftment of unseparated BM in a nonmyeloablative BMT setting.

Lung transplantation after hematopoietic stem cell transplantation

Pulmonary insufficiency following bone marrow transplant (BMT) is common and has significant associated mortality. Lung transplantation (LTX) is the only viable treatment for patients with end-stage pulmonary disease, but LTX after BMT is an uncommon event given the medical candidacy of the potential recipients. We sought to evaluate the short- and long-term outcomes of LTX in BMT recipients. We performed a retrospective evaluation of our institution's longitudinal LTX and BMT databases. Demographic and outcomes variables were collected. We identified 639 LTX from January 1, 1988, through December 31, 2009, and 5525 BMT from program inception, March 21, 1974, through December 31, 2009. From the cross-referenced cohort, we identified four patients who had BMT followed by LTX. Our series was composed of two men and two women, with a mean age of 32.3 yr (range, 20-59 yr). Single LTX were performed in two recipients (50%). All patients had significant and expected morbidities related to their transplant immunosuppression. Three patients (75%) required cardiopulmonary bypass at the time of LTX. The two recipients who underwent bilateral LTX required open chest management and subsequent tracheostomy. All patients are still alive at follow-up (range, 19-119 months, median 39.5). Our study demonstrates that LTX in the setting of BMT is a high-risk operation with the potential for a tumultuous perioperative course. Despite this, good outcomes and survival are obtainable in carefully selected patients. Selection factors include clinically stable patients without active sepsis and preoperative optimization of nutrition in anticipation of a prolonged recovery. An experienced multidisciplinary team approach and a protocol-driven management plan are paramount for successful outcomes in this challenging population.

Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.

AbstractAbstract 1593 Introduction:Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure. Disclosures:No relevant conflicts of interest to declare.

HDAC11 Inhibits T-Cell Response to Alloantigens and Reduces Gvhd In Mice.

AbstractAbstract 1466 Background:Histone acetyltransferases and histone deacetylases (HDAC) regulate gene expression through acetylation-deacetylation of histones. HDACs are the target of a family of compounds known as HDAC inhibitors, which have been shown to suppress pro-inflammatory cytokines and reduce acute graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect after allogeneic bone marrow transplantation (BMT) in mice. However, the role of individual HDAC members in the development of GVHD is not clear. Recently, HDAC11, the newest member of the HDAC family has emerged as an important transcriptional regulator of inflammatory responses in antigen-presenting cells (APCs)1. Here, we evaluated the role of HDAC11 on APCs and T cells in the allogeneic BMT setting in mice with genetic disruption of HDAC11. Method:Proliferation of wild-type (WT) and HDAC11 knock-out (KO) T cells in response to allogeneic antigens was compared by [H3] thymidine incorporation assay. Using the same method, we also tested the antigen presentation ability of WT and HDAC11 KO APCs. For in vivo studies, we used a clinical relevant mouse model of BMT: C57BL/6 (B6) ® BALB/c. To evaluate the role of HDAC11 in the function of T cells and APCs, WT and KO mice on B6 background were used as donors and recipients, respectively. Recipient survival was monitored daily and GVHD symptom was evaluated at least twice a week. HDAC11 KO mice were supplied by Merck and Co., Inc. Results:In vitro, HDAC11 KO T cells proliferated stronger than WT T cells under the stimulation of allogeneic APCs. Recipients of HDAC11 KO T cells lost significantly more body weight (p < 0.05), and died significantly sooner than those of WT T cells (p < 0.01). The pathologic score of KO recipients was higher than that of WT recipients in each of GVHD target organs including lung, liver, small intestine and colon. Mechanistically, we found that there were significantly more total and IFNγ-producing donor T cells in the recipients of KO cells than those of WT cells (p < 0.05). Collectively, HDAC11 KO T cells have higher activity in response to alloantigens in vitro and induced more severe GVHD in vivo compared to WT T cells. In contrast, KO and WT APCs had a similar ability to stimulate allogeneic T cells in vitro, and no significant difference in GVHD development was observed in WT or KO recipients after allogeneic BMT. Conclusion:HDAC11 negatively regulates T-cell function, but has no significant effect on APC function. This finding provides a rationale to promote T-cell immunity or tolerance by inhibiting or enhancing HDAC11, respectively.1 Villagra et al. Nature Immunology, 10:92-100, 2009. Disclosures:No relevant conflicts of interest to declare.

Successful Implementation of GM-CSF Cellular Immunotherapy for the Treatment of Multiple Myeloma In a Mouse Model of Autologous Bone Marrow Transplantation

AbstractAbstract 4288A modified murine model of multiple myeloma (MM), derived from the well established 5T33MM cell line (originated from spontaneously developed MM in aged C57BL/KalwRij (B6Rij; H2b haplotype)), was utilized to mimic minimal residual disease after bone marrow transplantation (BMT). The 5T33MM model has many of the characteristics associated with human MM, including homing and growth in the bone marrow compartment, hypercalcaemia and elevated tumor-associated IgG2bk in the circulation, with diffuse osteolytic lesions. In contrast to human MM however, the 5T33MM cells are also often found in the mouse spleen, since in this species the spleen is a bone marrow-like hematopoietic organ. Due to limitations on the availability of B6Rij mice and the vast array of mutant mice on the closely related C57BL/6 (B6) background, that could be useful in probing mechanisms of action for MM development or immune resistance to the tumor, it is highly desirable to adapt the MM model for B6 mice. However, 5T33MM cells do not cause disease in immunocompetent B6 mice, presumably because of allogeneic differences that cause rejection of the tumor cells. To overcome this problem, we found that 5T33MM cells will develop MM disease in lethally irradiated B6 recipients in a syngeneic BMT setting. Using this model, we tested for the first time the feasibility of using GM-CSF secreting vaccines in combination with tumor MHC-matched BMT from B6 mice for the treatment of MM. To this end, B6 Mice received a single subcutaneous infusion of 2×106 irradiated 5T33MM cells engineered to secrete GM-CSF (GM-5T33MM), or were left untreated. Seven days later, mice were lethally irradiated with 850 cGy and transplanted with 2×106 anti-T cell-depleted B6 BM cells. On day 1 post-BMT, recipients were challenged with 2×106 5T33MM cells (i.v). Mice that presented with the disease, developed hind limb paralysis and signs of morbidity, along with extensive plasmacytoma growth in the bone marrow and the spleen (as determined by H&E staining and flow cytometric analysis of BM and spleen samples). This pathology was similar to aged B6Rij mice that develop MM, as reported in the literature. Statistical comparisons using a non-parametric log-rank test showed enhanced survival rate in the GM-5T33MM treated group 70 days post-BMT (60% survival), compared to 0% survival, MST = 25 d (p = 0.020) in non-vaccinated recipients. These results indicate that a single pre-transplant administration of GM-5T33MM cells can significantly improve survival of transplanted/5T33MM challenged mice, even in the absence of donor lymphocytes. Furthermore, it suggests that immune cells exposed to GM-5T33MM vaccination, that survive host irradiation, constitute a significant component of this anti-tumor effect.The current experimental model should provide the basis to study the efficacy of GM-CSF secreting tumor vaccination in combination with BMT for the treatment of MM, as well as the cellular mechanisms associated with it. Disclosures:No relevant conflicts of interest to declare.