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GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is upregulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing mAb halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1, not IL-34. Both anti-CSF-1- and BLZ945-treatment greatly reduced numbers of monocyte-derived cells and microglia in the CNS. However, anti-CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti-CSF-1 treatments reduced the size of demyelinated lesions, and microglial activation in the grey matter. Lastly, we found that bone marrow-derived immune cells were the major mediators of CSF-1R-dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with total ablation of CSF-1R-dependent cells.Significance StatementMultiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by accumulation of myeloid immune cells into the central nervous system (CNS). Both harmful and beneficial myeloid cells are present in EAE/MS, and a goal of MS therapy is to preferentially remove harmful myeloid cells. The receptor for CSF-1 (CSF-1R) is found on myeloid cells and it is important for their survival. CSF-1R can bind two ligands, CSF-1 and IL-34, but is unknown whether their functions in EAE/MS differ. We found that blocking CSF-1 depleted only harmful myeloid cells in the CNS and suppressed EAE, whereas blocking IL-34 had no effect. Thus, we propose that blocking CSF-1 could be a novel therapy for MS.

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are inflammatory diseases of the central nervous system (CNS) in which T helper (Th) 17 cells play a major role in the disease pathogenesis. Th17 cells that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) are pathogenic and drive inflammation of the CNS. IL-9 is a cytokine with pleiotropic functions, and it has been suggested that it controls the pathogenic inflammation mediated by Th17 cells, and IL-9R(−/−) mice develop more severe EAE compared to wild-type (WT) counterparts. However, the underlying mechanism by which IL-9 suppresses EAE has not been clearly defined. Here we investigated how IL-9 modulates EAE development. By using mice knockout for IL-9 receptor (IL-9R), we show that more severe EAE in IL-9R(−/−) mice correlates with increased numbers of GM-CSF(+) CD4(+) T cells and inflammatory dendritic cells (DCs) in the CNS. Furthermore, DCs from IL-9R(−/−) mice induced more GM-CSF production by T cells and exacerbated EAE upon adoptive transfer than did WT DCs. Our results suggest that IL-9 reduces autoimmune neuroinflammation by suppressing GM-CSF production by CD4(+) T cells through the modulation of DCs.

PURPOSE: Advances in treatment have increased survival rates and quality of life of pediatric CNS tumor patients leading to a growing number of long-term survivors. However, there is sufficient clinical and scientific evidence for the need of a highly specialized lifelong follow-up care due to multidimensional late effects. Furthermore, adolescence and young adulthood are challenging age periods when patients frequently get lost to follow-up potentially having severe impact on health and well-being. Since 2020, we have established a structured transfer of long-term survivors older than 18 years to a newly founded highly specialized adult care follow-up setting for childhood cancer survivors (IONA). The aim of this study was to evaluate the current transition process. RESEARCH DESIGN: The standard of care transition process at the neuro-oncology unit of the MUV includes a joint appointment with the patient, a pediatric neuro-oncologist, psychologist and/or social worker and the team of the adult care facility (physician, psychologist). Different elements are used to end care safely in the pediatric structure and building trust in the upcoming out-patient-department at the same time. The transition process was evaluated statistically and analyzed qualitatively with regard to the factors that define a safe transition. RESULTS: After two years (01/2020-12/2021) 114 patients had a joint transition appointment, two patients contacted IONA directly. Shortly after the joint appointment all patients had a scheduled follow-up meeting at IONA. 102 patients (87.9%) showed up, seven patients (6%) already had a planned appointment, two patients (1.7%) were in contact with IONA but had no possibility to show up in person. Only five patients (4.3%) did not attend the appointment and were lost to follow-up. CONCLUSION: A structured interdisciplinary transition concept is a successful option to establish controlled and patient-safe transition from pediatric to adult care setting.

IFN-β has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-β therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-β therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-β therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-β suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-β signaling in monocytes was required for EAE suppression. IFN-β increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-β. IFN-β treatment suppressed IL-1β expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1β production by monocytes, and, in a positive feedback loop, IL-1β augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1β production by monocyte. IFN-β inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1β secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-β actions on monocytes disrupting this proinflammatory loop.

Interleukin (IL)-37, a novel member of the IL-1 family of cytokines, has anti-inflammatory properties. It was shown that IL-37 suppresses innate immunity and allergic reactions by modulating dendritic cells (DCs) and mast cells and interfering with the mTOR pathway. Although multiple sclerosis (MS) patients have increased serum levels of IL-37; it is currently unknown whether IL-37 has a protective role in autoimmune neuroinflammation. Here we show that IL-37 suppresses experimental autoimmune encephalomyelitis (EAE), the prototypical animal model of MS, by ultimately inducing IL-10+ B cells. B cell-derived IL-10 was critical for EAE suppression when mice were treated with IL-37; however, IL-37 did not directly induce IL-10 in B cells. Instead, IL-37 triggered the mTORC2 pathway in DCs, leading to production of the tissue repair-associated cytokine amphiregulin (Areg), which stimulated CD4+ T cells to produce IL-21 and DCs to produce IL-27. Blockade of Areg, IL-21 and IL-27 revoked the protective effects of IL-37 by precluding an increase in the frequency of IL-10+ B cells. Our results describe an IL-37-Areg-IL-10 transcellular circuit that suppresses CNS autoimmune inflammation that can be further explored in future therapies for MS and other neuroinflammatory conditions.

D-mannose (D-m) is a glucose epimer found in natural products, especially fruits. In mouse models of diabetes and airway inflammation, D-m supplementation via drinking water attenuated pathology by modifying cellular energy metabolism, leading to the activation of latent transforming growth factor beta (TGF-β), which in turn induced T regulatory cells (Tregs). Given that Tregs are important in controlling neuroinflammation in experimental autoimmune encephalomyelitis (EAE) and likely in multiple sclerosis (MS), we hypothesized that D-m could also suppress EAE. We found that D-m delayed disease onset and reduced disease severity in two models of EAE. Importantly, D-m treatment prevented relapses in a relapsing-remitting model of EAE, which mimics the most common clinical manifestation of MS. EAE suppression was accompanied by increased frequency of CD4+FoxP3+ Tregs in the central nervous system, suggesting that EAE suppression resulted from Treg cell induction by D-m. These findings suggest that D-m has the potential to be a safe and low-cost complementary therapy for MS.

Background Autologous stem cell transplantation (ASCT) following BEAM (BCNU, etoposide, cytarabine, melphalan) conditioning is standard of care in relapsed low- and high-grade B-cell lymphoma (DLBCL) and other lymphoproliferative disorders, but BCNU is associated with interstitial pneumonia and an increased mortality. A less toxic regimen might improve the outcome of patients with lymphoma after transplantation. Objectives We investigated the role of bendamustine replacing BCNU in the BEAM regimen in patients with lymphoma undergoing ASCT. Patients/methods The conditioning regimen BendaEAM consisted of bendamustine, cytarabine, etoposide, and melphalan and was used in patients with Hodgkin's disease (HD) and Non-Hodgkin lymphoma (NHL). Results Forty-one patients with HD (n = 9) or NHL (n = 32) were consecutively treated with Benda-BEAM replacing BCNU. No pulmonary or renal toxicities occurred, and no patient died related to transplant. After a median follow-up of 55 months, CR rate was 56%, 18 patients (44%) showed progression after a median time of 7 months after transplantation (range: 2-29 months), and 11 patients (24%) have died, all due to lymphoma progression. The 1-, 2-, and 4-year PFS are 73.2%, 58.6%, and 55.6% and the 1-, 2-, and 4-year OS 85.4%, 78.0%, and 72.6%, respectively. Conclusion BendaEAM seems to be feasible with a promising response rate and acceptable toxicity.

Autoimmune diseases such as multiple sclerosis (MS) develop because of failed peripheral immune tolerance for a specific self-antigen (Ag). Numerous approaches for Ag-specific suppression of autoimmune neuroinflammation have been proven effective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. One such approach is intravenous tolerance induction by injecting a myelin Ag used for triggering EAE. However, the translation of this and similar experimental strategies into therapy for MS has been hampered by uncertainty regarding relevant myelin Ags in MS patients. To address this issue, we developed a therapeutic strategy that relies on oligodendrocyte (Ol)-derived extracellular vesicles (Ol-EVs), which naturally contain multiple myelin Ags. Intravenous Ol-EV injection reduced disease pathophysiology in a myelin Ag-dependent manner, both prophylactically and therapeutically, in several EAE models. The treatment was safe and restored immune tolerance by inducing immunosuppressive monocytes and apoptosis of autoreactive CD4(+) T cells. Furthermore, we showed that human Ols also released EVs containing most relevant myelin Ags, providing a basis for their use in MS therapy. These findings introduce an approach for suppressing central nervous system (CNS) autoimmunity in a myelin Ag-specific manner, without the need to identify the target Ag.

Background: Primary CNS lymphoma is a highly aggressive and rare type of extranodal non-Hodgkin lymphoma. Although, new therapeutic approaches have led to improved survival, the management of the disease poses a challenge, practice patterns vary across institutions and countries, and remain ill-defined for vulnerable patient subgroups. Material and Methods: Using information from the Austrian Brain Tumor Registry we followed a population-based cohort of 189 patients newly diagnosed from 2005 to 2010 through various lines of treatment until death or last follow-up (12-31-2016). Prognostic factors and treatment-related data were integrated in a comprehensive survival analysis including conditional survival estimates. Results: We find variable patterns of first-line treatment with increasing use of rituximab and high-dose methotrexate (HDMTX)-based poly-chemotherapy after 2007, paralleled by an increase in median overall survival restricted to patients aged below 70 years. In the entire cohort, 5-year overall survival was 24.4% while 5-year conditional survival increased with every year postdiagnosis. Conclusion: In conclusion, we show that the use of poly-chemotherapy and immunotherapy has disseminated to community practice to a fair extent and survival has increased over time at least in younger patients. Annually increasing conditional survival rates provide clinicians with an adequate and encouraging prognostic measure.

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are neuroinflammatory diseases of the central nervous system (CNS), where leukocytes and CNS resident cells play important roles in disease development and pathogenesis. The antimalarial drug chloroquine (CQ) has been shown to suppress EAE by modulating dendritic cells (DCs) and Th17 cells. However, the mechanism of action by which CQ modulates EAE is far from being elucidated. Here, we comprehensively analyzed the CNS of CQ and PBS-treated EAE mice to identify and characterize the cells that are affected by CQ. Our results show that leukocytes are largely modulated by CQ and have a reduction in the expression of inflammatory markers. Intriguingly, CQ vastly modulated the CNS resident cells astrocytes, oligodendrocytes (OLs) and microglia (MG), with the latter producing IL-10 and IL-12p70. Overall, our results show a panoramic view of the cellular components that are affect by CQ and provide further evidence that drug repurposing of CQ will be beneficial to MS patients.

The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and invasion of neoplastic stem cells in various myeloid malignancies. Although mast cells (MCs) reportedly express CD44, little is known about the regulation and function of this receptor in neoplastic cells in systemic mastocytosis (SM). We found that clonal CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and CD117++/CD34- MCs invariably express CD44 in patients with indolent SM (ISM), SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia (MCL). In addition, all human MCL-like cell lines examined (HMC-1, ROSA, and MCPV-1) displayed cytoplasmic and cell-surface CD44. We also found that expression of CD44 in neoplastic MCs depends on RAS-MEK and STAT5 signaling and increases with the aggressiveness of SM. Correspondingly, higher levels of soluble CD44 were measured in the sera of patients with advanced SM compared with ISM or cutaneous mastocytosis and were found to correlate with overall and progression-free survival. To investigate the functional role of CD44, a xenotransplantation model was employed using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and a short hairpin RNA (shRNA) against CD44. In this model, the shRNA-mediated knockdown of CD44 resulted in reduced MC expansion and tumor formation and prolonged survival in SCID mice compared with HMC-1.2 cells transduced with control shRNA. Together, our data show that CD44 is a RAS-MEK/STAT5-driven MC invasion receptor that correlates with the aggressiveness of SM. Whether CD44 can serve as therapeutic target in advanced SM remains to be determined in forthcoming studies.

Objective and methods We performed a single center analysis of 60 adult patients (33 males and 27 females) with myelodysplastic syndromes (MDS) or secondary acute myeloid leukemia (AML) who underwent allogeneic hematopoietic stem cell transplantation (HCT) at our institution.

Data standards consisting of key data elements for clinical routine and trial documentation harmonize documentation within and across different health care institutions making documentation more efficient and improving scientific data analysis. This work focusses on the field of myeloid leukemia (ML), where a semantic core of common data elements (CDEs) in routine and trial documentation is established by automatic UMLS-based form analysis of existing documentation models. These CDEs (n = 227) were initially reviewed and commented by leukemia experts before they were systematically surveyed by an international voting process through seven hematologists of four countries. The total agreement score was 86%. 116 elements (51%) of these share an agreement score of 100%. This work generated CDEs with language-independent semantic codes and international clinical expert review to build a first approach towards an international data standard for ML. A first version of the CDE list is implemented in the data standard Operational Data Model and additional other data formats for reuse in different medical information systems.

Background: Expression of allergens in human cells is a prerequisite for the development of antigen-specific cell therapy in IgE-mediated allergy. We developed a strategy how the clinically relevant major grass pollen allergen Phl p 5 can be efficiently secreted or expressed on the surface of human cells with preserved allergenic activity. Methods: The cDNA of Phl p 5 was fused to a leader peptide with or without a transmembrane domain and both constructs were ligated into a mammalian expression vector. Transfection of these plasmids into human cells resulted in a membrane-anchored or secreted version of Phl p 5, respectively, as determined by ELISA or flow cytometric analysis. Results: Both the secreted and membrane-anchored Phl p 5 proteins bound IgE from allergic patients in an immunoblot assay and induced specific histamine release and CD203c upregulation in basophils of grass pollen-allergic patients. Proliferation of peripheral blood mononuclear cells from Phl p 5-allergic individuals was induced upon stimulation with both variants of Phl p 5 expressed in human cells similar to recombinant Phl p 5. Conclusions: Secreted and membrane-anchored Phl p 5 expressed in human cells preserved B cell as well as T cell epitopes and may be used to develop and test various cell-based strategies for allergen-specific immunomodulation and to delineate the tolerance mechanisms involved therein.

Background Recent data suggest that, among other factors, comorbidity may be an important prognostic variable in patients with myelodysplastic syndromes (MDS) who are eligible for haematopoietic stem cell transplantation (SCT). Patients and methods We examined the overall survival (OS) and underlying risk factors in 45 adult patients with MDS (n = 38), chronic myelomonocytic leukaemia (n = 1), or secondary acute myeloid leukaemia (AML) arising from MDS (n = 6), who underwent allogeneic SCT at our Institution. Results With a median follow-up of 37 months, OS for all patients was 23%, post-transplant relapse occurred in 11 patients, and 10 patients died from treatment-related complications. The overall outcome and survival was independent of cytogenetic abnormalities and International Prognostic Scoring System (IPSS). However, we identified comorbidity as defined by the haematopoietic cell transplantation specific comorbidity index (HCT-CI), as a significant adverse prognostic variable in our MDS patients. Conclusions Based on these data and similar published data we recommend selecting patients with MDS or secondary AML for SCT according to the presence of comorbidities.

Summary Background The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. Methods An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. Results In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments Mr L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P

Combinations of agents may improve outcomes among elderly acute myeloid leukaemia (AML) and high-risk myelodysplastic syndrome (MDS) patients. We performed an adaptive phase I/II trial for newly-diagnosed AML or high-risk MDS patients aged ≥50 years using a Bayesian approach to determine whether 1 of 3 doses of bendamustine (45, 60, 75 mg/m(2) days 1-3), together with idarubicin (12 mg/m(2) days 1-2), might provide a complete response (CR) rate ≥40% with30% grade 3-4 non-haematological toxicity. We treated 39 patients (34 AML; five MDS with10% marrow blasts; median age 73 years). None of the three bendamustine doses in combination with idarubicin met the required CR and toxicity rates; the 75 mg/m(2) dose because of excess toxicity (two of three patients) and the 60 mg/m(2) dose because of low efficacy (CR rate 10/33), although no grade 3-4 non-haematological toxicity was seen at this dose. Median survival was 7·2 months. All patients began treatment as outpatients but hospitalization was required in 90% (35/39). Although we did not find a dose of bendamustine combined with idarubicin that would provide a CR rate of40% with acceptable toxicity, bendamustine may have activity in AML/MDS patients, suggesting its addition to other regimens may be warranted.

CD44, also known as Hermes antigen, is a multifunctional invasion receptor that mediates homing and expansion of normal and neoplastic stem- and progenitor cells in various organs including the bone marrow (BM). Mast cells (MC) and their progenitors also express CD44. However, little is known about the impact and regulation of CD44 in neoplastic cells in systemic mastocytosis (SM). We examined the expression, regulation, and functional role of CD44 in neoplastic cells in SM. As assessed by multi-color flow cytometry, CD34+/CD38- stem cells (SC), CD34+/CD38+ progenitor cells (PC), and KIT+/CD34- MC invariably expressed CD44 in all SM variants, including patients with indolent SM (ISM, 11/11), SM with associated hematologic neoplasm (SM-AHN, 7/7), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 8/8). Expression of CD44 on SC, PC, and MC increased significantly with the aggressiveness of SM. Moreover, soluble CD44 levels measured in the sera of patients with SM by ELISA were found to correlate with the WHO type of SM. In particular, significantly higher levels of soluble CD44 were measured in advanced SM compared to ISM, cutaneous mastocytosis (CM), or healthy controls (p In conclusion, CD44 is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in patients with advanced SM. Our data also suggest that CD44 is an important mediator of evolution and of malignant spread of neoplastic MC into various organs in SM. Future studies will show whether CD44 can serve as a therapeutic target in patients with advanced SM. Disclosures Hoermann: Gilead: Research Funding; Novartis: Honoraria; Amgen: Honoraria; Ariad: Honoraria. Sperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Arock:Agensys, Inc: Research Funding. Valent:Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment option for various hematologic disorders. However, life-threatening adverse events resulting from treatment-related toxicity, severe infections, and/or graft-versus-host disease (GvHD) can occur. We report on a 64-year-old patient suffering from secondary acute myeloid leukemia (AML) who underwent successful allogeneic HSCT while on invasive mandatory ventilation (IMV). The patient received reduced intensity conditioning (RIC) according to the FLAMSA-protocol. Acute respiratory failure occurred one day before scheduled HSCT. Following emergency endotracheal intubation the patient was transferred to the intensive care unit (ICU). Because of respiratory deterioration, stem cell infusion was postponed. After stabilization of respiratory parameters, HSCT was performed during IMV which was continued for seven days. Following hematopoietic regeneration the patient was discharged in good condition on day 35 after HSCT. This case illustrates that intubation and mechanical ventilation do not necessarily exclude leukemic patients from HSCT.

Background The mammalian target of rapamycin (mTOR) has recently been identified as a potential target in acute myeloid leukemia (AML). Methods We treated 5 patients with chemotherapy-refractory AML with the mTOR-inhibitor rapamycin at 2 mg per os daily for 14 days, with dose adjustment allowed to reach a target serum rapamycin concentration of 10–20 ng/mL. Four of five patients received additional hydroxyurea at constant dose during treatment with rapamycin. Results Two patients achieved a leukocyte response, in one of them, a prolonged response was seen. In the other patients, blast counts remained stable or increased during rapamycin therapy. We did not observe severe hematologic or non-hematologic side effects of rapamycin. Conclusion Rapamycin at 2 mg per day acts mildly cytoreductive in a subgroup of patients with refractory AML. Higher doses and drug combinations may be required to obtain long lasting anti-leukemic effects in these patients.

Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of tissue mast cells (MC) in various organ systems, including bone marrow (BM). Indolent and advanced forms of SM have been described. Whereas patients with ISM have a normal or near normal life-expectancy, patients with advanced SM, including those suffering from mast cell leukemia (MCL) have a poor prognosis. In these patients, neoplastic MC are usually resistant against conventional drugs and various targeted drugs. In rapidly progressive aggressive SM (ASM) and MCL, polychemotherapy followed by allogeneic hematopoietic stem cell transplantation (alloHCT) has been proposed. However, outcome of alloHCT in advanced SM is unknown, and it also remains uncertain whether clinically relevant graft-versus-SM (GVSM) effects may occur in these patients, as only sporadic case reports have been published. We performed a retrospective multi-center analysis to evaluate the outcome of alloHCT in patients with advanced SM. Fifty-four advanced SM patients receiving SCT in 32 transplantation centers in Europe and America were identified between 1990 and 2013. The median patient age was 45 years. Donors were: HLA identical siblings (31), unrelated donors (URD) (15), umbilical cord blood donors (UCB) (2), and haploidentical donors (1). In 5 patients, stem cell source was not defined (5). Thirty-four patients received myeloablative conditioning (MAC) and 18 received reduced intensity conditioning regimens (RIC). In 2 patients, conditioning regimen was not specified. Indications for alloHCT were SM with an associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) (n=32), MCL (n=13, including one with MCL-AHNMD), 8 with ASM and 1 with myelomastocytic leukemia (MML). The most prevalent AHNMD was acute myeloid leukemia (AML, n=16). With follow-up of 35-6180 (median 365) days, SM responses (defined as ≥50% decrease in BM mast cells ± decrease in serum tryptase ± regression of other organ manifestations) were observed in 39 patients (72%), including complete responses (CR) documented in 12 patients (22%). Eleven patients had stable disease, whereas 4 patients (7%) progressed immediately after alloHCT (primary resistance). In addition, 10 patients progressed (5 of them within 100 days) after an initial response. Progression was most frequently seen in MCL patients (n=6, 50%). In the AHNMD group, only 8 patients relapsed/progressed (25%). The overall survival (OS) and SM progression-free survival (PFS) at 1 year were 63% and 50% for all patients, 77% and 68% for SM-AHNMD, 63% and 50% for ASM, and 25% and 17% for MCL, respectively. The strongest predictive variable associated with inferior survival was a diagnosis of MCL. Other factors associated with poor outcome were: Karnofsky performance status ≤70%, ≥2 SM regimens given before alloHCT (e.g., steroids, cladribine, chemotherapy, tyrosine kinase inhibitor), donor source (alternative donors-UCB and haploidentical compared to sibling or URD), SM progression within the first 100 days, normal cytogenetics (compared to t(8;21) (q22;q22), and RIC (compared to MAC). The following variables were not associated with poor outcome: patient and donor age, recipient-donor sex match status, graft source (BM vs. peripheral stem cells), BM mast cell percentage at time of alloHCT, and CR status of AML or SM response at time of alloHCT. This largest multi-center analysis of results in advanced SM provides evidence for clinical efficacy of alloHCT, presumably because of a GVSM effect of alloHCT (achieving CR, and response to donor-lymphocyte infusions and RIC alloHCT). However, responses varied among different SM categories: while patients with SM-AHNMD enjoyed excellent outcomes, the OS for MCL patients in general, was poor. Nevertheless it is remarkable that 3 of 13 patients with MCL – an otherwise fatal disease with a median survival of Disclosures: Vercellotti: Sangart Inc.: Research Funding; Seattle Genetics: Research Funding. Akin:Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding.

Abstract 4504 Patients (pts) with acute leukemias i.e. acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) suffering form primary refractory disease or refractory relapse have a very poor prognosis. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potential curative treatment option for such pts. However, disease-control prior to HSCT is essential for long term CR. In the present study we have retrospectively analyzed the outcome of pts who received clofarabine (10 mg/m2, d 1–4) and cyclophosphamide (200 mg/m2, d 1–4; ClofCy) to reduce the burden of leukemic cells prior to alloHSCT. A total number of 18 pts (females, n= 11; males, n= 7; median age; 37.5 years, range 21–64 years) with refractory leukaemias (AML, n=14; ALL, n=4) received ClofCy between December 2008 and January 2012 (1 cycle: 7 pts; 2 cycles: 3 pts; 3 cycles: 7 pts; 4 cycles: 1 patient). In all pts a marked decrease in leukemic cells was observed after a median of 2 cycles (range 1–4 cycles). Side effects included infections (n=7), moderate skin rush (n=4), transient increase in ALT and AST (n=2) and diarrhoea (n=1). AlloHSCT was performed in 13/18 pts. Five pts were not eligible for alloHSCT because of severe systemic fungal infections in 3 pts, clinical deterioration in 1 patient, or CNS relapse of leukemia in 1 patient. Myeloablative conditioning (cyclophosphamide/TBI) was administered in 9 pts, and dose-reduced conditioning (FLAMSA n=2; Fludarabin/Melphalan/Carmustin/ATG n=2) in 4 pts. Following stem cell infusion (median number of CD34+ cells/kg: 7.22×106) from a related (n=4) or unrelated (n=9) donor all pts showed rapid haematologic engraftment and full donor chimerism (median time to ANC > 0.5 G/l: 16 days; range: 12–25 days; median time to platelet >20G/l without substitution: 17 days; range: 13–32 days). As evidenced by bone marrow biopsy on day +28, all pts achieved CR following alloHSCT. After a median observation time of 262 days (range: 33–1496 days) 7 pts are alive. One patient died because of acute steroid-refractory graft-versus-host disease (day +48) and one from a systemic fungal infection (day +56). Four pts died after following reoccurrence of leukemia. Three pts had a hematologic relapse (days +383, +275, +141, respectively) and 1 patient developed a myelosarcoma on day +934. Of the 7 patients alive 5 are in continuous CR (691, 673, 555, 533 and 170 days post TX), two patients had a relapse and achieved a CR after DLI or a second HSCT, respectively. Together, we demonstrate that cytoreduction with ClofCy is a novel reasonable treatment approach for pts with refractory acute leukemias prior to alloHSCT. The regimen is relatively well-tolerated and resulted in a high response rate. Whether this novel debulking protocol will lead to improved long term outcome in pts with refractory leukemias remains to be determined in forthcoming studies with larger patient samples and longer observation periods. Disclosures: Valent: Phadia: Research Funding. Sperr:Genzyme: Speakers Bureau.

Heme oxygenase 1 (HO-1), also known as heat shock protein 32 (Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. Recent data suggest that HO-1/Hsp32 is also expressed in neoplastic cells in various malignancies. In the present study, we provide evidence that HO-1 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML, n=17) and in various AML cell lines such as HL60, KG1, KG1a, and U937. Expression of HO-1 mRNA was demonstrable by RT-PCR, and the HO-1 protein by immunocytochemistry and Western blotting. In addition, we were able to demonstrate expression of HO-1 mRNA and of HO-1 protein in the CD34+/CD38− progenitor/stem cell fraction in the leukemic clone in patients with AML. The HO-1 inductor hemin (10 μM) was found to promote expression of HO-1 in AML cells. Incubation with the HO-1-targeting drugs pegylated zink protoporphyrin (PEG-ZnPP) or styrene maleic acid-conjugated ZnPP (SMA-ZnPP), resulted in a dose-dependent inhibition of growth of leukemic cells at pharmacologic concentrations (IC50: 5–20 μM for cell lines and primary AML cells). The SMA-ZnPP-induced growth-inhibition of AML cells were found to be associated with induction of apoptosis as evidenced by light microscopy, electron microscopy, and by a Tunel assay. In consecutive experiments, combination experiments were performed using SMA-ZnPP and AML cell lines. In these experiments, SMA-ZnPP was found to synergize with cytarabine in producing growth inhibition in all AML cell lines tested. In summary, these data show that HO-1/Hsp32 is a novel survival factor and interesting target in AML. The clinical significance of this observation remains to be determined in forthcoming trials.

An increased lactate dehydrogenase (LDH) level at diagnosis is associated with a reduced probability of survival and an enhanced risk of AML development in primary (de novo) myelodysplastic syndromes (MDS). However, so far, little is known about the prognostic value of an increase in LDH levels during the follow up in these patients. We have serially determined LDH levels in 221 patients (102 males, 119 females) with de novo MDS (median age 70 years; FAB-types: RA, n=62; RARS, n=46; RAEB, n=48; RAEBT, n=36; CMML, n=29), and examined the prognostic value of LDH as a follow-up parameter. Confirming previous data, an elevated LDH level at diagnosis was found to be associated with a significantly increased probability of AML evolution and a significantly decreased probability of survival (p

Dasatinib (BMS354825) is a small molecule-type inhibitory drug that blocks several tyrosine kinases including PDGFR, KIT, BCR/ABL, and several src kinases. Dasatinib has recently been introduced as a new effective antileukemic drug in patients with imatinib-resistant chronic myeloid leukemia. However, because of the many targets of dasatinib and the broad range of potential target-cells in physiologic and pathologic tissues, dasatinib has recently been examined for its immunosuppressive and anti-inflammatory effects. We here report that dasatinib at 1 μM completely blocks the anti-IgE-induced release of histamine in human blood basophils in healthy subjects. The effects of dasatinib on histamine secretion in basophils were dose-dependent (IC50: 50–100 nM). Dasatinib was also found to block recombinant allergen-induced release of histamine from human basophils in sensitized individuals. In addition, as assessed by flow cytometry, dasatinib (1 μM) was found to inhibit the IgE-dependent upregulation of several activation-linked antigens, including aminopeptidase N (CD13), LAMP-3 (CD63), endolyn (CD164), and the basophil-specific ectoenzyme E-NPP3 (CD203c) on the cell surface of human basophils. The effects of dasatinib on upregulation of basophil differentiation antigens were found to be dose-dependent and were seen in normal subjects as well as in allergic individuals. Together, these data show that dasatinib blocks IgE-mediated activation and histamine release in human blood basophils. These anti-allergic effects of dasatinib may have clinical implications and may point to additional indications for this multifunctional drug.

Recent data suggest that CD44 may serve as a new therapeutic target in AML and possibly also in other myeloid neoplasms. Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in one or multiple organs. We have previously shown that normal tissue MC express CD44. In the present study, we asked whether CD44 is expressed on neoplastic human MC and whether CD44-ligation by the monoclonal antibody (mAb) A3D8 would be associated with inhibition of growth of neoplastic MC. As assessed by flow cytometry, primary neoplastic MC were found to express CD44 in all patients with SM analyzed (n=10). The human mast cell leukemia (MCL) cell line HMC-1 was also found to express CD44. As assessed by 3H-thymidine incorporation, the CD44 mAb A3D8 decreased the proliferation of HMC-1 cells in a dose-dependent manner (A3D8, 5 μg/ml: 46±26% of control=100%, p

The mammalian target of rapamycin (mTOR) has been described to be activated in BCR/ABL-transformed cells and to mediate rapamycin-induced inhibition of cell growth. We have recently shown that rapamycin downregulates expression of vascular endothelial growth factor (VEGF), a mediator of leukemia-associated angiogenesis, in primary CML cells. In the current study, we analyzed growth-inhibitory in vitro- and in vivo effects of rapamycin in leukemic cells in patients with imatinib-resistant CML, and asked, whether the rapamycin-induced suppression of VEGF in these cells is related to growth-inhibition. Rapamycin dose-dependently inhibited the in vitro growth of primary CML cells in all pts with imatinib-resistant CML tested (rapamycin, 10 nM: 50±17% of control; p20,000 to normal values (

In most patients with systemic mastocytosis (SM) including aggressive SM and mast cell leukemia (MCL), neoplastic cells express the oncogenic c-KIT mutation D816V. KIT-D816V is associated with constitutive tyrosine kinase (TK) activity and thus represents an attractive target of drug therapy. However, most available TK inhibitors including STI571=imatinib, fail to block TK-activity of KIT D816V at pharmacologic concentrations. We provide evidence that the novel TK-targeting drugs PKC412 and AMN107 decrease TK-activity of D816V-mutated KIT and counteract growth of Ba/F3 cells with doxycycline-induced expression of KIT D816V as well as growth of the human mast cell leukemia cell line HMC-1 expressing this c-KIT mutation. PKC412 was found to be the superior drug with IC50 values of 50–250 nM and without differences seen between HMC-1 cells exhibiting or lacking KIT D816V. By contrast, AMN107 exhibited potent effects only in the absence of KIT D816V in HMC-1 cells. Corresponding results were obtained with Ba/F3 cells exhibiting wild-type or the D816V-mutated variant of KIT. Moreover, we found that PKC412 and AMN107 inhibit growth of primary neoplastic MC in a patient with KIT D816V+ SM. The growth-inhibitory effects of PKC412 and AMN107 on HMC-1 cells were associated with TK-inhibition of KIT and with induction of apoptosis. In addition, PKC412 was found to downregulate expression of CD2 and CD63, two cell surface antigens upregulated in SM. In co-incubation experiments, PKC412 was found to synergize with AMN107, imatinib, and 2CdA in producing growth inhibition in HMC-1 cells lacking KIT D816V, whereas in KIT D816V+ HMC-1 cells, drug-interactions were additive rather than synergistic. Together, PKC412 and AMN107 alone and in combination counteract growth of neoplastic mast cells. Both drugs may therefore be considered as novel promising agents for targeted therapy in patients with aggressive SM or MCL.

The mammalian target of rapamycin (mTOR), a key-regulator of cell cycle progression, has recently been implicated in growth of neoplastic cells and tumor- and leukemia-associated angiogenesis. In addition, mTOR has been described as a regulator of expression of vascular endothelial growth factor (VEGF), a cytokine involved in tumor-related angiogenesis, in various neoplasms. We asked whether mTOR can be employed as a new therapeutic target in acute myeloid leukemia (AML) using the mTOR-targeting drug rapamycin and its derivatives RAD001 (everolimus) and CCI-779. As assessed by 3H-thymidine incorporation, rapamycin was found to counteract growth of AML cells in vitro in 9/11 patients examined as well as in the AML-derived cell lines U937, HL60, and KG1a. The effects of rapamycin on growth of AML cells were dose-dependent (primary AML cells: IC50: 10 pM - 1 nM; inhibition of 3H-thymidine uptake at 1 nM of rapamycin: 43±15 % of control; p

In patients (pts) with mast cell (MC) proliferative disorders, the clinical course, prognosis, and outcome vary depending on age, organ-involvement, and the disease-variant. We have retrospectively analyzed the clinical course and outcome in 56 pts with MC disorders refered to the University of Vienna between 1987 and 2004. Using WHO criteria, pts were found to have cutaneous mastocytosis (CM, n=6), indolent systemic mastocytosis (ISM, n=35), aggressive SM (ASM, n=3), mast cell leukaemia (MCL, n=1), SM with associated clonal hematologic non-MC-lineage disease (SM-AHNMD, n=6), and myelomastocytic leukemia (MML, n=5). These groups differed from each other in serum tryptase levels, hemoglobin, platelet counts, and lactate dehydrogenase (p5%) of MC in bone marrow smears, and iii. splenomegaly. Polychemotherapy resulted in complete remission in MML, but not in ASM or MCL. Interferon-alpha and 2CdA showed cytoreductive effects in some pts with ASM, but did not work in MCL. For the latter group of pts new therapies need to be developed.