ALBERT

Description: MgCO3·MgCl2·7H2O is the only known neutral magnesium carbonate containing chloride ions at ambient conditions. According to the literature, only small and twinned crystals of this double salt could be synthesised in a concentrated solution of MgCl2. For the crystal structure solution, single-crystal diffraction was carried out at a synchrotron radiation source. The monoclinic crystal structure (space group Cc) exhibits double chains of MgO octahedra linked by corners, connected by carbonate units and water molecules. The chloride ions are positioned between these double chains parallel to the (100) plane. Dry MgCO3·MgCl2·7H2O decomposes in the air to chlorartinite, Mg2(OH)Cl(CO3)·nH2O (n = 2 or 3). This work includes an extensive characterization of the title compound by powder X-ray diffraction, thermal analysis, SEM and vibrational spectroscopy.

Description: During evaporation of natural and synthetic K–Mg–Cl brines, the formation of almost square plate-like crystals of potassium carnallite (potassium chloride magnesium dichloride hexahydrate) was observed. A single-crystal structure analysis revealed a monoclinic cell [a = 9.251 (2), b = 9.516 (2), c = 13.217 (4) Å, β = 90.06 (2)° and space group C2/c]. The structure is isomorphous with other carnallite-type compounds, such as NH4Cl·MgCl2·6H2O. Until now, natural and synthetic carnallite, KCl·MgCl2·6H2O, was only known in its orthorhombic form [a = 16.0780 (3), b = 22.3850 (5), c = 9.5422 (2) Å and space group Pnna].

Description: Key Points GVHD elicits profound defects in DCs that prevent the priming of virus-specific T cells. Transfer of polyclonal T cells from immune donors at transplant provides effective antiviral immunity despite the presence of active GVHD.

Description: Background: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse cell surface antigen expression. Multidimensional flow cytometry (MDF) quantifies the expression of such surface antigens on neoplastic cells, permitting an objective means for sub-classification of this disorder. Objective: To investigate the relationship between the presenting immunophenotype and response to therapy in a large, controlled study of pediatric AML patients. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for MDF at diagnosis, (2) proper consent for specimen testing and (3) leukemia comprising 〉10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs. Unsupervised hierarchical clustering analysis was performed to mathematically define groups of patients with similar diagnostic IEPs. Selection of the appropriate number of clusters was accomplished by minimizing within-cluster variation while maximizing between-cluster variation. The rate of measurable residual disease (MRD) by MDF at the end of initial induction therapy (EOI1) and event free survival (EFS) was determined for each group and compared to the mean MRD rate and EFS for the entire cohort. Of the 769 total patients, 643 (84%) had bone marrow specimens submitted for MRD by MDF at EOI1. Results: Of the 643 patients with bone marrows submitted for MRD by MDF at EOI1, 223 (35%) were positive. The 5 year EFS rate was 49% for the study (N=769). Hierarchical clustering analysis of the IEPs defined 11 groups of patients (A-K) with similar quantitative diagnostic immunophenotypes. Five immunophenotypic groups of patients were associated with comparableEOI1 MRD rates (15%-36%) and EFS (50%-69%) to the study average. These 5 groups (A-E) consisted of immunophenotypes close to the normal counterpart of myeloid progenitor cells (Groups A, B, C; N=266, 77 and 106) or normal monocytes (Groups D and E; N=41 and 68). Two immunophenotypic groups (Group F and G) of patients were associated with significantly higher rates of MRD (Group F: 54%, p

Description: Background: Risk stratification of pediatric patients with acute myeloid leukemia (AML) combines cytogenetics and molecular markers at diagnosis with detection of measurable residual disease (MRD) by multidimensional flow cytometry (MDF) after initial induction chemotherapy (EOI1). However, using MDF to distinguish between favorable and poor risk groups by diagnostic immunophenotype has not been sufficiently reproducible for clinical implementation. Objective: We sought to characterize a recurrent, unique diagnostic immunophenotype identified among patients treated on Children's Oncology Group trial AAML0531 and determine the clinical characteristics, frequency, and outcome of patients with this immunophenotype in a large cohort. Patients and Methods: Of the 1022 newly diagnosed pediatric patients with de novo AML who enrolled in protocol AAML0531 (exclusive of patients with promyelocytic leukemia and Down syndrome), 812 who submitted a specimen for MDF at diagnosis and gave consent to MRD testing were eligible for the study. Results: A recurrent diagnostic phenotype was identified by a combination of high intensity CD56 expression, a lack of HLA-DR, dim to negative CD38 expression and dim to negative CD45 expression (designated as RAM phenotype). At diagnosis 19 patients (2.3%) expressed this phenotype and MRD was subsequently detected by MDF in 16/19 patients at EOI1 at a level of 0.02%-41% (median 0.3). Eleven patients with this phenotype submitted bone marrow specimens for MDF analysis at all 3 additional monitoring time points during therapy and in 8 patients MDF analysis revealed disease persistence throughout treatment. The experimental cohort (RAM cohort, N=19) and 2 control cohorts, one consisting of all other patients with CD56 positive AML (CD56+ control, N=166) and the other of patients with CD56 negative AML (CD56- control, N=627), were analyzed for clinical outcomes. Patients in the RAM cohort were significantly younger with a median age at diagnosis of 1.26 years [range 0.75-16.89] than those in the CD56+ control (median 7.57 years [range 0.01-29.8], p=0.002) and the CD56- control (median 10.64 [range 0.02-23.94], p

Description: Background: Acute myeloid leukemia (AML) is a heterogeneous disease with high genomic and phenotypic complexity. Although a relationship between phenotype and t(15;17) is recognized, the effect of other genotypic abnormalities on phenotype remains to be elucidated. Objective: To explore the relationships between genotypic and phenotypic abnormalities in diagnostic specimens in pediatric AML. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for multidimensional flow cytometry (MDF) at diagnosis, (2) consent to blood bank specimen testing and (3) leukemia comprising 〉10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs. Unsupervised hierarchical clustering was performed to mathematically cluster patients with similar IEPs. The underlying karyotypic and mutational profile of each patient was compared to the IEPs to identify clusters of patients with relationships between phenotype and identifiable genetic abnormalities. Supervised bagged tree-based models were used to quantify the importance of each cell-surface antigen in identifying these clusters. Levene's test was used to assess the homogeneity of mean cell-surface antigen expression for important markers. Results: Analysis of hierarchical clustering results revealed 7 immunophenotypic clusters (A-G) of patients with relationships between IEP and 4 common genetic abnormalities (Figure 1). Cluster A and Cluster B were both strongly associated with the t(8;21) karyotype (Figure 1). 54/62 patients in Cluster A (87%) and 24/26 patients in Cluster B (92%) were positive for t(8;21). The most important cell-surface antigens in identifying patients with the Cluster A associated phenotype were dim CD33, expression of CD56, expression of CD34 and expression of HLA-DR. The mean antigen expression of these markers was significantly more homogenous for patients in Cluster A compared to all other patients for CD33 (p = 0.02), CD34 (p 〈 0.01) and HLA-DR (p

Description: Introduction: Adenovirus (AdV) infection is a severe complication after hematopoietic stem cell transplantation, particularly in paediatric patients. Control of AdV infection particularly seems to be dependent on CD4+ T-cells but their main AdV antigenic targets have remained unknown so far. In order to design protocols for targeted adoptive immunotherapy the identification of the main AdV antigenic targets is a fundamental prerequisite. Methods: We here adapted a novel technique to directly assess the entire repertoire of CD4+ T-cells specific AdV antigens according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. The cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells were assessed by intracellular analysis of AdV-induced CD154+ CD4+ T-cells. Results: AdV-lysate-specific CD4+ T-cells reacted predominantly with AdV-hexon capsid protein. Furthermore, AdV-hexon (serogroup B) specific CD4+ T-cells crossreacted with recombinant hexon protein derived from various other AdV-serotypes and were characterized by a Th1-like cytokine profile. Conclusion: Our results prove the effectiveness of antigen-induced CD154-expression for assessment of the entire repertoire of CD4+ T-cells specific for pathogens, for the identification of immunodominant target antigen from pathogens. We demonstrate that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive CD4+ T-cells with a Th1-like cytokine profile for adoptive T-cell therapies.

Description: A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, improving hemophilia B treatment by allowing less frequent dosing than required with standard plasma-derived (pd) and recombinant (r) FIX products. The PROLONG-9FP clinical program has evaluated the use of rIX-FP for prophylaxis and on-demand treatment of bleeding in patients with severe hemophilia B. The clinical program is comprised of 5 clinical studies, including four completed studies (a Phase I pharmacokinetic (PK) study, a Phase II study, and two Phase III studies in adults and children), and an ongoing extension study, which includes previously untreated patients. Over 100 subjects from 42 hemophilia treatment centers in 12 countries have participated in the PROLONG-9FP clinical program. Here, we report on the long-term safety and efficacy of rIX-FP in 15 subjects who have continuously participated in 3 clinical studies (Phase II, Phase III and the on-going Phase III extension studies) over a period of 4 years. Subjects began either weekly prophylaxis treatment or on-demand treatment with rIX-FP in the Phase II 2004 study, and continued that treatment regimen in the Phase III 3001 study. The Phase III global study evaluated safety and efficacy of rIX-FP for prophylaxis treatment (PT) of every 7-, 10- and 14-days and on-demand treatment (ODT) of bleeding episodes. Subjects in the on-demand arm received only ODT for 6 months and then switched to 7-day PT. Subjects in the prophylaxis arm received 7-day PT for 6 months, and eligible subjects switched to 10- or 14-day PT interval. Upon completion of the 3001 study, subjects entered the on-going extension study 3003 and either continued the treatment regimen or switched to a longer prophylaxis interval of 10-, 14- or 21-days. Within subject comparisons of the annualized spontaneous bleeding rates (AsBR), total ABR over time and other efficacy parameters between regimens will be presented. Long-term use of rIX-FP is safe and well-tolerated. No subjects developed inhibitors to FIX or antibodies to rIX-FP during the 4 year treatment period, with a mean of 180 exposure days (EDs) for PT subjects and 125 EDs for ODT subjects. ABR decreased over time with rIX-FP prophylaxis, and longer treatment intervals were possible with no increase in consumption. Disclosures Lubetsky: CSL Behring: Consultancy. Martinowitz:CSL Behring: Honoraria, Speakers Bureau. Voigt:CSL Behring: Employment. Wolko:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Santagostino:Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Roche: Speakers Bureau; Octapharma: Speakers Bureau; Novo Nordisk: Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Bayer: Speakers Bureau; Biogen/Sobi: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; CSL Behring: Speakers Bureau.

Description: Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many hematological and immunological diseases but is hampered by the risk of graft failure or delayed engraftment. Clinical experience has shown that these problems can be overcome by transplantation of higher numbers of donor stem cells. This can be achieved by more efficient collection strategies (i.e. mobilization regimens) or by ex vivoexpansion. Here, we aim to transiently inhibit apoptosis in donor hematopoietic stem and progenitor cells (HSPCs) prior to transplantation in order to increase their numbers and their fitness. In previous studies we have identified two Bcl-2 proteins from the pro-apoptotic BH3-only subgroup, Bim and Bmf, to be central players in apoptosis induction of HSPCs during transplantation. Both proteins are efficiently repressed by the cytokines Flt3L and SCF and upregulated under cytokine deprivation in vitro. Lack of either protein or overexpression of their anti-apoptotic antagonists Bcl-2 or Bcl-xL strongly increased HSPC competitiveness during transplantation, both in murine transplantation and human xenotransplantation models. Moreover, less donor HSPCs were required for successful engraftment when Bim-mediated apoptosis was inhibited. In sum, our data indicated that modulation of Bim or Bmf levels inhibits apoptosis in murine and human HSPCs and that the resulting extended life span is beneficial during HSCT (Labi et al, 2013). Inhibition of the intrinsic apoptosis pathway could serve as a novel therapeutic approach to increase resistance of human HSPCs to factor deprivation and other types of stress caused during HSCT. However, permanent apoptosis inhibition in HSPCs can trigger their malignant transformation over time, especially when occurring together with activation of oncogenes promoting cell proliferation. Thus, such apoptosis inhibition needs to be transient when used therapeutically. Here we analyze whether transient apoptosis resistance lasting for only a limited time span is sufficient to increase competitiveness of HSPCs during HSCT. For overexpression of Bcl-xL we used adenoviral vectors known to act transiently. In proliferating murine HSPCs, adenoviral Bcl-xL persisted for 5-7 days, and for this period cells were protected from different stress stimuli engaging the intrinsic apoptosis pathway. Most importantly, adenoviral Bcl-xL overexpression increased the reconstitution potential of murine HSPCs in competitive transplantation experiments. Persistence of adenoviruses was excluded. As expected, transient Bcl-xL overexpression did not accelerate lymphomagenesis, neither on a wildtype nor on a premalignant murine background. However, adenoviral infection was associated with a relevant amount of toxicity to murine HSPCs. We thus performed transfection of full length Bcl-xL protein coupled to a protein transduction domain. Fluorescence microscopy indicated its mitochondrial localization, and functional tests revealed protection from apoptosis. However, when compared to adenoviral overexpression, Bcl-xL protein transduction proved to be less efficient due to the short protein half-life. In vivoexperiments are ongoing and will show whether such short-term apoptosis inhibition is sufficient to increase efficacy of transplantation. In sum, our studies will evaluate the benefit of therapeutic apoptosis inhibition in donor HSPCs during HSCT and contribute to on-going efforts aiming to improve transplantation medicine. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Under the auspices of the EC-Horizon 2020 project "TREGeneration", we are conducting five clinical phase I/II trials investigating the safety and efficacy of donor regulatory T cell (Treg) infusion in patients with chronic Graft-versus-Host Disease (cGvHD), a serious complication following hematopoietic stem cell transplantation (HSCT). Workpackage 4 evaluates T cell receptor (TCR) repertoire changes by high-throughput sequencing following the infusion of donor Tregs. Methods For repertoire analysis, four different T cell subsets (Treg, conventional CD4 T cells - Tcon, total CD4 and CD8 T cells) are FACS sorted from the infused product and from the cGvHD patients' peripheral blood before the infusion and at 10 time-points up to 12 months post infusion (Fig. 1A). To monitor TCR repertoire changes over time, we developed a novel quantitative "DeepImmune" technique for TCRβ amplification and sequencing. We amplify all TCRβ chain family transcripts from total RNA using a normalized primer set including molecular identifiers for unbiased and quantitative determination of TCRβ copy numbers. We have normalized the concentrations of our TCRβ primers using synthetic TCRβ gene sequences, thus minimizing amplification biases during RT-PCR to ≤3-fold differences among gene families. The inclusion of molecular identifiers during the RT-PCR step allows us to computationally remove additional biases introduced during the secondary PCR step for Illumina library preparation. Thus, our method results in ultra-deep characterization of immune receptor repertoires for sensitive detection of differences among samples and sample cohorts. Depending on T cell numbers in the samples, we routinely identify 1,000 - 150,000 clonotypes per sample. Results As first quality control for repertoire analysis, we cluster the top 200 TCRβ clonotypes from the different sequenced T cell subsets for each patient, resulting in heatmaps showing prominent repertoire differences among the analyzed T cell subsets (Fig. 1B; infused patient example shown). Next, we track the fate of infused donor Treg cells in the time course samples of the patients after Treg infusion, allowing us to quantify the number and frequency of TCRβ clonotypes that persist or expand in the patients after the infusion. We report that TCRβ clonotypes specific to the infused Treg cells (which were not present in the patients before Treg infusion) can be detected in the patients' blood after the infusion. Infusion-specific Treg clonotypes can reach up to 10-15% of the Treg repertoire of the patients and usually decline over time (Fig. 1C; boxplot shows clonotype overlap between infusion-specific Treg clonotypes and patient Treg clonotypes from five patients of the Lisbon cohort over 12 months). In the Tcon compartment of the patients, infusion-specific Treg clonotypes typically reach only 1-2% of the Tcon repertoire of the patients and also decline over time (Fig. 1D), similarly to what is observed in the CD8 T cell compartment (data not shown). This suggests that infused Tregs are not reverting to Tcons, an important control and promising result. We apply the same type of analysis to track clonotypes that were shared between the infused product and the patient prior to infusion, in addition to those clonotypes that were unique to the patient before infusion and not present in the infused product. Conclusion By applying our robust and sensitive TCRβ sequencing platform to samples from cGvHD patients infused with donor Treg, we are able to detect unique clonotypes of the infused product up to 1 year post-infusion. It remains to be be determined if there is any correlation between the level of detection of the infused clonotypes and the clinical responses of cGVHD to donor Treg therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.